ANTIBODIES THAT SPECIFICALLY BIND IRTA AND METHODS OF USE

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit U.S. Provisional Application No. 60/891,434, filed February 23, 2007, which is incorporated by reference herein in its entirety.

FIELD

This relates to the field of immunotherapy, specifically to antibodies that bind immunoglobulin superfamily receptor translocation associated 3 (IRT A3) or immunoglobulin superfamily receptor translocation associated 5 (IRT A5) and the use of these antibodies.

BACKGROUND Abnormalities of chromosome Iq21 are common in B cell malignancies, including B cell lymphoma and myeloma, but the genes whose expression is affected by these abnormalities are largely unknown. Chromosomal abnormalities involving band Iq21-q23 are frequent genetic lesions in both B cell non-Hodgkin's lymphoma and multiple myeloma. Among non-Hodgkin's lymphoma subtypes, translocation breakpoints at Iq21-q23, including translocations and duplications, have been reported, often as the single chromosomal abnormality in follicular and diffuse large B cell lymphoma (DLCL) in marginal-zone B cell lymphoma and in Burkitt's lymphoma.

By cloning the breakpoints of a t(l :14)(q21 :q32) chromosomal translocation in a myeloma cell line, two genes were identified, termed immunoglobulin superfamily receptor translocation associated (IRTA)I and IRT A2. IRT A2 is identical to sequences identified as BXMASl (Nakayama et al., Biochm. Biophys. Res. Commun. 285:830-7, 2001) and FcRH5 (Davis et al., Proc. Natl. Acad. ScL USA 98:9772-7, 2001). A series of genes homologous to IRTA2/BXMASl/FcRH5 have been identified in the same locus of the human genome, including the six IRTA family members thus far identified (for example, see Davis et al., Immunol. Rev. 190:123-36, 2002).

The IRTA molecules (also called Fc receptor homologues) are cell surface glycoproteins containing three to nine extracellular Ig-like domains. Five of the six family members (IRTAl, IRTA2, IRT A3, IRT A4 and IRTA5) are preferentially expressed during B cell differentiation and show topographically distinct patterns of expression in lymphoid organs, indicating a role in modulating normal B cell development and immune responses (see Miller et al., Blood 99:2662-2669, 1999). IRTAs are expressed in peripheral lymphoid tissues, including lymph nodes, tonsils, resting peripheral B cells and normal germinal center B cells (Davis et al., Proc. Natl. Acad. ScL USA 98:9772-7, 2001). IRTA2, IRTA3, IRTA4, and IRTA5 are all expressed at high levels in spleen, whereas, by comparison, IRTAl has been detected in lower levels in the spleen. IRTA expression has been analyzed within the B cell compartment of human tonsil tissue. IRTAl is expressed outside of lymphoid follicles in the marginal zone pattern and in intraepithelial lymphocytes. IRT A2 and 3 are expressed within the germinal center, with highest expression in the centocyte-rich light zone. IRT A4 and 5 are expressed highest within mantle zones, indicating expression in naive B cells.

B cell malignancies, which include various forms of leukemias and lymphomas as well as multiple myelomas, arise from neoplastic transformation of B cells at distinct stages of differentiation and result in significant morbidity and mortality. B cell-associated IRTA molecules are differentially expressed in various B cell malignancies. The IRTA genes have been shown to be highly expressed in B cell non-Hodgkin's lymphoma, chronic lymphocytic leukemias, follicular lymphomas, diffuse large cell lymphomas of B cell lineage, and multiple myelomas amongst others. There remains a need for methods and compositions which can be used to treat cancer, including B cell malignancies, such as non-Hodgkin's lymphoma, chronic lymphocytic leukemias, follicular lymphomas, diffuse large cell lymphomas of B lineage, and multiple myelomas B cell lymphoma and Burkitt's lymphoma.

SUMMARY

Monoclonal antibodies that specifically bind the extracellular domain of IRTA molecules (such as IRT A3 or IRT A5) are disclosed herein. In one

embodiment, the monoclonal antibody specifically binds the extracellular domain of IRT A3 and does not specifically bind IRTAl, IRT A2, IRT A4, IRT A5, or IRT A6. In another embodiment, the monoclonal antibody specifically binds the extracellular domain of IRTA5 and does not specifically bind IRTAl, IRTA2, IRTA3, IRTA4, or IRTA6.

Antibodies that bind IRTA molecules can be can be conjugated to effector molecules, including detectable labels, radionucleotides, toxins and chemotherapeutic agents. Also disclosed are nucleic acid molecules encoding the monoclonal antibodies that specifically bind IRTA molecules or fragments thereof, such as IRT A3 or IRT A5. Also disclosed are humanized monoclonal antibodies that specifically bind IRTA molecules and fragments thereof.

The antibodies disclosed herein have a number of uses. For example, the disclosed antibodies can be used to detect or confirm the diagnosis of a B cell malignancy. The antibodies disclosed herein are of use in treating a B cell malignancy in a subject, for example by administering to the subject a therapeutically effective amount of an antibody conjugated to an effector molecule, thereby treating the B cell malignancy The antibodies can also be used to detect and/or isolate Treg cells, such as non-proliferating Treg cells.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 is a table showing the selective cell binding characteristics and reactivity of anti-IRTA monoclonal antibodies (MAbs).

Figs. 2A-2B are plots of enzyme linked immunosorbent assay (ELISA) data and representative fluorescent activated cell sorting data showing the specific reactivity of anti-IRTA3 MAb H5 with IRT A3. Fig. 2A shows plots of an ELISA of anti-IRTA3 MAb H5 to IRTA 1-5-Fc-fusion proteins. Each Fc-fusion protein was coated on ELISA plates using secondary antibody (goat anti-human IgG). After incubation with a dilution series of anti-IRTA3 MAb H5, bound MAbs were detected by horseradish peroxidase- (HRP-) labeled goat anti-mouse IgG and tetramethylbenzidine (TMB) substrate. Fig. 2B is a set of histograms of FACS analysis of 293T cells transiently transfected with an expression plasmid for each IRTA. Two days after transfection, the cells were detached and incubated with anti-

IRT A3 MAb H5. The bound MAbs were detected by PE-conjugated goat anti- mouse IgG F(ab)2. Cells incubated with H5 and without H5 are shown in the histograms (log fluorescence versus cell number).

Figs. 3A-3D are representative plots of FACS data and a table showing the expression of IRT A3 on human peripheral blood mononuclear cells (PBMCs). PBMCs from normal donors were stained with combinations of MAbs specific to each marker and analyzed by a flow cytometer. Fig. 3A shows the expression of IRT A3 on B (CD 19+), T (CD3+) and NK (CD56+) cells. Each cell population identified with each marker in lymphocyte gates is shown in each histogram. Cells incubated with anti-IRTA3-PE or with mouse IgG2b-PE (the isotype controls) are shown. A small subset of CD3+ T cells expressed IRT A3. Fig. 3B shows the expression of IRTA3 on CD4+ and CD8+ T cell subsets. The analysis of the same sample as Fig. 3A is shown. Both some CD4+ T cells and some CD8+ T cells expressed IRT A3. Fig. 3 C shows the statistics of IRT A3 expression in T cell fractions of human PBMCs. Positive threshold for IRT A3 was determined so that fewer than 0.5% of cells in the isotype control. Fig. 3D shows the results of RT- PCR analysis of the FACS-sorted cells. PBMCs were sorted into 4 fractions (Pl- P4) based on the different staining levels of CD3 and IRT A3. RT-PCR products using an equal amount of total RNA from each fraction were run on agarose gels. IRTA3+CD3+ T cells expressed IRTA3 mRNA.

