HK1191033B - Polypeptides and polynucleotides, and uses thereof for treatment of immune related disorders and cancer - Google Patents

Abstract

This invention relates to LY6G6F, VSIG10, TMEM25 and LSR proteins, which are suitable targets for immunotherapy, treatment of cancer, infectious disorders, and/or immune related disorders, and drug development. This invention further relates to soluble LY6G6F, VSIG10, TMEM25 and LSR molecules, extracellular domains of LY6G6F, VSIG10, TMEM25 and LSR and conjugates, which are suitable drugs for immunotherapy, treatment of cancer, infectious disorders, and/or immune related disorders. This invention further relates to antibodies and antigen binding fragments and conjugates containing same, and/or alternative scaffolds, specific for LY6G6F, VSIG10, TMEM25 or LSR molecules, which are suitable drugs for immunotherapy, treatment of cancer, infectious disorders, and/or immune related disorders.

Description

Polypeptides and polynucleotides and their use for treating immune related disorders and cancer

Technical Field

The present invention relates to LY6G6F, VSIG10, TMEM25 and LSR proteins along with soluble molecules and conjugates and antibodies to such, LY6G6F, VSIG10, TMEM25 and LSR proteins are suitable targets for immunotherapy, treatment of cancer, infectious disorders, and/or immune related disorders, and drug development.

Background

In order to become activated productively, naive T cells must receive two independent signals from Antigen Presenting Cells (APCs). The first signal 1 is antigen specific and occurs when the T cell antigen receptor encounters the appropriate antigen-MHC complex on the APC. The fate of the immune response is determined by a second, antigen-independent signal (signal 2) that is delivered by a T cell costimulatory molecule that engages its APC-expressed ligand. This second signal may be either stimulatory (positive co-stimulation) or inhibitory (negative co-stimulation or co-suppression). In the absence of a costimulatory signal, or in the presence of a costimulatory signal, T-cell activation is impaired or failed, which can lead to a state of antigen-specific anergy (termed T-cell anergy), or can lead to apoptotic death of the T-cell.

The costimulatory molecule pair typically consists of a ligand expressed on the APC and its cognate receptor expressed on the T cell. The prototype ligand/receptor pairs of co-stimulatory molecules were B7/CD28 and CD40/CD 40L. The B7 family consists of structurally related cell surface protein ligands that can provide either stimulatory or inhibitory inputs to an immune response. Members of the B7 family are structurally related in that the extracellular domain comprises at least one variable or constant immunoglobulin domain.

Both positive and negative costimulatory signals play a key role in the regulation of cell-mediated immune responses, and molecules that mediate these signals have been shown to be effective targets for immune regulation. Based on this knowledge, several therapeutic approaches involving targeting of co-stimulatory molecules have been developed and shown to be useful for the prevention and treatment of cancer by turning on or preventing the turning off of the immune response in cancer patients, and for the prevention and treatment of autoimmune and inflammatory diseases, as well as rejection of allografts, each by turning off uncontrolled immune responses in subjects suffering from these pathological conditions, or by inducing a "turn off signal" via negative co-stimulation (or co-suppression).

The manipulation of these signals delivered by B7 ligands has been shown to be potential in the treatment of autoimmunity, inflammatory diseases, and transplant rejection. Therapeutic strategies include blocking of co-stimulation using monoclonal antibodies to the ligands or receptors of one co-stimulation pair, or using soluble fusion proteins consisting of co-stimulation receptors that can bind and block their appropriate ligands. Another approach is to use soluble fusion proteins of an inhibitory ligand to induce co-inhibition. These pathways rely, at least in part, on the eventual deletion of autoreactive or alloreactive T cells (which are responsible for the pathogenic processes in autoimmune diseases or transplantation, respectively), presumably because T cells become highly susceptible to induction of apoptosis in the absence of co-stimulation (induction of cell survival genes). Therefore, novel agents capable of modulating costimulatory signals without compromising the immune system's ability to fight pathogens would be highly advantageous for the treatment and prevention of such pathological conditions.

The co-stimulatory pathway plays an important role in tumor development. Interestingly, tumors have been shown to escape immune destruction by: blockade of T Cell activation by inhibition of costimulators in The B7-CD28 and TNF families, together with stimulation by attraction of Regulatory T cells that inhibit anti-Tumor T Cell Responses (see Wang (Wang) (2006) Immune supression by Tumor model Specific CD4+ Regulatory T cells in Cancer (immunosuppression by Tumor Specific CD4+ Regulatory T cells in Cancer), Semin. Cancer. biol. (symposium of Cancer biology) 16: 73-79; Greenwald et al, (2005) The B7 Family respiratory modified. Ann. Rev. Immunol. (Immunol.) (2007: 515-48; Watts (Watts) (2005) TNF/TNFR/Tumor model Co-proteins-Immune Responses (Compsoitems. of Cancer cells) 2007; stimulation of Tumor model et al (Cancer cells) 23: 23-48; TNF. Immunol. (clinical trials) TNF/TNFR clinical Co-protein Family 2007; Tumor model et al (Cancer cells) stimulation of Tumor model et al (Tumor model et al; Tumor model et al (Cancer cells) 23: 23. multidrug. (Cooperation et al Immunotherapy (immune signature of murine animals and humans reveals unique mechanisms of tumor escape and new targets for cancer Immunotherapy) clin. mine. res. ("clinical cancer research") 13 (13): 4016-4025). Such tumor-expressing co-stimulatory molecules have become attractive cancer biomarkers and can serve as tumor-associated antigens (TAAs). Furthermore, costimulatory pathways have been identified as immunological checkpoints that attenuate T cell-dependent immune responses at the initial level and effector function within tumor metastases. As engineered cancer vaccines continue to improve, it is becoming increasingly clear that such immunological checkpoints are a major obstacle to the ability of these vaccines to induce a therapeutic anti-tumor response. In that regard, co-stimulatory molecules may act as adjuvants for active (vaccination) and passive (antibody-mediated) cancer immunotherapy, providing strategies to thwart immune tolerance and stimulate the immune system.

In addition, such agents may be useful in other types of cancer immunotherapy, such as adoptive immunotherapy, in which a tumor-specific T cell population is expanded and directed to attack and kill tumor cells. Agents that can increase such anti-tumor responses have great therapeutic potential and may be valuable in attempts to overcome the hurdles of tumor immunotherapy. Recently, novel agents that do modulate several co-stimulatory pathways have been introduced into the clinic as cancer immunotherapy.

Emerging data from extensive research on acute and chronic infectious diseases support an important role for negative costimulatory receptors to also control infection. Memory CD 8T cells generated following acute viral infection are highly functional and constitute an important component of protective immunity. Modulation of the co-stimulatory pathway by limiting memory T cell responses within its protective capacity has also been shown to be effective in optimizing antiviral immunity (taijo et al, journal of immunology 2009: 182; 5430-). 5438). This has been demonstrated in a model of influenza infection by inhibiting CD28 co-stimulation with CTLA4-Ig suppressing the primary immune response in naive mice infected with influenza, but the memory CD 4T cell-mediated secondary response to influenza is significantly effective, leading to improved clinical outcome and increased survival to influenza challenge.

Chronic infectious diseases are often characterized by a variable degree of functional impairment of the virus-specific T-cell response, and this deficiency is a major cause of host inability to eliminate persistent pathogens. Although functional effector T cells are initially produced during the early stages of infection, they gradually lose function during chronic infection due to persistent exposure to foreign antigens, which leads to T cell depletion. Depleted T cells express high levels of multiple co-inhibitory receptors, such as CTLA-4, PD-1, and LAG3 (Crawford et al, Curr Opin Immunol 2009; 21: 179 minus 186; Kaufmann (Kaufmann) et al, J Immunol (journal of immunology) 2009; 182: 5891 minus 5897, Sharp (Sharp) et al, Nat Immunol (natural Immunol) 2007; 8: 239 minus 245). PD-1 overexpression due to depleted T cells was observed clinically in patients suffering from chronic viral infections including HIV, HCV and HBV (Crawford et al, Curr Opin Immunol. (Current Immunity) 2009; 21: 179-186; Kaufmann et al, J Immunol (J Immunol) 2009; 182: 5891-5897, Sharp et al, NatImmunol (Natural immunology) 2007; 8: 239-245). There have been some investigations into additional pathogens (including other viruses, bacteria, and parasites) in this pathway (Hofmeisi (Hofmeyer) et al, J biome Biotechnol. ((J. BioMed. Biotechnology. J.) -Vol 2011, Art. ID 451694, Paddra (Bhadra) et al, Proc Natl Acad. Sci. ((Proc. Natl. Acad. Sci.)) 2011; 108 (22): 9196-. For example, the PD-1 pathway has been shown to be involved in the control of bacterial infection using a model of sepsis induced by standard cecal ligation and perforation procedures. In this model, the absence of PD-1 in knockout mice protects against sepsis-induced death (Huang) et al, PNAS 2009: 106; 6303-.

T cell depletion can be reversed by blocking co-inhibitory pathways (e.g., PD-1 or CTLA-4) (riegas et al, J Immunol. (journal of immunology) 2009; 183: 4284-91; gold-meisen (Golden-Mason) et al, JVirol. ((journal of virology) 2009; 83: 9122-30; hofmeisi (Hofmeyer) et al, J biomeliotechnol. ((journal of biological and biological engineering) Vol 2011, art.id 451694), thus allowing for the restoration of antiviral immune function. The therapeutic potential of co-inhibitory blockade for the treatment of viral infections has been extensively studied by blocking the PD-L/PD-L1 pathway, which has been shown to be effective in several animal models of infection, including acute and chronic Simian Immunodeficiency Virus (SIV) infection in rhesus monkeys (Walu (Valu) et al, Nature (Nature) 2009; 458: 206-. In these models, PD-L/PD-L1 blockade improved the antiviral response and facilitated clearance of persistent virus. Furthermore, PD-L/PD-L1 blockade increased humoral immunity, as evidenced by increased production of specific anti-viral antibodies in plasma, which in combination with improved cellular responses results in a reduction in plasma viral load and increased survival.

Blocking negative-going signaling pathways (e.g., PD-1 and CTLA-4) can restore the host immune system, enabling it to respond to additional stimuli. Combining therapeutic vaccination with blocking of inhibitory signals synergistically enhances functional CD 8T-cell responses in chronically infected individuals and improves viral control to provide a promising strategy for the treatment of chronic viral infections, such as human immunodeficiency virus, hepatitis b virus and hepatitis c virus (Ha et al, Immunol Rev. (immunologic review) 2008/6 months; 223: 317-33). The results of a recent study indicate that blockade of the PD-1 pathway improves T cell responses to HBV vaccination in subjects with HCV infection and increases the likelihood that blockade of this pathway may improve the success of immunization in the context of chronic viral infection (Moorman et al, Vaccine, 4.12.2011; 29 (17): 3169-76). As promising candidates for combination with both prophylactic and therapeutic Vaccines, PD-1 and CTLA-4 antibodies are currently in clinical trials in chronic hepatitis c (newbolder and Obst, Expert Rev Vaccines (review by vaccine) march 2010 march; 9 (3): 243-7). PD-1 blockade also enhances the effectiveness of vaccination, leading to an increase in epitope-specific T cells (FinneFrock et al, J Immunol 2009; 182; 980-

In addition to the blockade of co-inhibitory pathways for the treatment of chronic infections, current studies using viral infection models have emphasized the importance of positive co-stimulatory signals in the course of memory responses to viruses. Costimulatory molecules (e.g., CD28, 4-1BB, and OX40) have also been implicated in the survival, production, maintenance, and quality of virus-specific memory CD8+ T cells. Delivery of costimulatory signals can help increase production and function of virus-specific memory CD8+ T cells. The use of co-stimulatory molecules as adjuvants in vaccines together with viral antigens may assist in the generation of an effective antigen-specific memory CD8+ T-cell response and may therefore lead to improved vaccines (data gupata (duttaguta) et al, Grit rev immunol. (critical review of immunology) 2009; 29 (6): 469-86).

A recent study also evaluated the effect of soluble PD-1(sPD-1), as a block of PD-1 and PD-L1, on vaccine-induced (vaccine-induced) antigen-specific T-cell responses in mice. Co-administration of sPD-1 with a DNA vaccine or with an adenovirus-based vaccine increased the antigen-specific CD8(+) T-cell response, indicating a vaccine type-independent adjuvant effect (vaccine type-independent adjuvant effect) of sPD-1 (Song et al, J Immunother, J.Immunotherapy J.2011.4 months; 34 (3): 297-. These and additional results of this study suggest that immunization strategies using the soluble extracellular domain (ECD) of a negative costimulatory protein as an adjuvant can be used to increase antigen-specific T-cell immunity resulting from vaccination.

B cells have also been considered to have a key role in the development and maintenance of many autoimmune diseases, such as Systemic Lupus Erythematosus (SLE) and heulans disease, through the production of pathogenic autoantibodies. However, it is clear that a number of other B cell functions are also critical in the pathogenesis of organ-specific autoimmune diseases, which were previously thought to be mainly T cell-mediated, such as Rheumatoid Arthritis (RA) and type 1 diabetes (T1D) (wang (Wong) et al 2010, Curr Opin Immunol [ current view of immunology ] 22: 723-. T cell helper B cells are a key process of the adaptive immune response. Follicular helper T (Tfh) cells are a subset of CD4+ T cells that are specialized in B cell help (reviewed by Crott (Crotty), Annu. Rev. Immunol. (Annu.) 29: 621-663, 2011). Tfh cells express the B cell homing chemokine receptor CXCR5, CXCR5 drives the migration of Tfh cells to the B cell follicle in the lymph node in a CXCL 13-dependent manner. Tfh cells first interact with cognate B cells at the T cell-B cell boundary and subsequently induce germinal center B cell differentiation and germinal center formation within the follicle (reviewed by crohn's (Crotty) Annu. Rev. Immunol. (annual Immunity) 29: 621-663, 2011). The requirement for Tfh cells for B cell helper and T cell dependent antibody responses suggests that this cell type is important for protective immunity against different types of infectious agents as well as for rational vaccine design. Not surprisingly, dysregulation and abnormal accumulation of Tfh cells have also been linked to autoimmune diseases (e.g., sjohn's disease and autoimmune arthritis) (Yu) and binnouesa (Vinuesa), 2010, cell. mol. immunol. ("cellular molecular immunology) 7: 198-" 203).

Tfh cells selectively express abundant surface proteins involved in their selective localization (e.g., CXCR5) and direct physical interaction with B cells to provide B cell help. In the latter group are several members of the family of co-stimulatory proteins highly expressed in Tfh cells, including the inducible co-stimulatory receptor ICOS, as well as the negative co-stimulatory molecules (inhibitory receptors) PD-1 and BTLA (Crotty), Annu. Rev. Immunol. ((Annu. Immunity Ann.) 29: 621-663, 2011), and therefore this cell subset can also be controlled by the modulation of co-stimulatory pathways and co-inhibitory pathways, which contribute to the effects on B-cell function.

Modulation of co-stimulation using agonists and/or antagonists of different co-stimulatory proteins has also been extensively studied as a strategy for treating autoimmune diseases, transplant rejection, allergies, and cancer. This field has been approved for the treatment of RA by CTLA4-Ig (abatacept,) Mutated CTLA4-Ig (belazepril,) And anti-CTLA 4 antibodies recently approved for the treatment of melanoma (lypima,) Is initiated clinically. Other co-stimulatory modulators are currently in an advanced stage of clinical development, including anti-PD-1 antibodies ((MDX-1106), anti-PD-1 antibodies in development for treatment of advanced/metastatic clear cell Renal Cell Carcinoma (RCC), and anti-CD 40L antibodies for treatment of kidney allograft transplantation (BG9588, ). In addition, such agents are also in clinical development for viral infections, such as anti-PD-1 Ab, MDX-1106, being tested for the treatment of hepatitis c, and anti-CTLA-4 Ab CP-675, 206 (tremelimumab) in clinical trials in patients with hepatocellular carcinoma, infected with hepatitis c virus; the purpose of this study was to test its effect on cancer and on viral replication.

Brief summary of the invention

In accordance with at least some embodiments, the present invention provides novel therapeutic and diagnostic compositions comprising: extracellular domains of LY6G6F, VSIG10, TMEM25 and/or LSR proteins or soluble or secreted forms thereof, and/or variants and/or orthologs and/or fragments, and/or conjugates comprising and/or nucleic acid sequences encoding same.

The full-length amino acid sequence of the known (wild-type) LY6G6F protein (lymphocyte antigen 6 complex locus protein G6f, Genbank accession No.: NP-001003693, SEQ ID NO: 1) is shown in FIG. 1A. The full-length amino acid sequence of a known (wild-type) VSIG10 protein (protein 10 comprising V-set and immunoglobulin domains, Genbank accession No.: NP-061959, SEQ ID NO: 3) and the amino acid sequence of a novel variant of VSIG10 (SEQ ID NO: 5) are shown in FIGS. 1B and 1C, respectively. The amino acid sequence alignment of the novel variant of VSIG10 (SEQ ID NO: 5) and the known (wild-type) VSIG10 protein (SEQ ID NO: 3) is shown in FIG. 2A. The full-length amino acid sequence of the known (wild-type) TMEM25 protein (transmembrane protein 25, Swiss-Prot accession number Q86YD3, SEQ ID NO: 7) is shown in FIG. 1D. The full-length amino acid sequence of the known (wild-type) LSR protein (lipoprotein receptor isoform 2 which stimulates lipolysis, Genbank accession No.: NP _991403) is provided in SEQ ID NO: 62 (c). LSR variant SEQ ID NO: 11. 13, 15, 16, 17 and 18 are shown in FIGS. 1E, 1F, 1G, 1H, 1I, and 1J, respectively. LSR variant SEQ ID NO: 11. 13, 15, 16, 17 and 18 are shown in FIGS. 2B, 2C, 2D, 2E, 2F, 2G, respectively, aligned with the amino acid sequence of a previously known LSR sequence (SEQ ID NOS: 62-67).

According to at least some embodiments, there is provided an isolated polypeptide comprising at least 98 amino acids of a soluble extracellular domain of a sequence selected from the group consisting of SEQ ID NOs: 11. 13, 15-18, 67, and 143; at least 62 amino acids of a soluble extracellular domain of a sequence selected from the group consisting of SEQ ID NOs: 1 and 58; at least 36 amino acids of a soluble extracellular domain of a sequence selected from the group consisting of SEQ ID NO: 3 and 5; or SEQ ID NO: 7, or a soluble extracellular domain consisting essentially of at least 46 amino acids as set forth in SEQ ID NO: 5 or a variant thereof having at least 95% sequence identity thereto; or a variant, or ortholog, or fragment thereof.

Optionally, such isolated polypeptide comprises only between 98 and 180 amino acids of a sequence selected from the group consisting of SEQ ID NO: 11. 13, 15-18, 67, and 143; between 62 and 228 amino acids of a sequence selected from the group consisting of SEQ ID NO: 1 and 58; between 36 and 393 amino acids of a sequence selected from the group consisting of SEQ ID NO: 3 and 5; or SEQ ID NO: between 46 and 216 amino acids of 7.

Optionally, the isolated polypeptide is selected from the group consisting of polypeptides comprising only: between 98 to 118, 135 to 155, and 160 to 180 amino acids of a sequence selected from the group consisting of SEQ ID NO: 11. 13, 15-18, 67, and 143; between 62 and 82, 95 and 115, 208 and 228 amino acids of a sequence selected from the group consisting of SEQ ID NO: 1 and 58; between 36 to 70, 80 to 100, 170 to 200, 265 to 290, 365 to 393 amino acids of a sequence selected from the group consisting of SEQ ID NO: 3 and 5; or SEQ ID NO: 7 between 46 and 66, 84 and 104, 196 and 216 amino acids.

Optionally, the isolated polypeptide comprises only: about 72, 106, or 218 amino acids of a sequence selected from the group consisting of SEQ ID NO: 1 and 58; about 108, 145, or 170 amino acids of a sequence selected from the group consisting of SEQ ID NOs: 11. 13, 15-18, 67, and 143; SEQ ID NO: about 56, 94, or 206 amino acids of 7; or SEQ ID NO: 3 and 5 of about 46, 49, 58, 60, 87, 89, 93, 94, 178, 182, 185, 187, 273, 279, 282, 374 or 383 amino acids.

Also optionally, such an isolated polypeptide consists essentially of a polypeptide sequence identical to that of any one of SEQ ID NOs: 12. 2, 4-6, 8, 14, 47-50, 10, 15-18, 22, 39, 59-61; the amino acid sequences recited in 81-102 have an amino acid sequence composition with at least 95% sequence identity. Optionally and preferably, such isolated polypeptide consists essentially of a polypeptide as set forth in any one of SEQ ID NOs: 12. 2, 4-6, 8, 14, 47-50, 10, 15-18, 22, 39, 59-61; 81-102, or a pharmaceutically acceptable salt thereof.

Optionally, the isolated polypeptide blocks or inhibits the interaction of LSR, TMEM25, VSIG10, LY6G6F, or a fragment or variant thereof with a corresponding functional counterpart. Optionally, the isolated polypeptide replaces or increases the interaction of LSR, TMEM25, VSIG10, LY6G6F, or a fragment or variant thereof with a corresponding functional counterpart.

Optionally, the isolated ortholog is a polypeptide selected from the group consisting of SEQ ID NO: 9 and 19-21.

According to at least some embodiments, the present invention provides isolated polypeptides comprising a discontinuous portion (fragment) of VSIG10 protein corresponding to:

A. an isolated chimeric polypeptide comprising a first amino acid sequence MAAGGSAPEPRVLVCLGALLAGWVAVGLEAVVIGEVHENVTLHCGNISGLRGQVTWYRNNSEPVFLLSSNSSLRPAEPRFSLVDATSLHIESLSLGDEGIYTCQEILNVT QWFQVWLQVA that is at least 95% homologous to amino acids 1-120 of a known VSIG10 protein (SEQ ID NO: 3) and to amino acids 1-120 of a VSIG10 variant (SEQ ID NO: 5); a second bridging amino acid sequence comprising N; and a third amino acid sequence PPPSAPQCWAQMASGSFMLQLTCRWDGGYPDPDFLWIEEPGGVIVGKSKLGVEMLSESQLSDGKKFKCVTSHIVGPESGASCMVQIRGPSLLSEPMKTCFTGGNVTLTCQVSGAYPPAKILWLRNLTQPEVIIQPSSRHLITQDGQNSTLTIHNCSQDLDEGYYICRADSPVGVREMEIWLSVKEPLNIGGIVGTIVSLLLLGLAIISGLLLHYSPVFCWKVGNTSRGQNMDDVMVLVDSEEEEEEEEEEEEDAAVGEQEGAREREELPKEIPKQDHIHRVTALVNGNIEQMGNGFQDLQDDSSEEQSDIVQEEDRPV that is at least 95% homologous to amino acids 223-540 of the known VSIG10 protein (SEQ ID NO: 3) and to amino acids 122-439 of the VSIG10 variant (SEQ ID NO: 5); wherein the first amino acid sequence, the second bridging amino acid sequence, and the third amino acid sequence are contiguous and in a sequential order.

Isolated polypeptides of the border portion of a VSIG10 variant (SEQ ID NO: 5), including a polypeptide having a length "n", wherein n is at least 10 amino acids long, optionally at least about 20 amino acids long, preferably at least about 30 amino acids long, more preferably at least about 40 amino acids long and most preferably at least about 50 amino acids long, wherein at least 3 amino acids include ANP (numbering according to the VSIG10 variant (SEQ ID NO: 5)) having the structure: a sequence starting at any one of amino acid numbers 120-x to 120 and ending at any one of amino acid numbers 122+ ((n-3) -x), wherein x varies from 0 to n-3.

According to at least some embodiments, the subject invention further provides an isolated polypeptide corresponding to the sequence of amino acid residues: discrete portions of the VSIG10 protein, novel junctions and border portions of the VSIG10 variant (SEQ ID NO: 5). The unique sequence of the new junction of the VSIG10 variant (SEQ ID NO: 5) is shown in the protein sequence alignment in FIG. 2A.

According to at least some embodiments, the subject invention provides isolated polypeptides comprising a discontinuous portion (fragment) of an LSR protein corresponding to:

A. an isolated chimeric polypeptide comprising a first amino acid sequence MALLAGGLSRGLGSHPAAAGRDAVVFVWLLLSTWCTAPARAIQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVLGRTSGVAELLPGFQAGPIE that is at least 95% homologous to amino acids 49-258 of a known LSR protein (SEQ ID NO: 62) and to amino acids 1-210 of LSR variant isoform f (SEQ ID NO: 18); a second bridging amino acid sequence comprising V; and a third amino acid sequence YAAGKAATSGVPSIYAPSTYAHLSPAKTPPPPAMIPMGPAYNGYPGGYPGDVDRSSSAGGQGSYVPLLRDTDSSVASEVRSGYRIQASQQDDSMRVLYYMEKELANFDPSRPGPPSGRVERAMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHSPRSPRGWDQEPAREQAGGGWRARRPRARSVDALDDLTPPSTAESGSRSPTSNGGRSRAYMPPRSRSRDDLYDQDDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTRDPRDNGSRSGDLPYDGRLLEEAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETDSQASRERRLKKNLALSRESLVV which is at least 95% homologous to amino acids 309-649 of the known LSR protein (SEQ ID NO: 62) and which also corresponds to amino acids 212-552 of the LSR variant isoform f (SEQ ID NO: 18); wherein the first amino acid sequence, the second bridging amino acid sequence, and the third amino acid sequence are contiguous and in a sequential order.

An isolated polypeptide of the border portion of LSR variant isoform f (SEQ ID NO: 18), comprising a polypeptide having a length "n", wherein n is at least 10 amino acids long, optionally at least about 20 amino acids long, preferably at least about 30 amino acids long, more preferably at least about 40 amino acids long and most preferably at least about 50 amino acids long, wherein at least 3 amino acids comprise EVY (numbering according to SEQ ID NO: 18) having the structure: a sequence starting at any one of amino acid numbers 210-x to 210 and ending at any one of amino acid numbers 212+ ((n-3) -x), wherein x varies from 0 to n-3.

C. An isolated chimeric polypeptide comprising a first amino acid sequence MALLAGGLSRGLGSHPAAAGRDAVVFVWLLLSTWCTAPARAIQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVL that is at least 95% homologous to amino acids 49-239 of a known LSR protein (SEQ ID NO: 66) and to amino acids 1-191 of LSR variant isoform f (SEQ ID NO: 18); a second amino acid sequence which is at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to the polypeptide having the sequence GRTSGVAELLPGFQAGPIE and which corresponds to amino acids 192-218 of the LSR variant isoform f (SEQ ID NO: 18); and a third amino acid sequence VYAAGKAATSGVPSIYAPSTYAHISPAKTPPPPAMIPMGPAYNGYPGGYPGDVDRSSSAGGQGSYVPLLRDTDSSVASEVRSGYRIQASQQDDSMRVLYYMEKELANFDPSRPGPPSGRVERAMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHSPRSPRGWDQEPAREQAGGGWRARRPRARSVDALDDLTPPSTAESGSRSPTSNGGRSRAYMPPRSRSRDDLYDQDDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTRDPRDNGSRSGDLPYDGRLLEEAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETDSQASRERRLKKNLALSRESLVV that corresponds to at least 95% homology to the known LSR protein SEQ ID NO: 66, which also corresponds to amino acid 211-552 of the LSR variant isoform f (SEQ ID NO: 18); wherein the first, second and third amino acid sequences are contiguous and in a sequential order.

D. An isolated polypeptide of the border portion of LSR variant isoform f (SEQ ID NO: 18) comprising an amino acid sequence that is at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to sequence GRTSGVAELLPGFQAGPIE of LSR variant isoform f (SEQ ID NO: 18).

According to at least some embodiments, the subject invention further provides an isolated polypeptide corresponding to the sequence of amino acid residues: discontinuous portions of LSR, new junctions and edge portions of LSR variant LSR isoform f (SEQ ID NO: 18). The unique sequence of the new junction of LSR isoform-f (SEQ ID NO: 18) is shown in the protein sequence alignment in FIG. 2G.

According to at least some embodiments, the subject invention provides polypeptides comprising a sequence of amino acid residues corresponding to a discontinuous portion of LY6G6F, VSIG10, TMEM25 and/or LSR proteins, including different portions of the extracellular domains corresponding to: residues 17-234 of LY6G6F (SEQ ID NO: 1), corresponding to the amino acid sequence depicted in SEQ ID NO: 2; residues 31-413 of VSIG10(SEQ ID NO: 3), corresponding to the amino acid sequence depicted in SEQ ID NO: 4; residues 31-312 of VSIG10(SEQ ID NO: 5), corresponding to the amino acid sequence depicted in SEQ ID NO: 6; residues 27-232 of TMEM25(SEQ ID NO: 7), corresponding to the amino acid sequence depicted in SEQ ID NO: 8; residues 42-211 of LSR (SEQ ID NO: 11, and/or SEQ ID NO: 143), corresponding to the amino acid sequence depicted in SEQ ID NO: 12; residues 42-192 of LSR (SEQ ID NO: 13), corresponding to the amino acid sequence depicted in SEQ ID NO: 14; residues 42-533 of LSR (SEQ ID NO: 15), corresponding to the amino acid sequence depicted in SEQ ID NO: 47; residues 42-532 of LSR (SEQ ID NO: 16), corresponding to the amino acid sequence depicted in SEQ ID NO: 48; residues 42-493 of LSR (SEQ ID NO: 17), corresponding to the amino acid sequence depicted in SEQ ID NO: 49; residues 42-552 of LSR (SEQ ID NO: 18), corresponding to the amino acid sequence depicted in SEQ ID NO: 50; and/or fragments and/or variants thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology thereto. According to still further embodiments, as described herein, the LY6G6F ECD fragment is selected from any one of seq id NOs: 81. 96 and variants thereof. According to still further embodiments, as described herein, the VSIG10 ECD fragment is selected from any one of seq id NOs: 82-93, 97-100 and variants thereof. According to still further embodiments, as described herein, the LSR ECD fragment is selected from any of SEQ ID NOs: 95. 102 and variants thereof. According to still further embodiments, as described herein, the TMEM25 ECD fragment is selected from any one of SEQ ID NOs: 94. 101 and variants thereof. According to yet further embodiments, the discontinuous portion of LY6G6F, VSIG10, TMEM25 and/or LSR proteins may or may not include a signal (leader) peptide (SP) sequence (fig. 1). In accordance with at least some embodiments of the present invention, examples of ECD moieties comprising the SP sequence of LY6G6F, VSIG10, TMEM25, and/or LSR proteins are provided. An example of an ECD portion of the SP sequence comprising LY6G6F protein (SEQ ID NO: 1) is the amino acid sequence set forth in SEQ ID NO: 59, or a pharmaceutically acceptable salt thereof. An example of an ECD portion of the SP sequence comprising the VSIG10 protein (SEQ ID NO: 3) is the amino acid sequence set forth in SEQ ID NO: 60, or a pharmaceutically acceptable salt thereof. An example of an ECD portion of the SP sequence comprising the VSIG10 protein (SEQ ID NO: 5) is the amino acid sequence set forth in SEQ ID NO: 61, or a pharmaceutically acceptable salt thereof. An example of an ECD portion of the SP sequence comprising the TMEM25 protein (SEQ ID NO: 7) is the amino acid sequence shown in SEQ ID NO: 39, or a pharmaceutically acceptable salt thereof. An example of an ECD portion of the SP sequence that includes the LSR protein (SEQ ID NO: 11) is the amino acid sequence set forth in SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof. An example of an ECD portion of the SP sequence that includes the LSR protein (SEQ ID NO: 14) is the amino acid sequence set forth in SEQ ID NO: 22, or a pharmaceutically acceptable salt thereof.

According to a further embodiment, the present invention provides a polypeptide comprising an amino acid sequence corresponding to SEQ ID NO: 18, including different portions thereof or variants thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology thereto. According to further embodiments, the present invention provides a polypeptide comprising a sequence corresponding to any one of SEQ ID NOs: 15-16, including different portions thereof or variants thereof having at least 95%, 96%, 97%, 98% or 99% sequence homology thereto. According to further embodiments, the present invention provides a polypeptide comprising a sequence corresponding to any one of SEQ ID NOs: 15-18, of the sequence of amino acid residues of the soluble LSR protein depicted in. According to still further embodiments, the sequence set forth in any one of SEQ ID NOs: 15-18 may or may not include a signal (leader) peptide sequence (fig. 1G, G, I and J).

According to yet further embodiments, the present invention provides polypeptides, particularly mouse orthologs (seq id NOs: 28, 29, 30, 31 and/or 32, respectively), comprising a sequence corresponding to amino acid residues of TMEM25, LY6G6F, VSIG10, LSR variant 1 and/or LSR variant 2 proteins, including but not limited to: corresponding to SEQ ID NO: 9. 19-21, or a portion or variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto.

According to still further embodiments, the present invention provides polypeptides comprising an amino acid sequence corresponding to a novel variant of any one of VSIG10(SEQ ID NO: 5) and LSR (SEQ ID NO: 11, 13, 15, 16 and 18).

According to at least some embodiments, the present invention provides a fusion protein comprising any one of the above polypeptides linked to a heterologous sequence. Optionally, such heterologous sequences include at least a portion of an immunoglobulin molecule. Optionally and preferably, the immunoglobulin molecule moiety is an immunoglobulin heavy chain constant region Fc fragment. Optionally and more preferably, the immunoglobulin heavy chain constant region is derived from an immunoglobulin isotype selected from the group consisting of: IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA, and IgD. Optionally and most preferably, such fusion protein has an amino acid sequence as set forth in any one of SEQ ID NOs: 71-80, 172-181 or as set forth in any one of SEQ ID NOs: 23-26, and optionally also modulates immune cell responses in vitro or in vivo.

According to at least some embodiments, the subject invention provides isolated nucleic acid sequences encoding: any one of the foregoing novel variants of TMEM25, VSIG10, and/or LSR, and/or any one of the foregoing LY6G6F, VSIG10, TMEM25 and/or LSR extracellular domain polypeptides or fragments or homologs or orthologs thereof.

According to at least some embodiments, there is provided an isolated nucleic acid sequence selected from the group consisting of seq id no: SEQ ID NO: 33-37, 40-46, 132, 155, 182, 198, or a variant thereof having at least 95% sequence identity thereto, or a degenerate variant thereof.

According to at least some embodiments, the subject invention provides an isolated polynucleotide encoding: a polypeptide comprising any of the sequences set forth in SEQ ID NO: 2. 4, 5, 6, 8-16, 18-22, 39, 47-50, 59-61, 143, a fragment or variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a degenerate variant thereof.

According to at least some embodiments, the subject invention provides an isolated polynucleotide comprising a sequence as set forth in any one of SEQ ID NOs: 33-37, 40-46, 132, 145, 155, 182, 188, or a sequence homologue or degenerate variant thereof. According to another embodiment, the isolated polynucleotide is substantially identical to the polynucleotide as set forth in any one of SEQ id nos: 33-37, 40-46, 145, at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous.

According to at least some embodiments, there is provided an expression vector or virus comprising at least one isolated nucleic acid sequence as described herein. According to at least some embodiments, there is provided a recombinant cell comprising an expression vector or virus comprising an isolated nucleic acid sequence as described herein, wherein the cell constitutively or inducibly expresses the polypeptide encoded by the DNA segment. According to at least some embodiments, there is provided a method of producing an LSR, TMEM25, VSIG10, LY6G6F soluble extracellular domain polypeptide, or fragment or fusion protein thereof, comprising culturing a recombinant cell as described herein under conditions whereby the cell expresses a polypeptide encoded by the DNA segment or nucleic acid, and recovering said polypeptide.

According to at least some embodiments of the present invention, there is provided a pharmaceutical composition comprising an isolated amino acid sequence of the extracellular domain of any one of LY6G6F, VSIG10, TMEM25, LSR proteins, or variants or orthologs or fragments or conjugates containing same or a soluble or secreted form thereof.

According to at least some embodiments, the present invention provides an isolated or purified amino acid sequence of, or a nucleic acid sequence encoding, the soluble and/or extracellular domain of LY6G6F, VSIG10, TMEM25 and/or LSR proteins, which may optionally be attached directly or indirectly to a non-LY 6G6F, VSIG10, TMEM25 and/or LSR protein or nucleic acid sequence, such as a soluble immunoglobulin functional region or fragment.

According to at least some embodiments, the present invention provides vectors (e.g., plasmids and recombinant viral vectors) and host cells comprising vectors that express secreted or soluble forms of LY6G6F, VSIG10, TMEM25 and/or LSR proteins and/or ECDs thereof, or fragments or variants thereof or orthologs thereof or polypeptide conjugates comprising any of the foregoing.

According to at least some embodiments, the present invention provides the use of these vectors (e.g. plasmids and recombinant viral vectors) and host cells comprising the vectors to express any one of LY6G6F, VSIG10, TMEM25 and/or LSR, secreted and/or soluble forms and/or ECD and/or fragments and/or variants thereof, and/or orthologs thereof and/or polypeptide conjugates comprising any one of the foregoing to produce said LY6G6F, VSIG10, TMEM25 and/or LSR proteins.

According to at least some embodiments, the present invention provides a pharmaceutical or diagnostic composition comprising any of the foregoing.

According to at least some embodiments, the present invention provides the use of any one of the following compounds or pharmaceutical compositions as a therapy for the treatment or prevention of cancer as exemplified herein, infectious disorders as exemplified herein, and/or immune-related disorders (including but not limited to autoimmune diseases, transplant rejection and graft-versus-host disease as exemplified herein), and/or for blocking or promoting immune co-stimulation mediated by any one of LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides, immune-related diseases as exemplified herein and/or for immunotherapy (promoting or inhibiting immune co-stimulation); these compounds comprise at least one of LY6G6F, VSIG10, TMEM25 and/or LSR extracellular domain, soluble or secreted form or fragment or ortholog or variant thereof, or conjugate, or nucleic acid sequence encoding same, including these compounds. According to at least some embodiments, the autoimmune disease includes any autoimmune disease, and may optionally and preferably include, but is not limited to, any one of the following types and subtypes: multiple sclerosis, rheumatoid arthritis, type I diabetes, psoriasis, systemic lupus erythematosus, inflammatory bowel disease, uveitis, or Creutzfeldt-Jakob syndrome.

According to at least some embodiments, the present invention provides the use of any one of the following compounds or pharmaceutical compositions for administration as an anti-cancer vaccine, as an adjuvant for an anti-cancer vaccine, and/or for adoptive immunotherapy, and/or for cancer immunotherapy as recited herein; these compounds comprise at least one of LY6G6F, VSIG10, TMEM25 and/or LSR extracellular domain, soluble or secreted form or fragment or ortholog or variant thereof, or conjugate, or nucleic acid sequence encoding same, including these compounds.

According to at least some embodiments, the present invention provides the use of any LY6G6F, VSIG10, TMEM25 and/or LSR protein, and/or nucleic acid sequence, as a target for drug development that specifically binds to any LY6G6F, VSIG10, TMEM25 and/or LSR protein and/or drug that agonizes or antagonizes the binding of other moieties to LY6G6F, VSIG10, TMEM25 and/or LSR protein.

In accordance with at least some embodiments, the present invention provides agents that modulate (agonize or antagonize) at least one of LY6G6F, VSIG10, TMEM25, and/or LSR related biological activities. By way of example, such agents include antibodies, small molecules, peptides, ribozymes, aptamers, antisense molecules, siRNA, and the like. These molecules may directly bind or modulate the activity caused by any LY6G6F, VSIG10, TMEM25 and/or LSR protein or LY6G6F, VSIG10, TMEM25 and/or LSR DNA or portions or variants thereof, or may indirectly modulate any LY6G6F, VSIG10, TMEM25 and/or LSR related activity, or bind to any LY6G6F, VSIG10, TMEM25 and/or LSR and portions and variants thereof, for example by modulating any LY6G6F, VSIG10, TMEM25 and/or LSR to its counter receptor (counter receptor) or endogenous ligand.

According to at least some embodiments, the present invention provides novel monoclonal or polyclonal antibodies and antigen binding fragments and conjugates comprising the same, and/or alternative scaffolds, that specifically bind to any one of LY6G6F, VSIG10, TMEM25 and/or LSR proteins, or polypeptides having at least 95% homology thereto, as described herein. Optionally, such antibodies bind to a protein selected from the group consisting of: SEQ ID NO: 1-8, 10-18, 22, 39, 47-50, 59-61, 9, 19-21, or a nucleic acid sequence corresponding to any one of SEQ ID NOs: 5 and 18, amino acid sequences of the unique borders; in particular, wherein the antibodies, antigen-binding fragments and conjugates comprising the same, and/or alternative scaffolds are adapted for use as therapeutic and/or diagnostic agents (in both in vitro and in vivo diagnostic methods), particularly for infectious disorders, and/or immune-related disorders as recited herein (including but not limited to autoimmune diseases as recited herein, immune-related diseases as recited herein, transplant rejection, and graft-versus-host disease); as well as the treatment and/or diagnosis of cancer and malignancies as recited herein.

According to at least some embodiments, antibodies are provided wherein the antigen binding site comprises a conformational or linear epitope and wherein the antigen binding site comprises about 3-7 contiguous or non-contiguous amino acids. Optionally, the antibody is a fully human antibody, a chimeric antibody, a humanized or primatized antibody.

Also optionally, the antibody is selected from the group consisting of: fab, Fab ', F (ab ') 2, F (ab '), F (ab), Fv or scFv fragments and minimal recognition units.

Optionally, the antibody is conjugated to a moiety selected from the group consisting of: a drug, radionuclide, fluorophore, enzyme, toxin, therapeutic agent, or chemotherapeutic agent; and wherein the detectable label is a radioisotope, metal chelator, enzyme, fluorescent compound, bioluminescent compound or chemiluminescent compound.

Also optionally, the antibody blocks or inhibits the interaction of any one of LSR, TMEM25, VSIG10, LY6G6F polypeptide, or fragment or variant thereof with a counterpart.

Also optionally, the antibody replaces or increases the interaction of LSR, TMEM25, VSIG10, LY6G6F polypeptide, or fragment or variant thereof with a counterpart.

Optionally, the antibody causes apoptosis or lysis of cancer cells expressing any one of LSR, TMEM25, VSIG10, LY6G6F proteins.

Optionally also, apoptosis or lysis involves CDC or ADCC activity of the antibody, wherein CDC (complement dependent cytotoxicity) or ADCC (antibody dependent cytotoxicity) activity is used to target the immune cells.

According to at least some embodiments, the invention provides antibodies and antigen-binding fragments directed against a discontinuous portion of LY6G6F protein, the discontinuous portion of LY6G6F protein comprising different portions of extracellular domains corresponding to: residues 17-234 of LY6G6F (SEQ ID NO: 1), set forth in SEQ ID NO: 2, and/or corresponds to any one of SEQ ID NOs: 81 and 96. According to further embodiments, the invention provides antibodies, antigen-binding fragments, and conjugates comprising the same, and/or alternative scaffolds, directed against a discontinuous portion of mouse LY6G6F protein (SEQ ID NO: 29), the discontinuous portion of mouse LY6G6F protein comprising a heavy chain variable region corresponding to SEQ ID NO: 20, or a different part of the extracellular domain of the polypeptide.

In accordance with at least some embodiments, the present invention provides antibodies and antigen-binding fragments, as well as conjugates comprising the same, and/or alternative scaffolds, directed against a discontinuous portion of VSIG10 protein, the discontinuous portion of VSIG10 protein comprising different portions corresponding to the following extracellular domains: amino acid residues 31-413 of VSIG10(SEQ ID NO: 3), depicted in SEQ ID NO: 4, performing the following steps; amino acid residues 31-312 of VSIG10(SEQ ID NO: 5), depicted in SEQ ID NO: 6, and/or corresponds to any one of SEQ ID NOs: 82-93, 97-100. According to further embodiments, the present invention provides antibodies, antigen-binding fragments, and conjugates comprising the same, and/or alternative scaffolds, directed against a discontinuous portion of mouse VSIG10 protein (SEQ ID NO: 30), the discontinuous portion of mouse VSIG10 protein comprising a sequence corresponding to SEQ ID NO: 19, or a different part of the extracellular domain of the polypeptide. In accordance with at least some embodiments, the present invention provides antibodies, antigen-binding fragments, and conjugates comprising the same, and/or alternative scaffolds, directed against a discontinuous portion of a VSIG10 protein, including the border portion of a variant of VSIG10(SEQ ID NO: 5), as described herein.

In accordance with at least some embodiments, the present invention provides antibodies, antigen-binding fragments, and conjugates comprising the same, and/or alternative scaffolds directed against a discontinuous portion of TMEM25 protein, the discontinuous portion of TMEM25 protein comprising different portions of extracellular domains corresponding to: amino acid residues 27-232 of TMEM25(SEQ ID NO: 7), depicted in SEQ ID NO: 8, and/or corresponds to any one of SEQ ID NOs: 94. 101, or a pharmaceutically acceptable salt thereof. According to further embodiments, the present invention provides antibodies and antigen-binding fragments and conjugates comprising the same, and/or alternative scaffolds, directed against a discontinuous portion of mouse TMEM25 protein (SEQ ID NO: 28), the discontinuous portion of mouse TMEM25 protein comprising SEQ ID NO: 9, or a different part of the extracellular domain as set forth in fig.

In accordance with at least some embodiments, the present invention provides antibodies and antigen-binding fragments, as well as conjugates comprising the same, and/or alternative scaffolds, directed against a discontinuous portion of an LSR protein comprising different portions of extracellular domains corresponding to: amino acid residues 42-211 of LSR (SEQ ID NO: 11), depicted in SEQ ID NO: 12, and (b); amino acid residues 42-192 of LSR (SEQ ID NO: 13), depicted in SEQ ID NO: 14, amino acid residues 42-533 of LSR (SEQ ID NO: 15), as depicted in SEQ ID NO: 47, amino acid residues 42-532 of LSR (SEQ ID NO: 16), depicted in SEQ ID NO: 48, amino acid residues 42-493 of LSR (SEQ ID NO: 17), depicted in SEQ ID NO: 49, amino acid residues 42-552 of LSR (SEQ ID NO: 18), depicted in SEQ ID NO: 50, and/or a sequence corresponding to any one of SEQ id nos: 95. 102, or a pharmaceutically acceptable salt thereof. According to further embodiments, the invention provides antibodies and antigen-binding fragments and conjugates comprising the same, and/or alternative scaffolds, directed against a discontinuous portion of mouse LY6G6F protein (SEQ ID NOS: 31-32), the discontinuous portion of mouse LY6G6F protein comprising a heavy chain variable region corresponding to SEQ ID NO: 21, or a different part of the extracellular domain of the same.

In accordance with at least some embodiments, the present invention provides antibodies and antigen-binding fragments, as well as conjugates comprising the same, and/or alternative scaffolds, directed against a discontinuous portion of an LSR protein that includes a unique border portion of LSR variant isoform-f (SEQ ID NO: 18).

In accordance with at least some embodiments, the present invention provides antibodies and antigen-binding fragments, as well as conjugates comprising the same, and/or alternative scaffolds, directed against a discontinuous portion of a soluble LSR protein, which comprises the amino acid sequence of SEQ ID NO: 15-18, 47-50.

According to at least some embodiments, the present invention relates to protein scaffolds having a specificity and affinity similar to the range of specific antibodies. According to at least some embodiments, the present invention relates to an antigen binding construct comprising a protein scaffold linked to one or more epitope binding domains. Such engineered protein scaffolds are typically obtained by designing a random library with mutagenesis focused on loop regions or additional allowable surface regions and by selecting variants for a given target via phage display or related techniques. In accordance with at least some embodiments, the present invention relates to alternative stents, including, but not limited to: anticalin, DARPin, armadillo repeat protein (armadilloreep proteins), protein a, lipocalin, fibronectin domains, ankyrin consensus repeat domain, thioredoxin, chemically constrained peptides, and the like. In accordance with at least some embodiments, the present invention relates to alternative stents for use as therapeutic agents in the treatment of: cancer as recited herein, immune-related diseases as recited herein, autoimmune diseases as recited herein, and infectious diseases, as recited herein, as well as for use in vivo diagnostics.

According to at least some embodiments of the present invention, there is provided a pharmaceutical composition comprising an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein, and further comprising a pharmaceutically acceptable diluent or carrier.

In accordance with at least some embodiments, there is provided use of any one of: any of the isolated polypeptides as described herein, or the fusion proteins as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein, or a pharmaceutical composition as described herein, wherein such administration to a subject inhibits or reduces T cell activation.

According to at least some embodiments, there is provided a use of any one of the following for treating cancer: any of the isolated polypeptides as described herein, or the fusion proteins as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein, or a pharmaceutical composition as described herein.

According to at least some embodiments, there is provided use of: an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein, or a pharmaceutical composition as described herein.

According to at least some embodiments, there is provided a method of performing one or more of the following on a subject:

a. up-regulation of cytokines;

b. inducing proliferation of T cells;

c. promoting antigen-specific T cell immunity;

d. promote CD4+ and/or CD8+ T cell activation; the method comprises administering to the subject any one of the isolated polypeptides as described herein, or the fusion proteins as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein, or a pharmaceutical composition as described herein.

According to at least some embodiments, there is provided a method for treating or preventing an immune system-related disorder, the method comprising administering to a subject in need thereof an effective amount of any of an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody or pharmaceutical composition as described herein.

Optionally, the immune system-related disorder comprises an immune-related disorder, autoimmune diseases as exemplified herein, transplant rejection and graft-versus-host disease and/or for blocking or promoting immune co-stimulation mediated by any one of LSR, TMEM25, VSIG10 and/or LY6G6F polypeptides, immune-related diseases as exemplified herein and/or for immunotherapy (promoting or inhibiting immune co-stimulation).

Optionally, such treatment is combined with another moiety used to treat an immune-related disorder.

Optionally, the moiety is selected from the group consisting of: immunosuppressants (e.g., corticosteroids, cyclosporine, cyclophosphamide, prednisone, azathioprine, methotrexate, sirolimus, tacrolimus), biologic agents (e.g., TNF- α blockers or antagonists, or any other biologic agent that targets any inflammatory cytokine), NSAID/Cox-2 inhibitors, hydroxychloroquine, sulfasalazine(sulphosalazolylpryine), sodium chloroaurate, etanercept, infliximab, mycophenolate mofetil, basiliximab, asexicept, rituximab, cyclophosphamide, interferon beta-la, interferon beta-lb, glatiramer acetate, mitoxantrone hydrochloride, anakinra and/or other biologicals and/or intravenous immunoglobulin (IVIG), interferons (e.g. IFN-beta-la (IVIG)), interferon species And) And IFN-beta-lb) (ii) a Glatiramer acetateA polypeptide; natalizumabMitoxantroneCytotoxic agents, calcineurin inhibitors (e.g., cyclosporine a or FK 506); an immunosuppressive macrolide, such as sirolimus or a derivative thereof; e.g., 40-O- (2-hydroxy) ethyl-sirolimus, a lymphocyte homing agent, e.g., FTY720 or an analog thereof, a corticosteroid; cyclophosphamide; azathioprine; methotrexate; leflunomide or an analog thereof; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyperguline or an analog thereof; immunosuppressive monoclonal antibodies, e.g., monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD11a/CD18, CD7, CD25, CD27, B7, CD40, CD45, CD58, CD137, ICOS, CD150(SLAM), OX40, 4-1BB or ligands thereof; or immunomodulatory compounds thereof, such as CTLA4-Ig (abatacept,) CD28-Ig, B7-H4-Ig, or other co-stimulatory agents, or adhesion molecule inhibitors such as mAbs or low molecular weight inhibitors (including LFA-1 antagonists, selectin antagonists, and VLA-4 antagonists), or another immunomodulatory agent.

Optionally, such an immune disorder is selected from an autoimmune disease, transplant rejection, or graft-versus-host disease.

Optionally, the autoimmune disease is selected from the group consisting of: multiple sclerosis, including relapsing multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis; psoriasis; rheumatoid arthritis; psoriatic arthritis, Systemic Lupus Erythematosus (SLE); ulcerative colitis; crohn's disease; benign lymphocytic vasculitis, thrombocytopenic purpura, idiopathic thrombocytopenia, idiopathic autoimmune hemolytic anemia, pure red blood cell regeneration disorder, Scheink's syndrome, rheumatic diseases, connective tissue diseases, inflammatory rheumatism, degenerative rheumatism, extraarticular rheumatism, juvenile rheumatoid arthritis, gouty arthritis (arthritis uratica), muscular rheumatism, chronic polyarthritis, cryoglobulinemic vasculitis (cryoglobulinemic vasculitis), ANCA-associated vasculitis, antiphospholipid syndrome, myasthenia gravis, autoimmune hemolytic anemia, Guillain-Barre syndrome, chronic immune polyneuropathy, autoimmune thyroiditis, insulin dependent diabetes mellitus, type I diabetes mellitus, Addison's disease, membranous nephropathy, Goodpasture's disease, autoimmune gastritis, autoimmune atrophic gastritis, pernicious anemia, pemphigus vulgaris, cirrhosis, primary biliary cirrhosis, dermatomyositis, polymyositis, fibromyositis, muscular sclerosis, celiac disease, immunoglobulin a nephropathy, allergic purpura, evans syndrome (evansyndrome), atopic dermatitis, psoriasis, arthropathic psoriasis (psoriasis arthopathia), Graves ' disease, Graves ' ophthalmopathy, scleroderma, systemic scleroderma, progressive systemic scleroderma, asthma, allergy, primary biliary cirrhosis, Hashimoto's thyroiditis (Hashimoto's thyroiditis), primary myxoedema, sympathetic ophthalmia, autoimmune uveitis, hepatitis, chronic active hepatitis, collagen disease, ankylosing spondylitis, scapulohumeral periarthritis, nodular systemic arteritis, calcinosis, wegener's granulomatosis, microscopic multiple vasculitis, chronic urticaria, bullous skin diseases, pemphigoid, atopic eczema, devike's disease, childhood autoimmune hemolytic anemia, refractory or chronic autoimmune cytopenia, prevention of development of autoimmune anti-factor VIII antibodies in acquired hemophilia a, cold agglutinin disease, neuromyelitis optica, stiff person syndrome, gingivitis, periodontitis, pancreatitis, myocarditis, vasculitis, gastritis, gout, gouty arthritis, and inflammatory skin disorders selected from the group consisting of: psoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne, noctomoplementic urticaria vasculitis, pericarditis, myositis, anti-synthetase syndrome, scleritis, macrophage activation syndrome, bekat syndrome, PAPA syndrome, bruise syndrome, gout, adult and juvenile stele's disease, cryropyrinopathy, Muckle-wills syndrome, familial cold-induced autoinflammatory syndrome, neonatal onset multisystem inflammatory disease, familial mediterranean fever, chronic pediatric nervous system, skin and joint syndromes, systemic juvenile idiopathic arthritis, Hyper IgD syndrome (Hyper IgD syndrome), Schnitzler's syndrome, autoimmune retinopathy, age-related macular degeneration, arteriosclerosis, chronic prostatitis, and TNF receptor-associated periodic syndrome (TRAPS).

Optionally, the autoimmune disease is selected from the group consisting of any type and subtype of any of the following: multiple sclerosis, rheumatoid arthritis, type I diabetes, psoriasis, systemic lupus erythematosus, inflammatory bowel disease, uveitis, and heulan syndrome.

According to at least some embodiments, there is provided a method for treating or preventing an infectious disease, the method comprising administering to a subject in need thereof an effective amount of any of an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody or pharmaceutical composition as described herein.

Optionally, the infectious disease is selected from diseases caused by bacterial infections, viral infections, fungal infections and/or other parasitic infections.

Optionally, the infectious disease is selected from hepatitis B, hepatitis C, infectious mononucleosis, EBV, cytomegalovirus, AIDS, HIV-1, HIV-2, tuberculosis, malaria, and schistosomiasis.

According to at least some embodiments, there is provided a method for treating or preventing cancer, the method comprising administering to a subject in need thereof an effective amount of any of an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody or pharmaceutical composition as described herein.

Optionally, such treatment is combined with another moiety or therapy for treating cancer.

Optionally, the therapy is radiotherapy, antibody therapy, chemotherapy, photodynamic therapy, adoptive T cell therapy, Treg depletion, surgery or combination therapy with conventional drugs.

Optionally, the moiety is selected from the group consisting of: immunosuppressants, cytotoxic drugs, tumor vaccines, antibodies (e.g., bevacizumab, erbitux), peptides, chloroplasts (pepti-bodies), small molecules, chemotherapeutic agents (e.g., cytotoxic and cytostatic agents (e.g., paclitaxel, cisplatin, vinorelbine, docetaxel, gemcitabine, temozolomide, irinotecan, 5FU, carboplatin)), immunological modifiers (e.g., interferons and interleukins), immunostimulatory antibodies, growth hormones or other cytokines, folic acid, vitamins, minerals, aromatase inhibitors, RNAi, histone deacetylase inhibitors, and protease inhibitors.

Optionally, the cancer is selected from the group consisting of: breast cancer, cervical cancer, ovarian cancer, endometrial cancer, melanoma, bladder cancer, lung cancer, pancreatic cancer, colon cancer, prostate cancer, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, myeloid leukemia, Acute Myelogenous Leukemia (AML), chronic myelogenous leukemia, thyroid cancer follicular carcinoma, myelodysplastic syndrome (MDS), fibrosarcoma and rhabdomyosarcoma, melanoma, uveal melanoma, teratocarcinoma, neuroblastoma, glioma, glioblastoma, benign tumors of the skin, keratoacanthoma, renal cancer, anaplastic large cell lymphoma, esophageal cancer, hepatocellular carcinoma, follicular dendritic cell carcinoma, intestinal cancer, Muscle invasive cancer, seminal vesicle tumor, epidermal carcinoma, spleen cancer, bladder cancer, head and neck cancer, stomach cancer, liver cancer, bone cancer, brain cancer, retina cancer, biliary tract cancer, small intestine cancer, salivary gland cancer, uterine cancer, testicular cancer, connective tissue cancer, prostatic hypertrophy, myelodysplasia, Waldenstrom's macroglobulinemia, nasopharyngeal cancer, neuroendocrine cancer, myelodysplastic syndrome, mesothelioma, angiosarcoma, kaposi's sarcoma, benign tumor, esophageal cancer, fallopian tube cancer, peritoneal cancer, papillary serous leigh cancer, malignant ascites, gastrointestinal stromal tumor (GIST), lifglamoru's syndrome, and von hippel-lindau syndrome (VHL), and wherein the cancer is non-metastatic, invasive or metastatic.

Optionally, the cancer is one of melanoma, liver cancer, kidney cancer, brain cancer, breast cancer, colon cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, multiple myeloma, hodgkin's lymphoma, non-hodgkin's lymphoma, acute and chronic lymphocytic leukemia, and acute and chronic myelogenous leukemia.

According to at least some embodiments, there is provided a method for enhancing a secondary immune response to an antigen in a patient, the method comprising administering to a subject an effective amount of any of an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody or pharmaceutical composition as described herein.

Optionally, the antigen is a cancer antigen, viral antigen or bacterial antigen, and wherein the patient has received treatment with an anti-cancer agent vaccine or viral vaccine.

An immunotherapy on a patient comprising:

in vivo or ex vivo tolerance induction, comprising administering to a patient or to leukocytes isolated from the patient an effective amount of any of: an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody or pharmaceutical composition as described herein;

Ex vivo enrichment and proliferation of said cells;

reinfusion of these tolerogenic regulatory cells into the patient.

A method of using at least one of the following as a cancer vaccine adjuvant: any of the isolated polypeptides as described herein, or the fusion proteins as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein; or a pharmaceutical composition, the method comprising administering to a patient an immunogenic amount of a tumor associated antigen preparation of interest; and a cancer vaccine adjuvant in a formulation suitable for immunization, wherein the immune response against the tumor-associated antigen is stronger in the presence of the cancer vaccine adjuvant than in the absence of the cancer vaccine adjuvant.

In accordance with at least some embodiments, there is provided a method of combining therapeutic vaccination with an antigen with administration of any one of: an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody or pharmaceutical composition as described herein.

According to at least some embodiments, to increase the immune response, a method is provided of combining any one of the following with an adjuvant, and an antigen in a vaccine: an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody or pharmaceutical composition as described herein.

Optionally, the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, and/or an antigen of another pathogen.

According to at least some embodiments, any of the foregoing therapeutic agents according to at least some embodiments of the invention may be used in adoptive immunotherapy, including antibodies and antigen-binding fragments and conjugates comprising them, and/or alternative scaffolds, directed against: any of LY6G6F, VSIG10, TMEM25 and/or LSR proteins; LY6G6F, VSIG10, TMEM25 and/or LSR secreted or soluble forms or ECD and/or variants, and/or orthologs, and/or conjugates. Immune tolerance (immunene toleranc)e or immunological tolerance) is a process by which the immune system does not attack an antigen. It may be 'natural' or 'self-tolerant', in which the body is not loaded with an immune response to self-antigens; or 'induce tolerance', where tolerance to external antigens can be generated by treating the immune system. It appears in three forms: central tolerance, peripheral tolerance, and acquired tolerance. Without wishing to be bound by a single theory, in addition to natural killer cells (NK), NKT cells, Dendritic Cells (DC), and cells, tolerance also employs regulatory immune cells-including Tregs-to directly suppress autoreactive cells, along with some other subpopulations of cells with immunomodulatory properties-including CD8 ⁺T cells and other types of CD4⁺T cells (Tr1, Tr 3).

Tolerance can be induced by blocking co-stimulation or by conjugating co-inhibitory B7 to its counter receptor. Transfer of tolerance involves the isolation of cells that have been used for induction of tolerance either in vivo (i.e., prior to cell isolation) or ex vivo, enrichment and proliferation of these cells ex vivo, followed by reinfusion of the proliferated cells into the patient. Such methods may be used to treat autoimmune diseases as recited herein, immune-related diseases as recited herein, transplant rejection (transplantation rejection). Thus, in accordance with at least some embodiments, to induce differentiation of tolerogenic regulatory cells, the present invention provides methods for tolerance induction, the methods comprising therapeutically administering to a patient, or to leukocytes isolated from the patient, in vivo or ex vivo, effective amounts of: any isolated soluble LY6G6F, VSIG10, TMEM25, LSR polypeptide, or a polypeptide comprising the extracellular domain of LY6G6F, VSIG10, TMEM25, LSR, or a fragment thereof, or fusion thereof to a heterologous sequence, and/or a polyclonal antibody or monoclonal antibody or antigen binding fragment specific for any LY6G6F, VSIG10, TMEM25 and/or LSR protein and conjugates comprising them, and/or alternative scaffolds, followed by ex vivo enrichment and proliferation of said cells and reinfusion of these tolerogenic regulatory cells into said patient.

According to at least some embodiments, the present invention provides an assay for detecting the presence of LY6G6F, VSIG10, TMEM25 and/or LSR proteins in a biological sample or individual in vitro or in vivo, comprising contacting the sample with an antibody and/or antigen binding fragment and/or conjugate comprising same specific for LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides, and/or an alternative scaffold, and detecting binding of LY6G6F, VSIG10, TMEM25 and/or LSR proteins in the sample and/or individual.

According to at least some embodiments, there is provided a method for detecting, in a sample, any nucleic acid having any one of SEQ ID NOs: 1-8, 11-18, 47-50, 58, 143, or a variant thereof that is at least 95% identical thereto.

According to at least some embodiments, there is provided a method for diagnosing a disease in a patient, comprising detecting in the subject or a sample obtained from said subject any nucleic acid having any one of SEQ ID NOs: 1-8, 11-18, 47-50, 58, 143, or a variant thereof that is at least 95% identical thereto, or a fragment thereof.

Optionally, the detection of the polypeptide is performed in vivo or in vitro.

Optionally, the detection is by immunoassay.

Optionally, the detection is performed using an antibody or fragment as described herein.

According to at least some embodiments, the present invention provides methods for detecting a disease, diagnosing a disease, monitoring disease progression or efficacy or relapse of a disease, or selecting a therapy for a disease, detecting cells infected with the foregoing disease, comprising detecting expression of LY6G6F, VSIG10, TMEM25 and/or LSR, wherein the disease is selected from cancer, an infectious disorder as recited herein, and/or an immune related disorder.

According to one embodiment, detecting the presence of such a polypeptide is indicative of the presence of the disease and/or its severity and/or its progression. According to another embodiment, a change in the expression and/or level of the polypeptide as compared to the expression and/or level thereof in a healthy subject or a sample obtained therefrom is indicative of the presence of the disease and/or its severity and/or its progression. According to a further embodiment, a change in the expression and/or level of the polypeptide as compared to the expression and/or level thereof in said subject or a sample obtained therefrom earlier is indicative of the progression of the disease. According to still further embodiments, detecting the presence of the polypeptide and/or its relative change in expression and/or level is useful for selecting a treatment and/or monitoring the treatment of the disease.

Brief description of the drawings

Figure 1 presents the following amino acid sequence: LY6G6F (FIG. 1A, SEQ ID NO: 1), VSIG10 (FIG. 1B, SEQ ID NO: 3, and 1C, SEQ ID NO: 5), TMEM25 (FIG. 1D, SEQ ID NO: 7), LSR (FIG. 1E (SEQ ID NO: 11), 1F (SEQ ID NO: 13), 1G (SEQ ID NO: 15), 1H (SEQ ID NO: 16), 1I (SEQ ID NO: 17), and 1J (SEQ ID NO: 18) proteins, fragments, ECDs and corresponding nucleic acid sequences encoding the same E, nucleic acid sequences of alternative exon skipping variants of skipping exons 3, 4 and 5) are presented in bold in FIGS. 1C, and 1I, respectively. The nucleic acid sequence corresponding to the transmembrane region (TM) is shown in bold and underlined form in FIG. 1C. The nucleic acid sequence corresponding to Signal Peptide (SP) appears in bold italics in FIGS. 1C, 1E, 1G, 1H, 1I, and 1J. The TGA stop codon is highlighted in FIGS. 1C and 1I.

