MX2007008260A - Irta-2 antibodies and their uses - Google Patents

Abstract

The present invention provides isolated monoclonal antibodies, particularly human monoclonal antibodies, that specifically bind to IRTA-2 with high affinity. Nucleic acid molecules encoding the antibodies of the invention, expression vectors, host cells and methods for expressing the antibodies of the invention are also provided. Immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies of the invention are also provided. The invention also provides methods for detecting IRTA-2, as well as methods for treating various B cell malignancies, including non-Hodgkin's lymphoma.

Description

IRTA-2 ANTIBODIES AND THEIR USES BACKGROUND OF THE INVENTION Genes / proteins associated with immune receptor translocation (IRTA), also known as homologous Fe receptor genes (FcRH), consist of a family of five members of cell surface receptors. similar to immunoglobulin (Miller et al, (2002) Blood 99: 2662; Davis et al, (2002) I munological Reviews 190: 123). The IRTAs were initially discovered by the analysis of the breakpoints of a multiple myeloma cell line that contained a rearrangement of chromosome Iq21 (Hatzivassiliou et al, (2001) Iw unity., 14: 277). Each of the IRTA glycoproteins contains between 3 to 9 extracellular Ig-like domains (Miller, 2002, supra). IRTAs are also characterized by having a cytoplasmic domain containing 3 to 5 tyrosine residues contained within particular portions, suggesting the presence of inhibitory immunotyrosine portions (ITIM) and portions similar to immunotyrosine activity (similar to ITAM) (Miller, 2002, supra, Hatzivassiliou, 2001, supra). IRTAs are commonly expressed in peripheral lymphoid tissues, which include lymph nodes, tonsils, peripheral B cells at rest and normal germinal center B cells (Davis et al, (2001) PNAS, 98: 9772). IRTA-2, -3, -4 and -5 are all expressed at high levels in spleen, whereas, in comparison, IRTA1 has been detected at lower levels in the spleen. The expression of IRTA has been analyzed within the B cell compartment of human amygdala tissue. IRTA-1 is expressed outside the lymphoid follicles in the marginal zone pattern and intraepithelial lymphocytes. IRTA-2 and -3 are expressed within the germinal center, with higher expression in the clear zone rich in centocytes. IRTA-4 and -5 are highly expressed within mantle zones, indicating expression in natural or simple B cells (Miller, 2002, supra). It has been shown that IRTA genes are highly expressed in non-B cell Hodgkin lymphoma, chronic lymphocytic leukemias, follicular lymphomas, diffuse large cell lymphomas of B lineage and multiple myelomas (Davis, 2001, supra).

BRIEF DESCRIPTION OF THE INVENTION The present invention provides isolated monoclonal antibodies, in particular human monoclonal antibodies, which bind to IRTA-2 and which exhibit numerous desirable properties. These properties include high affinity binding to human IRTA-2, but lacking substantial cross-reactivity with either IRTA-3 or human IRTA-4. Still further, it has been shown that the antibodies of the invention bind to a B-cell tumor cell line.

In preferred embodiments of the invention, human IRTA-2 comprises a polypeptide having an amino acid sequence as summarized in SEQ ID NO: 25 [Genbank Acc. No. NP_112571]; human IRTA-1 comprises a polypeptide having an amino acid sequence as summarized in SEQ ID NO: 26 [Genbank Acc. No. NP_112572]; human IRTA-3 comprises a polypeptide having an amino acid sequence as summarized in SEQ ID NO: 27 [Genbank Acc. No. AAL59390]; the human IRTA-4 comprises a polypeptide having an amino acid sequence as summarized in SEQ ID NO: 28 [Genbank Acc. No. AAL60249]; and / or the human IRTA-5 comprises a polypeptide having an amino acid sequence as summarized in SEQ ID NO: 29 [Genbank Acc. No. AAL60250]. In one aspect, the invention is concerned with an isolated monoclonal antibody or an antigen binding portion thereof, wherein the antibody: (a) binds to human IRTA-2 with ONE KD of lxlO "7 or less; ) binds human CHO cells transfected with IRTA-2, (c) does not bind substantially to human IRTA-3 or IRTA-4, and (d) binds to Granta 519 tumor cells and does not bind substantially to tumor cells of Ra io Ramos Preferably, the antibody is a human antibody, although in alternative embodiments the antibody can be a murine antibody, a chimeric antibody or a humanized antibody. In a preferred embodiment, the antibody does not bind substantially to Daudi tumor cells, IM-9, Karpas 1106P or SU-DHL-4. In more preferred embodiments, the antibody binds to human IRTA-2 with a KD of 5 x 10 ~ 8 M or less, binds to human IRTA-2 with a KD of 2 X 10"8 or less, binds to IRTA -2 human with a KD of 1 x 10"8 M or less, binds to human IRTA-2 with a KD of 5x10 ~ 9 M or less, binds to human IRTA-2 with a KD of 4x10 ~ 9 M or minor, bind to human IRTA-2 with a KD of 3x10"9 M or less or bind to human IRTA-2 with a KD of 2.1 x 10 ~ 9 or less.In another embodiment, the invention provides an isolated monoclonal antibody or an antigen binding portion thereof, wherein the antibody competes cross-linked by the binding to IRTA-2 with a reference antibody comprising: (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 14; and (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 16.

In various embodiments, the reference antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15; or the reference antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16. In another aspect, the invention is concerned with an isolated monoclonal antibody or an antigen binding portion thereof, comprising a region heavy chain variable which is the product of or derived from a human VH 3-23 gene, wherein the antibody binds specifically to IRTA-2. The invention also provides an isolated monoclonal antibody or an antigen binding portion thereof, comprising a heavy chain variable region that is the product of or derivative of a human VH 1-8 gene, wherein the antibody specifically binds to IRTA-2. The invention still further provides an isolated monoclonal antibody or an antigen binding portion thereof, comprising a light chain variable region that is the product of or derived from a human VK L6 gene, wherein the antibody binds specifically to IRTA -2. The invention still further provides an isolated monoclonal antibody or an antigen binding portion thereof, comprising a light chain variable region that is the product of or derived from a human V L18 gene, wherein the antibody specifically binds to IRTA -2. In a preferred embodiment, the invention provides an isolated monoclonal antibody or an antigen binding portion thereof, comprising: (a) a heavy chain variable region of a human VH3-23 or 1-8 gene; and (b) a light chain variable region of a human VK L6 or VK L18 gene; where the antibody binds specifically to IRTA-2. In a preferred embodiment, the antibody comprises a heavy chain variable region of a human VH 3-23 gene and a light chain variable region of a human VK L6 gene. In another preferred embodiment, the antibody comprises a heavy chain variable region of a human VH 1-8 gene and a light chain variable region of a human VK L18 gene. In another aspect, the invention provides an isolated monoclonal antibody or antigen binding portion thereof, comprising: a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences; and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein: (a) the heavy chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs : 5 and 6 and conservative modifications thereof; (b) the light chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequence of SEQ ID NOs: 11 and 12 and conservative modifications thereof; (c) the antibody binds to human IRTA-2 with a KD of lxlO "7 or less; (d) binds to human CHO cells transfected with IRTA-2; (e) the antibody does not bind substantially to IRTA-3 or human IRTA-4, and (f) the antibody binds to Granta 519 tumor cells and does not bind substantially to Raj io Ramos tumor cells.Preferably, the heavy chain variable region CDR2 sequence comprises a sequence of amino acids selected from the group consisting of amino acid sequences of SEQ ID NOs: 3 and 4 and conservative modifications thereof, and the light chain variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 9 and 10 and conservative modifications thereof Preferably, the heavy chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequences. noacids of SEQ ID NOs: 1 and 2 and conservative modifications thereof; and the light chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 7 and 8 and conservative modifications thereof. In a preferred embodiment, the antibody does not bind substantially to Daudi tumor cells, IM-9, Karpas 1106P or SU-DHL-4. In yet another aspect, the invention provides an isolated monoclonal antibody or antigen binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 14; (b) the light chain variable region comprises an amino acid sequence that is at least 80% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 16; (c) the antibody binds to human IRTA-2 with a KD of lXlO "7 M or less; (d) binds to human CHO cells transfected with IRTA-2; (e) the antibody does not bind substantially to IRTA -3 or human IRTA-4, and (f) the antibody binds to Granta 519 tumor cells and does not bind substantially to Raj io Ramos tumor cells.In a preferred embodiment, the antibody does not bind substantially to Daudi tumor, I-9, Karpas 1106P or SU-DHL-4 In preferred embodiments, the invention provides an isolated monoclonal antibody or antigen binding portion thereof, comprising: (a) a variable chain region CDR1 weighing comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2. (b) a heavy chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 4; (c) a heavy chain variable region CDR3 comprising of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 6; (d) a light chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 8; (e) a light chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 10; and (f) a light chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 and 12; wherein the antibody binds specifically to IRTA-2. A preferred combination comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 1; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 3; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 5; (d) a light chain variable region CDR1 comprising SEQ ID NO: 7; (e) a light chain variable region CDR2 comprising SEQ ID NO: 9; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 11. Another preferred combination comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 2; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 4; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 6; (d) a light chain variable region CDR1 comprising SEQ ID NO: 8; (e) a light chain variable region CDR2 comprising SEQ ID NO: 10; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 12. Other preferred antibodies of the invention or antigen binding portions thereof comprise: (a) a heavy chain variable region comprising a sequence of amino acids selected from the group consisting of SEQ ID NOs: 13 and 14; and (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 16; where the antibody binds specifically to IRTA-2. A preferred combination comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15.

Another preferred combination comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16. In another aspect of the invention, there are provided antibodies or antigen binding portions thereof, which compete for binding to IRTA -2 with any of the antibodies mentioned above. The antibodies of the invention can be, for example, full-length antibodies, for example of an IgGl or IgG4 isotype. Alternatively, the antibodies can be antibody fragments, such as Fab or Fab '2 fragments or single chain antibodies. The invention also provides an immunoconjugate comprising an antibody of the invention or antigen binding portion thereof, linked to a therapeutic agent, such as a cytotoxin or a radioactive isotope. The invention also provides a bispecific molecule comprising an antibody or antigen binding portion thereof, of the invention, linked to a second functional portion having a different binding specificity than the antibody or antigen binding portion thereof. Also provided are compositions comprising an antibody or antigen binding portion thereof or immunoconjugate or bispecific molecule of the invention and a pharmaceutically acceptable carrier. Nucleic acid molecules encoding the antibodies or antigen binding portions thereof, of the invention, are also encompassed by the invention, also as expression vectors comprising such nucleic acids and host cells comprising such expression vectors. In addition, the invention provides a transgenic mouse comprising human immunoglobulin heavy and light chain transgenes, wherein the mouse expresses an antibody of the invention, also as hybridomas prepared from such a mouse, wherein the hybridoma produces the antibody of the invention. In yet another aspect, the invention provides a method for treating a B cell malignancy in a subject in need of treatment comprising administering to the subject the antibody or antigen binding portion thereof, of the invention, such that the B cell malignancy in the subject is treated. The disease may be, for example, non-Hodgkin's lymphoma, chronic lymphocytic leukemias, follicular lymphocytes, diffuse large cell lymphomas of B lineage and multiple myelomas. The invention also provides methods for the manufacture of "second generation" anti-IRTA-2 antibodies based on the sequences of the anti-IRTA-2 antibodies provided herein. For example, the invention provides a method for the preparation of an anti-IRTA-2 antibody comprising: (a) providing: (i) a heavy chain variable region antibody sequence comprising a CDR1 sequence selected from the group consists of SEQ ID NOs: 1 and 2, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 3 and 4 and / or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 5 and 6; and / or (ii) a light chain variable region antibody sequence comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 7 and 8, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 9 and 10 and / or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 11 and 12; (b) altering at least one amino acid residue within the heavy chain variable region antibody sequence and / or the light chain variable region antibody sequence to create at least one altered antibody sequence; and (c) expressing the altered antibody sequence as a protein. Other elements and advantages of the present invention will become apparent from the following detailed description and examples which should not be construed as limiting. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

BRIEF DESCRIPTION OF THE FIGURES Figure 1A shows the nucleotide sequence (SEQ ID NO: 17) and amino acid sequence (SEQ ID NO: 13) of the heavy chain variable region of the human monoclonal antibody 9D12. The regions of CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 3) and CDR3 (SEQ ID NO: 5) are delineated and the derivations of germination line V, D and J are indicated. Figure IB shows the nucleotide sequence (SEQ ID NO: 19) and amino acid sequence (SEQ ID NO: 15) of the light chain variable region of the human monoclonal antibody 9D12. The regions of CDR1 (SEQ ID NO: 7), CDR2 (SEQ ID NO: 9) and CDR3 (SEQ ID NO: 11) are delineated and the derivations of germination line V and J are indicated. Figure 2A shows the nucleotide sequence (SEQ ID NO: 18) and amino acid sequence (SEQ ID NO: 14) of the heavy chain variable region of the human monoclonal antibody 8A1. The CDR1 (SEQ ID NO: 2), CDR2 (SEQ ID NO: 4) and CDR3 (SEQ ID NO: 6) regions are delineated and the germination line derivations V and J are indicated. Figure 2B shows the nucleotide sequence (SEQ ID NO: 20) and amino acid sequence (SEQ ID NO: 16) of the light chain variable region of the human monoclonal antibody 8A1. The regions of CDR1 (SEQ ID NO: 8), CDR2 (SEQ ID NO: 10) and CDR3 (SEQ ID NO: 12) are delineated and the derivations of germination line V and J are indicated. Figure 3 shows the alignment of the amino acid sequence of the heavy chain variable region of 9D12 with the amino acid sequence VH 3-23 of the human germination line (SEQ ID NO: 21). Figure 4 shows the alignment of the amino acid sequence of the heavy chain variable region of 8A1 with the human germ line VH 1-8 amino acid sequences (SEQ ID NO: 22). Figure 5 shows the alignment of the amino acid sequence of the light chain variable region of 9D12 with the amino acid sequence Vk L6 of the human germination line (SEQ ID NO: 23). Figure 6 shows the alignment of the amino acid sequence of the light chain variable region of 8A1 with the amino acid sequence of Vk L18 of human germination line (SEQ ID NO: 24). Figures 7A-C show the results of experiments demonstrating that human monoclonal antibodies, 9D12 and 8A1, directed against human IRTA-2, bind specifically to human IRTA-2. Figure 7A is a bar graph showing the binding to CHO cells transfected with IRTA-2. Figure 7B is a bar graph showing the lack of binding to CHO cells transfected with IRTA-3. Figure 7C is a bar graph showing the lack of binding to CHO cells transfected with IRTA-4. Figure 8 shows the results of flow cytometry experiments demonstrating that human monoclonal antibodies 9D12 and 8A1, directed against human IRTA-2, bind to CD19 + B cells. Figure 9 shows the results of flow cytometry experiments that demonstrate that human monoclonal antibodies, 9D12 and 8A1, directed against human IRTA-2, do not bind to the cell surface of the B-cell tumor cell lines of Ramos, Raji, Daudi or IM-9. Figure 10 shows the results of flow cytometry experiments demonstrating binding of human monoclonal antibodies 9D12 and 8A1, directed against human IRTA-2, to the cell lines of B Karpas 1106P, SU-DHL-4 and Granta 519 Figure 11 shows the results of flow cytometry experiments demonstrating binding of human monoclonal antibodies 9D12 and 8A1, directed against human IRTA-2, to B cell lines SU-DHL-6 and JEKO-1.

DETAILED DESCRIPTION OF THE INVENTION The present invention is concerned with isolated monoclonal antibodies, in particular human monoclonal antibodies, which specifically bind to IRTA-2 with high affinity. In certain embodiments, the antibodies of the invention are derived from particular heavy and light chain germination line sequences and / or comprise particular structural elements such as CDR regions comprising particular amino acid sequences. The invention provides isolated antibodies, methods of making such antibodies, immunoconjugates, and bispecific molecules comprising such antibodies and pharmaceutical compositions containing the antibodies, immunoconjugates, or bispecific molecules of the invention. The invention is also concerned with methods of using the antibodies, such as for detecting IRTA-2, as well as for treating diseases associated with the expression of IRTA-2, such as B-cell malignancies that express IRTA-2. Thus, the invention also provides methods of using the anti-IRTA-2 antibodies of the invention to treat B cell malignancies, for example, in the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemias, follicular lympholas, large diffuse cell of B lineage and multiple myelomas. In order for the present invention to be understood more easily, certain terms are defined first.