Figs. 4A-4D are representative plots of FACS data and a table showing the phenotypes of IRTA3+ cells in T-helper and T-cytotoxic cell fractions. Each panel shows an analysis of a representative sample. Expression of the indicated markers in the IRT A3 + or IRT A3- population in each subset is depicted in dot plots or histograms. Freshly isolated human PBMCs were stained with combinations of antibodies. In the flow cytometry analysis, the cells were gated on lymphocytes by scatter parameters followed by on CD3+ and then on CD4+ or on CD8+. IRTA3+ or IRTA3- population in each subset are depicted with CD62L and CD45RA (naϊve cell markers) (Fig. 4A); CD45RO (a memory maker) (Fig. 4B); or CD25 (activation or regulatory T cell marker) (Fig. 4C). Each panel shows an analysis of a representative sample. Fig. 4D the statistics of the multiple analyses (n=4 or 5). ***, PO.001; **, 0.00KPO.01; *, 0.0KPO.02; NS, 0.05<P by Student's T-test.

CD62L+CD45RA+ phenotype was determined according to the gates shown in Fig. 4 A. Threshold for CD25 was determined so that fewer than 0.1% of cells in the counter control without the CD25 MAb were included in the positive range.

Fig. 5 is a set of bar plots showing the expression kinetics of different activation markers and IRT A3 on CD4+ and CD8+ cells after T cell stimulation through TCR and CD28. Human PBMCs were stimulated with anti-CD3/CD28- coated beads. The cells were harvested at the indicated time and were then stained with a cocktail of markers. The stained cells were initially gated by CD3-side scatter profile to include large activated lymphocytes and then gated for CD4+ (Fig. 5A) or CD8+ (Fig. 5B). The percentages of CD3+ T cells with indicated phenotypes are shown. Threshold for CD25 was determined so that less than 0.1% of cells in the counter control without the CD25 MAb were included in the positive range. Early activation marker CD69 followed by late activation marker CD25 were induced in both CD4+ and CD8+ T cells. In contrast, the polyclonal T cell activation did not induce IRT A3 expression in any cell fractions including the activated cell fractions. CD4+, CD8+ and CD45RO+ cell ratios in CD3+ T cells were not significantly changed by the T cell activation. Representative data in three similar experiments is shown.

Fig. 6A-6E is a set of histograms and plots showing natural Treg cells in human PBMCs are subdivided into two subpopulations with different IRT A3 expression levels. PBMCs from normal donors were stained with CD 127, IRT A3, CD4 and CD25-specifϊc MAbs conjugated with different fluorochromes. The stained cells were fixed, permeabilized and the intracellular Foxp3 was stained with a MAb. One representative experiment is shown in Fig. 6 A-Fig. 6C. Fig. 6A shows three gating strategies (Foxp3+, CD25+CD1271ow, and CD25hi) to identify Treg-enriched cells in CD4+ cells. Positive thresholds for Foxp3 (between Pl and P2) and for CD25 (between P5 and P6) were determined so that fewer than 1% of cells in the counter control without the corresponding marker were included in the positive ranges. CD25 ^hl threshold (between P6 and P7) was drawn so that P7 includes 2% of CD4+ cells with highest CD25 signals. P3 and P4 gates are drawn so as to distinguish the CD25+CD127 ^low Treg population in P4 from other cells in P5. Fig. 6B shows the relationships among the three gated Treg populations.

Foxp3+ cells include most of CD25+CD127 ^low cells. CD25 ¹ " cells are a subpopulation of CD25+CD127 ^low cells that express Foxp3 at a high level. Fig. 6C shows expression of IRT A3 on the gated cells. Two peaks corresponding to IRTA3+ and IRTA3- cells were identified in each Tregs defined by the three gate strategy. Fig. 6D shows the frequency of each Treg population in CD4+ cells in four different specimens. Fig. 6E shows IRT A3 -expressing cells in each subpopulation. Using CD25+CD127 ^low or CD25 ¹ " gating strategy, about 40% of Treg cells were IRTA3-positive.

Fig. 7 is a set of bar graphs showing the cross reactivity of anti-IRTA monoclonal antibody (Mabs) to other IRTA Fc fusion proteins. Each Fc-fusion protein indicated on the top of the column was captured on ELISA plates using secondary antibody (goat anti-human IgG). After washing, 100 ng/50 ul/ well of MAbs were added, followed by horseradish peroxidase - (HRP) labeled goat anti- mouse IgG and TMB substrate. The signals in ELISA were normalized for the amounts of each Fc-fusion protein on the plates detected with HRP-goat anti-human Fc. The optical densities to each IRTA-Fc used for the normalization were within the range of 0.3-0.6. Anti-CD30 MAb BerH2 (IgGl) was used as a control.

Fig. 8A and 8B is a set of histograms, dot plots, bar charts and contour plots showing IRT A3 expression on nTregs is associated with CD25+CD1271ow phenotype rather than with Foxp3+ phenotype. PBMCs were stained with the markers as shown in Fig. 6. Marker expressions of CD4+Foxp3+ cells in lymphocytes gate were analyzed. Fig. 8A shows the association of CD25 positive and CD127 low levels with IRTA3 expression on CD4+Foxp3+ cells. As a result, IRTA3+Foxp3+ cells preferentially showed CD25+CD1271ow phenotype. Fig. 8B shows the percentages of Treg cells and non-Treg cells identified by combination of CD25 and CD127 levels in Foxp3+IRTA3+ and Foxp3+IRTA3- cells. Gatings for CD25+CD1271ow Treg cells were performed according to Fig. 6A. Bars represent averages of five independent experiments which are shown in different symbols. IRTA3+Foxp3+ cells were enriched in CD25+CD127 ^low natural regulatory T cell (nTreg) cell fraction.

Fig. 9A-9D is a set of dot plots graph and bar charts showing that both IRTA3+ and IRTA3- Treg cells suppress T cell proliferation in vitro. Fig. 9A shows

the sorting of cells. The CD4+ cells were divided into CD25+CD127 ^low Treg cells and CD25- CD127 ¹ " cells that were used as the responder cells (Teff) in the suppressor assays. The Treg cells were further divided into two sub-populations depending on IRT A3 expression. As shown in the post sort panels, these IRTA3+ and IRT A3- Treg populations had an equivalent level of CD25 and CD 127 expression. Fig. 9B shows the suppression of proliferation of Teffs by the sorted populations. The four sorted populations shown in Fig. 9A were added back to CFSE-labeled Teff cells (CD25-CD127 ¹¹¹ ) at 1 :2 ratio. Cells were cultured for 80 hr with or without stimulation by the beads that had been coated with anti-CD3 and anti-CD28 antibodies. After the incubation, fluorescence levels of the labeled Teff cells were analyzed to monitor CFSE dilution by cell dividing. The added back cell populations tested were CFSE-negative and excluded from the histograms by the background level of the fluorescence. Fig. 9C shows the kinetics of the suppression. After 80 hours and 96 hours of culture, CFSE-level of Teff cells in triplicate cultures were analyzed and converted division indexes (the average number of divisions that a responder cell has undergone) by Flow Jo program. Averages+SDs are shown. Fig. 9D shows the suppression by the different addback ratios of the sorted population from a different subject. All the cultures were stimulated by the antibodies coated beads and the cell division were monitored after 96 hours of incubation. Fig. 1OC and Fig. 1OD show representative data sets from four independent experiments using different human subjects.