Figure 2 presents an amino acid sequence comparison between: VSIG10 variant SEQ ID NO: 5 and the known VSIG10 protein, SEQ ID NO: 3(genbank accession No. NP _061959.2) (fig. 2A); LSR _ isoform-a, SEQ ID NO: 11 and the known LSR protein, genbank accession No. NP _991403 SEQ ID NO: 62 (FIG. 2B-1); LSR _ isoform-a, SEQ id no: 11 and the known LSR protein, genbank accession number XP — 002829104, SEQ ID NO: 68 (FIG. 2B-2); LSR _ isoform-b, SEQ ID NO: 13 and the known LSR protein, genbank accession No. NP _057009, SEQ ID NO: 63 (FIG. 2C-1); LSR _ isoform-b, SEQ ID NO: 13 and the known LSR protein, genbank accession number BAC11614, SEQ ID NO: 65 (FIG. 2C-2); LSR _ isoform-c, SEQ ID NO: 15 and the known LSR protein, genbank accession No. NP _991404, seq id NO: 66 (FIG. 2D-1); LSR _ isoform-c, SEQ ID NO: 15 and the known LSR protein, genbank accession number XP — 002829105.1, SEQ ID NO: 69 (FIG. 2D-2); LSR _ isoform-d, SEQ ID NO: 16 and the known LSR protein, genbank accession No. NP _991404, SEQ ID NO: 66 (FIG. 2E-1); LSR _ isoform-d, SEQ ID NO: 16 and the known LSR protein, genbank accession number XP 002829105.1, SEQ ID NO: 69 (FIG. 2E-2); LSR _ isoform-e, SEQ id no: 17 and the known LSR protein, genbank accession number BAG59226.1, SEQ ID NO: 67 (FIG. 2F); LSR _ isoform-f, SEQ ID NO: 18 and the known LSR protein, genbank accession No. NP _991403, SEQ ID NO: 62 (FIG. 2G-1); LSR _ isoform-f, SEQ ID NO: 18 and the known LSR protein, genbank accession No. NP _991404, SEQ ID NO: 66 (fig. 2G-2). The sequences of the unique border portions (unique junctions) of the VSIG10 variant (SEQ ID NO: 5) and LSR variant (SEQ ID NO: 18) are bold and highlighted (fig. 2A and 2G, respectively).

Figure 3 shows a scatter plot showing the expression of VSIG10 transcripts encoding VSIG10 protein on virtual panels of ALL tissues and disorders using the MED discovery engine, showing differential expression of VSIG10 transcripts in several groups of cells from the immune system (mainly in leukocytes), and in several groups of cells of different cancer disorders (e.g., CD10+ leukocytes from ALL and BM-CD34+ cells from AML).

Figure 4 shows a scatter plot showing the expression of LSR transcripts encoding LSR protein on a virtual panel of all tissues and conditions using MED discovery engine, showing differential expression of LSR transcripts in several groups of cells from the immune system (mainly in bone marrow cells), and in several groups of cells of cancerous conditions of different tissues (e.g. in breast, lung, ovarian, pancreatic, prostate and skin cancers).

FIG. 5A presents an amino acid sequence comparison of LY6G6F human (SEQ ID NO: 1) and mouse (reflNP-001156664.1, SEQ ID NO: 29). FIG. 5B presents an amino acid sequence comparison of VSIG10 human (SEQ ID NO: 3) and mouse (splD3YX43.2, SEQ ID NO: 30). FIG. 5C presents an amino acid sequence comparison of LSR human (SEQ ID NO: 11) and mouse (reflNP-059101.1, SEQ ID NO: 31) or mouse (reflNP-001157656.1, SEQ ID NO: 32). FIG. 5D shows an amino acid sequence comparison of TMEM25 human (SEQ ID NO: 7) and mouse (ref: lcll4109, SEQ ID NO: 28).

Fig. 6 presents a table summarizing the primers used to clone LY6G6F transcripts fused to EGFP. Gene specific sequences are shown in bold; the restriction site extension used for cloning the target is italicized; and the Kozak sequence is underlined.

Fig. 7 presents the full-length DNA sequence of LY6G6F fused to EGFP. The gene-specific sequence corresponding to the full-length sequence of LY6G6F is indicated in bold, and the gene-specific sequence corresponding to the EGFP sequence is underlined in non-bold italics.

Figure 8 presents the full-length amino acid sequence of the generated LY6G6F fused to EGFP. The gene-specific sequences corresponding to the full-length sequence of LY6G6F are indicated in bold; the gene specific sequences corresponding to the EGFP sequence are not bold italicized underlined.

Fig. 9A and 9B present the cellular localization of G6F _ EGFP fusion protein transiently expressed in HEK293T cells. This image was obtained using a 40x objective of a confocal microscope.

Figure 10 presents the mouse ECD fused to mouse IgG2a Fc as follows: mouse LY6G6F (also known as LY6G6F-Ig, FIG. 10A), mouse VSIG10 (FIG. 10B), mouse TMEM25 (also known as TMEM25-Ig, FIG. 10C) or mouse LSR (also known as LSR-Ig, FIG. 10D) ECD-mIgG2aFc fusion protein (SEQ ID NO: 23, 24, 25, or 26, respectively). The amino acid residues corresponding to the Signal Peptide (SP) are shown in italics. Amino acid residues corresponding to the ECD sequence are underlined. The amino acid residues corresponding to mouse IgG2a Fc are shown in bold type (SEQ ID NO: 27).

FIG. 11 presents the amino acid sequence of a human ECD fused to human IgG1 Fc, where the Cys at position 220 (according to position 5 in full-length human IgG1, SEQ ID NO: 70) was replaced by a Ser (SEQ ID NO: 156) as follows: human LY6G6F (fig. 11A), human VSIG10 (fig. 11B), human VSIG 10-skipping exon 3 variant (fig. 11C), human TMEM25 (fig. 11D), human LSR isoform a (fig. 11E), human LSR isoform B (fig. 11F), human LSR isoform C (fig. 11G), human LSR isoform D (fig. 11H), human LSR isoform E (fig. 11I), human LSR isoform F (fig. 11J) ECD (SEQ ID NOs: 71-80, respectively) fused to human IgG1 Fc. The amino acid residues corresponding to the Signal Peptide (SP) are shown in bold italics. Amino acid residues corresponding to the human ECD sequence are underlined. The amino acid residue corresponding to human IgG1 Fc is not indicated, where the Cys at position 220 was replaced by a Ser (SEQ ID NO: 156).

FIG. 12 is a histogram showing the overexpression of LSR transcripts detectable by or according to LSR _ seg24-36_200-309/310_ amplicon (SEQ ID NO: 140) in cancerous ovarian samples relative to normal samples.

FIG. 13 is a histogram showing the overexpression of LSR transcripts detectable by or according to LSR _ seg24-36_200-309/310_ amplicon (SEQ ID NO: 140) in cancerous breast samples relative to normal samples.

FIG. 14 is a histogram showing the overexpression of LSR transcripts detectable by or according to LSR _ seg24-36_200-309/310_ amplicon (SEQ ID NO: 140) in cancerous lung samples relative to normal samples.

FIG. 15 is a histogram showing the overexpression of LSR transcripts detectable by or according to LSR _ seg24-36_200-309/310_ amplicon (SEQ ID NO: 140) in normal tissue samples relative to cancerous samples.

FIG. 16 is a histogram showing the overexpression of LSR transcripts detectable by or according to LSR _ seg24-36_200-309/310_ amplicon (SEQ ID NO: 140) in cancerous kidney samples relative to normal samples.

FIG. 17 is a histogram showing the overexpression of LSR transcripts detectable by or according to LSR _ seg24-36_200-309/310_ amplicon (SEQ ID NO: 140) in cancerous liver samples relative to normal samples.

FIG. 18 shows a Western blot analysis of the expression of LSR _ P5a _ Flag _ m protein (SEQ ID NO: 144) in stably transfected recombinant HEK293T cells, as detected with anti-Flag (Sigma cat # A8592) (FIG. 18A) and anti-LSR antibodies as follows: abnova, cat # H00051599-B01P (FIG. 18B), Abeam, cat ab59646 (FIG. 18C) and Sigmacat # HPA007270 (FIG. 18D). Strip 1: HEK293T _ pIRESpuro 3; strip 2: HEK293T _ pIRESpuro3_ LSR _ P5a _ Flag.

Figure 19 shows the subcellular localization of LSR _ P5a _ Flag _ m. LSR _ P5a _ Flag _ m (SEQ ID NO: 144) predominantly localized to the cytoplasm of cells, but could also be detected on the cell surface as detected with anti-Flag (Sigma cat # A9594) (FIG. 19A) and anti-LSR antibodies as follows: abeam, cat ab59646 (FIG. 19B), Abnova, cat # H00051599-B01P (FIG. 19C) and Sigma cat # HPA007270 (FIG. 19D).

Figure 20 shows endogenous expression of LSR in different cell lines. A band of 72kDa corresponding to LSR was detected with anti-LSR antibodies in the following extracts: (1) caov3, (2) ES2, (3) OV-90, (4) OVCAR3, (5) SK-OV3, (6) TOV112D, (7) CaCo2, (8) HeLa, (9) Hep G2, (10) MCF-7, (11) SkBR3, and (12)293T _ LSR _ P5a _ Flag (FIG. 20A). anti-GAPDH (Abeam cat # ab9484) served as a loading control (fig. 20B).

FIG. 21 is a histogram showing the expression of TMEM25 transcript detectable by or according to eg21-27-TMEM25_ seg _21-27_200-344/346_ amplicon (SEQ ID NO: 123) in normal and cancerous breast tissue.

FIG. 22 is a histogram showing detectable expression of TMEM25 transcript by or according to eg21-27-TMEM25_ seg _21-27_200-344/346_ amplicon (SEQ ID NO: 123) in different normal tissues.

FIG. 23 presents Western blot results showing the specific interaction between (A) rabbit anti-TMEM 25 antibody and TMEM25_ P5 protein (SEQ ID NO: 7) and TMEM25_ P5_ Flag (SEQ ID NO: 129) instead of HEK _293T _ pRp 3. (B) Specific interaction between TMEM25_ P5_ Flag protein (SEQ ID NO: 129) and anti-Flag antibody. Strip 1: HEK293T _ pIRESpuro 3; strip 2: HEK293T _ pIRESpuro3_ TMEM 25-P5; the strip 3: HEK293T _ pIRESpuro3_ TMEM 25-P5-Flag.

FIG. 24 presents the cell surface localization of TMEM25_ P5(SEQ ID NO: 132) (FIG. 24A) and TMEM25_ P5_ Flag (SEQ ID NO: 129) (FIG. 24B) using anti-TMEM 25 antibody. FIG. 24C shows TMEM25_ P5_ Flag (SEQ ID NO: 129) localization using anti-Flag antibody (Sigma, cat # A9594).

Figure 25 shows that anti-TMEM 25 antibody binds to full-length TMEM25 protein in HEK293T recombinant cells expressing TMEM25_ P5_ Flag protein (1: 2250) (figure 25A), indicating membrane localization of TMEM25 protein, compared to mouse serum (1: 2250) (figure 25B) used as a negative control.

Figure 26 presents western blot results showing the expression of endogenous TMEM25 protein in different cell lines: (1): HEK293T _ pIRESpuro3, (2) HEK293T _ pIRESpuro3_ TMEM25-P5-Flag, (3) KARPAS, (4) G-361, (5) RPMI8226, (6) DAUDI, (7) Jurkat.

FIG. 27 shows the specific knock-down of TMEM25_ P5_ Flag protein (SEQ ID NO: 129) in HEK293T cells stably expressing TMEM25_ P5_ Flag (SEQ ID NO: 129), transfected with TMEM25_ P5 siRNA (L-018183-00-0005, Dhamacon) (lane 2) compared to HEK293T cells stably expressing TMEM25_ P5_ FLAG, transfected with Scrambled-SiRNA (lane 1) (Dharmacon, D-001810-10-05) using an anti-TMEM 25 antibody (Sigma, cat # HPA 012163).

Fig. 28 shows that anti-LSR (Cat No. ab59646, Abeam) at dose-dependent concentrations of 3ug/ml, 1ug/ml and 0.3ug/ml in sections of the positive control cell line (LSR _ P5a _ Flag _ m transfected HEK293T cells) compared to the negative control cell line empty vector HEK293T cells (column 2, fig. B, D and F) in the pH 9 antigen retrieval method showed specific immunoreactivity (column 1, fig. A, C and E).

Figure 29 shows that anti-TMEM 25(Cat No. hpaa012163, Sigma) (column 1, panels A, C and E) at dose-dependent concentrations of 3ug/ml, 1ug/ml and 0.3ug/ml in sections of the positive control cell line (TMEM25_ P5_ Flag transfected HEK293T cells), respectively, compared to the negative control cell line empty vector HEK293T cells (column 2, panels B, D and F) in the pH 9 antigen retrieval method shows specific immunoreactivity.

FIGS. 30A-E show the in vitro inhibitory effect of soluble LY6G6F-Ig (SEQ ID NO: 23), TMEM25-Ig (SEQ ID NO: 25), and LSR-Ig (SEQ ID NO: 26) on mouse T cell activation. Activation of T cells isolated from the spleen of DO11.10 mice was induced with 20ug/ml (FIG. 30A-C, E) or 2ug/ml (FIG. D) of OVA323-339 in the presence of irradiated splenocytes from Balb/c mice acting as APCs. In these studies, CTLA4-Ig or B7-H4-Ig were used as positive controls, while mouse IgG2a was used as the Ig control.

FIG. 31 shows the in vitro inhibitory effect of bead-bound LSR-Ig (SEQ ID NO: 26) on T cell proliferation induced by anti-CD 3 and anti-CD 28 coated beads.

FIG. 32 shows the effect of LY6G6F, VSIG10, TMEM25, and LSR fusion proteins (SEQ ID NOS: 23-26, respectively) on CD 4T cell activation as demonstrated by reduced expression of IFN γ secretion (A) and the activation marker CD69 (B). Each bar is the average of two cultures and error bars represent standard deviations (student t-test, P < 0.05, P < 0.01, compared to control mIgG2 a).

FIG. 33 shows the effect of stimulatory cells (a murine thymoma cell line, Bw5147, engineered to express membrane-bound anti-human CD3 antibody fragments) expressing cDNA encoding human LY6G6F, TMEM25, or LSR (SEQ ID NO: 1, 7, or 11, respectively) on the proliferation (CPM) of bulk human T cells (FIG. 33A), CD4+ human T cells (FIG. 33B), CD8+ human T cells (FIG. 33C), or naive CD4CD45RA + human T cells (FIG. 33D). Results are shown as mean +/-SEM of 6 (fig. 33A) or 3 (fig. 33B, C, and D) experiments. P < 0.05, P < 0.01, P < 0.001, and P < 0.0001 (student's t-test) represent significantly different results compared to the empty vector.

FIG. 34 shows the therapeutic effect of LSR-Ig (SEQ ID NO: 26) or TMEM25-Ig (SEQ ID NO: 25) on the treatment of PLP 139-151-induced R-EAE models in SJL mice. LSR-Ig (SEQ ID NO: 26) or TMEM25-Ig (SEQ ID NO: 25) were administered therapeutically from the onset of disease remission (day 18) with 100 microG/mouse, i.p. (i.p.) 3 times a week for two weeks. The therapeutic effect of LSR-Ig and TMEM25-Ig on clinical symptoms was shown as a reduction in mean clinical score (fig. 34A). Furthermore, on day 35 after R-EAE induction (FIG. 34B), LSR-Ig and TMEM25-Ig treatment inhibited DTH responses of either the induction epitope (PLP139-151) or the expansion epitope (PLP 178-191). In this study, the effect of LSR-Ig or TMEM25-Ig was studied compared to mIgG2a Ig negative control and CTLA4-Ig positive control given similar to the protocol for the test protein.

FIG. 35 shows the dose dependence and mode of action of TMEM25-Ig (SEQ ID NO: 25) in the R-EAE model in SJL mice. In this study, treatment was given at 100, 30 or 10 microrogs/mouse from the onset of remission (day 19), i.p. (i.p.) 3 times per week for two weeks, compared to the 10 microG/mouse IgG2a control given on a similar schedule, the effects of TMEM25-Ig treatment on disease progression (FIG. 35A), DTH responses to the expanded epitopes PLP178-191 and MBP84-104 on days 45 and 76 after R-EAE induction (FIG. 35B), ex vivo recall responses of splenocytes isolated on days 45 and 75 after disease induction (FIG. 35C), and LN cells isolated on day 45 after disease induction (FIG. 35D) are shown, as demonstrated by the effect of TMEM25-Ig treatment on cell proliferation and cytokine secretion (IFNg, IL-17, IL-10, and IL-4). The effect of TMEM25-Ig on cell counts of spleen, lymph nodes and CNS as well as different lineages present in the CNS (lines) when treated with 100 ug/dose of TMEM25-Ig is shown in fig. 35E.

FIG. 36 shows the therapeutic effect of VSIG10-Ig (SEQ ID NO: 24) treatment in the PLP 139-151-induced R-EAE model in SJL mice. VSIG10-Ig (SEQ ID NO: 24) was administered therapeutically at 100 microG/mouse starting from remission (day 19), i.p. (i.p.) 3 times a week for two weeks. The therapeutic effect of VSIG10-Ig on clinical symptoms was shown as a reduction in mean clinical score (fig. 36A). In addition, VSIG10-Ig treatment inhibited DTH responses to the epitope extensions (PLP178-191 and MBP MBP84-104) at day 45 and day 76 post-induction of R-EAE (FIG. 36B). Also shown is the effect of VSIG10-Ig on ex vivo recall responses (fig. 36C) of splenocytes isolated at days 45 and 75 post disease induction and LN cells isolated at day 45 post disease induction (fig. 36D), as demonstrated by the effect of VSIG10-Ig treatment on cell proliferation and cytokine (IFNg, IL-17, IL-10, and IL-4) secretion. The effect of VSIG10-Ig on cell counts of spleen, lymph nodes and CNS and the different lineages (lines) present within each of these tissues after treatment with 100 ug/dose of VSIG10-Ig is shown in fig. 36E. In this study, the effects of VSIG10-Ig were studied compared to mIgG2a Ig controls given at doses and schedules similar to VSIG 10-Ig.

FIG. 37 shows the therapeutic effect of LSR-Ig (SEQ ID NO: 26), i.p. (intraperitoneal injection) administered at 100 microG/mouse 3 times a week for 10 days in the collagen-induced arthritis (CIA) model of rheumatoid arthritis. Clinical scores (a), paw swelling (B) and histological lesions (C) were measured using CTLA4-Ig (100 microrogs/mouse) and TNFR-Ig (etanercept) as positive controls and mIgG2a Ig control (100 microrogs/mouse) as negative controls.

FIG. 38 shows the effect of treatment of LY6G6F-Ig (SEQ ID NO: 23), i.p. (intraperitoneal injection) administered at 25mg/kg 3 times per week for 2 weeks in a collagen-induced arthritis (CIA) model of rheumatoid arthritis, with measurements given in terms of clinical score.

For fig. 12-17, 21, 22, it is divided into separate parts "a", "B", etc., only for space reasons, so that all results can be shown.

Detailed description of the invention

In at least some embodiments, the invention relates to any one of the proteins designated LY6G6F, VSIG10, TMEM25 and/or LSR, and corresponding nucleic acid sequences thereof, and portions and variants thereof and fusion proteins and conjugates comprising the same, and/or polyclonal and monoclonal antibodies and/or antigen binding fragments and/or conjugates comprising the same, and/or a surrogate scaffold that binds LY6G6F, VSIG10, TMEM25 and/or LSR and/or portions and/or variants thereof, and their use as a therapeutic and/or diagnostic agent, and different uses as described herein.

U.S. patent application Nos. US 2009117566, US 20090017473, and other family members assigned to Genentech corporation disclose a 382 amino acid LY6G6F protein sequence having a transmembrane domain between residues 234-254 and 354-374 (DNA 234441, tumor associated antigen target (TAT) TAT201, in which SEQ ID NO: 92 is present). The '566,' 473 applications, as well as other applications from this patent family, disclose that TAT201 is overexpressed in colon and rectal cancers. PCT application nos. WO 2003083074 and WO 2004046342 disclose a 382 amino acid LY6G6F protein sequence as one of many genes that are overexpressed in colon cancer cells. These patent applications are further said to relate to methods for detecting and treating colon cancer using LY6G 6F. However, these patent applications do not teach or suggest or provide any motivation to guide the skilled person for the treatment and/or diagnosis of cancers other than colorectal cancer, and/or infectious disorders, and/or immune related disorders using antibodies specific for LY6G6F and/or LY6G6F ECD. These patent applications do not describe LY6G6F ECD and do not teach or suggest or provide any incentive to guide the skilled person to use LY6G6F ECD for the treatment of cancer and/or infectious disorders, and/or immune related disorders.

As one of many (hundreds to thousands) of proteins useful for diagnosing, preventing, and treating disorders associated with abnormal expression or activity of these proteins, TMEM25 is disclosed in PCT application nos. WO 9958642 and WO2003087300, and U.S. patent application nos. US 2007041963 and US 2005202526. However, these applications do not teach or suggest or provide any motivation to guide a skilled artisan to treat and/or diagnose cancer, and/or infectious disorders, and/or immune related disorders using antibodies specific for TMEM25 and/or TMEM25 ECD. TMEM25 is also disclosed in U.S. patent application No. US2004010134 as one of hundreds of albumin fusion proteins useful for diagnosing, treating, preventing or ameliorating a disease or disorder (e.g., cancer, anemia, arthritis, asthma, inflammatory bowel disease, or alzheimer's disease). However, this application does not teach or suggest or provide any motivation to guide a skilled artisan to treat and/or diagnose cancer, and/or infectious disorders, and/or immune related disorders using antibodies specific for TMEM25 and/or TMEM25 ECD. As a prognostic and predictive biomarker for breast cancer diagnosis, TMEM25 is also disclosed in dulland p (dolan p) et al, tumor biology (tumor Biol)30 (4): 200-9. However, this publication does not teach or suggest or provide any motivation to guide the skilled person to treat cancer, and/or infectious disorders, and/or immune related disorders using antibodies specific for TMEM25 and/or TMEM25 ECD.

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

As used herein, the term "isolated" refers to a compound of interest (e.g., a polynucleotide or polypeptide) that is in an environment different from that in which it naturally occurs, e.g., isolated from its natural environment, e.g., by concentrating a peptide into a concentrate that is not found in nature. "isolated" includes compounds in samples that are substantially enriched in a compound of interest and/or wherein such a compound of interest is partially or substantially purified.

By "immune cell" is meant any cell from a hematopoietic source, including but not limited to T cells, B cells, monocytes, dendritic cells, and macrophages.

As used herein, the term "polypeptide" refers to a chain of amino acids of any length, whether modified (e.g., phosphorylated or glycosylated).

As used herein, a "co-stimulatory polypeptide" or "co-stimulatory molecule" is a polypeptide that, upon interaction with a cell surface molecule on a T cell, modulates the T cell response.

As used herein, a "costimulatory signal" is a signaling activity derived from the interaction between a costimulatory polypeptide on an antigen presenting cell and its receptor on a T cell during an antigen-specific T cell response. Without wishing to be bound by a single hypothesis, it is believed that the antigen-specific T cell response is mediated by two signals: 1) conjugation of T Cell Receptors (TCR) with antigenic peptides presented in the context of MHC, (signal 1), and 2) a second antigen-independent signal (signal 2) delivered by contact between different costimulatory receptor/ligand pairs. Without wishing to be bound by a single hypothesis, this "second signal" is critical in determining the type of T cell response (activation vs inhibition) and the intensity and duration of this response, and is regulated by both positive and negative signals from co-stimulatory molecules (e.g., the B7 family of proteins).

As used herein, the term "B7" polypeptide refers to a member of the B7 protein family that co-stimulates T cells, including but not limited to: b7-1, B7-2, B7-DC, B7-H5, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-S3 and biologically active fragments and/or variants thereof. Representative biologically active fragments include the extracellular domain or extracellular domain fragment of a costimulatory T cell.

As used herein, a "variant" polypeptide comprises at least one amino acid sequence alteration as compared to the amino acid sequence of the corresponding wild-type polypeptide.

As used herein, a "conservative" amino acid substitution refers to a substitution in which the substituted amino acid has similar structural or chemical properties. As used herein, the term "host cell" refers to prokaryotic and eukaryotic cells into which a recombinant vector may be introduced.

As used herein, the term "border portion" or "new junction" refers to the junction between two portions of a splice variant according to the present invention that are not joined in the wild-type or known protein. For example, an edge can optionally occur due to the linkage between the portion of the "known protein" above and the tail of a variant, and/or an edge can occur if the internal portion of the wild-type sequence is no longer present, such that in the splice variant the two portions of the sequence are now contiguous, and in the known protein the two portions are not contiguous. "bridging" can optionally be a border portion as described above, but can also include a linkage between the head and the "known protein" portion of a variant, or a linkage between the tail and the "known protein" portion of a variant, or an insertion between a linkage and the "known protein" portion of a variant.

In some embodiments, the bridge between a tail or head or unique insertion and the "known protein" portion of a variant comprises at least about 10 amino acids, or in some embodiments at least about 20 amino acids, or in some embodiments at least about 30 amino acids, or in some embodiments at least about 40 amino acids, wherein at least one amino acid is from the tail/head/insertion and at least one amino acid is from the "known protein" portion of a variant. In some embodiments, the bridge can include any number of amino acids from about 10 to about 40 amino acids (e.g., 10, 11, 12, 13.. 37, 38, 39, 40 amino acids in length, or any number in between).

It should be noted that the bridges cannot extend beyond the length of the sequence in either direction, and it should be assumed that each bridge description should be interpreted in such a way that the length of the bridge does not extend beyond the sequence itself.

Furthermore, bridging is described with respect to sliding windows in some contexts below. For example, some descriptions of these bridges are characterized in the following format: the bridge between two edges (where a portion of a known protein is not present in the variant) can optionally be described as follows: a bridging moiety of contrg-NAME _ Pl (representing the NAME of the protein), comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids long, optionally at least about 20 amino acids long, at least about 30 amino acids long, at least about 40 amino acids long, or at least about 50 amino acids long, wherein at least two amino acids comprise XX (2 amino acids at the bridging center, one from each end of the border) having the structure (encoded according to the sequence of contrg-NAME _ Pl): a sequence starting at any amino acid number 49-x to 49 (for example) and ending at any amino acid number 50+ ((n-2) -x) (for example), where x varies from 0 to n-2. In this example, it should also be read to include bridges where n is any number of amino acids between 10-50 amino acids in length. Furthermore, the bridging polypeptide cannot extend beyond the sequence and therefore should be read such that 49-x (for example) is not less than 1 and 50+ ((n-2) -x) (for example) is not greater than the total sequence length.

As used herein, the term "cancer" should be understood to encompass any neoplastic disease (whether invasive or metastatic) characterized by abnormal and uncontrolled cell division leading to malignant growth or a tumor. Non-limiting examples of cancers that may be treated with compounds according to at least some embodiments of the present invention are solid tumors, sarcomas, and hematological malignancies, including but not limited to: breast cancer (e.g., breast tumor), cervical cancer, ovarian cancer (ovarian tumor), endometrial cancer, melanoma, bladder cancer (bladder tumor), lung cancer (e.g., adenocarcinoma and non-small cell lung cancer), pancreatic cancer (e.g., pancreatic tumor, e.g., exocrine pancreatic tumor), colon cancer (e.g., colorectal tumor, e.g., colon adenocarcinoma and colon adenoma), prostate cancer (including advanced disease), hematopoietic tumors of lymphoid lineage (including leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, B-cell lymphoma, burkitt's lymphoma, multiple myeloma, hodgkin's lymphoma, non-hodgkin's lymphoma), myeloid leukemia (e.g., Acute Myelogenous Leukemia (AML), chronic myelogenous leukemia, thyroid cancer, thyroid follicular cancer, myelodysplastic syndrome (MDS)), Tumors of mesenchymal origin (e.g. fibrosarcoma and rhabdomyosarcoma), melanoma, uveal melanoma, teratocarcinoma, neuroblastoma, glioma, glioblastoma, benign tumors of the skin (e.g. keratoacanthoma), renal carcinoma, anaplastic large-cell lymphoma, esophageal squamous cell carcinoma, hepatocellular carcinoma, follicular dendritic cell carcinoma, intestinal carcinoma, muscle-infiltrating carcinoma, seminal vesicle tumor, epidermal carcinoma, spleen carcinoma, bladder carcinoma, head and neck carcinoma, gastric carcinoma, liver carcinoma, bone carcinoma, brain carcinoma, retinal carcinoma, biliary tract carcinoma, small intestinal carcinoma, salivary gland carcinoma, uterine carcinoma, testicular carcinoma, connective tissue carcinoma, prostatic hypertrophy, myelodysplasia, Waldenstrom's macroglobulinemia, nasopharyngeal carcinoma, neuroendocrine carcinoma, myelodysplastic syndrome, mesothelioma, angiosarcoma, kaposi's sarcoma, benign tumors, esophageal carcinoma, cervical carcinoma, and cervical cancer, Fallopian tube cancer, peritoneal cancer, papillary serous lewy carcinoma, malignant ascites, gastrointestinal stromal tumors (GIST), and hereditary cancer syndromes such as lyverine syndrome and hippel-lindau syndrome (VHL), and wherein such cancers may be non-metastatic, invasive or metastatic.

According to at least some preferred embodiments of the invention, the cancer is selected from the group consisting of: melanoma, liver disorders, kidney disorders, brain disorders, breast disorders, colon disorders, lung disorders, ovarian disorders, pancreatic disorders, prostate disorders, stomach disorders, multiple myeloma, and hematopoietic cancers including, but not limited to, lymphoma (hodgkins and non-hodgkins), acute and chronic lymphocytic leukemias, and acute and chronic myelogenous leukemias, and wherein such cancers may be non-metastatic, invasive or metastatic.

As used herein, the term "autoimmune disease" should be understood to encompass any autoimmune disease as well as chronic inflammatory disorders. According to at least some embodiments of the present invention, these autoimmune diseases should be understood to encompass any disease disorder or condition selected from the group comprising, but not limited to: multiple sclerosis, including relapsing multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis; psoriasis; rheumatoid arthritis; psoriatic arthritis, Systemic Lupus Erythematosus (SLE); ulcerative colitis; crohn's disease; benign lymphocytic vasculitis, thrombocytopenic purpura, idiopathic thrombocytopenia, idiopathic autoimmune hemolytic anemia, pure red blood cell regeneration disorder, Scheink's syndrome, rheumatic diseases, connective tissue diseases, inflammatory rheumatism, degenerative rheumatism, extraarticular rheumatism, juvenile rheumatoid arthritis, gouty arthritis (arthritis uratica), muscular rheumatism, chronic polyarthritis, cryoglobulinemic vasculitis (cryoglobulinemic vasculitis), ANCA-associated vasculitis, antiphospholipid syndrome, myasthenia gravis, autoimmune hemolytic anemia, Guillain-Barre syndrome, chronic immune polyneuropathy, autoimmune thyroiditis, insulin dependent diabetes mellitus, type I diabetes mellitus, Addison's disease, membranous nephropathy, Goodpasture's disease, autoimmune gastritis, autoimmune atrophic gastritis, pernicious anemia, pemphigus vulgaris, cirrhosis, primary biliary cirrhosis, dermatomyositis, polymyositis, fibromyositis, muscular sclerosis, celiac disease, immunoglobulin a nephropathy, allergic purpura, Evans syndrome (Evans syndrome), atopic dermatitis, psoriasis, arthropathic psoriasis (psoriasis arthopathia), Graves ' disease, Graves ' ophthalmopathy, scleroderma, systemic scleroderma, progressive systemic scleroderma, asthma, allergy, primary biliary cirrhosis, Hashimoto's thyroiditis (Hashimoto's thyroiditis), primary myxoedema, inductive ophthalmia, autoimmune uveitis, hepatitis, chronic active hepatitis, collagen disease, ankylosing spondylitis, scapulohumeral periarthritis, nodular systemic arteritis, calcinosis, wegener's granulomatosis, microscopic polyangiitis, chronic urticaria, bullous skin diseases, pemphigoid, atopic eczema, devike's disease, childhood autoimmune hemolytic anemia, refractory or chronic autoimmune cytopenia, prevention of development of autoimmune anti-factor VIII antibodies in acquired hemophilia a, cold agglutinin disease, neuromyelitis optica, stiff person syndrome, gingivitis, periodontitis, pancreatitis, myocarditis, vasculitis, gastritis, gout, gouty arthritis, and inflammatory skin disorders selected from the group consisting of: psoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne, noctomoplementic urticaria vasculitis, pericarditis, myositis, anti-synthetase syndrome, scleritis, macrophage activation syndrome, bekat syndrome, PAPA syndrome, bruise syndrome, gout, adult and juvenile stele's disease, cryropyrinopathy, Muckle-wills syndrome, familial cold-induced autoinflammatory syndrome, neonatal onset multisystem inflammatory disease, familial mediterranean fever, chronic pediatric nervous system, skin and joint syndromes, systemic juvenile idiopathic arthritis, Hyper IgD syndrome (Hyper IgD syndrome), Schnitzler's syndrome, autoimmune retinopathy, age-related macular degeneration, arteriosclerosis, chronic prostatitis, and TNF receptor-associated periodic syndrome (TRAPS).

Optionally and preferably, the autoimmune disease includes, but is not limited to, any type and subtype of any of the following: multiple sclerosis, rheumatoid arthritis, type I diabetes, psoriasis, systemic lupus erythematosus, inflammatory bowel disease, uveitis, or Creutzfeldt-Jakob syndrome.

As used herein, "multiple sclerosis" includes one or more of the following: multiple sclerosis, benign multiple sclerosis, relapsing and remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, progressive relapsing multiple sclerosis, chronic progressive multiple sclerosis, transient/progressive multiple sclerosis, rapidly worsening multiple sclerosis, clinically definite multiple sclerosis, malignant multiple sclerosis (also known as Marburg's Variant), and acute multiple sclerosis. Optionally, "a condition involving multiple sclerosis" includes, for example, devike's disease (also known as neuromyelitis optica); acute disseminated encephalomyelitis, acute demyelinating optic neuritis, demyelinating transverse myelitis, Miller Fisher syndrome (Miller-Fisher syndrome), encephalomyelogenous radiculoneuropathy (encephamyradiculouropathy), acute demyelinating polyneuropathy, tumorous multiple sclerosis, and Barlow's multiple sclerosis.

As used herein, "rheumatoid arthritis" includes one or more of the following: rheumatoid arthritis, gout and pseudogout, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, still's disease, ankylosing spondylitis, rheumatoid vasculitis. Optionally, conditions associated with rheumatoid arthritis include, for example, osteoarthritis, sarcoidosis, Henoch-Schonlein purpura (Henoch-Schonlein purpura), psoriatic arthritis, reactive arthritis, spondyloarthropathies, septic arthritis, hemochromatosis, hepatitis, vasculitis, Wegener's granulomatosis, Lyme disease, familial mediterranean fever, hyper-immunoglobulin syndrome with recurrent fever D, TNF receptor-associated periodic syndrome, and enteropathic arthritis associated with inflammatory bowel disease.

As used herein, "uveitis" includes one or more of the following: uveitis, anterior uveitis (or iridocyclitis), intermediate uveitis (pars plana), posterior uveitis (or chorioretinitis), and panuveitis (panveitic).

As used herein, "inflammatory bowel disease" includes one or more of the following: inflammatory bowel disease, Crohn's disease, Ulcerative Colitis (UC), collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's disease, indeterminate colitis.

As used herein, "psoriasis" includes one or more of the following: psoriasis, non-pustular psoriasis (including psoriasis vulgaris and psoriatic erythroderma (erythroderma psoriasis)), pustular psoriasis (including generalized pustular psoriasis (pustular psoriasis of von Zumbusch)), palmoplantar pustulosis (persistent palmoplantar pustulosis, baber-type pustulosis, end-of-limb pustulosis), ringshaped pustulosis, continuous end-of-limb dermatitis, pustulosis herpetiformis. Optionally, the psoriasis-associated condition includes, for example, drug-induced psoriasis, skin fold psoriasis, diaper zone psoriasis, seborrheic dermatitis-like psoriasis, guttate psoriasis, nail psoriasis, psoriatic arthritis.

As used herein, "type 1 diabetes" includes one or more of the following: type 1 diabetes, insulin dependent diabetes, idiopathic diabetes, juvenile type 1 diabetes, maturity-onset diabetes of the young, latent autoimmune diabetes of adults, gestational diabetes. Conditions associated with type 1 diabetes include: neuropathy, including polyneuropathy, mononeuropathy, peripheral neuropathy, and autonomic neuropathy; ocular complications: glaucoma, cataract, retinopathy.

As used herein, "scholan syndrome" includes one or more of the following: scleroderma syndrome, primary and secondary Scleroderma syndrome, and conditions involving Scleroderma syndrome including connective tissue diseases such as rheumatoid arthritis, systemic lupus erythematosus, or scleroderma. Other complications include pneumonia, pulmonary fibrosis, interstitial nephritis, inflammation of the tissues surrounding the kidney filter, glomerulonephritis, renal tubular acidosis, carpal tunnel syndrome, peripheral neuropathy, cranial neuropathy, Primary Biliary Cirrhosis (PBC), cirrhosis, inflammation of the esophagus, stomach, pancreas, and liver (including hepatitis), polymyositis, Raynaud's phenomenon, vasculitis, autoimmune thyroid problems, lymphoma.

As used herein, "systemic lupus erythematosus" includes one or more of the following: systemic lupus erythematosus, discoid lupus, lupus arthritis, lupus pneumonia, lupus nephritis. Conditions associated with systemic lupus erythematosus include osteoarticular tuberculosis, antiphospholipid antibody syndromes, inflammation of various parts of the heart (e.g., pericarditis, myocarditis, and endocarditis), lung and pleural inflammation, pleuritis, pleural effusion, chronic diffuse interstitial lung disease, pulmonary hypertension, pulmonary embolism, pulmonary hemorrhage, and the symptoms of lung atrophy, lupus headache, Guillain-Barre syndrome, aseptic meningitis, demyelinating syndrome, mononeuropathy, mononeuritis multiplex, myasthenia gravis, myelopathy, cranial neuropathy, polyneuropathy, vasculitis.

As used herein, "immune-related disease (or disorder or condition)" should be understood to encompass any disease disorder or condition selected from the group consisting of, but not limited to: autoimmune diseases, inflammatory and immune disorders associated with graft transplant rejection, such as acute and chronic rejection of organ transplants, allogeneic stem cell transplants, autologous stem cell transplants, bone marrow transplants, and graft-versus-host diseases.

As used herein, the terms "inflammatory disorder" and/or "inflammation" used interchangeably include inflammatory abnormalities characterized by a dysregulated immune response to a noxious stimulus (e.g., a pathogen, damaged cells, or irritants). Inflammatory disorders underlie a very large number of human diseases. Non-immune diseases that have etiological origin in inflammatory processes include cancer, arteriosclerosis, and ischemic heart disease. Examples of disorders associated with inflammation include: chronic prostatitis, glomerulonephritis, hypersensitivity, pelvic inflammatory disease, reperfusion injury, sarcoidosis, vasculitis, interstitial cystitis, normocoplementic urticaria vasculitis, pericarditis, myositis, anti-synthetase syndrome, scleritis, macrophage activation syndrome, behcet's syndrome, PAPA syndrome, blautian syndrome, gout, adult and juvenile stele disease, cryropyrinopathy, Muckle-Wells syndrome, familial cold-induced autoinflammatory syndrome, neonatal onset multisystem inflammatory disease, familial mediterranean fever, chronic infantile nervous system, skin and joint syndrome, systemic juvenile idiopathic arthritis, Hyper IgD syndrome (Hyper IgD syndrome), senireler syndrome (schnitz's syndrome), TNF receptor-associated periodic syndrome (trap), gingivitis, periodontitis, hepatitis, cirrhosis, pancreatitis, myocarditis, vasculitis, gastritis, gout, gouty arthritis, and inflammatory skin disorders selected from the group consisting of: psoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne.

As used herein, the terms "infectious disorder and/or disease" and/or "infection" used interchangeably include any disorder, disease and/or condition resulting from the presence and/or growth of pathogenic biological agents in a single host organism. As used herein, the term "infection" includes disorders, diseases and/or conditions as above that exhibit clinically significant disease (i.e., typical medical signs and/or symptoms of disease) and/or are asymptomatic in many or all of their course. As used herein, the term "infection" also includes disorders, diseases and/or conditions resulting from the persistence of exogenous antigens that result in a depleted T cell phenotype characterized by reduced proliferation and impaired functionality as evidenced by cytokine production. As used herein, the terms "infectious disorder and/or disease" and/or "infection" further include any of the infectious disorders, diseases and/or conditions listed below resulting from bacterial infection, viral infection, fungal infection and/or parasitic infection.

As used herein, the term "viral infection" is any infection caused by a virus, and may optionally include, but is not limited to: retroviruses (e.g., human immunodeficiency viruses such as HIV-1 or HIV-2, Acquired Immunodeficiency (AIDS) (also known as HTLV-III, LAV or HTLV-III/LAV, or HIV-III); and other isolates such as HIV-LP; picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Caliciviridae (e.g., strains that cause gastroenteritis), Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus), Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, bovine virus); Paramyxoviridae (e., Mumps virus, measles virus, respiratory syncytial virus); orthomyxoviridae (e.g., influenza virus); bunyaviridae (e.g., hantavirus, bunyavirus, phlebovirus, and Nairo virus); arenaviridae (hemorrhagic fever virus); reoviridae (e.g., reoviruses, circoviruses, and rotaviruses); binuclear glyconucleoviridae; hepadnaviridae (hepatitis b virus); parvoviridae (parvoviruses); papovaviridae (papilloma virus, polyoma virus); adenoviridae (most adenoviruses); herpesviridae (herpesviridae) (herpes simplex viruses (HSV)1 and 2, varicella zoster virus, Cytomegalovirus (CMV), herpes virus); poxviridae (variola virus, vaccinia virus, pox virus); and iridoviridae (e.g., african swine fever virus); and unclassified viruses (e.g., etiological agents of spongiform encephalopathy, agents of hepatitis (believed to be defective satellites of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1-internally transmitted; class 2-parenterally transmitted (i.e., hepatitis C); Norwalk and related viruses, and astrovirus) along with severe acute respiratory syndrome virus and Respiratory Syncytial Virus (RSV).

As used herein, the term "fungal infection" includes any infection caused by a fungus, optionally including but not limited to: cryptococcus neoformans, histoplasma capsulatum, coccidioidomycosis immitis, blastomyces dermatitidis, chlamydia trachomatis, candida albicans.

As used herein, the term "parasitic infection" includes any infection caused by a parasite, and may optionally include, but is not limited to: protozoa, such as amoeba (Amebae), flagellates, plasmodium falciparum, Toxoplasma gondii, ciliates, coccidia, microsporidia, sporozoa; helminths, nematodes (roundworms), cestodes (tapeworms), trematodes (Fluke), arthropods, and abnormal proteins known as prions.

Infectious disturbances and/or diseases caused by bacteria can optionally include one or more of the following: septicemia, septic shock, sinusitis, skin infections, pneumonia, bronchitis, meningitis, bacterial vaginosis, urinary tract infections (UCI), bacterial gastroenteritis, impetigo and erysipelas, cellulitis, anthrax, pertussis, lyme's disease (lymeiasea), brucellosis, enteritis, acute enteritis, tetanus, diphtheria, pseudomembranous colitis, gas gangrene, acute food poisoning, anaerobic cellulitis, nosocomial infections, diarrhea, infantile meningitis, Traveller's diarrhea, hemorrhagic colitis, hemolytic uremic syndrome, tularemia, gastric ulcer, gastric and duodenal ulcers, kernell disease, pointiacridica fever, leptospirosis, leprosy (Hansen's disease), tuberculosis, gonorrhea, neonatal ophthalmia, septic arthritis, meningococcosis (including meningitis) Woo-fu syndrome), pseudomonas infection, rocky mountain spotted fever, typhial salmonellosis, gastroenteritis and enterocolibacillosis, bacillary dysentery/bacillary dysentery, coagulase-positive staphylococcus aureus infection (coagulo-positivestaphyloccal infection): topical skin infections, including diffuse skin infections (impetigo), deep-seated topical infections, acute infective endocarditis, sepsis, necrotizing pneumonia, Toxinoses (e.g., toxic shock syndrome and staphylococcal food poisoning), cystitis, endometritis, otitis media, streptococcal pharyngitis, scarlet fever, rheumatic fever, puerperium fever, necrotizing fasciitis, cholera, plagues (including bubonic plague and pneumonic plague); and any infection caused by a bacterium selected from, but not limited to: helicobacter pylori, Borrelia burgdorferi, Legionella pneumophila, Mycobacterium (e.g., Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium gordonae (M gordonae)), Staphylococcus aureus, gonococcus, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (group A Streptococcus), Streptococcus agalactiae (group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus (anaerobic genus), Streptococcus pneumoniae, pathogenic Campylobacter, enterococcus, Haemophilus influenzae, Bacillus anthracis (Bacillus antathracus), Corynebacterium diphtheriae, Corynebacterium, Rhodococcus erythropolis, Clostridium perfringens (Clostridium perfringens), Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, pasteurella multocida, Bacteroides, Fusobacterium nucleatum, Srepobacterium moniliformis, Treponema pallidum, Treponema elegans, Leptospira leptospira, and Actinomeyces israelii.

Non-limiting examples of infectious disorders and/or diseases caused by viruses are selected from the group consisting of, but not limited to: acquired Immunodeficiency (AIDS), West Nile encephalitis (West Nile encephalitis), coronavirus infection, rhinovirus infection, influenza, dengue fever, hemorrhagic fever; ear infections; severe Acute Respiratory Syndrome (SARS), acute febrile pharyngitis, pinkeye fever, epidemic keratoconjunctivitis, infantile gastroenteritis, infectious mononucleosis, burkitt's lymphoma, acute hepatitis, chronic hepatitis, cirrhosis, hepatocellular carcinoma, primary HSV-1 infection (child gingivitis, adult tonsillitis, and pharyngitis, keratoconjunctivitis), latent HSV-1 infection (herpes labialis, cold sore), aseptic meningitis, cytomegalovirus infection, giant cell inclusion body disease, kaposi's sarcoma, castleman's disease (castlemandease), primary exudative lymphoma, influenza, measles, encephalitis, post-infection encephalomyelitis, mumps, proliferative epithelial lesions (common plantar and anogenital warts, laryngeal papilloma, verrucous epidermodysplasia), membranous laryngitis, pneumonia, bronchiolitis, polio, rabies, bronchiolitis, pneumonia, german rubella, congenital rubella, hemorrhagic fever, chicken pox, dengue fever, ebola virus infection, echovirus infection, EBV infection, Fifth Disease, filovirus, flavivirus, hand-foot-and-mouth Disease, herpes zoster (shingles), human papilloma virus associated epidermoid lesions, lassa fever, lymphocytic choriomeningitis virus, parainfluenza virus infection, paramyxovirus, parvovirus B19 infection, picornavirus, poxvirus infection, rotavirus diarrhea, rubella, measles, chicken pox, smallpox infection.

Infectious disorders and/or diseases caused by fungi may optionally include, but are not limited to: allergic bronchopulmonary aspergillosis, aspergillosis (aspergilloma, aspergillosis), frog-dung mildew (basidiomycotosis), blastomycosis, candidiasis, chronic pulmonary aspergillosis, chytrid, coccidioidomycosis, ear mold (conidiomycotosis), covered smut (barley), cryptococcosis, dermatophytosis (dermataphyte), ringworm (dermataphytid), dermatomycosis, trichophyton (endothix), entomopathogenic fungi, epidemic lymphangitis, epidemic ulcer syndrome, esophageal candidiasis, exothermix, mycosis, histoplasmosis, lobeloblastomycosis (Lobomycosis), Massospora cicada, mycosis, strawberry mycosphaerella (mycosphaera), mycosphaerella, mycosis (mycosis), mycosis destructor, mycosis, myco. Non-limiting examples of infectious disorders and/or diseases caused by parasites are selected from the group consisting of, but not limited to: acanthamoeba, amebiasis, ascariasis, ancylostomiasis, anisakiasis, babesiasis, acanthosis, balantidiasis, baylisaciasis, blastocystisis, parasitic catfish (Candirru), trypanosomiasis, clonorchiasis, trypanosomiasis (Cochliomia), coccidiosis, cryptosporidiosis china hepatica, dinuclear amebiasis (Dientamoebiasis), schizocephaliasis, epizootic nematode infection (Dictophyme renalis infection), trichinosis, coccidiosis, elephantiasis, enterobiasis, fasciolosis, fasciolopsiasis, filariasis, giardiasis, palatopathy, membrana capsulosum, fasciolopsiasis syndrome (Halzounculomorphism), isosporosis, schistosomiasis (Kayatafferver), leiomycosis, lymphoblastosis, malaria, retrogonorrhea, malarial, fascioliasis, schistosomiasis, schizocysticercosis, nosema disease, onchocerciasis, taeniasis (the cause of cysticercosis), toxocariasis, toxoplasmosis, trichinosis, trichomoniasis, trichiasis, trypanosomiasis, and trichuria infection (Tapeworm infection).

A preferred example of an infectious disease is a disease caused by any one of: hepatitis B, hepatitis C, infectious mononucleosis, EBV, cytomegalovirus, AIDS, HIV-1, HIV-2, tuberculosis, malaria, and schistosomiasis.

As used herein, the term "vaccine" refers to a biological agent that improves immunity to a particular disease, wherein the vaccine includes an antigen, such as a pathogen in attenuated or killed form, that elicits an immune response against its toxin or one of its surface proteins. A vaccine typically includes an adjuvant that acts as an immunopotentiator to stimulate the immune system. As used herein, the terms "therapeutic vaccine" and/or "therapeutic vaccination" refer to a vaccine for the treatment of a persistent disease (e.g., infectious disease or cancer).

As used herein, the term "adjuvant" refers to an agent used to stimulate the immune system and increase the response to a vaccine without having any specific antigenic effect on its own.

As used herein, the terms LY6G6F and/or one or more LY6G6F proteins refer to the amino acid sequences set forth in SEQ ID NO: 1, and/or variants thereof, and/or orthologs and/or fragments thereof, and/or nucleic acid sequences encoding same, that function in cancer as exemplified herein and/or in an infectious disorder as exemplified herein, and/or in an immune-related disorder as exemplified herein, and/or in the etiology of cancer, and/or in an infectious disorder, and/or an immune-related disorder.

According to a preferred embodiment, a fragment of LY6G6F is included in any one of SEQ ID NOs: 2. 59, 81, 96, and/or a variant thereof. According to a preferred embodiment, an LY6G6F ortholog comprises any of SEQ ID NOs: 20. 29. According to a preferred embodiment, a nucleic acid sequence encoding a LY6G6F protein comprises SEQ id no: 33. 57 or 182.

As used herein, the terms VSIG10 and/or one or more VSIG10 proteins refer to a polypeptide encoded by any one of SEQ ID NOs: 3. 5, and/or variants thereof, and/or orthologs and/or fragments thereof, and/or nucleic acid sequences encoding same, that function in cancer as exemplified herein and/or in an infectious disorder as exemplified herein, and/or in an immune-related disorder as exemplified herein, and/or in the etiology of cancer and/or in an infectious disorder, and/or an immune-related disorder.

According to preferred embodiments, one VSIG10 fragment is included in any one of SEQ ID NOs: 4. 6, 60, 61, 82-93, 97-100, and/or a variant thereof, and/or an amino acid sequence comprising a unique border portion of the VSIG10 variant (SEQ ID NO: 5) shown in fig. 2A. According to preferred embodiments, a VSIG10 ortholog comprises any one of SEQ ID NOs: 19. 30, respectively. According to a preferred embodiment, a nucleic acid sequence encoding a VSIG10 protein includes any one of SEQ ID NOs: 34. 35, 36, 183, or 184.

As used herein, the terms TMEM25 and/or one or more TMEM25 proteins refer to proteins as set forth in any one of SEQ ID NOs: 7. 39, and/or variants thereof, and/or orthologs and/or fragments thereof, and/or nucleic acid sequences encoding same, that function in cancer as recited herein and/or in an infectious disorder as recited herein, and/or in an immune-related disorder as recited herein, and/or in the etiology of cancer and/or in an infectious disorder, and/or an immune-related disorder.

According to a preferred embodiment, one fragment of TMEM25 is included in any one of SEQ ID NOs: 8. 39, 94, 101, and/or variants thereof. According to a preferred embodiment, one TMEM25 ortholog comprises a polypeptide having an amino acid sequence according to any one of SEQ ID NOs: 9. and/or 28. According to a preferred embodiment, a nucleic acid sequence encoding TMEM25 protein comprises any one of SEQ ID NOs: 37 or 185.

As used herein, the term LSR and/or one or more LSR proteins refer to a sequence as set forth in any of SEQ ID NOs: 11. 13, 15-18, 143, and/or variants and/or orthologs and/or fragments thereof, and/or nucleic acid sequences encoding same, that play a role in cancer and/or in the etiology of infectious disorders, and/or immune-related disorders, as exemplified herein and/or in the differential expression in infectious disorders, as exemplified herein, and/or immune-related disorders, as exemplified herein.

According to a preferred embodiment, an LSR fragment is included in any of SEQ ID NOs: 10. 12, 14, 22, 47-50, 95, 102, and/or variants thereof, and/or amino acid sequences comprising unique border portions of the LSR variant (SEQ ID NO: 18) shown in figure 2G. An example of an LSR ortholog is presented in any of SEQ ID NOs: 21. 31, 32. According to a preferred embodiment, a nucleic acid sequence encoding an LSR protein comprises any of SEQ id nos: 40-46, 132, 155, 188, 186, 187, 145, 154.

Without wishing to be bound by a single hypothesis, according to at least some embodiments of the invention, each LY6G6F, VSIG10, TMEM25, and/or LSR protein is predicted to be an immune co-stimulatory protein, such as a member of the B7 protein family involved in B7 immune co-stimulation (including, for example, T cell responses raised against cancer cells), and a member of the B7 protein family that elicits an effect on immunity (e.g., triggering of an autoimmune effect).

As used herein, a "soluble extracellular domain (ECD)" or "extracellular domain" or "one or more soluble LY6G6F, VSIG10, TMEM25 and/or LSR protein/molecule" of LY6G6F, VSIG10, TMEM25 and/or LSR refers to a non-cell surface bound (i.e., circulating) LY6G6F, VSIG10, TMEM25 and/or LSR molecule or any portion thereof, including but not limited to: LY6G6F, VSIG10, TMEM25 and/or LSR-Ig fusion proteins in which the extracellular domain of LY6G6F, VSIG10, TMEM25 and/or LSR is fused to an immunoglobulin (Ig) moiety such that the fusion molecule is soluble, or fragments and derivatives thereof; a protein having the extracellular domain of LY6G6F, VSIG10, TMEM25 and/or LSR fused to or linked to a portion of a biologically or chemically active protein, such as the papillomavirus E7 gene product, melanoma associated antigen p97 or HIV env protein, or fragments and derivatives thereof; hybrid (chimeric) fusion proteins, such as LY6G6F, VSIG10, TMEM25 and/or LSR-Ig, or fragments and derivatives thereof. Such fusion proteins are described in more detail below.

"one or more soluble LY6G6F, VSIG10, TMEM25 and/or LSR proteins/molecules" also includes LY6G6F, VSIG10, TMEM25 and/or LSR molecules, or fragments and derivatives thereof, wherein the transmembrane domain is removed to render the protein soluble; a fragment, portion or derivative thereof, and a soluble LY6G6F, VSIG10, TMEM25 and/or LSR mutant molecule. The soluble LY6G6F, VSIG10, TMEM25 and/or LSR molecules used in methods according to at least some embodiments of the present invention may or may not include a signal (leader) peptide sequence.

Fragments of LY6G6F polypeptide

The term "soluble extracellular domain (ECD)" or "extracellular domain" or "soluble" form of LY6G6F also refers to a nucleic acid sequence encoding a corresponding protein of LY6G6F "soluble extracellular domain (ECD)" or "extracellular domain" or "soluble LY6G6F protein/molecule"). Optionally, LY6G6F ECD refers to any of the following polypeptide sequences and/or any of the polypeptide sequences listed in table a below, and/or a fragment or variant thereof, and/or a conjugate thereof, and/or a polynucleotide encoding same, that shares at least 80% sequence identity, more preferably at least 90% sequence identity, and even more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity with it:

SEQ ID NO: 2, amino acid residues 17-234 (excluding the signal peptide until transmembrane) (fig. 1A): ADNMQAIYVALGEAVELPCPSPPTLHGDEHLSWFCSPAAGSFTTLVAQVQVGRPAPDPGKPGRESRLRLLGNYSLWLEGSKEEDAGRYWCAVLGQHHNYQNWRVYDVLVLKGSQLSARAADGSPCNVLLCSVVPSRRMDSVTWQEGKGPVRGRVQSFWGSEAALLLVCPGEGLSEPRSRRPRIIRCLMTHNKGVSFSLAASIDASPALCAPSTGWDMP the flow of the air in the air conditioner,

and fragments and variants thereof that possess at least 80% sequence identity thereto, more preferably at least 90% sequence identity thereto, and even more preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto. SEQ ID NO: 59 represents an example of LY6G6FECD (including signal peptide).

TABLE A

Optionally, this fragment has the sequence set forth in SEQ ID NO: at least about 62, 63, 64, 65, etc. amino acids of the extracellular domain of a LY6G6F protein set forth in 1, up to 228 amino acids of the extracellular domain of a LY6G6F protein, and optionally including any integer value between 62 and 228 amino acids in length. Preferably, this fragment has at least about 62 and up to 82 amino acids of the extracellular domain of LY6G6F protein, and may optionally include any integer value between 62 and 82 amino acids in length. Also preferably, this fragment has at least about 95 up to 115 amino acids of the extracellular domain of LY6G6F protein, and may optionally include any integer value between 95 and 115 amino acids in length. Also preferably, this fragment has at least about 208 up to 228 amino acids of the extracellular domain of LY6G6F protein, and may optionally include any integer value between 208 and 228 amino acids in length. More preferably, this fragment is about 72 or 106 or 218 amino acids. In accordance with at least some embodiments of the invention, a LY6G6F fragment may or may not include a signal peptide sequence, and may or may not include 1, 2, 3, 4, or 5 consecutive amino acids from the transmembrane domain of LY6G 6F.

Specifically, the extracellular domain fragment of LY6G6F can include any portion corresponding to the extracellular domain of LY6G6F or any sequence including the IgV domain thereof, the fragment having any sequence corresponding to the residues of LY6G6F (SEQ ID NO: 1) starting anywhere between 14 and 27 and ending anywhere between 112 and 132.

The LY6G6F protein comprises an immunoglobulin domain within the extracellular domain, the IgV domain (or V domain) shown in box in fig. 1A, which is associated with the variable domain of an antibody. IgV domains can be responsible for receptor binding by analogy to other B7 family members. The Ig domain of the extracellular domain includes a disulfide bond formed between cysteine residues within the domain that is typical for this folding and can be important for structural function. In SEQ ID NO: 1, these cysteines are located at residues 35 and 106.

In one embodiment, a soluble fragment of LY6G6F is provided; such a soluble fragment can optionally be described as a first fusion partner, as described in more detail below with respect to the portions of the fusion protein. Useful fragments are those that retain the ability to bind to one or more of its native receptors and/or retain the ability to inhibit T cell activation. LY6G6F polypeptides that are fragments of full-length LY6G6F typically have at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100%, or even greater than 100% of the ability to bind one or more of its native receptors and/or the ability to inhibit T cell activation, as compared to full-length LY6G 6F. A soluble LY6G6F polypeptide fragment is a fragment of LY6G6F polypeptide that can be shed, secreted or otherwise extracted from a producer cell. In other embodiments, soluble fragments of LY6G6F polypeptides include fragments of the extracellular domain of LY6G6F that retain the biological activity of LY6G6F, e.g., fragments that retain the ability to bind to one or more of its native receptors and/or retain the ability to inhibit T cell activation. The extracellular domain may include 1, 2, 3, 4, or 5 contiguous amino acids from the transmembrane domain, and/or 1, 2, 3, 4, or 5 contiguous amino acids from the signal sequence. Alternatively, the extracellular domain may have 1, 2, 3, 4, 5, or more amino acids removed from the C-terminus, N-terminus, or both.

In some embodiments, the LY6G6F extracellular domain polypeptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 81, or a fragment or variant thereof, or a sequence located within the full-length protein of SEQ ID NO: 1 and the conserved cysteines of the IgV domain at residues 35 and 106, which corresponds to the amino acid sequence of SEQ ID NO: 96, the sequence set forth in seq id no: CPSPPTLHGDEHLSWFCSPAAGSFTTLVAQVQVGRPAPDPGKPGRESRLRLLGNYSLWLEGSKEEDAGRYWC are provided. In other embodiments, the LY6G6F extracellular domain polypeptide consists essentially of an amino acid sequence as set forth in any one of SEQ ID NOs: the amino acid sequence composition of the IgV domains as illustrated in 81 and 96.

Typically, the LY6G6F polypeptide fragment is expressed from a nucleic acid that includes a sequence that encodes a signal sequence. The signal sequence is typically cleaved from the immature polypeptide to produce a mature polypeptide lacking the signal sequence. The signal sequence of LY6G6F may be replaced by a signal sequence of another polypeptide using standard molecular biology techniques to affect the expression level, secretion, solubility, or other properties of the polypeptide. The signal peptide sequence used in place of the LY6G6F signal peptide sequence can be any signal peptide sequence known in the art.

Optionally, LY6G6F ECD refers to any nucleic acid sequence encoding a LY6G6F ECD polypeptide, optionally in SEQ ID NO: 33, or a fragment thereof and/or degenerate variant thereof, encoding a nucleic acid sequence as set forth in SEQ ID NO: 2, LY6G6F ECD polypeptide as set forth in claim 2.

Optionally, LY6G6F ECD refers to a direct ECD polypeptide. Optionally, LY6G6F ECD refers to SEQ id no: 20, and/or a mouse LY6G6F ECD polypeptide as set forth in SEQ ID NO: the mouse LY6G6F ECD-IgG2 a-Fc-fusion polypeptide set forth in 23.

Fragments of VSIG10 polypeptides

The term "soluble extracellular domain (ECD)" or "extracellular domain" or "soluble" form of VSIG10 also refers to a nucleic acid sequence encoding a corresponding protein of VSIG10 "soluble extracellular domain (ECD)" or "extracellular domain" or "soluble VSIG10 protein/molecule"). Optionally, VSIG10 ECD refers to any of the following polypeptide sequences and/or any of the polypeptide sequences listed in table B below, and/or fragments or variants thereof, and/or conjugates thereof, and/or polynucleotides encoding same, which possess at least 80% sequence identity, more preferably at least 90% sequence identity, and even more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity thereto: SEQ ID NO: 4, amino acid residues 31-413 (excluding the signal peptide until transmembrane) (fig. 1B): VVIGEVHENVTLHCGNISGLRGQVTWYRNNSEPVFLLSSNSSLRPAEPRFSLVDATSLHIESLSLGDEGIYTCQEILNVTQWFQVWLQVASGPYQIEVHIVATGTLPNGTLYAARGSQVDFSCNSSSRPPPVVEWWFQALNSSSESFGHNLTVNFFSLLLISPNLQGNYTCLALNQLSKRHRKVTTELLVYYPPPSAPQCWAQMASGSFMLQLTCRWDGGYPDPDFLWIEEPGGVIVGKSKLGVEMLSESQLSDGKKFKCVTSHIVGPESGASCMVQIRGPSLLSEPMKTCFTGGNVTLTCQVSGAYPPAKILWLRNLTQPEVIIQPSSRHLITQDGQNSTLTIHNCSQDLDEGYYICRADSPVGVREMEIWLSVKEPLNIGG, respectively;

SEQ ID NO: 6, amino acid residues 31-312 (skipping exon 3 variant, excluding the signal peptide, until transmembrane) (FIG. 1C): VVIGEVHENVTLHCGNISGLRGQVTWYRNNSEPVFLLSSNSSLRPAEPRFSLVDATSLHIESLSLGDEGIYTCQEILNVTQWFQVWLQVANPPPSAPQCWAQMASGSFMLQLTCRWDGGYPDPDFLWIEEPGGVIVGKSKLGVEMLSESQLSDGKKFKCVTSHIVGPESGASCMVQIRGPSLLSEPMKTCFTGGNVTLTCQVSGAYPPAKILWLRNLTQPEVIIQPSSRHLITQDGQNSTLTIHNCSQDLDEGYYICRADSPVGVREMEIWLSVKEPLNIGG the flow of the air in the air conditioner,

and variants thereof that possess at least 80% sequence identity thereto, more preferably at least 90% sequence identity thereto, and even more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity thereto. SEQ ID NO: 60-61 represent examples of VSIG10 ECD (including signal peptide).

Watch (A)B

Optionally, this fragment has the sequence set forth in SEQ ID NO: 3, up to 393 amino acids of the extracellular domain of the VSIG10 protein, and optionally including any integer value between 36 and 393 amino acids in length. Preferably, this fragment has at least about 36 up to 70 amino acids of the extracellular domain of VSIG10 protein, and may optionally include any integer value between 36 and 70 amino acids in length. Also preferably, this fragment has at least about 80 up to 100 amino acids of the extracellular domain of VSIG10 protein, and may optionally include any integer value between 80 and 100 amino acids in length. Also preferably, this fragment has at least about 170 up to 200 amino acids of the extracellular domain of VSIG10 protein, and may optionally include any integer value between 170 and 200 amino acids in length. Also preferably, this fragment has at least about 265 up to 290 amino acids of the extracellular domain of the VSIG10 protein, and may optionally include any integer value between 265 and 290 amino acids in length. Also preferably, this fragment has at least about 365 and up to 393 amino acids of the extracellular domain of VSIG10 protein, and may optionally include any integer value between 365 and 393 amino acids in length. More preferably, this fragment is about 46, 49, 58, 60, 87, 89, 93, 94, 178, 182, 185, 187, 273, 279, 282, 374, 383 amino acids. In accordance with at least some embodiments of the present invention, a VSIG10 fragment may or may not include a signal peptide sequence and may or may not include 1, 2, 3, 4, or 5 consecutive amino acids from the VSIG10 transmembrane domain.

Specifically, fragments of the extracellular domain of VSIG10 may include any portion of the extracellular domain corresponding to VSIG10 or sequences including one or more IgC2 domains thereof having any sequence of residues corresponding to VSIG10(seq id NO: 3) below: starting at any position between 28 and 41 and terminating at any position between 109 and 122 or starting at any position between 120 and 133 and terminating at any position between 205 and 222 or starting at any position between 216 and 233 and terminating at any position between 299 and 310 or starting at any position between 310 and 321 and terminating at any position between 394 and 414 or starting at any position between 28 and 41 and terminating at any position between 205 and 222 or starting at any position between 28 and 41 and terminating at any position between 299 and 310 or starting at any position between 28 and 41 and terminating at any position between 394 and 414 or starting at any position between 120 and 133 and terminating at any position between 299 and 310 or starting at any position between 120 and 133 and terminating at any position between 394 and 414 or starting at any position between 216 and 233 and terminating at any position between 394 and 414 (ii) a Or any sequence having residues corresponding to VSIG10_ variant _ skipping _ exon _3_ T95617_ P6(SEQ ID NO: 5) below: starting anywhere between 28 and 41 and ending anywhere between 198 and 209 or starting anywhere between 28 and 41 and ending anywhere between 293 and 313.