Additional definitions are summarized throughout the detailed description. The terms "gene 2 associated with the translocation of the immunoglobulin superfamily receptor" and "IRTA-2" are used interchangeably and include variants, isoforms and homologous species of human IRTA-2. Thus, the human antibodies of the invention can, in certain cases, cross-react with IRTA-2 from species other than human. In other cases, the antibodies may be completely specific for human IRTA-2 and may not exhibit species or other types of cross-reactivity. The complete amino acid sequence of human IRTA-2 has Genbank accession number NP_112571 (SEQ ID NO: 25). The terms "IRTA-1", "IRTA-3", "IRTA-4" and "IRTA-5" include variants, isoforms and homologues of species of "IRTA-1", "IRTA-3", "IRTA-4"and" IRTA-5"human, respectively. The complete amino acid sequence of human IRTA-1 has accession number of Genbank NP_112572 (SEQ ID NO: 26). The complete amino acid sequence of human IRTA-3 has Genbank accession number AAL59390 (SEQ ID NO: 27). The complete amino acid sequence of human IRTA-4 has Genbank accession number AAL60249 (SEQ ID NO: 28). The complete amino acid sequence of human IRTA-5 has accession number of Genbank AAL60250 (SEQ ID NO: 29). The term "immune response" refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes and soluble macromolecules produced by the anterior cells or the liver (in which antibodies, cytokines and complement are included) which result in selective damage to, destruction of or elimination of the human body from invading pathogens, cells or tissues infected with pathogens, cancer cells, or in cases of autoimmunity or pathological inflammation, normal human cells or tissues. A "signal transduction path" refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. As used herein, the phrase "cell surface receptor" includes, for example, molecules and complexes of molecules capable of receiving a signal and transmitting such signal through the plasma membrane of a cell. An example of a "cell surface receptor" of the present invention is the IRTA-2 receptor. The term "antibody" as referred to herein includes whole antibodies and any antigen binding fragment (ie, "antigen binding portion") or individual chains thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds or an antigen binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CHi, CH2 and < ¾ Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of a domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can moderate the binding of the immunoglobulin to tissues or host factors, in which several cells of the immune system (for example, effector cells) and the first component (CIq) of the classical complement system are included. The term "antigen binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retains the ability to specifically bind to an antigen ( for example, IRTA-2). It has been shown that the antigen binding function of an antibody can be effected by fragments of a full-length antibody. Examples of link fragments encompassed by the term "antigen binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHi domains; (ii) a F (ab ') 2 fragment / a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the engozone region; (iii) a Fd fragment consisting of the VH and CHi domains; (iv) a fragment of Fv consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) a region that determines isolated complementarity (CDR). In addition, although the two domains of the Fv, VL and VH fragment are encoded by separate genes, they can be linked, using recombinant methods, by a synthetic linker that allows them to be manufactured as a single protein chain in which the VL and VH pair to form monovalent molecules (known as a single Fv chain (scFv), see for example, Ave et al (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Nati. Acad. Sci. USA 85: 5879-5883). It is proposed that such single chain antibodies also be encompassed in the term "antigen binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art and the fragments are screened or filtered for utility in the same way as intact antibodies are. An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (eg, an isolated antibody that binds specifically to IRTA-2 is substantially free of antibodies that bind specifically to antigens other than IRTA-2). An isolated antibody that binds specifically to IRTA-2 may, however, have cross-reactivity with other antigens, such as IRTA-2 molecules of another species. In addition, an isolated antibody can be substantially free of other material and / or cellular chemical compounds. The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition shows a single binding specificity and affinity for a particular epitope. The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the structure and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (eg, randomly introduced mutations or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germination line of another mammalian species, such as a mouse, have been grafted onto sequences of human structure. The term "human monoclonal antibody" refers to antibodies that exhibit a single binding specificity that have variable regions in which both the structure and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma that includes a B cell obtained from a transgenic non-human animal, for example a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal to genes of human immunoglobulin or a hybridoma prepared therefrom (described further hereinbelow), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectome, (c) antibodies isolated from a recombinant combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means involving 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 an animal transgenic for human Ig sequences is used, somatic mutagenesis in vivo) and thus the amino acid sequences of the VH and VL regions of recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist in the repertoire of human antibody germination line in vivo. As used herein, "isotype" refers to an antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes. The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds an antigen." The term "human antibody derivatives" refers to any modified human antibody, for example, a conjugate of the antibody and another agent or antibody. The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germination line of another mammalian species, such as a mouse, have been grafted onto sequences of human structure. Additional structure region modifications can be made within the human structure sequences. The term "chimeric antibody" is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived of a mouse antibody and the constant region sequences are derived from a human antibody. As used herein, an antibody that "binds specifically to human IRTA-2" is intended to refer to an antibody that binds human IRTA-2 with a KD of 1 x 10"7 M or less, more preferably 5 x 10 ~ 8 M or less, more preferably 3 x 10"8 M or less, more preferably 1 x 10" 8 M or less, even more preferably 5 x 10"9 M or less. The term "KasS0c" or "Ka", as used herein, is intended to refer to the rate of association of a particular antibody-antigen interaction, while the term "KdiS" or "Ka", as used in the present, it is proposed to refer to the dissociation rate of a particular antibody-antigen interaction. The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of ¾ to Ka (ie, Kd / Ka) and is expressed as a molar concentration (M) . KD values for antibodies can be determined using methods well established in the art. A preferred method for determining the KD of an antibody is to use surface plasmon resonance, preferably using a biodetector system such as a Biacore® system. As used herein, the term "high affinity" for an IgG antibody refers to an antibody having a KD of 1 x 1 (7 M or less, more preferably 5 x 10"8 or less and even more preferably 5 x 10"9 M or less for a target antigen However, the" high affinity "linkage may vary for other antibody isotypes For example, the" high affinity "linkage for an IgM isotype refers to an antibody that has a KD of 10"6 M or less, more preferably 10" 7 or less, still more preferably 10"8 M or less As used herein, the term" subject "includes any human or non-human animal. The term "non-human animal" includes all vertebrates, for example, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Various aspects of the invention are described in additional detail in the following subsections.

Anti-IRTA-2 Antibodies The antibodies of the invention are characterized by particular elements or functional properties of the antibodies. For example, the antibodies specifically bind to human IRTA-2. Preferably, an antibody of the invention binds to IRTA-2 with high affinity, for example with a KD of 1 x 10 ~ 7 M or less. The anti-IRTA-2 antibodies of the invention preferably exhibit one or more of the following characteristics: (a) bind to human IRTA-2 with a KD of lxlCT7 M or less; (b) bind to human CHO cells transfected with IRTA-2; (c) are not substantially linked to human IRTA-3 or IRTA-4; and / or (d) bind to Granta 519 tumor cells and do not substantially bind to Raj i or Ramos tumor cells. Preferably, the antibody binds human IRTA-2 with a KD of 5 X 1 (T8 M or less, binds to human IRTA-2 with a KD of 2 X io ~ 8 M or less, binds to human IRTA-2 with a KD of 5 X 10"9 M or less, binds to human IRTA-2 with a KD of 4 X IO-9 M or less, binds to human IRTA-2 with a KD of 3 X IO-9 M or lower or bind to human IRTA-2 with a KD of 2.1 X 10 9 M or less In a preferred embodiment, the antibody does not bind substantially to tumor cells of Daudi, IM-9, Karpas 1106P or SU-DHL-4 Standard assays for evaluating the binding ability of antibodies to IRTA-2 are known in the art, which include, for example, ELISA, Western blots, RIA and flow cytometric analysis. Appropriate analyzes are described in detail in the examples.The binding kinetics (e.g., binding affinity) of the antibodies can also be determined by standard assays known in the art, such as by analysis e Biacore.

Monoclonal Antibodies 9D12 and 8A1 The preferred antibodies of the invention are the human monoclonal antibodies 9D12 and 8A1, isolated and structurally characterized as described in Examples 1 and 2. The VH amino acid sequences of 9D12 and 8A1 are shown in SEQ ID NOs: 13 and 14, respectively. The amino acid sequences of VL 9D12 and 8A1 are shown in SEQ ID NOs: 15 and 16, respectively. Since each of these antibodies can bind to IRTA-2, the VH and VL sequences can be "mixed and paired" to create other anti-IRTA-2 binding molecules of the invention. The IRTA-2 binding of such "mixed and paired" antibodies can be tested using the linkage assays described above and in the examples (e.g., ELISA). Preferably, when the VH and VL chains are mixed and paired, a VH sequence of a particular VH / VL match is replaced with a structurally similar VH sequence. Also, preferably a VL sequence of a particular VH / VL pairing is replaced with a structurally similar VL sequence.

Thus, in one aspect, the invention provides an isolated monoclonal antibody or antigen binding portion thereof comprising: (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: and 14; and (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 16; where the antibody binds specifically to IRTA-2, preferably human IRTA-2. Preferred heavy chain and light chain combinations include: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15; or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16. In another aspect, the invention provides antibodies comprising the CDR1, CDR2 and CDR3 of heavy chain and light chain of 9D12 and 8A1 or combinations thereof. The amino acid sequences of the VH CDR1's of 9D12 and 8A1 are shown in SEQ ID NOs: 1 and 2. The amino acid sequences of the VH CDR2's of 9D12 and 8A1 are shown in SEQ ID NOs: 3 and 4. The sequences of amino acids of the VH CDR3 of 9D12 and 8A1 are shown in SEQ ID NOs: 5 and 6. The Vk CDR1 amino acid sequences of 9D12 and 8A1 are shown in SEQ ID NOs: 7 and 8. The amino acid sequences of Vk CDR2 of 9D12 and 8A1 are shown in SEQ ID NOs: 9 and 10. The CDR3 amino acid sequences of 9D12 and 8A1 are shown in SEQ ID NOs: 11 and 12. The CDR regions are delineated using the Kabat (Kabat, EA, et al (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Since each of these antibodies can bind to IRTA-2 and that the antigen binding specificity is provided mainly by the CDR1, CDR2 and CDR3 regions, the CDR1, CDR2 and CDR3 sequences of VH and the CDR1 sequences , CDR2 and CDR3 of Vk can be "mixed and paired" (that is, the CDRs of different antibodies can be mixed and paired, although each antibody must contain a CDR1, CDR2 and CDR3 of VH and a CDR1, CDR2 and CDR3 of Vk ) to create other anti-IRTA-2 binding molecules of the invention. The IRTA-2 binding of such "mixed and paired" antibodies can be tested using the linkage assays described above and in the examples. { for example, ELISA, Biacore analysis). Preferably, when the VH CDR sequences are mixed and paired, the sequence of CDR1, CDR2 and / or CDR3 of a particular VH sequence is replaced with a structurally similar CDR sequence (s). Also, when the Vk CDR sequences are mixed and paired, the sequence of CDR1, CDR2 and / or CDR3 of a particular Vk sequence is preferably replaced with a structurally similar CDR sequence (s). It will be readily apparent to one skilled in the art that new VH and VL sequences can be created by substituting one or more VH and / or VL CDR region sequences with structurally similar sequences of the CDR sequences disclosed herein for antibodies monoclonal 9D12 and 8A1. Thus, in another aspect, the invention provides an isolated monoclonal antibody or antigen binding portion thereof comprising: (a) a heavy chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs : 1 and 2; (b) a heavy chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 4, - (c) a heavy chain variable region CDR3 comprising a selected amino acid sequence of the group consisting of SEQ ID NOs: 5 and 6; (d) a light chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 8; (e) a light chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 10; and (f) a light chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 and 12; wherein the antibody binds specifically to IRTA-2, preferably human IRTA-2. In a preferred embodiment, the antibody comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 1; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 3; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 5; (d) a light chain variable region CDR1 comprising SEQ ID NO: 7; (e) a light chain variable region CDR2 comprising SEQ ID NO: 9; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 11. In another preferred embodiment, the antibody comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 2; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 4; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 6; (d) a light chain variable region CDR1 comprising SEQ ID NO: 8; (e) a light chain variable region CDR2 comprising SEQ ID NO: 10; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 12.

Antibodies having particular germline sequences In certain embodiments, an antibody of the invention comprises a heavy chain variable region of a particular heavy chain line immunoglobulin gene and / or a light chain variable region of a light chain immunoglobulin of particular germination line. For example, in a preferred embodiment, the invention provides an isolated monoclonal antibody or an antigen binding portion thereof, comprising a heavy chain variable region that is the product of or derived from a human VH 3-23 gene, in where the antibody binds specifically to IRTA-2. In another preferred embodiment, the invention provides an isolated monoclonal antibody or an antigen binding portion thereof, comprising a heavy chain variable region that is the product of or derived from a human VH 1-8 gene, wherein the antibody binds specifically to IRTA-2. In still another preferred embodiment, the invention provides an isolated monoclonal antibody or an antigen binding portion thereof, comprising a light chain variable region that is the product of or derived from a human VK L6 human gene, wherein the antibody binds specifically to IRTA-2. In still another preferred embodiment, the invention provides an isolated monoclonal antibody or an antigen binding portion thereof, comprising a light chain variable region that is the product of or derived from a human VK L18 gene, wherein the antibody is binds specifically to IRTA-2. In yet another preferred embodiment, the invention provides an isolated monoclonal antibody or antigen binding portion thereof, wherein the antibody: (a) comprises a heavy chain variable region that is the product of or derived from a VH3 gene 23 or 1-8 human (such genes encode the amino acid sequences summarized in SEQ ID NOs: 21 and 22, respectively); (b) comprises a light chain variable region that is the product of or derived from a human VK L6 or human VK L18 (such genes encode the amino acid sequences summarized in SEQ ID NOs: 23 and 24, respectively); and (c) specifically binds to IRTA-2, preferably human IRTA-2. An example of an antibody having VH and VK of VH 3-23 and VK L6, respectively, is 9D12. An example of an antibody having VH and V of VH 1-8 and VK L18, respectively, is 8A1. As used herein, a human antibody comprises heavy or light chain variable regions that is "the product of" or "derivative of" a particular germination line sequence if the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin genes. Such systems include immunizing a transgenic mouse that carries human immunoglobulin genes with the antigen of interest or selection of a human immunoglobulin gene library displayed or displayed on the phage with the antigen of interest. A human antibody that is "the product of" or "derived 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 sequences of human germ line immunoglobulins. and selecting the human germline immunoglobulin sequence that is closest in sequence (ie,% major identity) to the human antibody sequence. A human antibody that is "the product of" or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germination line sequence, due to, for example, somatic mutations that occur in a stable manner in nature or intentional introduction of mutation directed to the site. However, a commonly selected human antibody is at least 90% identical in amino acid sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify human as human antibody when compared with the immunoglobulin amino acid sequences from the germination line of another species (for example, murine germination line sequences). In certain cases, a human antibody can be at least 95% 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 . Commonly, a human antibody derived from a particular human germ line sequence will exhibit no more than 10 amino acid differences of the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody can exhibit no more than 5 or even more than 4, 3, 2 or 1 amino acid difference of the amino acid sequence encoded by the germline immunoglobulin gene.