Figs. 10A- 1OD is a set of bar graphs and plots showing IRT A3 + Treg cells do not proliferate but IRT A3- Treg cells proliferate in the presence of IL-2 upon stimulation of their T cell receptor (TCR) and co-receptor. Fig. 1OA shows both IRTA3+ and IRTA3- nTreg cells show reduced proliferation without IL-2. Teff cells (CD25-CD127M), nTreg cells (CD25+CD1271ow) and nTreg cells with or without IRT A3 expression were isolated according to the sorting shown in Fig. 9A. Cells were labeled with CFSE and 5,000 cells were cultured for 80 hours with the indicated numbers of anti-CD3/CD28 beads. Treg populations showed reduced proliferation regardless of IRT A3 expression compared to the Teff cells even with the large excess of the beads to induce conventional T cells proliferation at the saturated level. Averages+SDs of triplicate cultures are shown. Fig. 1OB shows that

exogenous IL-2 conferred responsiveness to stimulation on IRT A3- Treg cells but not on IRT A3+ Treg cells. Sorted cells were cultured for 90 hours in the presence or absence of IL-2 and anti-CD3/CD28 beads. Fig. 1OC shows the time course of the proliferation of IRTA3+ and IRT A3- nTreg cells stimulated with anti- CD3/CD28 beads (2.5 beads/cell) in the presence or absence of IL-2 (10 ng/ml). Fig. 1OD shows that IL-10 secretion from IRT A3- Treg cells stimulated with anti- CD3/CD28 beads in the presence of exogenous IL-2. Each cytokine in the supernatant was measured after an 80-hour culture. Teffs secreted the indicated cytokines by the stimulation with anti-CD3/CD28 beads. However, Treg cells (CD25+CD 127 ^low ) didn't produce cytokines regardless of the expression of IRT A3. When the exogenous IL-2 was added in the culture, IRT A3- Treg cells secreted IL- 10 but IRTA3+ Treg cells did not produce IL-10. Fig. 10A-10D show a representative data set from three independent experiments using different human subjects. Fig. 11 is a set of dot plots showing no expression of IRT A3 on induced

Foxp3+ cells ex vivo from CD25-CD127 ¹ " Teff cells by TGF-β treatment. Teff cells (CD25-CD127 ^hl ) that had been sorted according to Fig. 9A were treated with anti- CD3/CD28-coated beads, TGF-β, IL-2 and their combinations for 4 days and analyzed with the expression of the indicated markers by flow cytometry. The quadrant gates for each column of panels are the same, which were determined with anti-CD3/CD28-treated cells using fluorescence minus one control. The percent number of cells in each quadrant gate is shown. Addition of TGF-β with anti- CD3/CD28-coated beads converted a large fraction of the stimulated cells to Foxp3+ cells. The induced Foxp3+ cells showed a marker profile similar to natural Treg in PBMCs, such as high levels of CD25 and CTLA-4 and rather lower levels of

CD127. The induced Foxp3+ cells, however, did not express IRTA3. Addition of exogenous IL-2 showed no effects on IRT A3 expression. The results are representative set of two experiments.

Fig. 12A-12C is a set of graphs, histograms, a table an electronic image of a gel showing the specific reactivity of anti-IRTA3 monoclonal antibody H5 with IRT A3 protein. Fig. 12A shows that H5 MAb specifically reacts with IRTA3-Fc fusion protein in ELISA. Different wells of an ELISA plate were coated with each

of IRTAl -6-Fc-fusion proteins and incubated with serially diluted H5 MAb. The bound MAbs were detected by peroxidase-labeled goat anti-mouse IgG and the enzyme substrate. H5 MAb bound to IRTA3-Fc protein and does not crossreact with other IRTA family members. The presence of each IRTA-Fc on the coated wells had been confirmed by a panel of previous MAbs to other IRTAs. Fig. 12B shows that H5 MAb reacts with IRTA3 protein transiently expressed on cell surface in flow cytometry analysis. 293T cells were transfected with each expression plasmid encoding each of IRTA5-6. Two days after transfection, the cells were detached and incubated with anti-IRTA3 MAb H5 followed by PE-conjugated goat anti-mouse IgG F(ab')2. Cells incubated with H5 (solid lines) and without H5 (the negative controls) are shown. H5 MAb bound to IRT A3 protein on cells and did not crossreact with other IRTA family members expressed on cells. The expression of each IRTA on the transfected cells had been confirmed by a panel of previous MAbs to other IRTAs. Fig. 12C shows that H5 MAb reacts with endogenous IRT A3 expressed on human cell lines. The indicated B cell lines were tested with the reactivity with H5 MAb and IRT A3 mRNA expression by RT-PCR. There was exact accordance between MAb reactivity and mRNA expression of IRT A3. The IRT A5 -6 cDNAs used in this study encode the same proteins as the following reference sequences: GENBANK® accession numbers, AF343659, AF343662, AF459027, AF459633, AF459634, and AY51366 1 as available February 23, 2007. H5 MAb did not cross reacted with FcγRl (CD64), FcγRIIb (CD32) or FcRIIIa (CD 16) in single or double colors flow cytometry using appropriate cell lines.

Fig. 13 is a set of bar graphs showing the non-association of CTLA4, GITR, HLA-DR, CD38 and CD 103 with IRT A3. Treg cells were identified as CD4+Foxp3+ population in these experiments. Non-association of the expression of the tested markers with IRT A3 was observed. Representative data of two independent experiments are shown. Fig. 14 is table Sl. Fig. 15 is a set of histograms of FACS data showing the cross reactivity of the anti-IRTA family MAbs to other IRTA family members when expressed on 293T cells. Two days after transfection with plasmids encoding IRTAs or CD30, the 293T cells were incubated with 200 ng/ 100 ul of each MAb, followed by PE-

conjugated goat anti-mouse IgG F(ab')2. Each panel shows the histogram versus log fluorescence with the negative control without primary antibodies. Anti-CD30 MAb BerH2 (IgGl) was used as a control.

Fig. 16 is a set of histograms of FACS data showing the reactivity of the anti-IRTA family MAbs to cell lines derived from B cells. Cells were incubated with 200 ng/ 100 ul of each MAb, followed by PE-conjugated goat anti-mouse IgG F(ab')2. Each panel shows the histogram versus log fluorescence with the negative control without primary antibodies. Anti-CD30 MAb BerH2 (IgGl) was used as a control. Fig. 17 is a sequence of alignment of the amino acid sequences of VH and

VL domains of the anti-IRTA3 antibodies E3 (SEQ ID NOs: 7 and 8) and E9 (SEQ ID NOs:9 and 10) and the germline sequences of E3 (SEQ ID NOs: 32 and 33) and E9 (SEQ ID NOs:34 and 35).

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 CF. R. § 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 is an exemplary amino acid sequence of IRT A3.