The VSIG10 protein contains an immunoglobulin domain, IgC2 domain (or Ig-like C2 domain or Ig C2-immobilization domain) in the extracellular domain that is associated with the constant domain of an antibody. These domains are illustrated in FIG. 1B (for SEQ ID NO: 3) and FIG. 1C (for SEQ ID NO: 5). The Ig domain of the extracellular domain includes a disulfide bond formed between cysteine residues within the domain that is typical for this folding and can be important for structural function. In SEQ ID NO: 3, these cysteines are at residues 44 and 103, and at residues 153 and 201, and at residues 245 and 290, and at residues 331 and 388. In SEQ ID NO: 5, these cysteines are located at residues 44 and 103, at residues 144 and 189, and at residues 230 and 287.

In one embodiment, a soluble fragment of VSIG10 is provided, which can optionally be described below with respect to the portion of the fusion protein as a first fusion partner. Useful fragments are those that retain the ability to bind to one or more of its receptors and/or retain the ability to inhibit T cell activation. In contrast to full-length VSIG10, VSIG10 polypeptides that are fragments of full-length VSIG10 typically have at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100%, or even greater than 100% of the ability to bind one or more of its native receptors and/or the ability to inhibit T cell activation. A soluble VSIG10 polypeptide fragment is a fragment of VSIG10 polypeptide that can be shed, secreted, or otherwise extracted from a producer cell. In other embodiments, soluble fragments of VSIG10 polypeptides include fragments of the VSIG10 extracellular domain that retain the biological activity of VSIG10, e.g., retain the ability to bind one or more of its native receptors and/or retain the ability to inhibit T cell activation. The extracellular domain may include 1, 2, 3, 4, or 5 contiguous amino acids from the transmembrane domain, and/or 1, 2, 3, 4, or 5 contiguous amino acids from the signal sequence. Alternatively, the extracellular domain may have 1, 2, 3, 4, 5, or more amino acids removed from the C-terminus, N-terminus, or both.

In some embodiments, the VSIG10 extracellular domain polypeptide includes a sequence as set forth in any one of SEQ ID NOs: 82. 83, 84 and 85, or a fragment or variant thereof, or a polypeptide located in at least one of the IgC2 domains as set forth in full-length proteins SEQ ID NOs: 3 and the conserved cysteine of the IgC2 domain at residue 44 and 103, corresponding to SEQ id no: 97 by the sequence set forth in seq id no: CGNISGLRGQVTWYRNNSEPVFLLSSNSSLRPAEPRFSLVDATSLHIESLSLGDEGIYTC, or the full-length protein of SEQ ID NO: 3 and the conserved cysteine of the IgC2 domain at residue 153 and 201, corresponding to SEQ ID NO: 98: CNSSSRPPPVVEWWFQALNSSSESFGHNLTVNFFSLLLISPNLQGNYTC or the full-length protein of SEQ ID NO: 3 and the conserved cysteine of the IgC2 domain at residue 245 and 209, which corresponds to the amino acid sequence of SEQ ID NO: 99 by the sequence set forth in seq id no: CRWDGGYPDPDFLWIEEPGGVIVGKSKLGVEMLSESQLSDGKKFKC or the full-length protein of SEQ ID NO: 3 and 388, which corresponds to the region between the conserved cysteines of the IgC2 domain at residues 331 and 388 of SEQ ID NO: 100, the sequence set forth in seq id no: CQVSGAYPPAKILWLRNLTQPEVIIQPSSRHLITQDGQNSTLTIHNCSQDLDEGYYIC are provided. In some further embodiments, the VSIG10 extracellular domain polypeptide consists essentially of SEQ ID NO: 82-93 and 97-100.

Typically, the VSIG10 polypeptide fragment is expressed from a nucleic acid that includes a sequence that encodes a signal sequence. The signal sequence is typically cleaved from the immature polypeptide to produce a mature polypeptide lacking the signal sequence. The signal sequence of VSIG10 can be replaced with a signal sequence of another polypeptide using standard molecular biology techniques to affect the level of expression, secretion, solubility, or other properties of the polypeptide. The signal peptide sequence used in place of the VSIG10 signal peptide sequence may be any signal peptide sequence known in the art.

Optionally, VSIG10 ECD also refers to any nucleic acid sequence encoding a VSIG10 ECD polypeptide, optionally referred to as a polypeptide as set forth in SEQ ID NO: 34. 36, or a fragment thereof and/or degenerate variant thereof, encoding a nucleic acid sequence as set forth in SEQ id no: 4. 6, the VSIG10 ECD polypeptide as set forth in fig.

Optionally, VSIG10 ECD refers to a direct ECD polypeptide. Optionally, VSIG10 ECD refers to SEQ id no: 19, and/or a mouse VSIG10 ECD polypeptide as set forth in SEQ ID NO: 24A mouse VSIG10 ECD-IgG2 a-Fc-fusion polypeptide.

Fragments of TMEM25 polypeptide

The term "soluble extracellular domain (ECD)" or "extracellular domain" or "soluble" form of TMEM25 also refers to a nucleic acid sequence that encodes a corresponding protein of TMEM25 "soluble extracellular domain (ECD)" or "extracellular domain" or "soluble TMEM25 protein/molecule"). Optionally, TMEM25 ECD refers to any of the following polypeptide sequences and/or any of the polypeptide sequences listed in table C below, and/or fragments or variants thereof, and/or conjugates thereof, and/or polynucleotides encoding same, that possess at least 80% sequence identity, more preferably at least 90% sequence identity, and even more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity thereto: SEQ ID NO: 8, amino acid residues 27-232 (excluding the signal peptide until transmembrane) (FIG. 1D): ELEPQIDGQTWAERALRENERHAFTCRVAGGPGTPRLAWYLDGQLQEASTSRLLSVGGEAFSGGTSTFTVTAHRAQHELNCSLQDPRSGRSANASVILNVQFKPEIAQVGAKYQEAQGPGLLVVLFALVRANPPANVTWIDQDGPVTVNTSDFLVLDAQNYPWLTNHTVQLQLRSLAHNLSVVATNDVGVTSASLPAPGLLATRVE the flow of the air in the air conditioner,

And variants thereof that possess at least 80% sequence identity thereto, more preferably at least 90% sequence identity thereto, and even more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity thereto. SEQ ID NO: 39 represents an example of TMEM25 ECD (including signal peptide).

Watch C

Optionally, this fragment has the sequence set forth in SEQ ID NO: at least about 46, 47, 48, 49, 50, 51, 52, etc. amino acids of the extracellular domain of TMEM25 protein set forth in 7, up to 216 amino acids of the extracellular domain of TMEM25 protein, may optionally include any integer value between 46 and 216 amino acids in length. Preferably, this fragment has at least about 46 up to 66 amino acids of the extracellular domain of TMEM25 protein, and may optionally include any integer value between 46 and 66 amino acids in length. Also preferably, this fragment has at least about 84 up to 104 amino acids of the extracellular domain of TMEM25 protein, and may optionally include any integer value between 84 and 104 amino acids in length. Also preferably, this fragment has at least about 196 up to 216 amino acids of the extracellular domain of TMEM25 protein, and may optionally include any integer value between 196 and 216 amino acids in length. More preferably, this fragment is about 56 or 94 or 206 amino acids. In accordance with at least some embodiments of the present invention, a fragment of TMEM25 may or may not include a signal peptide sequence, and may or may not include 1, 2, 3, 4, or 5 consecutive amino acids from the transmembrane domain of TMEM 25.

Specifically, the extracellular domain fragment of TMEM25 may include any portion corresponding to the extracellular domain of TMEM25 or a sequence including the IgC2 domain thereof, having any sequence corresponding to the residues of TMEM25(SEQ ID NO: 7) starting anywhere between 27 and 40 and ending anywhere between 113 and 133.

The TMEM25 protein comprises an immunoglobulin domain within the IgC2 domain (or Ig-like C2 domain or Ig C2-immobilization domain) of the extracellular domain, which is associated with the constant domain of an antibody. This domain is shown in box in FIG. 1D. The Ig domain of the extracellular domain includes a disulfide bond formed between cysteine residues within the domain that is typical for this folding and can be important for structural function. In SEQ ID NO: in 7, these cysteines are located at residues 52 and 107.

In one embodiment, a soluble fragment of TMEM25 is provided that can optionally be described as a first fusion partner, e.g., in the detailed section below with respect to fusion proteins. Useful fragments are those that retain the ability to bind to one or more of its receptors and/or retain the ability to inhibit T cell activation. TMEM25 polypeptides that are fragments of full-length TMEM25 typically have at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100%, or even greater than 100% of the ability to bind one or more of its native receptors and/or the ability to inhibit T cell activation, as compared to full-length TMEM 25. A soluble TMEM25 polypeptide fragment is a fragment of TMEM25 polypeptide that can be shed, secreted, or otherwise extracted from a producer cell. In other embodiments, soluble fragments of TMEM25 polypeptides include fragments of the TMEM25 extracellular domain that retain the biological activity of TMEM25, e.g., fragments that retain the ability to bind to one or more of its native receptors and/or retain the ability to inhibit T cell activation. The extracellular domain may include 1, 2, 3, 4, or 5 contiguous amino acids from the transmembrane domain, and/or 1, 2, 3, 4, or 5 contiguous amino acids from the signal sequence. Alternatively, the extracellular domain may have 1, 2, 3, 4, 5, or more amino acids removed from the C-terminus, N-terminus, or both.

In some embodiments, the TMEM25 extracellular domain polypeptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 94, or a fragment or variant thereof, or a polypeptide sequence located within the full-length protein of SEQ ID NO: 7 and the conserved cysteine of the IgC2 domain at residue 52 and 107, which corresponds to the amino acid sequence of SEQ ID NO: 101, the sequence set forth in seq id no: CRVAGGPGTPRLAWYLDGQLQEASTSRLLSVGGEAFSGGTSTFTVTAHRAQHELNC are provided. In other embodiments, the TMEM25 extracellular domain polypeptide consists essentially of a sequence as set forth in any one of SEQ ID NOs: 94 and 101, respectively, and the amino acid sequence of the IgC2 domain as set forth herein.

Typically, a TMEM25 polypeptide fragment is expressed from a nucleic acid that includes a sequence encoding a signal sequence. The signal sequence is typically cleaved from the immature polypeptide to produce a mature polypeptide lacking the signal sequence. The signal sequence of TMEM25 may be replaced with a signal sequence of another polypeptide using standard molecular biology techniques to affect the expression level, secretion, solubility, or other characteristics of the polypeptide. The signal peptide sequence used in place of the TMEM25 signal peptide sequence may be any signal peptide sequence known in the art.

Optionally, TMEM25 ECD further refers to any nucleic acid sequence encoding a TMEM25 ECD polypeptide, optionally referred to in SEQ ID NO: 37, or a fragment thereof and/or degenerate variant thereof, encoding a nucleic acid sequence as set forth in SEQ ID NO: the TMEM25 ECD polypeptide illustrated in 8.

Optionally, TMEM25 ECD refers to a direct ECD polypeptide. Optionally, TMEM25 ECD refers to SEQ id no: 9, and/or a mouse TMEM25 ECD polypeptide as set forth in SEQ ID NO: 25, mouse TMEM25 ECD-IgG2 a-Fc-fusion polypeptide.

Fragments of LSR polypeptides

The term "soluble extracellular domain (ECD)" or "extracellular domain" or "soluble" form of an LSR also refers to a nucleic acid sequence encoding a corresponding protein of the LSR "soluble extracellular domain (ECD)" or "extracellular domain" or "soluble LSR protein/molecule"). Optionally, LSR ECD refers to any of the following polypeptide sequences and/or any of the polypeptide sequences listed in table D below, and/or a fragment or variant thereof, and/or a conjugate thereof, and/or a polynucleotide encoding same, that shares at least 80% sequence identity, more preferably at least 90% sequence identity, and even more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity with it:

SEQ ID NO: 12, LSR isoform a ECD (excluding signal peptide, up to transmembrane) amino acid residues 42-211 (fig. 1E): IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVLGRTSGVAELLPG FQAGPIED, respectively;

SEQ ID NO: 14, LSR isoform B ECD (excluding signal peptide, up to transmembrane) amino acid residues 42-192 (fig. 1F): IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVLD, respectively;

SEQ ID NO: 47, LSR isoform C secretory variant, amino acid residues 42-533 (fig. 1G): IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVLVYAAGKAATSGVPSIYAPSTYAHLSPAKTPPPPAMIPMGPAYNGYPGGYPGDVDRSSSAGGQGSYVPLLRDTDSSVASEVRSGYRIQASQQDDSMRVLYYMEKELANFDPSRPGPPSGRVERAMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHSPRSPRGWDQEPAREQAGGGWRARRPRARSVDALDDLTPPSTAESGSRSPTSNGGRSRAYMPPRSRSRDDLYDQDDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTRDPRDNGSRSGDLPYDGRLLEEAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETDSQASRERRLKKNLALSRESLVV, respectively;

SEQ ID NO: 48, LSR isoform D secretory variant, amino acid residues 42-532 (fig. 1H): IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVLVYAAGKAATSGVPSIYAPSTYAHLSPAKTPPPPAMIPMGPAYNGYPGGYPGDVDRSSSAGGQGSYVPLLRDTDSSVASVRSGYRIQASQQDDSMRVLYYMEKELANFDPSRPGPPSGRVERAMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHSPRSPRGWDQEPAREQAGGGWRARRPRARSVDALDDLTPPSTAESGSRSPTSNGGRSRAYMPPRSRSRDDLYDQDDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTRDPRDNGSRSGDLPYDGRLLEEAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETDSQASRERRLKKNLALSRESLVV, respectively;

SEQ ID NO: 49, LSR isoform E secretory variant, amino acid residues 42-493 (fig. 1I): IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGMYAAGKAATSGVPSIYAPSTYAHLSPAKTPPPPAMIPMGPAYNGYPGGYPGDVDRSSSAGGQGSYVPLLRDTDSSVASEVRSGYRIQASQQDDSMRVLYYMEKELANFDPSRPGPPSGRVERAMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHSPRSPRGWDQEPAREQAGGGWRARRPRARSVDALDDLTPPSTAESGSRSPTSNGGRSRAYMPPRSRSRDDLYDQDDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTRDPRDNGSRSGDLPYDGRLLEEAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETDSQASRERRLKKNLALSRESLVV, respectively;

SEQ ID NO: 50, LSR isoform F secretory variant, amino acid residues 42-552 (fig. 1J): IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVLGRTSGVAELLPGFQAGPIEVYAAGKAATSGVPSIYAPSTYAHLSPAKTPPPPAMIPMGPAYNGYPGGYPGDVDRSSSAGGQGSYVPLLRDTDSSVASEVRSGYRIQASQQDDSMRVLYYMEKELANFDPSRPGPPSGRVERAMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHSPRSPRGWDQEPAREQAGGGWRARRPRARSVDALDDLTPPSTAESGSRSPTSNGGRSRAYMPPRSRSRDDLYDQDDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTRDPRDNGSRSGDLPYDGRLLEEAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETDSQASRERRLKKNLALSRESLVV the flow of the air in the air conditioner,

And variants thereof that possess at least 80% sequence identity thereto, more preferably at least 90% sequence identity thereto, and even more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity thereto. SEQ ID NO: 10. 22 represents an example of an LSR ECD (including a signal peptide).

Optionally, this fragment has the sequence set forth in SEQ ID NO: 11 and/or 143, up to 198 amino acids of the extracellular domain, and optionally including any integer value between 100 and 198 amino acids. In accordance with at least some embodiments of the present invention, an LSR fragment may or may not include a signal peptide sequence, and may or may not include 1, 2, 3, 4, or 5 consecutive amino acids from the LSR transmembrane domain.

Table D

Optionally, this fragment has the sequence set forth in SEQ ID NO: 11, up to 180 amino acids of the extracellular domain of the LSR protein, and optionally including any integer value between 98 and 180 amino acids in length. Preferably, this fragment has at least about 98 up to 118 amino acids of the extracellular domain of the LSR protein, and may optionally include any integer value between 98 and 118 amino acids in length. Also preferably, this fragment has at least about 135 up to 155 amino acids of the extracellular domain of the LSR protein, and may optionally include any integer value between 135 and 155 amino acids in length. Also preferably, this fragment has at least about 160 up to 180 amino acids of the extracellular domain of the LSR protein, and may optionally include any integer value between 160 and 180 amino acids in length. More preferably, this fragment is about 108 or 145 or 170 amino acids. In accordance with at least some embodiments of the present invention, an LSR fragment may or may not include a signal peptide sequence, and may or may not include 1, 2, 3, 4, or 5 consecutive amino acids from the LSR transmembrane domain.

The LSR protein comprises an immunoglobulin domain in the IgV domain (or V domain) of the extracellular domain, which is associated with the variable domain of the antibody. For SEQ ID NO: 11. the Ig domains of 13, 15, 16, and 18 are shown in boxes in FIGS. 1E, 1F, 1G, 1H, and 1J. The Ig domain of the extracellular domain includes a disulfide bond formed between cysteine residues within the domain that is typical for this folding and can be important for structural function. In SEQ ID NO: 11, these cysteines are located at residues 63 and 170.

In one embodiment, a soluble fragment of LSR is provided, which can optionally be described as a first fusion partner, e.g., below with respect to the portion of the fusion protein. Useful fragments are those that retain the ability to bind to one or more of its receptors and/or retain the ability to inhibit T cell activation. An LSR polypeptide that is a fragment of a full-length LSR typically has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100%, or even more than 100% of the ability to bind one or more of its native receptors and/or the ability to inhibit T cell activation, as compared to the full-length LSR. A soluble LSR polypeptide fragment is a fragment of an LSR polypeptide that may be shed, secreted or otherwise extracted from a producer cell. In other embodiments, soluble fragments of LSR polypeptides include fragments of LSR extracellular domains that retain the biological activity of LSR, such as fragments that retain the ability to bind to one or more of its native receptors and/or retain the ability to inhibit T cell activation. The extracellular domain may include 1, 2, 3, 4, or 5 contiguous amino acids from the transmembrane domain, and/or 1, 2, 3, 4, or 5 contiguous amino acids from the signal sequence. Alternatively, the extracellular domain may have 1, 2, 3, 4, 5, or more amino acids removed from the C-terminus, N-terminus, or both.

In some embodiments, the LSR extracellular domain polypeptide comprises an amino acid sequence as set forth in any of SEQ ID NOs: 95, or a fragment or variant thereof, or an amino acid sequence located in the full-length protein of SEQ ID NO: 11 and the conserved cysteine of the IgV domain at residues 63 and 170, which corresponds to the amino acid sequence of SEQ ID NO: 102: CTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAWGDSGVYYC are provided.

In some further embodiments, the LSR extracellular domain polypeptide consists essentially of a sequence as set forth in any of SEQ ID NOs: 95. and SEQ ID NO: 102, the amino acid composition of the IgV domain as set forth in seq id no.

Typically, an LSR polypeptide fragment is expressed from a nucleic acid that includes a sequence that encodes a signal sequence. The signal sequence is typically cleaved from the immature polypeptide to produce a mature polypeptide lacking the signal sequence. The signal sequence of an LSR may be replaced with a signal sequence of another polypeptide using standard molecular biology techniques to affect the expression level, secretion, solubility, or other characteristics of the polypeptide. The signal peptide sequence used in place of the LSR signal peptide sequence may be any signal peptide sequence known in the art.

Optionally, LSR ECD also refers to any nucleic acid sequence encoding an LSR ECD polypeptide, optionally to a polypeptide as set forth in seq id NO: 40. 41, 132, 44, 155, 188, or a fragment thereof and/or degenerate variant thereof, encoding a nucleic acid sequence as set forth in SEQ ID NO: 12. 14, 47, 48, 49, 50.

Optionally, the LSR ECD refers to a direct ECD polypeptide. Optionally, LSR ECD refers to SEQ ID NO: 21, and/or a mouse LSR ECD polypeptide as set forth in SEQ ID NO: 26A mouse LSR ECD-IgG2 a-Fc-fusion polypeptide as set forth in.

Variants of LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides

The invention encompasses useful variants of LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides, including those that increase biological activity, or increase the half-life or stability of the protein, as indicated by any of the assays described herein. Soluble LY6G6F, VSIG10, TMEM25 and/or LSR proteins or fragments thereof, or fusions thereof having LY6G6F, VSIG10, TMEM25 and/or LSR protein activity, respectively, may be engineered to increase biological activity. In a further embodiment, the LY6G6F, VSIG10, TMEM25 and/or LSR protein or fusion protein is modified by at least one amino acid substitution, deletion or insertion that increases the binding of the molecule to an immune cell (e.g., a T cell) and transmits an inhibitory signal to the T cell. Other optional variants are LY6G6F, VSIG10, TMEM25 and/or LSR proteins engineered to bind selectively to one type of T cell relative to other immune cells. For example, LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides may be engineered to optionally bind to Treg, Th0, Th1, Th17, Th2 or Th22 cells. Preferential binding refers to binding that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater for one type of cell over another type of cell. Still other variants of LY6G6F, VSIG10, TMEM25 and/or LSR proteins may be engineered to have reduced binding to immune cells relative to wild-type LY6G6F, VSIG10, TMEM25 and/or LSR proteins, respectively. These variants can be used in combination with variants with stronger binding properties to modulate immune responses with moderate impact.

Also optionally, the variant LY6G6F, VSIG10, TMEM25 and/or LSR protein may be engineered to have an increased half-life relative to wild-type. These variants are typically modified to be resistant to enzymatic degradation. Exemplary modifications include modified amino acid residues that are resistant to enzymatic degradation, as well as modified peptide bonds. Various modifications to achieve this goal are known in the art.

The LY6G6F protein (SEQ ID NO: 1) also has the following non-silent SNPs (single nucleotide polymorphisms) (given by its position on the amino acid sequence in which the alternative amino acids are listed) as listed in Table E, the SNPs present in the LY6G6F protein (SEQ ID NO: 1) sequence providing support for one or more alternative sequences to this protein according to the invention. SEQ ID NO: 58 is an example of such a substitute sequence, with the substituted amino acids of the portion using the following SNPs.

TABLE E-amino acid mutations

The LSR protein (SEQ ID NO: 11) also has the following non-silent SNPs (single nucleotide polymorphisms), (elucidated on the basis of its position on the amino acid sequence in which the alternative amino acids are listed), as listed in Table F, the SNPs present in the sequence of the LSR protein (SEQ ID NO: 11) providing support for one or more alternative sequences of this protein according to the invention. SEQ ID NO: 143 is an example of such a substitute sequence, with substitute amino acids for the portion using the following SNPs.

TABLE F amino acid mutations

The VSIG10 protein (SEQ ID NO: 3) also has the following non-silent SNPs (single nucleotide polymorphisms) (illustrated by their positions on the amino acid sequence in which the alternative amino acids are listed) as listed in Table G, and the SNPs present in the sequence of the VSIG10 protein (SEQ ID NO: 3) provide support for one or more alternative sequences to this protein according to the present invention.

TABLE G-amino acid mutations

Substitution of amino acids at SNP positions on amino acid sequences

333 V-＞M

435 H-＞Y

The TMEM25 protein (SEQ ID NO: 7) also has the following non-silent SNPs (single nucleotide polymorphisms), (illustrated by their positions on the amino acid sequence in which the alternative amino acids are listed), as listed in Table H, and the SNPs present in the TMEM25 protein (SEQ ID NO: 7) sequence provide support for one or more alternative sequences to such a protein according to the present invention.

TABLE H-amino acid mutations

Substitution of amino acids at SNP positions on amino acid sequences

25 W-＞C

342 Q-＞R

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

Nucleic acids

"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 refer to single or double stranded nucleic acid sequences that are isolated and provided as RNA sequences, complementary polynucleotide sequences (cDNA), genomic polynucleotide sequences, and/or composite polynucleotide sequences (e.g., combinations of the above).

Thus, the present invention encompasses the nucleic acid sequences described hereinabove; fragments thereof, sequences which hybridize therewith, sequences which are homologous thereto [ e.g. at least 90%, at least 95%, 96%, 97%, 98% or 99% or more identical to the nucleic acid sequences set forth herein ], sequences which encode similar polypeptides with different codon usage, altered sequences which are characterized by naturally occurring or random or artificially induced mutations, e.g. deletions, insertions or substitutions, of one or more nucleotides. The invention also encompasses homologous nucleic acid sequences (i.e., forming part of the polynucleotide sequences of the invention) that include sequence regions unique to the polynucleotides of the invention.

Thus, the invention also encompasses polypeptides encoded by the polynucleotide sequences of the invention. The present invention also encompasses homologs of these polypeptides, which homologs may be at least 90%, at least 95%, 96%, 97%, 98% or 99% or more homologous to the amino acid sequences set forth below, as may be determined using the BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters. As mentioned hereinabove, the biomolecule sequences of the present invention are useful as tissue or pathological markers and as putative drugs or drug targets for treating or preventing diseases.

Oligonucleotides designed to perform the methods of the invention (designed as described above) for any of the sequences provided herein can be generated according to any oligonucleotide synthesis method known in the art, such as enzymatic synthesis or solid phase synthesis. Oligonucleotides used according to this aspect of the invention are those having a length selected from the range of 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.

The oligonucleotides of the invention may comprise heterocyclic nucleosides consisting of purine and pyrimidine bases, which are bonded with 3 'to 5' phosphodiester linkages.

Preferred oligonucleotides are those that have been modified in the backbone, internucleoside linkages, or bases, as broadly described below. Such modifications can often promote oligonucleotide uptake and resistance to intracellular conditions.

Specific examples of preferred oligonucleotides for use according to this aspect of the invention include oligonucleotides comprising a modified backbone or non-natural internucleoside linkages. Oligonucleotides having a modified backbone include those that retain a phosphorus atom in the backbone, such as those described in U.S. patent nos.: 4,469,863; 4,476,301, respectively; 5,023,243; 5,177,196, respectively; 5,188,897, respectively; 5,264,423; 5,276,019; 5,278,302; 5,286,717, respectively; 5,321,131, respectively; 5,399,676, respectively; 5,405,939, respectively; 5,453,496, respectively; 5,455,233, respectively; 5,466,677, respectively; 5,476,925, respectively; 5,519,126, respectively; 5,536,821, respectively; 5,541,306, respectively; 5,550,111, respectively; 5,563,253, respectively; 5,571,799, respectively; 5,587,361, respectively; and 5,625,050.

Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3 '-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3' -amino phosphoramidates and aminoalkyl phosphoramidates), phosphorothioates, thioalkyl phosphonates, thioalkyl phosphonate triesters, and borane phosphates having the normal 3 '-5' linkages, 2 '-5' linked analogs of these linkages, and those borane phosphates having reversed polarity where adjacent pairs of nucleoside units are linked in 3 '-5' to 5 '-3' or 2 '-5' to 5 '-2'. Different salts, mixed salts and free acid forms may also be used.

Alternatively, the modified oligonucleotide backbone, wherein the phosphorus atom is not included, has a backbone formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatoms or heterocyclic internucleoside linkages. These backbones include those having morpholino linkages (formed in part from the sugar portion of the nucleoside); a siloxane backbone; sulfide, sulfoxide, and sulfone backbones; formyl and thiocarbonyl backbones; methylene formyl and thiocarbonyl backbones; a backbone comprising an olefin; a sulfamate backbone; methylene imino and methylene hydrazino backbones; sulfonate and sulfonamide backbones; an amide skeleton; and other backbones having mixed N, O, S and CH2 components, as described in U.S. patent nos. 5,034,506; 5,166,315, respectively; 5,185,444, respectively; 5,214,134, respectively; 5,216,141, respectively; 5,235,033, respectively; 5,264,562, respectively; 5,264,564, respectively; 5,405,938, respectively; 5,434,257, respectively; 5,466,677, respectively; 5,470,967, respectively; 5,489,677; 5,541,307, respectively; 5,561,225, respectively; 5,596,086, respectively; 5,602,240; 5,610,289, respectively; 5,602,240; 5,608,046, respectively; 5,610,289, respectively; 5,618,704, respectively; 5,623,070, respectively; 5,663,312, respectively; 5,633,360, respectively; 5,677,437, respectively; and 5,677,439.

Other oligonucleotides that can be used according to the invention are those in which the sugar and internucleoside linkages (i.e., the backbone) of the nucleotidic unit have been modified by replacement with a novel group. The base units are maintained so as to be complementary to the appropriate polynucleotide target. Examples of such oligonucleotide mimetics include Peptide Nucleic Acids (PNA). PNA oligonucleotides refer to oligonucleotides in which the sugar-backbone is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone. The base is maintained and is bound directly or indirectly to the aza nitrogen atom of the amide portion of the backbone. U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. nos. 5,539,082; 5,714,331; and 5,719,262, each of which is incorporated herein by reference. Other backbone modifications useful in the present invention are disclosed in U.S. patent nos.: 6,303,374, respectively.

The oligonucleotides of the invention may also include base modifications or substitutions. As used herein, "unmodified" or "natural" bases include the purine bases adenine (A) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified bases include, but are not limited to, other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and 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 (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-mercapto, 8-sulfanyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl 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. Additional bases are included in U.S. patent nos.: 3,687,808, in The encyclopedia Of Polymer Science and Engineering, pp 858-859, KloshenyUz (Kroschwitz), J.I. eds, John Wiley parent-publisher (John Wiley and Sons), those bases disclosed in 1990, in Englisch (Englisch), et al, applied chemistry (Angewandte Chemie, International edition, 1991, 30, 613, and those bases disclosed in Saigv (Sanghvi), Y.S., Chapter 15, Research and application (Antisearch and Applications), pp-302, Kruke (Crke), S.T. and Labule columns (Letee, Blou B), Antisense publications, 1993, those bases disclosed in. Such bases are particularly useful for increasing the binding affinity of oligomeric compounds according to at least some embodiments of the invention. These bases include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. It has been shown that 5-methylcytosine substitutions can increase nucleic acid duplex stability by 0.6-1.2 ℃ [ Sangiviv (Sanghvi) YS et al (1993) Antisense Research and Applications (Antisense Research and Applications), CRC Press, Bocardon (Boca Raton)276-278], and are presently preferred base substitutions, even more particularly when combined with 2' -O-methoxyethyl sugar modifications.

According to at least some embodiments of the invention, another modification of the oligonucleotide involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties (e.g., cholesterol moieties), cholic acids, thioethers (e.g., hexyl-S-trityl sulfide), mercaptocholesterol, fatty chains (e.g., dodecanediol or undecyl residues), phospholipids (e.g., di-hexadecyl-rac-glycerol or 1, 2-di-O-hexadecyl-rac-glycerol-3-H-phosphonic acid triethylammonium), polyamine or polyethylene glycol chains, or adamantane acetic acid, palmityl moieties, or octadecyl amine or hexylamino-carbonyl-hydroxycholesterol moieties, as described in U.S. patent nos.: 6,303,374, respectively.

It is not necessary that all positions in a given oligonucleotide molecule be uniformly modified, and indeed more than one of the above-described modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.

Peptides

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides can be modified, for example, by the addition of carbohydrate residues in order to form glycoproteins. The terms "polypeptide", "peptide" and "protein" include glycoproteins, as well as non-glycoproteins.

The polypeptide product can be synthesized, for example, by using standard solid phase techniques. Such methods include exclusion solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e. 10kDa) and/or when it cannot be produced by recombinant techniques (i.e. is not encoded by a nucleic acid sequence) and thus involves different chemistries.

Solid Phase polypeptide synthesis procedures are well known in the art and are further described by John morrosite (John Morrow Stewart) and Janis della hardwoods (Janis Dillaha Young), Solid Phase Peptide synthesis (Solid Phase Peptide Syntheses) (2 nd edition, Pierce Chemical Company, 1984).

Synthetic polypeptides can be purified by preparative high performance liquid chromatography [ cleton (Creighton T.) (1983) protein, structure and molecular principles (Proteins, structures and molecular principles).

Where a large number of polypeptides are desired, they may be produced, for example, by recombinant techniques as described below: bitter et al, (1987) Methods in enzymology (Methods in Enzymol), 153: 516, Studier et al (1990) Methods in enzymology (Methods in Enzymol), 185: 60-89 Brisson et al (1984) Nature 310: 511-: 307- & ltSUB & gt 311, Korozzi et al (1984) journal of the European society for molecular biology (EMBO J.) -3: 1671-: 838-843, Gurley (1986) molecular cell biology (mol.cell.biol.) 6: 559-565.

It will be appreciated that the peptides identified according to the teachings of the present invention may be degradation products, synthetic or recombinant peptides and peptidomimetics, typically synthetic peptides as peptide analogues as well as peptoids and semipeptoids, which may have modifications such as to make the peptides more stable or more permeable into cells when in vivo. Such modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, peptide bond modifications (including, but not limited to, CH2-NH, CH2-S, CH2-S ═ O, O ═ C-NH, CH2-O, CH2-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 specified, for example, in the Quantitative Drug Design (Quantitative Drug Design), c.a. ramsden (lamsden) Gd., chapter 17.2, f.choplin Pergamon Press (1992), which is incorporated herein by reference as if fully set forth herein. Further details of this aspect are provided below.

The peptide bond (-CO-NH-) within the peptide may for example be replaced by the following bond: n-methylated bonds (-N (CH3) -CO-), ester bonds (-C (R) H-C-O-C (R) -N-), ketomethylene bonds (-CO-CH2-), α -aza bonds (-NH-N (R) -CO-) (wherein R is any alkyl group such as methyl), carba bonds (-CH2-NH-), hydroxyethylidene (-CH (OH)) -CH2-), thioamide (-CS-NH-), olefinic double bonds (-CH ═ CH-), retro amide bonds (-NH-CO-), peptide derivatives (-N (R)) -CH2-CO-), wherein R is the "normal" side chain naturally present on a carbon atom.

These modifications may occur at any bond along the peptide chain and even at several (2-3) bonds at the same time.

The natural aromatic amino acids Trp, Tyr and Phe may be replaced by synthetic non-natural acids such as phenylglycine, TIC, naphthylalanine (Nol), a cyclomethylated derivative of Phe, a halogenated derivative of Phe or o-methyl-Tyr.

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

As used herein in the specification and in the claims section that follows, the term "one or more amino acids" should be understood to include 20 naturally occurring amino acids; those amino acids that are generally post-translationally modified in vivo, including, for example, hydroxyproline, phosphoserine, and phosphinothricine; and other unusual amino acids including, but not limited to, 2-amino fatty acids, hydroxylysine, isodesmosine, nor-valine, nor-leucine, and ornithine. Furthermore, the term "amino acid" includes both D-amino acids and L-amino acids.

Since the peptides of the invention are preferably used in therapeutic agents requiring the peptides to be in a soluble form, the peptides of the invention preferably comprise one or more non-natural or natural polar amino acids including, but not limited to, serine and threonine which are capable of increasing the solubility of the peptides due to their hydroxyl-containing side chains.

Expression system

In order to enable cellular expression of the polynucleotides of the invention, nucleic acid constructs according to the invention can be used which comprise at least one coding region of one of the above nucleic acid sequences and further comprise at least one cis-acting regulatory element. As used herein, the phrase "cis-acting regulatory element" refers to a polynucleotide sequence, preferably a promoter, that binds a trans-acting regulator and regulates transcription of a coding sequence located downstream thereof.

Any suitable promoter sequence may be used with the nucleic acid constructs of the present invention.

Preferably, the promoter used in the nucleic acid construct of the invention is active in the particular population of cells being transformed. Examples of cell type-specific and/or tissue-specific promoters include promoters such as liver-specific albumin [ Pinkert et al, (1987) Gene development (Genes Dev.) 1: 268-277], lymphoid specific promoter [ Carimox (Calame) et al, (1988) advanced immunology 43: 235-275 ]; in particular the T-cell receptor [ Winner diagram (Winto) et al, (1989) journal of the European society for molecular biology (EMBO J.) 8: 729-733] and a promoter for an immunoglobulin; [ Barnaci (Banerji) et al (1983) Cell (Cell)33729- > 740], neuron-specific promoters, such as neurofilament promoters [ Berne (Byrne) et al (1989) Proc. Natl. Acad. Sci., USA 86: 5473-5477], pancreas-specific promoters [ Edlanchi (Edlunch) et al (1985) Science 230: 912-916] or a mammary gland-specific promoter, such as the lactowhey promoter (U.S. Pat. No. 4,873,316 and European application publication No. 264,166). The nucleic acid construct of the invention may further comprise an enhancer, which may be adjacent to or remote from the promoter sequence and which may act so as to up-regulate transcription therefrom.

The nucleic acid construct of the present invention preferably further comprises a suitable selection marker and/or an origin of replication. Preferably, the nucleic acid construct used is a shuttle vector which can be propagated in E.coli (wherein the construct comprises the appropriate selection marker and origin of replication) and is suitable for propagation in cells or integration into selected genes and tissues. The construct according to the invention may be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.

Examples of suitable constructs include, but are not limited to, pcDNA3, pcDNA3.1(+/-), pGL3, PzeoSV2(+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto, each of which is commercially available from Invitrogen, Life technologies, Inc. (www.invitrogen.com). Examples of retroviral vectors and packaging systems are those sold by Clontech, San Diego, Calif. (Calif.), including the Retro-X vectors pLNCX and pLXSN, which allow cloning into multiple cloning sites and transcription of the transgene from the CMV promoter. Also included are vectors derived from Mo-MuLV, such as pBabe, in which the transgene will be transcribed from the 5' LTR promoter.

Presently 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. Useful lipids for lipid-mediated transfer of genes are, for example, DOTMA, DOPE, and DC-Choi [ Tonkinson et al, Cancer research, 14 (1): 54-65(1996)]. Most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses. Viral constructs, such as retroviral constructs, contain at least one transcriptional promoter/enhancer or locus defining element, or other element that controls gene expression by other means, such as alternative splicing, nuclear RNA export, or post-translational modification of messengers. Such vector constructs also include a packaging signal, Long Terminal Repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate for the virus used, unless it is already present in the viral construct. In addition, such constructs typically include a signal sequence for secretion of the peptide from the host cell in which it is placed. 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 to direct polyadenylation, and one or more restriction sites and a translation termination sequence. For example, such constructs 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 carriers that are not viral may be used, such as cationic lipids, polylysine, and dendrimers.

Recombinant expression vectors and host cells

Another aspect of the invention relates to a vector, preferably an expression vector, comprising a nucleic acid encoding a protein according to at least some embodiments of the invention, or a derivative, fragment, analogue or homologue thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Examples of vector types are plasmids and viral vectors. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors". The present invention is intended to include such forms of expression vectors, e.g., plasmids, viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

Recombinant expression vectors according to at least some embodiments of the present invention include nucleic acids according to at least some embodiments of the present invention in a form suitable for expression of the nucleic acids in a host cell, meaning that the recombinant expression vectors include one or more regulatory sequences selected based on the host cell to be used for expression, which are operably linked to the nucleic acid sequence to be expressed. In a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequences in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in superadel (Goeddel), "techniques for gene expression: the enzymology method comprises the following steps: (GeneExpression Technology: Methods in Enzymology)185, Academic Press (Academic Press), San Diego (San Lag., Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells as well as those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those of ordinary skill in the art that the design of the expression vector will depend on such factors as the choice of host cell to be transformed, the level of expression of the desired protein, etc. Expression vectors according to at least some embodiments of the present invention may be introduced into host cells to thereby produce proproteins or peptides (including fusion proteins or peptides) encoded by nucleic acids as described herein.

Recombinant expression vectors according to at least some embodiments of the present invention may be designed to produce variant proteins in prokaryotic or eukaryotic cells. For example, a protein according to at least some embodiments of the invention can be expressed in bacterial cells, such as E.coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are further described in Goeddel (high adel), "techniques for gene expression: methods in Enzymology (Gene expression technology: Methods in Enzymology)185, Academic Press (Academic Press), San Diego (San Diego), Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often performed in E.coli using vectors containing constitutive or inducible promoters that direct expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to the protein encoded therein, to the amino or C-terminus of the recombinant protein. Such fusion vectors are commonly used for three purposes: (i) increasing expression of the recombinant protein; (ii) increasing the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Typically, in fusion expression vectors, a protein cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety after purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include factor Xa, thrombin, PreScission, TEV, and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc.; Smith and Johnson (Johnson), 1988. "Gene 67: 31-40),. pMAL (New England Biolabs (New England laboratories), Beverly, Mass.), and pRIT5 (Pharmacia), Piscataway (Piscataway, N.J.) which respectively fuse glutathione S-transferase (GST), maltose E binding protein, or protein A to the targeted recombinant protein.

In another embodiment, the expression vector encoding the protein of the invention is a yeast expression vector. Examples of vectors for expression in Saccharomyces cerevisiae include pYepSecl (Baldari et al, 1987. J.Eur. Med. Biol.Proc.Soc. (EMBO J) 6: 229. 234), pMFa (Kurjan) and Herskowitz (Herskowitz), 1982. Cell 933 (Cell) 30: 943), pJRY88 (Schultz et al, 1987. Gene 54: 113. 123), pYES2(Invitrogen Corporation, Sangey, San Ego (Regto), Calif (Sanifornia), and picZ (Invitrogen Corp, Diego (Regto), Sandif, Calif).

Alternatively, the polypeptides of the invention may be produced in insect cells using baculovirus expression vectors. Baculovirus vectors that can be used for protein expression in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al, 1983, molecular cell biology (mol. cell. biol.) 3: 2156-2165) and the pVL series (Raklow) and Sammers (Summers), 1989, Virology (Virology) 170: 31-39).

In yet another embodiment, the nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Swed, 1987. (Nature 329: 840) and pMT2PC (Coofmann (Kaufman) et al, 1987. (journal of the European society of molecular biology) (EMBO J.) 6: 187-195), pIRESpuro (Clontech), pUB6 (Invitrogen), pCEP4 (Invitrogen), pREP4 (Invitrogen), DNA3 (Invitrogen). When used in mammalian cells, the control functions of the expression vector 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 both prokaryotic and eukaryotic cells see, for example, chapter 16 and chapter 17, chapter 2nd, of Molecular Cloning, Laboratory Manual (2 nd edition), of Sambrook et al, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., expression of the nucleic acid using tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of tissue-specific promoters include the albumin promoter (liver-specific; Pinkert (Pinkert) et al, 1987. Gene development (Genes Dev.) 1: 268-277), the lymph-specific promoters (Calames) and Eton (Eaton), 1988. advanced immunology (adv. Immunol) 43: 235-275), in particular the T-Cell receptor (Winto) and Bardimore (Baltimore), 1989. J.Eur. Med.Biol.J. (EMBO J) 8: 729-733) and the immunoglobulin (Banerji) et al, 1983. Cell (Cell) 33: 729-740; Queen (Queen) and Barmodore (Baltimore), 1983. Cell (Cell) 33: 729-748; e.g.nerve Cell promoter (Bddyre) (Burney) 741; Cell promoter specific for example), 1989. journal of the american academy of sciences (proc.natl.acad.sci USA) 86: 5473-: 912-916), and a mammary gland-specific promoter (e.g., the milk whey promoter; U.S. Pat. No. 4,873,316 and european application publication No. 264,166). Also contemplated are developmental regulatory promoters, for example, the murine homeobox (hox) promoter Kessel (Kessel) and Gruss (Gruss), 1990. Science (Science) 249: 374-379) and the alpha-fetoprotein promoter (Campis and Telfmann (Tilghman), 1989. Gene development (Genes Dev.) 3: 537-546).

According to at least some embodiments, the present invention further provides a recombinant expression vector comprising a DNA molecule according to at least some embodiments of the present invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner that allows expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to mRNA encoding a protein according to at least some embodiments of the invention. Regulatory sequences operably linked to a nucleic acid cloned in an antisense orientation can be selected to direct continuous expression of the antisense RNA molecule in various cell types, e.g., viral promoters and/or enhancers, or regulatory sequences can be selected to direct constitutive, tissue-specific, or cell type-specific expression of the antisense RNA. Antisense expression vectors can be in the form of recombinant plasmids, phagemids or attenuated viruses in which the antisense nucleic acid is produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the use of Antisense genes to regulate gene expression, see, e.g., Weintraub et al, Antisense RNA as a molecular tool for genetic analysis ("Antisense RNA for genetic analysis)", (Reviews of genetics-developments), Vol.1(1) 1986.

According to at least some embodiments, the present invention relates to a host cell into which a recombinant expression vector according to at least some embodiments of the present invention has been introduced. The terms "host cell" and "recombinant host cell" may be used interchangeably herein. It will be understood that such terms refer not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Because certain changes may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

The host cell may be any prokaryotic or eukaryotic cell. For example, a protein according to at least some embodiments of the invention may 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. As used herein, the terms "transformation" and "transfection" are intended to include a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al, Molecular Cloning, A Laboratory Manual, 2nd ed, second edition, Cold Spring Harbor Laboratory Press, N.Y., 1989, and other Laboratory manuals.

For stable transfection of mammalian cells, it is known that only a small fraction of cells can incorporate foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select these constitutive components (integrants), a gene encoding a selectable marker (e.g., antibiotic resistance) is typically introduced into the host cell along with the gene of interest. Various selectable markers include those that confer resistance to drugs such as G418, hygromycin, puromycin, blasticidin, and methotrexate. The nucleic acid encoding the selectable marker may be introduced into the host cell on the same vector as the nucleic acid encoding the protein according to at least some embodiments of the invention or may be introduced into a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells incorporating the selectable marker gene will survive, while other cells die).

Host cells according to at least some embodiments of the invention in culture, such as prokaryotic or eukaryotic host cells, may be used to produce (i.e., express) proteins according to at least some embodiments of the invention. Accordingly, the present invention further provides methods of using host cells according to at least some embodiments of the invention to produce proteins according to at least some embodiments of the invention. In one embodiment, the method comprises culturing a host cell of the invention into which a recombinant expression vector encoding a protein according to at least some embodiments of the invention has been introduced in a suitable medium such that a protein according to at least some embodiments of the invention is produced. In another embodiment, the method further comprises isolating the protein according to at least some embodiments of the invention from the culture medium or the host cell.

For efficient production of proteins, it is preferred that the nucleotide sequences encoding the proteins according to at least some embodiments of the present invention be placed under the control of expression control sequences, optimized for expression in the desired host. For example, these sequences may include optimized transcriptional and/or translational regulatory sequences (e.g., altered Kozak sequences).

It should be noted that, in accordance with at least some embodiments of the present invention, the LY6G6F, VSIG10, TMEM25 and/or LSR proteins described in accordance with at least some embodiments of the present invention may be isolated as naturally occurring polypeptides or from any source (whether natural, synthetic, semi-synthetic or recombinant). Thus, LY6G6F, VSIG10, TMEM25 and/or LSR proteins may be isolated from any species, particularly mammals, including cattle, sheep, pigs, murines, horses, and preferably humans, as naturally occurring proteins. Alternatively, LY6G6F, VSIG10, TMEM25 and/or LSR proteins may be isolated as recombinant polypeptides expressed in prokaryotic or eukaryotic host cells, or as chemically synthesized polypeptides.

The skilled person can readily employ standard separation methods to obtain isolated LY6G6F, VSIG10, TMEM25 and/or LSR proteins. The nature and extent of the separation depends on the source of the separated molecules and the intended use.

Transgenic animals and plants

According to at least some embodiments, the present invention also provides transgenic non-human animals and transgenic plants comprising one or more nucleic acid molecules according to at least some embodiments of the invention, which can be used to produce polypeptides according to at least some embodiments of the invention. These polypeptides may be produced in and recovered from a tissue or body fluid (e.g., milk, blood or urine) of a goat, cow, horse, pig, rat, mouse, rabbit, hamster or other mammal. See, for example, U.S. patent nos. 5,827,690, 5,756,687, 5,750,172, and 5,741,957.

Non-human transgenic animals and transgenic plants are produced by introducing one or more nucleic acid molecules according to at least some embodiments of the invention into the animal or plant via standard transgenic techniques. The transgenic cell used to make the transgenic animal may be an embryonic stem cell, a somatic cell, or a fertilized egg cell. The transgenic non-human organism can be chimeric, heterozygote of a non-chimeric, and homozygote of a non-chimeric. See, for example, Hogan et al, guide to Mouse Embryo manipulation (Manipulating the Mouse Embryo: A Laboratory Manual), Cold Spring Harbor Press, second edition (1999); jackson et al, genetics and transgenics in mice: utility methods (Mouse Genetics and Transgenics: A Practical Approach), Oxford University Press (Oxford University Press) (2000); and Pinketer (Pinkert), "A Laboratory Handbook of Transgenic Animal Technology", Academic Press (1999).

Gene therapy

According to at least some embodiments of the present invention, nucleic acid sequences encoding soluble LY6G6F, VSIG10, TMEM25 and/or LSR proteins may be used in gene therapy for the treatment of infectious, and/or immune related disorders, and/or cancer.

As used herein, "gene therapy" is the process of treating a disease by genetic manipulation such that a sequence of nucleic acid is transferred into a cell, which then expresses any gene product encoded by the nucleic acid. For example, as is well known to those of ordinary skill in the art, nucleic acid Transfer can be performed by inserting an Expression vector containing the nucleic acid of interest into cells ex vivo or in vitro via a variety of Methods, including, for example, calcium phosphate precipitation, diethylaminoethyl dextran, polyethylene glycol (PEG), electroporation, direct injection, lipofection, or viral infection (Sambrook et al, Molecular Cloning: A Laboratory Manual (Cold Spring harbor Laboratory Press 1989), Crigler (Krigler) M., Gene Transfer and Expression: A Laboratory Manual (W.H. Freueman (Freeman) and Co, New York, N.Y., 1993), and Wulogen (scientific), method of enzyme publication (Press), new York (New York, 1993). Alternatively, the nucleic acid sequence of interest can be transferred in vivo in various vectors and into cells by various methods including, for example, administering the nucleic acid directly into the subject, or inserting the nucleic acid into a viral vector and infecting the subject with the virus. Other methods for in vivo transfer include encapsulation of nucleic acids into liposomes, and direct transfer of liposomes, or liposomes in combination with red blood cell agglutinating Sendai virus (Sendai virus), to a subject. Transfected or infected cells express the protein product encoded by the nucleic acid in order to ameliorate the disease or disease symptoms.

Antibodies and immune system responses

As used herein, the term "immunological" (immunological) or "immune" response is the development of a beneficial humoral (antibody-mediated) and/or cellular (mediated by antigen-specific T cells or their secretory products) response directed against a peptide in a recipient patient. Such a response may be an active response induced by administration of an immunogen or a passive response induced by administration of antibodies or primed T-cells. Without wishing to be bound by a single hypothesis, the cellular immune response is elicited by binding to MHC class I or class II molecules to present polypeptide epitopes in order to activate antigen-specific CD4+ T helper cells and/or CD8+ cytotoxic T cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, neutrophils or other components of innate immunity or recruitment. The presence of a cell-mediated immune response can be determined by a proliferation assay (CD4+ T cells) or CTL (cytotoxic T lymphocytes) assay. The relative contribution of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by isolating antibodies and T-cells separately from the immunized syngeneic animal and measuring the protective or therapeutic effect in a second subject.

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

"Signal transduction pathway" refers to the biochemical relationship between a variety of signal transduction molecules that function in transmitting a signal from one part of a cell to another part of a cell.

As used herein, the phrase "cell surface receptor" includes, for example, molecules and complexes of molecules that are capable of receiving a signal and transmitting such a signal across the plasma membrane of a cell.

The term "antibody" as referred to herein includes all polyclonal and monoclonal antibodies as well as any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region includes three domains, CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region includes one domain, CL. The VH and VL regions may be further subdivided into regions of high variability (designated Complementarity Determining Regions (CDRs)) interspersed with more conserved regions (designated Framework Regions (FRs)). Each VH and VL includes three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of these antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including different cells of the immune system (e.g., effector cells) as well as the first component of the classical complement system (Clq).

As used herein, the term "antigen-binding portion" (or simply "antibody portion") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., LY6G6F, VSIG10, TMEM25 and/or LSR molecules, and/or fragments thereof). It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) fab fragment, a monovalent fragment consisting of the V light, V heavy, Constant Light (CL) and CH1 domains; (ii) a F (ab'). 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) fv fragments, consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragments (Ward et al, (1989) Nature 341: 544-546) consisting of VH domains; and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods via a synthetic linker that enables them to be prepared as a single protein chain in which the VL and VH regions form a pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Border (Bird) et al (1988) Science 242: 423-. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of ordinary skill in the art and are screened for utility in the same manner as intact antibodies.

As used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to LY6G6F, VSIG10, TMEM25 or LSR proteins and/or fragments thereof, respectively, and substantially free of antibodies that specifically bind antigens other than LY6G6F, VSIG10, TMEM25 or LSR). However, isolated antibodies that specifically bind to LY6G6F, VSIG10, TMEM25, or LSR proteins, respectively, may be cross-reactive to other antigens (e.g., LY6G6F, VSIG10, TMEM25, or LSR molecules from other species). Furthermore, the 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 consisting of a single molecule. The monoclonal antibody compositions exhibit a single binding specificity and binding 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 comprises a constant region, then the constant region is also derived from human germline immunoglobulin sequences. Human antibodies according to at least some embodiments of the invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which the CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences.

The term "human monoclonal antibody" refers to an antibody having variable regions exhibiting a single binding specificity, wherein both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a transgenic non-human animal (e.g., a transgenic mouse) having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes, or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells transformed to express human antibodies (e.g., from transfectomas); (c) antibodies isolated from a recombinant, combinatorial human antibody library; and (d) antibodies prepared, expressed, produced or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. 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 can be subjected to in vitro mutagenesis (or, when using animals that are transgenic for human Ig sequences, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of these recombinant antibodies (although derived from and related to human germline VH and VL sequences) may not naturally occur in the in vivo human antibody germline expression profile.

As used herein, "isotype" refers to the class of antibodies encoded by the heavy chain constant region (e.g., IgM or IgG 1).

The phrases "antibody recognizing an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody specifically binding to an antigen".

As used herein, an antibody that "specifically binds human LY6G6F, VSIG10, TMEM25, and/or LSR protein" is intended to refer to an antibody that binds LY6G6F, VSIG10, TMEM25, or LSR protein, respectively, e.g., an antibody having a KD of 5X10-8M, 3X10-8M, 1X10-9M, or less.

As used herein, the term "K-assoc" or "Ka" is intended to refer to the binding rate of a particular antibody-antigen interaction; rather, as used herein, the term "Kdiss" or "Kd" is intended to refer to the rate of dissociation of a particular antibody-antigen interaction. As used herein, the term "KD" is intended to refer to the dissociation constant, which is obtained from the ratio of KD to Ka (i.e., KD/Ka) and is expressed as molar concentration (M). The KD value of an antibody can be determined using well established methods in the art. A preferred method of determining the KD value of an antibody is by using surface plasmon resonance, preferably using a biosensor system, such as the biacore rtm system.

As used herein, the term "high affinity" for an IgG antibody refers to an antibody having a KD of 10 "8M or less, more preferably 10" 9M or less, and even more preferably 10 "10M or less, against a target antigen. However, "high affinity" binding to different antibody isotypes can vary. For example, "high affinity" binding to an IgM isotype refers to antibodies having a KD of 10-7M or less, more preferably 10-8M or less.

As used herein, the term "subject" or "patient" includes any human or non-human animal. The term "non-human animal" includes all vertebrate hair-profit mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like.

anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and anti-LSR antibodies

Antibodies according to at least some embodiments of the invention include those with specific germline sequences, homologous antibodies, antibodies with conservative modifications, engineered and modified antibodies, characterized by specific functional characteristics or properties of these antibodies. For example, these antibodies specifically bind to human LY6G6F, VSIG10, TMEM25, or LSR. Preferably, an antibody according to at least some embodiments of the invention binds with high affinity, e.g., with a KD of 10 "8M or less or 10" 9M or less or even 10 "10M or less, to the corresponding LY6G6F, VSIG10, TMEM25 or LSR. The anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and anti-LSR antibodies described in accordance with at least some embodiments of the present invention exhibit one or more of the following characteristics:

(i) Binds to the corresponding human LY6G6F, VSIG10, TMEM25 or LSR with a KD of 5.X10-8M or less;

(ii) modulating (enhancing or inhibiting) B7 immune co-stimulation and associated activities and functions, such T cell responses involving anti-tumor immunity and autoimmunity; and/or

(iii) Binds to LY6G6F, VSIG10, TMEM25 or LSR antigen expressed by cancer cells including, for example, melanoma, liver cancer, kidney cancer, brain cancer, breast cancer, colon cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, multiple myeloma, and hematopoietic cell cancers including but not limited to lymphoma (hodgkins and non-hodgkins), acute and chronic lymphatic leukemia, and acute and chronic myeloid leukemia, but does not substantially bind to normal cells. In addition, preferably, these antibodies and conjugates thereof will be effective in eliciting selective killing of such cancer cells and in modulating immune responses involved in autoimmunity and cancer.

More preferably, the antibody binds to the corresponding human LY6G6F, VSIG10, TMEM25 or LSR antigen with a KD of 3X10-8M or less, or with a KD of 1X10-9M or less, or with a KD of 0.1X10-9M or less, or with a KD of 0.05X10-9M or less, or with a KD between 1X10-9 and 1X 10-11M.

Standard assays for assessing the binding ability of these antibodies to LY6G6F, VSIG10, TMEM25 or LSR 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 (e.g., binding affinity) of these antibodies can also be assessed by standard assays known in the art, e.g., by Biacore analysis.

Once anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25, and anti-LSR antibodies are generated, sequences from the antibodies can be bound to LY6G6F, VSIG10, TMEM25, or LSR, and VH and VL sequences can be "mixed and matched" to generate other anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25, and anti-LSR binding molecules as described in accordance with at least some embodiments of the present invention. LY6G6F, VSIG10, TMEM25 or LSR binding of such "mixed and matched" antibodies can be tested using the binding assays described above (e.g., ELISA). Preferably, when VH and VL chains are mixed and matched, VH sequences from a particular VH/VL pairing are replaced by a structurally similar VH sequence. Also, preferably, the VL sequence from a particular VH/VL pairing is replaced by a structurally similar VL sequence. For example, the VH and VL sequences of a cognate antibody are particularly suitable for mixing and matching.

Antibodies with specific germline sequences

In certain embodiments, an antibody of the invention comprises a heavy chain variable region from a particular germline heavy chain immunoglobulin gene and/or a light chain variable region from a particular germline light chain immunoglobulin gene.

As used herein, if the variable region of an antibody is obtained from a system using human germline immunoglobulin genes, such human antibodies include the heavy or light chain variable region as the "product" of, or "originating from," a particular germline sequence. Such systems include immunization of transgenic mice carrying human immunoglobulin genes with the antigen of interest, or screening of phage-displayed human immunoglobulin gene libraries with the antigen of interest. A human antibody that is the "product" of, or "originates from," a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequence of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., greatest percent identity) to the sequence of the human antibody.

A human antibody that is the "product" of or "originates from" a particular human germline immunoglobulin sequence may comprise amino acid differences compared to the germline sequence, e.g., due to deliberate introduction of naturally-occurring somatic mutations or site-directed mutations. However, the selected human antibody is typically at least 90% identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene and comprises amino acid residues that identify the human antibody as being human when compared to germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In certain instances, the human antibody may typically be at least 95%, 96%, 97%, 98%, or 99%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will exhibit no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain instances, such a human antibody may exhibit no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

Homologous antibodies

In yet another embodiment, the antibodies of the invention comprise heavy and light chain variable regions comprising amino acid sequences homologous to the isolated anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 or anti-LSR amino acid sequences of the preferred anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 or anti-LSR antibodies, respectively, wherein the antibodies retain the desired functional properties of the parent anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 or anti-LSR antibodies.

As used herein, the percent homology between two amino acid sequences is equal to the percent identity between the two sequences. The percent identity between these two sequences is a function of the number of identical positions shared by these sequences (i.e., percent homology-number of identical positions/total number of positions X100) taking into account the number of clefts and the length of each cleft, which needs to be introduced in an optimized alignment of the two sequences. Comparison of sequences and percent identity between two sequences can be accomplished using mathematical algorithms, as described in the non-limiting examples below.

The percent identity between two amino acid sequences can be determined using the algorithms of meyeres (e.meyers) and miller (w.miller) ("computer applications in bioscience" (comput.appl.biosci.), 4: 11-17(1988)), which have been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithms, which have been incorporated into the GAP program in the GCG software package (commercially available) using either the Blossum62 matrix or the PAM250 matrix, and with a GAP weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the invention may be further used as "query sequences" to perform searches against public databases, for example to identify related sequences. Such a search can be performed using alchol (Altschul) et al (1990) journal of molecular biology (J mol. biol.) 215: XBLAST programs 403-10. BLAST protein searches may be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the antibody molecules described in accordance with at least some embodiments of the present invention. To obtain gapped alignments for comparison purposes, gapped BLAST as described in Aldhill et al, (1997) Nucleic Acids research (Nucleic Acids Res.) can be used. When utilizing BLAST and gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used.

Antibodies with conservative modifications

In certain embodiments, the antibodies of the invention comprise a heavy chain variable region (comprising CDR1, CDR2, and CDR3 sequences) and a light chain variable region (comprising CDR1, CDR2, and CDR3 sequences), wherein one or more of the CDR sequences comprises a particular amino acid sequence based on the preferred anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25, or anti-LSR antibodies isolated and produced using the methods herein; or conservative modifications thereof, and wherein these antibodies retain the desired functional properties of the anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 or anti-LSR antibodies, respectively, according to at least some embodiments of the present invention.

In various embodiments, the anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25, or anti-LSR antibody may be, for example, a human, humanized, or chimeric antibody.

As used herein, the term "conservative sequence modification" is intended to refer to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into antibodies according to at least some examples 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 an 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 amino acids with the following: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), side chains without electrical polarity (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CDR region of an antibody according to at least some embodiments of the present invention may be replaced with amino acid residues from the same side chain family, and this altered antibody may be tested for retained function (i.e., the functions set forth above in (c) through (j)) using the functional assays described herein.

An antibody that binds to the same epitope as anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 or anti-LSR described in accordance with at least some embodiments of the present invention.

In another embodiment, the invention provides antibodies that bind to a preferred epitope on human LY6G6F, VSIG10, TMEM25 or LSR, which possess desirable functional properties, such as modulation of B7 co-stimulation and related functions. Other antibodies can be selected that have the desired epitope specificity and will have the ability to cross-compete with the desired antibody for binding to LY6G6F, VSIG10, TMEM25 or LSR antigens.

Engineered and modified antibodies

Antibodies according to at least some embodiments of the invention may further be prepared using antibodies having one or more VH and/or VL sequences derived from anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 or anti-LSR antibody starting materials to engineer a modified antibody that may have altered properties from the starting antibody. An antibody may be engineered by modifying one or more residues within one or both variable regions (i.e., VH and/or VL), e.g., within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody may be engineered by modifying residues within the constant region, for example to alter the effector function of the antibody.

One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with the target antigen primarily through amino acid residues located in the six heavy and light chain Complementarity Determining Regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than sequences outside the CDRs. Because the CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing expression vectors that include the CDR sequences from that particular naturally occurring antibody grafted onto framework sequences from different antibodies with different properties (see, e.g., Riechmann, L. (Rickman, L.) (1998) Nature (Nature) 332: 323-containing 327; Jones, P.) (1986) Nature (Nature) 321: 522-containing 525; Queen, C.) (1989) Proc. Natl. Acad. U.S.A. (Proc. Natl. A. U.S.A.) see 86: 10029-containing 10033; U.S. Pat. No. 5,225,539 to Wett (Win.) (Winter.) (U.S.S.A.;. No. 5,762; U.S.S.S.A.; 5,699; Queen) U.S.S.A.;,699; U.S.S.S.S.A.; 5,699; 085,699; and U.S.S.S.S.A.; 085,585; U.No. 6; U.

Suitable framework sequences can be obtained from public DNA databases or published references including 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), along with the following: kabat (Kabat, e.a.) et al (1991) Sequences of Proteins of Immunological Interest (Sequences of Proteins of Immunological Interest), Fifth Edition (Fifth Edition), U.S. department of Health and Human Services (U.S. department of Health and Human Services), NIH publication Nos. 91-3242; thomlinson (Tomlinson, I.M.) et al (1992) "about Fifty sets of expression profiles of human germline VH Sequences revealed by VH Segments with different Hypervariable Loops" ("The repertile of humamgermine VH Sequences dive about filing Groups of VH Segments with differential Hypervariable Loops") "journal of molecular biology (j.mol.biol.) 227: 776-798; and Cox (Cox, j.p.l.) et al (1994) "catalogue of Human germline VH regions which have revealed Strong preferences in their use" ("adorrection of Human gemm-line VH Segments a Strong Bias in the same usage") -european journal of immunology (eur.j immunological.) 24: 827-836; the contents of each of which are expressly incorporated herein by reference).

Another type of variable region modification is mutation of amino acid residues within the VH and/or VL CDR1, CDR2, and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest. To introduce mutations, site-directed mutagenesis or PCR-mediated mutagenesis can be performed, and antibody binding, or other functional properties of interest, can be appropriately assessed in vitro or in vivo assays. Preferably, conservative modifications (as discussed above) are introduced. These mutations may be amino acid substitutions, additions or deletions, but are preferably substitutions. Furthermore, it is typical to alter no more than one, two, three, four or five residues within a CDR region.

Engineered antibodies according to at least some embodiments of the invention include those in which framework residues within the VH and/or VL are modified, e.g., to improve the properties of the antibody. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "back mutate" one or more framework residues of the corresponding germline sequence. Rather, an antibody that has undergone somatic mutation may comprise framework residues that differ from the germline sequence from which the antibody originates. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody originates.

In addition to, or in the alternative to, modifications made within the framework or CDR regions, antibodies according to at least some embodiments of the present invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, antibodies according to at least some embodiments of the invention may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or may be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Such embodiments are described further below. The numbering of residues in the Fc region is that of the EU index of Kabat (Kabat).