Homologous Antibodies In yet another embodiment, an antibody of the invention comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the preferred antibodies described herein and wherein the antibodies retain the functional properties of the anti-IRTA-2 antibodies of the invention. For example, the invention provides an isolated monoclonal antibody or antigen binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises a sequence of amino acids that is at least 80% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOS: 13 and 14; (b) the light chain variable region comprises an amino acid sequence that is at least 80% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 16; (c) the antibody binds a human IRTA-2 with a KD of lxlO "7 M or less; (d) the antibody binds human CHO cells transfected with IRTA-2; (e) the antibody does not bind substantially to human IRTA-3 or IRTA-4; and (f) the antibody binds to Granta 519 tumor cells and does not bind substantially to Raj i or Ramos tumor cells. In various embodiments, the antibody can be, for example, a human antibody, a humanized antibody or a chimeric antibody. In a preferred embodiment, the antibody does not bind substantially to Daudi tumor cells, IM-9, Karpas 1106P or SU-DHL-4. In other embodiments, the amino acid sequences of VH and / or VL may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences summarized above. An antibody that has VH and VL regions that have high homology (ie, 80% or greater) to the VH and VL regions of the sequences summarized above, can be obtained by mutagenesis (e.g., site-directed mutagenesis or mutagenesis). PCR-modeled) of nucleic acid molecules encoding SEQ ID NOs: 17, 18, 19 and 20, followed by tests of the altered antibody encoded for retained function (ie, the functions summarized in (c) to (f) above) using the functional analyzes described herein. As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the sequences is a function of the number of identical positions shared by the sequences. { that is,% of homology # of identical positions / total of # of positions x 100), taking into account the number of spaces and the length of each space, which need to be introduced for an optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences is carried out using a mathematical algorithm, as described in the non-limiting examples below. The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and. Mi11er. { Comput. Appl. Biosci. , 4: 11-17 (1988)) that has been incorporated into the ALIGN program (version 2.0), using a weight residue table PAM 120, a space length penalty of 12 and a space penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970)) algorithm that has been incorporated into the GAP program in the elements package of GCG programming (available at http://www.gcg.com), using either a Blossum 62 matrix or a PA 250 matrix and a space weight of 16, 14, 12, 10, 8, 6 or 4 and a weight of length of 1, 2, 3, 4, 5 or 6. Additionally or alternatively, the protein sequences of the present invention can be additionally used as an "interrogation sequence" to perform a search against public databases for, for example, identify related sequences. Such searches can be carried out using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 211: 403-10. Searches of BLAST proteins can be carried out with the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain alignments with spaces for comparison purposes, BLAST with spaces can be used as described in Altschul et al, (1997) Nucleic Acids Res. 25 (17): 3389-3402. When BLAST and BLAST programs are used with spaces, the default parameters of the respective programs (for example, XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Antibodies with Conservative Modifications In certain embodiments, an antibody of the invention comprises a heavy chain variable region comprising sequences of CDR1, CDR2 and CDR3 and a light chain variable region comprising sequences of CDR1, CDR2 and CDR3, wherein one or more of these CDR sequences comprise amino acid sections specified on the basis of the preferred antibodies described herein (eg, 9D12 or 8A1) or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the anti-human antibodies. -IRTA-2 of the invention. Thus, the invention provides an isolated monoclonal antibody or an antigen binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences and a light chain variable region comprising CDR1, CDR2 sequences. and CDR3, wherein: (a) the heavy chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 5 and 6 and conservative modifications thereof; (b) the light chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NOs: 11 and 12 and conservative modifications thereof; (c) the antibody binds to human IRTA-2 with a KD of lXlO "7 M or less; (d) the antibody binds to human CHO cells transfected with IRTA-2; (e) the antibody does not bind substantially to human IRTA-3 or IRTA-4, and (f) the antibody binds to Granta 519 tumor cells and does not bind substantially to Raj io Ramos tumor cells, In a preferred embodiment, the variable region CDR2 sequence. The heavy chain comprises an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 3 and 4 and conservative modifications thereof, and the light chain variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of group consisting of amino acid sequences of SEQ ID NOs: 9 and 10 and conservative modifications thereof In another preferred embodiment, the heavy chain variable region CDR1 sequence comprises a selected amino acid sequence of the group consisting of amino acid sequences of SEQ ID NOs: 1 and 2 and conservative modifications thereof; and the light chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 7 and 8 and conservative modifications thereof. In various embodiments, the antibodies may be, for example, human antibodies, humanized antibodies or chimeric antibodies. In a preferred embodiment, the antibody does not bind substantially to tumor cells of Daudi, I-9, Karpas 1106P or SU-DHL-4. As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include substitution, additions and cancellations of amino acids. Modifications can be introduced to an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and moderate mutagenesis by PCR. Conservative amino acid substitutions are those in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues that have similar side chains have been defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acid side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (for example, threonine, valine, isoleucine) and aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of the invention can be replaced with other amino acid residues of the same side chain family and the altered antibody can be tested for retained function (ie, the functions summarized in (c) through (f) above) using the functional analyzes described herein.

Antibodies that bind to the same epitope as the anti-IRTA-2 antibodies of the invention. In another embodiment, the invention provides antibodies that bind to the same epitope on human IRTA-2 as any of the monoclonal antibodies IRTA-2 of the invention (i.e., antibodies that have the ability to cross-compete by binding to IRTA). -2 with any of the monoclonal antibodies of the invention). In preferred embodiments, the antibody referenced for cross-competition studies may be monoclonal antibody 9D12 (which has VH and VL sequences as shown in SEQ ID NOs: 13 and 15, respectively) or monoclonal antibody 8A1 ( having the sequences VH and VL as shown in SEQ ID NOs: 14 and 16, respectively). Such cross-competing antibodies can be identified based on their ability to cross-compete with 9D12 or 8A1 in standard IRTA-2 binding assays. For example, BIAcore analysis, ELISA analysis or flow cytometry can be used to demonstrate cross-competition with the antibodies of the present invention. The ability of a test antibody to inhibit the binding of, for example 9D12 or 8A1, to human IRTA-2 demonstrates that the test antibody can compete with 9D12 or 8A1 for the binding to human IRTA-2 and thus binds to it. epitope on human IRTA-2 as 9D12 or 8A1. In a preferred embodiment, the antibody that binds to the same epitope on human IRTA-2 as 9D12 or 8A1 is a human monoclonal antibody. Such human monoclonal antibodies can be prepared and isolated as described in the examples.

Antibodies designed and modified An antibody of the invention can be further prepared using an antibody having one or more of the VH and / or VL sequences disclosed herein as a starting material for designing a modified antibody, such modified antibody can have properties altered from the starting antibody. An antibody can be designed by modifying one or more residues within one or both of the variable regions (ie, VH and / or VL), for example within one or more regions of CDR and / or within one or more framework regions.

Additionally or alternatively, an antibody can be designed by modifying residues within the constant region (s), for example to alter the effector function (s) of the antibody. One type of variable region design that can be effected is CDR grafting. The antibodies interact with target antigens predominantly by means of amino acid residues that are located in the six regions that determine the heavy and light chain complementarity (CDR). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than the 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 antibodies that occur stably in nature by constructing expression vectors that include CDR of the antibody that is stably presented in the specific native grafted on structure sequences of a different antibody with different properties (see, for example, Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P. et al (1986) Nature 321: 522-525; Queen, C. et al. (1989) Proc. Nati. Acad. See, USA 86: 10029-10033; US Patent No. 5,225,539 issued to Winter and Patents US Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 issued to Queen et al.) Thus, another embodiment of the invention is concerned with an isolated monoclonal antibody or antigen binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4 and SEQ ID NOs: 5 and 6, respectively and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10 and SEQ ID NOs: 11 and 12, respectively. Thus, such antibodies contain the CDR sequences of VH and VL of monoclonal antibodies 9D12 or 8A1 may still contain different structure sequences of these antibodies. Such structure sequences may be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the human germination line sequence database "VBase" (available on the Internet at www.mrc-cpe. cam.ac.uk/vbase). Also as in Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I.M., et al. (1992) "The Repertoire of Human Germline VH Sequences Reveal about Fifty Groups of VH Segments with Different Hypervariable Loops" J ", Mol. Biol. 227: 776-798, and Cox, JPL et al. (1994)" A Directory of Human Germ-line VH Segments Reveal Strong Bias in their Usage "Eur. J. Immuno1., 24: 827-836, the contents of each of which is expressly incorporated herein by reference. As another example, the sequences of Germination line DNA for human heavy and light chain variable region genes can be found in the Genbank database.For example, the following heavy chain germination line sequences found in the HuMAb mouse HCo7 are available in the numbers Genbank Accession Attachments: 1-69 (NG_0010109, NT_024637 and BC070333), 3-33 (NG_0010109 and NT_024637) and 3-7 (NG_0010109 and NT_024637) As another example, the following heavy chain germination line sequences found in the mouse HCol2 HuMAb is They are available in the attached Genbank access numbers: 1-69 (NG_0010109, NT_024637 and BC070333), 5-51 (NG_0010109 and NT_024637), 4-34 (NG_0010109 and NT_024637), 3-30.3 (?) and 3-23 (AJ406678). Preferred structure sequences for use in the antibodies of the invention are those that are structurally similar to the structure sequences used by antibodies selected from the invention, for example, similar to the sequences of structure VH 3-23 (SEQ ID NO: 21 ) and / or the VH structure sequences 1-8 (SEQ ID NO: 22) and / or the structure sequences VK L6 (SEQ ID NO: 23) and / or the structure sequence of Vk L18 (SEQ ID NO: 24) used by preferred monoclonal antibodies of the invention. The VR CDR1, CDR2 and CDR3 sequences and the VK CDR1, CDR2 and CDR3 sequences can be grafted onto regions of structure having the identical sequence as that found in the germline immunoglobulin gene of which the sequence of The structure is derived or the CDR sequences can be grafted onto regions of structure containing one or more mutations compared to the germination line sequence. For example, it has been found that in certain instances it is beneficial to mutate residues within structure regions to maintain or enhance the ability to bind antigen antigen (see, for example, U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370 issued to Queen et al). Another type of variable region modification is to mutate amino acid residues in the regions of CDR1, CDR2 and / or CDR3 of VH and / or V to thereby improve one or more binding properties (eg, affinity) of the antibody of interest. Site-directed mutagenesis or moderate mutagenesis by PCR can be performed to introduce the mutation (s) and the effect on the antibody binding or other functional property of interest, can be evaluated in in vitro or in vivo assays as described in the present and provided in the examples. Preferably, conservative modifications are introduced (as discussed above). The mutations can substitutions, additions or cancellations of amino acids, but are preferably substitutions. Commonly, no more than one, two, three, four or five residues within a CDR region are altered. Thus, in another embodiment, the invention provides isolated anti-IRTA-2 monoclonal antibodies or antigen binding portions thereof, comprising a heavy chain variable region comprising: (a) a VH CDR1 region comprising a amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions in comparison with SEQ ID NOs: 1 and 2; (b) a VH CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 4 or an amino acid sequence having one, two, three, four or five amino acid substitutions, cancellations or additions compared to SEQ ID NOs: 3 and 4; (c) a VH CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 6 or an amino acid sequence having one, two, three, four or five amino acid substitutions, cancellations or additions compared to SEQ ID NOs: 5 and 6; (d) a VK CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 8 or an amino acid sequence having one, two, three, four or five amino acid substitutions, cancellations or additions compared to SEQ ID NOs: 7 and 8; (e) a VK CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 10 or an amino acid sequence having one, two, three, four or five amino acid substitutions, cancellations or additions compared to SEQ ID NOs: 9 and 10; and (f) a VK CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 and 12 or an amino acid sequence having one, two, three, four or five amino acid substitutions, cancellations or additions compared to SEQ ID NOs: 11 and 12. The designed antibodies of the invention include those in which modifications have been made to residues from within VH and / or VK, for example to improve the properties of the antibody. Commonly such structural modifications are made to decrease the immunogenicity of the antibody. For example, one method is to "retromue" one or more structure residues to the corresponding germination line sequence. More specifically, an antibody that has undergone somatic mutation may contain structure residues that differ from the germination line sequence from which the antibody is derived. Such residues can be identified by comparing the antibody structure sequences with the germination line sequences from which the antibody is derived. For example, for 9D12, amino acid residue # 88 (within FR3) of VH is a valine while its residue in the corresponding germination line sequence VH 3-23 is an alanine. In order to return the structure region sequences to their germination line configuration, somatic mutations can be "retromutated" to the germination line sequence by, for example, site-directed mutagenesis or moderate mutagenesis by PCR (e.g., residue). # 88 (residue # 22 of FR3) of VH of 9D12 can be "retromutated" from valine to alanine). As another example, for 9D12, amino acid residue # 91 (within FR3) of VH is an alanine, while this residue in the corresponding germination line sequence of VH 3-23 is a threonine. To return the region of structure sequences to their germination line line configuration, for example, residue # 91 (residue # 25 of FR3) of VH of 9D12 can be "retromutated" from alanine to threonine. It is intended that such "retromutated" antibodies be encompassed by the invention. As another example, for 9D12, the amino acid residue # 93 (within FR3) of VH is a leucine, while this residue in the corresponding germination line sequence of VH 3-23 is a valine. To return the structure region sequences to their germination line configuration, for example, residue # 93 (residue # 27 of FR3) of the VH of 9D12 can be "retromutated" from leucine to valine. It is intended that such "retromutated" antibodies also be encompassed by the invention. As yet another example, for 8A1, amino acid residue # 2 (within FR1) of VH is a methionine, while this residue in the corresponding VH 1-8 germination line sequence is a valine. To return the structure region sequences to their germination line configuration, for example, residue # 2 within FR1 of VH of 8A1 can be "retromutated" from methionine to valine. It is intended that such "retromutated" antibodies also be encompassed by the invention. As yet another example, for 8A1, amino acid residue # 30 (within FR1) of VH is an isoleucine, while this residue in the corresponding VH 1-8 germination line sequence is a threonine. In order to return the structure region sequences to their germination line configuration, for example, residue # 30 within FR1 of VH of 8A1 can be "retromutated" from isoleucine to threonine. It is intended that such "retromutated" antibodies also be encompassed by the invention. Another type of structure modification involves mutation of one or more residues within the framework region or even within one or more regions of CDR, to remove the T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This procedure is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 of Carr et al. Additional or alternative modifications effected within the framework or CDR regions, the antibodies of the invention can be designed to include modifications within the Fe region, commonly to alter one or more functional properties of the antibody, such as serum half-life. , complement fixation, Fe receptor binding, and / or antigen-dependent cellular cytotoxicity. In addition, an antibody of the invention can be chemically modified. { for example, one or more chemical moieties can be attached to the antibody) or modified to alter their glycosylation, again to alter one or more functional properties of the antibody. Each of these modalities is described in further detail later herein. The numbering of waste in the region of Fe is that of the EU index of Kabat. In one embodiment, the engozyne region of CH1 is modified such that the number of cysteine residues in the engozne region is altered, for example, increased or decreased. This procedure is further described in U.S. Patent No. 5,677,425 issued to Bodmer et al. The number of cysteine residues in the engozne region of CH1 is altered to, for example, facilitate the assembly of the light or heavy chains or to increase or decrease the stability of the antibody. In another embodiment, the Fe-engozne region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the interface region of the CH2-CH3 domain of the Fe-engozne fragment, such that the antibody has Staphylococcylase Protein A (SpA) linkage impaired with respect to the Engozne domain faith link of natural SpA. This procedure is described in further detail in U.S. Patent No. 6,165,745 issued to Ward et al. In another embodiment, the antibody is modified to increase its biological half-life. Several procedures 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 issued to Ward. Alternatively, to increase the biological half-life, the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fe region of an IgG, such as it is described in U.S. Patent Nos. 5,869,046 and 6,121,022 issued to Presta et al. In still other embodiments, the Fe region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function (s) of the antibody. For example, one or more amino acids selected from the amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue, such that the antibody has an altered affinity for a ligand effector but retains the antigen binding ability of the original antibody. The effector ligand to which the affinity is altered, can be, for example, a Fe receptor or the Cl component of complement. This process is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both issued to Winter et al. In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue, such that the antibody has altered Clq binding and / or reduced or abolished complement-dependent cytotoxicity. (CDC). This procedure is described in further detail in U.S. Patent No. 6,194,551 issued to Idusogie et al. In another example, one or more amino acid residues at amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This method is further described in PCT publication WO 94/29351 by Bodmer et al. In yet another example, the Fe region is modified to increase the ability of the antibody to moderate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for an FCY receptor by modifying one or more amino acids in 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 process is further described in PCT publication WO 00/42072 by Presta. In addition, binding sites on ++++ IgGl for human FcyRl, FcyRJI, FCYRIII and FcRn have been mapped and variants with enhanced binding have been described (see Shields, RL et al (2001) J. Biol. Che. 276 : 6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 were shown to improve binding to FcyRIII. Additionally, it was demonstrated that the following combination mutants improve the binding of FC7RIII: T256A / S298A, S298A / E333A, S298A / K224A and S298A / E333A / K334A. In yet another embodiment, the glycosylation of an antibody is modified. For example, an aglycosylated antibody can be elaborated (that is, the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be effected, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions may be made that result in the removal of one or more glycosylation sites of variable region structure to thereby eliminate glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such a procedure is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al. Additionally or alternatively, an antibody can be made into an antibody having an altered type of glycosylation, such as a hypophosphorylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisection GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modifications can be effected, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which recombinant antibodies of the invention are expressed to thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705 and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), such that antibodies expressed in the cell lines of s704, Ms705 and Ms709 lack fucose in their carbohydrates . The cell lines Ms704, s705 and Ms709 FUT8_ / "were created by the targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 of Yamane et al and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87: 614-22.) As another example, EP 1,176,195 to Hanai et al. Describes a cell line with a functionally altered FUT8 gene, which encodes a fucosyl transferase, such that the antibodies expressed in such a Cell line exhibits hypophosphorylation by reducing or eliminating the alpha-linked linkage enzyme, Hanai et al also describes cell lines that have low enzyme activity to add fucose to the N-acetylglucosamine that binds to the Fe region of the antibody or does not have the activity of enzyme, for example the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). Presta PCT publication O 03/035835 describes a CHO cell line variant, cell as Lecl3, with reduced ability to attach fucose to Asn (297) -linked carbohydrates, also resulted in hypophosphorylation of antibodies expressed in that host cell (see also Shields, R.L. et al. (2002) J ". Biol. Chem. 277: 26733-26740.) PCT publication WO 99/54342 to Umana et al. Describes cell lines designed to express glycoprotein-modifying glycosyl transferases. (Eg, beta (1 , 4) -N-acetylglucosaminyltransferase III (GnTIII)) in such a way that the antibodies expressed in the designed cell lines exhibit bisection GlcNac structures which also results in increased ADCC activity of the antibodies (see also Umana et al (1999) Nat. Biotech 17: 176 -180). Alternatively, the fucose residues of the antibody can be excised using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes residues of fucosyl from antibodies (Tarentino, A.L. et al. (1975) Biochem.14: 5516-23). Another modification of the antibodies in the present which is contemplated by the invention is pegylation. An antibody can be pegylated to, for example, increase the biological half-life. { for example, in the serum) of the antibody. To pegylate an antibody, the antibody or fragment thereof, is commonly reacted with polyethylene glycol (PEG), such as a reactive ester or PEG aldehyde derivative, under conditions in which one or more PEG groups are attached to the antibody or fragment of antibody. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the PEG forms that have been used to derive 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 an aglycosylated antibody. Methods for protein pegylation are known in the art and can be applied to the antibodies of the invention. See, for example, EP 0 154 316 of Nishimura et al. and EP 0 401 384 of Ishikawa et al.