SEQ ID NO: 2 is an exemplary amino acid sequence of IRTA5. SEQ ID NO: 3 is an exemplary amino acid sequence of IRTAl.

SEQ ID NO: 4 is an exemplary amino acid sequence of IRTA2.

SEQ ID NO: 5 is an exemplary amino acid sequence of IRTA4.

SEQ ID NO: 6 is an exemplary amino acid sequence of IRTA6.

SEQ ID NO: 7 is the amino acid sequence of the V _H of the anti-IRTA5 MAb E3.

SEQ ID NO: 8 is the amino acid sequence of the V _L of the anti-IRTA5 MAb E3.

SEQ ID NO: 9 is the amino acid sequence of the V _H of the anti-IRTA5 MAb E9.

SEQ ID NO: 10 is the amino acid sequence of the V _L of the anti-IRTA5 MAb E9. SEQ ID NOs: 11-18 are exemplary frameworks of human Mabs.

SEQ ID NO: 19 is an exemplary nucleic acid sequence of Pseudomonas exotoxin.

SEQ ID NOs: 20-21 are amino acid sequences of exemplary carboxy terminal modifications to Pseudomonas exotoxin. SEQ ID NO: 22 is an exemplary nucleic acid sequence of the V _H of the anti-

IRT A5 MAb E3.

SEQ ID NO: 23 is an exemplary nucleic acid sequence of the V _L of the anti- IRT A5 MAb E3.

SEQ ID NO: 24 is an exemplary nucleic acid sequence of the V _H of the anti- IRT A5 MAb E9.

SEQ ID NO: 25 is an exemplary nucleic acid sequence of the V _L of the anti- IRTA5 MAb E9.

SEQ ID NOs: 26-31 are nucleic acid sequences of PCR primers.

SEQ ID NO: 32 is the amino acid sequence of the germline V _H of the anti- IRT A5 MAb E3.

SEQ ID NO: 33 is the amino acid sequence of the germline V _L of the anti- IRT A5 MAb E3.

SEQ ID NO: 34 is the amino acid sequence of the germline V _H of the anti- IRTA5 MAb E9. SEQ ID NO: 35 is the amino acid sequence of the germline V _L of the anti-

IRTA5 MAb E9.

DETAILED DESCRIPTION

/. Abbreviations ATCC: American Type Culture Collection

CDR: complementarity determining region CTL: cytotoxic T lymphocyte

dsFv: disulfide stabilized fragment of a variable region

DLCL: diffuse large B cell lymphoma

DT: diphtheria toxin

EF-2: elongation factor 2 ELISA: enzyme-linked immunosorbent assay

EM: effector molecule (moiety)

FACS: fluorescence activated cell sorter

HCDR: heavy chain complementarity determining region

HRP: horseradish peroxidase Ig: Immunoglobulin

IL-2: Interleukin 2

IL-IO: Interleukin 10

IRTA: immunoglobulin superfamily receptor translocation associated iTreg: induced regulatory T cells kDa: kilodaltons

LCDR: light chain complementarity determining region

MAb: monoclonal antibody

MALT: mucosa-associated lymphoid tissue nTreg: natural regulatory T cells NK: Natural Killer

PBMC: peripheral blood mononuclear cell

PCR: polymerase chain reaction

PE: Pseudomonas exotoxin

RCA: Ricinus communis agglutinin scFv: single chain fragment of a variable region

TCR: T-cell receptor

Treg: Regulatory T cell

Teff: T effector cell

TMB: trimethoxybenzene V _R : variable region of a heavy chain

V _L : variable region of a light chain

//. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341). As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Also, as used herein, the term "comprises" means "includes." Hence "comprising A or B" means including A, B, or A and B. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

To facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

Administration: The introduction of a composition into a subject by a chosen route. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject.

Amplification: Of a nucleic acid molecule (such as a DNA or RNA molecule) refers to use of a technique that increases the number of copies of a nucleic acid molecule in a sample, such as a biological sample. An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to a nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. The product of amplification may be characterized by

electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing using standard techniques. Other examples of amplification include strand displacement amplification, as disclosed in U.S. Patent No. 5,744,311; transcription-free isothermal amplification, as disclosed in U.S. Patent No. 6,033,881; repair chain reaction amplification, as disclosed in PCT Publication No. WO 90/01069; ligase chain reaction amplification, as disclosed in European Patent No. EP-A-320 308; gap filling ligase chain reaction amplification, as disclosed in U.S. Patent No. 5,427,930; and NASBA™ RNA transcription-free amplification, as disclosed in U.S. Patent No. 6,025,134. Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.

Antibody: A polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen, such as IRT A3 or a fragment thereof or IRT A5 or a fragment thereof. This includes intact immunoglobulins and the variants and portions of them well known in the art, such as Fab' fragments, F(ab)' ₂ fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3 ^rd Ed., W.H. Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. References to "V _H " or "VH" refer to the variable region of an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab. References to "V _L " or "VL" refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

There are two types of light chain, lambda (λ) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains"). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs". The extent of the framework region and CDRs have been defined (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5 ^th Edition, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, MD (NIH Publication No. 91-3242), which is hereby incorporated by reference). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three- dimensional space, for example to hold the CDRs in an appropriate orientation for antigen binding.

The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDRl , CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a V _H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a V _L CDRl is the CDRl from the variable domain of the light chain of the antibody in which it is found.

A "monoclonal antibody" is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody- forming cells from a fusion of myeloma cells with immune spleen cells. These fused cells and their progeny are termed "hybridomas." Monoclonal antibodies include humanized monoclonal antibodies.

A "humanized" immunoglobulin, such as a humanized IRTA3 or IRT A5 monoclonal antibody, is an immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a "donor," and the human immunoglobulin providing the framework is termed an "acceptor." In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they should be substantially identical to human immunoglobulin constant regions, for example, at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A "humanized antibody" is an antibody, such as a humanized IRT A3 or IRT A5 monoclonal antibody, comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by means of genetic engineering (for example see U.S. Patent No. 5,585,089).

Autoimmune Disease: A disease in which the immune system produces an immune response (for example, a B cell or a T cell response) against an antigen that is part of the normal host (that is, an autoantigen), with consequent injury to tissues. An autoantigen may be derived from a host cell, or may be derived from a commensal organism such as the micro-organisms (known as commensal organisms) that normally colonize mucosal surfaces.

Exemplary autoimmune diseases affecting mammals include rheumatoid arthritis, juvenile oligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease (for example, Crohn's disease, ulcerative colitis), autoimmune gastric atrophy, pemphigus vulgaris, psoriasis,

vitiligo, type 1 diabetes, non-obese diabetes, myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemic lupus erythematosis, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, Addison's disease, systemic sclerosis, polymyositis, dermatomyositis, autoimmune hemolytic anemia, pernicious anemia, and the like.

Binding affinity: Affinity of an antibody for an antigen. In one embodiment, affinity is calculated by a modification of the Scatchard method described by Frankel et ah, MoI. Immunol., 16:101-106, 1979. In another embodiment, binding affinity is measured by an antigen/antibody dissociation rate. In yet another embodiment, a high binding affinity is measured by a competition radioimmunoassay. In several examples, a high binding affinity is at least about

1 x 10 ^" M. In other embodiments, a high binding affinity is at least about 1.5 x 10 ^" , at least about 2.0 x 10 ^"8 , at least about 2.5 x 10 ^"8 , at least about 3.0 x 10 ^"8 , at least about 3.5 x 10 ^" , at least about 4.0 x 10 ^" , at least about 4.5 x 10 ^" , or at least about 5.O x IO ⁸ M.