In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This pathway is further described in U.S. Pat. No. 5,677,425 to Bordermol et al. The number of cysteine residues in the hinge region of CH1 is altered, for example, to assist in the assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced in the interfacial region of the CH2-CH3 domain of the Fc-hinge fragment such that the antibody has impaired staphylococcal protein a (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Pat. No. 6,165,745 to Ward et al.

In another embodiment, the antibody is modified to increase its biological half-life. Different approaches are possible. For example, one or more of the following mutations may be introduced: T252L, T254S, T256F, as described in U.S. patent No. 6,277,375 to Ward (Ward). Alternatively, to increase biological half-life, the antibody may be altered within the CH1 or CL region to include a salvage receptor binding epitope in both loops of the CH2 domain taken from the Fc region of IgG, as described in U.S. patent nos. 5,869,046 and 6,121,022 to hangul et al.

In still other embodiments, the Fc region is altered by substituting at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 can be substituted with a different amino acid such that the antibody has an altered affinity for the effector ligand, but retains the antigen-binding ability of the parent antibody. The altered affinity effector ligand may be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in both U.S. Pat. nos. 5,624,821 and 5,648,260 to Winter et al.

In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be substituted with a different amino acid such that the antibody has altered Clq binding and/or reduced or abrogated Complement Dependent Cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 to Idusogie et al.

In another example, amino acid residues within one or more of amino acid positions 231 and 239 can be altered to thereby alter the ability of the antibody to fix complement. This pathway is further described in PCT publication WO 94/29351 to Bordermol et al.

In yet another example, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or increase the affinity of the antibody for the Fey receptor by modifying one or more of the amino acids at the following positions: 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, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is further described in PCT publication WO 00/42072 to hangul (Presta). Furthermore, binding sites for Fc γ RI, Fc γ RII, Fc γ RIII and FcRn have been mapped in human IgG1, and variants with improved binding have been described (see hilz (Shields, r.l.) et al (2001) journal of biochemistry (j.biol. chem.) 276: 6591-6604). It is shown that specific mutations at positions 256, 290, 298, 333, 334 and 339 can improve binding to FcyRIII. Additionally, the following combinatorial mutants were shown to improve Fc γ RIII binding: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A. Furthermore, mutations (e.g., M252Y/S254T/T256E or M428L/N434S) improve binding to FcRn and increase the circulating half-life of the antibody (see Chen (Chan CA) and Kater (Carter PJ) (2010) Nature RevImmunol 10: 301-316).

In yet another embodiment, the glycosylation of the antibody is modified. For example, deglycosylated (aglycosylated) antibodies may be prepared (i.e., the antibodies lack glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions may be made which result in the elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such deglycosylation (aglycosylation) may increase the affinity of the antibody for the antigen. Such an approach is described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 to kouzu (Co) et al.

Additionally or alternatively, an antibody with altered types of glycosylation can be prepared, for example, a low fucosylated (hypofucosylated) antibody with reduced amounts of fucose (fucosyl) residues or an antibody with an increased bisecting (bisecting) GlcNac structure. Such altered glycosylation patterns have been shown to increase the ADCC capacity of the antibody. Such carbohydrate modifications can be accomplished, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells having altered glycosylation machinery have been described in the art and can be used as host cells in which recombinant antibodies according to at least some embodiments of the present invention are expressed to thereby produce antibodies having altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucose transferase (fucosyltransferase) gene, FUT8(α (1, 6) fucose transferase (α (1, 6) fucosyltransferase)), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose with respect to their carbohydrates. The Ms704, Ms705, and Ms709 FUT 8.7-cell lines were generated by targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. patent publication No. 20040110704 to Yamane et al and Yamane to Ohnuki et al (2004) Biotechnology and bioengineering (Biotechnol Bioeng) 87: 614-22). As another example, EP 1,176,195 to Kaanel (Hanai) et al describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such a cell line exhibit low fucosylation (hypofucosylation) by reducing or eliminating the alpha 1, 6 bond-related enzyme (alpha 1, 6 bond-related enzyme). Kaempferia (Hanai) et al also describe cell lines that have low or no enzymatic activity for adding fucose to N-acetylglucosamine bound to the Fc region of the antibody, such as the rat myeloma cell line YB2/0(ATCC CRL 1662). PCT publication WO 03/035835 to Korea (Presta) describes a variant CHO cell line, Led 3 cell, with reduced ability to attach fucose to Asn (297) -linked carbohydrates, which also results in low fucosylation of antibodies expressed in that host cell (see also Hilz (Shields, R.L.) et al (2002) J. Biol. chem. 277: 26733) 26740). PCT publication WO 99/54342 to Umama (Umana) et al describes a cell line engineered to express a glycoprotein-modified glycosyltransferase (e.g., β (1, 4) -N-acetylglucosaminyltransferase (GnTIII)) such that antibodies expressed in the engineered cell line exhibit an increased bisecting GlcNac structure, which results in increased ADCC activity of these antibodies (see also Umama (Umana) et al (1999) Nature Biotech 17: 176-180). Alternatively, the fucose residue of the antibody may be cleaved off using a fucosidase. For example, fucosidase α -L-fucosidase removes fucose residues from antibodies (Tarentino, A.L.) et al (1975) biochemistry (Biochem.) 14: 5516-23).

Another modification of antibodies contemplated herein by the present invention is pegylation. An antibody can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG) (e.g., a reactive ester or aldehyde derivative of PEG) under conditions in which one or more PEG groups are attached to the antibody or fragment thereof. Preferably, pegylation is performed via an acylation reaction or alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy-or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is a deglycosylated antibody. Methods for pegylating proteins are known in the art and may be applied to antibodies according to at least some embodiments of the present invention. See, for example, EP 0154316 to west village (Nishimura) et al and EP 0401384 to Ishikawa et al.

Methods of engineering antibodies

As discussed above, anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 or anti-LSR antibodies having VH and VK sequences disclosed herein can be made by modifying the VH and/or VL sequences, or constant regions attached thereto, to generate novel anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 or anti-LSR antibodies, respectively. Thus, in another aspect according to at least some embodiments of the present invention, the structural features of an anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 or anti-LSR antibody according to at least some embodiments of the present invention may be used to generate structurally related anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 or anti-LSR antibodies that retain at least one functional property of an antibody according to at least some embodiments of the present invention, for example binding to human LY6G6F, VSIG10, TMEM25 or LSR, respectively. For example, one or more CDR regions of a LY6G6F, VSIG10, TMEM25 or LSR antibody or mutant thereof may be recombinantly combined with known framework regions and/or other CDRs to produce additional, recombinantly engineered, anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 or anti-LSR antibodies as discussed above in accordance with at least some embodiments of the present invention. Other types of modifications include those described in the previous section. Starting materials for the engineering method are one or more VH and/or VK sequences, or one or more CDR regions thereof, provided herein. In order to produce engineered antibodies, it is not necessary to actually prepare (i.e., express as a protein) an antibody having one or more of the VH and/or VK sequences provided herein, or one or more CDR regions thereof. Of course, the information contained in these sequences is used as a starting material for the generation of "second generation" sequences derived from the original sequence, and these "second generation" sequences are prepared and expressed as a protein.

Altered antibody sequences can be prepared and expressed using standard molecular biology techniques.

Preferably, the antibody encoded by the altered antibody sequence is an antibody produced thereby in the provided methods and sequences that retains one, some or all of the functional properties of an anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 or anti-LSR antibody, respectively, these functional properties include binding to LY6G6F, VSIG10, TMEM25 or LSR antigen at a particular KD level or less and/or modulating B7 co-stimulation and/or selectively binding to a desired target cell expressing LY6G6F, VSIG10, TMEM25 and/or LSR antigen, such target cells are, for example, melanoma, liver disorders, kidney disorders, brain disorders, breast disorders, colon disorders, lung disorders, ovarian disorders, pancreatic disorders, prostate disorders, stomach disorders, multiple myeloma, and hematopoietic cancers, including but not limited to lymphomas (hodgkins and non-hodgkins), acute and chronic lymphatic leukemias, and acute and chronic myeloid leukemias.

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

In certain embodiments of the methods of engineering antibodies according to at least some embodiments of the present invention, mutations may be introduced randomly or selectively along all or part of the anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25, or anti-LSR antibody coding sequence, and the resulting modified anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25, or anti-LSR antibodies may be screened for binding activity and/or other desired functional properties.

Methods of mutagenesis have been described in the art. For example, PCT publication WO 02/092780 to Short describes methods for generating and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, PCT publication WO 03/074679 to Lazar et al describes methods for optimizing physicochemical properties of antibodies using computational screening methods.

Nucleic acid molecules encoding antibodies

Another aspect of the invention relates to nucleic acid molecules encoding antibodies according to at least some embodiments of the invention. These nucleic acids may be present in whole cells, in cell lysates, or in partially purified or substantially pure form. Nucleic acids are "isolated" or "appear substantially pure" when purified from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) by standard techniques, including alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See, Osubel (F. Ausubel) et al, eds (1987) molecular biology laboratory Manual, Greene Publishing and Wiley Interscience (Willigerin Press across sciences), New York. Nucleic acids according to at least some embodiments of the invention may be, for example, DNA or RNA, and may or may not comprise intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

Nucleic acids according to at least some embodiments of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by a hybridoma (e.g., a hybridoma prepared from a transgenic mouse carrying human immunoglobulin genes as described further below), cdnas encoding the light and heavy chains of the antibody prepared from the hybridoma can be obtained by standard PCR amplification or cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), the nucleic acid encoding the antibody can be recovered from the library.

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

As used in this context, the term "operably linked" is intended to mean that the two DNA fragments are linked such that the amino acid sequences encoded by the two DNA fragments remain in frame.

Isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the DNA encoding the VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH 3). The sequence of the Human heavy chain constant region gene is known in the art (see, e.g., Kabat (Kabat, e.a.) et al (1991) Sequences of Proteins of Immunological Interest (Sequences of Proteins of Immunological Interest), Fifth Edition (Fifth Edition), U.S. department of Health and Human Services (U.S. department of Health and Human Services), NIH publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG1 or IgG4 constant region. For the Fab fragment heavy chain gene, the DNA encoding VH may be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted to a full-length light chain gene (along with the Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding a light chain constant region, CL. The sequence of the Human light chain constant region gene is known in the art (see, e.g., Kabat (Kabat, e.a.) et al (1991) Sequences of Proteins of Immunological Interest (Sequences of Proteins of Immunological Interest), Fifth Edition (Fifth Edition), U.S. department of Health and Human Services (U.S. department of Health and Human Services), NIH publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region, but is most preferably a kappa constant region.

To generate the scFv gene, the DNA fragment encoding VH and encoding VL is operably linked to another fragment encoding a flexible linker (e.g., encoding the amino acid sequence (Gly4-Ser)3) such that the VH and VL sequences can be expressed as a contiguous single-chain protein by which the VH and VL regions are linked (see, e.g., Border (Bird) et al (1988) Science 242: 423 + 426; Huston (Huston) et al (1988) Proc. Natl. Acad. Sci. USA 85: 5879 + 5883; McCafferty et al, (1990) Nature 348: 552 + 554).

Production of anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 or anti-LSR monoclonal antibodies

Monoclonal antibodies (mabs) of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodologies, such as Kohler (Kohler) and Milstein (Milstein) (1975) Nature 256: 495 by standard somatic hybridization technique. Although in principle a somatic hybridization procedure is preferred, other techniques for producing monoclonal antibodies may be employed, such as viral or oncogenic transformation of B lymphocytes.

One 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 immune splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

The chimeric antibody or humanized antibody of the present invention can be prepared based on the sequence of the murine monoclonal antibody prepared as described above. DNA encoding the heavy and light chain immunoglobulins can be obtained from a murine hybridoma of interest and can be engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to make chimeric antibodies, murine variable regions can be linked to human constant regions using methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 to cabbieuy (CabiUy) et al). To make humanized antibodies, murine CDR regions can be inserted into human frameworks using methods known in the art (see, e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762, and 6,180,370 to Queen et al).

According to at least some embodiments of the invention, the antibodies are human monoclonal antibodies. Such human monoclonal antibodies directed against LY6G6F, VSIG10, TMEM25 and/or LSR may be produced using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomal mice are referred to herein as HuMAb mice RTM and KM mice RTM, respectively, and are collectively referred to herein as "human Ig mice". HuMAb mouse TM (Metarex. Inc.) contains the human immunoglobulin gene minilocus (minioci) encoding the unrearranged human heavy (μ and γ) and kappa light chain immunoglobulin sequences, along with targeted mutations that inactivate the endogenous μ and kappa chain loci (see, e.g., Lanbolger (Lonberg) et al (1994) Nature 368 (6474): 856-859). Thus, these mice display reduced expression of mouse IgM or kappa and, in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgG kappa monoclonal antibodies (reviewed in Lonberg, N.) (1994), supra; review in Lonberg, N.) (1994) handbook of Experimental Pharmacology (Handbokof Experimental Pharmacology) 113: 49-101; Lonberg, N.) (Huszar, D.) (1995) International immunological review (Intern. Rev. Immunol.) -13: 65-93, and Harding, F.) and Lanbogerg (Lonberg, N.) (1995) Ann. Scien. 764.546.764). The preparation and use of HuMab mice RTM and the genomic modifications carried by such mice are further described in: taylor (Taylor, L.) et al (1992) Nucleic Acids Research (Nucleic Acids Research) 20: 6287-6295; chen (Chen, J.) et al (1993) International Immunology 5: 647-656; guan (Tuaiuon) et al (1993) Proc. Natl. Acad. Sci. USA 90: 3720-3724; catalyst (Choi) et al (1993) Nature Genetics (Nature Genetics) 4: 117-; chen (Chen, J.) et al (1993) journal of the European Association of molecular biology (EMBO J.) 12: 821-830; roman (Tuaiuon) et al (1994) journal of immunology 152: 2912-2920; taylor (Taylor, L.) et al (1994) International Immunology 6: 579-; and fisherwald (fisherworld, D.) et al (1996) Nature Biotechnology (Nature Biotechnology) 14: 845-851, the entire contents of which are hereby expressly incorporated by reference in their entirety. See further, U.S. Pat. nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, and 5,770,429, all of which are loberg (Lonberg) and ka (Kay); U.S. patent No. 5,545,807 to Surani (Surani), et al; PCT publications WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962 all belonging to lanborger (Lonberg) and ka (Kay); and Yuanbing (Korman) et al PCT publication No. WO 01/14424.

In another example, human antibodies according to at least some examples of the invention can be cultured using mice carrying human immunoglobulin sequences on transgenes and transchromosomes (e.g., mice carrying a human heavy chain transgene and a human light chain transchromosome). Such mice, referred to herein as "KM mice TM.", are described in detail in PCT publication WO 02/43478 to the stephania praecox (Ishida) et al.

Furthermore, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and may be used to culture anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR antibodies according to at least some embodiments of the present invention. For example, an alternative transgenic system known as Xenomouse (Abgenix corporation) may be used; such mice are described, for example, in U.S. Pat. nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963 to Kucherlapati (Kucherlapati) et al.

Furthermore, alternative transchromosomal animal systems expressing human immunoglobulin genes are available in the art and may be used to culture anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR antibodies according to at least some embodiments of the present invention. For example, what are referred to as "TC mice," mice carrying both human light chain transchromosomes and human heavy chain transchromosomes may be used; such mice are described in tsukau (Tomizuka) et al (2000) journal of the american college of sciences (proc.natl.acad sci.usa) 97: 722- > 727. Furthermore, cattle carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al (2002) Natural Biotechnology (Nature Biotechnology) 20: 889-894) and can be used to culture anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR antibodies according to at least some embodiments of the present invention.

Human monoclonal antibodies according to at least some embodiments of the invention can also be prepared using phage display methods that screen human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies are well established in the art. See, for example: U.S. Pat. nos. 5,223,409, 5,403,484, and 5,571,698 to Ladner et al; U.S. Pat. nos. 5,427,908 and 5,580,717 to dowier et al; maccafetti (McCafferty) et al, U.S. patent nos. 5,969,108 and 6,172,197; and U.S. patent nos. 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081 to Griffiths et al.

Human monoclonal antibodies according to at least some embodiments of the invention can also be prepared using SCID mice in which human immune cells have been reconstituted such that a human antibody response can be generated when immunized. Such mice are described, for example, in U.S. patent nos. 5,476,996 and 5,698,767 to Wilson et al.

Immunization of HUMAN IG MICE (HUMAN IG MICE)

When human Ig mice are used to culture human antibodies according to at least some embodiments of the invention, such mice may be immunized with purified or concentrated preparations of LY6G6F, VSIG10, TMEM25 and/or LSR antigens and/or recombinant LY6G6F, VSIG10, TMEM25 and/or LSR, or LY6G6F, VSIG10, TMEM25 and/or LSR fusion proteins as described below: lanbourg L (onberg, N.) et al, (1994) Nature 368 (6474): 856-859; fishwield (D.) et al, (1996) Nature Biotechnology (Nature Biotechnology) 14: 845-; and PCT publications WO 98/24884 and WO 01/14424. Preferably, at the time of the first infusion, these mice are 6-16 weeks old. For example, purified or recombinant preparations (5-50.mu.g) of LY6G6F, VSIG10, TMEM25 and/or LSR antigens may be used to immunize human Ig mice intraperitoneally.

Other previous experience with different antigens has shown that transgenic mice respond when first immunized Intraperitoneally (IP) with the antigen in complete freund's adjuvant, followed by IP immunization every other week (total of 6) with the antigen in complete freund's adjuvant. However, adjuvants other than Freund's adjuvant have been found to be effective. Furthermore, whole cells in the absence of adjuvant were found to be highly immunogenic. The immune response can be monitored during the course of an immunization protocol by plasma samples obtained by retroorbital bleeds. Plasma can be screened by ELISA (as described below), and mice with sufficient titers of anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25, and/or anti-LSR human immunoglobulin can be used for fusion. Mice can be boosted intravenously with antigen 3 days prior to sacrifice and removal of the spleen. It is expected that 2-3 fusions will be required for each immunization. Between 6 and 24 mice were typically immunized for each antigen. Two lines, HCo7 and HCo12, are commonly used. In addition, the two transgenes HCo7 and HCo12 can be bred together into a single mouse with two different human heavy chain transgenes (HCo7/HCo 12). Alternatively or additionally, KM mouse rtm. strain may be used.

Production of human monoclonal antibody-producing hybridomas

To generate hybridomas that produce human monoclonal antibodies according to at least some embodiments of the present invention, spleen cells and/or lymph node cells can be isolated from immunized mice and can be fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. These resulting hybridomas can be screened for the production of antigen-specific antibodies. For example, a single cell suspension of splenic lymphocytes from immunized mice can be fused with 50% PEG to one-sixth the number of P3X63-Ag8.653 non-secreting mouse myeloma cells (ATCC, CRL 1580). Cells were plated on flat-bottomed microtiter plates at approximately 2X10-5, followed by two weeks of incubation in selection medium containing: 20% fetal Clone Serum (total Clone Serum), 18% "653" conditioned medium, 5% origen (IGEN), 4mM L-glutamine, 1mM sodium pyruvate, 5mM HEPES, 0.055mM 2-mercaptoethanol, 50 units/ml penicillin, 50mg/ml streptomycin, 50mg/ml gentamicin, and 1 XHAT (Sigma, HAT added 24 hours after fusion). After about two weeks, the cells can be cultured in a medium in which HAT is replaced with HT. Individual wells can then be screened by ELISA for human monoclonal IgM and IgG antibodies. Once a large number of hybridoma growths have occurred, the media can be observed, usually after 10-14 days. Antibody secreting hybridomas can be replated, screened again, and if still positive for human IgG, these monoclonal antibodies can be subcloned at least twice by limiting dilution. The stable subclones can then be cultured in vitro to produce small amounts of antibody in the tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas were grown in two-liter roller bottles for purification of monoclonal antibodies. The supernatant may be filtered and concentrated prior to affinity chromatography on protein a-sepharose (Pharmacia), Piscataway, new jersey). To ensure purity, the eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography. The buffer solution can be changed to PBS and the concentration can be determined by OD280 using an extinction coefficient of 1.43. These monoclonal antibodies can be aliquoted at-80 ℃ and stored.

Generation of transfectomas producing monoclonal antibodies

Antibodies according to at least some embodiments of the invention can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art (e.g., Morrison, S.) (1985) Science 229: 1202).

For example, to express these antibodies, or antibody fragments thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using hybridomas expressing the antibodies of interest), and these DNAs can be inserted into expression vectors such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operatively linked" is intended to mean that the antibody gene is linked to a vector such that transcriptional and translational control sequences within the vector exert their intended functions of regulating transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene may be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., ligation of the antibody gene fragment and complementary restriction sites on the vector, or blunt-ended ligation if no restriction sites are present). The light and heavy chain variable regions of the antibodies described herein can be used to generate full length antibody genes of any antibody isotype by inserting them into expression vectors that already encode the heavy and light chain constant regions of the desired isotype, such that VH segments are operatively linked to CH segments within the vector and VK segments are operatively linked to CL segments within the vector. Additionally or alternatively, such a recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody from the host cell. The antibody chain gene may be cloned into the vector such that a signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a protein other than an immunoglobulin).

In addition to antibody chain genes, recombinant expression vectors according to at least some embodiments of the present invention carry regulatory sequences that control the expression of these antibody chain genes in host cells. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of these antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Goeddel) Gene expression technology: methods in Enzymology (Gene expression Technology: Methods in Enzymology)185, Academic Press (Academic Press), San Diego (San Diego), Calif. (1990). It will be understood by those of ordinary skill in the art that the design of the expression vector, including the choice of regulatory sequences, will depend on such factors as the choice of the host cell to be transformed, the level of expression of the desired protein, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from: cytomegalovirus (CMV), simian virus 40(SV40), adenoviruses (e.g., adenovirus major late promoter (AdMLP), and polyoma virus. alternatively, non-viral regulatory sequences such as ubiquitin protein promoter or beta globin promoter can be used, again, the regulatory elements consist of sequences from diverse sources, such as SR α, promoter systems comprising sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe, Y.) et al (1988) molecular cell biology (mol.cell.biol.) 8: 466-.

In addition to these antibody chain genes and regulatory sequences, recombinant expression vectors according to at least some embodiments of the invention may carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., an origin of replication) and a selectable marker gene. The selectable marker gene aids in the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all to Ackerel et al). For example, typically, a selectable marker gene confers resistance to a drug, such as G418, hygromycin or methotrexate, to a host cell into which the vector has been introduced. 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 expression of the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into the host cell by standard techniques. The term "transfection" in different forms is intended to encompass a wide variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is theoretically possible to express antibodies according to at least some embodiments of the invention in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, and most preferably mammalian host cells, is most preferred because such eukaryotic cells, and in particular mammalian cells, are easier to assemble and secrete properly folded and immunologically active antibodies than prokaryotic cells. Prokaryotic expression of antibody genes has been reported to be ineffective for producing high yields of active antibodies (Boss, M.A) and Wood (Wood, C.R.) (1985) Today's Immunology 6: 12-13).

Preferred mammalian host cells for expression of recombinant antibodies according to at least some embodiments of the invention include Chinese hamster ovary cells (CHO cells) (including DHFR-CHO cells as described in Urlaub and Chason (Chasin) for use with DHFR selectable markers such as those described in Koffman (R.J.Kaufman) and Sharp (P.A.Sharp) (1982) molecular biology 159: 601-621, (1980) Proc. Natl.Acad.Sci.USA 77: 4216-4220), NSO myeloma cells, COS cells, and SP2 cells. In particular, another preferred expression system for use with NSO myeloma cells is the GS gene expression system disclosed in WO87/04462, WO 89/01036 and EP 338,841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a time sufficient to allow expression of the antibody in the cells or, more preferably, to allow secretion of the antibody into the medium in which the host cells are grown. The antibody can be recovered from the culture medium using standard protein purification methods.

Characterization of antibodies binding to antigens

Antibodies according to at least some embodiments of the present invention may be tested for binding to LY6G6F, VSIG10, TMEM25 and/or LSR by standard ELISA. Briefly, microtiter plates were coated with purified LY6G6F, VSIG10, TMEM25, and/or LSR at 0.25. mu.g/ml in PBS, and then blocked with 5% bovine serum albumin in PBS. Dilutions of the antibody (e.g., dilutions of plasma from immunized mice) are added to each well and incubated for 1-2 hours at 37 ℃. These plates were washed with PBS/Tween and then incubated with a secondary reagent coupled to alkaline phosphatase (e.g., for human antibodies, a goat anti-human IgG Fc-specific polyclonal reagent) for 1 hour at 37 ℃. After washing, the plates were developed with pNPP substrate (1mg/ml) and analyzed at OD 405-650. Preferably, the mouse that showed the highest titer will be used for fusion.

Hybridomas showing positive reactivity with LY6G6F, VSIG10, TMEM25 and/or LSR immunogens can also be screened using an ELISA assay as described above. Hybridomas that bound to LY6G6F, VSIG10, TMEM25, and/or LSR with high affinity were subcloned and further characterized. One clone from each hybridoma that retains the reactivity of the maternal cells (by ELISA) can be selected for the preparation of a 5-10 vial cell bank stored at-140 ℃ and used for antibody purification.

To purify the anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR antibodies, selected hybridomas were grown in two liter roller bottles for purification of monoclonal antibodies. The supernatant may be filtered and concentrated prior to affinity chromatography on protein a-sepharose (Pharmacia), Piscataway, new jersey). To ensure purity, the eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography. The buffer solution can be changed to PBS and the concentration can be determined by OD280 using an extinction coefficient of 1.43. These monoclonal antibodies can be aliquoted at-80 ℃ and stored.

To determine whether selected anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR monoclonal antibodies bind to a unique epitope, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, I11). Competition studies using unlabeled and biotinylated monoclonal antibodies can be performed using LY6G6F, VSIG10, TMEM25 and/or LSR coated-ELISA plates as described above. Biotinylated mAb binding can be detected with a streptavidin basic phosphatase probe.

To determine the isotype of the purified antibody, an isotype ELISA may be performed using reagents specific for one particular isotype of antibody. For example, to determine the isotype of a human monoclonal antibody, the wells of a microtiter plate may be coated with 1 μ g/ml of anti-human immunoglobulin overnight at 4 ℃. After blocking with 1% BSA, the plates were reacted with 1 μ g/ml or less of the test monoclonal antibody or purified isotype control for one to two hours at ambient temperature. These wells can then be reacted with either human IgG1 or human IgM-specific alkaline phosphatase conjugated probes. The plates were developed and analyzed as described above.

anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR IgG can be further tested by western blotting for reactivity with LY6G6F, VSIG10, TMEM25 and/or LSR antigens, respectively. Briefly, LY6G6F, VSIG10, TMEM25 and/or LSR antigens may be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the isolated antigens were transferred to nitrocellulose filters, blocked with 10% fetal bovine serum, and probed with the monoclonal antibody to be tested. Human IgG binding can be detected using anti-human IgG alkaline phosphatase and visualized with a BCIP/NBT substrate tablet (Sigma chemical, inc. (Sigma chem. co.), st louis, missous).

Replacement support

According to at least some embodiments, the present invention relates to protein scaffolds having a specificity and affinity similar to the range of specific antibodies. According to at least some embodiments, the present invention relates to an antigen binding construct comprising a protein scaffold linked to one or more epitope binding domains. Such engineered protein scaffolds are typically obtained by designing a pool of random mutations focused on the loop regions or additional allowable surface regions and by selecting variants for a given target via phage display or related techniques. In accordance with at least some embodiments, the present invention relates to alternative stents, including, but not limited to: anticalin, DARPin, Armadillo repeat proteins (Armadillo repeat proteins), protein a, lipocalin, fibronectin domains, ankyrin consensus repeat domains (ankyrin consensus repeats domains), thioredoxin, chemically constrained peptides (chemically constrained peptides), and the like. According to at least some embodiments, the present invention relates to alternative scaffolds that can be used as therapeutic agents for the treatment of cancer, autoimmunity, and infectious diseases, as well as for in vivo diagnostics.

According to at least some embodiments, the present invention further provides a pharmaceutical composition comprising an antigen binding construct as described herein, a pharmaceutically acceptable carrier.

As used herein, the term "protein scaffold" includes, but is not limited to, immunoglobulin (Ig) scaffolds, such as IgG scaffolds, which may be four-chain or double-chain antibodies, or may comprise only the Fc region of an antibody, or may comprise one or more constant regions from an antibody (which constant regions may be of human or primate origin), or may be a human and primate artificial chimera of constant regions. Such protein scaffolds may include an antigen binding site in addition to one or more constant regions, for example where the protein scaffold includes an intact IgG. Such protein scaffolds can be linked to other protein domains, for example protein domains with antigen binding sites, such as epitope binding domains or ScFv domains.

A "domain" is a folded protein structure having a tertiary structure that is independent of the rest of the protein. In general, domains are responsible for discrete functional properties of a protein and, in many cases, can be added to, removed from, or transferred to other proteins without losing the remainder of the protein and/or the function of the domain. A "single antibody variable domain" is a folded polypeptide domain that includes the sequence features of an antibody variable domain. Thus, it includes full antibody variable domains and modified variable domains, e.g., antibody variable domains in which one or more loops have been replaced by sequences that are not characteristic of the antibody variable domain, or that have been truncated or include N-terminal or C-terminal extensions, along with folded fragments of the variable domain that retain at least the binding activity and specificity of the full-length domain.

The term "immunoglobulin single variable domain" refers to an antibody variable domain (VH, V HH, V L) that specifically binds to an antigen or epitope independently of different V regions or domains. An immunoglobulin single variable domain can exist together with other different variable regions or variable domains (e.g., homo-or hetero-multimers), wherein the single immunoglobulin variable domain does not require these other regions or domains to bind antigen (i.e., wherein the immunoglobulin single variable domain binds antigen independently of additional variable domains). A "domain antibody" or "dAb" is identical to an "immunoglobulin single variable domain" that is capable of binding antigen, as used herein. The immunoglobulin single variable domain may be a human antibody variable domain, but also includes single antibody variable domains from other species, such as rodents (e.g. as disclosed in WO 00/29004), hinged sharks and camelids vhh dabs. Camelid vhh is an immunoglobulin single variable domain polypeptide derived from a heavy chain antibody which naturally lacks a light chain, including the following species: camels, llamas, alpacas, dromedary camels, and guanacos. Such V HH domains may be humanized according to standard techniques available in the art, and such domains are still considered "domain antibodies" according to the invention. As used herein, "VH" includes camelid V HH domains. NARV is another class of immunoglobulin single variable domains identified in cartilaginous fish species, including the game shark. These domains are also known as Novel Antigen Receptor (Novel Antigen Receptor) variable domains (often abbreviated as v (nar) or NARV). For further details, see molecular immunology 44, 656-665(2006) and US 20050043519 a.

The term "epitope binding domain" refers to a domain that specifically binds to an antigen or epitope independently of a different V region or domain, which may be a domain antibody (dAb) (e.g. a human, camelid or shark immunoglobulin single variable domain) or it may be a domain that is a derivative of a scaffold selected from the group consisting of: CTLA-4 (Evibody); a lipocalin; protein A derived molecules, such as the Z-domain (Affinibody, SpA), A-domain (Avimer/Maxibody) of protein A; heat shock proteins such as GroEI and GroES; transferase (trans-body); ankyrin repeat protein (DARPin); a peptide aptamer; a C-lectin like domain (tetranectin); human & # 947-crystallin and human ubiquitin protein (affilins); a PDZ domain; the scorpion toxin kunitz-type domain of a human protease inhibitor; armadillo repeat sequence proteins, thioredoxin, and fibronectin (adnectin); the domain has been subjected to protein engineering to obtain binding to ligands other than the natural ligand.

The loops corresponding to the CDRs of the antibody may be substituted with heterologous sequences to confer different binding properties, i.e., evibodes. For further details, see Journal of Immunological Methods 248(1-2), 31-45 (2001). Lipocalins are a family of extracellular proteins that transport hydrophobic small molecules (e.g., steroids, bile pigments, retinoids, and lipids). They have a rigid secondary structure with multiple loops at the open end of a conical structure, which can be engineered to bind to different target antigens. Anticalin is between 160-180 amino acids in size and is derived from lipocalins. For further details, see journal of biochemistry and biophysics (Biochim Biophyacta) 1482: 337-350(2000), US 7250297B 1 and US 20070224633. affibody is a scaffold of protein a from staphylococcus aureus that can be engineered to bind antigen. This domain consists of a triple helix bundle of about 58 amino acids. Libraries have been generated by randomization of surface residues. For further details, see Protein engineering and selection (Protein Eng. Des. sel.)17, 455-462(2004) and EP 1641818A 1. Avimer is a multi-domain protein derived from the A-domain scaffold family. The natural domain of about 35 amino acids assumes a defined disulfide-bonded structure. Diversity is generated by shuffling of natural variations displayed by the a-domain family. For further details, see Nature Biotechnology 23(12), 1556-. Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered to bind to unused target antigens by inserting peptide sequences into the free surface loops. Examples of engineered transferrin scaffolds include the Trans-body. For further details, see journal of biochemistry (J.biol.chem)274, 24066-24073 (1999).

Ankyrin repeat proteins (darpins) were designed from ankyrin, a family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. The single ankyrin repeat is a repeat consisting of two alpha helices; a 33-residue motif consisting of β -turns. They can be engineered by randomizing residues in the first alpha-helix and beta-turn of each repeat sequence to bind different target antigens. The binding interface can be increased by increasing the number of modules (method of affinity maturation). For further details, see journal of molecular biology (J.MoT Biol.)332, 489-503(2003), PNAS100(4), 1700-1705(2003) and journal of molecular biology (J.MoT Biol.)369, 1015-1028(2007) and US 20040132028A 1.

Fibronectin is a scaffold that can be engineered to bind antigens. Adnectin consists of a 15 repeat unit backbone of the native amino acid sequence of domain 10 of human type III fibronectin (FN 3). The three loops at one end of the β -sandwich may be engineered to enable the Adnectin to specifically recognize a therapeutic target of interest. For further details, see Protein engineering design and selection (Protein Eng. Des. sel.)18, 435-444(2005), US20080139791, WO 2005056764, and US 6818418B 1.

Peptide aptamers are combinatorial recognition molecules consisting of a constant scaffold protein, typically thioredoxin (TrxA), which contains a constrained variable peptide loop inserted at the active site. For further details, see Expert opinion on biotherapy (Expert opin in biol. ther.)5.783-797 (2005).

Microbodies are derived from naturally occurring microproteins of 25-50 amino acids in length, examples of which include 3-4 cysteine bridges-microproteins include KalataBI and Konoxin (cono toxin) and knottin. These miniproteins have a loop that can be engineered to include up to 25 amino acids without affecting the overall folding of the miniprotein. For further details of engineering knottin domains see WO 2008098796.

Other epitopes of the binding domain include proteins that have been used as scaffolds to engineer different target antigen binding properties, including human & # 947; beta-crystallin and human ubiquitin (affilin), kunitz-type domain of human protease inhibitor, PDZ-domain of Ras-binding Protein AF-6, scorpion toxin (charybdotoxin), C-type lectin domain (tetranectin), reviewed in chapter 7-Non-antibody scaffolds from Handbook of Therapeutic Antibodies (Non-antibody scans affolds of Therapeutic Antibodies) (2007, edited by stevens debel) and "Protein Science" 15: 14-27 (Protein Science 2006.) the epitope binding domain of the invention may be derived from any of these alternative Protein domains.

Conjugates or immunoconjugates

The invention encompasses conjugates for use in immunotherapy comprising LY6G6F, VSIG10, TMEM25 and/or LSR antigens and soluble portions thereof including extracellular domains or portions or variants thereof. For example, the invention encompasses conjugates wherein the ECD of LY6G6F, VSIG10, TMEM25 and/or LSR antigen is attached to an immunoglobulin or fragment thereof. The present invention contemplates their use for promoting or inhibiting LY6G6F, VSIG10, TMEM25 and/or LSR antigenic activity (e.g., immune co-stimulation) and their use in the treatment of transplantation, autoimmunity, and cancer indications as described herein.

In another aspect, the invention features antibody-drug conjugates consisting of an antibody (or antibody fragment, e.g., single chain variable fragment) linked to a payload drug (typically cytotoxic) for use in, e.g., the treatment of cancer. The antibody causes the ADC to bind to the target cancer cell. Typically the ADC is then internalized by the cell and the drug is released into the cell. Due to the targeting, side effects are reduced and a wider therapeutic window is given. Hydrophilic linkers (e.g., PEG4Mal) help prevent the drug from being pumped out by resistant cancer cells through MDR (multidrug resistance) transporters. Although ADC based cleavable linkers are considered to have a more unfavorable therapeutic window, targets that are not efficiently internalized (tumor cell surface antigens) appear to be more suitable as cleavable linkers.

In another aspect, the invention features immunoconjugates that include an anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 or anti-LSR antibody, or fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, drug (e.g., immunosuppressant) or radiotoxin. Such conjugates are referred to herein as "immunoconjugates". Immunoconjugates comprising one or more cytotoxins are referred to as "immunotoxins". The cytotoxin or cytotoxic agent includes any agent that is harmful (e.g., kills) on the cell. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthrax dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, proparacaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil aminoenamine), alkylating agents (e.g., mechlorethamine, thiopea, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozocin, mitomycin C, and cis-dichlorodiammineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and Atramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that may be conjugated to antibodies according to at least some embodiments of the present invention include duocarmycin, calicheamicin, maytansine and auristatin, and derivatives thereof. An example of a calicheamicin antibody conjugate is commercially available (Mylotarg. TM.; Whitman pharmaceutical factory (Wyeth)).

Cytotoxins may be conjugated to antibodies according to at least some embodiments of the present invention using linker technology available in the art. Examples of types of linkers that have been used to couple cytotoxins to antibodies include, but are not limited to: hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. Linkers can be selected that are, for example, susceptible to cleavage by low pH within lysosomal compartments or by proteases, such as proteases preferentially expressed in tumor tissue, e.g., cathepsins (e.g., cathepsin B, C, D).

To further discuss the type of cytotoxin, linkers and methods for coupling therapeutic agents to antibodies are also described in ziteng (Saito, G.) et al (2003) review on advanced drug delivery (adv. drug deliv. rev.) 55: 199-; treel (Trail, p.a.) et al (2003) Cancer immunology, immunotherapy 52: 328-337; payne (Payne, G.) (2003) "Cancer cells" (Cancer Cell) 3: 207-212; allen (Allen, t.m.) (2002) natural reviews of cancer (nat. rev. cancer) 2: 750- > 763; pascal (patan, I.) and kraettman (Kreitman, R.J.) (2002) research drugs novelts (curr. opin. investig. drugs) 3: 1089-; senter (Senter, P.D.) and Springger (Springer, C.J.) (2001) review on advanced drug delivery (adv. drug Deliv. Rev.) 53: 247-264.

The antibodies of the invention may also be conjugated to radioisotopes to produce cytotoxic radiopharmaceuticals, also known as radioimmunoconjugates. Examples of 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. Examples of radioimmunoconjugates are commercially available, including Zevalin (IDEC pharmaceuticals) and Bexxar (Corixa pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using antibodies according to at least some embodiments of the present invention.

Antibody conjugates according to at least some embodiments of the invention may be used to modify a given biological response, and the drug moiety is not to be understood as being limited to classical chemotherapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, enzymatically active toxins or active fragments thereof, such as abrin, ricin a, pseudomonas exotoxin, or diphtheria toxin; a protein, such as tumor necrosis factor or interferon-gamma; or, biological response modifiers, such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

Techniques For coupling such therapeutic moieties to Antibodies are well known, see, For example, alunn et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy" ("Monoclonal Antibodies For Immunotargeting Drugs In Cancer Therapy"), reisfield et al (ed.), pp.243-56 (allen R leiss, Inc.)1985) In Monoclonal Antibodies And Cancer Therapy "; hersterlon (Hellstrom) et al, "Antibodies For Drug Delivery" ("Antibodies For Drug Delivery"), Robinson et al (eds.), pp.623-53 (Marcel Dekker, Inc.)1987, in Controlled Drug Delivery (second edition); monoclonal antibody' 84: biological And Clinical Applications Soppe (Thorpe) In Monochromatic antibodies' 84: Biological And Clinical Applications, "antibodies Of Cytotoxic Agents In Cancer Therapy: a Review "(" antibody carrier for cytotoxic agents in tumor therapy: Review "), Pinkhara (Pincher) et al (ed.), pp.475-506 (1985); "Analysis, Results, And Future developments Of Therapeutic uses Of Radiolabeled Antibodies In Cancer Therapy" ("Analysis, Results, And Future developments Of Radiolabeled Antibodies In Monoclonal Antibodies For Cancer Detection And Therapy"), Boldwen et al (eds.), pp.303-16(academic Press) 1985), And Sorpet al, "The Preparation Of Antibody-Toxin Conjugates And cytotoxic properties" ("Immunol 62"). 119-58(1982).

Bispecific molecules

In another aspect, the invention features bispecific molecules that include anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR antibodies, or fragments thereof, according to at least some embodiments of the invention. An antibody, or antigen-binding portion thereof, according to at least some embodiments of the invention can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand of a receptor), to produce a bispecific molecule that binds to at least two different binding sites or target molecules. Antibodies according to at least some embodiments of the invention may also be actually derivatized or linked to one other functional molecule to produce multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To produce a bispecific molecule according to at least some embodiments of the invention, an antibody may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other binding molecules, e.g., another antibody, antibody fragment, peptide, or binding mimetic, such that a bispecific molecule is produced.

Accordingly, the present invention includes bispecific molecules comprising at least one first binding specificity for LY6G6F, VSIG10, TMEM25 and/or LSR and a second binding specificity for a second targeting epitope. According to at least some embodiments of the invention, such a second targeting epitope is an Fc receptor, such as a human fcyri (CD64) or a human fca receptor (CD 89). Accordingly, the invention includes bispecific molecules capable of binding to both Fc γ R, Fc ar or FcR expressing effector cells (e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)), and to target cells expressing LY6G6F, VSIG10, TMEM25 and/or LSR, respectively. These bispecific molecules target LY6G6F, VSIG10, TMEM25 and/or LSR expressing cells to effector cells and trigger Fc receptor mediated effector cell activity, such as phagocytosis, antibody dependent cell mediated cytotoxicity (ADCC), cytokine release, or production of superoxide anion of LY6G6F, VSIG10, TMEM25 and/or LSR expressing cells.

According to at least some embodiments of the invention, wherein the bispecific molecule is multispecific, the molecule may further comprise a third binding specificity in addition to the anti-Fc binding specificity and the anti-6 f binding specificity. In one embodiment, the third binding specificity is an anti-Enhancer Factor (EF) protein, e.g., a molecule that binds to a surface protein involved in cytotoxic activity and thereby increases the immune response to the target cell.

The term "anti-enhancer moiety" can be an antibody, functional antibody fragment or ligand that binds to a given molecule (e.g., antigen or receptor) and thereby results in an enhancement of the effect of the binding determinant of the Fc receptor or target cell antigen. The "anti-enhancer moiety" may bind to an Fc receptor or a target cell antigen. Alternatively, the anti-enhancer moiety may be bound to an entity that is different from the entity to which the first and second binding specificities bind. For example, the anti-enhancer element moiety can bind to cytotoxic T-cells (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1, or other immune cells that result in an increased immune response against the target cell).

According to at least some embodiments of the invention, the bispecific molecules comprise as binding specificity at least one antibody, or antibody fragment thereof, including Fab, Fab ', F (ab') 2, Fv, or single chain Fv. Such antibodies may also be light or heavy chain dimers, or any minimal fragment thereof, such as Fv or single chain constructs as described in U.S. Pat. No. 4,946,778 to Ladner et al, the contents of which are specifically incorporated by reference.

In one embodiment, the binding specificity for the Fey receptor is provided by a monoclonal antibody, which binding is not blocked by human immunoglobulin g (igg). As used herein, the term "IgG receptor" refers to any of the eight gamma-chain genes located on chromosome 1. These genes encode a total of twelve transmembrane or soluble receptor isoforms, which are divided into three Fc γ receptor classes: fc γ Rl (CD64), Fc γ RII (CD32), and Fc γ RIII (CD 16). In a preferred embodiment, the Fc γ receptor is a human high affinity Fc γ RI. Human Fc γ RI is a 72kDa molecule showing high affinity for monomeric IgG (108-10-9 m. -1).

The production and characterization of certain preferred anti-Fc γ monoclonal antibodies is described in PCT publication WO 88/00052 to Fanger et al and in U.S. patent No. 4,954,617, the teachings of which are incorporated herein by reference in their entirety. These antibodies bind to an epitope of fcyrl, FcyRII or FcyRIII at a site different from the Fc γ binding site of the receptor, and thus, their binding is not substantially blocked by physiological levels of IgG. Specific anti-Fc γ Rl antibodies useful in the present invention are mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. The hybridoma producing mAb 32 is available from the american germplasm preservation center, ATCC accession No. HB 9469. In other embodiments, the anti-Fcy receptor antibody is a humanized form of monoclonal antibody 22 (H22). Production and characterization of the H22 antibody is described in genino (Graziano, r.f.) et al (1995) journal of immunology (j.immunol.)155 (10): 4996 and PCT publication WO 94/10332. The cell line producing the H22 antibody was deposited under the name HA022CLI of the american germplasm preservation center and HAs accession number CRL 11177.

In still other preferred embodiments, the binding specificity for an Fc receptor is provided by an antibody that binds to a human IgA receptor (e.g., Fc-alpha receptor (Fc α RI (CD89))), the binding of which is preferably not blocked by human immunoglobulin a (IgA). The term "IgA receptor" is intended to include the gene product of the α -gene (Fc α RI) located on chromosome 19. This gene is known to encode several alternatively spliced transmembrane isoforms of 55kDa to 10 kDa.

Fc α RI (CD89) is constitutively expressed on monocytes/macrophages, eosinophils, and neutrophils, but not on non-effector cell populations. Fc α RI has a moderate affinity (about 5X 10-7M-l) for both IgA1 and IgA2, which increases when exposed to cytokines such as G-CSF or GM-CSF (Morton, H.C.) et al (1996) Critical Reviews in Immunology 16: 423-440). Four Fc α RI-specific monoclonal antibodies that bind Fc α RI outside the IgA ligand binding domain, identified as A3, a59, a62, and a77, have been described (Monteiro, r.c.) et al (1992) journal of immunology 148: 1764.

Fc α RI and Fc γ RI are preferred trigger receptors for use in bispecific molecules according to at least some embodiments of the present invention because they (1) are expressed predominantly in immune effector cells, e.g., monocytes, PMNs, macrophages, and dendritic cells; (2) at high levels (e.g., 5,000-100,000/cell); (3) are mediators of cytotoxic activity (e.g., ADCC, phagocytosis); (4) mediate enhanced antigen presentation of antigens, including targeting their self-antigens.

Although human monoclonal antibodies are preferred, other antibodies that may be employed in bispecific molecules according to at least some embodiments of the present invention are murine, chimeric, and humanized monoclonal antibodies.

Bispecific molecules of the invention can be prepared by coupling component binding specificities, e.g., anti-FcR and anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR binding specificities, using methods known in the art. For example, each binding specificity of a bispecific molecule can be generated separately and then coupled to each other. When the binding specificity is a protein or peptide, a variety of coupling or crosslinking agents can be used for covalent conjugation. Examples of crosslinking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5' -dithiobis (2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3- (2-pyridyl-disulfide) propionate (SPDP), and thiosuccinimide 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (thio-SMCC) (see, e.g., Capofsky (Karpovsky) et al (1984) J.Immunol.Med.160: 1686; Liu (Liu, M A) et al (1985) Proc.Natl.Acad.Sci.USA) 82: 8648). Other methods include Pulu (Paulus) (1985) Berlin institute communications (Behring ins. Mitt.) No.78, 118-; brennan et al (1985) Science 229: 81-83), and Geranii (Glennie) et al (1987) J.Immunol 139: 2367-. Preferred coupling agents are SATA and thio-SMCC, both available from Pierce Chemical Co (rockford, I11).

When the binding specificities are antibodies, they may be coupled via thiol bonding of the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of thiol residues, preferably one, prior to conjugation.

Alternatively, both binding specificities may be encoded in the same vector and expressed and assembled in the same host cell. This approach is particularly useful when the bispecific molecule is a mAbXmAb, mAbXFab, fabxfa (ab') 2, or ligand XFab fusion protein. A bispecific molecule according to at least some embodiments of the present invention may be a single chain molecule comprising a single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific molecules may comprise at least two single chain molecules. Methods for making bispecific molecules are described, for example, in U.S. Pat. nos. 5,260,203; U.S. patent nos. 5,455,030; U.S. patent nos. 4,881,175; U.S. Pat. nos. 5,132,405; U.S. Pat. nos. 5,091,513; U.S. patent nos. 5,476,786; U.S. patent nos. 5,013,653; U.S. Pat. nos. 5,258,498; and U.S. patent No. 5,482,858.

Binding of these bispecific molecules to their specific targets can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (IA), FACS analysis, bioassay (e.g., growth inhibition), or western blot assay. Each of these assays generally detects the presence of a protein-antibody complex of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, the FcR-antibody complex can be detected using, for example, an enzymatically linked antibody or antibody fragment that recognizes and specifically binds to the antibody-FcR complex. Alternatively, a variety of other immunoassays may be used to detect these complexes. For example, the antibody can be radiolabeled and used in Radioimmunoassays (RIA) (see, e.g., wenttaub (Weintraub, B.), Principles of radioimmunoassays, seven Training courts on radioligand Assay technology, the endocrine Society, 3 months 1986, which is incorporated herein by reference). The detection of the radioisotope may be performed by such means as the use of a gamma counter or scintillation counter or by autoradiography.

Protein modification

Fusion proteins

According to at least some embodiments, the LY6G6F, VSIG10, TMEM25 and/or LSR fusion polypeptide has a first fusion partner comprising all or a portion of LY6G6F, VSIG10, TMEM25 and/or LSR proteins fused to a second polypeptide, either directly or via a linker peptide sequence or chemical linker useful for linking the two proteins. LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides can optionally be fused to a second polypeptide to form fusion proteins as described herein. The presence of this second polypeptide can alter the solubility, stability, affinity, and/or valency of LY6G6F, VSIG10, TMEM25, and/or LSR fusion polypeptides. As used herein, "valency" refers to the number of binding sites available per molecule. In one embodiment, the second polypeptide is a polypeptide from a different source or a different protein.

In accordance with at least some embodiments, LY6G6F, VSIG10, TMEM25 and/or LSR protein or fragment is selected for its activity for the treatment of immune and/or infectious disorders, and/or cancers as described herein.

In one embodiment, the second polypeptide comprises one or more domains of an immunoglobulin heavy chain constant region, preferably having an amino acid sequence corresponding to the hinge of the human immunoglobulin C γ 1, C γ 2, C γ 3 or C γ 4 chain, the CH2 and CH3 regions or the hinge corresponding to the C γ 2a chain of a murine immunoglobulin, the CH2 and CH3 regions. SEQ ID NO: exemplary sequences of the hinge, CH2, and CH3 regions of human immunoglobulin C γ 1 are provided at 70.

According to at least some embodiments, the fusion protein is a dimeric fusion protein. In an optional dimeric fusion protein, the dimer results from covalent bonding of Cys residues in the hinge region of two Ig heavy chains, which are the same Cys residues in a normal Ig heavy chain that dimerizes, which are disulfide-linked. Such proteins are referred to as LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides, or fragments or fusion proteins thereof.

In one embodiment, the immunoglobulin constant domain may comprise one or more amino acid insertions, deletions or substitutions that enhance binding to a particular cell type, increase its bioavailability, or increase its stability of LY6G6F, VSIG10, TMEM25, and/or LSR polypeptides, fusion proteins or fragments thereof. Suitable amino acid substitutions include conservative as well as non-conservative substitutions as described above.

These fusion proteins optionally comprise a domain that functions to dimerize or multimerize two or more fusion proteins. The peptide/polypeptide linker domain may be a separate domain or, alternatively, may be comprised within one of the other domains of the fusion protein (LY6G6F, VSIG10, TMEM25 and/or LSR polypeptide or second polypeptide). Similarly, the domain that functions to dimerize or multimerize the fusion protein may be a separate domain, or alternatively, may be contained within one of the other domains of the fusion protein (LY6G6F, VSIG10, TMEM25 and/or LSR polypeptide, second polypeptide or peptide/polypeptide linker domain). In one embodiment, the dimerization/multimerization domain and the peptide/polypeptide linker domain are the same. Further specific, illustrative and non-limiting examples of dimerization/multimerization domains and linkers are set forth below.

According to at least some embodiments of the invention, the fusion proteins disclosed herein have formula I: N-R1-R2-R3-C, wherein "N" represents the N-terminus and "C" represents the C-terminus of the fusion protein. In further embodiments, "R1" is LY6G6F, VSIG10, TMEM25, and/or LSR polypeptide, "R2" is an optional peptide/polypeptide or chemical linker domain, and "R3" is a second polypeptide. Alternatively, R3 may be LY6G6F, VSIG10, TMEM25 and/or LSR polypeptide and R1 may be a second polypeptide. Various non-limiting examples of linkers are described in more detail below.

Optionally, the fusion protein comprises LY6G6F, VSIG10, TMEM25, and/or LSR polypeptide fragments as described herein, which may be fused to one or more "half-life extending moieties" optionally via a linker peptide of one or more amino acids (e.g., GS). A "half-life extending moiety" is any moiety that, when attached to a protein, extends the in vivo half-life of the protein in a subject's body (e.g., in the subject's plasma), e.g., a polypeptide, small molecule, or polymer, e.g., a half-life extending moiety is polyethylene glycol (PEG), monomethoxypeg (mpeg), or immunoglobulin (Ig) in one embodiment of the invention. In one embodiment of the invention, the PEG is a 5, 10, 12, 20, 30, 40, or 50kDa moiety or greater or comprises about 12000 ethylene glycol units (PEG 12000).

Fusion proteins can also optionally be prepared by chemical synthesis methods and "linked" chemically in synthetic or post-synthetic processes. Cross-linking and other such methods (optionally also using the gene-level fusion methods described above) can optionally be used, as described, for example, in U.S. patent No. 5,547,853 to Wallner et al, which is incorporated herein by reference as a non-limiting example only, to the extent it is fully set forth herein.

According to the present invention, a fusion protein can be prepared by fusing the protein of the present invention with a portion of an immunoglobulin comprising a constant region of one immunoglobulin. More preferably, the portion of the immunoglobulin comprises a heavy chain constant region, 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 optionally and most preferably an Fc chain, most preferably an IgG Fc fragment comprising the hinge, CH2, and CH3 domains. The Fc chain can optionally be a known or "wild-type" Fc chain, or alternatively can be mutated or truncated. The Fc portion of the fusion protein may optionally differ by isotype or subclass, may be chimeric or hybrid, and/or may be modified to, for example, improve effector function, control half-life, tissue accessibility, enhance biophysical properties such as stability, and improve production efficiency (and at a lower cost). Many modifications useful in constructing the disclosed fusion proteins and methods for making them are known in the art, see, e.g., muller (Mueller), et al, molecular immunology, 34 (6): 441-: 493-499(2008), and Presta (Presta), "Current opinion in immunology" (Cur. Opin. Immun.) 20: 460-470(2008). In some embodiments, the Fc region is a native IgG1, IgG2, or IgG4 Fc region. In some embodiments, the Fc region is a hybrid, e.g., a chimera consisting of an IgG2/IgG4 Fc constant region.

Modifications of the Fc region include, but are not limited to: IgG4 was modified to prevent binding to Fc γ receptors and complement, IgG1 was modified to improve binding to one or more Fc γ receptors, IgG1 was modified to minimize effector function (amino acid changes), IgG1 with altered/no polysaccharides (typically by altering the expression host or Asn at substitution position 297), and IgG1 with altered pH-dependent binding to FcRn. The Fc region may include the entire hinge region, or less than the entire hinge region.

In another embodiment, the Fc domain may comprise one or more amino acid insertions, deletions, or substitutions that reduce binding to the low affinity inhibitory Fc receptor CD32B (fcyriib) and maintain wild-type levels of binding to or enhance binding to the low affinity activating Fc receptor CD16A (fcyriiia).

Another example includes IgG2-4 hybrids and IgG4 mutants that have reduced binding to FcR (Fc receptor) thereby increasing half-life. Representative IgG2-4 hybrids and IgG4 mutants are described in Angal (Angal, S.) et al, Molecular Immunology, 30 (1): 105-108 (1993); muller (Mueller, J.) et al, Molecular Immunology, 34 (6): 441-452 (1997); and king (Wang) et al, U.S. patent No. 6,982,323. In some embodiments, the IgG1 and/or IgG2 domain is deleted; for example, Angal et al describe IgG1 and IgG2 with a proline to serine 241.

In further embodiments, the Fc domain comprises an amino acid insertion, deletion, or substitution that enhances binding to CD 16A. Numerous substitutions that increase binding to CD16A and decrease binding to CD32B within the Fc domain of human IgG1 are known in the art and described in Stavenhagen et al, Cancer research (Cancer Res.), 57 (18): 8882-90 (2007). Exemplary variants of the human IgG1 Fc domain with reduced binding to CD32B and/or increased binding to CD16A comprise F243L, R929P, Y300L, V305I, or P296L substitutions. These amino acid substitutions may be present in any combination in the human IgG1 Fc domain.

In one embodiment, the human IgG1 Fc domain variant comprises F243L, R929P, and Y300L substitutions. In another embodiment, the human IgG1 Fc domain variant comprises F243L, R929P, Y300L, V305I, and P296L substitutions. In another embodiment, the human IgG1 Fc domain variant comprises an N297A/Q substitution, as these mutations abrogate Fc γ R binding. A non-limiting, illustrative, exemplary type of mutation is described in U.S. patent application No. 20060034852, published 2006, 2, 16, which is hereby incorporated by reference to the same extent as if fully set forth herein. The term "Fc chain" may also optionally include any type of Fc fragment.

Several specific amino acid residues have been identified that are important for antibody constant region-mediated activity in IgG subclasses. Thus, the inclusion, substitution or exclusion of these specific amino acids allows the inclusion or exclusion of specific immunoglobulin constant region-mediated activities. In addition, specific changes may result in, for example, deglycosylation and/or other desired changes in the Fc chain. At least some changes may optionally be made in order to block Fc function that is considered undesirable, such as undesirable immune system effects, as described in more detail below.

Non-limiting, illustrative examples of changes that can be mutated to Fc to modulate the activity of the fusion protein include the following changes (given with respect to Fc sequence nomenclature as given by Kabat (Kabat), from Kabat et al: "Sequences of Proteins of Immunological Interest" (Sequences of Proteins of Immunological Interest), USdepartment of Health and Human Services (U.S. department of Health and Human Services), NIH, 1991): 220C- > S; 233-238ELLGGP- > EAEGAP; 265D- > A, preferably in combination with 434N- > A; 297N- > A (e.g., to block N-glycosylation); 318 and 322 EYKCK- > AYACA; 330-331AP- > SS; or combinations thereof (see, e.g., Clark (M.Clark), "Chemical Immunol and Antibody Engineering", pp 1-31 for an explanation of these mutations and their effects). Constructs featuring Fc chains of the above variations optionally and preferably include a hinge region in combination with CH2 and CH3 domains.

The above mutations may optionally be implemented in order to enhance a desired property or alternatively to block an undesired property. For example, deglycosylation of antibodies has been shown to maintain desirable binding functionality while blocking depletion of T-cells or triggering cytokine release, which may optionally be undesirable (see m.clark ("chemical immunology and Antibody Engineering"), pp 1-31). Substitution of 331 proline to serine can block the ability to activate complement, which can optionally be considered an undesirable function (see m.clark, "Chemical Immunol and Antibody Engineering," pp 1-31). Combining this change to change 330 alanine to serine can also enhance the desired effect of blocking the ability to activate complement.

Residues 235 and 237 have been shown to be involved in antibody-dependent cell-mediated cytotoxicity (ADCC) such that altering the 233-238 residue block as described can also block this activity if ADCC is considered an undesirable function.

Residue 220 is typically a cysteine from the Fc of IgG1, which is the site where the heavy chain forms a covalent bond with the light chain. Optionally, this residue can be changed to another amino acid residue (e.g., serine) to avoid any type of covalent bond (see clark (m. clark), "Chemical Immunol and Antibody Engineering", pp 1-31) or by deletion or truncation.

The above changes at residues 265 and 434 can optionally be performed in order to reduce or block Binding to Fc Receptors, which can optionally block the undesired functions of Fc associated with its immune system function (see "Binding site on human IgG1 for Fc Receptors", "Binding site for Fc Receptors on human IgG 1", hilz (Shields) et al, Vol276, pp 6591-6604, 2001).

The above variations are merely illustrative of optional variations and are not intended to be limiting in any way. Furthermore, the above explanations are provided for descriptive purposes only and are not intended to be limited by a single hypothesis.

In a further embodiment, the fusion protein comprises the extracellular domain of LY6G6F, or a fragment thereof, fused to an Ig Fc region. Recombinant IgLY6G6F polypeptide, fragments thereof or fusion proteins, which can be prepared by fusing the coding region of the extracellular domain of LY6G6F or fragments thereof to the Fc region of human IgG1 or mouse IgG2a as described previously (Chapoval et al, Methods of molecular pharmaceuticals (Methods MoT Med), 45: 247-255 (2000)).

Optionally, LY6G6F ECD also refers to a fusion protein that includes the amino acid sequence of human LY6G6F ECD fused to a human immunoglobulin Fc. Optionally, the fusion protein comprises a polypeptide fused to any one of SEQ ID NOs: 70. 156 of human IgG1 Fc SEQ ID NO: 2, the amino acid sequence of human LY6G6F ECD. Optionally, the amino acid sequence of the fusion protein is given in SEQ ID NO: 71 or SEQ ID NO: 172.

In a further embodiment, the fusion protein comprises an extracellular domain of VSIG10, or a fragment thereof, fused to an Ig Fc region. Recombinant IgVSIG10 polypeptide, fragments thereof or fusion proteins, which can be prepared by fusing the coding region of the extracellular domain of VSIG10 or fragments thereof to the Fc region of human IgG1 or mouse IgG2a as described previously (Chapoval et al, Methods mol. Med. (Methods for molecular pharmaceuticals), 45: 247-255 (2000)).

Optionally, VSIG10 ECD also refers to a fusion protein comprising the amino acid sequence of human VSIG10 ECD fused to a human immunoglobulin Fc. Optionally, the fusion protein comprises a polypeptide fused to any one of SEQ ID NOs: 70. 156 of human IgG1 Fc selected from any one of SEQ ID NOs: 4 and 6, the amino acid sequence of human VSIG10 ECD. Optionally, the amino acid sequence of the fusion protein is given in any one of SEQ ID NOs: 72. 73, 173 and 174.

In a further embodiment, the fusion protein comprises an extracellular domain of TMEM25, or a fragment thereof, fused to an Ig Fc region. Recombinant IgTMEM25 polypeptide, fragments thereof or fusion proteins, which can be prepared by fusing the coding region of the extracellular domain of TMEM25 or fragments thereof to the Fc region of human IgG1 or mouse IgG2a as described previously (Chapoval et al, Methods mol. Med. (Methods in molecular pharmaceuticals), 45: 247-255 (2000)).

Optionally, TMEM25 ECD also refers to a fusion protein comprising the amino acid sequence of human TMEM25 ECD fused to a human immunoglobulin Fc. Optionally, the fusion protein comprises a polypeptide fused to any one of SEQ ID NOs: 70. 156 of human IgG1 Fc SEQ ID NO: the amino acid sequence of human TMEM25 ECD as set forth in 8. Optionally, the amino acid sequence of the fusion protein is given in any one of SEQ ID NOs: 74. 175.

In a further embodiment, the fusion protein comprises an extracellular domain of LSR, or a fragment thereof, fused to an Ig Fc region. Recombinant Ig LSR polypeptide, fragments thereof or fusion proteins, which can be prepared by fusing the coding region of the extracellular domain of LSR or a fragment thereof to the Fc region of human IgG1 or mouse IgG2a as described previously (Chapoval et al, Methods mol. Med. ("Methods of molecular pharmaceuticals"), 45: 247-.

Optionally, the LSR ECD also refers to a fusion protein comprising the amino acid sequence of a human LSR ECD fused to a human immunoglobulin Fc. Optionally, the fusion protein comprises a polypeptide fused to any one of SEQ ID NOs: 70. 156 of human IgG1 Fc selected from any one of SEQ ID NOs: 12. 14, 15, 16, 17, 18, 47, 48, 49 and 50, or an amino acid sequence of the amino acid sequence set forth in seq id No. 14, 15, 16, 17, 18, 47, 48, 49 and 50. Optionally, the amino acid sequence of the fusion protein is given in any one of seq id NOs: 75. 76, 77, 78, 79, 80, 176, 177, 178, 179, 180, and 181.

The foregoing exemplary fusion proteins can incorporate any combination of the variants described herein. In another embodiment, the terminal lysine of the foregoing exemplary fusion protein is deleted.

The disclosed fusion proteins can be isolated using standard molecular biology techniques. For example, an expression vector comprising a DNA sequence encoding LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides, fragments or fusion proteins thereof is transfected into 293 cells by calcium phosphate precipitation and cultured in serum-free DMEM. The supernatant was collected at 72h and passed through protein G, or preferably protein AThe fusion protein was purified by column chromatography (Pharmacia, Uppsala, Sweden). Optionally, encoding LY6G6F,DNA sequences of VSIG10, TMEM25 and/or LSR polypeptides, fragments or fusion proteins thereofIn retroviral vectors and after four rounds of retroviral vector transduction, expression in CHO-S cells. The protein was purified from the supernatant by using protein a chromatography.

In another embodiment, the second polypeptide may have a coupling domain through which additional molecules may bind to LY6G6F, VSIG10, TMEM25 and/or LSR fusion proteins. In one such embodiment, the conjugated molecule is capable of targeted delivery of the fusion protein to a specific organ or tissue; further specific, illustrative, non-limiting examples of such targeting domains and/or molecules are set forth below.

In another such embodiment, the coupling molecule is another immunomodulatory agent that may enhance or augment the effect of LY6G6F, VSIG10, TMEM25 and/or LSR fusion proteins. In another embodiment, the coupling molecule is polyethylene glycol (PEG).