Methods for designing antibodies As discussed above, anti-IRTA-2 antibodies having VH and VK sequences disclosed herein can be used to create new anti-IRTA-2 antibodies by modifying the VH and / or VK sequences or the constant region (s) attached to them. Thus, in another aspect of the invention, the structural elements of an anti-IRTA-2 antibody of the invention, for example 9D12 or 8A1, are used to create structurally related anti-IRTA-2 antibodies that retain at least one functional property. of the antibodies of the invention, such as binding to human IRTA-2. For example, one or more CDR regions of 9D12 or 8A1 or mutations thereof, can be combined recombinantly with regions of known structure and / or other CDRs to create additional recombinantly designed anti-IRTA-2 antibodies of the invention, as discuss above. Other types of modifications include those described in the previous section. The starting material for the design method is one or more of the VH and / or VK sequences provided herein or one or more CDR regions thereof. To create the designed antibody, it is not necessary to actually prepare. { that is, expressing 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. Rather, the information contained in the sequence (s) is used as the starting material to create a "second generation" sequence (s) derived from the original sequence (s) (is ) and then the "second generation" sequence (s) is (are) prepared and expressed as a protein. Thus, in another embodiment, the invention provides a method for the preparation of an anti-IRTA-2 antibody comprising: (a) providing: (i) a heavy chain variable region antibody sequence comprising a selected CDR1 sequence of the group consisting of SEQ ID NOs: 1 and 2, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 3 and 4 and / or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 5 and 6; and / or (ii) a light chain variable region antibody sequence comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 7 and 8, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 9 and 10 and / or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 11 and 12; (b) altering at least one amino acid residue within the heavy chain variable region antibody sequence and / or the light chain variable region antibody sequence to create at least one altered antibody sequence; and (c) expressing the altered antibody sequence as a protein. Standard molecular biology techniques can be used to prepare and express the altered antibody sequence. Preferably, the antibody encoded by the altered antibody sequence (s) is one that retains one, some or all of the functional properties of the anti-IRTA-2 antibodies described herein, such functional properties include, but are not limited to, are limited to: (i) binds to human IRTA-2 with a KD of lxlO "7 M or less, (ii) binds to human CHO cells transfected with IRTA-2, (iii) does not bind substantially to IRTA- 3 or human IRTA-4, and / or (iv) binds to Granta 519 tumor cells and does not bind substantially to Raji or Ramos tumor cells, In a preferred embodiment, the antibody does not bind substantially to human cells. Daudi tumor, IM-9, Karpas 1106P or SU-DHL-4 The functional properties of the altered antibodies can be determined using standard analyzes available in art and / or described herein, such as those summarized in the examples ( example, flow cytometry, link analysis). In certain embodiments of the methods for designing antibodies of the invention, mutations can be randomly or selectively introduced throughout all or part of an anti-IRTA-2 antibody coding sequence and the resulting modified anti-IRTA-2 antibodies can be selected in terms of link activity and / or other functional properties as described herein. Mutational methods have been described in the art. For example, PCT publication O 02/092780 by Short discloses methods for creating and selecting 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 using computational selection methods to optimize physicochemical properties of antibodies.

Nucleic Acid Molecules Encoding Antibodies of the Invention Another aspect of the invention is concerned with nucleic acid molecules encoding the antibodies of the invention. The nucleic acids may be present in whole cells, in a cell lysate or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when it is purified when it is purified from other cellular compounds or other contaminants, eg, other cellular nucleic acids or proteins, by standard techniques, in which alkaline / SDS treatment is included , CsCl bands, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., Ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. A nucleic acid of the invention may be, for example, DNA or RNA and may or may not contain intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule. The nucleic acids of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described hereinafter), cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by amplification. of standard PCR or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), the nucleic acid encoding the antibody can be recovered from the library. Preferred nucleic acid molecules of the invention are those encoding the VH and VL sequences of monoclonal antibodies 9D12 or 8A1. DNA sequences encoding the VH sequences of 9D12 and 8A1 are shown in SEQ ID NOs: 17 and 18, respectively. DNA sequences encoding the VL sequences of 9D12 and 8A1 are shown in SEQ ID NOs: 19 and 20, respectively. Once the DNA fragments encoding 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 to full length antibody chain genes , to Fab fragment genes or to a scFv gene. In these manipulations, a DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked", as used in this context, means that the two DNA fragments are linked in such a way that the amino acid sequences encoded by the two DNA fragments remain in frame. The isolated DNA encoding the VH region can be converted to a heavy chain gene by operably linking the DNA encoding VH to another DNA molecule encoding heavy chain constant regions (CH1, CH2, and CH3). The human heavy chain constant region gene sequences are known in the art (see for example, Kabat, EA, et al. (1991) Sequences of Protein of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments spanning these regions can be obtained by standard PCR amplification. The heavy chain constant region may be a constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD, but more preferably is a constant region of IgG1 or IgG4. For a heavy chain gene of Fab fragment, the DNA encoding VH can be operably linked to another DNA molecule that encodes only the heavy chain CH1 constant region. The isolated DNA encoding the VL region can be converted to a full length light chain gene (also as to a Fab light chain gene) by operably linking the VL encoding DNA to another DNA molecule encoding the region light chain constant, CL. The sequences of human light chain constant region genes are known in the art (see for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments spanning these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region, but more preferably it is a kappa constant region. To create a scFv gene, the DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker, for example, encoding the amino acid sequence (GIy4-Ser) 3, such that the sequences VH and VL can be expressed as a contiguous single chain protein, with the VL and VH regions linked by the flexible linker (see for example, Bird et al. (1988) Science 242: 423-426: Huston et al. (1988) Proc. Nati, Acad. Sci. USA 85: 5879-5883; McCafferty et al, (1990) Nature 348: 552-554).

Production of monoclonal antibodies of the invention The monoclonal antibodies (mAbs) of the present invention can be produced by a variety of techniques, in which conventional monoclonal antibody methodology is included, for example the Kohler standard somatic cell hybridization technique and Milstein (1975) Nature 256: 495. Although somatic cell hybridization methods are preferred, in principle, other techniques for producing monoclonal antibody can be used, for example, viral or oncogenic transformation of B lymphocytes. The preferred animal system for The preparation of hybridomas is the murine system. The production of hybridoma in the mouse is a very well established procedure. Immunization protocols and techniques for the isolation of splenocytes immunized for fusion are known in the art. Fusion partners (eg, murine myeloma cells) and fusion procedures are also known. Chimeric or humanized antibodies of the present invention may be prepared based on the sequence of a murine monoclonal antibody prepared as described above. The DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and designed to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, murine variable regions can be linked to human constant regions using methods known in the art (see for example, U.S. Patent No. 4,816,567 issued to Cabilly et al.). To create a humanized antibody, the murine CDR regions can be inserted into a human structure using methods known in the art (see for example, U.S. Patent No. 5,225,539 issued to US Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 issued to Queen et al). In a preferred embodiment, the antibodies of the invention are human monoclonal antibodies. Such human monoclonal antibodies directed against IRTA-2 can be generated using transgenic or transchromosomal mice that carry parts of the human immune system in place of the mouse system. These transgenic and transchromosomal mice include mice referred to herein as the HuMAb® mouse and KM® mouse, respectively and are collectively referred to herein as "human Ig mice". The HuMAb® mouse (Medarex®, Inc.) contains minisites of human immunoglobulin gene encoding heavy chain immunoglobulin sequences (μ and?) And? lightly human without accommodation, together with targeted mutations that inactivate the μ and? chain sites. endogenous (see for example, Lonberg, et al. (1994) Nature 368 (6474): 856-859). Thus, mice exhibit reduced expression of mouse IgM or? , and in response to immunization, introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate human IgG of high monoclonal affinity (Lonberg, N. et al. (1994), supra, reviewed in Lonberg. , N. (1994) Handbook of Experimental Pharology 113: 49-101; Lonberg, N. and Huszar, D. (1995) Intern., Rev. Immunol., 13: 65-93 and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci. 764: 536-546). The preparation and use of the HuMAb® mouse and the genomic modifications made by such mice is further described in Taylor, L. et al. (1992) Nucleic Acids Research 20: 6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Nati Acad. Sci. USA 90: 3720-3724; Choi et al. (1993) Nature Genetics 4: 117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J ". Immunol., 152: 2912-2920; Taylor, L. et al. (1994) International Immunology 6: 579-591; and Fishwild, D. et al (1996) Nature Biotechnology 14: 845-851, the contents of all of which are hereby specifically incorporated by reference in their entirety See also, U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; ,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all issued to Lonberg and Kay; U.S. Patent No. 5,545,807 issued to Surani et al .; PCT publications Nos. O 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, all issued to Lonberg and Kay; and PCT publication No. WO 01/14424 issued to Korman et al. In another embodiment, human antibodies of the invention can be created using a mouse that carries human immunoglobulin sequences on transgenes and transchromosomes, such as a mouse carrying a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as "KM mice ™", are described in detail in PCT publication WO 02/43478 of Ishida et al. Still further, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to create anti-IRTA-2 antibodies of the invention. For example, an alternative transgenic system referred to as Xenoraton (Abgenix, Inc.) can be used; such mice are described, for example, in U.S. Patent Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963 issued to Kucherlapati et al. In addition, alternative transchromosomal animal systems expressing human immunoglobulin genes are available in the art and can be used for ++++ to create anti-IRTA-2 antibodies of the invention. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, referred to as "TC mice" can be used; such mice are described in Tomizuka et al. (2000) Proc. Nati Acad. Sci. USA 97: 722-727. In addition, cows carrying human heavy and light chain transeromosomes have been described in the art (Kuroiwa et al (2002) Nature Biotechnology 20: 889-894) and can be used to create anti-IRTA-2 antibodies of the invention. The human monoclonal antibodies of the invention can also be prepared using phage display methods to select libraries of human immunoglobulin genes. Such methods of phage display to isolate human antibodies are established in the art. See for example: U.S. Patent Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al .; U.S. Patent Nos. 5,427,908 and 5,580,717 to Dower et al; U.S. Patent Nos. 5,969,108 and 6,172,197 to cCafferty et al; 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 of the invention can also be prepared using SCID mice to which human immune cells have been reconstituted, such that a human antibody response can be generated after immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 issued to Wilson et al.

Immunization of human Ig mice When human Ig mice are used to create human antibodies of the invention, such mice can be immunized with a purified or enriched preparation of IRTA-2 and / or recombinant IRTA-2 antigen or an IRTA fusion protein. -2, as described by Lonberg, N. et al. (1994) Nature 368 (6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851; and PCT publication WO 98/24884 and WO 01/14424. Preferably, the mice will be 6-16 weeks of age after the first infusion. For example, a purified or recombinant preparation (5-50 μL) of the IRTA-2 antigen can be used to immunize human Ig mice intraperitoneally. Detailed procedures for generating fully human monoclonal antibodies to IRTA-2 are described in the example below. Cumulative experience with several antigens has shown that transgenic mice respond when they are initially immunized intraperitoneally (IP) with antigen in complete Freund's adjuvant, followed by IP immunizations one week after another (up to a total of 6) with antigen in incomplete Freund's adjuvant. However, adjuvants other than Freund are also effective. In addition, it is found that whole cells in the absence of adjuvant are highly immunogenic. The immune response can be monitored in the course of the immunization protocol with plasma samples being obtained by retro-orbital bleeding or bleedings. Plasma can be selected by ELISA (as described hereinafter) and mice with sufficient anti-IRTA-2 human immunoglobulin titres can be used for fusions. The mice can be strengthened intravenously with antigen 3 days before sacrifice and removal of the spleen. It is expected that 2-3 fusions for each immunization can be made. Between 6 and 24 mice are commonly immunized for each antigen. Usually both strains HCo7 and HCol2 are used. In addition, both the HCo7 and HCol2 transgene can be cross-linked together to a single mouse having two different human heavy chain transgenes (HCo7 / HCol2). Alternatively or additionally, KM mouse strain can be used, as described in example 1.

Generation of Hybridomas Producing Human Monoclonal Antibodies of the Invention To generate hybridomas that produce human monoclonal antibodies of the invention, splenocytes and / or lymph node cells of immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a line Mouse myeloma cell, The resulting hybridomas can be selected for the production of antigen-speci fi c antibodies. For example, single-cell suspensions of splenic lynocytes from immunized mice can be fused to one sixth of the number of mouse myeloma cells that do not secrete P3X63 -Ag8.653 (ATCC, CRL 1580) with 50% PEG. Cells are deposited at approximately 2 x 105 in a flat bottom microtiter plate, followed by a two week incubation in a selective medium containing 20% fetal clone serum, 18% "653" conditioned media, 5% of origin (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units / ml penicillin, 50 mg / ml streptomycin, 50 mg / ml gentamicin and IX HAT (Sigma, the HAT is added 24 hours after the merger). After approximately two weeks, the cells can be cultured in the medium in which the HAT is replaced with HT. Then the individual cavities can be selected by ELISA for human monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, the medium can usually be observed after 10-14 days. Antibody-secreting hybridomas can be re-deposited, selected again and if they are still positive for human IgG, the monoclonal antibodies can be subcloned at least twice by limiting dilution. Then the stable subclones can be cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization. To purify human monoclonal antibodies, selected hybridomas can be cultured in two liter spinner flasks for the purification of monoclonal antibody. The supernatants can be filtered and concentrated before affinity chromatography with protein A-sepharose (Pharmacia, Piscataway, NJ.). IgG eluted can be verified by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged to PBS and the concentration can be determined by OD280 using an extinction coefficient of 1.43. The monoclonal antibodies can be dosed in aliquots and stored at -80 ° C.

Generation of Transfectomes Producing Monoclonal Antibodies of the Invention The antibodies of the invention can also be produced in a host cell transfectoma using for example a combination of recombinant DNA techniques and genetic transfection methods as is well known in the art (e.g. , Orrison, S. (1985) Science 229: 1202). For example, to express the antibodies or antibody fragments thereof, DNA encoding partial length or full length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or clinical cDNA cloning). using a hybridoma expressing the antibody of interest) and the 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 "operably linked" means that an antibody gene is ligated to a vector such that the transcriptional and translational control sequences within the vector serve its supposed function of regulating the transcription and translation of the antibody. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into a separate vector or more commonly, both genes are inserted into the same expression vector. Antibody genes are inserted into the expression vector by standard methods (eg, ligation of complementary restriction sites on the antibody and vector gene fragment or blunt end ligation if no restriction site is present). The light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors that already encode heavy chain constant regions and chain constant regions. light of the desired isotype, such that the VH segment is operatively linked to the CH segment (s) within the vector and the VK segment is operatively linked to the LC segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates the secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is bound in frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide. { that is, a signal peptide from a non-immunoglobulin protein). In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. 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 the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology, Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). It will be appreciated by those skilled in the art that the design of the expression vector, in which the regulatory sequence selection is included, may depend on factors such as the choice of the host cell to be transformed, the level of protein expression desired , etc. Preferred regulatory sequences for expression of host cell mamifera 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), adenovirus (eg. For example, the adenovirus major late promoter (AdMLP) and polyoma, alternatively, non-viral regulatory sequences can be used, such as the ubiquitin promoter or 3-globin promoter.Furthermore, regulatory elements composed of sequences from different sources, such as as the SRa promoter system, which contains sequences of the SV40 premature promoter and the long terminal repeat of type 1 human T cell leukemia virus (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8: 466 -472) In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention can carry additional sequences, such as sequences that regulate the replication of the vector in host cells. { for example, origins of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells to which the vector has been introduced (see, for example, U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017, all from Axel et al). For example, the selectable marker gene confers resistance to drugs such as G418, hygromycin or methotrexate, on a host cell to which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with selection / amplification of methotrexate) and the neo gene (for selection of G418). For the expression of light and heavy chains, he (the) expression vector (s) encoding the heavy and light chains is (are) transfected (s) to a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, for example, electroporation, calcium phosphate precipitation, transfection of DEAE-dextran, and similar. Although it is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, the expression of antibodies in eukaryotic cells and more preferably mammalian host cells, is most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete an appropriately folded and immunologically active antibody. It has been reported that prokaryotic expression of antibody genes is not effective for the production of high yields of active antibody (Boss, M.A. and Wood, C.R. (1985) Immunology Today 6: 12-13). Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese hamster ovary (CHO cells) (in which dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Nati. Acad. Sci. USA) are included. 77: 4216-4220, used with a DHFR selectable marker, for example as described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159: 601-621), NSO myeloma cells, COS cells and cells SP2 In particular, for use with NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in O 87/04462, O 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 period of time sufficient to allow expression of the antibody in the host cells or more preferably, secretion of the antibody to the host cells. culture medium to which the host cells are cultured. The antibodies can be recovered from the culture medium using standard protein purification methods.