CD4: Cluster of differentiation factor 4. A T cell surface protein that mediates interaction with MHC class II molecules. This cell surface antigen is also known as T4, Leu-3, OKT4 or L3T4. CD4 is a 55 kDa transmembrane glycoprotein belonging to the immunoglobulin superfamily. A T cell that expresses CD4 is a "CD4+" T cell. Likewise, a T cell that does not express CD4 is a "CD4-" T cell.

CD25: Cluster of differentiation factor 25, the IL-2 receptor alpha chain. A T cell that expresses CD25 is a "CD25+" T cell.

Chemotherapeutic agents: Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth such as psoriasis. In one embodiment, a chemotherapeutic agent is an agent of use in treating a lymphoma, leukemia, or another tumor. In one embodiment, a chemotherapeutic agent is a radioactive compound. One of skill in the art can readily identify a chemotherapeutic agent of use (for example see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al, Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2 ^nd ed., © 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery,

R. (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, D.S., Knobf, M.F., Durivage, HJ. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one agent to treat cancer. One example is the administration of an antibody that binds IRT A3 or an antibody that binds IRT A5, or a fragment of these antibodies, used in combination with a radioactive agent or chemical compound.

Chimeric antibody: An antibody which includes sequences derived from two different antibodies, which typically are of different species. Most typically, chimeric antibodies include human and murine antibody domains, generally human constant regions and murine variable regions, murine CDRs and/or murine SDRs.

Conservative variants: "Conservative" amino acid substitutions are those substitutions that do not substantially affect or decrease an activity and/or antigenicity of a polypeptide, such as IRT A3 or IRT A5. For example, an IRT A3 or IRT A5 polypeptide can include at most about 1, at most about 2, at most about 5, and most about 10, or at most about 15 conservative substitutions and specifically bind an antibody that binds the original IRTA polypeptide. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibodies raised antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide. Non-conservative substitutions are those that reduce an activity or antigenicity.

Contacting: Placement in direct physical association, which includes both in solid and in liquid form.

Cytokine: The term "cytokine" is used as a generic name for a diverse group of soluble proteins and peptides that act as humoral regulators at nano- to picomolar concentrations and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. These proteins also mediate interactions between cells directly and regulate processes taking place in the extracellular environment. Many cytokines act as cellular survival factors by preventing programmed cell death. Cytokines include both naturally occurring peptides and variants that retain full or partial biological activity.

Cytotoxicity: The toxicity of a molecule, such as an immunotoxin (for example an IRTA monoclonal antibody, such as a IRT A3 or IRT A5 antibody, conjugated to an effector molecule), to the cells intended to be targeted, as opposed to the cells of the rest of an organism. In contrast, the term "toxicity" refers to toxicity of an immunotoxin to cells other than those that are the cells intended to be targeted by the targeting moiety of the immunotoxin, and the term "animal toxicity" refers to toxicity of the immunotoxin to an animal by toxicity of the immunotoxin to cells other than those intended to be targeted by the immunotoxin.

Degenerate variant: A polynucleotide encoding an IRT A3 or IRT A5 polypeptide includes a sequence that is degenerate as a result of the genetic code. Similarly a polynucleotide encoding an anti-IRTA3 antibody or an anti-IRTA5 antibody includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the IRTA polypeptide or antibody encoded by the nucleotide sequence is unchanged.

Effector molecule: The portion of a chimeric molecule that is intended to have a desired effect on a cell to which the chimeric molecule is targeted. An effector molecule is also known as an effector moiety (EM), therapeutic agent, or diagnostic agent, or similar terms.

Therapeutic agents include such compounds as toxins, nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides. Alternatively, the molecule linked to a targeting moiety, such as an anti-IRTA3 antibody or anti-IRTA5 antibody, may be an encapsulation system, such as a liposome or micelle that contains a therapeutic composition such as a drug, a nucleic acid (such as an antisense nucleic acid), or another therapeutic moiety that can be shielded from direct exposure to the circulatory system. Means of preparing liposomes attached to antibodies are well known to those of skill in the art. See, for example, U.S. Patent No. 4,957,735; and Connor et al, Pharm. Ther. 28:341-365,

1985. Diagnostic agents or moieties include radioisotopes and other detectable labels. Detectable labels useful for such purposes are also well known in the art, and include radioactive isotopes such as ³² P, ¹²⁵ I, and ¹³¹ I, fluorophores, chemiluminescent agents, and enzymes. Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic. Thus, they can elicit a specific immune response. An antibody specifically binds a particular antigenic epitope on a polypeptide.

Expression: The translation of a nucleic acid into a protein. Proteins may be expressed and remain intracellular, become a component of the cell surface membrane, or be secreted into the extracellular matrix or medium

Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter. A promoter is an array of nucleic acid control sequences that directs transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. Both constitutive and inducible promoters are included (see for example, Bitter et ah, Methods in Enzymology 153:516-544, 1987).

Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue- specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included (see for example, Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (such as the metallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences.

A polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.

FoxP3: A transcription factor also known as "FKH ^sf " or "scurfin." Exemplary nucleic acids encoding FoxP3, and exemplary amino acids sequences of FoxP3 polypeptide are disclosed in published PCT Application No. 02/090600 A2, which is incorporated herein by reference. The FoxP3 transcription factor is predominately expressed by Treg cells. FoxP3 is a regulator of cytokine production and cell to cell contact dependent inhibition of T effector cell activation. Mutations in FoxP3 have been shown to be involved in scurfy mice and in humans with IPEX (Immunodysregulation, Polyendocrinopathy, and Enteropathy, X-linked). FoxP3 expression confers suppressive function to peripheral CD4+CD25+ Treg cells and/or CD127CD4+CD25+ Treg cells. In some examples, IRT A3 is expressed on FoxP3 expressing Treg cells. Host cells: Cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be

identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used. Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies. An immune response can include any cell of the body involved in a host defense response for example, an epithelial cell that secretes interferon or a cytokine.

Immunoconjugate: A covalent linkage of an effector molecule to an antibody, for example an IRTA antibody, such as an IRT A3 or IRT A5 antibody. The effector molecule can be a detectable label or an immunotoxin. Specific, non- limiting examples of toxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, or modified toxins thereof, or other toxic agents that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds that typically bring about death through liver toxicity. PE and DT, however, can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (such as domain Ia of PE and the B chain of DT) and replacing it with a different targeting moiety, such as an antibody. In one embodiment, an antibody is joined to an effector molecule (EM). In another embodiment, an antibody joined to an effector molecule is further joined to a lipid or other molecule to a protein or peptide to increase its half-life in the body. The linkage can be either by chemical or recombinant means. "Chemical means" refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) can optionally be included between the antibody and the effector molecule. Because immunoconjugates were originally prepared from two molecules with separate functionalities, such as an antibody and an effector molecule, they are also sometimes referred to as "chimeric molecules."