Peptide or polypeptide linker domains

The disclosed LY6G6F, VSIG10, TMEM25 and/or LSR fusion proteins can optionally comprise a peptide or polypeptide linker domain that separates the LY6G6F, VSIG10, TMEM25 and/or LSR polypeptide from a second polypeptide. In one embodiment, the linker domain comprises an immunoglobulin hinge region. In a further embodiment, the hinge region is derived from a human immunoglobulin. Suitable human immunoglobulins from which the hinge may be derived include IgG, IgD and IgA. In a further embodiment, the hinge region is derived from a human IgG. The amino acid sequences of immunoglobulin hinge regions, as well as other domains, are well known in the art. In one embodiment, the LY6G6F, VSIG10, TMEM25 and/or LSR fusion polypeptide comprises a polypeptide sequence that differs from seq id NO: 70, the hinge region, the CH2 region, and the CH3 region of the human immunoglobulin C γ 1 chain having at least 85%, 90%, 95%, 99%, or 100% sequence homology, optionally wherein the Cys at position 220 (according to full-length human IgG1, position 5 in SEQ ID NO: 70) is replaced by Ser (SEQ ID NO: 156): EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The hinge can be further shortened to remove any one of SEQ ID NOs: 70 or 156, amino acid 1, 2, 3, 4, 5, or a combination thereof. In one embodiment, any one of SEQ ID NOs: 70 or 156 is deleted from amino acids 1-5. Exemplary LY6G6F, VSIG10, TMEM25 and/or LSR fusion polypeptides comprising the hinge region of human immunoglobulin C γ 1 chain with the Cys at position 220 replaced by Ser, the CH2 region, and the CH3 region are given in SEQ ID NO: 71. 72, 73, 74, 75, 76, 77, 78, 79, 80.

In another embodiment, the LY6G6F, VSIG10, TMEM25 and/or LSR fusion polypeptide comprises a sequence identical to SEQ ID NO: 157, having at least 85%, 90%, 95%, 99% or 100% sequence homology to the CH2 region and the CH3 region of the human immunoglobulin C γ 1 chain: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In another embodiment, the LY6G6F, VSIG10, TMEM25 and/or LSR fusion polypeptide comprises a sequence identical to SEQ ID NO: 158, having at least 85%, 90%, 95%, 99% or 100% sequence homology to the murine immunoglobulin C γ 2a chain, the CH2 region and the CH3 region: EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK are provided. In another embodiment, the linker domain comprises a hinge region of an immunoglobulin as described above, and further comprises one or more additional immunoglobulin domains.

Other suitable peptide/polypeptide linker domains include naturally occurring or non-naturally occurring peptides or polypeptides. The peptide linker sequence has a length of at least 2 amino acids. Optionally, the peptide or polypeptide domain is a flexible peptide or polypeptide. By "flexible linker" is meant herein a peptide or polypeptide comprising two or more amino acid residues linked by one or more peptide bonds, which provides two polypeptides linked thereby with an increased degree of rotational freedom compared to the degree of rotational freedom that the two linked polypeptides would have in the absence of the flexible linker. This rotational freedom allows the two or more antigen binding sites linked by the flexible linker to each more effectively access one or more target antigens. Exemplary flexible peptides/polypeptides include, but are not limited to, the following amino acid sequences: Gly-Ser (SEQ ID NO: 159), Gly-Ser-Gly-Ser (SEQ ID NO: 160), Ala-Ser (SEQ ID NO: 161), Gly-Gly-Gly-Ser (SEQ ID NO: 162), Gly4-Ser (SEQ ID NO: 163), (Gly4-Ser)2(SEQ ID NO: 164), (Gly4-Ser)3(SEQ ID NO: 165) and (Gly4-Ser)4(SEQ ID NO: 166). Additional flexible peptide/polypeptide sequences are well known in the art. Other suitable linker domains include helix-forming linkers, such as Ala- (Glu-Ala-Lys) n-Ala (n ═ 1-5). Additional helix-forming peptide/polypeptide sequences are well known in the art. Non-limiting examples of such linkers are depicted in SEQ ID NO: 167-.

Dimerization, multimerization and targeting domains

The fusion proteins disclosed herein optionally comprise a dimerization or multimerization domain that functions to dimerize or multimerize two or more fusion proteins. This domain, which functions to dimerize or multimerize the fusion protein, may be a separate domain, or alternatively, may be contained within one of the other domains of the fusion protein (LY6G6F, VSIG10, TMEM25 and/or LSR polypeptide, second polypeptide, or peptide/polypeptide linker domain).

Dimerization or multimerization may occur between two or more fusion proteins via a dimerization or multimerization domain. Alternatively, dimerization or multimerization of the fusion protein may occur by chemical crosslinking. The dimers or multimers formed may be homodimers/homomultimers or heterodimers/heteromultimers. The second polypeptide "partner" in the LY6G6F, VSIG10, TMEM25 and/or LSR polypeptide may be composed of one or more other proteins, protein fragments or peptides as described herein, including but not limited to any immunoglobulin (Ig) protein or portion thereof (preferably the Fc region), or portion of a biologically or chemically active protein, such as the papillomavirus E7 gene product, melanoma associated antigen p97, and HIV env protein (gp 120). The "partner" may optionally be selected to provide a soluble dimer/multimer and/or one or more of the other biological activities described herein.

A "dimerization domain" is formed from at least two amino acid residues or from at least two peptides or polypeptides (which may have the same, or different, amino acid sequences). The peptides or polypeptides may interact with each other via covalent and/or non-covalent associations. The optional dimerization domain comprises at least one cysteine capable of forming an intermolecular disulfide bond with a cysteine on the partner fusion protein. The dimerization domain may comprise one or more cysteine residues such that one or more disulfide bonds may be formed between the partner fusion proteins. In one embodiment, the dimerization domain comprises one, two, or three to about ten cysteine residues. In a further embodiment, the dimerization domain is an immunoglobulin hinge region.

Additional exemplary dimerization domains may be any domain known in the art and include, but are not limited to: coiled coil, acid patch (acid patch), zinc finger, calcium hand (calnium hand), CHI-CL pair, as described in U.S. Pat. No. 5,821,333, "interface" (interface) with engineered "nodules" (knob) and/or "protuberances" (protuberance), leucine zipper (e.g., from jun and/or fos) (U.S. Pat. No. 5,932,448), and/or yeast transcription activators GCN4, SH2(src homology 2), SH3(src homology 3) (Vidal et al, Biochemistry, 43, 7344-44 (2004)), phosphotyrosine binding (PTB) (Zhou et al, Nature: 592 (378)), WW (Sudol, Physics and molecular biology, Biochemistry, Kichy et al, Biophys L113, Pharma, 132, Biophys, Pharma, 132, WO (Pharma, Pharma, nature, 378: 85-88 (1995)); keman (Komau) et al, Science (Science), 269.1737-1740(1995))14-3-3, WD40 (Hu5 et al, J Biol Chem (J Biol Chem.), 273, 33489-33494(1998)) EH, Lim, isoleucine zipper, receptor dimer pairs (e.g., interleukin-8 receptor (IL-8R); and integrin heterodimers such as LFA-I and GPIIIb/IIIa, or one or more dimerization domains thereof, dimer ligand polypeptides such as Nerve Growth Factor (NGF), neurotrophin-3 (NT-3), interleukins (IL-8), Vascular Endothelial Growth Factor (VEGF), VEGF-C, VEGF-D, PDGF members, and brain-derived neurotrophic factor (BDNF) (Arakawa et al, J. Biol. chem., 269 (45)), 27833 27839(1994), and Yersiki et al, Radejejejewski (Biochem.), 32 (48): 1350(1993)) and may also be variants of these domains in which affinity is altered. These pairs of polypeptides can be identified by methods known in the art, including yeast two-hybrid screening. Yeast two-hybrid screens are described in U.S. patent nos. 5,283,173 and 6,562,576. The affinity between a pair of interacting domains can be determined using methods known in the art, including as described in Bangpi (Katahira) et al, J.Biol Chem, 277, 9242-9246 (2002). Alternatively, a library of peptide sequences may be screened for heterodimerization, for example using the method described in WO 01/00814. Useful methods of protein-protein interaction are also described in U.S. Pat. No. 6,790,624.

A "multimerization domain" is a domain that causes three or more peptides or polypeptides to interact through one or more covalent associations and/or non-covalent associations. Suitable multimerization domains include, but are not limited to, coiled-coil domains. Coiled coils are peptide sequences having a continuous pattern of predominantly hydrophobic residues spaced 3 and 4 residues apart, usually in the form of sequences of seven amino acids (heptad repeats) or eleven amino acids (eleven repeats), which are assembled (folded) to form a multimeric helical bundle. Coiled coils having a certain irregularly distributed sequence comprising 3 and 4 residues apart are also contemplated. The hydrophobic residues are specifically the hydrophobic amino acids Val, He, Leu, Met, Tyr, Phe and Trp. By "predominantly hydrophobic" is meant that at least 50% of the residues must be selected from the hydrophobic amino acids mentioned.

The coiled coil domain may be derived from laminin. In the extracellular space, heterotrimeric coiled-coil protein laminins play an important role in the formation of the basement membrane. Clearly, multifunctional oligomeric structures are required for laminin function. The coiled-coil domain may also be derived from thrombospondin in which three (TSP-I and TSP-2) or five (TSP-3, TSP-4 and TSP-5) chains are linked; or from COMP (COMPcc) (Guo) et al, J.European society for molecular biology (EMBO J), 1998, 17: 5265-. Additional non-limiting examples of coiled-coil domains derived from other proteins, as well as other domains that mediate multimerization of polypeptides, are known in the art, such as vasodilator-stimulating phosphoprotein (VASP) domains, maternal protein-1 (CMP), viral fusion peptides, soluble NSFs (N-ethylmaleimide-sensitive factor), adsorbed protein receptor (SNARE) complexes, leucine-rich repeats, certain tRNA synthetases suitable for use in the disclosed fusion proteins.

In another example, LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides, fusion proteins or fragments thereof may be induced by binding to a second multivalent polypeptide, such as an antibody, so as to form a multimer. Antibodies suitable for use in multimerizing LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides, fusion proteins or fragments thereof include, but are not limited to, IgM antibodies and cross-linked multivalent IgG, IgA, IgD, or IgE complexes.

Dimerization or multimerization may occur between two or more fusion proteins via dimerization or multimerization domains, including those described above. Alternatively, dimerization or multimerization of the fusion protein may occur by chemical crosslinking. The fusion protein dimer may be a homodimer or a heterodimer. The fusion protein multimer can be a homomultimer or a heteromultimer. The fusion protein dimers as disclosed herein have formula II: N-R1-R2-R3-C N-R4-R5-R6-C or alternatively of formula III: N-R1-R2-R3-C C-R4-R5-R6-N, wherein the dimeric fusion protein provided by formula II is defined as being in a parallel orientation and the dimeric fusion protein provided by formula III is defined as being in an anti-parallel orientation. The parallel and antiparallel dimers are also known as cis and trans dimers, respectively. "N" and "C" denote the N-terminus and C-terminus of the fusion protein, respectively. The components "R1", "R2" and "R3" of the fusion protein are as defined above with respect to formula I. With respect to both formula II and formula III, "R4" is LY6G6F, VSIG10, TMEM25 and/or LSR polypeptide or second polypeptide, "R5" is an optional peptide/polypeptide linker domain and "R6" is LY6G6F, VSIG10, TMEM25 and/or LSR polypeptide or second polypeptide, wherein when "R4" is the second polypeptide, "R6" is LY6G6F, VSIG10, TMEM25 and/or LSR polypeptide and when "R4" is LY6G6F, VSIG10, TMEM25 and/or LSR polypeptide, "R6" is the second polypeptide. In one embodiment, "R1" is a LY6G6F, VSIG10, TMEM25, and/or LSR polypeptide, "R4" is also a LY6G6F, VSIG10, TMEM25, and/or LSR polypeptide, and both "R3" and "R6" are second polypeptides.

When "R1" ═ R4 "," R2 "═ R5" and "R3" ═ R6 ", the fusion protein dimers having formula II are defined as homodimers. Similarly, when "R1" ═ R6 "," R2 "═ R5" and "R3" ═ R4 ", the fusion protein dimer having formula III is defined as a homodimer. When these conditions are not met for any reason, the fusion protein dimer is defined as a heterodimer. For example, the heterodimer may comprise a domain orientation that satisfies these conditions (i.e., for dimers according to formula II, "R1" and "R4" are both LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides, "R2" and "R5" are both peptide/polypeptide linker domains and "R3" and "R6" are both second polypeptides), however the identity of one or more of these domains is different. For example, although both "R3" and "R6" may be LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides, one polypeptide may comprise a wild-type LY6G6F, VSIG10, TMEM25 and/or LSR amino acid sequence and the other polypeptide may be a variant LY6G6F, VSIG10, TMEM25 and/or LSR polypeptide. Exemplary variants LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides are LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides that have been modified to have increased or decreased binding to target cells, increased activity against immune cells, increased or decreased half-life or stability. Fusion protein dimers comprising a CHI or CL region of an immunoglobulin as part of a polypeptide linker domain preferably form heterodimers wherein one fusion protein of the dimer comprises the CHI region and the other fusion protein of the dimer comprises the CL region.

Fusion proteins can also be used to form multimers. Like dimers, multimers can be parallel multimers, in which all fusion proteins of the multimer are aligned in the same orientation relative to their N-and C-termini. The multimers can be antiparallel multimers, wherein the fusion proteins of the multimers can alternatively be aligned in opposite orientations relative to their N-and C-termini. Multimers (parallel or antiparallel) can be homomultimers or heteromultimers. The fusion protein is optionally produced in dimeric form; more preferably, the fusion is performed at the gene level by ligating the polynucleotide sequences corresponding to the two (or more) proteins, protein portions and/or peptides, such that the ligated or fused proteins are produced by the cell according to the ligated polynucleotide sequences. The preparation of such fusion proteins is described with reference to U.S. patent No. 5,851,795 to Linsley et al, which is incorporated by reference as a non-limiting example only, to the same extent as if fully set forth herein.

Targeting domains

LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides and fusion proteins may comprise a targeting domain that targets the molecule to a specific site within the body. The optional targeting domain targets the molecule to the area of inflammation. Exemplary targeting domains are antibodies or antigen-binding fragments thereof that are specific for inflamed tissue or proinflammatory cytokines including, but not limited to, IL17, IL-4, IL-6, IL-12, IL-21, IL-22, and IL-23. In the case of neurological disorders, such as multiple sclerosis, the targeting domain may target the molecule to the CNS or may bind to VCAM-I on the vascular epithelium. The additional targeting domain may be a peptide aptamer specific for a pro-inflammatory molecule. In other embodiments, LY6G6F, VSIG10, TMEM25 and/or LSR fusion proteins may include binding partners specific for polypeptides displayed on the surface of immune cells, such as T cells. In still other embodiments, the targeting domain specifically targets activated immune cells. Optional immune cells to be targeted include Th0, Th1, Th17, Th2 and Th 22T cells, other cells that secrete or cause other cells to secrete inflammatory molecules including, but not limited to: IL-l beta, TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6, IL-23, IL-22, IL-21, and MMP, and Treg. For example, the targeting domain of tregs can specifically bind to CD 25. The above variations are merely illustrative of optional variations and are not intended to be limiting in any way. Furthermore, the above explanations are provided for descriptive purposes only and are not intended to be limited by a single hypothesis.

Addition of radicals

If the protein according to the invention is a linear molecule, it is possible to place different functional groups that are sensitive to or suitable for chemical modification at different points on the linear molecule. Functional groups may be added to the ends of the linear form of the proteins according to at least some embodiments of the present invention. In some embodiments, the functional groups improve the activity of the protein with respect to one or more characteristics, including but not limited to: stability, permeability (across cell membranes and/or tissue barriers), tissue localization, improvement in therapeutic efficacy, reduced clearance, reduced toxicity, improved selectivity, improved resistance to cell pump (cellular pump) expulsion, and the like. For convenience and without intending to be limiting, the free N-terminus of one of the sequences contained in the compositions according to at least some embodiments of the present invention will be referred to as the N-terminus of the composition, and the free C-terminus of the sequence will be referred to as the C-terminus of the composition. Either the C-terminus or the N-terminus, or both, of the sequence may be attached to a carboxylic acid functional group or an amino functional group, respectively.

Non-limiting examples of suitable functional groups are described in Green (Green) and Wuts ("protecting groups in Organic Synthesis"), John Wiley and Sons (John Willi-Giraffe), chapters 5 and 7, 1991, the teachings of which are incorporated herein by reference. Preferred protecting groups are those which facilitate transport of the active ingredient attached to the protecting group into the cell, for example by reducing the hydrophilicity of the active ingredient and increasing its lipophilicity, these being examples of "transport moieties across cell membranes".

These moieties can optionally and preferably be cleaved in vivo by intracellular hydrolysis or enzymatically. (Ditter et al, J.Pharm. Sci., 57: 783(1968), Ditter (Ditter) et al, J.Pharm. Sci., 57: 828(1968), Ditter (Ditter) et al, J.Pharm. Sci., 58: 557(1969), gold (King) et al, Biochemistry (Biochemistry) 26: 2294(1987), Lindberg et al, Drug Metabolism and configuration (Drug Metabolism and Disposition) 17: 311(1989), and Denk (Tunek) et al, Biochemistry (biochem. Pharm Pharma., 3837: anson, 1988), and Biochem (biochem) 239: Apochem, FAS. et al, biochem. J.anshen et al, biochem. FAS. 1985, Biochem et al, biochem. J.anshen et al, biochem. FAS. 1987, and Biochem. et al, biochem. J.P.. Hydroxyl protecting groups include ester, carbonate, and carbamate protecting groups. Amino protecting groups include alkoxycarbonyl and aryloxycarbonyl groups as described above for the N-terminal protecting group. Carboxylic acid protecting groups include aliphatic esters, benzylic esters, and aryl esters as described above for the C-terminal protecting group. In one embodiment, the carboxylic acid groups in the side chain of one or more glutamic or aspartic acid residues in the composition of the invention are preferably protected with a methyl, ethyl, benzyl or substituted benzyl ester, more preferably as a benzyl ester.

Non-limiting, illustrative examples of N-terminal protecting groups include acyl (-CO-R1) and alkoxycarbonyl or aryloxycarbonyl (-CO-O-R1), wherein R1 is an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aromatic, or substituted aromatic group. Specific examples of acyl groups include, but are not limited to: acetyl, (ethyl) -CO-, n-propyl-CO-, isopropyl-CO-, n-butyl-CO-, sec-butyl-CO-, tert-butyl-CO-, hexyl, lauroyl, palmitoyl, myristoyl, stearoyl, oleoylphenyl-CO-, 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, tert-butyl-O-CO-, phenyl-O-CO-, substituted phenyl-O-CO-, and benzyl-O-CO-, (substituted benzyl) -O-CO-, adamantane, naphthyl (naphylene), myristylene (myristoleyl), toluene, biphenyl, cinnamoyl (cinnamyl), nitrobenzoyl, toluoyl, furoyl, benzoyl, cyclohexane, norbornane, or Z-hexanal. To assist in N-acylation, one to four glycine residues may be present at the N-terminus of the molecule.

The carboxyl group at the C-terminal end of the compound may be protected, for example, by groups including, but not limited to: amides (i.e. hydroxyl at the C-terminus is replaced by-NH)₂、-NHR₂and-NR₂R₃Substituted) OR esters (i.e. the hydroxyl group at the C-terminus is replaced by-OR₂Instead). Optionally, R₂And R₃Independently is an aliphatic radical, a substituted aliphatic radical, a benzyl radical, a substituted benzyl radical, an aryl radical or a substituted aromatic radicalA radical group. In addition, together with the nitrogen atom, R₂And R₃A C4 to C8 heterocyclic ring can optionally be formed having from about 0-2 additional heteroatoms (e.g., nitrogen, oxygen, or sulfur). Non-limiting suitable examples of suitable heterocycles include piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl (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- (tert-butyl), -O- (n-butyl), -O-benzylethyl-O-phenyl.

By peptido-mimetic moieties

The "peptidomimetic organic moiety" can optionally replace an amino acid residue in the compositions of the invention as both a conservative substitution and a non-conservative substitution. These moieties are also called "unnatural amino acids" and can optionally replace amino acid residues, amino acids, or serve as spacer groups within peptides that replace (in lieu of) deleted amino acids. These peptidomimetic organic moieties can optionally and preferably have similar steric, electronic, or conformational properties as the amino acid being replaced, and such peptidomimetics are used to replace amino acids in the necessary positions and are considered conservative substitutions. However, such similarity is not required. According to a preferred embodiment of the invention, one or more peptide mimetics are selected such that the composition at least substantially retains its physiological activity compared to the native protein according to the invention.

Peptidomimetics can optionally be used to inhibit degradation of peptides by enzymatic or other degradation processes. These peptidomimetics can optionally and preferably be produced by organic synthesis techniques. Non-limiting examples of suitable peptidomimetics include the D amino acid of the corresponding L amino acid, tetrazole (Zaburlo (Zabrocki) et al, J.Am.chem.Soc., 110: 5875-5880 (1988)); isosteres of amide bonds (Jones et al Tetrahedron letters 29: 3853-3856 (1988)); LL-3-amino-2-properdine-6-carboxylic acid (LL-Acp) (Kempu et al, J.Org.chem., 50: 5834-5838 (1985)). Similar analogs are shown in Kempp et al, Tetrahedron letters 29: 5081-5082(1988) and Kempu et al, Tetrahedron letters 29: 5057-5060(1988), Kempu (Kemp), et al, Tetrahedron letters 29: 4935-4938(1988) and Kemp et al, J.Org.chem., 54: 109, 115 (1987). Other suitable but exemplary peptide mimetics are shown in the long well (Nagai) and zoste (Sato), "Tetrahedron letters" (Tetrahedron Lett.) 26: 647-650 (1985); diume (Di Maio), et al, journal of the british chemical society, department of the general gold (j. chem. soc. perkin Trans.), 1687 (1985); kahn (Kahn), et al, Tetrahedron letters 30: 2317 (1989); olsen (Olson) et al, journal of the american society for chemistry (j.am.chem.soc.) 112: 323-333 (1990); jiawei (Garvey) et al, journal of organic chemistry (j. org. chem.) 56: 436 (1990). Additional suitable exemplary peptidomimetics include hydroxy-1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid ester (Miyake et al, J.Wutian research laboratories 43: 53-76 (1989)); 1, 2, 3, 4-tetrahydro-isoquinoline-3-carboxylic acid ester (Ktzmierski et al, J.Am.chem.Soc.) -133: 2275-2283 (1991)); histidine isoquinolinone carboxylic acid (HIC) (Zechel et al, international journal of peptide protein research (int.j.pep.proteinres.)43 (1991)); (2S, 3S) -methyl-phenylalanine, (2S, 3R) -methyl-phenylalanine, (2R, 3S) -methyl-phenylalanine, and (2R, 3R) -methyl-phenylalanine (Katzmierski and Heruby, Tetrahedron letters (1991)).

Exemplary, illustrative, but non-limiting, unnatural amino acids include beta-amino acids (beta 3 and beta 2), homo-amino acids, cyclic amino acids, aromatic amino acids, Pro and Pyr derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted Phe and Tyr derivatives, linear core amino acids (linear core amino acids), or diamino acids. They are available from a number of suppliers, such as, for example, Sigma-Aldrich (USA).

Chemical modification of proteins

In the present invention, any portion of the proteins according to at least some embodiments of the present invention can optionally be chemically modified, i.e., altered by the addition of functional groups. For example, the flanking amino acid residues present in the native sequence may optionally be modified, but alternatively, as described below, other portions of the protein may optionally be modified in addition to or in place of the flanking amino acid residues. If a chemical synthesis procedure is followed, modifications may optionally be made during synthesis of the molecule, for example by addition of chemically modified amino acids. However, chemical modifications of amino acids already present in the molecule ("in situ" modifications) are also possible.

The amino acids of any sequence region of the molecule can optionally be modified according to any of the following exemplary types of modifications (considered "chemically modified" in the peptide concept). Non-limiting exemplary types of modifications include carboxymethylation, acylation, phosphorylation, glycosylation, or fatty acylation. Ether linkages can optionally be used to link a serine or threonine hydroxyl group to a hydroxyl group of the sugar. Amide bonds may optionally be used to attach glutamic or aspartic carboxyl groups to amino groups on sugars (Geerg and Jeanloz, Advances in carbohydrate Chemistry and Biochemistry, Vol.43, Academic Press (1985); Konz (Kunz), applied Chemistry, Ang.chem., International edition English 26: 294-308 (1987)). Acetal and ketal linkages can also optionally be formed between amino acids and carbohydrates. Fatty acid acyl derivatives can optionally be prepared, for example, by acylation of a free amino group (e.g., lysine) (Toth et al, Peptides: Chemistry, Structure and Biology (Peptides: Chemistry, Structure and Biology), Riville (Rivier) and Marsa (Marshal), eds., ESCOM Publ. Press, Leiden (Leiden), 1078-1079 (1990)).

As used herein, the term "chemically modified" when referring to a protein or peptide according to the present invention refers to a protein or peptide in which at least one of the amino acid residues in the protein or peptide is modified by natural processes (e.g., processing or other post-translational modifications) or by chemical modification techniques well known in the art. Examples of many known modifications typically include, but are not limited to: acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation (GPI anchor formation), covalent attachment of a lipid or lipid derivative, methylation, myristylation, pegylation, prenylation, phosphorylation, ubiquitination, or any similar process.

Other types of modifications optionally include the addition of a cycloalkane moiety to a biomolecule, such as a protein as described in PCT application No. WO2006/050262, which is incorporated herein by reference as if fully set forth herein. These moieties are designed for use with biomolecules and may optionally be used to impart different properties to proteins.

Furthermore, optionally any point on the protein may be modified. For example, pegylation of a glycosylated moiety on a protein can optionally be performed as described in PCT application No. WO 2006/050247, which is incorporated herein by reference as if fully set forth herein. One or more polyethylene glycol (PEG) groups may optionally be added to the O-linked and/or N-linked glycosylation. The PEG group may optionally be branched or linear. Optionally, any type of water-soluble polymer may be attached to the glycosylation site on the protein via a glycosyl linker.

Altered glycosylation

Proteins according to at least some embodiments of the invention may be modified so as to have an altered glycosylation pattern (i.e., altered from the original or native glycosylation pattern). As used herein, "altered" means that one or more carbohydrate moieties are deleted from the original protein and/or at least one glycosylation site is added to the original protein.

Glycosylation of proteins is usually either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. Where X is a tripeptide sequence of any amino acid except proline, asparagine-X-serine and asparagine-X-threonine are recognition sequences for enzymatic attachment of a carbohydrate moiety to an asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to a protein according to at least some embodiments of the invention is conveniently accomplished by altering the amino acid sequence of the protein such that it comprises one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Alterations may also be caused by the addition or substitution of one or more serine or threonine residues in the sequence of the original protein (for O-linked glycosylation sites). The amino acid sequence of the protein can also be altered by introducing changes at the DNA level.

Another means of increasing the number of carbohydrate moieties on a protein is by chemically or enzymatically coupling glycosides to amino acid residues of the protein. Depending on the coupling mode used, the sugar may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those of cysteine, (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline, (e) aromatic residues, such as those of phenylalanine, tyrosine or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87/05330 and Aplin (ebolin) and Wriston (riston), crccrit. rev. biochem. (key review on CRC biochemistry), 22: 259-306 (1981).

Removal of any carbohydrate moieties present on the proteins according to at least some embodiments of the present invention may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposing the protein to trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all of the sugars except the linked sugar (N-acetylglucosamine or N-acetylgalactosamine), leaving the amino acid sequence intact.

Chemical deglycosylation was performed by hakutan (Hakimuddin) et al, biochemical and biophysical archives (arch, biochem, biophysis), 259: 52 (1987); and arg (Edge), et al, analytical biochemistry (anal. biochem.), 118: 131 (1981). Enzymatic cleavage of the carbohydrate moiety on proteins can be performed using various endo-and exo-glycosidases such as sotachura (Thotakura), et al, methods in enzymology (meth. enzymol.), 138: 350 (1987).

Application method

As used herein, a "therapeutic agent" is any LY6G6F, VSIG10, TMEM25 and/or LSR protein and polypeptide, or orthologs, or fragments thereof, particularly the extracellular domain of LY6G6F, VSIG10, TMEM25 and/or LSR proteins or secreted forms thereof, and/or fusion proteins, and/or multimeric proteins comprising the same, or the nucleic acid sequences of LY6G6F, VSIG10, TMEM25 and/or LSR or fragments thereof, and drugs that specifically bind to LY6G6F, VSIG10, em tm 25 and/or LSR proteins, and/or drugs that agonize or antagonize the binding of other moieties to LY6G6F, VSIG10, TMEM25 and/or LSR proteins, and/or drugs that modulate (agonize or antagonize) the activity of at least one of LY6G6F, VSIG10, TMEM25 and/or LSR related drugs. Such drugs include monoclonal and/or polyclonal antibodies, and/or antigen binding fragments, and/or conjugates comprising the same, and/or a surrogate scaffold thereof that includes an antigen binding site that specifically binds to any one of LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides or epitopes thereof. By way of example, such agents also include small molecules, peptides, ribozymes, aptamers, antisense molecules, siRNA, and the like.

Stimulation of the activity of LY6G6F, VSIG10, TMEM25 and/or LSR is desirable in the following situations: wherein LY6G6F, VSIG10, TMEM25 and/or LSR are abnormally downregulated and/or wherein increased activity of LY6G6F, VSIG10, TMEM25 and/or LSR may have a beneficial effect. Likewise, inhibition of the activity of LY6G6F, VSIG10, TMEM25 and/or LSR is desirable in the following situations: wherein LY6G6F, VSIG10, TMEM25 and/or LSR are abnormally upregulated, and/or wherein decreased activity of LY6G6F, VSIG10, TMEM25 and/or LSR may have a beneficial effect.

As mentioned above, these therapeutic agents may be used to treat immune-related disorders as exemplified herein, and/or autoimmune disorders as exemplified herein, and/or infectious disorders as exemplified herein, and/or carcinomas as exemplified herein, and/or to block and/or promote immune co-stimulation mediated by any of LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides.

According to an additional aspect of the invention, these therapeutic agents may be used to prevent pathological inhibition of T cell activity, e.g., directed against cancer cells or chronically infected T cells; and/or preventing pathological stimulation of T cell activity, such as T cells directed against autoantigens in autoimmune diseases. For example, these molecules can be administered to cells in culture, in vitro or ex vivo, or to human subjects (e.g., in vivo) to treat, prevent and diagnose a variety of disorders. Preferred subjects include human patients having disorders mediated by cells expressing LY6G6F, VSIG10, TMEM25 and/or LSR proteins, and cells possessing LY6G6F, VSIG10, TMEM25 and/or LSR activity.

According to an additional aspect of the invention, these therapeutic agents may be used to inhibit T cell activation, as may be evidenced, for example, by T cell proliferation and cytokine secretion.

According to an additional aspect of the invention, the therapeutic agents may be used to elicit one or more of the following biological activities in vivo or in vitro: inhibiting the growth and/or killing of cells expressing LY6G6F, VSIG10, TMEM25 and/or LSR; mediating phagocytosis or ADCC of cells expressing LY6G6F, VSIG10, TMEM25 and/or LSR in the presence of human effector cells; or block binding of LY6G6F, VSIG10, TMEM25 and/or LSR ligands to LY6G6F, VSIG10, TMEM25 and/or LSR, respectively.

Thus, according to an additional aspect of the present invention, there is provided a method of treating an immune-related disorder as exemplified herein, and/or an autoimmune disorder as exemplified herein, and/or an infectious disorder as exemplified herein, and/or a carcinoma as exemplified herein, and/or blocking or promoting immune co-stimulation mediated by LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides in a subject by: administering to a subject in need thereof an effective amount of any therapeutic agent and/or a pharmaceutical composition comprising any therapeutic agent and further comprising a pharmaceutically acceptable diluent or carrier.

The subject according to the invention is a mammal, preferably a human being diagnosed as suffering from one of the diseases, disorders or conditions described above, or alternatively susceptible to at least one type of cancer and/or infectious disorder, and/or immune-related disorder.

As used herein, the term "treatment" refers to the prevention, delay of onset, cure, recovery, attenuation, alleviation, minimization, inhibition, or cessation (suppression) of the deleterious effects of the aforementioned disease, disorder, or condition. It also includes the control (management) of diseases as described above. By "controlling" is meant reducing the severity of the disease, reducing the frequency of episodes of the disease, reducing the duration of such episodes, reducing the severity of such episodes, and the like.

According to the present invention, treatment may be achieved by specifically up-regulating the expression of at least one polypeptide of the invention in a subject.

It will be appreciated that the treatment of the above-mentioned diseases according to the invention may be combined with other therapeutic methods known in the art (i.e. combination therapy). Thus, the therapeutic agents and/or pharmaceutical compositions comprising them recited herein in accordance with at least some embodiments of the present invention may be used in combination with one or more of the following agents to modulate immune responses: soluble gp39 (also known as CD40 ligand (CD40L), CD154, T-BAM, TRAP), soluble CD29, soluble CD40, soluble CD80 (e.g., ATCC 68627), soluble CD86, soluble CD28 (e.g., 68628), soluble CD56, soluble Thy-1, soluble CD3, soluble TCR, soluble VLA-4, soluble VCAM-1, soluble LECAM-1, soluble ELAM-1, soluble CD44, antibodies reactive with gp39 (e.g., ATCC HB-10916, ATCC HB-12055, and ATCC HB-12056), antibodies reactive with CD40 (e.g., ATCC HB-9110), antibodies reactive with B7 (e.g., ATCC HB-253, ATCC CRL-2223, ATCC CRL-2226, ATCC HB-301, ATCC HB-11341, etc.), antibodies reactive with CD28 (e.g., ATCC HB-11944 or 9.3 mAb), antibodies reactive with LFA-1 (e.g., ATCC HB-9579 and ATCC TIB-213), antibodies reactive with LFA-2, with the reactive antibody IL-2, with the reactive antibody IL-12, with the reactive antibody IFN-. gamma., antibodies reactive with CD2, antibodies reactive with CD48, antibodies reactive with any ICAM (e.g., ICAM-1(ATCC CRL-2252), ICAM-2, and ICAM-3), antibodies reactive with CTLA4 (e.g., ATCC HB-304), antibodies reactive with Thy-1, an antibody reactive with CD56, an antibody reactive with CD3, an antibody reactive with CD29, an antibody reactive with TCR, an antibody reactive with VLA-4, an antibody reactive with VCAM-1, antibody LECAM-1 reactive, antibody ELAM-1 reactive, antibody reactive with CD 44; l104EA29YIg, CD80 monoclonal antibody (mAb), CD86 mAb, gp39 mAb, CD40 mAb, CD28 mAb; anti-lfallmab, antibodies or other agents that target the mechanisms of the immune system, such as CD52 (alemtuzumab), CD25 (darlizumab), VLA-4 (natalizumab), CD20 (rituximab), IL2R (darlizumab), and MS4a1 (ocrelizumab); novel oral immunomodulators that have been shown to prevent lymphocyte recirculation from lymphoid organs, such as fingolimod (FTY720) or novel oral immunomodulators that cause lymphocyte depletion, such as mylinax (oral cladribine) or teriflunomide; and agents that prevent immune activation, such as panclar (dimethyl fumarate BG-12) or laquinimod (ABR 216062). Other combinations can be readily appreciated by one of ordinary skill in the art. In some embodiments, these therapeutic agents may be used to attenuate or reverse the activity of pro-inflammatory drugs (pro-inflammatory drugs), and/or limit the side effects of such drugs.

As will be readily understood by one of ordinary skill in the art, the combination may include therapeutic agents according to at least some embodiments of the present invention and/or pharmaceutical compositions comprising them, as well as an additional immunosuppressive agent; therapeutic agents and/or pharmaceutical compositions comprising them as listed herein and two other immunosuppressive agents; therapeutic agents and/or pharmaceutical compositions containing them as exemplified herein, as well as three other immunosuppressive agents, and the like. Determination of the optimal combination and dosage can be determined and optimized using methods well known in the art.

The therapeutic agent according to the invention and one or more other therapeutic agents may be administered either sequentially or simultaneously. Other therapeutic agents are, for example, cytotoxic agents, radiotoxic agents or immunosuppressive agents. The composition may be linked to the agent (as an immunocomplex) or may be administered separately from the agent. In the latter case (administered separately), the composition may be administered before, after or simultaneously with the agent, or may be co-administered with other known treatments, such as anti-cancer treatments (e.g. radiation). Such therapeutic agents include, among others, antineoplastic agents, such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, onconine, and cyclophosphamide hydroxyurea, which are themselves effective only at toxic or sub-toxic levels to the patient. Cisplatin was administered intravenously at a dose of 100 mg/dose every four weeks, and doxorubicin at a dose of 60-75mg/ml every 21 days.

Co-administration of a human anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 or anti-LSR antibody, and antigen-binding fragments thereof, according to at least some embodiments of the present invention, with a chemotherapeutic agent provides two anti-cancer agents that act via different mechanisms, which produce cytotoxic effects on human tumor cells. Such co-administration can solve the problems attributed to the development of resistance of tumor cells to drugs or changes in their antigenicity that render them unresponsive to the antibody. Target-specific effector cells, such as effector cells linked to compositions according to at least some embodiments of the invention (e.g., human antibodies, multispecific molecules, and bispecific molecules) can also be used as therapeutic agents. The effector cells for targeting may be human leukocytes, such as macrophages, neutrophils or monocytes. Other cells include eosinophils, natural killer cells, and other cells with IgG-or IgA-receptors. If desired, effector cells may be obtained from the subject to be treated. The target-specific effector cells may be administered as a suspension of cells in a physiologically acceptable solution. The number of cells may be about 10-8 to 10-9, but will vary depending on the purpose of the treatment. Typically, this amount is sufficient to obtain localization at the target cell (e.g. a tumor cell expressing LY6G6F, VSIG10, TMEM25 and/or LSR proteins) and sufficient to achieve cell killing by e.g. phagocytosis. The route of administration may also vary.

Treatment with target-specific effector cells may be performed in conjunction with other techniques for removing target cells. For example, anti-tumor therapies using compositions according to at least some embodiments of the present invention (e.g., human antibodies, multispecific molecules, and bispecific molecules) and/or effector cells equipped with these compositions can be used in combination with chemotherapy. Additionally, combination immunotherapy can be used to direct two distinct cytotoxic effector populations to tumor cell rejection. For example, an anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 or anti-LSR antibody linked to an anti-Fc- γ RI or anti-CD 3 may be used in combination with an IgG-or IgA-receptor specific binding agent.

Bispecific and multispecific molecules according to at least some embodiments of the invention may also be used to modulate fcyr or fcyr levels on effector cells, for example by capping and eliminating receptors on the cell surface. Mixtures of anti-Fc receptors may also be used for this purpose.

The invention also encompasses the use of a composition according to at least some embodiments of the invention in combination with other pharmaceutical agents for the treatment of immune system disordersUse is provided. For example, autoimmune diseases may be treated with molecules according to at least some embodiments of the invention in combination with, but not limited to: immunosuppressive agents, such as corticosteroids, cyclosporine, cyclophosphamide, prednisone, azathioprine, methotrexate, sirolimus, tacrolimus, biologic agents such as TNF-alpha blockers or antagonists, or any other biologic agent that targets any inflammatory cytokine, nonsteroidal anti-inflammatory drugs/Cox-2 inhibitors, hydroxychloroquine, sulfasalazine (sulphazolapryine), sodium chloroaurate, etanercept, infliximab, mycophenolate mofetil, basiliximab, asecept, rituximab, cyclophosphamide, interferon beta-la, interferon beta-lb, glatiramer acetate, mitoxantrone hydrochloride, anakinra, and/or other biologic agents and/or intravenous immunoglobulin (IVIG). Non-limiting examples of such known therapeutic agents include interferons, such as IFN- β -la (R) ((R)) And) And IFN-beta-lbGlatiramer acetateA polypeptide; natalizumabAnd mitoxantroneA cytotoxic agent is provided.

Thus, the use of an agent according to at least some embodiments of the invention to treat multiple sclerosis may be combined with any known therapeutic agent or method, e.g., to treat multiple sclerosis. Such treatments are known for the treatment of multiple sclerosisNon-limiting examples of agents or methods include interferons, IFN- β -la (And) And IFN-beta-lbGlatiramer acetateA polypeptide; natalizumabAnd mitoxantroneA cytotoxic agent, aminopyridineOther drugs include corticosteroids, methotrexate, cyclophosphamide, azathioprine, and intravenous immunoglobulin (IVIG), inosine, Ocrelizumab (Ocreluzumab; R1594), Michelson (Mylinax; Caldripine), alemtuzumab (alemtuzumab; Campath), daclizumab (daclizumab; Zenapax), Panaclar/dimethylfumarate (BG-12), Teriflunomide (Teriflunomide; HMR1726), fingolimod (fingolimod; FTY720), laquinimod (Laquinimod; ABR216062), and hematopoietic stem cell transplantation, Neuromax, Rituximab (Rituximab; Rituxan), BCG vaccine, low dose naltrexone (naltrexolone), anthelmintic therapy, hemocathartic, stents, and alternative therapies such as vitamin D, polyunsaturated fatty acids, cannabis.

Thus, the use of agents according to at least some embodiments of the invention to treat rheumatoid arthritis may be combined with any known therapeutic agent or method, e.g., for treating rheumatoid arthritis. Non-limiting examples of such known therapeutic agents or methods for treating rheumatoid arthritis include glucocorticoids, non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., salicylates, or cyclooxygenase-2 inhibitors, ibuprofen, and naproxen, diclofenac, indomethacin, etodolac), Disease-modifying anti-rheumatic drugs (DMARDs) -oral DMARDs: auranofin (Auranofin, ridaudara), Azathioprine (Azathioprine, Imuran), Cyclosporine (Cyclosporine, sandimmumee, genraf, Neoral, non-commercial), D-Penicillamine (Penicillamine, cuprinine), Hydroxychloroquine (Hydroxychloroquine, Plaquenil), Gold sodium thiomalate (IM Gold thiomamlate, Myochrysine), chlorothioglucose (Aurothioglucose, solanal), Leflunomide (Leflunomide, Arava), Methotrexate (Methotrexate, rheumatrix), Minocycline (Minocycline ), staphylococcal protein a immunoadsorbent (prosorb column), Sulfasalazine (lfasalazine, Azulfidine). Biological DMARD: TNF- α blockers, including Adalimumab (Adalimumab, Humira), Etanercept (Etanercept, Enbrel), Infliximab (Infliximab, Remicade), golimumab (golimumab, Simponi), certolizumab (certolizumab pegol, Cimzia), and other biological DMARDs, such as Anakinra (Anakinra, Kineret), Rituximab (Rituximab, Rituxan), tositumomab (Tocilizumab, actermra), CD28 inhibitors, including Abatacept (abatacecept, Orencia), and belicept.

Thus, treatment of IBD using the agents according to at least some embodiments of the present invention may be combined with, for example, any known therapeutic agent or method of treating IBD. Non-limiting examples of such known therapeutic agents or methods for treating IBD include immunosuppression to control symptoms, such as prednisone (prednisone), mesalamine (Mesalazine; including Saxaceae (Asacol), Peresan (Pentasia), Lialda, Aspiro), Azathioprine (Imuran), methotrexate or 6-mercaptopurine, steroids, Ondansetron (Ondandrotron), TNF- α blockers (including Riximab, Adamantimumab, Golimumab, Cetollizumab (cetomab pegol)), Abasipu (Orencia, Abatacept), Ultramumab (Ulteki)numab)Briakiumab (ABT-874) and certolizumab ozogamicinITF2357(givinostat), Natalizumab (Tysabri), filstert (SB-683699), infliximab (Remicade, infliximab), vedolizumab (MLN0002), and other drugs including GSK1605786 CCX282-B (Traficet-EN), AJM300, Ultecumab (Stelara, usekinumab), Sesamimod (CNI-1493), tasocitinib (CP-690550), LMW Heparin MMX (LMW Heparin MMX), budesonide MMX, golimumab (Simponi, golimumab), Gardcib HPV vaccines (Gardasil HPVvaccine), epsipros (Epaxal Berna) (virosomal hepatitis a vaccines), surgery, such as enterotomy, stricture plasty (strictureplasty) or temporary or permanent colostomy or ileostomy antifungals, such as nystatin (broad-spectrum intestinal antifungals) and itraconazole (itraconazole, Sporanox) or fluconazole (Diflucan); alternative drugs, prebiotics and probiotics, cannabis, anthelmintic treatment of the Trichuris suis helminth parasite (trichosuris helminth) or the treatment of its egg cells.

Thus, the use of agents according to at least some embodiments of the invention to treat psoriasis may be combined with any known therapeutic agent or method of treating psoriasis, for example. Non-limiting examples of such known therapeutic agents for treating psoriasis include topical agents typically used for mild conditions, phototherapy for moderate conditions, and systemic agents for severe conditions. Non-limiting examples of external agents: bath solutions and moisturizers, mineral oil and petrolatum; ointments and creams comprising: coal tar, anthratriphenol (anthralin), corticosteroids such as desoximetasone (Topicort), Betamethasone (Betamethasone), fluocinonide (fluocinonide), vitamin D3 analogs (e.g., calcipotriol), and retinoids. Non-limiting examples of phototherapy: sunlight; psoralen with a wavelength of 311-313nm and long-wave ultraviolet Phototherapy (PUVA). Non-limiting examples of systemic agents: biological agents, such as interleukin antagonists, TNF- α blockers, including antibodies, such as infliximab (Remicade), adalimumab (admira, Humira), golimumab, certolizumab, and recombinant TNF- α decoy receptor, etanercept (Enbrel); t cell targeting agents such as efavirenzab (Xannelim/Raptiva), alfacacet (amevacpt), dendritic cell-like efavirenzab (dendritic cells suchmelizumab); cytokine-targeting monoclonal antibodies (mabs) including anti-IL-12/IL-23 (ustekumab (brand name stellara)) and anti-interleukin-17; briakiumab (ABT-874); small molecules, including but not limited to ISA 247; immunosuppressants, such as methotrexate, cyclosporine; vitamin a and retinoids (synthetic forms of vitamin a); and alternative therapies, such as changes in diet and lifestyle, fasting periods (fostering period), low energy diet and vegetarian diet, diet supplemented with cod liver oil rich in vitamin a and vitamin D (e.g. cod liver oil), diet of cod liver oil rich in two omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and comprising vitamin E; ichthyotherapy, hypnotherapy, marijuana.

Thus, the use of agents according to at least some embodiments of the present invention to treat type 1 diabetes may be combined with any known therapeutic agent or method, e.g., to treat type 1 diabetes. Non-limiting examples of such known therapeutic agents for the treatment of type 1 diabetes include insulin, insulin analogs, islet transplantation, stem cell therapy (including) Non-insulin therapies, e.g. il-l beta inhibitors, including anakinraAbiraypuDiamyld, AlfaSaite (alefacept)Otelixizumab, DiaPep277(Hsp60 derived peptide), alpha 1-antitrypsin, prednisone, azathioprine, cyclosporine, El-INT (an injectable islet neotherapy comprising epidermal growth factor analogs and gastrin analogs), statins, includingSitagliptin (dipeptidyl peptidase (DPP-4) inhibitor), anti-CD 3 mAb (e.g., Teplizumab); CTLA4-Ig (abacavir), anti-IL-I β (conatinumab), anti-CD 20mAb (e.g., rituximab).

Thus, the use of agents according to at least some embodiments of the invention to treat uveitis may be combined with any known therapeutic agent or method, e.g., for treating uveitis. Non-limiting examples of such known therapeutic agents for the treatment of uveitis include corticosteroids, external cycloplegics such as atropine (atropine) or homatropine (homatropine), or PSTTA injections (triamcinolone acetate under the posterior tenon capsule), antimetabolite drugs such as methotrexate, TNF-alpha blockers (including infliximab, adalimumab, etanercept, golimumab, trastuzumab).

Thus, the use of an agent according to at least some embodiments of the invention to treat sjogren's syndrome may be combined with any known therapeutic agent or method, e.g., for treating sjogren's syndrome. Non-limiting examples of such known therapeutic agents for the treatment of Hugrand syndrome include cyclosporine, pilocarpine (Salagen) and cevimeline (cevimeline; Evoxac), hydroxychloroquine (Plaquenil), cortisone (cortisone; prednisone and others) and/or azathioprine (Imuran) or cyclophosphamide (Cytoxan), dexamethasone, thalidomide, dehydroepiandrosterone, NGX267, Rebamipide, FID114657, Etanercept (Etanercept), Raptiva, belimumab, MabThera (rituximab); anakinra, intravenous immunoglobulin (IVIG), allogeneic bone marrow mesenchymal stem cells (AlloMSC), automated neuroelectrical stimulation of "aliwell Crown".

Thus, the use of agents according to at least some embodiments of the invention to treat systemic lupus erythematosus can be combined with any known therapeutic agent or method, for example, to treat systemic lupus erythematosus. Non-limiting examples of such known therapeutic agents for the treatment of systemic lupus erythematosus include corticosteroids and disease modifying antirheumatic drugs (DMARD), drugs commonly resistant to malaria such as prasuquinic (plaquenil) and immunosuppressants such as methotrexate and azathioprine, hydroxychloroquine, cytotoxic drugs such as cyclophosphamide and mycophenolate mofetil, Hydroxychloroquine (HCQ), Benlysta (belimumab), non-steroidal anti-inflammatory drugs, prednisone, fampricipil (Cellcept), Prograf (Prograff), asecept (Atacpt), Lupuzor, intravenous immunoglobulin (IVIG), CellCept (mycophenolate mofetil), Orrisci (Orecina), 4-4 m RG (2077), rituximab, Olymphaduzumab, Epiguzumab, Centab 136, Siffamumab (Melimumab; CTLA-623), CTLA-623, AMG-R-D (AMG-R), and AMR (AMR-D), and Lu-D-, Prazimod (paquinimod; ABR-215757), LY2127399, CEP-33457, dehydroepiandrosterone, levothyroxine, sodium abepimox (LJP 394), memantine, opioids, sirolimus, kidney transplantation, stem cell transplantation.

Therapeutic agents and/or pharmaceutical compositions comprising them according to at least some embodiments of the invention as exemplified herein may be administered as the sole active ingredient or in conjunction with other drugs or other anti-inflammatory agents in an immunomodulating regimen, e.g. for the treatment or prevention of allograft or xenograft acute or chronic rejection or inflammatory or autoimmune conditions, or for the induction of tolerance.

For example, it may be used in combination with: calcineurin inhibitors, such as cyclosporine a or FK 506; immune suppressionMacrolides such as sirolimus or derivatives thereof; e.g., 40-O- (2-hydroxy) ethyl-rapamycin, lymphocyte homing agents, e.g., FTY720 or analogs thereof, corticosteroids; cyclophosphamide; azathioprine; methotrexate; leflunomide or an analog thereof; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualin or an analog thereof; immunosuppressive monoclonal antibodies, e.g., monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD11a/CD18, CD7, CD25, CD27, B7, CD40, CD45, CD58, CD137, ICOS, CD150(SLAM), OX40, 4-1BB or ligands thereof; or other immunomodulatory compounds, such as CTLA4/-Ig (Albapulin, Or belief), CD28-Ig, B7-H4-Ig, or other co-stimulatory agents, or adhesion molecule inhibitors such as mabs or low molecular weight inhibitors, including LFA-1 antagonists, selectin antagonists, and VLA-4 antagonists.

When therapeutic agents and/or pharmaceutical compositions comprising them according to at least some embodiments of the invention as recited herein are administered in combination with other immunosuppressive/immunomodulatory or anti-inflammatory therapies, such as those specified above, the dosage of the immunosuppressive, immunomodulatory or anti-inflammatory compound co-administered will of course vary depending on the type of co-drug used (e.g. whether it is a steroid or cyclosporin), the particular drug used, the condition being treated, etc.

Treatment of malignant tumors using these agents of the invention may be combined with other treatments well known in the art, such as one or more of the following: radiotherapy, antibody therapy, chemotherapy, photodynamic therapy, surgery or combined therapy with conventional drugs (e.g. immunosuppressive or cytotoxic drugs),

therapeutic agents or pharmaceutical compositions according to at least some embodiments of the invention may also be administered in combination with other compounds or immunotherapy. For example, combination therapy may include a compound of the invention in combination with at least one other therapeutic or immunomodulatory agent, or immunostimulatory strategy, including but not limited to: tumor vaccines, adoptive T cell therapy, Treg depletion, antibodies (e.g., bevacizumab, erbitux), peptides, chloroplasts (pepti-bodies), small molecules, chemotherapeutic agents, such as cytotoxic and cytostatic agents (e.g., paclitaxel, cisplatin, vinorelbine, docetaxel, gemcitabine, temozolomide, irinotecan, 5FU, carboplatin), immunological modifiers, such as interferons and interleukins, immunostimulatory antibodies, growth hormones or other cytokines, folic acid, vitamins, minerals, aromatase inhibitors, RNAi, histone deacetylase inhibitors, proteasome inhibitors, and the like.

According to at least some embodiments of the present invention, there is provided the use of a therapeutic agent as exemplified herein and/or pharmaceutical compositions comprising them in combination with one known therapeutic agent effective in the treatment of infections.

The therapeutic agents and/or pharmaceutical compositions comprising them as exemplified herein may be administered in combination with one or more additional therapeutic agents for the treatment of bacterial infections, including but not limited to: antibiotics, including aminoglycosides, carbapenems, cephalosporins, macrolides, lincosamides, nitrofurans, penicillins, peptides, quinolones, sulfonamides, tetracyclines; drugs against mycobacteria including but not limited to clofazimine, cycloserine, rifabutin, rifapentine, streptomycin; and other antibacterial agents such as chloramphenicol, fosfomycin, metronidazole, mupirocin, and tinidazole.

The therapeutic agents and/or pharmaceutical compositions comprising them as exemplified herein may be administered in combination with one or more additional therapeutic agents for the treatment of viral infections, including but not limited to: antiviral drugs, such as oseltamivir (brand name Tamiflu) and zanamivir (brand name Relenza) arbidol-adamantane derivatives (amantadine, rimantadine) -neuraminidase inhibitors (oseltamivir, laninamivir, peramivir, zanamivir) nucleoside analog reverse transcriptase inhibitors, including purine analogs guanine (acyclovir (Aciclovir) #/Valacyclovir (Valacyclovir), Ganciclovir (Ganciclovir)/Valganciclovir (Valganciclovir), Penciclovir (Penciclovir)/Famciclovir (Famciclovir)) and adenine (vidarabine), pyrimidine analogs, uridine (idoxuridine, trifluridine), thymine (brivudine), cytosine (cytarabine); phosphonoformic acid; nucleoside analogue/NARTI: entecavir (Entecavir), lamivudine, telbivudine, cladribine; nucleoside analogs/NtRTIs: adefovir (Adefovir), Tenofovir (Tenofovir); nucleic acid inhibitors, such as Cidofovir (Cidofovir); interferon alpha-2 b, pegylated interferon alpha-2 a; ribavirin (Ribavirin) #/Taribavirin; antiretroviral drugs including zidovudine, lamivudine, abacavir (abacavir), lopinavir (lopinavir), ritonavir (ritonavir), tenofovir (tenofovir)/emtricitabine (emtricitabine), efavirenz (efavirenz), alone or in various combinations, gp41 (envivirtide), latiravir (Raltegravir); protease inhibitors, such as Fosamprenavir (Fosamprenavir), Lopinavir (Lopinavir) and Atazanavir (Atazanavir), methazone, behenyl alcohol, Fomivirsen (fosivirsen), triamantadine.

The therapeutic agents and/or pharmaceutical compositions comprising them as exemplified herein may be administered in combination with one or more additional therapeutic agents for the treatment of fungal infections, including but not limited to: antifungal agents of polyene antifungal agents, imidazole, triazole, and thiazole antifungal agents, allylamine, echinocandin, or other antifungal agents.

Alternatively or additionally, the up-regulation method can optionally be achieved by specifically up-regulating (optionally expressing) the amount of at least one polypeptide of the invention or an active part thereof in the subject.

As mentioned above and in the examples section that follows, the biomolecule sequences of this aspect of the invention can be used as valuable therapeutic tools in the treatment of diseases, disorders or conditions in which altered activity or expression of a known wild-type gene product (known protein) contributes to the onset or progression of the disease, disorder or condition. For example, where a disease is caused by overexpression of a membrane-bound receptor, a soluble variant thereof may be used as an antagonist that competes with the receptor for binding to the ligand to thereby terminate signal transduction of the receptor.

According to at least some embodiments, immune cells (preferably T cells) can be contacted with a therapeutic agent in vivo or ex vivo to modulate an immune response. The T cell contacted with the therapeutic agent can be any cell that expresses a T cell receptor (including alpha/beta and gamma/T cell receptors). T-cells include all cells expressing CD3, including subsets of T-cells that also express CD4 and CDS. T-cells include primary and memory cells as well as effector cells, such as CTLs. T-cells also include cells such as Th1, Tc1, Th2, Tc2, Th3, Th17, Th22, Treg, and Tr1 cells. T-cells also include NKT-cells and similarly distinct classes of T-cell lineages.

Inhibiting epitope spreading

Epitope spreading refers to the ability of B and T cell immune responses to be diverse at a specific level from a single determinant on self-antigens to many sites, and at the level of V gene usage (Mannnus (Monneaux, F.), et al, Arthritis and Rheumatism, 46 (6): 1430-1438 (2002)). Epitope spreading is not limited to systemic autoimmune diseases. It has been described in humans, as well as EAE with various myelin proteins, induces T cell-dependent organ-specific diseases, such as type 1 diabetes and multiple sclerosis, in experimental animals.

Epitope spreading involves the acquired recognition of new epitopes in the same self-molecule together with epitopes present in related proteins in the same macromolecular complex. Epitope spreading can be assessed by measuring delayed-type hypersensitivity (DTH) responses, methods of which are known in the art.

One embodiment provides a method of inhibiting or reducing epitope spreading in a subject by administering to the subject an effective amount of a therapeutic agent. In a further embodiment, any one of these therapeutic agents inhibits epitope spreading in a subject having multiple sclerosis. Preferably, these therapeutic agents inhibit or block multiple points of the inflammatory pathway.

Yet another embodiment provides a method of inhibiting or reducing epitope spreading in a subject having multiple sclerosis by administering to the subject an effective amount of a therapeutic agent so as to inhibit or reduce differentiation, proliferation, activity and/or cytokine production and/or secretion of Th1, Th17, Th22 and/or other cells that secrete or cause other cells to secrete inflammatory molecules, including but not limited to IL-l β, TNF- α, TGF- β, IFN- γ, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs.

Use of a therapeutic agent according to at least some embodiments of the invention as an adjuvant in cancer vaccination:

immunization against tumor-associated antigens (TAAs) is a promising approach for cancer treatment and prevention, but it faces several challenges and limitations, such as tolerance mechanisms associated with self-antigens expressed by tumor cells. Costimulatory molecules such as B7.1(CD80) and B7.2(CD86) have improved the efficacy of gene-based and cell-based vaccines in animal models and are under investigation in clinical trials as adjuvants. This adjuvant activity can be achieved either by enhancing the co-stimulatory signal or by blocking the inhibitory signal, which is transmitted by negative co-stimulatory molecules expressed by the tumor cells (Neighbors et al, 2008. J. Immunotherapy; 31 (7): 644-55). According to at least some embodiments of the invention, a secreted or soluble form or ECD and/or variant, and/or ortholog, and/or conjugate of any LY6G6F, VSIG10, TMEM25 and/or LSR, and/or polyclonal or monoclonal antibodies and/or antigen binding fragments and/or conjugates comprising the same, and/or alternative scaffolds, specific for any LY6G6F, VSIG10, TMEM25 and/or LSR protein may be used as an adjuvant for cancer vaccination. According to at least some embodiments, there is provided a method for improving the immune effect against TAA comprising administering to a patient an effective amount of a secreted or soluble form or ECD and/or variant thereof, and/or ortholog, and/or conjugate of any one of LY6G6F, VSIG10, TMEM25 and/or LSR, and/or a polyclonal antibody or monoclonal antibody and/or antigen binding fragment and/or conjugate comprising same, and/or surrogate scaffold, specific for any one of LY6G6F, VSIG10, TMEM25 and/or LSR proteins.

Use of a therapeutic agent according to at least some embodiments of the invention for adoptive immunotherapy:

one of the essential features of some tolerance models is that once this tolerance state has been established, it can be permanently extended by adoptive transfer of donor-specific regulatory cells to the natural recipient. Such adoptive transfer studies have also addressed the ability of T-cell subsets and non-T-cell transfer tolerance. Such tolerance can be induced by blocking co-stimulation or by engaging co-inhibition with its counter-receptor. This approach, which has been successfully applied in animals and is being evaluated in clinical trials in humans (scaelapino KJ and dah DI PLoS one 2009; 4 (6): e 6031; leiy et al, Immunity, 2009; 30 (5): 656-665) offers a promising therapeutic option for autoimmune disorders and transplants. According to at least some embodiments of the invention, secreted or soluble forms of LY6G6F, VSIG10, TMEM25 and/or LSR or ECD and/or variants, and/or orthologs, and/or conjugates thereof, and/or polyclonal or monoclonal antibodies and/or antigen binding fragments and/or conjugates comprising the same specific for any one of LY6G6F, VSIG10, TMEM25 and/or LSR proteins, and/or alternative scaffolds may be used for adoptive immunotherapy. Thus, in accordance with at least some embodiments, to induce differentiation of tolerogenic regulatory cells, the present invention provides methods for in vivo or ex vivo tolerance induction comprising administering to a patient or to leukocytes isolated from the patient an effective amount of: secreted or soluble forms of LY6G6F, VSIG10, TMEM25 and/or LSR or ECD and/or variants, and/or orthologs, and/or conjugates thereof, and/or polyclonal or monoclonal antibodies specific for any one of LY6G6F, VSIG10, TMEM25 and/or LSR proteins, or and/or antigen binding fragments and/or conjugates comprising them, and/or replacement scaffolds; this is followed by ex vivo enrichment and proliferation of the cells and reinfusion of these tolerogenic regulatory cells into the patient.

Alternatively, the immune response of a patient may be enhanced by: removing the immune cells from the patient, contacting the immune cells with an agent that inhibits the activity of LY6G6F, VSIG10, TMEM25 and/or LSR, and/or with an agent that inhibits the interaction of LY6G6F, VSIG10, TMEM25 and/or LSR with its natural binding partner, and reintroducing the ex vivo stimulated immune cells into the patient. In another embodiment, the method of modulating an immune response involves isolating immune cells from a patient, transfecting them with a nucleic acid molecule encoding a form of LY6G6F, VSIG10, TMEM25 and/or LSR such that the cells express all or a portion of LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides according to various embodiments of the present invention on their surface, and reintroducing the transfected cells into the patient. These transfected cells have the ability to modulate an immune response in a patient.

Use of therapeutic agents according to at least some embodiments of the invention for immune enhancement

1. Treatment of cancer

The therapeutic agents provided herein are generally useful as immune response stimulating therapeutic agents in vivo and ex vivo. In general, the disclosed therapeutic compositions are useful for treating a subject suffering from or susceptible to any disease or disorder for which the subject's immune system mount an immune response. The ability of the therapeutic agent to modulate the LY6G6F, VSIG10, TMEM25 and/or LSR immune signals enables a more robust immune response. Therapeutic agents according to at least some embodiments of the present invention are useful for stimulating or enhancing immune responses involving immune cells (e.g., T cells).

Therapeutic agents according to at least some embodiments of the invention are useful for stimulating or enhancing an immune response in a host to treat cancer by administering to the subject an amount of the therapeutic agent effective to stimulate T cells in the subject.

2. Use of therapeutic agents in vaccines

The therapeutic agents according to at least some embodiments of the present invention are administered alone or in combination with any other suitable therapy. In one embodiment, these therapeutic agents may be administered in combination with, or as a component of, a vaccine composition as described above. Therapeutic agents according to at least some embodiments of the invention may be administered prior to, concurrently with, or subsequent to the administration of the vaccine. In one embodiment, the therapeutic agent is administered concurrently with the administration of the vaccine.

Pharmaceutical composition

In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, comprising a therapeutic agent according to at least some embodiments of the present invention, or a combination thereof.

Accordingly, the invention features a pharmaceutical composition that includes a therapeutically effective amount of a therapeutic agent according to at least some embodiments of the invention.

The pharmaceutical compositions according to at least some embodiments of the present invention are further preferably used for the treatment of cancer, immune-related disorders and/or infectious disorders, wherein such cancer may be non-metastatic, invasive or metastatic.

"treatment" refers to both therapeutic treatment and prophylactic (preventative) measures. Those in need of treatment include those already suffering from such disorders as well as those in which such diseases are to be prevented. Thus, the mammal to be treated may have been diagnosed as suffering from such a disorder or may be susceptible to or susceptible to such a disorder. "mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo, sports, or pet animals, such as dogs, horses, cats, cattle, etc. Preferably, such mammal is a human.

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

The therapeutic agents of the present invention may be provided to a subject alone, or as part of a pharmaceutical composition in which they are mixed with a pharmaceutically acceptable carrier.

The pharmaceutical compositions according to at least some embodiments of the present invention may also be administered in combination therapy, i.e., combined with other agents. For example, the combination therapy may include the following in combination with at least one other therapeutic agent or immunomodulator: anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR antibodies or LY6G6F, VSIG10, TMEM25 and/or LSR modulators according to at least some embodiments of the present invention, such as soluble polypeptide conjugates comprising an extracellular domain of LY6G6F, VSIG10, TMEM25 and/or LSR antigens or a small molecule (e.g., peptide, ribozyme, aptamer, siRNA), or other drug that binds to LY6G6F, VSIG10, TMEM25 and/or LSR.

Such compositions are referred to as "pharmaceutically acceptable carriers" if administration thereof is tolerated by a recipient patient. As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Such compositions include sterile water, buffered saline of pH and ionic strength (e.g., Tris-HCl, acetate, phosphate), and optional additives such as detergents and solubilizers (e.g., polysorbate 20, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosol, benzyl alcohol), and bulking substances (e.g., lactitol, mannitol). Non-aqueous solvents or vehicles may also be used as described in detail below.