Characterization of antibody binding to antigen The antibodies of the invention can be tested for binding to IRTA-2 by, for example, standard ELISA. Briefly, microtiter plates are coated with IRTA-2 purified at 0.25 μ / tt? 1 in PBS and then blocked with 5% bovine serum albumin in PBS. Antibody dilutions (eg, plasma dilutions of IRTA-2-immunized mice) are added to each well and incubated for 1-2 hours at 37 ° C. The plates are washed with PBS / Tween and then incubated with secondary reagent (for example, for human antibodies, a goat-anti-human IgG polyclonal reagent) conjugated to alkaline phosphatase for 1 hour at 37 ° C. After washing, the plates are developed with pNPP substrate (1 mg / ml) and analyzed at OD of 405-650. Preferably, the mice that develop the highest titers are used for infusions. An ELISA analysis as described above can also be used to select hybridomas that show positive reactivity with the IRTA-2 immunogen. Hybridomas that bind with high avidity to IRTA-2 are subcloned and further characterized. A clone of each hybridoma, which retains the reactivity of the original cells (by ELISA) can be chosen for the preparation of a cell bank of 5-10 flasks stored at -140 ° C and for antibody purification. To purify anti-IRTA-2 antibodies, selected hybridomas can be cultured in two-liter spinner flasks for monoclonal antibody purification. The supernatants can be filtered and concentrated before affinity chromatography with protein A-sepharose (Pharmacia, Piscataway, NJ). IgG eluted can be verified by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged to PBS and the concentration can be determined by OD2eo using an extinction coefficient of 1.43. The monoclonal antibodies can be dosed in aliquots and stored at -80 ° C. To determine whether the selected anti-IRTA-2 monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). These competition using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using coated IRTA-2 ELISA plates as described above. The biotinylated mAb linkage can be detected with a streptavidin-alkaline phosphatase probe. To determine the isotype of antibodies. purified, isotype ELISA can be performed using specific reagents for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, microtiter plate cavities can be coated with 1 μg / ml anti-human immunoglobulin overnight at 4 ° C. After blocking with 1% BSA, the plates are reacted with 1 xg / ml or less of test monoclonal antibodies or purified isotype controls, at room temperature, for one to two hours. The cavities can then be reacted with either human IgGl or conjugated Ig-specific alkaline phosphatase probes. The plates are developed and analyzed as described above. Human IgG anti-IRTA-2 can be further tested for reactivity with IRTA-2 antigen by Western blotting. Briefly, IRTA-2 can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked with 10% fetal calf serum and tested with the monoclonal antibodies to be tested. The human IgG binding can be detected using anti-human IgG alkaline phosphatase and developed with BCIP / NBT substrate tablets (Sigma Chem. Co., St. Louis, OR.).

Immunoconjugates In another aspect, the present invention comprises an anti-IRTA-2 antibody or fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin. Such conjugates are referred to herein as "immunoconjugates". Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins". A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells (e.g., exterminates). Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantron, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine , 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 decarbazine), alkylating agents (e.g., mechlorethamine, chlorambucil thioepa, melphalan, carmustine (BSNU), and lomustine). (CC U), cyclotosfamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamin platinum (II) (DDP) cisplatin), anthracyclines (eg, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin ( formerly actinomycin), bleomycin, mithramycin and anthramycin (AC)) and anti-mitotic agents (e.g., vincristine and vinblastine). Other preferred examples of therapeutic cytotoxins that can be conjugated to an antibody of the invention include duocarmycins, calicheamicins, maytansines and auristatins, and derivatives thereof. An example of a calicheamicin antibody conjugate is commercially available (Mylotarg ™; Wyeth-Ayerst). The cytotoxins can be conjugated to antibodies of the invention using linker technology available in the art. Examples of types of linker that have been used to conjugate a cytotoxin to an antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers. A linker can be chosen which is, for example, susceptible to cleavage by low pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferably expressed in tumor tissue such as cathepsins (eg, cathepsins B, C, D) ). For an additional discussion of types of cytotoxins, linkers and methods for conjugating therapeutic agents to antibodies, see also Saito, G. et al. (2003) Adv. Drug Deliv. Rev. 55: 199-215; Trail, P.A. et al. (2003) Cancer I nunol. Immunother. 52: 328-337; Payne, G. (2003) Cancer Cell 3: 207-212; Alien, T.M. (2002) Nat. Rev. Cancer 2: 750-763; Pastan, I. and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3: 1089-1091; Senter, P.D. and Springer, CJ. (2001) Adv. Drug Deliv. Rev. 53: 247-264. The antibodies of the present invention can also be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine131, indium111, yttrium90 and lutetium177. Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, in which Zevalin ™ (IDEC Pharmaceuticals) and Bexxar ™ (Corixa Pharmaceuticals) are included and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the invention. The antibody conjugates of the invention can be used to modify a given biological response and the drug portion will not be construed as limited to classical chemical therapeutic agents. For example, the drug portion may be a protein or polypeptide that possesses a desired biological activity. Such proteins may include, for example, an enzymatically active toxin or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin or diphtheria toxin.; a protein such as tumor necrosis factor or interferon- ?; or biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), colony stimulating factor granulocyte macrophage ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors. Techniques for conjugating such therapeutic portions to antibodies are well known, see, for example, Arnon et al, "Onoclonal Antibodies for Immunotherapy of Drugs in Cancer Therapy," in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al, "Antibodies for Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological and Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy", in Monoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985) and Thorpe et al, "The Preparation and Cytotoxic Properties of Antibody-Toxin Conjugates", Immunol.

Rev., 62: 119-58 (1982) BISISpecific Molecules In another aspect, the present invention comprises bisespecific molecules comprising an anti-IRTA-2 antibody or fragment thereof, of the invention. An antibody of the invention or antigen binding portions thereof, can be derived or linked to another functional molecule, for example another peptide or protein (eg, another antibody or ligand for a receptor) to generate a bispecific molecule that is links to at least two different binding sites or target molecules. The antibody of the invention can in fact be derived or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and / or target molecules; such multispecific molecules are also proposed to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule of the invention, an antibody of the invention can be functionally linked. { for example, by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that it is result a bispecific molecule.

Thus, the present invention includes bisespecific molecules comprising at least a first binding specificity for IRTA-2 and a second binding specificity for a target epitope. In a particular embodiment of the invention, the second target epitope is a Fe receptor, for example, a human FcyRI receptor (CD64) or a human Fea receptor (CD89). Accordingly, the invention includes bisespecific molecules capable of binding to both FcyR and FcaR expressing effector cells (e.g., monocytes, macrophages or polymorphonuclear cells (PMN)) and to targeting cells expressing IRTA-2. These bispecific molecules target cells that express IRTA-2 to effector cells and trigger effector cell activities moderated by the Fe receptor such as phagocytosis of cells expressing IRTA-2, moderate cell-dependent antibody cytotoxicity (ADCC), cytokine release or superoxide anion generation. In one embodiment of the invention in which the bispecific molecule is multispecific, the molecule can further include a third binding specificity, in addition to an anti-Fc binding specificity and an anti-IRTA-2 binding specificity. In one embodiment, the third binding specificity is a portion of anti-enhancement factor (EF), for example, a molecule that binds to a protein surface involved in cytotoxic activity and thereby increases the immune response against the target cell . The "anti-enhancement factor portion" can be an antibody, functional antibody fragment or a ligand that binds to a given molecule, for example, an antigen or a receptor and thereby results in an improvement of the effect of the determinants of binding to the Fc receptor or target cell antigen. The "anti-enhancement factor portion" can be linked to an Fc receptor or a target cell antigen. Alternatively, the anti-enhancement factor portion may be linked to an entity that is different from the entity to which the first and second link specificities are linked. For example, the anti-enhancement factor portion can be linked to a cytotoxic T cell (eg, via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or another immune cell which results in an increased immune response against the target cell). In one embodiment, the bisespecific molecules of the invention comprise a binding specificity of at least one antibody or an antibody fragment thereof, in which, for example, a Fab, Fab ', F (ab') 2 is included. / Fv ° Fv of a single chain. The antibody can also be a light chain or heavy chain dimer or any minimal fragment thereof such as an Fv or a single chain construct as described in US Pat. No. 4, 946,778 issued to Ladner et al., The content of which is incorporated by reference. In one embodiment, the binding specificity for a Fcy receptor is provided by a monoclonal antibody, the binding of which is not blocked by human immunoglobulin G (IgG). As used herein, the term "IgG receptor" refers to any of the eight chain genes? located on chromosome 1. These genes encode a total of twelve transmembrane isoforms or soluble receptor that are grouped into three classes of FCY receptor: FCYRI (CD64), FCY RIKCD32) and FCYRIII (CD16). In a preferred embodiment, the Fcy receptor is a FCYRI of high human affinity. The human FcyRI is a 72 kDa molecule, which shows high affinity for monomeric IgG (108-109 M "1) The production and characterization of certain preferred anti-FcY monoclonal antibodies are described by Fanger et al. PCT WO 88/00052 and in U.S. Patent No. 4,954,617, the teachings of which are fully incorporated herein by reference These antibodies bind to an epitope of, FCYRII or FCYRIII at a site that is distinct from the FCY link site. of the receptor and thus, its binding is not substantially blocked by physiological levels of IgG Specific anti-FcyRI antibodies useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. Hybridoma producing mAb 32 is available from the American Type Culture Collection, Accession No. ATCC HB9469. In other embodiments, the anti-FcY receptor antibody is a humanized form of the monoclonal antibody 22 (H22) .The production and characterization of the anti H22 bodies are described in Graziano, R.F. et al. (1995) J. Immunol 155 (10): 4996-5002 and PCT publication WO 94/10332. The cell line that produces H22 antibody was deposited in the American Type Culture Collection under the designation HA022CL1 and has Accession No. CRL 11177. In still other preferred embodiments, the binding specificity for a receptor Fe receptor is provided by an antibody that it binds to a human IgA receptor, for example, an Fc-alpha receptor (Fea RI (CD89)), the linkage of which is preferably not blocked by human immunoglobulin A (IgA). The term "IgA receptor" is intended to include the genetic product of the a (Fea RI) gene located on chromosome 19. It is known that this gene encodes several transmembrane isoforms alternatively spliced from 55 to 110 kDa. FcaRI (CD89) is constitutively expressed monocytes / macrophages, eosinophilic and neutrophilic granulocytes, but not on non-effector cell populations. FcaRI has medium affinity (= 5 x 107 M-1) for both IgAl and IgA2, which is increased in exposure to cytokines such as G-CSF or GM-CSF (Morton, HC et al. (1996) Critical Reviews in Immunology , 16: 423-440). Four specific FcaRI monoclonal antibodies, identified as A3, A59, A62 and A77, which bind to FcaRI outside the IgA ligand binding domain, have been described (Monteiro, RC et al. (1992) J. "I munol. 148: 1764) FcaRI and FcyRI are preferred trigger receptors for use in the bispecific molecules of the invention because they are (1) expressed primarily on immune effector cells, eg, monocytes, PM, macrophages and dendritic cells.; (2) expressed at high levels. { for example, 5, 000-100, 000 / cell); (3) mediators of cytotoxic activities (eg, ADCC, phagocytosis); (4) they moderate the presentation of improved antigen of antigens, in which anti-antigens are included, pointed to them. While human monoclonal antibodies are preferred, other antibodies that can be used in the bispecific molecules of the invention are murine, chimeric and humanized monoclonal antibodies. The bispecific molecules of the present invention can be prepared by conjugating the constituent binding specificities, for example, the binding specificities anti-FcR and anti-IRTA-2, using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to each other. When the binding specificities are proteins or peptides, a variety of coupling agents 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-pyridylthio) ropionate (SPDP) and sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohaxan-1-carboxylate (sulfo-SMCC) (see, for example, Karpovsky et al. (1984) J. "Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Nati, Acad. Sci. USA 82: 8648) Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229: 81-83) and Glennie et al. (1987) J. Im uno.1 139: 2367-2375). Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce. Chemical Co. (Rockford, Ill.) When the binding specificities are antibodies, they can be conjugated via the sulfhydryl linkage of the C-terminus engozne regions of the two heavy chains. The engozne is modified to contain an odd number of sulfhydryl residues, preferably one before conjugation. Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific molecule is mAb x mAb, mAb x Fab, Fab x F (ab ') 2 or ligand fusion protein x Fab. A bispecific molecule of the invention can be a single chain molecule comprising a single chain antibody and a binding determinant or a single chain bispecific molecule comprising two binding determinants. The bisespecific molecules can comprise at least two molecules of a single chain. Methods for preparing bispecific molecules are described for example in U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. Patent No. 5,482,858. Binding of the bispecific molecules to their specific targets can be confirmed for example by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioanalysis (eg, growth inhibition) or Western Blot analysis. Each of these analyzes generally detects the presence of protein-antibody complexes of particular interest when employing a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, Fcr-antibody complexes can be detected using for example, an enzyme-linked antibody or antibody fragment that recognizes and binds specifically to the antibody-FcR complexes. Alternatively, the complexes can be detected using any of a variety of other immunoassays. For example, the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassay, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated in the present by reference). The radioactive isotope can be detected by means such as the use of a gamma counter or a scintillation counter or by autoradiography.

Pharmaceutical compositions In another aspect, the present invention provides a composition, for example, a pharmaceutical composition, containing one or a combination of monoclonal antibodies or antigen binding portion (s) thereof, of the present invention, formulated in conjunction with a pharmaceutically acceptable carrier. Such compositions may include one or a combination of (eg, two or more different) antibodies or immunoconjugates or bispecific molecules of the invention. For example, a pharmaceutical composition of the invention may comprise a combination of antibodies (or immunoconjugates or bispecific) that bind to different different epitopes on the target antigen or that have complementary activities.

The pharmaceutical compositions of the invention can also be administered in combination therapy, that is, combined with other agents. For example, the combination therapy may include an anti-IRTA-2 antibody of the present invention combined with at least one other anti-inflammatory agent or immunosuppressive agent. Examples of therapeutic agents that can be used in combination therapy are described in greater detail later in the section as to uses of the antibodies of the invention. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarding agents and the like which are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, ie, antibody, immunoconjugate or bispecific molecule, can be coated in a material to protect the compound from the action of acids and other natural conditions that can deactivate the compound. The pharmaceutical compounds of the invention may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impair any undesirable toxicological effects (see, eg, Berge, SM, et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, bromic, iodic, phosphorous and the like, as well as from non-toxic organic acids, such as aliphatic mono- and dicarboxylic acids , phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. The base addition salts include those derived from alkaline earth metal salts, such as sodium, potassium, magnesium, calcium and the like, as well as from non-toxic organic amines, such as α, β-dibenzylethylenediamine, N-methylglucamine, chloroprocaine , choline, diethanolamine, ethylenediamine, procaine and the like. A pharmaceutical composition of the 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 bisulfate, 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, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) and appropriate mixtures thereof, vegetable oils, such as olive oil and injectable organic esters, such as ethyl oleate. The 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. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. The prevention of the presence of microorganisms can be ensured both by sterilization procedures, supra and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and those similar to the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be effected by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin. Pharmaceutically acceptable carriers include sterile aqueous solutions or sterile dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except as regards any conventional medium or agent is incompatible with the active compound, the use thereof is contemplated in the pharmaceutical compositions of the invention. Complementary active compounds can also be incorporated into the compositions. Therapeutic compositions commonly must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure appropriate to the high concentration of the drug. 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 appropriate mixtures thereof. The proper fluidity can be maintained, for example by the use of coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable 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 effected by including in the composition a delay in absorption, for example, monostearate and gelatin salts. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients listed above, as required, followed by microfiltration of sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the other ingredients required from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum-dried and freeze-dried (lyophilization) which produce a powder of the active ingredient plus any additional desired ingredient of a sterile filtered solution previously from the same The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on 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 in general will be that amount of a composition that produces a therapeutic effect. In general, of one hundred percent, this amount will range from about 0.01 percent to about 99 percent active ingredient, preferably from about 0.1 percent to about 70 percent, more preferably from about 1 percent to about 30 percent. percent active ingredient in combination with a pharmaceutically acceptable carrier. Dosage regimens are adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered in time or the dose can be reduced or increased proportionally as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. The unit dosage form as used herein refers to physically discrete units suitable as unit dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the unit dosage forms of the invention are determined by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be obtained, and (b) the limitations inherent in the art of combining such Active compound for the treatment of sensitivity in individuals. For administration of the antibody, the dosage ranges from about 0.0001 to 100 mg / kg and more usually 0.01 to 5 mg / kg, of the body weight of the host. For example, the doses may be 0.3 mg / kg of body weight, 1 mg / kg of body weight, 3 mg / kg of body weight, 5 mg / kg of body weight or 10 mg / kg of body weight, or in the range of 1-10 mg / kg. An exemplary treatment regimen covers administration 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 3 to 6 months. Preferred dosage regimens for an anti-IRTA-2 antibody of the invention include 1 mg / kg of body weight or 3 mg / kg of body weight via intravenous administration, the antibody is given using one of the following dosing schedules: ( i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg / kg of body weight once followed by 1 mg / kg of body weight every three weeks. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the indicated ranges. The antibody is usually administered on multiple occasions. The intervals between individual dosages can be for example weekly, monthly, every three months or annually. The intervals may also be irregular as indicated by measuring blood levels of antibody to the target antigen in the patient. In some methods, the dosage is adjusted to obtain an antibody concentration in the plasma of approximately 1-1000 g / ml and in the same methods approximately 25-300 mg / ml. Alternatively, the antibody can be administered as a sustained release formulation, in which case less frequent administration is required. The dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies and non-human antibodies. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until the progression of the disease is reduced or terminated and preferably until the patient shows partial or complete improvement of the symptoms of the disease. After this, a prophylactic regimen can be administered to the patient. The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied to obtain an amount of the active ingredient that is effective to obtain the desired therapeutic response for a particular patient, composition and mode of administration, without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors which include the activity of the particular compositions of the present invention used or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound that is employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions used, age, sex, weight, condition, general health and medical history of the patient being treated and similar factors well known in the medical arts.