The term "chimeric molecule," as used herein, therefore refers to a targeting moiety, such as a ligand or an antibody, conjugated (coupled) to an effector molecule.

The terms "conjugating," "joining," "bonding" or "linking" refer to making two polypeptides into one contiguous polypeptide molecule, or to covalently attaching a radionuclide or other molecule to a polypeptide, such as an scFv. In the specific context, the terms include reference to joining a ligand, such as an antibody moiety, to an effector molecule ("EM").

Immunogenic peptide: A peptide which comprises an allele-specific motif or other sequence, such as an N-terminal repeat, such that the peptide will bind an MHC molecule and induce a cytotoxic T lymphocyte ("CTL") response, or a B cell response (such as antibody production) against the antigen from which the immunogenic peptide is derived.

In one embodiment, immunogenic peptides are identified using sequence motifs or other methods, such as neural net or polynomial determinations, known in the art. Typically, algorithms are used to determine the "binding threshold" of peptides to select those with scores that give them a high probability of binding at a certain affinity and will be immunogenic. The algorithms are based either on the effects on MHC binding of a particular amino acid at a particular position, the effects on antibody binding of a particular amino acid at a particular position, or the effects on binding of a particular substitution in a motif-containing peptide. Within the context of an immunogenic peptide, a "conserved residue" is one which appears in a significantly higher frequency than would be expected by random distribution at a particular position in a peptide. In one embodiment, a conserved residue is one where the MHC structure may provide a contact point with the immunogenic peptide. In one specific non-limiting example, an immunogenic polypeptide includes a region of IRT A3 or IRT A5, or a fragment thereof, wherein the polypeptide that is expressed on the cell surface of a host cell that expresses the full- length IRT A3 or IRT A5 polypeptide.

Immunogenic composition: A composition comprising an IRTA3 or IRTA5 polypeptide that induces a measurable CTL response against cells expressing IRT A3 or IRTA5 polypeptide or induces a measurable B cell response (such as production of antibodies) against an IRT A3 or IRT A5 polypeptide, respectively. It

further refers to isolated nucleic acids encoding an IRT A3 or IRT A5 polypeptide that can be used to express the IRT A3 or IRTA5 polypeptide (and thus be used to elicit an immune response against this polypeptide). For in vitro use, the immunogenic composition may be the isolated protein or peptide. For in vivo use, the immunogenic composition will typically comprise the protein or peptide in pharmaceutically acceptable carriers, and/or other agents. Any particular peptide, such as an IRT A3 polypeptide or an IRTA5 polypeptide or a fragment thereof, or nucleic acid encoding the polypeptide, can be readily tested for its ability to induce a CTL or B cell response by art-recognized assays. Immunogenic compositions can include adjuvants, which are well known to one of skill in the art.

Immunologically reactive conditions: Includes reference to conditions which allow an antibody raised against a particular epitope to bind to that epitope to a detectably greater degree than, and/or to the substantial exclusion of, binding to substantially all other epitopes. Immunologically reactive conditions are dependent upon the format of the antibody binding reaction and typically are those utilized in immunoassay protocols or those conditions encountered in vivo. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988) for a description of immunoassay formats and conditions. The immunologically reactive conditions employed in the methods are "physiological conditions" which include reference to conditions (for example, temperature, osmolality, pH) that are typical inside a living mammal or a mammalian cell. While it is recognized that some organs are subject to extreme conditions, the intra- organismal and intracellular environment normally lies around pH 7 (for example., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the predominant solvent, and exists at a temperature above 0 ⁰ C and below 50 ⁰ C.

Osmolality is within the range that is supportive of cell viability and proliferation.

Immunosuppression: Nonspecific unresponsiveness of cellular and/or humoral immunity. Immunosuppression refers to the prevention or diminution of an immune response and occurs when T and/or B cells are depleted in number or suppressed in their reactivity, expansion or differentiation. Immunosuppression may arise from activation of specific or non-specific Treg cells, from cytokine signaling,

in response to irradiation, or by drugs that have generalized immunosuppressive effects on T and B cells.

Immunosuppressive agent: A molecule, such as a chemical compound, small molecule, steroid, nucleic acid molecule, or other biological agent, that can decrease an immune response such as an inflammatory reaction.

Immunosuppressive agents include, but are not limited to an agent of use in treating an autoimmune disorder. Specific, non-limiting examples of immunosuppressive agents are non-steroidal anti-inflammatory agents, cyclosporine A, FK506, and anti-CD4. In additional examples, the agent is a biological response modifier, such as KINERET® (anakinra), ENBREL® (etanercept), or

REMICADE® (infliximab), a disease-modifying antirheumatic drug (DMARD), such as ARA V A® (leflunomide), a nonsteroidal anti-inflammatory drug (NSAIDs), specifically a Cyclo-Oxygenase-2 (COX-2) inhibitor, such as CELEBREX® (celecoxib) and VIOXX® (rofecoxib), or another product, such as HYALGAN® (hyaluronan) and SYNVISC® (hylan G-F20). In other embodiments, an immunosuppressive agent as an antibody that specifically binds IRT A3

Inhibiting or treating a disease: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as a tumor (for example, a leukemia or a lymphoma, such as a B cell lymphoma). "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term "ameliorating," with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of metastases, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.

Immunoglobulin superfamily receptor translocation associated (IRTA):

There are six known IRTAs, termed IRTAl, IRT A2, IRT A3, IRT A4, IRT A5 and IRT A6, five of which (IRTAl -5) are important in B cell development. Dysregulation of several IRTAs perturbs B cell immunological responses and are involved in B cell malignancy. Exemplary amino acid sequence of IRTAs, and nucleic acids encoding IRTA polypeptides are disclosed in published PCT Application No. WO 01/38490, which is incorporated herein by reference. IRTA1-6 are alternatively referred to as Fc receptor- like protein 1-6 (FCRL 1-6). However, the number designation is not identical, for example, IRTAl is referred to as FCRL4, IRT A2 is referred to as FCRL5, IRT A3 is referred to as FCRL3, IRTA4 is referred to as FCRL2, IRT A5 is referred to as FCRLl , and IRT A6 is referred to as FCRL6.

The cytoplasmic domains of the IRTAs contain consensus immunoreceptor tyrosine-based activation motifs (ITAMs) and/or immunoreceptor tyrosine -based inhibitory motifs (ITIMs). Expression of IRTAs was investigated in human tissues at the RNA level by Northern blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and by in situ hybridization (for example, see Hatzivassiliou et al., Immunity 14:277-89, 2001).

Isolated: An "isolated" biological component (such as a nucleic acid or protein (such as an antibody, for example an IRT A3 or IRT A5 antibody) or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, for example, other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. "Isolated" can also describe an isolated sample such as cells (for example T cells, such as IRTA3+ T cells) taken from a subject. Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate

detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes.

Ligand: Any molecule which specifically binds an IRT A3 protein or an IRT A5 protein and includes, inter alia, antibodies that specifically bind an IRTA3 protein or an IRTA5 protein. In alternative embodiments, the ligand is a protein or a small molecule (one with a molecular weight less than 6 kiloDaltons).