Examples of suitable aqueous and nonaqueous carriers that may be used in the pharmaceutical compositions according to at least some embodiments of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Depending on the route of administration, the active compound (i.e., a soluble polypeptide conjugate comprising the extracellular domain of LY6G6F, VSIG10, TMEM25 and/or LSR antigens, monoclonal or polyclonal antibodies and antigen binding fragments and conjugates comprising them, and/or alternative scaffolds, or bispecific molecules that specifically bind any one of LY6G6F, VSIG10, TMEM25 and/or LSR proteins) may be coated in a material that serves to protect the compound from the action of acids and the disruption of other natural conditions that may inactivate the compound. Pharmaceutical compositions according to at least some examples of the invention may include one or more pharmaceutically 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 toxicological effects (see, e.g., Berge, s.m., bolger S.M et al (1977) journal of pharmacy (j.pharm.sci.) 66: 1-19). Examples of such salts include acid addition salts as well as base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphoric and the like, as well as those derived from non-toxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, as well as salts derived from non-toxic organic amines, such as N, N' -dibenzylethylenediamine, N-methylglucamine, chloroprocaine (chloroprocaine), choline, diethanolamine, ethylenediamine, procaine (procaine), and the like.

Pharmaceutical compositions according to at least some embodiments of the present invention may also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogensulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Prevention of the presence of microorganisms can be ensured by the sterilization procedures described above, as well as by the inclusion of various antibacterial and antifungal agents, such as parabens (parabens), chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents as pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions described in accordance with at least some embodiments of the invention is contemplated. Auxiliary active compounds may also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols 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 delaying absorption, for example, monostearate salts and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization and microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which 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 methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization and microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which 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 methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

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 is generally that amount of the composition which produces a therapeutic effect. Typically, this amount ranges from about 0.01 to about ninety nine percent, preferably from about 0.1 to about 70 percent, most preferably from about 1 to about 30 percent of the active ingredient, in one hundred percent, in combination with a pharmaceutically acceptable carrier.

The dosage regimen is adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the doses may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for convenient administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for dosage unit forms according to at least some embodiments of the invention are dictated by and directly dependent on the following factors: (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) limitations inherent in the art of formulating such an active compound to treat an individual's susceptibility.

For administration of the antibody, the dose will range from about 0.0001 to 100mg/kg, and more usually 0.01 to 5mg/kg of the host body weight. For example, the dose may be 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight or 10mg/kg body weight or in the range of 1-10mg/kg body weight. An exemplary treatment regimen entails the following administrations: once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every three months to once every 6 months. A preferred dosage regimen for an antibody according to at least some embodiments of the invention comprises 1mg/kg body weight or 3mg/kg body weight administered intravenously, using one of the following dosing regimens: (i) six doses every four weeks, then every three weeks; (ii) every three weeks; (iii) once 3mg/kg body weight, followed by 1mg/kg body weight every three weeks.

A similar dosage regimen may optionally be followed for a fusion protein as described herein; alternatively, the fusion proteins can optionally be administered in an amount between 0.0001 to 100mg/kg of patient body weight per day, preferably between 0.001 to 10.0 mg/kg/day, according to any suitable timing scheme. Therapeutic compositions according to at least some embodiments of the invention may be administered, for example, three times daily, twice daily, once daily, three times weekly, twice weekly or once weekly, biweekly or once every 3, 4, 5, 6, 7 or 8 weeks. In addition, the composition may be administered for a short or long period of time (e.g., 1 week, 1 month, 1 year, 5 years).

In some methods, two or more monoclonal antibodies with different binding specificities can be administered simultaneously, in which case the dose of each antibody administered falls within the indicated range. Antibodies are typically administered on a variety of occasions. The interval between single doses may be, for example, weekly, monthly, every three months, or yearly. The intervals may also be irregular, as indicated by measuring blood levels of antibodies to the target antigen in the patient. In some methods, the dose is adjusted to achieve a plasma antibody concentration of about 1-1000mug/ml, and in some methods about 25-300. mu.g/ml.

Alternatively, the therapeutic agent may be administered in a sustained release formulation, in which case less frequent administration is required. The dosage and frequency will vary depending on the half-life of the therapeutic agent in the patient. Overall, human antibodies exhibit the longest half-life, followed by humanized, chimeric, and non-human antibodies. The half-life of the fusion protein can vary widely. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the remainder of the life. In therapeutic applications, relatively higher doses at relatively shorter intervals are sometimes required until disease progression is reduced or terminated, and preferably until the patient exhibits partial or complete improvement in disease symptoms. Thereafter, a prophylactic regimen may be administered to the patient.

The actual dosage level of the active ingredient in the pharmaceutical compositions of the invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends on various pharmacokinetic factors including the activity of the specific composition of the invention or its ester, salt or amide used, the route of administration, the time of administration, the rate of excretion of the specific compound used, the duration of the treatment, other drugs, compounds and/or materials used in combination with the specific composition used, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A "therapeutically effective dose" of LY6G6F, VSIG10, TMEM25 and/or LSR soluble protein or LY6G6F, VSIG10, TMEM25 and/or LSR extracellular domain or fusion proteins containing them, or anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR antibodies described in accordance with at least some embodiments of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, an increase in lifespan, alleviation of disease, or prevention or reduction of injury or disability due to affliction with disease. For example, for treatment of LY6G6F, VSIG10, TMEM25 and/or LSR positive tumors (e.g., melanoma, liver disease, kidney disease, brain disease, breast disease, colon disease, lung disease, ovarian disease, pancreatic disease, prostate disease, stomach disease, multiple myeloma, and hematopoietic cancers including but not limited to lymphomas (hodgkins and non-hodgkins), acute and chronic lymphatic leukemias, and acute and chronic myeloid leukemias), a "therapeutically effective dose" preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to an untreated subject. The ability of a compound to inhibit tumor growth can be assessed in an animal model that predicts therapeutic efficacy in human tumors. Alternatively, such a property of the composition can be assessed by examining the ability of the compound to inhibit, such in vitro inhibition being by assays known to skilled practitioners. A therapeutically effective amount of a therapeutic compound can reduce tumor size, or otherwise ameliorate a symptom in a subject.

One of ordinary skill in the art will be able to determine a therapeutically effective amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

The compositions of the present invention may be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route of administration and/or mode of administration will vary depending on the desired result. Preferred routes of administration of therapeutic agents according to at least some embodiments of the present invention include intravascular delivery (e.g., injection or infusion), intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, oral, enteral, rectal, pulmonary (e.g., inhalation), nasal, topical (including transdermal, buccal, and sublingual), intravesical, intravitreal, intraperitoneal, vaginal, cerebral delivery (e.g., intracerebroventricular, intracerebral, and convection-enhanced diffusion), CNS delivery (e.g., intrathecal, perimedullary, and intraspinal), or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), transmucosal (e.g., sublingual administration) or via implant, or other parenteral routes of administration, such as by injection or infusion, or other routes of delivery and/or forms of administration known in the art. As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. In a particular embodiment, the protein, therapeutic agent or pharmaceutical composition according to at least some embodiments of the present invention may be administered intraperitoneally or intravenously.

Alternatively, LY6G6F, VSIG10, TMEM25 and/or LSR specific antibodies or other LY6G6F, VSIG10, TMEM25 and/or LSR drugs or molecules that modulate LY6G6F, VSIG10, TMEM25 and/or LSR protein activity and conjugates comprising the same and combinations thereof may be administered via a parenteral route, such as a topical, epidermal or mucosal route of administration, e.g. intranasally, bucally, vaginally, rectally, sublingually or topically.

The active compounds may be prepared with carriers that protect the compound from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are patented or generally known to those of ordinary skill in the art. See, e.g., Sustaineedand Controlled Release Drug Delivery Systems, Robinson (J.R. Robinson), e editors, Marcel Dekker, Inc. (Massel Dekker), New York (New York), 1978.

The therapeutic composition may be administered using medical devices known in the art. For example, in one preferred embodiment, therapeutic compositions according to at least some embodiments of the present invention may be administered using a needle-like hypodermic injection device, such as those described in U.S. Pat. nos. 5,399,163; 5,383,851, respectively; 5,312,335, respectively; 5,064,413, respectively; 4,941,880, respectively; 4,790,824, respectively; or 4,596,556. Examples of well-known implants and modules suitable for use in the present invention include: U.S. patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing a drug at a controlled rate; U.S. patent No. 4,486,194, which discloses a therapeutic device for administering a drug transdermally; U.S. Pat. No. 4,447,233, which discloses a drug infusion pump that delivers a drug at a precise infusion rate; U.S. patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chambered compartments; and U.S. patent No. 4,475,196, which discloses osmotic drug delivery systems. 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 antibody, LY6G6F, VSIG10, TMEM25 and/or LSR soluble protein, ectodomain, and/or fusion protein may be formulated to ensure proper distribution in vivo. For example, the Blood Brain Barrier (BBB) excludes many highly hydrophilic compounds. To ensure that therapeutic compounds according to at least some embodiments of the invention cross the BBB (if desired), they may be formulated, for example, in liposomes. Methods of making liposomes are described, for example, in U.S. Pat. nos. 4,522,811; 5,374,548, respectively; and 5,399,331. Liposomes can include one or more moieties that are selectively transported into a particular cell or organ, thereby enhancing targeted drug delivery (see, e.g., landed (v.v. ranade) (1989) journal of clinical pharmacology (j.clin.pharmacol.) 29: 685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to lauan (Low) et al); mannoside (Meize (Umezawa) et al, (1988) Command of the biochemistry and biophysics research (biochem. Biophys. Res. Commun), 153: 1038); antibodies (blautiman (p.g. bloeman) et al (1995) FEBS lett.357: 140; ovusis (m.owas) et al (1995) antimicrobial and chemotherapy (anti. microbial. agents chemicotherapy) 39: 180); the surfactant protein a receptor (Briscoe et al (1995) physiological miscellaneous us (am. j Physiol.) 1233: 134); pl20 (Schreier et al (1994)' J. Biol. chem.) 269: 9090); see also kleinan (k.keinanen); labucanan (m.l. laukkanen) (1994) FEBS lett.346: 123; kirilon (j.j.killion); fiddler (i.j. fidler) (1994)' methods for immunization (immunology) 4: 273.

anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25, and anti-LSR antibodies according to at least some embodiments of the present invention may be used as neutralizing antibodies. A neutralizing antibody (Nab) is an antibody that is capable of binding to and neutralizing or inhibiting a specific antigen, thereby inhibiting its biological action, for example by blocking a receptor on a cell or virus, inhibiting the binding of the virus to a host cell. Nab will partially or completely abolish the biological action of an agent either by blocking important surface molecules required for its activity or by interfering with the binding of the agent to its receptor on the target cell.

In yet another example, immunoconjugates of the invention can be used to target such compounds (e.g., therapeutic agents, labels, cytotoxins, radiotoxins, immunosuppressive agents, etc.) to cells having LY6G6F, VSIG10, TMEM25 and/or LSR cell surface receptors by linking the compounds to antibodies. Accordingly, the invention also provides methods for localizing (e.g., with a detectable label, such as a radioisotope, fluorescent compound, enzyme, or enzyme cofactor) ex vivo or in vivo a cell expressing LY6G6F, VSIG10, TMEM25, and/or LSR. Alternatively, these immunoconjugates can be used to kill cells having LY6G6F, VSIG10, TMEM25 and/or LSR cell surface receptors by targeting a cytotoxin or a radiotoxin to LY6G6F, VSIG10, TMEM25 and/or LSR antigens.

Diagnostic use of LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides and corresponding polynucleotides

According to some embodiments, the sample taken from the subject (patient) for performing the diagnostic assay according to at least some embodiments of the invention is selected from the group consisting of: bodily fluids or secretions, including, but not limited to, blood, serum, urine, plasma, prostatic fluid, semen (semen), external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, cerebrospinal fluid, synovial fluid, sputum, saliva, milk, peritoneal fluid, pleural fluid, cystic fluid, the ductal system of the breast (and/or lavages thereof), bronchoalveolar lavages, lavages of the reproductive system, and lavages of any other part of the body or system in the body; a sample of any organ (including isolated cells or tissue), wherein the cells or tissue may be obtained from an organ selected from the group consisting of, but not limited to: lung, colon, ovarian and/or breast tissue; stool or tissue samples, or any combination thereof. In some embodiments, this term encompasses a sample of an in vivo cell culture component. The sample can optionally be diluted with a suitable eluent before being subjected to the diagnostic assay.

In some embodiments, the phrase "marker" in the context of the present invention refers to a nucleic acid fragment, peptide, or polypeptide that is differentially present in a sample taken from a patient (subject) having one of the diseases or conditions described herein, as compared to a comparable sample taken from a subject not having one of the diseases or conditions described above.

In some embodiments, the phrase "differentially present" refers to a difference in the amount or mass of marker present in a sample taken from a patient having one of the diseases or conditions described herein as compared to a comparable sample taken from a patient not having one of the diseases or conditions described herein. For example, if the amount of nucleic acid fragments in one sample is significantly different from the amount of nucleic acid fragments in another sample, the nucleic acid fragments can optionally be present differentially between the two samples, e.g., as measured by hybridization and/or NAT-based assays. If the amount of polypeptide in one sample is significantly different from the amount of polypeptide in another sample, then the polypeptide can optionally be differentially present between the two samples. It should be noted that a label may be considered to be differentially present if it is detectable in one sample but not in another. Optionally, a relatively low amount of upregulation can serve as the marker, as described herein. Such relative levels of labeling can be readily determined by one of ordinary skill in the art; further guidance is provided in the description of each individual marker below.

In some embodiments, the phrase "diagnostic" means identifying the presence or nature of a pathological condition. Diagnostic methods vary in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive ("percentage of true positives"). Diseased individuals not detected by this assay are "false negatives". Subjects who are not ill and who are negative in this assay are called "true negatives". The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those that test positive but do not suffer from the disease. Although a particular diagnostic method may not provide a definitive diagnosis of a disorder, it is acceptable if the method provides a positive indication of assistance in the diagnosis.

As used herein, the term "diagnosis" refers to the process of identifying a medical condition or disease by its signs, symptoms, and in particular from the results of different diagnostic procedures, including, for example, detecting the expression of a nucleic acid or polypeptide according to at least some embodiments of the invention in a biological sample (e.g., in cells, tissues, or serum, as defined below) obtained from an individual. Furthermore, as used herein, the term "diagnosis" encompasses the following: screening for a disease, detecting the presence or severity of a disease, providing a prognosis for a disease, monitoring the progression or recurrence of a disease, together with an assessment of the efficacy and/or recurrence of a disease, disorder or condition, together with selecting a therapy and/or treatment for a disease, optimization of a given disease therapy, monitoring the treatment of a disease, and/or predicting the suitability of a therapy for a particular patient or subpopulation or determining the appropriate dosing of a therapeutic product in a patient or subpopulation. Diagnostic procedures can be performed in vivo or in vitro.

In some embodiments, the phrase "qualitatively" when used in reference to differences in expression levels of polynucleotides or polypeptides as described herein refers to the presence versus absence of expression; or, in some embodiments, to the temporal regulation of expression; or in some embodiments, refers to the timing of expression; or in some embodiments, refers to post-translational modification of the expressed molecule; and others as understood by those of ordinary skill in the art. In some embodiments, the phrase "quantitatively," when in reference to a difference in the level of expression of a polynucleotide or polypeptide as described herein, refers to an absolute difference in the amount of expression, as determined by any means known in the art; or in other embodiments, relative differences that may be statistically significant; or in some embodiments, indicate a trend in terms of differences in expression, when considered as a whole or over an extended period of time, etc.

In some embodiments, the term "diagnosing" refers to staging a disease or symptom, determining the severity of a disease, monitoring the progression of a disease, predicting the outcome of a disease and/or the prospect of recovery. The term "detecting" can also optionally encompass any of the above.

In some embodiments, diagnosis of a disease according to the invention may be affected by determining the level of a polynucleotide or polypeptide of the invention in a biological sample obtained from the subject, wherein the determined level may be associated with a predisposition to, or the presence or absence of the disease. It should be noted that "a biological sample obtained from a subject" can also optionally include a sample that is not removed from the subject, as described in more detail below.

In some embodiments, the term "level" refers to the level of expression of RNA and/or protein, or to 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 (i.e., 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).

To determine the level of labeled DNA, RNA, and/or polypeptide of interest in a subject, a biological sample can be collected from the subject using a number of well-known tissue or fluid collection methods.

Examples include, but are not limited to: fine needle biopsy, core needle biopsy, and surgical biopsy (e.g., brain biopsy), as well as lavage. Regardless of the procedure used, once a biopsy/sample is obtained, the level of this marker can be determined and a diagnosis can be made accordingly.

Determining the level of the same marker in normal tissues of the same origin is preferably effected in parallel, to detect increased expression and/or amplification and/or decreased expression of the marker in comparison to normal tissues.

In some embodiments, the term "test amount" of a marker refers to the amount of the marker in a sample of a subject consistent with a diagnosis of a particular disease or disorder. The test amount can be an absolute amount (e.g., micrograms/ml) or a relative amount (e.g., relative intensity of a signal).

In some embodiments, the term "control amount" of a marker can be any amount or series 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 the marker in a patient having a particular disease or condition or a person not having such a disease or condition. The control amount can be either an absolute amount (e.g., micrograms/ml) or a relative amount (e.g., relative intensity of the signal).

In some embodiments, the term "detecting" refers to identifying the presence, absence, or amount of a target to be detected.

In some embodiments, the term "label" includes any moiety or event that is detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful tags include 32P, 35S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in ELISA), biotin-streptavidin, dioxigenin, haptens and proteins obtainable from antisera or monoclonal antibodies thereto, or nucleic acid molecules having sequences complementary to the target. The label typically produces a measurable signal, such as a radioactive signal, a chromogenic signal, or a fluorescent signal, that can be used to quantify the amount of bound label in the sample. The tag may be incorporated into or attached to a primer or probe covalently, or also by ionic, van der waals or hydrogen bonding, for example with a radionuclide recognized by streptavidin, or a biotinylated nucleotide. The label may be directly or indirectly detectable. Indirect detection may involve a second label directly or indirectly bound to the first label. For example, the tag may be a ligand for a binding partner, such as biotin, which is a binding partner for streptavidin, or a nucleotide sequence for a binding partner which is a complementary sequence to which it can specifically hybridize. The binding partner may itself be directly detectable, e.g., the antibody itself may be labeled with a fluorescent molecule. The binding partner may also be indirectly detectable, e.g., a nucleic acid having a complementary nucleotide sequence may be part of a branched DNA molecule, which in turn is detectable by hybridization with other labeled nucleic acid molecules (see, e.g., french lander (p.d. fahrlander) and clausium (a.klausner), "biology/Technology (Bio/Technology) 6: 1165 (1988)). Quantification of the signal can be achieved by, for example, scintillation counting, densitometry, or flow cytometry.

Exemplary detectable labels that may optionally and preferably be used in conjunction with immunoassays include, but are not limited to, magnetic beads, fluorescent dyes, radioactive labels, enzymes (e.g., horseradish peroxide, alkaline phosphatase, and other substances commonly used in ELISA), and calorimetric labels (e.g., colloidal gold or colored glass or plastic beads). Alternatively, the label in the sample may be detected using an indirect assay, wherein the label-binding specific antibody is detected, for example using a second labeled antibody; and/or competition or inhibition assays, wherein for example monoclonal antibodies binding to different epitopes of the label are incubated with the mixture simultaneously.

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

When referring to a protein or peptide (or other epitope), in some embodiments, the term "specifically (or selectively) binds" or has "specific (or selective) immunoreactivity" or "specifically interacts or binds" with an antibody refers to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins as well as other biologies. Thus, under the conditions of a particular immunoassay, a specific antibody binds to a particular protein at least two times greater than background (non-specific signal) and does not substantially bind in significant amounts to other proteins present in the sample. Under such conditions, specific binding to the antibody may require selection of the antibody for its specificity for a particular protein. For example, polyclonal antibodies raised against seminal basic proteins from a particular species (e.g., rat, mouse, or human) can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with seminal basic proteins and not with other proteins (except polymorphic variants and alleles of seminal basic proteins). This selection can be achieved by subtracting antibodies that cross-react with seminal basic proteins from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid phase ELISA immunoassays are routinely used to select Antibodies specifically immunoreactive with a protein (see, e.g., Harlow and Lane, Antibodies, a laboratory manual, (1988) which provide an indication of the manner and conditions of an immunoassay that can be used to determine specific immunoreactivity). Typically, the specific or selective reaction will be at least twice background signal or background noise, and more typically greater than 10-fold to 100-fold background.

In another embodiment, the present invention provides a method for detecting a polypeptide of the present invention in a biological sample, comprising: contacting a biological sample with an antibody that specifically recognizes a polypeptide according to the invention and detecting said interaction; wherein the presence of the interaction is correlated with the presence of the polypeptide in the biological sample.

In some embodiments of the invention, the polypeptides described herein are non-limiting examples of markers useful for diagnosing diseases and/or indicating disorders. Each marker of the present invention may be used, individually or in combination, for different uses, including but not limited to prognosis, prediction, screening, early diagnosis, determining progression, therapy selection, and therapy monitoring of a disease and/or condition.

In a related aspect, the detected diseases include cancers, such as non-solid and solid tumors, sarcomas, and hematologic malignancies.

In another related object, these detected diseases include autoimmune disorders, rejection of any organ transplants, and/or graft-versus-host disease.

Each polypeptide/polynucleotide of the invention may be used, individually or in combination, for different uses, including but not limited to prognosis, prediction, screening, early diagnosis, determining progression, therapy selection, and therapy monitoring of a disease and/or condition indicative, as detailed above.

Such a combination can optionally include any subcombination of labels, and/or a combination characterized by at least one other label (e.g., a known label). Further, such a combination can optionally and preferably be used as described above with respect to determining a ratio of quantitative or semi-quantitative measurements between any of the markers described herein and any of the other markers described herein, and/or any other known markers, and/or any other markers.

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

Alternatively, such correlation can optionally include determining a concentration for each of the plurality of markers, calculating a single index value based on the concentration for each of the plurality of markers, and comparing this index value to a threshold level.

Still alternatively, such associating can optionally include determining a temporal change in the at least one marker, and wherein the temporal change is used in the associating step.

Still alternatively, such association can optionally include determining whether at least an "X" number of the plurality of markers have a concentration outside a predetermined range and/or above or below a threshold (as described above). The value of "X" can optionally be one marker, multiple markers, or all markers; alternatively or additionally, rather than including any markers in the count of "X", one or more specific markers of a plurality of markers can optionally be required to be associated with the disease or disorder (according to a range and/or cutoff).

Still alternatively, such correlation can optionally include determining whether the ratio of marker concentrations for the two markers is outside of a range and/or above or below threshold. Optionally, if the ratio is above or below the critical level and/or outside a range, then the ratio is associated with the disease or condition.

Optionally, a combination of two or more of these correlations may be used with a single graph and/or for association between multiple graphs.

Optionally, the method distinguishes a disease or disorder with a sensitivity of at least 70%, specificity of at least 85%, when compared to a normal subject. As used herein, sensitivity relates to the number of positive (diseased) samples detected/total number of positive samples present; specificity relates to the number of true negative (non-diseased) samples detected/total number of negative samples present. Preferably, such a method discriminates between a disease or disorder with a sensitivity of at least 80%, specificity of at least 90%, when compared to a normal subject. More preferably, such methods distinguish a disease or disorder with a sensitivity of at least 90%, specificity of at least 90%, when compared to a normal subject. Still more preferably, such a method distinguishes a disease or disorder with a sensitivity of at least 70%, a specificity of at least 85%, when compared to a subject exhibiting symptoms that mimic the symptoms of the disease or disorder.

The marking panel may be analyzed in a variety of ways well known to those of ordinary skill in the art. For example, each member of the panel may be compared to a "normal" value, or a value indicative of a particular outcome. The specific diagnosis/prognosis may depend on the comparison of each marker to this value; alternatively, if only a subset of the markers are outside the normal range, then this subset may indicate a particular diagnosis/prognosis. The skilled artisan will also appreciate that diagnostic markers, differential diagnostic markers, prognostic markers, time to marker onset, markers of disease or condition differentiation, and the like can be combined in a single assay or device. The marking may also be used for a plurality of purposes in general, for different purposes, for example by applying different thresholds or different weighting factors to the marking.

In one embodiment, the panels include indicia for the following targets: diagnosis of disease; diagnosis of the disease and indications if the disease is in the acute phase and/or an acute episode of the disease has occurred; diagnosis of the disease and indications if the disease is in a non-acute phase and/or a non-acute episode of the disease has occurred; indicating whether a combination of acute and non-acute phases or episodes has occurred; diagnosis of the disease and subsequent prognosis of adverse outcomes; diagnosis of the disease and the prognosis of the subsequent acute or non-acute phase or onset; progression of the disease (e.g., for cancer, such progression may include, for example, the occurrence or recurrence of metastasis).

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

In certain embodiments, one or more diagnostic or prognostic indicators are associated with a condition or disease only by the presence or absence of these indicators. In other embodiments, critical levels of diagnostic or prognostic indicators can be established, and the levels of these indicators in a patient sample can simply be compared to these critical levels. The sensitivity and specificity of a diagnostic and/or prognostic test depends not only on the analytical "quality" of the test-they also depend on the definition of what constitutes an abnormal result. In practice, the receiver operating characteristic curve, or "ROC" curve, is typically calculated by plotting the values of the variables for their relative frequencies in the "normal" and "diseased" populations, and/or by comparing the results obtained from the subject before, during and/or after treatment.

According to at least some embodiments of the invention, LY6G6F, VSIG10, TMEM25 and/or LSR proteins, polynucleotides or fragments thereof can be characterized as biomarkers for detecting disease and/or indicative of a condition, as detailed above.

According to still other embodiments, the invention may optionally and preferably encompass any amino acid sequence encoded by a nucleic acid sequence corresponding to LY6G6F, VSIG10, TMEM25 and/or LSR as described herein or a fragment thereof. Any oligopeptide or peptide related to such amino acid sequences or fragments thereof can optionally (additionally or alternatively) also be used as biomarker.

In still other embodiments, the invention provides methods of detecting a polynucleotide of the invention in a biological sample using a NAT-based assay, comprising: hybridizing the isolated nucleic acid molecules or at least about one oligonucleotide fragment of minimal length to nucleic acid material of a biological sample and detecting the hybridization complexes; wherein the presence of the hybridization 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. Also within the scope of the invention are kits comprising LY6G6F, VSIG10, TMEM25 and/or LSR protein or LY6G6F, VSIG10, TMEM25 and/or LSR conjugate or antibody composition (e.g., human antibody, bispecific or multispecific molecule, or immunoconjugate) of the invention and instructions for use. The kit may further comprise one or more additional agents, such as an immunosuppressive, cytotoxic or radiotoxic agent, or one or more human antibodies according to at least some embodiments of the invention (e.g., human antibodies having complementary activity that bind to an epitope on an antigen different from the first human antibody).

Nucleic Acid Technology (NAT) based assays:

detection of a nucleic acid of interest in a biological sample can also optionally be achieved by NAT-based assays involving nucleic acid amplification techniques, such as PCR, for example (or variations thereof, such as real-time PCR, for example). As used herein, a "primer" defines an oligonucleotide that is capable of annealing to hybridize to a target sequence, thereby generating a double-stranded region that serves as a point of initiation of DNA synthesis under suitable conditions. Amplification of the selected, or targeted, nucleic acid sequence may be performed by a variety of suitable methods known in the art. Non-limiting examples of amplification techniques include Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), transcription based amplification, q3 replicase and NASBA (fruit (Kwoh) et al, 1989, Proc. Natl.Acad.Sci.USA)86, 1173-. Non-limiting examples of nucleic acid technology based assays are selected from the group consisting of: PCR, real-time PCR, LCR, self-sustained synthesis reaction, Q-beta replicase, cycling probe reaction, branched DNA, RFLP analysis, DGGE/TGGE, single strand conformation polymorphism, dideoxy fingerprinting, microarray, fluorescence in situ hybridization, and comparative genomic hybridization. The term "amplification pair" (or "primer pair") as used herein refers to a pair of oligonucleotides (oligos) of the invention that are selected for use together in amplifying a selected nucleic acid sequence by one of a variety of types of amplification processes, preferably polymerase chain reaction. These oligos are designed to bind to complementary sequences under selected conditions, as is generally known in the art. In a particular embodiment, amplification of a nucleic acid sample from a patient is performed under conditions that favor amplification of the nucleic acids that are most differentially expressed. In a preferred embodiment, RT-PCR is performed on mRNA samples from patients under conditions that favor the amplification of the most abundant mRNA. In another preferred embodiment, amplification of the differentially expressed nucleic acids is performed simultaneously. One of ordinary skill in the art will recognize that such methods may be modified for detection of differentially expressed proteins rather than differentially expressed nucleic acid sequences. Nucleic acids (i.e., DNA or RNA) for use in 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 particular needs and targeted genome employed. Optionally, these oligonucleotide primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules in length, and they may be modified to be particularly suitable for the selected nucleic acid amplification system. As is generally known in the art, these oligonucleotide primers can be designed by considering the melting point at which it hybridizes to its targeting sequence (Sambrook et al, 1989, Molecular Cloning-analysis Manual, 2 nd edition, CSH laboratories, Ausubel et al, 1989, John Wiley and Sons, Inc., in Current Protocols in Molecular Biology, N.Y.).

Immunoassay method

In another embodiment of the invention, an immunoassay may be used to qualitatively or quantitatively detect and analyze the marker in the sample. Such a method comprises: providing an antibody that specifically binds to the label; contacting the sample with the antibody; and detecting the presence of the complex of the antibody bound to the label in the sample.

To prepare antibodies that specifically bind to the label, a purified protein label can be used. Antibodies that specifically bind to a protein label can be prepared using any suitable method known in the art.

After the antibody is provided, the label can be detected and/or quantified using any of a variety of recognized immunological binding assays. Useful assays include, for example, Enzyme Immunoassays (EIAs), such as enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (IA), Western blot assays, or dot blot assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). Typically, a sample obtained from a subject can be contacted with an antibody that specifically binds to the label.

Optionally, the antibody may be immobilized to a solid support to facilitate washing and subsequent separation of the complex prior to contacting the antibody with the sample. Examples of solid supports include, but are not limited to, glass or plastic, for example in the form of: a microtiter plate, a rod, a bead, or a microbead. Antibodies can also be attached to a solid support.

After incubating the sample with the antibody, the mixture is washed and the formed antibody-labeled complex can be detected. This can be achieved by incubating the washed mixture with a detection reagent. Alternatively, the label in the sample may be detected using an indirect assay, wherein a second, labeled antibody is used to detect the labeled binding-specific antibody, for example; and/or competition or inhibition assays, wherein for example monoclonal antibodies binding to different epitopes of the label are incubated with the mixture simultaneously.

Throughout these assays, incubation and/or washing steps are required after each combination of reagents. The incubation step may vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time depends on the assay format, label, volume of solution, concentration, etc. Typically, although these measurements can be made over a range of temperatures (e.g., 10 ℃ to 40 ℃), they will be made at ambient temperatures.

Immunoassays can be used to determine the test amount of a label in a sample from a subject. First, the immunoassay method described above can be used to detect the test amount of the label in the sample. If a label is present in the sample, it will form an antibody-label complex with the antibody that specifically binds to the label under suitable incubation conditions as described above. The amount of antibody-labeled complex can optionally be determined by comparison to a standard. As noted above, the test amount of label need not be measured in absolute units, so long as the units of measurement can be compared to the control amount and/or signal.

Radio-immunoassay (RIA): in one version, this method involves precipitation of the desired substrate and involves the methods described in detail herein below, wherein specific antibodies and radiolabeled antibody binding proteins (e.g., protein a labeled with I125) are immobilized on a precipitable support (e.g., agarose beads). The number of counts of precipitated pellets is proportional to the amount of substrate.

In an alternate version of RIA, a labeled substrate and an unlabeled antibody binding protein are employed. Samples containing unknown amounts of substrate were added in varying amounts. The decrease in the precipitation count 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 (e.g., immobilized cells or protein solution) containing a protein substrate to a surface (e.g., a well of a microtiter plate). A substrate-specific antibody coupled to an enzyme is applied and allowed to bind to the substrate. The presence of the antibody is then detected and quantified by a colorimetric reaction using an enzyme coupled to the antibody. Enzymes commonly used in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of the reaction, the amount of substrate present in the sample is proportional to the amount of color produced. To improve the accuracy of quantitation, substrate standards are often employed.

Western blotting: this method involves the separation of the substrate from other proteins by means of an acrylamide gel, followed by transfer of the substrate to a membrane (e.g., nylon or PVDF). The presence of the substrate is then detected by an antibody that specifically binds to the substrate, which in turn is detected by an antibody binding reagent. The antibody binding agent may be, for example, protein a, or other antibodies. The antibody binding reagent may be radiolabeled or enzyme linked as described above. Detection may be by autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantification and determination of the identity of the substrate by the relative position on the membrane, which is an indication of the migration distance in the acrylamide gel during electrophoresis.

Immunohistochemical analysis: this method involves in situ detection of a substrate in immobilized cells by substrate-specific antibodies. The substrate-specific antibody may be enzyme-linked or linked to a fluorophore. Detection was performed by microscopy and subjective evaluation. If an enzyme-linked antibody is used, a colorimetric reaction is required.

Fluorescence Activated Cell Sorting (FACS): this method involves the in situ detection of a substrate in a cell by a substrate-specific antibody. These substrate-specific antibodies are linked to fluorophores. Detection is performed by means of a cell sorting device which reads the wavelength of the light emitted by each cell as it passes through a beam of light. Such methods may employ two or more antibodies simultaneously.

Wireless imaging method

These methods 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, detecting cancerous cells. Unlike PECT, SPECT can optionally be used with both labels simultaneously. SPECT also has some other advantages, for example, with respect to the cost and type of tags that can be used. For example, U.S. patent No. 6,696,686 describes the use of SPECT for detecting breast cancer, and is incorporated herein by reference as if fully set forth herein.

Theranostics:

the term theranostics describes the following uses of diagnostic tests: for diagnosing a disease based on the results of the diagnostic test, selecting the correct treatment regimen, and/or monitoring the patient's response to treatment based on the results of the diagnostic test. Theranostic tests can be used to select treatments for patients that are particularly likely to benefit them and are less likely to produce side effects. They may also provide an early and objective indication of the efficacy of an individual patient, such that (if necessary) such treatment can be altered with minimal delay. For example: DAKO and Genentech (genetech) together produced HercepTest and Herceptin (Herceptin) for the treatment of breast cancer, the first theranostic test that was simultaneously approved with a new therapeutic drug. In addition to HercepTest (an immunohistochemical test), other theranostic tests are under development, using traditional clinical chemistry, immunoassays, cell-based techniques, and nucleic acid testing. PPGx' S recently introduced a TPMT (mercaptopurine S-methyltransferase) test that enabled physicians to identify patients at risk for potentially fatal adverse reactions to 6-mercaptopurine, an agent used in the treatment of leukemia. Furthermore, Nova Molecular company opened up SNP genotyping of the apolipoprotein E gene to predict the response of alzheimer's patients to treatment with cholinergic agents, and is now widely used in clinical trials for new drugs for this indication. Thus, the field of theranostics represents a cross-over point for diagnostic test information that predicts a patient's response to treatment as well as selecting the appropriate treatment for that particular patient.

Substitution labeling:

a surrogate marker is one that is detectable in the laboratory and/or based on a patient's physiological signal or symptoms, and is used as a surrogate for a clinically meaningful endpoint in a therapeutic trial. The surrogate marker is a direct measure of how the patient feels, functions, or survives, and it is expected that it predicts the effect of the treatment. The need for such markers is greatly increased when surrogate markers can be measured earlier, more conveniently, or more frequently than the endpoint of interest (referred to as the clinical endpoint) in terms of the therapeutic effect of the patient. Ideally, surrogate markers should be biologically plausible, predictive of disease progression, and measurable by normalization assays (including but not limited to traditional clinical chemistry, immunoassays, cell-based techniques, nucleic acid assays, and imaging modalities).

Therapeutic compositions (e.g., human antibodies, multispecific and bispecific molecules, and immunoconjugates) having a complement binding site (e.g., from a portion of IgG1, IgG2, or IgG3 or IgM that binds complement) according to at least some embodiments of the invention can also be used in the presence of complement. In one embodiment, ex vivo treatment of a cell population comprising target cells with a binding agent according to at least some embodiments of the invention and appropriate effector cells may be supplemented by the addition of complement or complement-containing serum. Phagocytosis of target cells coated with a binding agent according to at least some embodiments of the invention can be improved by the binding of complement proteins. In another embodiment, target cells coated with a composition according to at least some embodiments of the invention (e.g., human antibodies, multispecific molecules, and bispecific molecules) can also be lysed by complement. In yet another embodiment, the compositions according to at least some embodiments of the invention do not activate complement.

Therapeutic compositions (e.g., human antibodies, multispecific and bispecific molecules, and immunoconjugates) according to at least some embodiments of the invention can also be administered with complement. Thus, in accordance with at least some embodiments of the present invention, there are compositions comprising a human antibody, a multispecific or bispecific molecule, and serum or complement. These compositions are advantageous because the complement is located in close proximity to the human antibodies, multispecific molecules, or bispecific molecules. Alternatively, the human antibody, multispecific molecule, or bispecific molecule according to at least some embodiments of the invention may be administered separately from complement or serum.

The invention is further illustrated by the following examples. This information and examples are illustrative and should not be considered as further limiting. The contents of all figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

Examples of the invention

Example 1:

expression patterns of proteins according to at least some embodiments of the invention using the MED discovery engine:

MED is a proprietary software platform for collecting common gene expression data, normalizing, annotating and executing different queries. Expression data from the most widely used Affymetrix microarrays can be downloaded from the Gene expression Cart (GeneExpression Omnibus) (GEO-www.ncbi.nlm.nih.gov/GEO). The data was normalized by multiplication by setting 95 percent to a constant value (normalized expression 1200) and the noise was filtered by setting the lower limit to 30% to 0. The experiments were annotated, first automatically and then manually, to identify tissues and disorders, and the chips were grouped according to this annotation, and cross-validation of this grouping was by comparing the overall expression pattern of the genes of each chip with the overall average expression pattern of the genes in this group. Each probeset in each group is assigned an expression value that is the median of the expression levels of the probesets included in all chips in the group. The vectors of the expression of all probesets within a certain group form the virtual chips of that group, and the set of all such virtual chips is a virtual panel. The panel (or sub-panel) can be queried to identify a probe set (e.g., specific expression in a subset of tissues, or differential expression between diseased and healthy tissues) that has a desired behavior. These probe sets were ligated to the LEADS contig and RefSeq (http:// www.ncbi.nlm.nih.gov/RefSeq /) by probe-level mapping for further analysis.

The Affymetrix platforms downloaded are HG-U95A and HG-U133 families (A, B, A2.0 and PLUS 2.0). Three virtual panels are generated: u95 and U133Plus 2.0 based on the corresponding Affymetrix platform, and U133 using a set of common probe sets for HG-U133A, HG-U133A2.0 and HG-U133 PLUS 2.0 +.

The results of the MED discovery engine are presented in a scatter plot. A scatter plot is a compact representation of a given panel (set of groups). The y-axis is a (normalized) expression and the x-axis describes the groups in the panel. For each group, the median expression was represented by a solid mark, and the expression values for the different chips in the group were represented by small dashes ("-"). These groups are arranged and labeled as follows-the "other" groups (e.g., benign, non-cancerous disease, etc.) use triangles, the treated cells use squares, the normal use circles, the matching use crosses, and the cancer uses diamonds. The number of chips in each group is also written next to its name.

The MED discovery engine was used to assess the expression of VSIG10 transcripts. The expression data for Affymetrix probe set 220137 is shown in FIG. 3 (for all the graphs associated with the MED discovery engine, the division into "A", "B", etc. is for spatial reasons only, and thus can show all probe results) when representing the VSIG10 gene data. As is evident from the scatter plot presented in fig. 3, the expression of VSIG10 transcripts detectable with the above probe set was observed in several groups of cells from the immune system (mainly leukocytes). Differential expression is observed in different cancer disorders, for example on CD10+ leukocytes from ALL (acute lymphoblastic leukemia) and BM-CD34+ cells from AML (acute myeloid leukemia) cells.

Figure 3 shows a scatter plot demonstrating the expression of VSIG10 transcripts encoding VSIG10 protein on a virtual panel of all tissues and disorders using the MED discovery engine.

The MED discovery engine was used to evaluate expression of LSR transcripts. Expression data for Affymetrix probe set 208190_ s, when representing LSR gene data, is shown in figure 4. As is evident from the scatter plot presented in fig. 4, the expression of LSR transcripts detectable with the above probe set was observed in several groups of cells from the immune system (mainly bone marrow cells). High expression of LSR transcript is also observed in tissues of different cancerous conditions, e.g. breast, lung, ovarian, pancreatic, prostate and skin cancer.

Figure 4 shows a scatter plot demonstrating the expression of LSR transcripts encoding LSR proteins on virtual panels of all tissues and disorders using the MED discovery engine.

Example 2

Method for analyzing expression of RNA encoding LY6G6F, VSIG10, TMEM25 and/or LSR proteins

Targets according to at least some embodiments of the present invention are tested for their expression in different cancerous as well as non-cancerous tissue samples. A description of the samples used in the ovarian cancer test panels is provided in table 1 below. A description of the samples used in the breast cancer test panel is provided in table 2 below. An illustration of the samples used in the lung cancer test panel is provided in table 3. A description of the samples used in the health test panel is provided in table 4. A description of the samples used in the kidney cancer test panel is provided in table 5. A description of the samples used in the liver cancer test panel is provided in table 6. The test was then performed as described in the materials and methods section below.

Materials and methods

RNA preparation-RNA was obtained from: ABS (Wilmington, DE 19801, USA, http:// www.absbioreagents.com), BioChain Inst.Inc. (Biochain research institute Co.) (Haywo, CA 94545 USA, www.biochain.com), GOG-temporal Cooperative Human Tissue network for ovarian samples (pediatric Cooperative Human Tissue network), Gynecologic Oncology Group Tissue Bank (Gynecologic Oncology Group Tissue Bank), Columbus's Children Hospital (Columbus, OH 43205 IcUSA), Ambion (Austin), TX 78744 USA, http:// www.ambion.com), Analytical biologicals service Inc. (Analyzer Biological services Inc.) (Wilmington, DE 19801 USA, www.absbioreagents.com), Assiterd (bottom specific 4820, MI 202, USA 63), Genencortis 6778) (Special services Inc. (European corporation) (Avicular corporation, Inc. (Seville corporation, USA 8678), ISRAEL, www.tasmc.org.il/e /) and The Chaim Sheba medical center (Harim Barm medical center) (Tel-Hashomer, ISRAEL, eng. RNA samples were obtained from patients or from autopsy. All total RNA samples were treated with dnasel (ambion).

RT-PCR for ovarian, renal and health panels-10 ug of purified RNA was mixed with random hexamer primers (Applied Biosystems) according to the manufacturer's instructions), 4mM dNTPs, 12.5. mu.l of 10 XMultiScripte^TMBuffers (Applied Biosystems), 6. mu.l (50U/. mu.l) of RNase (Promega)) and 6. mu.l (50U/. mu.l) of MultiScripte (Applied Biosystems)) were mixed in a total volume of 125. mu.l. The reaction was incubated at 25 ℃ for 10min, followed by a further incubation at 37 ℃ for 2 hours. The mixture was then inactivated at 85 ℃ for 5 seconds. The resulting cDNA was diluted 1: 10 to 1: 40 in TE buffer (10mM tris pH 8, 1mM EDTA pH 8) (depending on panel calibration).

Real-time (RT) PCR analysis was performed as follows-cDNA prepared as described above (5 μ Ι) was used as template for real-time PCR reaction (final volume of 20 μ Ι), determined using SYBR Green I (PE Applied Biosystem) together with specific primers and UNG enzyme (Eurogentech or ABI or Roche). Amplification was achieved as follows: 50 ℃ for 2min, 95 ℃ for 10min, and then 95 ℃ for 15sec, 40 cycles, followed by 60 ℃ for 30sec, followed by a dissociation step. Detection was performed using PE Applied Biosystem SDS 7000(PE Applied biosystems SDS 7000). The cycle of the reaction in which a critical level of fluorescence is reached (Ct ═ critical cycle, described in detail below) is recorded and used to calculate the relative transcript number in the RT reaction. The relative quantity is calculated using the equation Q ═ efficiency ^ -Ct. The efficiency of the PCR reaction was calculated from a standard curve generated by using several different dilutions of the Reverse Transcription (RT) reaction. To minimize the inherent differences in RT reactions, the resulting relative quantities were normalized using a normalization factor calculated in the following manner:

Expression of several Housekeeping (HSKP) genes was examined in each panel. The relative number of each housekeeping gene (Q) in each sample calculated as described above was divided by the median number of such genes in all panel samples to obtain "relative Q vs med (relative Q rel to med)". Then, for each sample, the median of "relative Q versus med (relative Q rel to med") "of the selected housekeeping genes was calculated and served as a normalization factor for such samples for further calculations.

For each RT sample, the expression level of a particular amplicon was normalized to a normalization factor calculated from the expression levels of different housekeeping genes. Housekeeping genes (HSKG) for ovary, kidney, lung, liver, breast and healthy panels are listed in table 7.

The HSKG used for ovarian and healthy panel calibration was: HPRT1, SDHA and G6 PD; the HSKP genes used for kidney and liver panel calibration were: g6PD, PBGD and SDHA; the HSKP genes used for lung panel calibration were: UBC, PBGD, HPRT and SDHA; the HSKP genes used for breast panel calibration were: g6PD, PBGD, RPL19, and SDHA.

Table 1: details of ovarian RNA:

table 2: details of mammary RNA:

table 3: details of lung panel RNA

Table 4: healthy panel RNA details:

table 5: kidney Panel RNA details

Table 6: liver Panel RNA details

Table 7: housekeeping gene

Specific primers and amplicons for expression analysis of LSR transcripts are provided in table 8.

Table 8: LSR primers and amplicons

Specific primers and amplicons for expression analysis of TMEM25 transcripts are provided in table 9.

Table 9: TMEM25 primers and amplicons

Expression data for LSR _ seg24-36_200-309/310_ amplicon (SEQ ID: 140) are described in examples 3-9 below.

Example 3

Expression of LSR _ transcript detectable by the amplicon as depicted in the sequence name LSR _ SEG24-36_200-309/310 in normal and cancerous ovarian tissue

Expression of the LSR transcript detectable by or according to the LSR _ seg24-36_200-309/310_ amplicon (SEQ ID: 140) and the primers LSR _ seg24F _ 200-. The Ct value of the undetected sample (sample number 28) was designated as 41 and calculated accordingly. In parallel, similar measurements were made of the expression of several housekeeping genes-SDHA (SEQ ID: 103) (GenBank accession NM-004168; amplicon-SDHA-amplicon (SEQ ID: 106)), HPRTl (SEQ ID: 107) (GenBank accession NM-000194; HPRTl-amplicon (SEQ ID: 110)), and G6PD (SEQ ID: 111) (GenBank accession NM-000402; G6 PD-amplicon (SEQ ID: 1114)). For each RT sample, the expression of the amplicons above was normalized to the normalization factor calculated in the expression of these housekeeping genes as described in normalization method 2 of the materials and methods section. The normalized number of each RT sample was then divided by the median of the number of normal samples (sample numbers 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, and 64, table 1 above) to obtain an upregulated value for each sample relative to the median of the normal samples.

Figure 12 is a histogram showing overexpression of the LSR transcript indicated above in cancerous ovarian samples relative to normal samples.

As is evident from fig. 12, expression of LSR transcript detectable by the above amplicons was significantly higher in serous, mucinous, and adenocarcinoma samples than in non-cancerous samples (sample numbers 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, and 64, table 1 above). Notably, at least 5-fold overexpression was found in the serous carcinoma sample of 21/27, the mucinous carcinoma sample of 7/9, and the intimal carcinoma sample of 7/8.

Statistical analysis was applied to test the significance of these results, as described below.

The P value for the difference in expression level of LSR transcript detectable by the above amplicon in ovarian cancer versus normal tissue samples was determined by T-test to be 2.22 e-002. The P-value for the difference in expression level of LSR transcript detectable by the above amplicon in ovarian mucinous carcinoma samples versus normal tissue samples was determined by T-test to be 6.84 e-004. The P-value for the difference in expression levels of LSR transcript detectable by the above amplicon in the intima-like cancer versus normal tissue samples of the ovaries was determined by T-test to be 4.61 e-003. The P-value for the difference in expression level of LSR transcript detectable by the above amplicon in ovarian adenocarcinoma samples versus normal tissue samples was determined by T-test to be 5.68 e-004.

The 5-fold threshold for overexpression was found to distinguish serous carcinomas from normal samples with a P value of 2.59e-009 as examined by the exact Fisher test. The 5-fold threshold for overexpression was found to distinguish plasma mucinous carcinomas from normal samples with a P value of 8.43e-006 as examined by the exact Fisher test. A5-fold threshold for overexpression was found to distinguish plasma intimal carcinoma from normal samples with a P value of 2.38e-006 as examined by the exact Fisher test. A5-fold threshold for overexpression was found to distinguish plasma adenocarcinoma samples from normal samples with a P value of 7.28e-012 as examined by the exact Fisher test.

The above values demonstrate the statistical significance of these results.

Primer pairs may also optionally and preferably be encompassed within the present invention; for example, for the above experiments, the following primer pair was used as only one non-limiting illustrative example of a suitable primer pair: LSR _ seg24F _ 200-; and LSR _ seg36R _200 _ 310(SEQ ID: 142).

The invention also preferably encompasses any amplicon obtained by using any suitable primer pair; for example, for the above experiments, the following amplicons were obtained as only one non-limiting illustrative example of suitable amplicons: LSR _ seg24-36_200-309/310_ amplicon (SEQ ID: 140).

Example 4

Expression of LSR _ transcript detectable by the amplicon as depicted in the sequence name LSR _ SEG24-36_200-309/310 in normal and cancerous breast tissue

Expression of the LSR transcript detectable by or according to the seg24-36FR-LSR _ seg24-36_200-309/310_ amplicon (SEQ ID: 140) and the primers LSR _ seg24F _200 _ 309(SEQ ID: 141) and LSR _ seg36R _200 _ 310(SEQ ID: 142) was measured by real-time PCR. The Ct value of the detected sample (sample number 81) was designated 41 and calculated accordingly. In parallel, similar measurements were made of the expression of several housekeeping genes-G6 PD (SEQ ID: 111) (GenBank accession NM-000402; G6 PD-amplicon), PBGD (SEQ ID: 115) (GenBank accession BC 019323; PB GD-amplicon), RPL19(SEQ ID: 119) (GenBank accession NM-000981 RPL 19-amplicon), and SDHA (SEQ ID: 103) (GenBank accession NM-004168 SDHA-amplicon). For each RT sample, the expression of the amplicons above was normalized to the normalization factor calculated in the expression of these housekeeping genes as described in normalization method 2 of the materials and methods section. The normalized number of each RT sample was then divided by the median of the number of normal samples (sample numbers 43, 45, 46, 47, 48, 49, 50, 51, 52, 54, 56, 58, 59, 60, 61, 62, 63, 64, 66, 67, 68, and 69, table 2 above) to obtain a compromise upshifting relative to the median of the normal samples for each sample.

Figure 13 is a histogram showing overexpression of the LSR transcript indicated above in a cancerous breast sample relative to a normal sample.

As is evident from fig. 13, in the cancer samples, the expression of LSR transcript detectable by the above amplicons was higher than in the non-cancerous samples (sample numbers 43, 45, 46, 47, 48, 49, 50, 51, 52, 54, 56, 58, 59, 60, 61, 62, 63, 64, 66, 67, 68 and 69, table 2 above). Notably, at least 5-fold overexpression was found in the 9/53 adenocarcinoma sample.

Primer pairs may also optionally and preferably be encompassed within the present invention; for example, for the above experiments, the following primer pair was used as only one non-limiting illustrative example of a suitable primer pair: LSR _ seg24F _ 200-; and LSR _ seg36R _200 _ 310(SEQ ID: 142).

The invention also preferably encompasses any amplicon obtained by using any suitable primer pair; for example, for the above experiments, the following amplicons were obtained as only one non-limiting illustrative example of suitable amplicons: LSR _ seg24-36_200-309/310_ amplicon (SEQ ID: 140).

Example 5

Expression of LSR _ transcript detectable by the amplicon as depicted in the sequence name LSR _ SEG24-36_200-309/310 in normal and cancerous lung tissue

Expression of the LSR transcript detectable by or according to seg24-36FR LSR _ seg24-36_200-309/310_ amplicon (SEQ ID: 140) and primers LSR _ seg24F _200 _ 309(SEQ ID: 141) and LSR _ seg36R _200 _ 310(SEQ ID: 142) was measured by real-time PCR. In parallel, similar measurements were made of the expression of several housekeeping genes-HPRT 1(SEQ ID: 107) (GenBank accession NM-000194 HPRTl-amplicon (SEQ ID: 110)), PBGD (SEQ ID: 115) (GenBank accession BC 019323; PB GD-amplicon (SEQ ID: 118)), SDHA (SEQ ID: 103) (GenBank accession NM-004168; SDHA-amplicon (SEQ ID: 106)), and ubiquitin (SEQ ID: 133) (GenBank accession BC 000449; ubiquitin-amplicon (SEQ ID: 136)). For each RT sample, the expression of the amplicons above was normalized to the normalization factor calculated in the expression of these housekeeping genes as described in normalization method 2 of the materials and methods section. The normalized number of each RT sample was then divided by the median of the number of normal samples (sample numbers 51, 52, 53, 54, 56, 57, 58, 59, 60, 61, 62, 63, 64, 69, and 70, table 3 above) to obtain an upregulated value for each sample relative to the median of the normal samples.

Figure 14 is a histogram showing overexpression of the LSR transcript indicated above in cancerous lung samples relative to normal samples.

As is evident from fig. 14, expression of LSR or transcript detectable by the above amplicons is significantly higher in adenocarcinoma and non-small cell carcinoma samples than in non-cancerous samples (sample numbers 51, 52, 53, 54, 56, 57, 58, 59, 60, 61, 62, 63, 64, 69 and 70, table 3 above), and in some squamous cell carcinoma samples than in non-cancerous samples. Notably, at least 5-fold overexpression was found in the adenocarcinoma sample of 7/15, the squamous cell carcinoma sample of 3/18, and the non-small cell carcinoma sample of 10/40.

Statistical analysis was applied to test the significance of these results, as described below.

The P-value for the difference in expression levels of homo sapiens lipolysis activated lipoprotein receptor transcripts in lung adenocarcinoma versus normal tissue samples detectable by the above amplicon was determined by T-test to be 2.98 e-005. The P-value for the difference in expression level of LSR transcript detectable by the above amplicon in lung squamous cell carcinoma samples versus normal tissue samples was determined by T-test to be 7.42 e-003. The P value for the difference in expression levels of homo sapiens lipolysis activated lipoprotein receptor transcripts detectable by the above amplicon in the lung large cell carcinoma sample versus the normal tissue sample was determined by the T-test to be 1.76 e-002. The P value for the difference in expression level of homo sapiens lipolysis activated lipoprotein receptor transcripts detectable by the above amplicon in lung small cell carcinoma samples versus normal tissue samples was determined by T-test to be 4.35 e-002. The P-value for the difference in expression level of homo sapiens lipolysis activated lipoprotein receptor transcripts in lung non-small cell carcinoma samples versus normal tissue samples detectable by the above amplicon was determined by T-test to be 4.31 e-006.

The 5-fold threshold for overexpression was found to distinguish plasma adenocarcinoma from normal samples with a P value of 3.16e-003 as examined by the exact Fisher test. The 5-fold threshold for overexpression was found to distinguish non-small cell carcinomas from normal samples with a P value of 2.90e-002 as examined by the exact Fisher test.

The above values demonstrate the statistical significance of these results.

Primer pairs may also optionally and preferably be encompassed within the present invention; for example, for the above experiments, the following primer pair was used as only one non-limiting illustrative example of a suitable primer pair: LSR _ seg24F _ 200-; and LSR _ seg36R _200 _ 310(SEQ ID: 142).

The invention also preferably encompasses any amplicon obtained by using any suitable primer pair; for example, for the above experiments, the following amplicons were obtained as only one non-limiting illustrative example of suitable amplicons: LSR _ seg24-36_200-309/310_ amplicon (SEQ ID: 140).

Example 6

Expression of LSR _ transcript in different normal tissues detectable by the amplicon as depicted in the sequence name LSR _ SEG24-36_200-309/310

Expression of the LSR transcript detectable by or according to seg24-36FR LSR _ seg24-36_200-309/310_ amplicon (SEQ ID: 140) and primers LSR _ seg24F _200 _ 309(SEQ ID: 141) and LSR _ seg36R _200 _ 310(SEQ ID: 142) was measured by real-time PCR. In parallel, similar measurements were made of the expression of several housekeeping genes-SDHA (SEQ ID: 103) (GenBank accession NM-004168; SDHA-amplicon (SEQ ID: 106)), HPRT1(SEQ ID: 107) (GenBank accession NM-000194; HPRTl-amplicon (SEQ ID: 110)), and G6PD (SEQ ID: 111) (GenBank accession NM-000402; G6 PD-amplicon (SEQ ID: 114)). For each RT sample, the expression of the amplicons above was normalized to the normalization factor calculated in the expression of these housekeeping genes as described in normalization method 2 of the materials and methods section. The normalized number of each RT sample was then divided by the median of the number of ovarian samples (sample numbers 20, 21, 22, and 23, table 4 above) to obtain a relative expression value for each sample relative to the median of the ovarian samples.

FIG. 15 is a histogram showing the expression of the LSR transcript indicated above in normal tissue samples relative to ovarian samples.

Example 7

Expression of LSR _ transcript detectable by the amplicon as depicted in the sequence name LSR _ SEG24-36_200-309/310 in normal and cancerous renal tissue

Expression of the LSR transcript detectable by or according to the seg24-36FR-LSR _ seg24-36_200-309/310_ amplicon (SEQ ID: 140) and the primers LSR _ seg24F _200 _ 309(SEQ ID: 141) and LSR _ seg36R _200 _ 310(SEQ ID: 142) was measured by real-time PCR. In parallel, similar measurements were made of the expression of several housekeeping genes-SDHA (SEQ ID: 103) (GenBank accession NM-004168; SDHA-amplicon (SEQ ID: 106)), G6PD (SEQ ID: 111) (GenBank accession NM-000402; G6 PD-amplicon (SEQ ID: 114)) and PBGD (SEQ ID: 115) (GenBank accession BC 019323; PB GD-amplicon (SEQ ID: 118)). For each RT sample, the expression of the amplicons above was normalized to the normalization factor calculated in the expression of these housekeeping genes as described in normalization method 2 of the materials and methods section. The normalized number of each RT sample was then divided by the median of the number of normal samples (sample numbers 1, 2, 3, 4, and 19, table 5 above) to obtain an upregulated value for each sample relative to the median of the normal samples.

Figure 16 is a histogram showing the down-regulation of homo sapiens lipolysis-activated lipoprotein receptor transcripts indicated above in cancerous kidney samples relative to normal samples.

As is evident from fig. 16, expression of LSR transcript detectable by the above amplicon is significantly lower in cancerous kidney samples than in non-cancerous kidney samples (sample numbers 1, 2, 3, 4 and 19, table 5 above).

Statistical analysis was applied to test the significance of these results, as described below.

The P-value for the difference in expression levels of LSR transcript detectable by the above amplicon in cancerous kidney samples versus normal tissue samples was determined by T-test to be 1.25 e-01.

Primer pairs may also optionally and preferably be encompassed within the present invention; for example, for the above experiments, the following primer pair was used as only one non-limiting illustrative example of a suitable primer pair: LSR _ seg24F _ 200-; and LSR _ seg36R _200 _ 310(SEQ ID: 142).

The invention also preferably encompasses any amplicon obtained by using any suitable primer pair; for example, for the above experiments, the following amplicons were obtained as only one non-limiting illustrative example of suitable amplicons: LSR _ seg24-36_200-309/310_ amplicon (SEQ ID: 140).

Example 8

Expression of LSR _ transcript detectable by the amplicon as depicted in the sequence name LSR _ SEG24-36_200-309/310 in normal and cancerous liver tissue

Expression of the LSR transcript detectable by or according to seg24-36FR _ seg24-36_200-309/310_ amplicon (SEQ ID: 140) and primers LSR _ seg24F _200 _ 309(SEQ ID: 141) and LSR _ seg36R _200 _ 310(SEQ ID: 142) was measured by real-time PCR. In parallel, similar measurements were made of the expression of several housekeeping genes-SDHA (SEQ ID: 103) (GenBank accession NM-004168; SDHA-amplicon (SEQ ID: 106)), G6PD (SEQ ID: 111) (GenBank accession NM-000402; G6 PD-amplicon (SEQ ID: 114)) and PBGD (SEQ ID: 115) (GenBank accession BC 019323; -PBGD-amplicon (SEQ ID: 118)). For each RT sample, the expression of the amplicons above was normalized to the normalization factor calculated in the expression of these housekeeping genes as described in normalization method 2 of the materials and methods section. The normalized number of each RT sample was then divided by the median of the number of normal samples (sample numbers 41, 42, 43, 44, and 45, table 6 above) to obtain an upregulated value for each sample relative to the median of the normal samples.

Figure 17 is a histogram showing the expression of the LSR transcript indicated above in a cancerous liver sample relative to a normal sample.

Primer pairs may also optionally and preferably be encompassed within the present invention; for example, for the above experiments, the following primer pair was used as only one non-limiting illustrative example of a suitable primer pair: LSR _ seg24F _ 200-; and the LSR _ seg36R _200-310(SEQ ID: 142) reverse primer.

The invention also preferably encompasses any amplicon obtained by using any suitable primer pair; for example, for the above experiments, the following amplicons were obtained as only one non-limiting illustrative example of suitable amplicons: LSR _ seg24-36_200-309/310_ amplicon (SEQ ID: 140).

Example 9

Cloning of LSR _ Tl _ P5a ORF fused to FLAG tag

Cloning of LSR _ Tl _ P5a Open Reading Frame (ORF) (SEQ ID NO: 154) fused to FLAG (amino acid sequence: DYKDDDDK, SEQ ID NO: 153) to generate LSR _ P5a protein (SEQ ID NO: 11) fused to FLAG was performed by PCR as follows.

A3-step PCR reaction was performed using Pfuultra II fusion HS DNA polymerase (Agilent, Cat. 600670) under the following conditions: in the first step, 1. mu.l of undiluted ovarian sample (IDPZQXH) from ovarian panels (Table 1) served as template for the PCR reaction with 0.5. mu.l of each primer 200_369_ LSR _ Kozak _ NheI (SEQ ID NO: 147) and 200_379_ LSR _ Rev (SEQ ID NO: 148) and a total reaction volume of 25. mu.l. The reaction conditions are as follows: 5 minutes, 98 ℃; 35 cycles: 20 seconds, 98 ℃, 30 seconds, 55 ℃ and 1.5 minutes, 72 ℃; then 5 minutes, 72 ℃. The PCR products were diluted 1: 20 in DDW and 1ul was used as template for each PCR reaction in step 2.

For the second step, the 5' portion of the LSR was amplified with 0.5ul of each primer 200_369_ LSR _ Kozak _ NheI (10. mu.M) (SEQ ID NO: 147) and 200_371_ LSR _ seg36R (10. mu.M) (SEQ ID NO: 149) in a total reaction volume of 25. mu.l. The 3' portion of the LSR was amplified using 0.5ul of each primer 200_370_ LSR _ seg36F (10. mu.M) (SEQ ID NO: 150) and 200 373_ LSR _ Flag _ BamHI _ Rev (10. mu.M) (SEQ ID NO: 151) in a total reaction volume of 25. mu.l. The reaction conditions for both reactions were: 5 minutes, 98 ℃; 35 cycles: 20 seconds, 98 ℃, 15 seconds, 60 ℃ and 1.5 minutes, 72 ℃; then 5 minutes, 72 ℃. The products of each reaction were separated on a 1% agarose gel and Qiaquick was used^TMGel Extraction Kit(Qiaquick^TMGel extraction kit) (Qiagene, catalog No. 28706) it was purified from the gel. 100ng of 5 'product and 100ng of 3' product were used as templates for the third PCR reaction in which the full LSR-Flag sequence was amplified. Each of the primers 200_369_ LSR _ Kozak _ NheI (SEQ ID NO: 147) and 200-373_ LSR _ Flag _ BamHI _ Rev (SEQ ID NO: 151) was 0.5. mu.l, and the total reaction volume was 25. mu.l. The reaction conditions are as follows: 5 minutes, 98 ℃; 35 cycles: 20 seconds, 98 ℃, 30 seconds, 55 ℃ and 1.5 minutes, 72 ℃; then 5 minutes, 72 ℃. All primers used included gene specific sequences, restriction endonuclease sites, Kozak sequences, and FLAG tag sequences. The PCR product of step 3 was separated on a 1% agarose gel. After verifying the size of the expected band, Qiaquick was used ^TMGel Extraction Kit(Qiaquick^TMGel extraction kit) to purify the PCR product.

The purified full-length PCR product was digested with NheI and BamHI restriction enzymes (new england Biolabs, beverly, ma, usa). After digestion, the DNA was run on a 1% agarose gelAnd (5) separating. Bands of the desired size were excised from the gel as described above and extracted. The digested DNA was then ligated into the NheI and BamHI digested pIRESpuro3 vector as described above, treated with Antarctic Phosphatase (Antarctic Phosphomutase) (New England Biolabs, Beverley, MA, U.S. A., Cat. No. M0289L) at 37 ℃ for 30 minutes, and used Qiaquick as described above^TMGel Extraction Kit(Qiaquick^TMGel extraction kit) it was purified from a 1% agarose gel. Ligation was performed using T4 DNA ligase (Promega, Cat. No. M180A).

Example 10

Cloning of LSR _ Tl _ P5a ORF

Cloning of the LSR _ Tl _ P5a Open Reading Frame (ORF) (SEQ ID NO: 154) was performed by PCR to generate the LSR _ P5a protein (SEQ ID NO: 11) as described below.

The PCR reaction was carried out using Pfuultra II fusion HS DNA polymerase (Agilent, Cat. 600670) under the following conditions: 50ng of the above pIRES _ puro3_ LSR _ Tl _ P5a _ Flag construct served as template for the PCR reaction, with 0.5. mu.l of each primer 200_369_ LSR _ Kozak _ NheI (SEQ ID NO: 147) and 200_ 372_ LSR _ BamHI _ Rev (SEQ ID NO: 152) and a total reaction volume of 25. mu.l. The reaction conditions are as follows: 5 minutes, 98 ℃; 35 cycles: 20 seconds, 98 ℃, 30 seconds, 55 ℃ and 1.5 minutes, 72 ℃; then 10 minutes, 72 ℃. All primers used included gene specific sequences, restriction endonuclease sites, and Kozak sequences. PCR products were separated on a 1% agarose gel after verifying the size of the expected bands, using Qiaquick as described above ^TMGel Extraction Kit(Qiaquick^TMGel extraction kit) to purify the PCR product.