A "therapeutically effective dosage" of an anti-IRTA-2 antibody of the invention preferably results in a decrease in the severity of the symptoms of the disease, an increase in frequency and duration of disease-free periods or a prevention of impairment or disability due to the affliction of the disease. For example, for the treatment of IRTA-2 + tumors, a "therapeutically effective dosage" preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by less about 60% and still more preferably at least about 80% relative to the untreated subjects. The ability of a compound to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit such inhibition in vitro by analyzes known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can decrease the size of the tumor or otherwise improve symptoms in a subject. Those of ordinary skill in the art would be able to determine such quantities 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 composition of the present invention can 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 experienced technician, the route and / or mode of administration will vary depending on the desired results. Preferred routes of administration for antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other routes of parenteral administration routes, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal , transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, an antibody of the invention can be administered via a non-parenteral route, such as a route of topical, epidermal or mucosal administration, for example intranasal, oral, vaginal, rectal, sublingual or topically. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, in which implants, transdermal patches and microencapsulated delivery systems are included. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Many methods for the preparation of such formulations are patented or are generally known to those skilled in the art. See, for example, Sustained and Controlled Relay Drug Delivery Systems, J.R. Robinson, ed. , Marcel Dekker, Inc., New York, 1978. Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the invention can be administered with a hypodermic injection device without needles, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for medical delivery at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medication through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for administering the medical device at an accurate infusion rate, U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous administration of drug; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system, these patents are incorporated herein by reference. Many other such implants, administration systems and modules are known to those skilled in the art. In certain embodiments, the human monoclonal antibodies of the invention can be formulated to ensure proper in vivo distribution. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For liposome manufacturing methods, see, e.g., U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes may comprise one or more portions that are selectively transported to specific cells or organs, thus improving the administration of the targeted drug (see, for example, V. V. Ranade (1989) J. Clin Pharmacol 29: 685). Exemplary targeting moieties include foliate or biotin (see, for example, U.S. Patent No. 5,416,016 to Low et al); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357: 140; Owais et al. (1995) Antimicrob., Agents Chemother., 39: 180); surfactant protein A receptor (Briscoe et al (1995) Am. J. Physiol. 1233: 134); p 120 (Schreier et al. (1994) J. Biol. Chem. 269: 9090); see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346: 123; JJ Killion; I.J. Fidler (1994) Immuno ethods 4: 273.

USES AND METHODS OF THE INVENTION Antibodies, particularly human antibodies, antibody compositions and methods of the present invention have numerous diagnostic and therapeutic utilities in vitro and in vivo that involve the diagnosis and treatment of diseases moderated by IRTA-2. For example, these molecules can be administered to cells in culture, in vitro or ex vivo or to human subjects, for example, in vivo, to treat, prevent and to diagnose a variety of disorders. As used herein, the term "subject" is intended to include human and non-human animals. Non-human animals include all vertebrates, for example, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians and reptiles. Preferred subjects include human patients who have moderate alterations by the activity of IRTA-2. The methods are particularly suitable for the treatment of human patients who have an alteration associated with the expression of aberrant IRTA-2. When antibodies to IRTA-2 are co-administered with another agent, the two can be administered either in one or the other order or simultaneously. Given the specific binding of the antibodies of the invention to IRTA-2, compared to IRTA-3 or IRTA-4, the antibodies of the invention can be used to specifically detect the expression of IRTA-2 on the cell surface and in addition , can be used to purify IRTA-2 via immunoaffinity purification. In addition, given the expression of IRTA-2 on several tumor cells (Davis et al, (2002) Immunological Reviews 190: 123), human antibodies, antibody compositions and methods of the present invention can be used to treat a subject with a tumorigenic alteration, for example, an alteration characterized by the presence of tumor cells expressing IRTA-2 in which are included, for example, Burkitt's lymphoma, anaplastic large cell lympholas (ALCL), cutaneous T-cell lympholas , nodular small cell lymph nodes, lymphocytic lymphocytes, peripheral T-cell lympholas, Lennert's lympholas, immunoblastic lympholas, T cell leukemia / lympholas (ATLL), adult T-cell leukemia (T-ALL), entroblastic follicular lymphoma cancers / centrocytes (cb / cc), diffuse large cell lymphomas of lineage B, T-cell lymphoma resembling angioimmunoblastic lymphadenopathy (AILD), lymphomas based on ca associated body virus HIV, embryonal carcinomas, undifferentiated carcinomas of the rhino-pharynx (eg, Schmincke tumor), Castleman's disease, Kaposi's sarcoma and other B-cell lymphomas. In one embodiment, antibodies (e.g. human monoclonal, multispecific and bispecific molecules and compositions) of the invention can be used to detect levels of IRTA-2 or levels of cells containing IRTA-2 on their membrane surface, such levels can then be linked to certain disease symptoms. Alternatively, the antibodies can be used to inhibit or block the function of IRTA-2 which in turn can be linked to the prevention or improvement of certain symptoms of the disease, thereby implicating IRTA-2 as a mediator of the disease. This can be obtained by contacting a sample and a control sample with the anti-IRTA-2 antibody under conditions that allow the formation of a complex between the antibody and IRTA-2. Any complexes formed between the antibody and IRTA-2 are detected and compared in the sample and the control. In another embodiment, the antibodies (e.g., human antibodies, multispecific and bispecific molecules and compositions) of the invention can be tested initially for binding activity associated with therapeutic or in vitro diagnostic use. For example, the compositions of the invention can be tested using the flow cytometric analyzes described in the examples hereinafter. Antibodies (for example, human antibodies, multispecific and bispecific molecules, immunoconjugates and compositions) of the invention have additional utility in therapy and diagnosis of IRTA-2-related patients. For example, human monoclonal antibodies, multispecific or bispecific molecules and immunoconjugates can be used to produce in vivo or in vitro one or more of the following biological activities: inhibit the growth of and / or kill a cell that expresses IRTA-2; moderate phagocytosis or ADCC of a cell that expresses IRTA-2 in the presence of human effector cells or block the binding of ligand IRTA-2 to IRTA-2. In a particular embodiment, antibodies (eg, human antibodies, multispecific and bispecific molecules and compositions) "are used in vivo to treat, prevent or diagnose a variety of related IRTA-2 diseases. Examples of related IRTA-2 diseases include, among others, cancer, non-Hodgkin's lymphoma, Burkitt's lymphoma, anaplastic large cell lymphomas (ALCL), cutaneous T-cell lymphomas, nodular small-cell excised lympholas, lymphocytic lymphocytes, lymphomas of peripheral T cells, Lennert's lymphomas, immunoblastic lymphomas, T cell / lymphoma leukemia (ATLL), adult T cell leukemia (T-ALL), entroblastic / centrocytic follicular lymphoma cancers (cb / cc), large cell lymphomas diffuse lineage B, T-cell lymphoma resembling angioimmunoblastic lymphadenopathy (AILD), lymphomas based on associated HIV body cavity, embryonal carcinomas, undifferentiated carcinomas of the rhino-pharynx (for example, Schmincke tumor), Castleman's disease, Kaposi's sarcoma and other B-cell lymphomas. Appropriate routes of administration of antibody compositions (e.g., human monoclonal antibodies, multispecific and bispecific and immunoconjugated molecules) of the invention in vivo and in vitro are well known in the art and can be selected by those of ordinary skill. For example, antibody compositions can be administered by injection (e.g., intravenous or subcutaneous). Appropriate dosages of the molecules used will depend on the age and weight of the subject and the concentration and / or formulation of the antibody composition. As previously described, the human anti-IRTA-2 antibodies of the invention can be co-administered with one or more other therapeutic agents, for example, a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent. The antibody can be linked to the agent (such as an immunocomplex) or can be administered separately from the agent. In the latter case (separate administration), the antibody can be administered before, after or concurrently with the agent or can be co-administered with other known therapies, for example, an anti-cancer therapy, such as radiation. Such therapeutic agents include, among others, anti-neoplastic agents such as doxorubicin (adriamycin), cisplatin sulfate bleomycin, carmustine, chlorambucil and cyclophosphamide hydroxyurea which, by themselves, are only effective at levels that are toxic or subtoxic to a patient. Cisplatin is administered intravenously as a dose of 100 mg / dose once every four weeks and adriamycin is administered intravenously as 60-75 mg / ml dose once every 21 days. The co-administration of human anti-IRTA-2 antibodies or antigen binding fragments thereof, of the present invention with chemotherapeutic agents provides two anticancer agents that operate via different mechanisms that produce a cytotoxic effect on human tumor cells. Such co-administration can solve problems due to the development of drug resistance or a change in the antigenicity of the tumor cells that would render them non-reactive with the antibody.

Target-specific effector cells, for example, effector cells linked to compositions (eg, human antibodies, multispecific and bispecific molecules) of the invention can also be used as therapeutic agents. Effector cells for targeting can be human leukocytes such as macrophages, neutrophils or monocytes. Other cells include eosinophils, natural killer cells and other cells that carry IgG or IgA receptor. If desired, effector cells can be obtained from the subject to be treated. Target-specific effector cells can be administered as a suspension of cells in a physiologically acceptable solution. The number of cells administered may be of the order of 108-109 but will vary depending on the therapeutic purpose. In general, the amount will be sufficient to obtain localization in the target cell, for example, a tumor cell that expresses IRTA-2 and to effect cell killing, for example by phagocytosis. The routes of administration may also vary. Therapy with objective-specific effector cells can be carried out in conjunction with other techniques for the removal of targeted cells. For example, anti-tumor therapy using the compositions (eg, human antibodies, multispecific and bispecific molecules) of the invention and / or effector cells armed with these compositions can be used in conjunction with chemotherapy. Additionally, combination therapy can be used to direct two distinct cytotoxic effector populations toward tumor cell rejection. For example, anti-IRTA-2 antibodies linked to anti-Fc-gamma RI or anti-CD3 can be used in conjunction with specific IgG or IgA receptor binding agents. The bisespecific and multispecific molecules of the invention can also be used to modulate the levels of FcyR or FcyR on effector cells, such as by coronation and elimination of receptors on the cell surface. Mixtures of anti-Fc receptors can also be used for this purpose. Compositions (eg, human, humanized or chimeric antibodies, multispecific and bispecific and immunoconjugate molecules) of the invention having complement binding sites, such as portions of IgG1, -2 or -3 or IgM that link complement, can also be used in the presence of complement. In one embodiment, ex vivo treatment of a population of cells comprising target cells with a binding agent of the invention and appropriate effector cells can be complemented by the addition of complement or complement containing serum. Phagocytosis of target cells coated with a binding agent of the invention can be improved by binding of complement proteins. In another embodiment, target cells coated with the compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can also be lysed by complement. In yet another embodiment, the compositions of the invention do not activate the complement. The compositions (eg, human, humanized or chimeric antibodies, multispecific and bispecific and immunoconjugate molecules) of the invention can also be administered together with complement. Thus, in the scope of the invention, there are compositions comprising human antibodies, multispecific or bispecific molecules and serum or complement. These compositions are advantageous in that the complement is located in close proximity to human antibodies, multispecific or bispecific molecules. Alternatively, the human antibodies, multispecific or bispecific molecules of the invention and the complement or serum can be administered separately. Also within the scope of the invention are equipment comprising the antibody compositions of the invention (e.g., human antibodies, bisespecific or multispecific or immunoconjugated molecules) and instructions for use. The kit may additionally contain one or more additional reagents, such as an immunosuppressive agent, a cytotoxic agent or a radiootoxic agent or one or more additional human antibodies of the invention (eg, a human antibody having a complementary activity that binds to an epitope on the IRTA-2 antigen different from the first human antibody). Thus, patients treated with the antibody compositions of the invention can be further administered (before, simultaneously with or following the administration of a human antibody of the invention) with another therapeutic agent, such as a cytotoxic or radiotoxic agent, which improves or increases the therapeutic effect of human antibodies. In other embodiments, the subject can be further treated with an agent that modulates, for example, enhances or inhibits, the expression or activity of FCY or FCY receptors for example, by treating the subject with a cytokine. Preferred cytokines for administration during treatment with the multispecific molecule include granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-? (IFN-?) And tumor necrosis factor (TNF). The compositions (eg, human antibodies, multispecific and bispecific molecules) of the invention can also be used to target cells expressing FCYR or IRTA-2, for example to label such cells. For such use, the binding agent can be linked to a molecule that can be detected. Thus, the invention provides methods for localization of ex vivo or in vitro cells expressing Fe receptors, such as FcyR or IRTA-2. The detectable label can be, for example, a radioisotope, a fluorescent compound, an enzyme or an enzyme co-factor. In a particular embodiment, the invention provides methods for detecting the presence of IRTA-2 antigen in a sample or measuring the amount of IRTA-2 antigen, comprising contacting the sample and a control sample, with an antibody. human monoclonal or an antigen binding portion thereof, which specifically bind to IRTA-2, under conditions that allow the formation of a complex between the antibody or a portion thereof and IRTA-2. Then the formation of a complex is detected, where a difference in complex formation between the sample compared to the control sample is indicative of the presence of IRTA-2 antigen in the sample. In other embodiments, the invention provides methods for treating a moderate alteration by IRTA-2 in a subject, eg, cancer, non-Hodgkin's lymphoma, Burkitt's lymphoma, anaplastic large cell lympholas (ALCL), cutaneous T-cell lymphomas. , nodular small excised cell lymphomas, lymphocytic lymphomas, peripheral T-cell lymphomas, Lennert's lymphomas, immunoblastic lymphomas, T-cell leukemia / lymphomas (ATLL), adult T-cell leukemia (T-ALL), entroblastic follicular lymphoma cancers / centrocytic (cb / cc), diffuse large cell lymphomas of lineage B, B-cell lymphoma similar to angioimmunoblastic lymphadenopathy (AILD), lymphomas based on HIV-associated body cavity, embryonal carcinomas, carcinomas without differentiating the rhino-pharynx (eg, Schmincke tumor), Castleman's disease, Kaposi's sarcoma and other B-cell lymphomas, by administering human antibodies to the subject previously written Such antibodies and derivatives thereof are used to inhibit the activities induced by I TA-2 associated with certain alterations, for example, proliferation and differentiation. By contacting the antibody with IRTA-2 (for example, by administering the antibody to a subject), the ability of IRTA-2 to induce such activities is inhibited and thus, the associated alteration is treated. The antibody composition can be administered alone or together with another therapeutic agent, such as a cytotoxic agent or a radiotoxic agent that acts in conjunction with or synergistically with the antibody composition to treat or prevent disease moderated by IRTA-2. In still another embodiment, immunoconjugates of the invention can be used to target compounds. { for example, therapeutic agents, markers, cytotoxins, radiotoxin immunosuppressants, etc.) to cells having IRTA-2 cell surface receptors by binding such compounds to the antibody. Thus, the invention also provides methods for ex vivo or in vivo localization of cells expressing IRTA-2 (for example, with a detectable marker, such as a radioisotope, a fluorescent compound, an enzyme or an enzyme co-factor). . Alternatively, immunoconjugates can be used to kill cells that have IRTA-2 cell surface receptors by targeting cytotoxins or radiotoxins to IRTA-2. The present invention is further illustrated by the following examples which should not be construed as additional limitations. The contents of all figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

EXAMPLES Example 1; Generation of human monoclonal antibodies against IRTA-2 Antigen A recombinant fusion protein composed of extracellular domain of IRTA-2 bound to a polypeptide without IRTA-2 was generated by standard recombinant methods and used as an antigen for immunization. - < s ^ ®,, HuMAb mouse and transgenic KM mouse Fully human monoclonal antibodies to IRTA-2 were prepared using HCo7 / HCol2 strains of the transgenic HuMAb® mouse and the KM strain of transgenic transchromosomal mice, each of which expresses human antibody genes . In each of these mouse strains, the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al. (1993) 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. Each of these mouse strains carries a human kappa light chain transgene, KCo5, as described in Fishwild et al. (1996) Nature Biotechnology 14: 845-851. The HCo7 strain carries the HCo7 human heavy chain transgene as described in U.S. Patent Nos. 5,545,806; 5,625,825; and 5,545,807. The HCol2 strain carries the human heavy chain transgene HCol2 as described in example 2 of PCT publication WO 01/09187. The strain of Mouse KM contains transchromosome SC20 as described in PCT publication WO 02/43478.