Lymphocytes: A type of white blood cell that is involved in the immune defenses of the body. There are two main types of lymphocytes: B cells and T cells. T cells are white blood cells critical to the immune response. T cells include, but are not limited to, CD4 ⁺ T cells and CD8 ⁺ T cells. A CD4 ⁺ T lymphocyte is an immune cell that carries a marker on its surface known as "cluster of differentiation 4" (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8 ⁺ T cells carry the "cluster of differentiation 8" (CD8) marker. In one embodiment, CD8 T cells are cytotoxic T lymphocytes. In another embodiment, a CD8 cell is a suppressor T cell. B cells are white blood cells critical to the antibody response. B cells mature within the bone marrow and leave the marrow expressing an antigen binding antibody on their cell surface. When a naϊve B cell encounters the antigen for which its membrane-bound antibody is specific, the cell begins to divide rapidly and its progeny differentiate into memory B cells and effector cells termed "plasma cells." Memory B cells have a longer life span and continue to express membrane- bound antibody with the same specificity as the original parent cell. Plasma cells do not produce membrane-bound antibody but instead produce the antibody in a form that can be secreted. Secreted antibodies are the major effector of humoral immunity.

Major Histocompatibility Complex or MHC: Generic designation meant to encompass the histocompatibility antigen systems described in different species, including the human leukocyte antigens ("HLA"). The term "motif refers to the pattern of residues in a peptide of defined length, usually about 8 to about 11 amino acids, which is recognized by a particular MHC allele. The peptide motifs are typically different for each MHC allele and differ in the pattern of the highly conserved residues and negative binding residues.

Neoplasia and Tumor: The product of neoplasia is a neoplasm (a tumor), which is an abnormal growth of tissue that results from excessive cell division. A tumor that does not grow into an unlimited manner to invade surrounding tissue and metastasize is referred to as "benign." A tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant." Examples of hematological tumors include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Leukemias and lymphomas are also classified by cellular origin, such as from B cells or T cells. Examples of B cell origin neoplasms include neoplasms originating from B cell precursors such as precursor B lymphoblastic leukemia and lymphoma. In other examples, the B cell origin neoplasm is from derived from mature B cells (also called peripheral B cells). Examples of mature B cell neoplasms include B cell chronic lymphocytic leukemia, small lymphocytic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B cell lymphoma, nodal marginal zone lymphoma, extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue (MALT) type, Hairy cell leukemia, plasma cell myeloma/plasmacytoma follicular lymphoma, mantle cell lymphoma, diffuse large cell B cell lymphoma, (such as mediastinal large B cell lymphoma, intravascular large B cell lymphoma, and primary effusion lymphoma), Burkitt's lymphoma/Burkitt's cell leukemia, and B cell proliferations of uncertain malignant potential such as lymphomatoid granulomatosis and post- transplant lymphoproliferative disorder.

Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer,

breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma). Nucleic acid: A polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T. "

Conventional notation is used herein to describe nucleotide sequences: the left-hand end of a single-stranded nucleotide sequence is the 5 '-end; the left-hand direction of a double-stranded nucleotide sequence is referred to as the 5'-direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand;" sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5'-end of the RNA transcript are referred to as "upstream sequences;" sequences on the DNA strand having the same sequence as the RNA

and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences."

"cDNA" refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form. "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. "Recombinant nucleic acid" refers to a nucleic acid having nucleotide sequences that are not naturally joined together and can be made by artificially combining two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. Recombinant nucleic acids include nucleic acid vectors comprising an amplified or assembled nucleic acid which can be used to transform a suitable host cell. A host cell that comprises the recombinant nucleic acid is referred to as a "recombinant host cell." The gene is then expressed in the recombinant host cell to produce, such as, a "recombinant polypeptide." A recombinant nucleic acid can also serve a non-coding function (such as, promoter, origin of replication, ribo some-binding site, etc.) as well.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter, such as the IRT A3 or IRTA5 promoter, is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence or a promoter operably linked to a nucleic acid sequence expressing an antibody that specifically binds IRTA 3 or IRT A5, such as the antibodies disclosed herein. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. Peptide: A chain of amino acids of between 3 and 30 amino acids in length.

In one embodiment, a peptide is from about 10 to about 25 amino acids in length. In yet another embodiment, a peptide is from about 11 to about 20 amino acids in length. In yet another embodiment, a peptide is about 12 amino acids in length.

An "IRT A3 peptide" is a series of contiguous amino acid residues from an IRT A3 protein, such as a fragment of IRT A3 protein of about 10 to about 25 amino acids in length, such as about 11 to about 20 amino acid in length, such as about 12 consecutive amino acids of an IRT A3 protein. In one example, with respect to immunogenic compositions comprising an IRT A3 peptide, the term further refers to variations of these peptides in which there are conservative substitutions of amino acids, so long as the variations do not alter by more than about 20% (such as no more than about 1%, about 5%, or about 10%) the ability of the peptide to produce a B cell response, or, when bound to a Major Histocompatibility Complex Class I molecule, to activate cytotoxic T lymphocytes against cells expressing wild-type IRT A3 protein. For example, induction of CTLs using synthetic peptides and CTL cytotoxicity assays are taught in U.S. Patent No. 5,662,907. Similarly, an "IRTA5 peptide" is a series of contiguous amino acid residues from an IRT A5 protein, such as a fragment of IRTA5 protein of about 10 to about 25 amino acids in length, such as about 11 to about 20 amino acid in length, such as about 12 consecutive amino acids of an IRT A5 protein. In one example, with respect to immunogenic compositions comprising an IRT A5 peptide, the term further refers to variations of these peptides in which there are conservative substitutions of amino acids, so long as the variations do not alter by more than about 20% (such as no more than about

1%, about 5%, or about 10%) the ability of the peptide to produce a B cell response, or, when bound to a Major Histocompatibility Complex Class I molecule, to activate cytotoxic T lymphocytes against cells expressing wild-type IRT A5 protein. As noted above, induction of CTLs using synthetic peptides and CTL cytotoxicity assays are taught in U.S. Patent No. 5,662,907.

"Soluble" forms of IRT A3 or IRT A5 are secreted and can bind to the antibodies disclosed herein. Soluble IRT A3 or IRTA5 may be, for example, a product resulting from alternative splicing of the IRT A3 or IRT A5 gene, respectively, or a soluble extracellular domain that has been shed from cell surface- expressed full-length IRT A3 or IRT A5.

Polypeptide: Any chain of amino acids, regardless of length or post- translational modification (for example by glycosylation or phosphorylation). In one embodiment, the polypeptide is IRTA3 polypeptide. In another embodiment, the polypeptide is an IRTA5 polypeptide. A "residue" refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic. A polypeptide has an amino terminal (N-terminal) end and a carboxy terminal end.

Pharmaceutical agent: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or a cell.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975, describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch,

or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell. In one embodiment, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation. The IRT A3 and IRT A5 polypeptides and antibodies to these peptides disclosed herein can be purified by any of the means known in the art. See, for example, Guide to Protein Purification, ed. Deutscher, Meth. Enzymol. 185, Academic Press, San Diego, 1990; and Scopes, Protein Purification: Principles and Practice, Springer Verlag, New York, 1982. Substantial purification denotes purification from other proteins or cellular components. A substantially purified protein (such as an antibody) is at least 60%, 70%, 80%, 90%, 95% or 98% pure. Thus, in one specific, non-limiting example, a substantially purified protein is 90% free of other proteins or cellular components.