The purified PCR product was digested with NheI and BamHI restriction enzymes (New England Biolabs, Beverly, Mass., USA). After digestion, the DNA was separated on a 1% agarose gel. To take a desired size from a band as described aboveThe gel was excised and extracted. The digested DNA was then ligated into the NheI and BamHI digested pIRESpuro3 vector as described above, incubated with Antarctic Phosphatase (Antarctic Phosphomutase) (New England Biolabs, Beverley, MA, U.S. A., Cat. No. M0289L) at 37 ℃ for 30 minutes and using Qiaquick as described above^TMGel Extraction Kit(Qiaquick^TMGel extraction kit) it was purified from a 1% agarose gel. Ligation was performed using T4 DNA ligase (Promega, Cat. No. M180A).

Sequence verification was performed for both the above-described labeled and unlabeled constructs (Hylabs, Raohortt, Israel). Two nucleotide mismatches were identified, as follows: SEQ ID NO: 154, and a is paired with a from SEQ ID NO: 154, which results in seq id NO: 145 and SEQ ID NO: 146; this results in a peptide set forth in SEQ ID NO: 301 with an amino acid mismatch of I and M at amino acid position 209, which results in a polypeptide having the amino acid sequence of SEQ ID NO: 143 and a protein having the amino acid sequence set forth in SEQ ID NO: 144, or a pharmaceutically acceptable salt thereof.

The above recombinant plasmids were treated to generate stable pools as described below.

Example 11

Establishment of stable library of recombinant HEK293T cell expressing LSR _ P5a _ FLAG _ M protein

HEK-293T cells were stably transfected with LSR _ Tl _ P5a _ Flag _ m (SEQ ID NO: 146) and pIRESpuro3 empty vector plasmid as follows:

HEK-293T (ATCC, CRL-11268) cells were plated on sterile 6-well plates suitable for tissue culture, comprising: 2ml of prewarmed complete medium, DMEM [ Dulbecco's modified Eagle's Media (Dulbecco's modified Igor medium), Biotech Industries (Beiit Ha' Emek, Israel, Cat. No.: 01-055-1A) ] + 10% FBS [ fetal bovine serum, Biotech Industries (Biologic Industries) (BeHa 'Emek, Israel, Cat. No.: 04-001-1A) ] +4mM L-glutamine (Biologic Industries (Beit Ha' Emek, Israel), Cat. No.: 03-020-1A). 500,000 cells per well were transfected with 2. mu.g of the DNA construct using 6. mu.l of FuGENE 6 reagent (Roche, Cat. No.: 11-814-443-001) diluted to 94ul of DMEM. The mixture was incubated at room temperature for 15 minutes. The complex mixture was added dropwise to the cells. These cells were placed in an incubator maintained at 37 ℃ with a CO2 content of 5%. 48 hours after transfection, these cells were transferred to 75cm2 tissue culture flasks containing 15ml of selection medium (complete medium supplemented with 5. mu.g \ ml puromycin (Sigma Co., Cat. No. P8833)). Cells were placed in an incubator and medium was replaced every 3-4 days until colony formation was observed.

Example 12

Ectopic expression analysis of LSR _ P5a _ FLAG _ M in stably transfected HEK293T cells

Expression of LSR _ P5a _ Flag _ m (SEQ ID NO: 144) in stably transfected HEK293T cells was determined by Western blot analysis of cell lysates using anti-LSR antibodies and anti-Flag antibodies as indicated in Table 9.

Cells were dissociated from the plate using PBS-Based Enzyme-free Cell Dissociation Buffer (Gibco, 13151-014), washed in Dulbecco's Phosphate Buffered Saline (PBS) (Biologic industries, 02 023-1A) and centrifuged at 1200g for 5 min. Whole cell extraction was performed by resuspending the cells in 50mM Tris-HCl pH7.4, 150mM NaCl, 1mM EDTA, 1% Triton X-100 supplemented with 25X complete EDTA without protease inhibitor cocktail (Roche, 11873580001) and vortexing for 20 seconds. Cell extracts were collected after centrifugation at 20,000g for 20 minutes at 4 ℃ and protein concentration was determined using the Bradford Biorad protein assay (Biorad cat # 500-. Equivalent protein amounts were analyzed by SDS-PAGE (Invitrogen, NuPAGE 4-12% NuPAGEbis Tris, Cat # NP0335, NP0322) and transferred to nitrocellulose membranes (BA83, 0.2 μm, Schleicher and Schuell, Cat # 401385). Membranes were blocked with TTBS (Biolab, Cat #: 20892323)/10% skim milk (Difco, Cat #232100) and incubated for 16 hours at 4 ℃ with indicated primary antibody (FIG. 18) diluted in TTBS/5% BSA (Sigma-Aldrich, A4503) at the indicated concentration (Table 9). After 3 washes with TTBS, the membrane was further incubated for 1 hour at room temperature with secondary conjugated antibody diluted in TTBS as indicated. Chemiluminescence was performed using ECL Western Blotting Detection Reagent (GE Healthcare, Cat # RPN2209) and the membrane was exposed to Super RX Fuji X-Ray film (catalog No. 4741008389).

FIG. 18 shows the expression of LSR _ P5a _ Flag _ m protein (SEQ ID NO: 144) in recombinant HEK293T cells of the expected band size-70 k Da, detected using Flag (Sigma cat # A8592) (FIG. 18A) and anti-LSR antibody as follows: abnova, cat # H00051599-B01P (FIG. 18B), Abeam, cat ab59646 (FIG. 18C) and Sigma cat # HPA007270 (FIG. 18D).

Example 13

Determination of the subcellular localization of ectopic LSR _ P5a _ FLAG _ M in HEK293T cells

The subcellular localization of LSR _ P5a _ Flag _ m protein (SEQ ID NO: 144) in stably transfected cells was determined by confocal microscopy.

Stably transfected recombinant HEK293T cells expressing LSR _ P5a _ Flag _ m (SEQ ID NO: 144) described above were plated on cover glass pre-coated with poly-L-lysine (Sigma, Cat. No. P4832). After 24 hours, the cells were processed for immunostaining and analyzed by confocal microscopy. The coverslip was washed in Phosphate Buffered Saline (PBS) and then fixed in a solution of PBS/3.7% Paraformaldehyde (PFA) (EMS, Cat. No.: 15710)/3% glucose (Sigma, Cat. No.: G5767) for 15 minutes. PFA was quenched with PBS/3mM glycine (Sigma, cat # G7126) for 5 min. After 5 min of washing in two PBS's, the cells were permeabilized using PBS/0.1% Triton-X100 for 5 min at room temperature and washed twice in PBS. Then, blocking of the nonspecific region was performed using PBS/Bovine Serum Albumin (BSA) (Sigma, cat # A4503) for 20 minutes. The coverslip was then incubated with various primary antibodies for 1 hour in a humid heat test chamber (humid chamber), each primary antibody used being diluted as indicated in PBS/5% BSA, followed by three 5 minute washes in PBS. The coverslips were then incubated for 30 minutes with the corresponding secondary antibody diluted in PBS/2.5% BSA at the indicated dilution. The antibodies and dilutions used are specified in table 9. After a prewashing in Hank's Balanced Salt Solutions W/o phenol red (HBSS) (Biological Industries, Cat. No. 02-016-1), the coverslips were incubated for 10min with WGA-Alexa 488 (Invitrogen, Cat. W11261) diluted 1: 200 in HBSS, washed in HBSS and incubated in BISBENZIMIDE H33258 (BISBAZIMIDE H33258) (Sigma, Cat. No. 14530) diluted 1: 1000 in HBSS. The coverslips were then mounted on slides with Gel Mount Aqueous medium (Sigma, catalog No. G0918) and the cells were observed for the presence of fluorescent product using a confocal microscope.

Subcellular localization of LSR _ P5a _ Flag _ m is shown in FIG. 19, with LSR _ P5a _ Flag _ m (SEQ ID NO: 144) being predominantly localized to the cytoplasm of the cell, but also detectable at the cell surface, using anti-Flag (Sigma cat # A9594) (FIG. 19A) and anti-LSR antibodies as follows: abcam, cat ab59646 (FIG. 19B), Abnova, cat # H00051599-B01P (FIG. 19C) and Sigma cat # HPA007270 (FIG. 19D).

Example 14

Analysis of expression of endogenous LSR proteins in different cell lines

Expression of endogenous LSR protein in different cell lines was analyzed by western blot as follows.

Cell extracts of SK-OV3(ATCC number HTB-77), Caov3(ATCC number HTB-75), OVCAR3(ATCC number HTB-161), ES-2(ATCC number CRL-1978), OV-90(ATCC number CRL-11732), TOV112D (ATCC number CRL-11731), and Hep G2(ATCC number HB-8065) were prepared as described above.

Cell extracts of HeLa (catalog No. sc-2200), MCF-7 (catalog No. sc-2206), CaCo2 (catalog No. sc-2262), and SKBR3 (catalog No. sc-2218) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz Biotechnology).

Equal amounts of protein were analyzed by SDS-PAGE and transferred to nitrocellulose membranes as described above. Membranes were blocked with TTBS (Biolab, Cat #: 20892323)/10% skim milk (Difco, Cat #232100) and incubated for 16 hours at 4 ℃ with anti-LSR antibody (Abcam, Cat # ab59646) diluted in TTBS/5% BSA (Sigma-Aldrich, A4503) at the indicated concentration (Table 9). After 3 washes with TTBS, the membrane was further incubated for 1 hour at room temperature with secondary conjugated antibody (table 9) diluted in TTBS as indicated. Chemiluminescence was performed using ECL western Blotting Detection Reagent (ECL western Blotting Detection Reagent) (GE Healthcare, Cat # RPN2209) and the membrane was exposed to Super RX Fuji X-Ray film (Super RX Fuji X-Ray film) (catalog No. 4741008389).

Figure 20 shows endogenous expression of LSR in different cell lines. Bands at 72kDa corresponding to LSR were detected with anti-LSR antibodies in extracts of SK-OV3, Caov3, OVCAR3, OV-90, Hep G2, HeLa, CaCo2, and SkBR3 (FIG. 20A). anti-GAPDH (Abeam cat # ab9484) served as a loading control (fig. 20B).

Table 9: first and second antibodies

Example 15

Expression of TMEM25_ transcript detectable by the amplicon as depicted in sequence name TMEM25_ SEG21-27 in normal and cancerous breast tissue

Expression of TMEM25 transcript detectable by or according to seg21-27-TMEM25_ seg _21-27_200-344/346_ amplicon (SEQ ID NO: 123) and primers TMEM25_ seg21F _200 and 344(SEQ ID NO: 124) and TMEM25_ seg27R _200 and 346(SEQ ID NO: 125) was measured by real-time PCR. In parallel, similar measurements were made of the expression of several housekeeping genes-G6 PD (GenBank accession NM-000402; (SEQ ID NO: 111) G6 PD-amplicon (SEQ ID NO: 114)), RPL19(GenBank accession NM 000981; (SEQ ID NO: 119) -RPL 19-amplicon (SEQ ID NO: 122)), PBGD (GenBank accession BC 019323; (SEQ ID NO: 115) PB GD-amplicon (SEQ ID NO: 118)), and SDHA (GenBank accession NM-004168; (SEQ ID NO: 103) SDHA-amplicon (SEQ ID NO: 106)). For each RT sample, the expression of the amplicons above was normalized to the normalization factor calculated in the expression of these housekeeping genes as described in the materials and methods section. The normalized number of each RT sample was then divided by the median of the number of normal samples (sample numbers 43, 45, 46, 47, 48, 49, 50, 51, 52, 54, 56, 58, 59, 60, 61, 62, 63, 64, 66, 67, 68, and 69, table 1 above) to obtain one fold value of differential expression for each sample relative to the median of the normal samples.

In both experiments performed, no differential expression was observed for cancerous versus normal samples (fig. 21).

Primer pairs may also optionally and preferably be encompassed within the present invention; for example, for the above experiments, the following primer pair was used as only one non-limiting illustrative example of a suitable primer pair: TMEM25_ seg21F _200 and 344(SEQ ID NO.124) forward primers; and TMEM25_ seg27R _200 _ 346(SEQ ID NO.125) reverse primer.

The invention also preferably encompasses any amplicon obtained by using any suitable primer pair; for example, for the above experiments, the following amplicons were obtained as only one non-limiting illustrative example of suitable amplicons: TMEM25_ seg _21-27_200-344/346_ amplicon (SEQ ID NO: 123).

Example 16

Expression of TMEM25_ transcript detectable by the amplicon as depicted in the sequence name TMEM25_ SEG21-27 in different normal tissues

Expression of TMEM25 transcript detectable by or according to seg21-27-TMEM25_ seg _21-27_200-344/346_ amplicon (SEQ ID NO: 123) and primers TMEM25_ seg21F _200 and 344(SEQ ID NO: 124) and TMEM25_ seg27R _200 and 346(SEQ ID NO: 125) was measured by real-time PCR. In parallel, similar measurements were made of the expression of several housekeeping genes-SDHA (GenBank accession NM-004168; (SEQ ID NO: 103) SDHA _ amplicon (SEQ ID NO: 106)), G6PD (GenBank accession NM-000402; (SEQ ID NO: 111) G6PD _ amplicon (SEQ ID NO: 114)), and HPRT1(GenBank accession NM-000194; (SEQ ID NO: 107) HPRTl _ amplicon (SEQ ID NO: 110)). For each RT sample, the expression of the amplicons above was normalized to the normalization factor calculated in the expression of housekeeping genes as described in the normalization method 2 of the materials and methods section. The normalized number of each RT sample was then divided by the median of the number of breast samples (sample numbers 30, 31, 32, and 33, table 2 above) to obtain a relative expression value for each sample relative to the median of the breast samples (fig. 22).

Example 17

Cloning of the TMEM25 protein

Cloning of TMEM25_ T0_ P5 ORF fused to FLAG tag

Cloning of TMEM25_ T0_ P5 Open Reading Frame (ORF) (SEQ ID NO: 130) fused to FLAG (SEQ ID NO: 153) was performed by RT PCR as follows.

Mu.l of undiluted colon cancer pool DNA served as template for the PCR reaction. PCR was performed using KAPA HifiDNA polymerase (KAPABIOSYSTEM, Cat. No. KK2101) under the following conditions: 1. mu.l of the above cDNA; mu.l (25. mu.M) -each of the primers 200 and 374_ TMEM25_ NheI _ Kozak _ seg5F (SEQ ID NO: 127) and 200 and 375_ TMEM25_ Flag _ STOP _ EcoRI _ seg43R (SEQ ID NO: 128), total reaction volume of 50. mu.l; the reaction procedure is as follows: 5 minutes, 95 ℃; 40 cycles: 20 seconds, 98 ℃, 15 seconds, 55 ℃, 1 minute, 72 ℃; then 5 minutes, 72 ℃. Primers used included gene specific sequences; a restriction enzyme site; kozak sequence and FLAG tag.

25 μ l of the PCR product was loaded on a 1.5% agarose gel stained with ethidium bromide, electrophoresed at 100V in 1 × TAE solution, and visualized using UV light. After verifying the size of the expected band, 1 μ l of the PCR product on the upper template served as the template for re-amplification. PCR was performed using KAPA Hifi DNA polymerase (KAPABIOSYSTEM, catalog No. KK2101) under the same conditions as described above.

Using QIAquick^TMGel Extraction Kit(QIAquick^TMGel extraction kit) (Qiagene, catalog No.: 28707) The PCR product was purified from the gel.

The purified PCR product was digested with NheI and BamHI restriction enzymes (new england Biolabs, beverly, MA, u.s.a.). The digested DNA was then ligated into pIRESpuro3(pRp) vector (Clontech, cat No: 631619) previously digested with the above restriction enzymes, using T4DNA ligase (Promega, Prologeg, Cat. No: M1801). The resulting DNA was transformed into competent E.coli bacterium DH5 alpha (RBC Bioscience, Inc., Taipei, Taiwan, Cat: RH816) according to the manufacturer's instructions, then plated on agar plates for selection of recombinant plasmids, and incubated overnight at 37 ℃. The next day, the pair was PCR-paired using pIRESpuro3 vector-specific primers and gene-specific primersPositive colonies were screened (data not shown). PCR products were analyzed as described above using a 2% agarose gel. After verifying the size of the expected bands, positive colonies were grown in 5ml of stock Broth (Terrific Broth) supplemented with 10. mu.g/ml ampicillin, shaking overnight at 37 ℃. Using Qiaprep ^TMSpin miniprep kit (Qiaprep)^TMSpin Miniprep Kit) (Qiagene, catalog No.: 27106) Plasmid DNA was isolated from bacterial cultures. The exact clones were verified by sequencing the inserts (Hylabs, Raoholtt, Israel). When error-free colonies were detected (i.e., no mutations within the ORF), the recombinant plasmids were processed for further analysis.

Cloning of unlabeled TMEM25_ T0_ P5 ORF

Cloning of the unlabeled Open Reading Frame (ORF) of TMEM25_ T0_ P5 (SEQ ID NO: 130) was performed by RT PCR as follows.

Mu.l of undiluted colon cancer pool DNA served as template for the PCR reaction. PCR was performed using KAPA HifiDNA polymerase (KAPABIOSYSTEM, Cat. No. KK2101) under the following conditions: 1. mu.l of the above cDNA; mu.l (25. mu.M) -each of the primers 200-374-TMEM 25-NheI-Kozak-seg 5F (SEQ ID NO: 127) and 200-377-TMEM 25-STOP-EcoRI-seg 43R (SEQ ID NO: 131) in a total reaction volume of 50. mu.l; the reaction procedure is as follows: 5 minutes, 95 ℃; 40 cycles: 20 seconds, 98 ℃, 15 seconds, 55 ℃, 1 minute, 72 ℃; then 5 minutes, 72 ℃. Primers used included gene specific sequences; restriction enzyme sites and Kozak sequence.

25 μ l of the PCR product was loaded on a 1.5% agarose gel stained with ethidium bromide, electrophoresed at 100V in 1 × TAE solution, and visualized using UV light. After verifying the size of the expected band, 5 μ l of the PCR product on the upper template served as the template for re-amplification. PCR was performed using KAPA Hifi DNA polymerase (KAPABIOSYSTEM, catalog No. KK2101) under the same conditions as described above.

Using QIAquick^TMGel Extraction Kit(QIAquick^TMGel extractionKit) (Qiagene company, catalog No.: 28707) The PCR product was purified from the gel.

The purified PCR product was digested with NheI and BamHI restriction enzymes (New England Biolabs, Beverly, Mass., USA). The digested DNA was then ligated into pIRESpuro3(pRp) vector (Clontech, cat No: 631619) previously digested with the above restriction enzymes, using T4 DNA ligase (Promega, Prologeg, Cat. No: M1801). The resulting DNA was transformed into competent E.coli bacterium DH5 alpha (RBC Bioscience, Inc., Taipei, Taiwan, Cat: RH816) according to the manufacturer's instructions, then plated on agar plates for selection of recombinant plasmids, and incubated overnight at 37 ℃. The next day, positive colonies were screened by PCR using pIRESpuro3 vector-specific primers and gene-specific primers (data not shown). PCR products were analyzed as described above using a 2% agarose gel. After verifying the size of the expected bands, positive colonies were grown in 5ml of stock Broth (Terrific Broth) supplemented with 100. mu.g/ml ampicillin, shaking overnight at 37 ℃. Using Qiaprep ^TMSpin miniprep kit (Qiaprep)^TMSpin Miniprep Kit) (Qiagene, catalog No.: 27106) Plasmid DNA was isolated from bacterial cultures. The exact clone was verified by sequencing the insert (Hylabs, Rehowter, Israel). When error-free colonies were detected (i.e., no mutations within the ORF), the recombinant plasmids were processed for further study.

Example 18

Generation of a Stable library expressing TMEM25_ P5 and TMEM25_ P5_ FLAG proteins

TMEM25_ T0_ P5(SEQ ID NO: 130) and TMEM25_ T0_ P5_ FLAG (SEQ ID NO: 126) pIRESpuro3 constructs or pIRESpuro3 empty vectors were stably transfected into HEK-293T cells as follows:

HEK-293T (ATCC, CRL-11268) cells were plated on sterile 6-well plates suitable for tissue culture using: 2ml of prewarmed complete medium, DMEM [ Dulbecco's modified Eagle's Media (Dulbecco's modified Igor medium), Biotech Industries (Beiit Ha' Emek, Israel, Cat. No.: 01-055-1A) ] + 10% FBS [ fetal bovine serum, Biotech Industries (Biologic Industries) (BeHa 'Emek, Israel, Cat. No.: 04-001-1A) ] +4mM L-glutamine (Biologic Industries (Beit Ha' Emek, Israel), Cat. No.: 03-020-1A). 350,000 cells per well were transfected with 2. mu.g of the DNA construct using 6. mu.l of FuGENE 6 reagent (Roche, Cat. No.: 11-814-443-001) diluted to 94ul of DMEM. The mixture was incubated at room temperature for 15 minutes. The complex mixture was added dropwise to the cells and swirled. The cells were placed in an incubator maintained at 37 ℃ with a CO2 content of 5%. 48 hours after transfection, the transfected cells were transferred to 75cm2 tissue culture flasks containing 15ml of selection medium (complete medium supplemented with 5. mu.g \ ml puromycin (Sigma, Cat. No. P8833)). Cells were placed in an incubator and the medium was changed every 3-4 days until colony formation was observed.

When a sufficient number of cells passed selection, 3-5 million cells were harvested. Cells were lysed in 300. mu.l of RIPA buffer (50mM TrisHC1 pH 8, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (Roche, Cat.: 11873580001) at 4 ℃. After centrifugation at 14,000x rpm for 10 minutes at 4 ℃, the clarified supernatant was transferred to a clean tube and used for the WB step: mu.g of the lysate was mixed with DTT 1, 4-dithiothreitol (DTT; a reducing agent) to a final concentration of 100 mM.

In addition, the samples were incubated at 100 ℃ for 10 minutes, followed by spinning at 14,000x rpm for 1 minute. When 30. mu.l of the sample is loaded 4-12% according to the manufacturer's instructionsBis-Tris gels (Bis-Trisgel) (Invitrogen, catalog No.: NP0321), SDS-PAGE (lemmli (Laemmli), uk, Nature 1970; 227; 680, 685) and the gel was washed in 1x MES SDS running buffer (Invitrogen, catalog No.: NP0060) Middling glue, using XCell SureLock^TMMini-Cell (Invitrogen, catalog number E10001). Using XCell according to manufacturer's instructions ^TMII blotting device (Cell)^TMII blotting appatatus) (Invitrogen, catalog No. E19051) the isolated proteins were transferred to nitrocellulose membranes (Schleicher and Schuell, catalog No.: 401385).

The membrane containing the imprinted protein was treated for antibody detection as follows:

the non-specific regions of the membrane were blocked by incubation for 1 hour at room temperature in 5% skim milk diluted in Phosphate Buffered Saline (PBS) supplemented with 0.05% tween-20 (PBST) (all subsequent incubations occurred at room temperature for 1 hour). The blocking solution was then replaced with a first rabbit anti-TMEM 25 antibody (Cat No. HPA012163, Sigma) diluted 1: 500 in 5% Bovine Serum Albumin (BSA) (Sigma, Cat # A4503) diluted in PBS. After 1 hour incubation, three 5 minute washes with the second antibody: affinity purification of Goat anti-Rabbit IgG coupled to peroxidase conjugated Goat anti-Rabbit IgG (Goat anti-Rabbit concugated to peroxidase conjugated Goat anti-Rabbit IgG) diluted 1: 20,000 in blocking solution (Jackson, Cat. 111. sub. 035-00). The proteins were also detected by mouse anti-Flag M2-peroxidase (Sigma, cat # A8592) diluted 1: 1000 in blocking solution. After 1 hour incubation, 3X5 minutes of washing, ECL substrate (PIERCE, Cat: PIR-34080) was applied for 1 minute, followed by exposure to X-ray film (Fuji, Cat: 100 NIF). The results are presented in fig. 23.

FIG. 23A demonstrates that the rabbit anti-TMEM 25_ P5 specifically recognizes TMEM25_ P5 protein (SEQ ID NO: 7) and TMEM25_ P5_ Flag (SEQ ID NO: 129) at the expected band size of about 40.2kDa, but not HEK _293T _ pRp 3.

FIG. 23B demonstrates that the TMEM25_ P5_ Flag protein (SEQ ID NO: 129) at the expected band size of about 40.2kDa is specifically recognized by anti-Flag.

Example 19

Determination of subcellular localization of ectopic TMEM25_ P5 and TMEM25_ P5_ FLAG in HEK293T cells by immunofluorescence

Using confocal microscopy, protein localization of TMEM25_ P5(SEQ ID NO: 7) and TMEM25_ P5_ FLAG (SEQ ID NO: 129) was observed in stable transfections as described above.

Stably transfected recombinant HEK293T cells expressing TMEM25_ P5(SEQ ID NO: 7) and TMEM25_ P5_ FLAG (SEQ ID NO: 129) were plated on poly-L-lysine (Sigma, cat # P4832) pre-coated coverslips. After 24 hours, the cells were processed for immunostaining and analyzed by confocal microscopy.

The coverslip was washed in Phosphate Buffered Saline (PBS) and then fixed for 15 minutes using a solution of 3.7% Paraformaldehyde (PFA) (Sigma, Cat: P-6148)/3% glucose (Sigma, Cat: G5767) diluted in PBS. Quenching of PFA was performed by 5 min incubation in 3mM glycine (Sigma, Cat: G7126) diluted in PBS. After a 5-minute wash in both PBS, non-specific regions were blocked using 5% Bovine Serum Albumin (BSA) (Sigma, Cat: A4503) (diluted in PBS) for 20 minutes.

The coverslip was then incubated with the antibody in a moist heat test chamber (humid chamber) for 1 hour using either mouse anti-FLAG-Cy 3 antibody (Sigma, Cat: A9594) in 5% BSA diluted 1: 200 in PBS or rabbit anti-TMEM 25(Cat No. HPA012163, Sigma) in 5% BSA diluted 1: 50 in PBS followed by three 5 minute washes in PBS. Only for the anti-TMEM 25 Ab, a second Ab is required: donkey anti-rabbit cy3(cat #711-165-152, Jackson (Jackson) diluted 1: 200 in 5% BSA in PBS) was incubated in a humid heat test chamber (humidchamber) for 1 hour followed by three 5 min washes in PBS. After a pre-wash with BISBENZIMIDE H33258 (BISBENZIMIDE H33258) (HBSS) (Sigma, cat # 14530), the coverslips were incubated for 10min with WGA-Alexa 488 (Invitrogen, cat # W11261) diluted 1: 200 in HBSS, followed by two washes in HBSS and an incubation in H33258 (BISBENZIMIDE H33258) (HBSS) (Sigma, cat # 14530) diluted 1: 1000 in HBSS. The coverslip was then mounted on a slide with Gel Mount Aqueous medium (Sigma company, catalog No. G0918) and the cells were observed for the presence of fluorescent product using a confocal microscope.

The subcellular localization of TMEM25_ P5(SEQ ID NO: 132) and TMEM25_ P5_ Flag (SEQ ID NO: 129) using anti-TMEM 25 Ab are shown in FIGS. 24A and 24B, respectively. FIG. 24C shows TMEM25_ P5_ FLAG (SEQ ID NO: 129) localization using anti-FLAG Ab (Sigma, Cat: A9594). The TMEM25_ P5 protein was localized to the cell surface.

Example 20

Cellular localization of TMEM25_ P5_ Flag by FACS

Membrane localization of the TMEM25_ P5_ Flag protein (SEQ ID NO: 129) was observed in the above stable transfection, by flow cytometry analysis using anti-TMEM 25 antibody (Abl628, Yomics) and by normal mouse serum as negative control (015-000-120, Jackson). Recombinant HEK293T cells expressing TMEM25_ P5_ Flag were stained with Anti-TMEM 25 antibody (a) or by normal Mouse serum (B), followed by Donkey Anti-Mouse-DyLight 549 conjugated secondary Ab (Donkey Anti Mouse-DyLight 549 conjugated secondary Ab) (Jackson, 715-506-150) and observed for the presence of fluorescent signal.

Recombinant HEK293T-TMEM25_ P5_ Flag cells were dissociated from the plate using PBS-Based Enzyme-free Cell dissociation buffer (Gibco Corp.; 13151-014), washed in FACS buffer [ Dulbecco's Phosphate Buffered Saline (PBS) (Bioindustrial Industries, 02 023-1A)/1% bovine albumin (Sigma, A7030) ] and counted. 0.5X 10^6 cells were resuspended in 100. mu.l of antibody solution at a dilution of 1: 2250ul and incubated on ice for 1 hour. The cells were washed with ice cold FACS buffer and incubated on ice for 1 hour using the secondary antibody as indicated. The cells were washed with ice cold FACS buffer and resuspended in 500 μ l FACS buffer before analysis on a FACS apparatus (FACSCalibur, BD). Data were obtained and analyzed using Cellquest Pro ver.5.2.

The results presented in figure 25 demonstrate that anti-TMEM 25 antibody (a) binds to full-length TMEM25 protein in HEK293T recombinant cells expressing TMEM25_ P5_ Flag protein, compared to mouse serum (B) used as a negative control, indicating membrane localization of TMEM25 protein.

Example 21

Analysis of the expression of endogenous TMEM25 protein in different cell lines

The expression of endogenous TMEM25 protein in different cell lines was analyzed by western blotting as follows.

Cell extracts of JurKAT (ATCC No. TIB-152), Daudi (ATCC No. CCL-213), RPMI8226(ATCC No. CCL-155), G-361(ATCC No. CRL-1424), KARPAS (ATCC No. VR-702) were prepared as described above (lanes 3-7 in FIG. 26-see figure for corresponding lane/material arrangement).

Whole cell lysates were prepared as described above and analyzed by western blot. Equal amounts of protein were analyzed by SDS-PAGE and transferred to nitrocellulose membranes as described above.

Membranes were blocked by incubation for 1 hour at room temperature in 5% skim milk diluted in Phosphate Buffered Saline (PBS) supplemented with 0.05% tween-20 (PBST) (all subsequent incubations occurred at room temperature for 1 hour). The blocking solution was then replaced with a first rabbit anti-TMEM 25 antibody (Cat No. HPA012163, Sigma) diluted 1: 500 in 5% Bovine Serum Albumin (BSA) (Sigma, Cat # A4503) diluted in PBS. After 1 hour incubation, three 5 minute washes with the second antibody: affinity purification of Goat anti-Rabbit IgG coupled to peroxidase conjugated Goat anti-Rabbit IgG (Goat anti-Rabbit conjugated to peroxidase conjugated Rabbit anti-Rabbit IgG) diluted 1: 20,000 in blocking solution (Jackson, Cat. No.: 111-035-00) the protein was also detected by mouse anti-Flag M2-peroxidase (Sigma, Cat. No.: A8592) diluted 1: 1000 in blocking solution. After 1 hour incubation, 3X5 minutes of washing, ECL substrate (PIERCE, Cat: PIR-34080) was applied for 1 minute, followed by exposure to X-ray film (Fuji, Cat: 100 NIF).

Figure 26 shows the endogenous expression of TMEM25 in different cell lines. A protein of 40.2kDa as observed in HEK293T cells expressing TMEM25_ P5_ Flag (lane 2; lane 1 shows a control without Flag) was detected with anti-TMEM 25 antibodies in extracts of RPMI8226 (lane 5), Daudi (lane 6) and Jurkat (lane 7).

Example 22

Transfection of stable HEK293T _ TMEM25 with siRNA into TMEM25

In the aforementioned transfection with TMEM25_ P5-SiRNA, specific knockdown of TMEM25_ P5_ Flag protein (SEQ ID NO: 129) was observed in HEK293T cells stably expressing TMEM25_ P5_ Flag (SEQ ID NO: 129).

siRNAs purchased from Dharmacon corporation were as follows: TMEM25(L-018183-00-0005, Dharmacon, on TARGET plus SMART library, human TMEM25(84866), 5nmol) and scrambled SiRNA (Dharmacon, D-001810-10-05) as a negative control.

Cells were plated at 50% -70% cell confluence (confluency) 24hr prior to transfection. 250pmol of siRNA complex was added to 250ul of reduced serum Opti-MEM (cat 31985, GIBCO). Concurrently, liposome 2000 reagent (cat # 11668019, Invitrogen) was mixed; 5ul was added to 250ul of reduced serum Opti-MEM (cat 31985, GIBCO). Tubes were pooled and incubated at RT for 15-30min for adequate complex formation; the material was then distributed over the cells and incubated for 48 hr. Cells were harvested and cell lysates prepared as described above and detected by anti-TMEM 25(Cat No. hpaj012163, Sigma), followed by a second donkey anti-rabbit conjugated to peroxidase.

FIG. 27 demonstrates the reduced specificity of TMEM25_ P5_ FLAG protein (SEQ ID NO: 129) in HEK293T cells stably expressing TMEM25_ P5_ FLAG (SEQ ID NO: 129), transfected with TMEM25_ P5 siRNA (L-018183-00-0005, Dharmacon) (lane 2), compared to HEK293T cells stably expressing TMEM25_ P5_ FLAG, transfected with Scrambled-SiRNA (lane 1) (Dharmacon, D-001810-10-05), using an anti-TMEM 25 antibody (Sigma, cat # HPA 012163).

Example 23

Immunohistochemistry (IHC) Using anti-LSR and anti-TMEM 25 polyclonal antibodies

To evaluate the tissue binding profile, anti-LSR (Abeam, Cat No. ab59646) and anti-TMEM 25(Cat No. HPA012163, Sigma Co.) were applied to the panels of tumor Tissue Microarrays (TMA) as detailed in Table 10.

HEK-293 cells expressing LSR _ P5a _ Flag _ m (SEQ ID NO: 144) or TMEM25_ P5_ Flag (SEQ ID NO: 129) were used as positive controls for calibration of stained pAb. HEK293T cells transfected with the empty vector were used as negative controls along with rabbit serum IgG antibodies.

Immunohistochemical detection by antibodies anti-LSR and correspondingly anti-LSR _ P5a _ Flag _ m (SEQ ID NO: 144) or TMEM25_ P5_ Flag (SEQ ID NO: 129) of TMEM25 was calibrated in Formalin Fixed Paraffin Embedded (FFPE) sections. Two antigen retrieval methods were used: pH 6.1 and pH 9.0 at three Ab concentrations (3. mu.g/ml, 1. mu.g/ml, 0.3. mu.g/ml).

The antigen retrieval method was performed as follows. The FFPE sections were deparaffinized, antigen retrieved and rehydrated using a pH 6.1 or pH 9.0 Flex + three-in-one (3-in-l) antigen retrieval buffer and a PT Link automated antigen retrieval system at 95 ℃ with automatic heating and cooling for 20 min.

After antigen retrieval, sections were washed 2x5min in distilled water and then loaded into DAKO Autostainer plus (DAKO Autostainer plus). Sections were then incubated for 10min with Flex + peroxidase blocking reagent, rinsed twice in 50mM Tris.HCl, 300mM NaCl, 0.1% Tween-20, pH 7.6(TBST), followed by incubation for 10min with protein blocking reagent (DAKO X0909).

Sections were incubated for 30min with primary antibody diluted in DAKO Envision Flex antibody diluent (DAKO Cytomation, Cat # K8006). After incubation with primary antibody, sections were then rinsed twice in FLEX buffer, incubated with anti-mouse/rabbit FLEX + HRP for 20min, rinsed twice in FLEX buffer and then incubated with Diaminobenzidine (DAB) substrate for 10 min. The color reaction was stopped by rinsing the slide with distilled water.

After pigment formation, sections were counterstained with hematoxylin, dehydrated in increasing series of ethanol (90% -99% -100%), washed in three changes of xylene and mounted under DePeX. Stained sections were analyzed using an Olympus BX51 microscope with a Leica DFC290 camera.

Figure 28 shows that anti-LSR antibodies (cat No. ab59646, Abeam) (panels A, C and E) at dose-dependent concentrations of 3ug/ml, 1ug/ml and 0.3ug/ml in sections of positive control cell lines (panels B, D and F), respectively, show specific immunoreactivity at pH 9, as compared to negative control cell lines (panels A, C and F), according to the antigen retrieval method described previously.

Figure 29 shows that specific immunoreactivity was shown at pH 9 in anti-TMEM 25(cat No. hpaa012163, Sigma) (panels A, C and E) at dose-dependent concentrations of 3ug/ml, 1ug/ml and 0.3ug/ml, respectively, in sections of positive control cell lines (panels B, D and F) compared to negative control cell lines (panels A, C and F), according to the antigen retrieval method described previously.

Table 10: summary of tissue samples included in Tissue Microarrays (TMAs).

Example 24

full-Length confirmation of transcripts encoding LY6G6F

The full-length transcript encoding LY6G6F (SEQ ID NO: 1) was confirmed as follows:

1. the reverse transcription reaction was performed as follows: mu.g of purified RNA (lung normal) was mixed with 150ng of random hexamer primer (Invitrogen, Calsbarda, CA, USA, Cat. No.: 48190-. The mixture was incubated at 65 ℃ for 5min and then rapidly frozen on ice. Thereafter, 50. mu.l of 5 XSuperScriptII first strand buffer (SuperScriptII first strand buffer) (Invitrogen, Cat.: 18064-014, Cat.: Y00146), 24. mu.l of 0.1M DTT, and 400 units of RNase inhibitor (RNase) (Promega, Melowky, WS, USA, Cat.: N2511) were added, and the mixture was incubated at 25 ℃ for 10min, followed by further incubation at 42 ℃ for 2 min. Then, 10. mu.l (2000 units) of SuperScriptII (Invitrogen, Cat. No.: 18064-014) was added, and the reaction (final volume 250. mu.l) was further incubated at 42 ℃ for 50min and then inactivated at 70 ℃ for 15 min. The resulting cDNA was diluted 1: 20 in TE buffer (10mM Tris, 1mM EDTA pH 8)

2. PCR was performed using 2 XGoTaq ReadyMix (Promega, catalog No. M7122.) under the following conditions: 12.5ul of GoTaq ready mix; 5ul cDNA from above; 1ul of 10uM forward primer 100-690(SEQ ID NO: 51); : 1ul of 10uM reverse primer 100-691(SEQ ID NO: 52) and 5.5ul of H20, the total reaction volume being 25 ul; the reaction procedure is as follows: 5 minutes, 95 ℃; 35 cycles: 30 seconds, 94 ℃, 30 seconds, 53 ℃, 50 seconds, 72 ℃; then 10 minutes, 72 ℃. Details regarding the primers are presented in table 11 below.

The above PCR products were loaded on a 1.2% agarose gel stained with ethidium bromide, electrophoresed at 100V in 1 × TAE solution, and visualized using UV light. Bands of the expected size were used with QiaQuick^TMGelExtraction kit(QiaQuick^TMGel extraction kit) (Qiagene, catalog No.: 28707) Excised from the gel and extracted. The purified DNA was then sequenced using the above primers (university of Telaviv (Tel-Avivuniversity), Israel) and tested against the full-length LY6G 6F-encoding transcript (SEQ ID NO: 1).

Example 25

Cloning of full-Length transcript encoding LY6G6F fused to EGFP

Cloning of the full-length transcript encoding LY6G6F fused to EGFP (enhanced green fluorescent protein) was performed as follows.

First, an EGFP expression vector was constructed, and then a gene encoding SEQ ID NO: the LY6G6F open reading frame (SEQ ID NO: 57) of the amino acid sequence set forth in 58 was cloned. EGFP was subcloned into pIRESpuro3(Clontech, Cat. No.: 631619) as follows: the EGFP-N1 vector (Clontech, Cat: 6085-1) was digested with NheI and NotI to excise the EGFP gene. To obtain the EGFP-pIRESpuro3 vector, the EGFP insert was then ligated into pIRESpuro3(Clontech, Cat. No.: 631619) previously digested with the same enzymes.

Platinum PFX was used under the following conditions^TM(Invitrogen, Calsbad, CA, USA, Cat. No.: 1178-: 5 μ l of Platinum PFX 10 × buffer; 2 μ l-confirmed DNA from the above purification; 1. mu.l-10 mM dNTPs (2.5 mM each nucleotide); 1. mu.l of a Platinum PFX enzyme; 37. mu.l-H2O; 1 μ l of 10uM forward primer 100-729(SEQ ID NO: 53); 1ul of 10uM reverse primer 100-730(SEQ ID NO: 54) (10 uM each), total reaction volume of 50. mu.l; the reaction procedure is as follows: 5 minutes, 95 ℃; 35 cycles: 30 seconds, 94 ℃, 30 seconds, 55 ℃, 60 seconds and 68 ℃; then 10 minutes, 68 ℃. The primers used included gene specific sequences corresponding to the desired coordinates of the protein as well as restriction endonuclease sites and Kozak sequences, as listed in table 11 below and in fig. 6. Bold letters in table 11 indicate gene-specific sequences, whereas the restriction site extensions used for cloning the target are italicized and the Kozak sequence is underlined.

The above 5ul PCR product was loaded on a 1.2% agarose gel stained with ethidium bromide, electrophoresed at 100V in 1 × TAE solution, and visualized using UV light. After verifying the expected size of the band, the Qiaquick PCR purification kit (Qiaquick PCR purification kit) (Qiagen)^TMValencia, CA, u.s.a., catalog No. 28106) the remaining PCR product was processed for DNA purification. The extracted PCR products were digested with NheI and BamHI restriction enzymes (New England Biolabs, beverly, MA, u.s.a.) as listed in table 11. After digestion, the DNA was loaded on a 1.2% agarose gel as described above. Bands of the expected size were used with QiaQuick^TMGel Extraction kit(QiaQuick^TMGel extraction kit) (Qiagene, catalog No.: 28707) Excised from the gel and extracted.

The digested DNA was ligated into EGFP _ PIRESpuro3 vector previously digested with NheI and EcoRI restriction enzymes using the LigaFastTM Rapid DNA ligation System (Promega, Prologeg., catalog No.: M8221). The resulting DNA was transformed into competent E.coli bacterium DH5 alpha (RBC Bioscience, Inc., Taipei, Taiwan, Cat: RH816) according to the manufacturer's instructions, then plated on agar plates for selection of recombinant plasmids, and incubated overnight at 37 ℃.

GoTaq was usedReady Mix (Promega, catalog No. M7122) was screened for positive clones by PCR. Anodal clones were grown in 5ml of stock broth (terrifickoth) supplemented with 100. mu.g/ml ampicillin, and shaken overnight at 37 ℃. Using Qiaprep^TMSpin miniprep kit (Qiaprep)^TMSpinMiniprep Kit) (Qiagene, catalog No.: 27106) Plasmid DNA was isolated from bacterial cultures. The exact clone was verified by sequencing the insert (university of Telaviv, Israel). When error-free colonies were detected (i.e., no mutations within the ORF), the recombinant plasmids were processed for further analysis.

The full-length DNA sequence of the generated LY6G6F fused to EGFP (SEQ ID NO: 55) is shown in FIG. 7. In fig. 7, the gene-specific sequence corresponding to the full-length sequence of LY6G6F is marked in bold type, while the EGFP sequence is marked in italics and underlined. The full-length amino acid sequence of the generated LY6G6F fused to EGFP (SEQ ID NO: 56) is shown in FIG. 8; the gene-specific sequence corresponding to the full-length sequence of LY6G6F is marked in bold type, while the EGFP sequence is marked in italics and underlining.

Table 11: details of the primers

Example 26

Determination of the cellular localization of LY6G6F

To determine the cellular localization of the LY6G6F protein, the LY6G6F-EGFP fusion protein (SEQ ID NO: 56) was used. Localization of LY6G6F protein was observed in transient transfections using confocal microscopy (Chen et al, Molecular Vision 2002; 8; 372-388). 48 hours after transfection, these cells were observed for the presence of fluorescent products.

The above LY6G6F-EGFP pIRESpuro3 construct was transiently transfected into HEK-293T cells as follows:

2ml of pre-warmed DMEM [ Dulbecco's modified Eagle's Media (Dulbecco's modified Igor Medium), Biological Industries (Beit Ha' Emek, Israel), Cat. No.: 01-055-1A ] + 10% FBS (fetal bovine serum) +4mM L-glutamine, HEK-293T (ATCC, CRL-11268) cells were plated onto 13mM diameter sterile glass coverslips (Marienfeld, Cat. No.: 0111530) which were placed in 6-well plates. 500,000 cells per well were transfected with 2. mu.g of the DNA construct using 6. mu.l of FuGENE 6 reagent (Roche, Cat. No.: 11-814-443-001) diluted to 94ul of DMEM. The mixture was incubated at room temperature for 15 minutes. The complex mixture was added dropwise to the cells and swirled. The cells were placed in an incubator maintained at 37 ℃ with a CO2 content of 5%.

48 hours after transient transfection, these cells were further processed for analysis in a confocal microscope. The coverslip in Phosphate Buffered Saline (PBS) washing 3 times, and using 3.7% paraformaldehyde (Sigma, catalog number: P-6148) fixed 15 minutes. After 2 washes in PBS, the fixed coverslips were glued to the slides using mounting solution (Sigma, Cat # G0918) and the cells were observed for the presence of fluorescent product using a confocal microscope. The results are presented in fig. 9A and 9B.

FIGS. 9A and 9B demonstrate that LY6G6F _ EGFP (SEQ ID NO: 56) fusion protein localizes to the cell membrane when expressed in HEK 293T cells. This image was obtained using a 40x objective of a confocal microscope.

Example 27

Cloning and expression of LY6G6F, VSIG10, TMEM25, and LSR ECD-mouse IGg2A-FC fusion proteins

Mouse orthologs of human LY6G6F, VSIG10, TMEM25, and LSR proteins were identified using the BlastP software of the National Center for Biotechnology Information (NCBI) (using default parameters) and used to obtain experimental evidence of concepts related to the functionality of LY6G6F, VSIG10, TMEM25, and/or LSR Ig fusion proteins in animal models. The mouse orthologs corresponding to human LY6G6F, VSIG10, TMEM25 and LSR proteins are shown in SEQ ID NOs: 20. 19, 9 and 21. Amino acid alignments and comparisons of human LY6G6F, VSIG10, LSR, and TMEM25 proteins with the corresponding mouse orthologs are shown in fig. 5A, 5B, 5C, and 5D, respectively.

The cDNA sequences of each of the mouse TMEM25(SEQ ID NO: 9), LY6G6F (SEQ ID NO: 20), VSIG10(SEQ ID NO: 19), and LSR (SEQ ID NO: 21) proteins were fused to the Fc domain of mouse IgG2aFc (SEQ ID NO: 27). In all cases, for each ECD, the naturally corresponding signal peptide was used. The LY6G6F, VSIG10, TMEM25, or LSR ECD-mIgG2aFc Ig fusion proteins (SEQ ID NOS: 23, 24, 25, or 26, respectively) generated are shown in FIGS. 10A-D, respectively.

LY6G6F, VSIG10, TMEM25 or LSR ECD-mIgG2aFc fusion protein (SEQ ID NO: 23, 24, 25, or 26, respectively) was cloned into a retroviral vector (retrovector) followed by transduction of the retroviral vector into Catalent' S "autologous" (in-house) CHO-S cell lines. Pooled (pooled) populations were generated and fertility was confirmed. The library is then expanded and the relative fertility and relative copy number of the library are determined. Cell culture supernatants were analyzed by Catalent's Fc ELISA assay to confirm the production of LY6G6F, VSIG10, TMEM25 or LSR ECD-mIgG2aFc fusion proteins.

Protein solutions were tested for bioburden (bioburden) and endotoxin. Human fusion proteins consisting of LY6G6F, VSIG10, TMEM25 or the human ECD of LSR ECD fused to human IgG1 (as depicted in figure 11) were also expressed using a similar system. LY6G6F, VSIG10, TMEM25 or LSR ECD-Ig fusion proteins for mouse and human T cells Evaluation of the effect of in vitro activation:

example 28:

effect of LY6G6F, VSIG10, TMEM25 or LSR ECD-IG fusion proteins on activation of DO11.10 primary CD4+ T cells by OVA peptides

The original CD4 was isolated from the spleen of five DO11.10 mice (Jackson) by automax sorting⁺T cell: CD 4-negative sorting (Miltenyi, Cat # 130-. Balb/c whole splenocytes were also collected from one mouse and irradiated using 3000rads to serve as DO11.10 CD4⁺Antigen Presenting Cells (APCs) of T cells. Will be the original CD4⁺T cells to irradiated APCs at a ratio of 1: 1(APC to T cells) at 5X10 per well⁵The cells were cultured in flat bottom 96 well plates in 200ul HL-1 medium and activated with 20ug/ml or 2ug/ml OVA323-339 in the presence of one of TMEM25-ECD-Ig (SEQ ID NO: 25), LSR-ECD-Ig (SEQ ID NO: 26), LY6G6F-ECD-Ig (SEQ ID NO: 23) at the indicated concentrations. As positive controls, B7-H4-Ig (R and D systems) or CTA4-Ig (mouse ECD fused to mIgG2a Fc) were used. Isotype control Ig (mIgG2a, BioXCell, Cat. # BE0085) was used as a negative control. These cells were pulsed with 1uCi of tritiated thymidine for 24 hours and harvested at 72 hours.

As shown in FIG. 30, TMEM25-ECD-Ig, LSR-ECD-Ig, and LY6G6F-ECD-Ig caused dose-dependent inhibition of T cell activation. This is shown as inhibition of T cell proliferation induced by OVA323-339 at 20ug/ml (fig. 30A-C, E) or 2ug/ml (fig. 30D).

The VSIG10-ECD-Ig fusion protein (SEQ ID NO: 24) did not show activity in three experiments performed in similar assays.

Example 29:

effect of LY6G6F, VSIG10, TMEM25 or LSR ECD-IG fusion proteins on activation of naive CD4+ T cells by anti-CD 3/anti-CD 28 coated beads

Isolation from 5 SJL (Harlan Co.) mice by automax sorting as described in the previous sectionGo out original CD4⁺T cells. The beads were coated following the manufacturer's protocol (Dynabeads M-450Epoxy Cat.140.11, Invitrogen) with anti-CD 3(0.5 ug/ml; clone 2C11) and anti-CD 28(2 ug/ml; clone 37.51eBioscience) and increasing concentrations of LSR-ECD-Ig or mIgG2a isotype control (mIgG2a, BioXCell Cat. # BE 85) (0.1-10 ug/ml). The total amount of protein used to coat the beads with LSR-ECD-Ig was made up to 10ug/ml (with control Ig). Naive CD4+ T cells (0.5X 10)⁶Per well) was activated with coated beads at a ratio of 1: 2 (beads to T cells). After 24 hours, the cells were pulsed with 1uCi of tritiated thymidine and harvested after 72 hours.

LSR-ECD-Ig (SEQ ID NO: 26) significantly inhibited the proliferation of T cell proliferation and caused its effects in a dose-dependent manner (FIG. 31).

TMEM25, LY6G6F, and VSIG10 ECD Ig fusion proteins shown in figures 10 and 11 were tested in a similar assay with similar results.

Example 30

Dose response effects of LY6G6F, VSIG10, TMEM25, and LSR ECD Ig fusion proteins on anti-CDS bound plate-activated mouse CD4+ T cells as demonstrated in cytokine production and expression of the activation marker CD 69.

Untounched CD4+ CD25-T cells were isolated from a pool of spleen and lymph node cells of BALB/C mice by negative selection using the CD4+ CD62L + T cell isolation Kit (CD4+ CD62L + T cell isolation Kit) (Miltenyi, Cat #130-093-227) according to the manufacturer's instructions. The purity obtained was > 95 percent.

Tissue culture 96-well plates were coated with 2ug/ml anti-CD 3mAb (clone 145-2C11) overnight at 4 ℃ in the presence of 1ug/ml, 5ug/ml, and 10ug/ml LY6G6F, VSIG10, TMEM25, and LSR ECD-Ig fusion proteins (SEQ ID NOS: 23, 24, 25, and 26, respectively). To complement the total protein concentration of 12. mu.g/ml per well, control mIgG2a (clone C1.18.4 from BioXCell; Cat # BE0085) was added to each well. The wells were arranged at 1x per well 10⁵CD4+ CD25-T cells were plated. 48hr after stimulation, culture supernatants were collected and analyzed using a mouse IFN γ ELISA kit, and these cells were analyzed by flow cytometry for expression of the activation marker CD 69.

The results shown in figure 32 demonstrate the inhibitory effect of LY6G6F, VSIG10, TMEM25 and LSR ECD-Ig fusion proteins on CD 4T cell activation, as demonstrated by reduced IFN γ secretion (figure 32A) and reduced CD69 expression (figure 32B) in TCR stimulation, compared to control mIgG2A and CTLA 4-Ig.

Example 31

Effect of LY6G6F, VSIG10, TMEM25 or LSR ECD-IG fusion proteins on CD4+ T cell differentiation in vitro.

To test the ability of LY6G6F, VSIG10, TMEM25, and LSR Ig fusion proteins to inhibit CD4+ T cell differentiation, naive CD4+ T cells were isolated from DO11.10 mice that were transgenic for T Cell Receptor (TCR) specific for OVA323-339 peptide. Using DO 11.10T cells, both polyclonal (anti-CD 3/anti-CD 28 mAb) and peptide-specific responses to the same population of CD4+ T cells were able to be studied. Naive CD4+ T cells were isolated from DO11.10 mice and activated in culture in the presence of anti-CD 3/anti-CD 28 coated beads or OVA323-339 peptide plus irradiated BALB/c spleen cells in the presence of LY6G6F, VSIG10, TMEM25 or LSR ECD-Ig fusion protein, control Ig, or B7-H4 Ig. These cells were activated in the presence of Th driving conditions as follows: th0 cells (IL-2), Th1 cells (IL-2+ IL-12), Th2 cells (IL-2+ IL-4), or Th17 cells (TGF-. beta. + IL-6+ IL-23+ anti-IL-2). The effects on T cell differentiation and Th-specific responses were assessed by measuring cell proliferation and production of subtype-specific cytokines: IL-4, IL-5, IL-10, IL-17, IFN-gamma.

Example 32

Evaluation of the effect of LY6G6F, VSIG10, TMEM25 or LSR ECD IG fusion proteins on human T cell activation.

The effect of LY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion proteins on human T cell responses was tested by two different in vitro assays using purified human T cells. In the first assay, human T cells were activated by anti-CD 3 and anti-CD 28 coated beads, and in the other assay, activation was performed using anti-CD 3 and anti-CD 28 antibodies in the presence of autologous, radiation-treated PBMCs. The regulatory activity of LY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion proteins on human T cell activation was assessed by measuring cell proliferation and cytokine release.

Study I-activation of human T cells by anti-CD 3 and anti-CD 28-coated beads is inhibited by fusion proteins

Naive CD4+ T cells were isolated from 4 healthy human donors and activated with anti-CD 3 mAb/anti-CD 28 mAb coated beads in the presence of control mIgG2a, or either LY6G6F, VSIG10, TMEM25, or LSR ECD Ig fusion protein. Establishing two parallel culture groups; one culture was pulsed with tritiated thymidine for 24 hours and harvested at 72 hours, while the second plate was harvested at 96 hours for cytokine production via LiquiChip.

Study II-activation of human T cells by radiation-treated autologous PBMC is inhibited by fusion protein

Total PBMCs were isolated from fresh blood of healthy human donors using ficoll gradients. Mix 10x10⁶Total PBMCs were resuspended in Ex-Vivo 20 medium and irradiated at 3000 rad. These cells are used to activate isolated T cells in vitro by presenting anti-CD 3, anti-CD 28, or any of the test proteins. The balance of PBMCs was used for T cell Isolation via the use of CD4+ T cell Isolation Kit II (CD4+ T cell Isolation Kit II) from Miltenyi.

For activation, at 5X10⁵In the presence of autologous, radiation-treated PBMC, 5x10⁵The isolated T cells are cultured. anti-CD 3 (0.5. mu.g/ml), anti-CD 28 (2. mu.g/ml) and LY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion proteins or control Ig (mIgG2a) were added in soluble form. Cultures were pulsed with 1uCi of tritiated thymidine for 24 hours, and proliferation was measured at 72 hours.

Example 33

Effect of LY6G6F, TMEM25 and LSR proteins on human T cell response in ectopic expression in APC-like cells

The effect of LY6G6F, TMEM25 and LSR on human T cell responses was assessed as their ectopic expression in 'T cell stimulator' cells: a murine thymoma cell line, Bw5147, these 'T cell stimulator' cells are engineered to express a membrane-bound anti-human CD3 antibody fragment (with or without co-expression of putative co-stimulatory or co-inhibitory ligands) that triggers TCR-complexes on human T cells.

Gene-synthesis (gene-synthesis) encodes codon-optimized cDNA for LY6G6F (SEQ ID NO: 1), TMEM25(SEQ ID NO: 7) and LSR (SEQ ID NO: 11) and was directionally cloned into retroviral vector pCJK2 via Sfi-I sites. A monocistronic expression construct was generated. The constructs were confirmed by agarose gel electrophoresis and expressed in Bw5147 cells displaying high levels of membrane bound anti-CD 3 antibody (Bw-3/2) (Leitner et al, 2010). Bw5147 cells transduced with the "empty" vector (pCJK2) served as negative controls. In addition, Bw-3/2 cells expressing costimulatory molecules (ICOSL and CD70) and Bw-3/2 cells expressing costimulatory molecules (B7-H3 and B7-H1/PD-L1) were also used as controls. The uniform high expression of stimulated membrane-bound (stimulated membrane-bound) anti-CD 3 antibody was confirmed by FACS using DyLight-649 anti-mouse IgG (H + L) antibody, which reacted with murine single chain antibody that stimulated cell expression. The presence and high level of transcription of the monocistronic construct in the corresponding stimulated cells was confirmed by qPCR.

T cells were purified from buffy coats or heparinized blood derived from healthy volunteer donors and mononuclear fractions were obtained by standard density centrifugation using Ficoll-Paque (GE-Healthcare). Untouched bulk human T cells with the corresponding biotinylated mAb bound to paramagnetic streptavidin beads were obtained by MACS-depletion of CD11b, CD14, CD16, CD19, CD33, and MHC class II-bearing cells (MHC-class II-bearing cells), 2009. Purified CD 8T cells and CD 4T cells were obtained by adding biotinylated CD4 and CD8 mAb to the pool. The original CD4+ T cell Isolation Kit II (naivcd 4+ T cell Isolation Kit II) was used to isolate the original CD4+ T cells. After isolation, cells were analyzed by FACS for purity, and samples with sufficient purity (> 90%) were used for the experiments.

Stimulated cells were harvested, counted, irradiated (2x3000rad) and seeded in flat bottom 96-well plates (20,000 cells/well). MACS-purified T cells stored in liquid nitrogen were thawed, counted and added to wells at 100,000 cells per well; the total volume was 200. mu.l/well. Triplicate wells were established for each condition. After 48 hours of co-cultivation, the wells were added³H-thymidine (final concentration of 0.025 mCi; PerkinElmer/New England Nuclear Corporation, Perkin Elmer/New England Nuclear industries group Co., Welsli, Mass.). After further incubation for 18 hours, the plates were harvested on filter plates and determined as described in Firstel Sammer (Pfistershammer) et al, 2004³Binding of H-thymidine. In addition, a series of experiments were performed on MACS-purified T cell subsets (CD 8T cells, CD 4T cells, and primary CD45RA-p positive CD 4T cells). Additional controls in all experiments included wells with individual stimulated cells for microscopic evaluation of cells and also for determining the basis of stimulated cells w/o T cells³Binding of H-thymidine. The results of stimulated cells that rapidly disintegrate after irradiation treatment are excluded from the analysis.

The results shown in figure 33 are the average of several experiments and show the effect of stimulatory cells expressing LY6G6F, TMEM25 or LSR on the proliferation of human bulk T cells (figure 33A), CD4+ T cells (figure 33B), CD8+ T cells (figure 33C), or naive CD4CD45RA + T cells (figure 33D). Expression of the control co-stimulatory molecules (ICOSL and CD70) resulted in consistent and significant stimulation of proliferation of all cell subtypes. Similar to the expression of control co-inhibitory molecules (B7-H3 and B7-H1/PD-L1), which resulted in mild inhibition of proliferation of different T cell subtypes, the expression of LY6G6F, TMEM25, and LSR also resulted in mild inhibition of T cell proliferation, displaying the most significant inhibition on CD8+ T cells.

Example 34

Characterization of target cells of LY6G6F, VSIG10, TMEM25 and/or LSR proteins by determination of their binding characteristics to immune cells

Splenocytes from DO11.10 mice (transgenic mice in which all CD4+ T cells express a T cell receptor specific for OVA323-339 peptide) were activated in the presence of OVA323-339 peptide, and cells were collected at T0, 6, 12, 24, and 48 hours after initial activation to determine which cell type was expressing the receptor of LY6G6F, VSIG10, TMEM25, and/or LSR over time. Cells were then co-stained for CD3, CD4, CD8, B220, CD19, CD11B, and CD11 c.

Example 35

Evaluation of the Effect of LY6G6F, VSIG10, TMEM25 or LSR ECD IG fusion proteins on B cell class switching and antibody secretion

Dormant B cells were isolated from naive C57BL/6 mice and activated in vitro in the presence of anti-CD 40 plus (i) no exogenous cytokine, (ii) IL-4, or (iii) IFN- γ. At the time of culture line establishment, cell cultures received control Ig (mIgG2a), anti-CD 86 mAb (as a positive control for increased Ig production), or any of the LY6G6F, VSIG10, TMEM25, and LSR ECD fusion proteins described in this example 5, and were cultured for 5 days. Each LY6G6F, VSIG10, TMEM25 and LSR ECD fusion protein was tested at three concentrations. At the end of the incubation, the supernatants were tested for the presence of IgM, IgG1 and IgG2a via ELISA. If the B cells appear to have altered ability to do class switching for one isotype of antibody relative to another, the number of class-switched B cells is determined via ELISPOT. If the number of antibody producing cells is altered, it is also determined whether the levels of gamma 1-and gamma 2 a-sterile transcripts for IgG1 and IgG2a are altered relative to the level of mature transcripts.

Assessment of the therapeutic efficacy of Ly6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion proteins for the treatment of autoimmune diseases

Example 36:

therapeutic efficacy of LY6G6F, VSIG10, TMEM25 or LSR ECD IG fusion proteins in mouse R-EAE model with multiple sclerosis

The therapeutic effect of TMEM25-ECD-Ig, LSR-ECD-Ig, and VSIG10-ECD-Ig fusion proteins (SEQ ID NOS: 25, 26, and 24, respectively) for treating autoimmune diseases is in the presence of multiple sclerosis; tested in a mouse model of relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE):

female SJL mice, 6 weeks old, were purchased from Harlan and maintained in the CCM facility for 1 week prior to starting the experiment. Mice were randomly assigned to groups of 10 animals each and sensitized with 50 μ g PLP139-151/CFA on day 0. Mice that received 6 i.p. (i.p.) doses of 100 ug/dose of TMEM25-ECD-Ig (SEQ ID NO: 25), LSR-ECD-Ig (SEQ ID NO: 26), mIgG2a isotype control, or CTLA4-Ig (mouse ECD fused to mouse IgG2a Fc) served as positive controls. Treatment was initiated at the onset of disease remission and was given 3 times per week for 2 weeks. Mice are subject to disease symptoms. On day 35, (during the peak of disease recurrence), the DTH (delayed type hypersensitivity) response against the disease inducing epitope (PLP139-151) and against the recurrence-associated myelin epitope (PLP178-191) was determined in 5 mice in each group via injection of 10 μ g of PLP139-151 into one ear and PLP178-191 into the other ear. Ear swelling levels were measured 24 hours after challenge.

This example shows a significant reduction in disease severity in R-EAE-induced mice when treated with TMEM25-ECD-Ig (SEQ ID NO: 25) or (SEQ ID NO: 26) in a 3 weekly, 100 ug/dose treatment model, as shown in FIG. 34A. The level of inhibition was similar to that of CTLA 4-Ig.

Furthermore, on day 35, R-EAE mice treated with TMEM25-ECD-Ig (SEQ ID NO: 25) or (SEQ ID NO: 26) significantly inhibited DTH responses against disease induced epitopes (PLP139-151) and against relapse-associated myelin epitopes (PLP178-191) (FIG. 34B).

To test the dose dependence of the efficacy of TMEM25-ECD-Ig (SEQ ID NO: 25) in the PLP-induced R-EAE model and its mode of action, disease was induced as described above and mice were treated 3 times a week for two weeks at 100, 30 or 10 ug/dose starting from disease remission. TMEM25-ECD-Ig reduced the severity level of the disease in an indicated dose-dependent manner with a milder effect as observed at the lowest tested dose (10 ug/dose), which is significantly different from the effect of the high dose (100 ug/dose) (fig. 35A). On days 45 and 76, TMEM25-ECD-Ig also inhibited the DTH response to the expanded epitopes PLP178-191 and MBP84-104 (FIG. 35B). In addition, TMEM25-ECD-Ig inhibited recall responses (recall responses) to PLP139-151, PLP178-191, and MBP84-104 by splenocytes at day 45 and day 76 and cervical lymph node cells at day 45 (FIGS. 35C and 35D). This is mainly shown in the inhibition of proliferation together with a reduction in IFN γ and IL-17 release. TMEM25-ECD-Ig also inhibited the release of IL-4 and IL-10 from cervical lymph node cells of mice treated with 30 ug/dose of TMEM 25-ECD-Ig. Under these conditions, there was no consistent effect on IL-4 and IL-10 release from splenocytes.

The beneficial effects of TMEM25-ECD-Ig (SEQ ID NO: 25) in the R-EAE model were also accompanied by a significant reduction in the infiltration of immune cells into the CNS (FIG. 35E). Although there was no significant change in the lineage tested in the CNS, there was a clear trend of a decrease in CD4+ T cells and Dc (CD11C +) and some increase in the B cell (CD19+) population, although this did not reach statistical significance (fig. 35E).