Immunizations of HuMab and KM; To generate fully human monoclonal antibodies to IRTA-2, mouse mice from HuMAb * and mouse K were immunized with purified recombinant IRTA-2 fusion protein derived from NSO cells that have been transfected with an expression vector containing the gene encoding the fusion protein. General immunization schemes for the HuMAb mouse are described in Lonberg, N. et al (1994; Nature 368 (6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851 and publication of PCT WO 98/24884 Mice were 6-16 weeks of age at the first antigen infusion A purified recombinant preparation (5-50 μg) of IRTA-2 fusion protein was used to immunize HuMab mice and KM mice * intraperitoneally, subcutaneously (Se) or via hindquarter injection Transgenic mice were immunized twice with antigen in complete Freund's adjuvant or Ribi adjuvant either intraperitoneally (IP), subcutaneously (Se) or via hindquarter (FP) , followed by IP, Se or FP immunization (this is a total of 11 immunizations) with the antigen in incomplete Freund's adjuvant or Ribi's adjuvant.The immune response was monitored by retro-orbital bleeds.The plasma was selected by ELISA (as described). ribe later in the present) and mice with sufficient titers of anti-IRTA-2 human immunoglobulin were used for fusions. The mice were reinforced intravenously with antigen 3 and 2 days before being sacrificed and removal of the spleen. Typically, 10-35 fusions were performed for each antigen. Several dozens of mice were immunized for each antigen.

- ® Selection of HuMab or KM mice that produce anti-IRTA-2 antibodies; To select HuMab or KM mice that produce antibodies that bind to IRTA-2, sera from immunized mice were tested by ELISA as described by Fishwild, D. et al. (nineteen ninety six). Briefly, microtiter plates were coated with recombinant IRTA-2 purified at 1-2 μg / ml in PBS, 50 μ? / Wells incubated 4 ° C overnight then blocked with 200 μ? / Well of chicken serum at 5 ° C. % in PBS / Tween (0.05%). Plasma dilutions of IRTA-2-immunized mice were added to each well and incubated for 1-2 hours at room temperature. The plates were washed with PBS / Tween and then incubated with a goat-anti-human IgG polyclonal antibody conjugated with horseradish peroxidase (HRP) for 1 hour at room temperature. After washing, the plates were developed with ABTS substrate (Sigma, A-1888, 0.22 mg / ml) and analyzed by spectrophotomere at OD 415-495. Mice that revealed the highest titers of anti-IRTA-2 antibodies were used for fusions. The fusions were carried out as described hereinafter and the hybridoma supernatants were tested for anti-IRTA-2 activity by ELISA.

Generation of hybridomas that produce human monoclonal antibodies to IRTA-2 Mouse splenocytes, isolated from HuMab and KM® mice, were fused with PEG to a mouse myeloma cell line based on standard protocols. Then the resulting hybridomas were selected for the production of antigen-specific antibodies. Single cell suspension of splenic lymphocytes from immunized mice were fused to a quarter of the number of mouse myeloma cells that do not secrete SP2 / 0 (ATCC, CRL 1581) with 50% PEG (Sigma). Cells were deposited at approximately lxl05 / cavity in the flat bottom microtiter plate, followed by approximately a two week incubation in selective medium containing 10% fetal bovine serum, 10% P388D1 conditioned medium (ATCC, CRL TIB -63), 3-5% of origin (IGEN) in DME (Mediatech, CRL 10013, with high content of glucose, L-glutamine and sodium pyruvate) plus 5 mM HEPES, 2-mercaptoethanol 0.055 mM, 50 mg / ml of gentamicin and lx HAT (Sigma, CRL P-7185). After 1-2 weeks, the cells were cultured in medium in which the HAT was replaced with HT. Then the individual cavities were selected by ELISA (described above) for anti-human IRTA-2 monoclonal IgG antibodies. Once extensive hybridoma growth occurred, the medium was usually monitored after 10-14 days. Hybridomas secreting antibody were re-deposited, selected again and if they are still positive for human IgG, the anti-IRTA-2 monoclonal antibodies were subcloned at least twice by limiting dilution. Then the stable subclones were cultured in vitro to generate small amounts of antibody in tissue culture medium for further characterization. Hybridoma clones 9D12, generated from a HuMAb * and 8A1 mouse, generated from a KM * mouse, were selected for further analysis.

Example 2; Structural characterization of human monoclonal antibodies 9D12 and 8A1 The DNA sequences encoding the heavy and light chain variable regions of monoclonal antibodies 9D12 and 8A1 were obtained from hybridomas 9D12 and 8A1, respectively, using standard PCR techniques and were sequenced using standard DNA sequence techniques. The nucleotide sequences and amino acid sequences of the heavy chain variable region of 9D12 are shown in Figure 1A and in SEQ ID NOs: 13 and 17, respectively. The nucleotide and amino acid sequences of the light chain variable region of 9D12 are shown in Figure IB and in SEQ ID Nos: 15 and 19, respectively. Comparison of the 9D12 heavy chain immunoglobulin sequence with the known human germ line immunoglobulin heavy chain sequences showed that the heavy chain of 9D12 uses a VH fragment from the germination line VH 3-23, a segment D of the human germination line 7-27 and a segment JH of the human germination line JH 3b. Alignment of the 9D12 sequence with the germination line VH 3-23 sequence is shown in Figure 3. Further analysis of the 9D12 VH sequence using the Kabat system of the determination of the CDR region drove to delineation of the heavy chain CDR1, CDR2 and CD3 regions as shown in Figures 1A and 3 and in SEQ ID NOs: 1, 3 and 5, respectively. Comparison of the light chain immunoglobulin sequence of 9D12 with the known human germline immunoglobulin light chain sequences showed that the light chain of 9D12 uses a VL fragment from the human germination line VK L6 and a JK segment of the JK human germination line 1. The sequence alignment of 9D12 VL to the germination line VK L6 sequence is shown in Figure 5. Additional analysis of the 9D12 VL sequence using the Kabat system of the determination of the CDR region led to the delineation of the CDR1 regions, CDR2 and CD3 as light chain as shown in Figures IB and 5 and in SEQ ID NOs: 7, 9 and 11, respectively. The nucleotide and amino acid sequences of the heavy chain variable region of 8A1 are shown in FIGS. 2A and SEQ ID NOs: 14 and 18, respectively. The amino acid nucleotide sequences of the light chain variable region of 8A1 are shown in Figure 2B and in SEQ ID NOs: 16 and 20, respectively. Comparison of the 8A1 heavy chain immunoglobulin sequence with the known human germline immunoglobulin heavy chain sequences showed that the heavy chain of 8A1 uses a VH fragment from the human germination line VH 1-8, a segment D of the human germination line 3-10 and a segment JH of the human germination line JH 6b. Alignment of the 8A1 VH sequence with the germination line VH 1-8 sequence is shown in Figure 4. Further analysis of the 8A1 VH sequence using the Kabat system of the determination of the CDR region led to the delineation of the heavy chain CDR1, CDR2 and CD3 regions as shown in Figures 2A and 4 and in SEQ ID NOs: 2, 4 and 6, respectively. Comparison of the light chain immunoglobulin sequence of 8A1 with the known human germline immunoglobulin light chain sequences showed that the light chain of 8A1 uses a VL segment of the human germination line VK L18 and a segment of JK of the human germination line JK 2. The alignment of the sequence of 8A1 VL with the sequence of VK L18 of germination line is shown in figure 6. The additional analysis of the sequence of 8A1 VL using the Kabat system of the determination of the CDR region led to delineation of the light chain CDR1, CDR2 and CD3 regions as shown in Figures 2B and 6 and in SEQ ID NOs: 8, 10 and 12, respectively.

Example 3; Characterization of binding specificity and binding kinetics of human anti-IRTA-2 monoclonal antibodies In this example, binding affinity and binding kinetics of anti-IRTA-2 antibodies were examined by Biacore analysis. Also, the binding specificity was examined by flow cytometry.

Affinity and binding kinetics Anti-IRTA-2 antibodies were characterized for affinities and binding kinetics by Biacore analysis (Biacore AB, Uppsala, Sweden). Purified anti-IRTA-2 monoclonal antibodies were captured by anti-human IgG antibody covalently linked to a CM5 chip (chip coated with carboxy methyl dextran) via primary amines, using standard amine coupling chemistry and equipment provided by Biacore. The binding was measured by flowing the IRTA-2-Fc antigen (a fusion protein composed of the extracellular domain of IRTA-2 bound to the Fe portion of human IgGl) in pH buffer HBS EP (provided by Biacore AB) at concentrations of 400, 300, 200, 100 and 50 nM at a flow rate of 12 μ? / min. The kinetics of antigen-antibody association was followed either for 20 minutes (for 8A1) or 5 minutes (for 9D12) and the dissociation kinetics was followed either for 10 minutes (for 8A1) or 5 minutes (for 9D12). The association and dissociation curves were adjusted to a 1: 1 Langmuir link model using BIA evaluation elements.

(Biacore AB). The values of KD, kon and kQff that were determined are shown in table 1. Table 1. Biacore link data for IRTA-2 HuMAbs Linkage Specificity by Flow Cytometry Chinese hamster ovary (CHO) cell lines expressing either the IRTA-2, IRTA-3 or IRTA-4 protein on the cell surface were developed and used to determine the specificity of monoclonal antibodies IRTA-2 by flow cytometry. CHO cells were transfected with expression plasmids containing full length cDNA encoding transmembrane forms of IRTA-2, IRTA-3 or IRTA-4. In addition, the transfected proteins contained a myc tag at the N-terminus for detection by anti-myc antibody. The analysis of the two anti-IRTA-2 monoclonal antibodies was determined by incubating the transfected cells with each of the IRTA-2 Abs at a concentration of 10 μg / ml. The cells were washed and the binding was detected with an anti-human IgG Ab labeled with phycoerythrin. A murine anti-myc Ab followed by labeled anti-murine IgG was used as a positive control. The secondary antibody was only used as a negative control. The results are illustrated in Figures 7A-C. The monoclonal antibodies of IRTA-2 9D12 and 8A1 bound to the CHO line transfected with IRTA-2 but not to CHO lines expressing IRTA-3 or -4, as measured by the mean fluorescent intensity (MFI) of the dye. These data demonstrate the specificity of monoclonal antibodies for IRTA-2.

Example 4; Linkage of IRTA-2 antibodies to normal B cells and to tumor lines derived from B cell Two-color fluorescence and flow cytometry were used to demonstrate the binding of the HuMAbs from IRTA-2 to peripheral blood B cells. CD19 is a cell surface marker that can be used to distinguish B lymphocytes from peripheral blood. Human peripheral blood mononuclear cells were incubated with biotinylated 9D12, biotinylated 8A1 or a biotinylated Ab human isotype control. Cells were washed and incubated with FITC-labeled streptavidin together with an anti-CD19 phycoerythrin-labeled antibody. The cells were washed and analyzed by flow cytometry. The results are illustrated in Figure 8. The monoclonal antibodies IRTA-2 9D12 and 8A1 show increased binding to CD 19+ cells compared to control isotype antibody, as measured by the mean fluorescent intensity (MFI) of the dyeing. These data demonstrate that the IRTA-2 protein, as determined by monoclonal antibodies 9D12 and 8A1, is expressed by normal peripheral blood B lymphocytes. The binding of the IRMA-2 HuMAbs by flow cytometry to the Ramos B-cell tumor lines (ATCC CRL-1596), Raji (ATCC CCL-86), Daudi (ATCC CCL-213), IM-9 ( ATCC CCL-159), Karpas 1106P (DSMZ ACC 545), SU-DHL-4 (DSMZ ACC 495), Granta 519 (DSMZ ACC 342), SU-DHL-6 (DSMZ ACC 572) and JEKO-1 (DSMZ ACC 553) was determined. B-cell tumor lines from Ramos, Raji, Daudi and IM-9 were each incubated with the HuMAbs of IRTA-2, an anti-B cell positive control antibody or an isotype-matched negative human control antibody, washed and detected by a phycoerythrin-labeled anti-human secondary antibody. The cells were washed and analyzed by flow cytometry. The results for the binding of antibodies 9D12 and 8A1 are shown in Figure 9. The HuMAbs of IRTA-2 do not bind substantially to any of the B-cell tumor lines of Ramos, Raji, Daudi or IM-9. The low level of binding observed for 9D12 and 8A1 to the IM-9 cell line was comparable to the binding level seen for the isotype control antibody, thus demonstrating that the link was not specific. The cell lines of Karpas 1106P, SU-DHL-4, Granta 519, SU-DHL-6 and JEKO-1 were incubated with each of the HuMAbs of IRTA-2 or a human control antibody, washed and detected by an antibody. secondary anti-human phycoerythrin-labeled. The results for the binding of antibodies 9D12 and 8A1 are shown in Figures 10 and 11. These data show that anti-IRTA-2 antibodies bind to tumor cells of Granta 519, SU-DHL-6 and JEKO-1. in comparison to the human control antibody, but it does not bind substantially to Karpas 1106P or SU-DHL- tumor cells. Together, these data demonstrate that certain B-cell tumor lines express the IRTA-2 protein on the cell surface.