VSIG10-ECD-Ig (SEQ ID NO: 24) was also tested in the PLP-induced R-EAE model described above. Treatment was started on the day of onset of remission and given at 100 ug/dose, 3 x/week for 2 weeks. VSIG10-ECD-Ig significantly reduced the severity of the disease as shown on the reduction of disease score (fig. 36A). The beneficial effects of VSIG10-ECD-Ig in this model were also accompanied by inhibition of DTH responses to the expanded epitopes PLP178-191 and MBP84-104 on days 45 and 76 (FIG. 36B). Furthermore, on day 45 of ingestion, VSIG10-ECD-Ig (SEQ ID NO: 24) inhibited the recall response (call response) of splenocytes and draining (cervical) lymph node cells in response to activation by induction of epitope PLP139-151, or extended epitopes PLP178-191 and MBP84-104 (FIGS. 36C and 36D). This is shown in the inhibition of cell proliferation in conjunction with secretion of IFNg, IL-17, IL-4 and IL-10.

Interestingly, on day 76, VSIG10-ECD-Ig (SEQ ID NO: 24) only inhibited the proliferation of spleen cells induced by MBP84-104, but not by earlier myelin epitopes (FIG. 36C). Treatment of VSIG10-ECD-Ig in the R-EAE model also significantly reduced the penetration of immune cells into the CNS, which was accompanied by a significant visible, but not significant, increase in the number of cells in the lymph nodes (fig. 36E). The major subset of cells that are reduced in the CNS is CD4+ T cells, however, there is also a clear trend in the CNS for the reduction of CD19+ B cells and CD11c + Dcs. All these immune cell subtypes were significantly elevated in lymph nodes, suggesting that VSIG10-ECD-Ig may inhibit the transport of immune cells from lymph nodes to the CNS.

LY6G6F-ECD-Ig fusion protein was studied in a similar model with multiple sclerosis.

Example 37:

efficacy of LY6G6F, VSIG10, TMEM25 or LSR ECD IG fusion proteins in mouse CIA model with rheumatoid arthritis

Study I: LSR-ECD-Ig (SEQ ID NO: 26) was tested in a mouse model with collagen-induced arthritis (CIA) as a model with rheumatoid arthritis. Male DBA/1 mice were housed in groups of 8-10 mice and maintained at 21 ℃ plus or minus 2 ℃ for 12h light/dark cycle, with food and water ad libitum. Arthritis was induced by immunization with type II collagen emulsified in complete freund's adjuvant. Mice were monitored on a daily basis for signs of arthritis. At the onset of arthritis (day 1), LSR-ECD-Ig (SEQ ID NO: 26), mIgG2a isotype control or CTLA4-Ig (mouse ECD fused to mouse IgG2 aFc) were initially administered 3 times weekly as positive controls (100 ug/dose each) for 10 days. The posterior footpad swelling was measured (using a micrometer caliper), along with the number and extent of joint involvement in all extremities. The two measurements (clinical score and footpad thickness) generated can be used for statistical evaluation.

At the end of the treatment period, mice were bled and sacrificed. For histological analysis, the claws were removed after necropsy, fixed in buffered formalin (10% v/v), and then decalcified in EDTA in buffered formalin (5.5% w/v). Tissues were then embedded in paraffin, sectioned and stained with hematoxylin and eosin. The scoring system is as follows:

0 is normal; synovitis, but cartilage loss and bone erosion are absent or limited to discrete lesions; synovitis and presents significant erosion, but the normal joint architecture is intact; synovitis, extensive erosion, and destruction of the joint architecture.

This example shows that treatment of mice with established CIA with LSR-ECD-Ig at 100 ug/dose, 3 times per week, for 10 days, resulted in effective reduction of clinical score (fig. 37A) as well as paw swelling (fig. 37B) and histological lesions (fig. 37C). The efficacy of LSR-ECD-Ig (SEQ ID NO: 26) was similar to that obtained with CTLA 4-Ig.

The efficacy of TMEM25-ECD-Ig, VSIG10-ECD-Ig, and LY6G6F-ECD-Ig was evaluated in this CIA model.

Treatment with TMEM25-ECD-Ig (SEQ ID NO: 25) or with LSR-ECD-Ig (SEQ ID NO: 26) showed NO efficacy in the more severe CIA model, where a supplemental dose of type II collagen emulsified in complete Freund's adjuvant was administered on day 21. In this severe CIA Enbrel, the positive control given at the same regimen and dose had very weak efficacy. Treatment with TMEM25-ECD-Ig also showed no therapeutic effect in the CIA model, which was given a supplemental dose of type II collagen without adjuvant on day 21.

Study II: the efficacy of LY6G6F ECD Ig fusion protein was studied in the CIA model using the following modified CIA model: female DBA/1 mice (Taconic farm, 9-11 weeks old) were acclimated for 7 days. On day 0, mice were immunized with chicken collagen/CFA, 0.05mL EK-0210 emulsion/mouse (Hooke Laboratories, Inc.), and on day 20, a booster dose with chicken collagen/IFA, 0.05mL EK-0211 emulsion/mouse (Hooke Laboratories, Inc.). Mice were scored daily and scores were enrolled into one of the following treatment groups on the day of arthritis onset:

group 1: LY6G6F-ECD-Ig (SEQ ID NO: 23), i.p. (intraperitoneal injection), Q2D, 30mg/kg, for 2 weeks, 10 mL/kg.

Group 2: vehicle (PBS) Q2D, for 2 weeks, 10mL/kg (negative control).

From the time of enrollment, mice were scored every other day for clinical signs and joint stiffness according to the following scoring system:

clinical score:

joint stiffness score:

fraction of paw	Clinical observations
		0	No joint stiffness
1	Mild ankylosis
		2	Stiffness of the middle joint
3	Severe ankylosis

This example shows that treatment of mice with established CIA with 30mg/kg LY6G6F-PCD-Ig Q2D for 2 weeks, from the onset of arthritis, resulted in a reduction in disease as evidenced by a reduction in disease score (fig. 38).

Efficacy of VSIG10-ECD-Ig (SEQ ID NO: 24) and TMEM25-ECD-Ig (SEQ ID NO: 25) were evaluated in a similar model.

Study III: effect of LY6G6F, VSIG10, TMEM25 and LSR ECD-Ig fusion proteins on tolerance induction in a metastatic model with CIA

To further understand the effect of LY6G6F, VSIG10, TMEM25, and LSR ECD-Ig fusion proteins on immune modulation, the ability of these proteins to induce tolerance in a transfer model was analyzed.

Briefly, spleen and LN cells from arthritic DBA/1 mice treated with LY6G6F, VSIG10, TMEM25, and LSR ECD Ig fusion proteins (SEQ ID NOS: 23, 24, 25, and 26, respectively) for 10 days were removed and i.p (i.p.) injected into T-cell deficient C.B-17SCID recipients. The mice then received an injection of 100 μ g of type II collagen (without CFA), which is required for successful arthritis metastasis. Arthritis was then monitored in SCID mice. Histology was performed and anti-collagen antibody levels were measured to determine that LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion protein treatment conferred long-term disease protection.

Example 38

Evaluation of the role of LY6G6F, VSIG10, TMEM25 and LSR ECD-IG fusion proteins in a model of viral infection with TMEV

The Taylor Murine Encephalomyelitis Virus (TMEV) is a natural endemic pathogen in mice that causes an induced demyelinating disease (TMEV-IDD) in the mouse's susceptible line (SJL/J, H-2KS), similar to the primary progressive form of MS (Munz) et al, Nat Rev Immunol 2009; 9: 246-58). TMEV infection leads to a life-long lasting viral infection of the CNS, which leads to the development of chronic T cell-mediated autoimmune demyelination triggered (i.e., epitope spreading) via reactivation of CD 4T cell responses to endogenous myelin epitopes in the inflamed CNS (Miller et al, Nature medicine (Nat Med) 1997; 3: 1133-6; Katz-Levy et al, J Clin Invest 1999; 104: 599-610).

Within 21 days after infection, SJL mice cleared most of the virus, whereas latent viral infection was maintained and infected microglia, astrocytes, and neurons. Disease symptoms manifest approximately 25-30 days after infection.

The effect of treatment with Y6G6F, VSIG10, TMEM25 or LSR Ig fusion proteins (SEQ ID NOS: 23, 24, 25 and 26, respectively) on the acute and chronic stages of viral infection was studied in the TMEV-IDD model by assessing viral clearance and disease severity.

The method comprises the following steps:

by intracranial inoculation of 30ul of 3x10 in serum-free medium in the right hemisphere⁷Bean strain 8386 of TMEV in Plaque Forming Units (PFU), female SJL/J mice (5-6 weeks) were infected with TMEV. From day 2 post-infection, mice were treated with control Ig, Y6G6F, VSIG10, TMEM25, or LSR ECD-Ig fusion protein, each at 100 ug/dose; 3 doses/week for 2 weeks.

Mice followed clinical scores. At day 7 and 14 after infection (after 3 and 6 treatments, respectively), brains and spinal cords were collected from 5 mice of each treatment group for plaque assay. These tissues were weighed so that after plaque assays were completed, the ratio of PFU/mg of CNS tissue could be calculated.

TMEV plaque assay:

at day 7 and 14 after infection, the brains and spinal cords of mice treated with control Ig (mouse IgG2a), or with each of Y6G6F, VSIG10, TMEM25, or LSR ECD-Ig fusion proteins (SEQ ID NOS: 23, 24, 25, and 26, respectively) were collected from non-perfused anesthetized mice. The brain and spinal cord were weighed and homogenized. CNS homogenates were serially diluted in DMEM and added to tissue culture-treated plates (tissue culture-treated plates) with confluent BHK-21 cells, incubated for 1h at room temperature with periodic mild shaking.

The medium/agar solution was mixed 1: 1 (vol: vol), added to the cells and allowed to solidify at room temperature. These plates were then incubated at 34 ℃ for 5 days. At the end of the culture, 1ml of formalin was added and incubated at room temperature for 1h to fix the monolayer of cells. Formalin was poured into the waste container and the agar was removed from the plates. Plaques were visualized by staining with crystal violet for 5min, and the plates were gently rinsed with diH 2O. To determine PFU/ml homogenate, the number of plaques on each plate was multiplied by the dilution factor of the homogenate and divided by the amount of homogenate added in each plate. PFU/mg tissue was calculated by dividing PFU/ml by the weight of the tissue.

Example 39

Assessment of the effects of Y6G6F, VSIG10, TMEM25 and LSR ECD-IG fusion proteins on primary and secondary immune responses to viral infection in a mouse model with influenza

To test the effect of Y6G6F, VSIG10, TMEM25 or LSR ECD-Ig fusion proteins (SEQ ID NOs: 23, 24, 25 and 26, respectively) on primary and secondary immune responses to viral infection, BALB/c naive mice (for primary immune response) and 'HA-memory mice', along with 'polyclonal influenza memory mice' (for assessing secondary responses mediated by memory CD4T cells) were used, as specified in taijean et al, journal of immunology (JImmunol), 2009: 182, respectively; 5430-5438 and is described below.

To obtain ` HA-memory mice `, HA-specific memory CD 4T cells were first generated, and primary CD 4T cells were BALB/c-HA mice expressing a transgenic T receptor (TCR) specific for the influenza Hemagglutinin (HA) peptide (110-119) from HA-TCR mice]Purified from the spleen of (a), and sensitized in vitro by culturing for 3 days at 37 ℃ splenocytes treated with 5.0microG/ml HA peptide and mitomycin C-depleted T-depleted BALB/C as APC. Transfer of the resulting activated HA-specific effector cells to the congenic BALB/c (Thyl.1) host (5X 10)⁶Cells/mouse) to generate "HA-memory mice" with a stable population of HA-specific memory CD 4T cells.

To obtain 'polyclonal-memory mice', polyclonal influenza-specific memory CD 4T cells were first generated by intranasal infection of BALB/c mice with sublethal doses of PR8 influenza, CD 4T cells were isolated 2-4 months after infection, and the frequency of influenza-specific memory CD 4T cells was determined by ELISPOT. CD 4T cells from previously primed mice were transferred into BALB/c hosts to generate "polyclonal influenza-memory" mice with endogenous T cells of sufficient complement.

Primary and secondary responses to influenza virus were tested by infecting naive BALB/c mice or BALB/c-HA memory mice, as well as BALB/c 'polyclonal influenza-memory mice', with sublethal or lethal doses of PR8 influenza via intranasal administration.

Mice were treated with Y6G6F, VSIG10, TMEM25 or LSR ECD-Ig fusion protein or with mIgG2a control before and after influenza challenge. Weight loss and mortality were monitored daily. Six days after challenge, the virus content in bronchoalveolar lavage (BAL) was analyzed by collecting the lavage fluid and test supernatants, as determined by tissue culture infectious dose 50% (TCID50) in MDCK cells. In addition, lung histopathology was performed.

To test the effect of Y6G6F, VSIG10, TMEM25 and LSR ECD-Ig fusion proteins on T cell proliferation, BALB/c or BALB/c-HA memory mice or BALB/c 'polyclonal influenza-memory mice' were infected as above and BrdU (1 mg/dose) was administered on days 3, 4 and 5 after infection. On day 6, spleen and lung were harvested and BrdU binding was estimated. Cytokine production by lung memory CD 4T was also studied during influenza challenge in HA-specific memory CD 4T cells stimulated in vitro with HA peptides or with IgG2a in the presence of Y6G6F, VSIG10, TMEM25 or LSR ECD-Ig fusion proteins for 18 hours.

Example 40

Assessment of the effects of Y6G6F, VSIG10, TMEM25 and LSR ECD-IG fusion proteins on primary CD 8T cell response and secondary CD 8T cell response to viral infection in a mouse model with influenza

The effect of Y6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins (SEQ ID NO: 23, 24, 25 and 26, respectively) on the primary CD 8T cell response to influenza virus was studied using C57BL/6 mice infected with influenza A HKx31 via intranasal or intraperitoneal administration according to methods as described in, e.g., Henderks et al, J Immunol 2005; 175; 1665-1676; Bertran et al, J Immunol 2004; 172: 981-8. During sensitization, Y6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion protein or mIgG2a controls were given. Animals were monitored daily for weight loss and mortality. To follow virus-specific CD8+ T cells, MHC H-2Db tetramer, NP366-374 peptide loaded with a major CD 8T cell epitope was used. Virus-specific H-2D in the lungs, draining lymph nodes, and spleen is expected^bthe/Np 366-374+ CD8+ T cells peaked about 8-10 days after infection and thereafter dropped to only 1.5% of virus-specific CD8+ T cells (Hendriks et al, J. Immunol 2005; 175; 1665-1676; Bertran & lt & gten & lten & gt 2005)(Bertram) et al, J Immunol 2002; 168: 3777-85; bertran (Bertram) et al, J.Immunol (J.) 2004; 172: 981-8). Therefore, mice were sacrificed at day 8 and day 21 post-infection and virus-specific CD8+ T cells in the lungs, draining lymph nodes, and spleen were evaluated. Viral clearance was assessed. CD 8T cell responses were assessed in spleen cell suspensions and included intracellular IFN- γ staining and CTL activity as previously described (Bertran (Bertram) et al, J Immunol 2004; 172: 981-8) and detailed below.

Cells were surface stained with FITC-conjugated anti-mouse CD62L, PE-conjugated anti-mouse CD8 to measure CD8+ activated T cells (or anti-mouse CD4 to track CD4+ cells). In addition to these Abs, allophycocyanin-labeled tetramers (from the murine class I MHC molecule H-2D) were used^b、β₂-microglobulin, and influenza NP peptide NP_366-374Composition) to measure influenza-specific CD8+ T cells. For intracellular IFN-gamma staining, 1. mu.l of NP was used_366-374Peptide and GolgiStop (BDPharMingen, san Diego, Calif.) cell suspensions were restimulated in culture medium at 37 ℃ for 6 h. Cells were then harvested, resuspended in PBS/2% FCS/azide, and surface stained with PE-anti-CD 8 and FITC-anti-CD 62L as described above. After surface staining, cells were fixed in Cytofix/Cytoperm solution (BD PharMingen), and then stained with allophycocyanin-conjugated anti-mouse IFN-. gamma.diluted in 1 Xperm wash (BD PharMingen). The samples were analyzed by flow cytometry.

For cytotoxicity testing (CTL response), splenocytes from influenza infected mice were incubated at 37 ℃ for 2h to remove adherent cells. To is directed at⁵¹Anti-influenza NP of Cr-labeled EL4 cells _366-374Specific CTL Activity, determination of effectors at 3-fold serial dilutions, these⁵¹Cr-labeled EL4 cells like NP with 50uM as described by Bertonin (Bertram) et al 2002 and Bertran (Bertram) et al 2004_366-374The peptide was pulsed for 6 h.

Some mice were re-challenged 3 weeks after infection with a serologically different influenza A/PR8/34(PR8), which shares the NP gene with influenza A HKx31, but differs in hemagglutinin and neuraminidase, such that neutralizing Ab does not limit secondary CTL responses. Mice were sacrificed on days 5 and 7 after restimulation and assessed for the number of virus-specific CD 8T cells in the lungs, draining lymph nodes and spleen by Hendriks et al and Bertran (Bertram) et al (Hendriks et al, J Immunol 2005; 175; 1665. sup. 1676; Bertran (Bertram) et al, Immunol 2004; 172: 981-8) and as detailed above. Again CD 8T cell responses (including intracellular IFN- γ staining and CTL activity) were evaluated in spleen cell suspensions of mice at days 5 and 7 after virus re-challenge as described above.

To determine the effect of Y6G6F, VSIG10, TMEM25 and LSR ECD-Ig fusion proteins on the proliferation and accumulation of memory CD8+ T cells during the re-response, adoptive transfer experiments were performed according to the methods previously described (Hendriks et al, J Immunol 2005; 175; 1665-1676; Bertran (Bertram) et al, J Immunol 2004; 172: 981-8): mice were immunized with influenza a HKx 31. Twenty-one days later, T cells were purified from the spleen on a T cell enrichment immune column (CEDARLANE laboratory, hornsbie, ontario, canada) and labeled with CFSE (alternatively, Thy 1.1 congenic mice were used as recipients). Equal numbers of tetramer-positive T cells were injected through the tail vein of recipient mice. Mice were re-challenged with influenza virus as described above, and 7 days later splenocytes were evaluated for donor virus-specific CD 8T cells as described above.

Example 41

Protein expression in depleted T cells, and binding to depleted T cells and evaluation of their effect on reversion to depleted T cell phenotype of Y6G6F, VSIG10, TMEM25 and LSR ECD-IG fusion proteins

Acute viral infection after chronic viral infection, memory CD 8T cell differentiation occurs along different pathways (kleinerman (Klenerman) and bill (Hill), nature immunology (Nat Immunol)6, 873-. Memory CD 8T cells generated following acute viral infection are highly functional and constitute an important component of protective immunity. In contrast, chronic infection is often characterized by a variable degree of functional impairment of the virus-specific T-cell response, and this deficiency is a major cause of host anergy to eliminate persistent pathogens. Although functional effector T cells are initially produced during the initial stages of infection, they gradually lose function during chronic infection, which leads to a depleted phenotype characterized by impaired T cell functionality.

Study i. during acute and chronic viral infections, Y6G6F, VSIG10, TMEM25 and LSR ECDIg fusion proteins effect on clearance of viral infections and on T cell function.

According to journal of virology by hui (Wherry) et al (j.virol.) 77: 4911 the effects of Y6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins (SEQ ID NOs: 23, 24, 25 and 26, respectively) on acute and chronic viral infections were evaluated in a mouse model infected with LCMV (lymphocytic choriomeningitis virus) using the methodology described in Nature, 2006 by Barber et al, Nature, 2003, and Barber, and described in detail below.

Use of two LCMV strains that can cause either acute or chronic infection in adult mice; the Armstrong (Armstrong) line was cleared within a week, while establishment of persistently infected clone 13 line could last for months. Since these two lines, which retain all known T cell epitopes, differ by only two amino acids, it is possible to follow the same CD 8T cell response after acute or chronic viral infection. In contrast to the generation of highly potent memory CD 8T cells following acute Armstrong infection, LCMV-specific CD 8T cells became depleted during persistent clone 13 infection (Wherry et al J. Virol. 77: 4911. about. 4927, 2003; Barber et al Nature (Nature) 2006; 439: 682-7).

By 2X10⁵Acute infection was initiated by intraperitoneal infection of mice with Armstrong strain of LCMV from PFU or 2X10⁶Mice were infected intravenously with CI-13 from PFU to elicit chronic infection. Mice were treated i.p. (i.p.) with Y6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion protein or with mIgG2a control and with specific anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25, anti-LSR-antibody or isotype control.

Using virus-specific MHC tetrameric epitopes, e.g. D, which differs in acute or chronic infection^bNP_396-404And D^bGP_33-41Mice were monitored for the number of virus-specific CD 8T cells in the spleen. According to huili (Wherry) et al, journal of virology (j.virol.) 77: 4911 CD 8T cell function assays, such as intracellular cytokine levels and CTL activity, were performed as described in 4927, 2003 and analogously to those described in example 40. Additional assays include the production of splenocytes following stimulation with virus-specific epitopes; and assessment of viral titers in serum and in spleen, liver, lung and kidney (Wherry et al, J.Virol., 77: 4911-.

To assess the modulation of these protein or their counterpart receptors during depletion of T cells, the binding of Y6G6F, VSIG10, TMEM25 and LSR expression and Y6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins to depleted T cells was assessed:

t cells were isolated from mice with chronic LCMV infection induced by the CI-13 strain. These cells were co-stained with fluorescently labeled anti-PD-1 Ab (as positive control) (depleted T cells highly expressing PD-1) and biotinylated Y6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins or biotinylated anti-Y6G 6F, anti-VSIG 10, anti-TMEM 25 and anti-LSR fusion protein antibodies, and corresponding isotype controls. Binding was detected by FACS analysis using fluorescently labeled streptavidin.

Example 42

Evaluation of the expression of LY6G6F, VSIG10, TMEM25 and/or LSR proteins in follicular helper T (TFH) cells and the binding of IG fusion proteins to TFH cells

Follicular helper T (Tfh) cells are a subset of CD4+ T cells that are specialized in B cell help (reviewed by Crott (Crotty), Annu. Rev. Immunol. (Annu.) 29: 621-663, 2011). Tfh cells migrate into B-cell follicles in lymph nodes and interact with cognate B-cells at T-cell-B-cell borders and subsequently induce germinal center B-cell differentiation and germinal center formation in the follicles (reviewed by Crotty (crohn's disease), annu. rev. immunol. (annual meeting of immunology) 29: 621663, 2011). The requirement for Tfh cells for B cell helper and T cell dependent antibody responses suggests that this cell type is important for protective immunity against different types of infectious agents and for rational vaccine design.

As CXCR5^hiSLAM^loBTLA^hiPDl^hiBcl6⁺Tfh cells, virus-specific CD4+ T cells, were readily identifiable at the highest point of response to CD4+ T cells infected with acute lymphocytic choriomeningitis virus (LCMV) (Tot (Choi) et al 2011, Immunity 34: 932-946). From patients with intraperitoneal administration of 2X10 ⁵T cells were isolated from acute LCMV infected mice induced by Armstrong (Armstrong) strain of LCMV from PFU. These cells were co-stained with a fluorescent-labeled antibody directed against a marker for Tfh (CXCR5, PD1, BTLA, Bcl6) that is highly expressed by Tfh cells, and biotinylated LY6G6F, VSIG10, TMEM25, and LSR ECD-Ig fusion proteins or biotinylated antibodies specific for LY6G6F, VSIG10, TMEM25, and LSR, and corresponding isotype controls. Fc fusion protein by FACS analysis using fluorescently labeled streptavidinOr binding of an antibody.

Example 43

Evaluation of the Effect of LY6G6F, VSIG10, TMEM25 and LSR IG fusion proteins on the production and Activity of follicular helper T (TFH) cells

To investigate the effect of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins on Tfh differentiation and development of B cell immunity in vivo, C57BL/6 was treated with LY6G6F, VSIG10, TMEM25 and LSR Ig fusion proteins and isotype control throughout the course of acute viral infection of Armstrong (Armstrong) strains of LCMV (lymphocytic choriomeningitis virus). Tfh differentiation and Bcl6 protein expression were assessed by FACS analysis as described by ero (Eto) et al 2011(PLoS One 6: e 17739). 8 days after LCMV infection, splenocytes were analyzed for Tfh production by FACS analysis (CD 44) ^hiCXCR5^hiSLAM^lo) And Bcl6 expression were evaluated. Furthermore, the effects of LY6G6F, VSIG10, TMEM25, and LSRECD Ig fusion proteins (SEQ ID NOS: 23, 24, 25, and 26, respectively) on antigen-specific B cell responses were evaluated as described by Epigram (Eto) et al 2011(PLoS One 6: e17739), including titers of anti-LCMV IgG in sera on day 8 post-LCMV infection, and plasma cells gating CD 19+ splenocytes (CD 138) by FACS analysis on day 8 post-infection⁺IgD^-) Quantification of development.

Example 44

Effect of LY6G6F, VSIG10, TMEM25 and LSR ECD IG fusion proteins on type 1 diabetes in NOD mice, CD28-KO NOD, and B7-2-KO NOD

The effects of LY6G6F, VSIG10, TMEM25 and LSRECD Ig fusion proteins were studied in a widely used mouse model with type 1 diabetes: non-obese diabetic (NOD) mice develop spontaneous In NOD mice, spontaneous insulitis, overt pathological lesions through the evolution of several characteristic stages, starting with near insulitis and ending with invasive and destructive insulitis and overt diabetes. Proximal insulitis was first observed at 3-4 weeks of age, invasive insulitis was observed at 8-10 weeks, and destructive insulitis occurred just before onset of clinical diabetes, the earliest cases being 10-12 weeks. At 20 weeks of age, 70% -80% of female NOD mice become diabetic (ansali et al 2003 journal of experimental medicine (j.exp.med.) 198: 63-69).

Two KO mice were also used: CD-28-KO NOD mice and B7-1/B7-2 Dual KO NOD mice-develop accelerated diabetes (Lunhouse (Lenschow) et al 1996 Immunity 5: 285-.

Study I: NOD mice were treated with LY6G6F, VSIG10, TMEM25 or LSR ECD-Ig fusion proteins (SEQ ID NOS: 23, 24, 25 and 26, respectively) at early and late stages during the course of diabetes evolution, before or after the onset of disease, to examine the effects of these compounds on disease pathogenesis and to demonstrate that such treatment reduced the onset of disease and improved pathogenesis. To study the effect on insulitis, blood glucose levels were measured 3 times per week for up to 25 weeks (ansali, et al 2003 journal of experimental medicine (j.exp.med.) 198: 63-69).

Experimental evaluation by individual immune cell types investigated the mechanism and mode of action of disease changes: pancreas, pancreatic LN, and spleen will be harvested to obtain tregs, Th subtypes, and CD8T cells, DCs, and B cells. The effect of cytokine secretion from cells isolated from pancreas, pancreatic LN, and spleen was analyzed, focusing on IFNg, IL-17, IL-4, IL-10, and TGFb. For the effect of the test compounds, by examining individual immune cell types (including tregs, Th and CD8T cells, DCs and B cells); cytokines (IFNg, IL-17, IL-4, IL-10 and TGFb) and histology were used to study disease alteration mechanisms. Histological analysis of the pancreas was performed to compare the onset of insulitis and lymphocyte infiltration.

Study II-Effect of LY6G6F IG fusion protein in the modulation of type 1 diabetes in an adoptive transfer model

To further investigate the mode of action of Ig fusion proteins, an adoptive transfer model of diabetes was used. T cells from diabetic or pre-diabetic NOD donors were transferred to NOD SCID recipient mice. These mice were monitored for the development of diabetes. Urine glucose and blood glucose, and histological evaluation of the pancreas, as well as T cell responses were monitored as described in the previous examples.

Study III: diabetes was also induced by transferring activated CD4+ CD62L + CD25-BDC 2.5T cells (TCR recognition transgenic cells of islet-specific peptide 1040-p31 activated by incubation with 1040-p 31) to NOD recipients. Mice were treated with LY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion protein, control mIgG2a or positive control. Treatment began 1 day after transfer. 10-28 days after transfer, mice were tracked for glucose levels (Boolean-Jordan et al, J Clin Invest. 2004; 114 (7): 979-87).

Seven days after treatment, pancreas, spleen, pancreatic LN, and peripheral lymph node cells of different immune cell populations were extracted and examined. In addition, recall responses were measured by testing ex vivo proliferation and cytokine secretion in response to the p31 peptide.

In the above studies, LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins prevent or reduce the onset of disease or its severity.

Example 45

Role of LY6G6F, VSIG10, TMEM25 and LSR ECD IG fusion proteins in lupus mouse model

Study I: a mouse model of lupus-prone infection, (NZB x NZW) F1(B/W) was used. Cyclophosphamide (CTX) is the main drug for diffuse proliferative glomerulonephritis in patients with lupus, and dack (Daikh) and Wofsy (Wofsy) report that the combined treatment of CTX with CTLA4-Ig is more effective than either agent alone in reducing renal disease and extending survival of NZB/NZW fls mice with end-stage nephritis (dack (Daikh) and Wofsy (Wofsy), "journal of immunology (j.immunol), 166 (5): 2913-6 (2001)). In proof-of-concept (proof-of-concept) studies, treatments using LY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion proteins and CTX either alone or in combination were tested.

Blood samples were collected 3 days prior to protein treatment and then plasma anti-dsDNA autoantibodies were analyzed by ELISA during and every other week after treatment. Glomerulonephritis is assessed by histological analysis of the kidney. Proteinuria was measured by testing fresh urine samples using urine analysis paper.

LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins (SEQ ID NOS: 23, 24, 25 and 26, respectively) have at least a beneficial effect in alleviating lupus nephritis.

Study II: the NZM 2410-derived b6.slel. sle2.sle3 mouse model of SLE was used.

NZM2410 is a recombinant inbred line produced by NZB and NZW developing a highly osmotic lupus-like disease with earlier disease onset. The effects of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins were studied in this model by assessment of proteinuria and autoantibodies as described above.

Study III: an induced lupus model was used. This model is based on chronic graft versus host (cGVH) disease induced by transfer of Ia-incompatible spleen cells from one normal mouse strain (e.g., B6.C-H2(bml2)/KIEg (bml2)) to another mouse strain (e.g., C57BL/6), which results in autoimmune syndromes like Systemic Lupus Erythematosus (SLE), including anti-double stranded DNA (anti-dsDNA) autoantibodies and immune complex proliferative glomerulonephritis (Appley et al, clinical and experimental immunology (Clin. Exp. Immunol.) 198978: 449 453; Essenberg (Eisberg) and Choudhuri (Choudhury)2004 molecular medicine Methods (Methods mol. Med. 102: 273-284).

Lupus was induced in this model after spleen cells from bml2 mice were injected into C57BL/6 recipients. The effects of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins were studied in this model by assessment of proteinuria and autoantibodies as described above. T cell responses and B cell responses will also be evaluated.

Study IV: a mouse model of MRL/lpr susceptible lupus is used. The effects of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins were studied in this model by assessment of proteinuria and autoantibodies as described above.

Example 46

Role of LY6G6F, VSIG10, TMEM25 and LSR ECD IG fusion proteins in the control of enteritis.

Use of an adoptive transfer mouse model with colitis in mice, whereby CD45RB from BALB/c mice^{Height of}Transfer of-CD 4+ naive T cells to syngeneic SCID mice resulted in the development of IBD-like syndrome 6-10 weeks after T cell reconstitution, similar to human Crohn's disease.

SCID mice were injected i.p. (i.p.) with syngeneic CD45RB^{Height of}-CD4⁺Reconstitution with T cells, either alone or in combination with syngeneic CD45RB^{Is low in}-CD4⁺Or CD25⁺CD4⁺Cells (4X 10 per cell population)⁵Mice) cotransfer (Liu) et al, J Immunol 2001; 167(3): 1830-8). Syngeneic CD45RB from the spleen of normal mice will be used ^{Height of}CD4⁺T cell reconstituted colitis SCID mice were treated twice weekly with LY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion protein or Ig isotype control i.p. (i.p.) starting at the beginning of T cell transfer for up to 8 weeks. All mice were monitored weekly for weight, loose stools or diarrhea, and rectal prolapse. All mice were sacrificed at 8 weeks after T cell transfer or when they exhibited 20% of the original body weight loss. Colon tissue was collected for histological and cytological examinations. LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins have at least a beneficial effect in the alleviation of inflammatory bowel disease.

Example 47

Role of LY6G6F, VSIG10, TMEM25 and LSR ECD IG fusion proteins in mouse model with psoriasis

Study I: and establishing a psoriasis SCID xenograft model.

Human psoriatic plaques were transplanted onto SCID mice. A scratch biopsy (2.5_2.5cm) was obtained from patients with psoriasis vulgaris involving 5% -10% of the total skin who did not receive any systemic treatment or phototherapy for 6 months and did not receive any topical formulation other than emollient for 6 weeks. Biopsies are obtained from active plaques located on the thigh or arm. Divide each biopsy into about 1cm ²Four equal parts of size. Each sheet was transplanted to a separate mouse.

Under general anesthesia, vascular plexus on the fascia covering the underlying back muscle was kept intact by removing a full thickness skin sample, while generating about 1cm on the shaved area of the back of 7-to 8-week-old CB 17 SCID mice²The graft bed of (1). The partial thickness human skin obtained by the shave biopsy is then transferred in situ to the graft bed. Liquid Veterinary bandage Nexaband (Veterinary Products Laboratories, Phoenix, arizona (Phoenix, AZ)) was used to attach human skin to mouse skin and antibiotic ointment (subtilisin) was applied. Mice were treated intraperitoneally with LY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion protein, isotype control or CTLA4-Ig (positive control) three times a week for four weeks.

Drill biopsies (2mm) were obtained at day 0 of the study period (before treatment) and at day 28 (after treatment). Biopsies were flash frozen and cryosectioned for histopathology and immunohistochemistry studies. Efficacy was determined by comparing pre-and post-treatment data: (i) epidermal process length to determine effect on epidermal thickness, and (ii) level of lymphomonocyte permeate to determine effect on inflammatory cell permeate. (Raychaudhuri et al, 2008, journal of research dermatology (J Invest Dermatol.); 128 (8): 1969-76; Boenk (Boehnck.) et al, 1999, dermatological research archive (Arch Dermatol Res) 291: 104-6).

LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins (SEQ ID NOS: 23, 24, 25 and 26, respectively) have at least a beneficial effect in alleviating psoriasis.

Study II: role of LY6G6F, VSIG10, TMEM25 and LSR in psoriasis and colitis models by adoptive transfer of CD45RBHI CD4+ T cells in SCID mice

Intravenous (i.v.) injection of 0.3_10 into immunocompromised mice⁶CD4+ CD45RBhi cells. On the day after cell adoptive transfer, mice were injected i.p. (i.p.) with 10 μ g of staphylococcal enterotoxin B (devonpot et al, international immunopharmacology (Int Immunopharmacol) 2002 for 4 months; 2 (5): 653-72). Recipient mice were treated with LY6G6F, VSIG10, TMEM25, or LSR ECD-Ig fusion proteins (SEQ ID NOS: 23, 24, 25, and 26, respectively), isotype control, or CTLA4-Ig (positive control). Mice were assessed weekly for weight loss and the presence of skin lesions for 8 weeks.

The results obtained were similar to those described above.

Example 48

Role of LY6G6F, VSIG10, TMEM25 and LSR ECD IG fusion proteins in modulating graft rejection.

Study I: role of LY6G6F, VSIG10, TMEM25 and LSR in allogeneic islet transplantation in diabetic mice. To test the effect of LY6G6F, VSIG10, TMEM25, and LSR ECD Ig fusion proteins (SEQ ID NOS: 23, 24, 25, and 26, respectively) on graft rejection, a model of allogeneic islet transplantation was used. Diabetes was induced in C57BL/6 mice by treatment with streptozotocin. Seven days later, islets with BALB/c isolated donor mice were transplanted under the mouse kidney capsule. Recipient mice were treated with LY6G6F, VSIG10, TMEM25 or LSR ECD Ig fusion protein or with mIgG2a as a negative control. Donor splenocytes that were resistant to ECDI-fixation were used as positive controls for successful modulation of islet transplant rejection. Blood glucose levels in recipient mice were monitored as a measure of graft acceptance/rejection (Roo (Luo) et al, PNAS, 2008, 23 d. vol.105_ No.38_ 14527-.

Study II: role of LY6G6F, VSIG10, TMEM25 and LSR in the HYA-model of skin graft rejection.

In humans and some strains of laboratory mice, male tissue is recognized as a foreign body and is destroyed by the female immune system via recognition of histocompatibility-the antigen encoded by the Y chromosome (Hya). Male tissue destruction is thought to be accomplished by cytotoxic T lymphocytes in a helper-dependent manner.

To test the effect of LY6G6F, VSIG10, TMEM25, and LSR ECD Ig fusion proteins (SEQ ID NOS: 23, 24, 25, and 26, respectively) on transplantation, a Hya model system was used in which female C57BL/6 mice received tail skin grafts from male C57BL/6 donors.

In this study, female C57BL/6 mice were transplanted with split-thickness in situ (split-thickness) tail skin from age-matched male C57BL/6 mice. Mice were treated with LY6G6F, VSIG10, TMEM25 or LSR ECDIg fusion proteins, isotype control mIgG2 a. The immunodominant Hya-encoded CD4 epitope (Dby) attached to female spleen leukocytes (Dby-SP) was used as a positive control for successful regulation of graft rejection (Martin et al, J Immunol) 9/15 days 2010; 185 (6): 3326-3336). Skin grafts were scored daily for edema, pigment loss, and hair loss. Rejection is defined as complete hair loss and more than 80% pigment loss. In addition, T cell recall responses from cells isolated from spleen and draining lymph nodes at different time points were studied in response to specific CD4 epitopes (Dby), CD8 epitopes (Uty and Smcy) or irrelevant peptides (OVA 323-339), while anti-CD 3 stimulation was used as a positive control for proliferation and cytokine secretion.

Study III: the effects of LY6G6F, VSIG10, TMEM25 and LSR ECD Ig fusion proteins were studied in murine models of syngeneic bone marrow cell transplantation using the Hya model system described above. Male hematopoietic cells expressing the CD45.1 marker are transplanted into female host mice expressing the CD45.1 congener marker. Female hosts were treated with LY6G6F, VSIG10, TMEM25 or LSR ECDIg fusion proteins or with isotype control mIgG2 a. The female host was followed over time and monitored for the presence of CD45.1+ cells.

Example 49

Establishment of the role of LY6G6F, VSIG10, TMEM25 and/or LSR proteins as modulators of cancer immune surveillance according to at least some embodiments of the present invention:

1) proof of concept in vivo

a)Mouse cancer syngeneic model:

(i) tumor cells overexpressing either LY6G6F, VSIG10, TMEM25 and/or LSR proteins or non-related control proteins were transplanted into genetically matched mice. Tumor volume (and tumor weight after sacrifice) as well as ex vivo analysis of immune cells from tumor draining lymph nodes or spleen were then examined to demonstrate rejection of the tumor to be delayed (i.e., tumors overexpressing LY6G6F, VSIG10, TMEM25 and/or LSR grew faster than tumors overexpressing non-relevant control proteins). Ex vivo analysis of immune cells from tumor draining lymph nodes is expected to reveal an increase in the frequency of regulatory T cells and a decrease in the responsiveness of effector T cells to stimuli. (J.Exp.Med.)2011 Vol.208No. 3577-592).

(ii) An in vivo homology model using additional cellular domains of mouse orthologs of any of LY6G6F, VSIG10, TMEM25 and/or LSR proteins (SEQ ID NOS: 23, 24, 25 and 26, respectively) fused to an antibody Fc fragment (mouse ECD-Fc) was tested as follows. After tumor establishment, C57BL/6 mouse IV (i.v.) was injected with mouse ECD-FC at 3-4 day intervals, as described in journal of immunology (J Immunol) 2010; 185 of the formula (I); 2747, 2753. Tumor volume (and tumor weight after sacrifice) and ex vivo analysis of immune cells from tumor draining lymph nodes or spleen were then examined. As a result of mouse ECD-FC IV treatment with LY6G6F, VSIG10, TMEM25 and/or LSR, rejection of the tumor was delayed (i.e., the tumor grew faster in mice treated with mouse ECD-FC with LY6G6F, VSIG10, TMEM25 and/or LSR than in mice treated with non-related control protein). Ex vivo analysis of immune cells from tumor draining lymph nodes revealed an increase in the frequency of regulatory T cells and a decrease in the responsiveness of effector T cells to stimuli.

(iii) Establishment of syngeneic tumors and treatment with neutralizing antibodies directed against any one of LY6G6F, VSIG10, TMEM25 and/or LSR proteins (1, 3, 5, 7, 11, 143, 13, 15-17, 18, 28, 29-32). Tumor cells were transplanted into genetically identical mice. After tumor establishment, mice were injected IV (intravenously) with different doses of neutralizing antibodies against any of LY6G6F, VSIG10, TMEM25 and/or LSR proteins. Rejection of the tumor was increased as a result of treatment with neutralizing antibody IV specific for any LY6G6F, VSIG10, TMEM25 and/or LSR protein (i.e., the tumor grew more slowly in mice treated with neutralizing antibody to any LY6G6F, VSIG10, TMEM25 and/or LSR protein than in mice treated with non-related antibody). Ex vivo analysis of immune cells from tumor draining lymph nodes revealed a decrease in the frequency of regulatory T cells and an increase in the responsiveness of effector T cells to stimuli.

b) Human cancer xenograft model:

(i) the tumor immune response was reconstituted in a model of immunocompromised nod. cg-Prkdcscid I12rgtmlWjl/SzJ mice (Jackson laboratory), "NSG" mice. Establishing a human tumor in an NSG model and preloading APCs with tumor antigens, or/and T cells (transferring CD 8T cells pre-activated with cancer target cells to NSG mice bearing tumors (all transplanted cells/injected cells from cancer patients). this model consists of 1. APC overexpressing any LY6G6F, VSIG10, TMEM25 and/or LSR protein, 2.APC silencing of any LY6G6F, VSIG10, TMEM25 and/or LSR protein (siRNA or ShRNA), 3. cancer cells overexpressing any LY6G6F, VSIG10, TMEM25 and/or LSR protein, and 4. silencing of any LY6G F, VSIG10, TMEM25 and/or LSR protein on cancer cells (siRNA or ShRNA). including positive control (e.g. B7-H25, PD-1) and/or LSR protein (ex. J. tumor volume) and then examining tumor volume alone (ex. J. 1. tumor volume) -881). Overexpression of either LY6G6F, VSIG10, TMEM25 and/or LSR protein on either the APC or tumor cells resulted in delayed rejection of the tumor (i.e., the tumor grew faster in mice treated with either APC or tumor cells overexpressing either LY6G6F, VSIG10, TMEM25 and/or LSR protein than in mice treated with a non-relevant control protein). Silencing (with SiRNA or SHRNA) of either LY6G6F, VSIG10, TMEM25 and/or LSR on either the APC or tumor cell results in enhanced rejection of the tumor.

(ii) NSG cancer xenografts were established as described above (without genetic manipulation of APC and/or cancer cells) and treated with neutralizing antibodies directed against any of LY6G6F, VSIG10, TMEM25 and/or LSR proteins. Treatment of NSG xenografts with neutralizing antibodies directed against either LY6G6F, VSIG10, TMEM25 and/or LSR resulted in enhanced rejection of tumors.

2) In vitro confirmation of Natural Killer (NK) cell Activity

a)Binding assay:

(i) such as journal of immunology (J Immunol) 2005; 174, and (b) a; 6692 in 6701, binding assays for human LY6G6F, VSIG10, TMEM25 and/or LSR ECD-FC proteins on activated primary cultured NK cells. If the anti-receptors for LY6G6F, VSIG10, TMEM25 and/or LSR are expressed on NK cells, binding of LY6G6F, VSIG10, TMEM25 and/or LSR ECD-Fc can be observed.

(ii) Such as PNAS, 2009, vol.109; binding assays for specific antibodies directed against any of LY6G6F, VSIG10, TMEM25 and/or LSR proteins were performed on activated primary cultured NK cells as described in 17858 and 17863. If any of LY6G6F, VSIG10, TMEM25 and/or LSR is expressed on NK cells, binding of LY6G6F, VSIG10, TMEM25 and/or LSR specific antibodies, respectively, can be observed.

(ii) As described in journal of immunology (J Immunol) 2006; 176; 6762-6769 binding assays for human LY6G6F, VSIG10, TMEM25 and/or LSR ECD-FC proteins were performed on different human cancer cell lines that could serve as target cells for NK killing. If the anti-receptor of any of LY6G6F, VSIG10, TMEM25 and/or LSR is expressed on a cancer target cell, binding of LY6G6F, VSIG10, TMEM25 and/or LSR ECD-Fc can be observed, respectively.

b)Functional killing assay:

(i) killing assays were performed using an overexpression system (either NK cells overexpressing either LY6G6F, VSIG10, TMEM25 and/or LSR proteins or cancer target cells). Such as PNAS, 2009, vol.109; 17858-17863, cells NK (effector; e) were co-incubated with radioactively (S35) -labeled cancer target cells (target; t) at different e: t ratios. The lysis of target cells by NK killing activity was then assessed by measurement of radioactive emissions. Overexpression of any of LY6G6F, VSIG10, TMEM25 and/or LSR proteins on cancer target cells and/or NK cell lines results in down-regulation of NK-mediated killing activity.

(ii) Such as PLoS ONE; 2010; vol.5; killing assays were performed in the presence of human LY6G6F, VSIG10, TMEM25 and/or LSR ECD-FC proteins as described in p.1-10. Treatment with the ECD-Fc of any LY6G6F, VSIG10, TMEM25 and/or LSR interferes with the interaction of LY6G6F, VSIG10, TMEM25 and/or LSR with its counter-receptor and thus reduces its inhibitory activity, which results in enhanced killing activity.

(iii) Such as PNAS, 2009, vol.109; killing assays were performed in the presence of neutralizing antibodies directed against any of LY6G6F, VSIG10, TMEM25 and/or LSR proteins as described in 17858-17863. Treatment with neutralizing antibodies directed against either LY6G6F, VSIG10, TMEM25 and/or LSR resulted in enhanced NK killing activity.

(iv) The "redirected killing assay" (Re-directed killing assay) was performed as follows: such as PNAS, 2009, vol.109; 17858 17863 cancer target cells expressing high density of Fc receptors are coated with activating antibodies directed against either LY6G6F, VSIG10, TMEM25 and/or LSR proteins and exposed to NK cells (expressing the indicated LY6G6F, VSIG10, TMEM25 and/or LSR proteins). Cross-linking of any one of LY6G6F, VSIG10, TMEM25 and/or LSR with an active antibody causes reduced NK-mediated killing activity.

3) Expression analysis

a) Expression of LY6G6F, VSIG10, TMEM25 and/or LSR proteins on cells isolated from human tumor biopsy

i) Confirmation of expression of any LY6G6F, VSIG10, TMEM25 and/or LSR protein was performed on an isolated population of cells from a tumor using specific antibodies directed against any LY6G6F, VSIG10, TMEM25 and/or LSR protein, respectively. As in journal of experimental medicine (j.exp.med.); 2006; vol.203; p.871-881 and Cancer research 2007 (Cancer res.); 67; 8900-8905, different cell populations, (e.g., tumor cells, endothelium, tumor-associated macrophages (TAMs) and DCs, B cells, and different T cells (CD4, CD8, and tregs) were freshly isolated from tumor biopsies to demonstrate the expression of any one of LY6G6F, VSIG10, TMEM25, and/or LSR in tumor cells, as well as tumor stroma and immunopermeabilities.

ii) binding assays to human LY6G6F, VSIG10, TMEM25 and/or LSRECD-FC proteins on an isolated population of cells from a tumor. As in journal of experimental medicine (j.exp.med.); 2006; vol.203; p.871-881 and Cancer research 2007 (Cancer res.); 67; 8900-8905, different cell populations, (e.g., tumor cells, endothelium, tumor-associated macrophages (TAMs) and DCs, B cells and different T cells (CD4, CD8 and tregs) were isolated from tumor biopsies to show the expression of any of the anti-receptors for LY6G6F, VSIG10, TMEM25 and/or LSR in tumor cells as well as on tumor stroma and immune cells.

b) LY6G6F, VSIG10, TMEM25 and/or LSR proteins in draining lymph nodes and spleen isolated from tumor-bearing mice Is expressed on the cell

(i) Confirmation of expression of LY6G6F, VSIG10, TMEM25 and/or LSR proteins on epithelial Cancer cells and immune cells of the spleen from tumor draining lymph nodes from C57 tumor-bearing mice was performed using specific antibodies directed against LY6G6F, VSIG10, TMEM25 and/or LSR proteins, respectively, as described in Clinical Cancer Research 1996 Vol.2, 811-820. Three different cancer types are being tested: b16 (melanoma), ID8 (ovary), and MC38 (colon)) to show expression of any one of LY6G6F, VSIG10, TMEM25 and/or LSR in tumor cells and immune cells in tumor draining lymph nodes.

ii) binding assays of mouse LY6G6F, VSIG10, TMEM25 and/or LSR ECD-FC proteins were performed on cells of tumor draining lymph nodes isolated from C57 tumor bearing mice versus epithelial cancer cells of the spleen as well as immune cells to show the expression of anti-receptors of any one of LY6G6F, VSIG10, TMEM25 and/or LSR in tumor cells and immune cells in tumor draining lymph nodes.

c) Expression of LY6G6F, VSIG10, TMEM25 and/or LSR proteins on M2 polarized macrophages

(i) As in natural immunology (nat. immunol.) 2010; vol.11; validation of expression of LY6G6F, VSIG10, TMEM25 and/or LSR proteins was performed on primary monocytes isolated from peripheral blood, differentiated into macrophages and exposed to "M2 driver stimuli" (e.g., IL4, IL10, glucocorticoids, TGF β) using specific antibodies directed against LY6G6F, VSIG10, TMEM25 and/or LSR proteins, respectively, to show expression of any of LY6G6F, VSIG10, TMEM25 and/or LSR in M2 differentiated macrophages.

ii) as described above, binding assays for LY6G6F, VSIG10, TMEM25 and/or LSR human ECD-FC proteins were performed on primary monocytes isolated from peripheral blood, differentiated into macrophages and exposed to a "M2 driving stimulus" (e.g., IL4, IL10, glucocorticoids, TGF β) to show the expression of any one of the anti-receptors for LY6G6F, VSIG10, TMEM25 and/or LSR in M2 differentiated macrophages.

Example 50

Development of fully human anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR antibodies

Production of human monoclonal antibodies against LY6G6F, VSIG10, TMEM25 and/or LSR antigens

Fusion proteins consisting of extracellular domains of LY6G6F, VSIG10, TMEM25 and/or LSR linked to a mouse IgG2 Fc polypeptide were produced by standard recombinant methods and used as antigens for immunization.

Transgenic HuMab mice.

Fully human monoclonal antibodies against LY6G6F, VSIG10, TMEM25 and/or LSR were prepared using mice from the HCo7 strain of transgenic HuMab mice rtm. In this mouse strain, the kappa light chain gene of endogenous mice has been described in Chen (Chen) et al (1993) journal of the European society of molecular biology (EMBO J.) 12: 811-820 and the endogenous mouse heavy chain gene has been homozygously disrupted as described in example 1 of PCT publication WO 01/09187. In addition, this mouse strain carries the human kappa light chain transgene KCo5, as described by fisherwald (fisherwild) et al (1996) Nature Biotechnology (Nature Biotechnology) 14: 845-851; and human heavy chain transgenes, as described in U.S. Pat. nos. 5,545,806, 5,625,825, and 5,545,807.

HuMab immunoassay:

to generate fully human monoclonal antibodies against LY6G6F, VSIG10, TMEM25 and/or LSR, mice of the HCo7 HuMab mouse strain can be immunized with purified recombinant LY6G6F, VSIG10, TMEM25 and/or LSR fusion proteins derived from mammalian cells transfected with an expression vector containing the gene encoding the fusion protein. Systemic immunization protocols against HuMab mice are described in lamberg (Lonberg, N.) et al (1994): Nature 368 (6474): 856-859; fishweld (Fishwild, D.) et al (1996) Nature Biotechnology (Nature Biotechnology) 14: 845, 851 and PCT publication WO 98/24884. These mice were 6-16 weeks old when the antigen was first infused. HuMab mice were immunized intraperitoneally with purified recombinant LY6G6F, VSIG10, TMEM25 and/or LSR antigen preparations (5-50.mu.g, purified from transfected mammalian cells expressing LY6G6F, VSIG10, TMEM25 and/or LSR fusion proteins).

Transgenic mice were immunized IP with antigen in complete freund's adjuvant or Ribi adjuvant followed by 3-21 days of IP immunization with antigen in complete freund's adjuvant or Ribi adjuvant (up to a total of 11 immunizations). The immune response was monitored by retroorbital bleeding. Plasma can be screened by ELISA (as described below) and mice with sufficient titers of anti-LY 6G6F, VSIG10, TMEM25 and/or LSR human immunoglobulin are used for fusion. The antigen was used to immunize mice intravenously 3 days before sacrifice and removal of the spleen.

Selection of HuMab mice that produce anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR antibodies:

to select HuMab mice that produce antibodies that bind to LY6G6F, VSIG10, TMEM25 and/or LSR, sera from immunized mice were tested by modified ELISA as originally described by fisherwald (fisherwild, D.) et al (1996). Briefly, microtiter plates were coated with purified recombinant LY6G6F, VSIG10, TMEM25 and/or LSR fusion proteins in PBS at 1-2. mu.g/ml, incubated overnight at 50. mu.l/well at 4 ℃ and then blocked with 5% BSA in PBS at 200. mu.l/well. Dilutions of plasma from LY6G6F, VSIG10, TMEM25 and/or LSR-immunized mice were added to each well and incubated for 1-2 hours at ambient temperature. The plates were washed with PBS/tween and then incubated with goat-anti-human kappa light chain polyclonal antibody coupled with alkaline phosphatase for 1 hour at room temperature. After washing, the plates were developed with pNPP substrate and analyzed by spectrophotometer at OD 415-. Mice showing the highest titers of anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR antibodies were used for fusion. Fusions were performed as described below and hybridoma supernatants were tested for anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR activity by ELISA.

Production of hybridomas producing human monoclonal antibodies against LY6G6F, VSIG10, TMEM25 and/or LSR.

Mouse splenocytes isolated from HuMab mice were fused with PEG to a mouse myeloma cell line based on standard protocols. These resulting hybridomas are then screened for the production of antigen-specific antibodies. A single cell suspension of splenic lymphocytes from immunized mice was fused with 50% PEG (Sigma) into one-fourth the number of P3X63 ag8.6.53 non-secreting mouse myeloma cells (ATCC, CRL 1580). Subjecting the cells to a treatment at about 1X 10^-5The/well plates were plated in flat-bottom microtiter plates followed by incubation for about two weeks in selective medium supplemented with 10% fetal bovine serum: origen in RPMI (IGEN0, L-glutamine, sodium pyruvate, HEPES, penicillin, streptomycin, gentamicin, 1 XHAT, and β -mercaptoethanol after 1-2 weeks, cells were cultured in media in which HAT was replaced with HT. Individual wells were then screened by ELISA (described above) for human anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25, and/or anti-LSR monoclonal IgG antibodies. Once significant hybridoma growth had occurred, media could be monitored, typically after 10-14 days.the antibody secreting hybridomas were replated, rescreened, and if positive for human IgG, anti-VSIG 6G6F, anti-VSIG 10, anti-TMEM 25, and/or anti-LSR monoclonal antibodies were subcloned at least twice by limiting dilution.the stable subclones were then cultured in vitro, to produce small amounts of antibody in the tissue culture medium for further characterization.

Hybridoma clones were selected for further analysis.

Structural characterization of the desired anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR human monoclonal antibodies

The cDNA sequences encoding the heavy chain variable region and the light chain variable region of the obtained anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR monoclonal antibodies were obtained from the resulting hybridomas, respectively, 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 were identified. These sequences can be compared to the light chain and heavy chain sequences of known human germline immunoglobulins and the CDRs of each heavy and light chain of the identified obtained anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR sequences.

Characterization of the binding specificity and binding kinetics of anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR human monoclonal antibodies

The binding affinity, binding kinetics, binding specificity and cross-competition of anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR antibodies were examined by Biacore analysis. Binding specificity was also examined by flow cytometry.

Binding affinity and binding kinetics

anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR antibodies generated according to the present invention were characterized for affinity and binding kinetics by Biacore analysis (Biacore AB, uppsala, sweden). Purified recombinant human LY6G6F, VSIG10, TMEM25 and/or LSR fusion proteins were covalently linked to a CM5 chip (carboxymethyl dextran coated chip) via primary amines using standard amine coupling chemistry and by a kit providing Biacore. Binding was measured by flowing the antibody in HBS EP buffer (supplied by BIAcore AB) at a concentration of 267nM at a flow rate of 50 μ l/min. The association kinetics of the antigen-associated antibody were followed for 3 minutes, while the dissociation kinetics were followed for 7 minutes. The association and dissociation curves were fitted into a 1: 1 Langmuir binding model (Langmuir binding model) using BlA evaluation (BlAevaluation) software (Biacore AB). To minimize the effect of affinity in the estimation of the binding constant, only the data corresponding to the initial segments of the association and dissociation phases were used for the fit.

Epitope mapping of the obtained anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR antibodies

Biacore was used to determine the epitope classification of anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSRHuMAb. The obtained anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR antibodies were used to localize their epitopes on LY6G6F, VSIG10, TMEM25 and/or LSR antigens, respectively. These different antibodies were coated on three different surfaces of the same chip to 8000RU each. Dilutions of each mAb (starting at 10 μ G/mL) were prepared and incubated for one hour with Fc-fusion LY6G6F, VSIG10, TMEM25 and/or LSR (50 nM). The incubation complexes were injected simultaneously to all three surfaces (and blank surfaces) at a flow rate of 20 μ L/min for 1.5 minutes. At the end of 1.5 minutes, the signal from each surface after subtraction of the appropriate blanks was plotted against the concentration of mAb in the complex. In analyzing the data, anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR antibodies were classified into different epitope groups depending on the epitope mapping results. Their functional characteristics were also compared.

Chinese hamster ovary Cells (CHO) expressing LY6G6F, VSIG10, TMEM25 and/or LSR proteins on the cell surface were developed and used to determine the specificity of LY6G6F, VSIG10, TMEM25 and/or LSR HuMAb by flow cytometry. CHO cells were transfected with expression plasmids containing the full-length cDNA encoding the transmembrane-type LY6G6F, VSIG10, TMEM25 and/or LSR antigens or variants thereof. The transfected protein containing an epitope tag at the N-terminus is used for the detection of antibodies specific for the epitope. The binding of anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR MAb was assessed by incubating each LY6G6F, VSIG10, TMEM25 and/or LSR Ab with transfected cells at a concentration of 10 μ G/ml. These cells were washed and binding was detected with FITC-labeled anti-human IgG Ab. A murine anti-epitope tag Ab followed by a labeled anti-murine IgG was used as a positive control. Non-specific human and murine abs were used as negative controls. The data obtained were used to assess the specificity of humabs with LY6G6F, VSIG10, TMEM25 and/or LSR antigen targets.

These antibodies and other antibodies specific for LY6G6F, VSIG10, TMEM25 and/or LSR may be used in the aforementioned anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR related treatments, such as the treatment of cancer, where LY6G6F, VSIG10, TMEM25 and/or LSR antigens are differentially expressed; and/or modulating (enhancing or inhibiting) B7 immune co-stimulation involving LY6G6F, VSIG10, TMEM25 and/or LSR antigens, for example in the treatment of cancer and autoimmune diseases, wherein such antibodies for example prevent negative stimulation of T cell activity against a desired target cancer cell or prevent positive stimulation of T cell activity, thereby eliciting a desired anti-autoimmune effect.

The present invention has been described and provides various embodiments regarding the manufacture and selection of desired anti-LY 6G6F, anti-VSIG 10, anti-TMEM 25 and/or anti-LSR antibodies for use as therapeutics and diagnostics where the disease or condition is associated with LY6G6F, VSIG10, TMEM25 and/or LSR antigens. Various embodiments can optionally be combined in any suitable manner beyond the explicit combinations and sub-combinations shown herein. The invention will now be further described by the following claims.

Claims (30)

1. An isolated polypeptide consisting of SEQ ID NO 11 or 12.

2. A fusion protein comprising the polypeptide of claim 1 linked to a heterologous sequence, wherein the heterologous sequence is human IgG1 Fc.

3. The fusion protein of claim 2, wherein the amino acid sequence of the fusion protein is as set forth in SEQ ID NO: 75-80, 176, 181, respectively.

4. The fusion protein of claim 2, which modulates an immune cell response.

5. A nucleic acid sequence encoding the polypeptide of claim 1 or the fusion protein of any one of claims 2-4.

6. The nucleic acid sequence according to claim 5, selected from the group consisting of SEQ ID NO: SEQ ID NO: 40-46, 132, 44, 155, 186, 188, 193, 198.

7. An expression vector or virus comprising at least one nucleic acid sequence according to claim 5.

8. A recombinant cell comprising the expression vector or virus of claim 7, wherein the cell constitutively or inducibly expresses the polypeptide encoded by the nucleic acid sequence.

9. A method of producing an LSR soluble ectodomain polypeptide, or a fragment or fusion protein thereof, comprising culturing the recombinant cell of claim 8 under conditions whereby the cell expresses the polypeptide encoded by the nucleic acid sequence, and recovering said polypeptide.

10. A pharmaceutical composition comprising: the isolated polypeptide of claim 1, or the fusion protein of claim 2, and further comprising a pharmaceutically acceptable diluent or carrier.

11. Use of any one of the isolated polypeptide of claim 1 or the fusion protein of claim 2 in the manufacture of a medicament for treating an immune system-related disorder, wherein the immune system-related disorder comprises an autoimmune disease and transplant rejection, wherein the autoimmune disease is selected from the group consisting of: multiple sclerosis; rheumatoid arthritis; systemic Lupus Erythematosus (SLE); ulcerative colitis; crohn's disease; type I diabetes, and psoriasis.

12. The use of claim 11, wherein the multiple sclerosis comprises relapsing multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis.

13. Use of any one of the isolated polypeptide of claim 1 or the fusion protein of claim 2 in the preparation of a medicament for inhibiting or reducing activation of T cells, wherein the medicament is administered to a subject.

14. Use of any one of the isolated polypeptide of claim 1 or the fusion protein of claim 2 in the manufacture of a medicament for treating an immune system-related disorder, wherein the immune system-related disorder comprises an autoimmune disease and transplant rejection, wherein the autoimmune disease is selected from the group consisting of: multiple sclerosis, rheumatoid arthritis; systemic Lupus Erythematosus (SLE); ulcerative colitis; crohn's disease; type I diabetes, and psoriasis.

15. The use of claim 14, wherein the treatment is combined with an additional therapy for treating an immune-related disorder.

16. The use of claim 15, wherein the therapy is selected from the group consisting of: immunosuppressants, non-steroidal anti-inflammatory drugs, hydroxychloroquine, sulfasalazine (sulphosalazopryine), sodium chloroaurate, etanercept, infliximab, basiliximab, asecept, rituximab, carcinoxane (cytoxan), mitoxantrone hydrochloride, anakinra, and/or other biological agents.

17. The use of claim 15, wherein the therapy is selected from the group consisting of: intravenous immunoglobulin (IVIG), interferons; glatiramer acetate NatalizumabMitoxantroneCytotoxic agents, calcineurin inhibitors; immunosuppressive macrolides, lymphocyte homing agents, corticosteroids; cyclophosphamide; azathioprine; methotrexate; leflunomide or an analog thereof; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyperguline or an analog thereof; immunosuppressive monoclonal antibodies, or ligands for leukocyte receptors; or other immunomodulatory compound or another immunomodulatory agent.

18. The use of claim 15, wherein the therapy is an organism.

19. The use of claim 15, wherein the therapy is a Cox-2 inhibitor.

20. The use of claim 15, wherein the therapy is a polypeptide.

21. The use of any one of claims 16-19, wherein the immunosuppressive agent is a corticosteroid, cyclosporine, cyclophosphamide, prednisone, azathioprine, methotrexate, rapamycin, or tacrolimus; and/or the biological agent is a TNF-alpha blocker or antagonist, or a targetAny other biological agent that is directed against any inflammatory cytokine; and/or the interferon is IFN-beta-la ( And) Or IFN-beta-lbAnd/or the calcineurin inhibitor is cyclosporine a or FK 506; and/or the immunosuppressive macrolide is rapamycin or a derivative thereof; and/or said lymphocyte homing agent is FTY720 or an analog thereof; and/or the immunosuppressive monoclonal antibody is a monoclonal antibody to a leukocyte receptor; and/or the other immunomodulatory compound is CTLA4-Ig (abatacept,) CD28-Ig, B7-H4-Ig, or other costimulatory agents, or adhesion molecule inhibitors.

22. The use according to claim 21, wherein the derivative of rapamycin is 40-O- (2-hydroxy) ethyl-rapamycin; and/or the adhesion molecule inhibitor is a mAb or a low molecular weight inhibitor.

23. The use of claim 22, wherein the low molecular weight inhibitor comprises an LFA-1 antagonist, a selectin antagonist and a VLA-4 antagonist.

24. The use of claim 17, wherein the leukocyte receptor is MHC, CD2, CD3, CD4, CD11a/CD18, CD7, CD25, CD27, B7, CD40, CD45, CD58, CD137, ICOS, CD150(SLAM), OX40, or 4-1 BB.

25. The use of claim 21, wherein the leukocyte receptor is MHC, CD2, CD3, CD4, CD11a/CD18, CD7, CD25, CD27, B7, CD40, CD45, CD58, CD137, ICOS, CD150(SLAM), OX40, or 4-1 BB.

26. The use of any one of claims 16-20 and 22-25, wherein the immune disorder is selected from an autoimmune disease or transplant rejection.

27. The use of claim 21, wherein the immune disorder is selected from an autoimmune disease or transplant rejection.

28. The use of claim 26, wherein the autoimmune disease is selected from the group consisting of: multiple sclerosis, rheumatoid arthritis; systemic Lupus Erythematosus (SLE); ulcerative colitis; crohn's disease; type I diabetes and psoriasis.

29. The use of claim 27, wherein the autoimmune disease is selected from the group consisting of: multiple sclerosis, rheumatoid arthritis; systemic Lupus Erythematosus (SLE); ulcerative colitis; crohn's disease; type I diabetes and psoriasis.

30. The use according to claim 28 or 29, wherein the autoimmune disease is selected from the group consisting of any type and subtype of any of the following diseases: multiple sclerosis, rheumatoid arthritis, type I diabetes, psoriasis, and systemic lupus erythematosus.