SEQUENCE LISTING Amino acid sequence of VH CDR1 of 9D12 (SEQ ID NO: 1) 1 ssams VH CDR1 amino acid sequence of 8A1 (SEQ ID NO: 2) 1 sydin VH CDR2 amino acid sequence of 9D12 (SEQ ID NO: 3) 1 sisgsgdtty yadsvkg Amino Acid Sequence of VH CDR2 of 8A1 (SEQ ID NO: 4) 1 wmhpnsgntg yaqkfqg Amino Acid Sequence of VH CDR3 of 9D12 (SEQ ID NO: 5) 1 nwgaafdi Amino Acid Sequence of VH CDR3 of 8A1 (SEQ ID NO: 6) 1 grgitmvrgg iiyygmdv VK amino acid sequence CDR1 of 9D12 (SEQ ID NO: 7) 1 rasqsvssyl a VK CDR1 amino acid sequence of 8A1 (SEQ ID NO: 8) 1 rasqgissal a VK amino acid sequence CDR2 of 9D12 (SEQ ID NO: 9) ldasnrat Amino acid sequence of VK CDR2 of 8A1 (SEQ ID NO: 10) 1 dassles Amino acid sequence of VK CDR 3 of 9D12 (SEQ ID NO: 11) 1 qqrsnwprwt Amino acid sequence of VK CDR3 of 8A1 (SEQ ID NO: 12) 1 qqfnsypht Amino acid sequence of VH of 9D12 (SEQ ID NO: 13) 1 evqllesggg lvqpggslrl scaa sgftfs ssamswvrqa pgkglewvss isgsgdttyy 61 adsvkgrfti srdnskntly lqmnslrved aalyycaanw gaafdiwgqg tmvtvss Amino acid sequence of VH of 8A1 (SEQ ID NO: 14) 1 qmqlvqsgae vkkpgasvkv sckasgytfi sydinwvrqa tgqglewmgw mhpnsgntgy 61 aqkfqgrvtm trntsistay melsslrsed tavyycargr gitmvrggii yygmdvwgqg ttvtvss Amino acid sequence of VK of 9D12 (SEQ ID NO: 15) 1 eivltqspat lslspgerat lscrasqsvs sylawyqqkp gqaprlliyd asnratgipa 61 rfsgsgsgtd ftltisslep edfavyycqq rsnwprwtfg qgtkveik amino acid sequence of VK of 8A1 (SEQ ID NO: 16) 1 aiqltqspss lsasvgdrvt itcrasqgis salawyqqkp gkapklliyd asslesgvps 61 rfsgsgsgtd ftltisslqp edfatyycqq fnsyphtfgq gtkleik Nucleotide Sequence of VH of 9D12 (SEQ ID NO: 17) 1 GAG GTG CAA CTG TTG GAG TCT GGG GGA GGC TTG GTA CAG CCT GGG GGG TCC CTG AGA CTC TCC TGT GCC GCC TCT GGA TTC ACC TTT AGC AGC TCT GCC ATG AGC TGG GTC CGC CAG GCT CGA GGG AAG GGG CTG GAG TGG GTC TCA TCT ATT AGT GGT AGT GGT GAT ACC ACA TAC TAC GCA GAC TCC GTG AAG GGC CGG T TC ACC ATC TCC AGA GAC AAT TCC AAG AAT ACG CTG TAT CTG CAA ATG AAC AGC CTG AGA GTC GAG GAC GCG GCC TTA TAT TAC TGT GCG GCT AAC TGG GGA GCT GCT TTT GAT ATC TGG GGC CAA GGG ACA ATG GTC ACC GTC TCT TCA Nucleotide VH Sequence of 8A1 (SEQ ID NO: 18) CAG ATG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG CCT GGG GCC TCA GTG AAG GTC TCC TGC AAG GCT TCT GGA TAC ACC TTC ATT AGT TAT GAT ATC AAC TGG GTG CGA CAG GCC ACT GGA CAA GGG CTT GAG TGG ATG GGA TGG ATG CAC CCT AAC AGT GGT AAC ACA GGC TAT GCA CAG AAG TTC CAG GGC AGA GTC ACC ATG ACC AGG AAC ACC TCC ATA AGC ACA GCC TAC ATG GAA CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG TAT TAC TGT GCG AGA GGC CGA GGA ATT ACT ATG GTT CGG GGA GGT ATT ATC TAC TAC GGT ATG GAC GTC TGG GGC CAA GGG ACC ACG GTC ACC GTC TCC TCA Nucleotide Sequence of VK of 9D12 (SEQ ID NO: 19) GAA ATT GTG TTG ACA CAG TCT CCA GCC ACC CTG TCT TTG TCT CCA GGG GAA AGA GCC ACCCCTC TCC TGC AGG GCC AGT CAG AGT GTT AGC AGC TAC TTA GCC TGG TAC CAA CAG AAA CCT GCC CAG GCT CCC AGG CTC CTC ATC TAT GAT GCA TCC AAC AGG GCC ACT GGC ATC ACA GCC AGG TTC AGT GGC AGT GGG TCT GGG ACA GAC TTC ACT CTC ACC ATC AGC AGC CTA GAG CCT GAA GAT TTT GCT GTT TAT TAC TGT CAG CAG CGT AGC AAC TGG CCT CGG TGG ACG TTC GGC CAA GGG ACC AAG G TG GAA ATC AAA Nucleotide VK sequence of 8D1 (SEQ ID NO: 20) GCC ATC CAG TTG ACC CAG TCT CCA TCC TCC CTG TCT GTC TCT GTA GGA GAC AGA GTC ACC ATC ACT TGG CGG GCA AGT CAG GGC ATT AGC AGT GCT TTA GCC TGG TAT CAG CAG AAA CCA GGG AAA GCT CCT AAG CTC CTG ATC TAT GAT GCC TCC AGT TTG GAA AGT GGG GTC CCA TCA AGG TTC AGC GGC AGT GGA TCT GGG ACA GAT TTC ACT CTC ACC ATC AGC AGC CTG CAG CCT GAA GAT TTT GCA ACT TAT TAC TGT CAA CAG TTT AAT AGT TAC CCT CAC ACT TTT GGC CAG GGG ACC AAG CTG GAG ATC AAA Amino acid sequence of germination line of VH 3-23 (SEQ ID NO: 21) 1 evqllesggg lvqpggslrl scaasgftfs sylamswvrq apgkglewvs aisgsggsty 61 yadsvkgrft isrdnskntl ylqmnslrae dtavyyca Amino acid sequence of germination line of VH 1-8 (SEQ ID NO: 22) 1 qvqlvqsgae vkkpgasvkv kvsckasgy tftsydinwv rqatgqglew mgwmnpnsgn 61 tgyaqkfqgr vtmtrntsis taymelsslr sedtavyycar g Amino acid sequence of germination line of VK L6 (SEQ ID NO: 23) 1 eivltqspat lslspgerat lscrasqsvs sylawyqqkp gqaprlliyd asnratgipa 61 rfsgsgsgtd ftltisslep edfavyycqq rsnwp Amino acid sequence of line germination VK L18 (SEQ ID NO: 24) 1 aiqltqspss lsasvgdrvt itcrasqgis salawyqqkp gkapklliyd asslesgvps 61 rfsgsgsgtd ftltisslqp edfatyycqq fhsyp amino acid sequence IRTA-2 (SEQ ID NO: 25) 1 mllwvillvl apvsgqfart prpiiflqpp wttvfqgerv tltckgfrfy spqktkwyhr 61 ylgkeilret pdnilevqes geyrcqaqgs plsspvhldf ssaslilqap lsvfegdsw 121 lrcrakaevt lnntiykndn vlaflnkrtd fhiphaclkd ngayrctgyk esccpvssnt 181 vkiqvqepft rpvlrassfq pisgnpvtlt cetqlslers dvplrfrffr ddqtlglgws 241 lspnfqitam wskdsgf wc kaatmphsvi sdsprswiqv qipashpvlt lspekalnfe 301 gtkvtlhcet qedslrtlyr fyhegvplrh ksvrcergas isfslttens gnyyctadng 361 lgakpskavs lsvtvpvshp vlnlsspedl ifegakvtlh ceaqrgslpi lyqfhhedaa 421 lerrsansag gvaisfslta ehsgnyycta dngfgpqrsk avslsitvpv shpvltlssa 481 ealtfegatv tlhcevqrgs pqilyqfyhe dmplwssstp svgrvsfsfs lteghsgnyy 54 1 ctadngfgpq rsewslfvt vpvsrpiltl rvpraqawg dllelhceap rgsppilywf 601 yhedvtlgss sapsggeasf nlsltaehsg nysceanngl vaqhsdtisl svivpvsrpi 661 ltfrapraqa wgdllelhc ealrgsspil ywfyhedvtl gkisapsggg asfnlsltte 721 hsgiyscead ngpeaqrsem vtlkvavpvs rpvltlrapg thaavgdlle lhcealrgsp 781 lilyrffhed vtlgnrssps ggaslnlslt aehsgnysce adnglgaqrs etvtlyitgl 841 tanrsgpfat gvaggllsia glaagallly cwlsrkagrk pasdparspp dsdsqeptyh 901 nvpaweelqp vytnanprge nwysevrii qekkkhavas piiysevkva dprhlrnkgs 961 stpvsgslfl assaphr amino acid sequence of IRTA-1 (SEQ ID NO: 26) 1 apvcgqsaaa mllwasllaf hkpvisvhpp wttffkgerv tltcngfqfy atekttwyhr 61 hywgekltlt pgntlevres glyrcqargs prsnpvrllf ssdslilqap ysvfegdtlv 121 lrchrrrkek ltavkytwng nilsisnksw dllipqassn nngnyrcigy gdendvfrsn 181 fkiikiqelf phpelkatds qptegnsvnl scetqlpper sdtplhfnff rdgevilsdw 241 stypelqlpt vwrensgsyw cgaetvrgni hkhspslqih vqripvsgvl letqpsggqa 301 vegemlvlvc svaegtgdtt fswhredmqe slgrktqrsl raelelpair qshaggyyct 361 adnsyg pvqs mvlnvtvret gatggllsal pgnrdglvaa llavallfhc wrrrksgvgf 421 lgdetrlppa pgpgesshsi cpaqvelqsl yvdvhpkkgd lvyseiqttq lgeeeeants 481 rtlledkdvs wysevktqh pdnsagkiss kdees Amino acid sequence of IRTA-3 (SEQ ID NO: 27) 1 tpgreqsgva mllwllllil pkavlllnpp wstafkgekv alicssishs laqgdty yh 61 dekllkikhd kiqitepgny qcktrgssls davhvefspd wlilqalhpv fegdnvilrc 121 qgkdnknthq kvyykdgkql pnsynlekit vnsvsrdnsk yhctayrkfy ildievtskp 181 lniqvqelfl hpvlrassst piegspmtlt cetqlspqrp dvqlqfslfr dsqtlglgws 241 rsprlqipam wtedsgsywc evetvthsik krslrsqirv qrvpvsnvnl eirptggqli 301 egenmvlics vaqgsgtvtf swhkegrvrs lgrktqrsll aelhvltvke sdagryycaa 361 dnvhspilst wirvtvripv shpvltfrap rahtwgdll elhceslrgs ppilyrfyhe 421 dvtlgnssap sgggasfhls ltaehsgnys cdadnglgaq hshgvslrvt vpvsrpvltl 481 rapgaqawg dllelhcesl rgsfpilywfyheddtlgni sahsgggasf nlslttehsg 541 nysceadngl gaqhskwtl nvtgtsrnrt gltaagitgl vlsilvlaaa aallhyarar 601 rkpgglsatg tsshspsecq epsssrpsri dpqepthskp lapmelepmy snvnpgdsnp 661 iysqiwsiqh tkensancpm mhqeheeltv lyselkkthp ddsageassr graheeddee 721 nyenvprvll ASDH Amino acid sequence of IRTA-4 (SEQ ID NO: 28) 1 mllwsllvif davteqadsl tlvapssvfe gdsivlkcqg eqnwkiqkma yhkdnkelsv 61 fkkfsdfliq savlsdsgny fcstkgqlfl wdktsnivki kvqelfqrpv ltassfqpie 121 ggpvslkcet rlspqrldvq lqfcffrenq vlgsgwsssp elqisavwse dtgsywckae 181 tvthrirkqs lqsqihvqri pisnvsleir apggqvtegq klillcsvag gtgnvtfswy 241 reatgtsmgk ktqrslsael eipavkesda gkyycradng hvpiqskwn ipvripvsrp 301 vltlrspgaq aavgdllelh cealrgsppi lyqfyhedvt lgnssapsgg gasfnlslta 361 ehsgnyscea nnglgaqcse avpvsisgpd gyrrdlmtag vlwglfgvlg ftgvalllya 421 lfhkisgess atneprgasr pnpqeftyss ptpdmeelqp vyvnvgsvdv dwysqvwsm 481 qqpessanir tllenkdsqv iyssvkks amino acid sequence of IRTA-5 (SEQ ID NO: 29) 1 mlprllllic aplcepaelf liaspshpte gspvtltckm pflqssdaqf qfcffrdtra 61 Igpgwssspk lqiaamwked tgsywceaqt maskvlrsrr sqinvhrvpv advsletqpp 121 ggqvmegdrl vlicsvamgt gditflwykg avglnlqskt qrsltaeyei psvresda 181 eq yycvaengyg pspsglvsit vripvsrpil mlrapraqaa vedvlelhce alrgsppily 241 wfyheditlg srsapsggga sftilslteeh sgnysceann glgaqrseav tlnftvptga 301 rsnhltsgvi egllstlgpa tvallfcygl krkigrrsar dplrslpspl pqeftylnsp 361 tpgqlqpiye nvnwsgdev yslayynqpe qesvaaetlg thmedkvsld iysrlrkani 421 tdvdyedam SUMMARY OF LIQUID SEQUENCE

Claims (38)

1. CLAIMS 1. An isolated monoclonal antibody or an antigen binding portion thereof, characterized in that the antibody: (a) binds to human IRTA-2 with a KD of lXlO "7 M or less, (b) does not bind substantially to human IRTA-3 or IRTA-4, and (c) binds to Granta tumor cells 519 and does not bind substantially to Raj io Ramos tumor cells
2. 2. The antibody according to claim 1, characterized in that it is a human antibody
3. 3. The antibody according to the claim 1, characterized in that it is a chimeric or humanized antibody.
4. 4. The antibody in accordance with the claim 2, characterized in that it is a full length antibody of an IgGl or IgG4 isotype.
5. 5. The antibody according to claim 2, characterized in that it is an antibody fragment or a single chain antibody.
6. 6. The antibody according to claim 2, characterized in that the antibody binds to human IRTA-2 with a KD of 5x10"8 M or less
7. 7. The antibody according to claim 2, characterized in that the antibody binds to human IRTA-2 with a KD of 5 xlO "8 M or less.
8. 8. The antibody according to claim 2, characterized in that the human IRTA-2 comprises a polypeptide having an amino acid sequence as summarized in SEQ ID NO: 25 [No. of Access Genbank NP_112571].
9. 9. The antibody according to claim 2, characterized in that human IRTA-3 comprises a polypeptide having an amino acid sequence as summarized in SEQ ID NO: 27 [No. of Access Genbank AAL59390].
10. 10. The antibody according to claim 2, characterized in that human IRTA-4 comprises a polypeptide having an amino acid sequence as summarized in SEQ ID NO: 28 [No. of Access Genbank AAL60249].
11. 11. The antibody according to claim 2, characterized in that the antibody binds substantially to tumor cells of SU-DHL-6 or JEKO-1.
12. 12. The antibody according to claim 2, characterized in that the antibody does not bind substantially to tumor cells of Daudi, IM-9, Karpas 1106P or SU-DHL-4.
13. 13. An isolated monoclonal antibody or antigen binding portion thereof, wherein the antibody competes cross-linked by the binding to IRTA-2 with a reference antibody, characterized in that the antibody: (a) binds to IRTA-2 human with a KD constant of lxlO "7 M or less, (b) does not bind substantially to human IRTA-3 or IRTA-4, and (c) binds to Granta tumor cells 519 and does not bind substantially to Raji or Ramos tumor cells
14. 14. The antibody according to claim 13, characterized in that the reference antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13; (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15.
15. 15. The antibody according to claim 13, characterized in that the reference antibody comprises: (a) a variable region of heavy chain which comprises the sequence a of amino acids of SEQ ID NO: 14; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.
16. 16. The antibody according to claim 13, characterized in that human IRTA-2 comprises a polypeptide having an amino acid sequence. as summarized in SEQ ID NO: 25 [No. of Access Genbank NP_12571].
17. 17. The antibody according to claim 13, characterized in that the human IRTA-3 comprises a polypeptide having the amino acid sequence as summarized in SEQ ID NO: 27 [No. of Access Genbank AAL59390].
18. 18. The antibody according to claim 13, characterized in that human IRTA-4 comprises a polypeptide having an amino acid sequence as summarized in SEQ ID NO: 28 [No. of Access Genbank AAL60249].
19. 19. An isolated monoclonal antibody or antigen binding portion thereof, characterized in that it comprises a heavy chain variable region which is the product of or derived from a human VH 3-23 gene or a human VH 1-8 gene, wherein the antibody binds specifically to IRTA-2.
20. 20. An isolated monoclonal antibody or antigen binding portion thereof, characterized in that it comprises a light chain variable region that is the product of or derived from a human VK L6 gene or a human VK L18 gene, wherein the antibody is linked specifically to IRTA-2.
21. 21. The isolated monoclonal antibody or antigen binding portion thereof according to claim 20, characterized in that it further comprises a heavy chain variable region that is the product of or derived from a human VH 3-23 gene or a gene of VH 1-8 human.
22. 22. The antibody according to claim 1, characterized in that it comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 1; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 3; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 5; (d) a light chain variable region CDR1 comprising SEQ ID NO: 7; (e) a light chain variable region CDR2 comprising SEQ ID NO: 9; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 11.
23. 23. The antibody according to claim 1, characterized in that it comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 2; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 4; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 6; (d) a light chain variable region CDR1 comprising SEQ ID NO: 8; (e) a light chain variable region CDR2 comprising SEQ ID NO: 10; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 12.
24. 24. An isolated monoclonal antibody or antigen binding portion thereof, characterized in that it comprises: (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 14; and (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 16; where the antibody binds specifically to IRTA-2.
25. 25. An isolated monoclonal antibody or antigen binding portion thereof, characterized in that it comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15.
26. 26. An isolated monoclonal antibody or antigen binding portion thereof, characterized in that it comprises: (a) a variable chain region heavy comprising the amino acid sequence of SEQ ID NO: 14; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.
27. 27. A composition characterized in that it comprises the antibody according to claim 1 or antigen binding portion thereof and an acceptable carrier. pharmaceutically
28. 28. An immunoconjugate characterized in that it comprises the antibody according to claim 1 or antigen binding portion thereof, linked to a therapeutic agent.
29. 29. A composition characterized in that it comprises the immunoconjugate according to claim 28 and a pharmaceutically acceptable carrier.
30. 30. The immunoconjugate according to claim 28, characterized in that the therapeutic agent is a cytotoxin.
31. 31. A composition characterized in that it comprises the immunoconjugate according to claim 30 and a pharmaceutically acceptable carrier.
32. 32. The immunoconjugate according to claim 28, characterized in that the therapeutic agent is a radioactive isotope.
33. 33. A composition characterized in that it comprises the immunoconjugate according to claim 32 and a pharmaceutically acceptable carrier.
34. 34. An isolated nucleic acid molecule characterized in that it encodes the antibody according to claim 1 or antigen binding portion thereof.
35. 35. An expression vector characterized in that it comprises the nucleic acid molecule according to claim 34.
36. 36. A host cell characterized in that it comprises the expression vector according to claim 35.
37. 37. A method for the preparation of an antibody anti-IRTA-2 characterized in that it comprises expressing the antibody in the host cell according to claim 36 and isolating the antibody from the host cell.
38. 38. A method for inhibiting the growth of tumor cells expressing IRTA-2, characterized in that it comprises contacting the cells with the antibody according to claim 1 or antigen binding portion thereof, in an amount effective to inhibit the growth of tumor cells.