Pulsatile Dose: A dose administered as a bolus. A pulsatile dose can be administered to a subject as a single administration, such as by direct injection or by an intravenous infusion during a specified time period. Thus, the pulsatile dose can be a "push" or rapid dose, but need not be, as it can be administered over a defined time period, such as in an infusion. Repeated pulsatile doses can be administered to a subject, such as a bolus administered repeatedly, such as about every one, two, or three months, or about every one, two, three or four weeks or about every one, two or three days in a therapeutic regimen. In this embodiment, the administered dose can be the same amount of an agent, or can be different amounts administered at several time points separated by periods wherein the agent is not administered to the subject, or wherein a decreased amount of the agent is administered to the subject. Regulatory T Cells (Treg): CD4+CD25+ T cells that prevent the activation and/or expansion of other cell populations, for example CD4+CD25- responder T cells. Reduction or functional alteration of Treg cells leads to the spontaneous

development of various organ-specific autoimmune diseases, including, for example, autoimmune thyroiditis, gastritis, and type 1 diabetes (see, for example, Sakaguchi et ah, J. Immunol. 155:1151-64, 1995; Suri-Payer et ah, J. Immunol. 160: 1212-18, 1998; Itoh et ah, J. Immunol. 162:5317-26, 1999). The FoxP3 transcription factor is predominantly expressed by the Treg cell lineage (Fontenot et at., Nature Immunol. 4:330-36, 2003; Hori et ah, Science 299:1057-61, 2003). There are classes of Treg cells, such as natural T regulatory (nTreg) and induced Treg (iTreg) cells. nTreg cells are selected in the thymus as an anti-self repertoire. In addition to their role in autoimmunity, they are believed to exert regulatory function in infection, in transplantation immunity as well as in tumor immunity. iTreg cells are induced in the periphery as Treg cells and hence, they are termed as induced Treg (iTreg) cells.

Responder T Cells: A subpopulation of mature T cells that facilitate an immune response through cell activation and/or the secretion of cytokines. In one embodiment, the responder T cells are CD4+CD25- T cells. In another embodiment, the responder T cells are CD8+CD25- T cells. One specific, non- limiting example of a responder T cell is a T lymphocyte that proliferates upon stimulation by antigen or a stimulator cell, such as an allogenic stimulator cell. Another specific, non-limiting example of a responder T cell is a T lymphocyte whose responsiveness to stimulation can be suppressed by Treg cells. Sample: A portion, piece, or segment that is representative of a whole. This term encompasses any material, including for instance samples obtained from a subject.

A "biological sample" is a sample obtained from a subject including, but not limited to, cells; tissues; bodily fluids, such as blood, derivatives and fractions of blood, such as serum, and lymphocytes (such as B cells, T cell, and subtractions thereof); and biopsied or surgically removed tissue, including tissues that are, for example, unfixed, frozen, fixed in formalin and/or embedded in paraffin. In particular embodiments, the biological sample is obtained from a subject, such as blood or serum. In one example a sample is an IRTA3+ T cell sample. In other examples a sample is an IRTA3+ or IRTA5+ B cell sample.

Selectively hybridize: Hybridization under moderately or highly stringent conditions that excludes non-related nucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (for example, GC versus AT content), and nucleic acid type (for example, RNA versus DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.

A specific, non-limiting example of progressively higher stringency conditions is as follows: 2 x SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2 x SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2 x SSC/0.1% SDS at about 42 ⁰ C (moderate stringency conditions); and 0.1 x SSC at about 68 ⁰ C (high stringency conditions). One of skill in the art can readily determine variations on these conditions (for example, Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Washing can be carried out using only one of these conditions, for example, high stringency conditions, or each of the conditions can be used, for example, for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.

Sequence identity: The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. For example, homologs or variants of an IRT A3 or IRT A5 polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. MoI. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic

Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et al, Nature Genet. 6: 119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. MoI. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet. Specific binding agent: An agent that binds substantially only to a defined target. Thus an IRT A3 specific binding agent is an agent that binds substantially to an IRT A3 polypeptide or antigenic fragment thereof. In one embodiment, the specific binding agent is a monoclonal or polyclonal antibody that specifically binds the IRT A3 polypeptide or antigenic fragment thereof. Similarly, an IRT A5 specific binding agent is an agent that binds specifically to an IRT A5 polypeptide or antigenic fragment thereof. In one embodiment, the specific binding agent is a monoclonal or polyclonal antibody that specifically binds the IRTA5 polypeptide or antigenic fragment thereof.

The term "specifically binds" refers, with respect to an antigen such as an IRTA (for example, IRT A3 or IRT A5), to the preferential association of an antibody or other ligand, in whole or part, with a cell or tissue bearing that antigen and not to cells or tissues lacking a detectable amount of that antigen. It is, of course, recognized that a certain degree of non-specific interaction may occur between a molecule and a non-target cell or tissue. Nevertheless, specific binding may be distinguished as mediated through specific recognition of the antigen. Although selectively reactive antibodies bind antigen, they may do so with low affinity. On the other hand, specific binding results in a much stronger association between the antibody (or other ligand) and cells bearing the antigen than between the bound antibody (or other ligand) and cells lacking the antigen. Specific binding typically results in greater than 2-fold, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody or other ligand (per unit time) to a cell or tissue bearing the IRT A3 or IRT A5 polypeptide as compared to a

cell or tissue lacking the RT A3 or IRT A5 polypeptide respectively. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats are appropriate for selecting antibodies or other ligands specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein.

Targeting moiety: A portion of a chimeric molecule intended to provide the molecule with the ability to bind specifically to the IRT A3 or IRT A5 polypeptide. A "ligand" is a targeting molecule specific for the IRT A3 or IRT A5 polypeptide and is generally synonymous with "targeting moiety." An antibody , such as an antibody that specifically binds IRT A3 or ITRA5, is a version of a targeting moiety.

Therapeutically effective amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit or suppress growth of a tumor. In one embodiment, a therapeutically effective amount is the amount necessary to eliminate a tumor. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in tumors) that has been shown to achieve a desired in vitro effect. Toxin: A molecule that is cytotoxic for a cell. Toxins include abrin, ricin,

Pseudomonas exotoxin (PE), diphtheria toxin (DT), botulinum toxin, saporin, restrictocin or gelonin, or modified toxins thereof. For example, PE and DT are highly toxic compounds that typically bring about death through liver toxicity. PE and DT, however, can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (for example, domain Ia of PE or the B chain of DT) and replacing it with a different targeting moiety, such as an antibody.

Transduced: A transduced cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term transduction encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation

with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art.

///. Specific Embodiments Antibodies that bind IRT A3 or IRT A5

Antibodies are disclosed herein that bind immunoglobulin (Ig) superfamily receptor translocation associated 3 (IRT A3, also known as FCRH3) or that bind immunoglobulin superfamily receptor translocation associated 5 (IRT A5 also known as FCRHl). Monoclonal antibodies are disclosed herein that bind IRT A3, such as antibodies that specifically bind the extracellular domain of IRT A3. In one example, IRTA3 has an amino acid sequence set forth as: MLLWLLLLIL TPGREQSGVA PKAVLLLNP P WS TAFKGEKV AL I C S S I S HS

LAQGDTYWYH DEKLLKIKHD KIQITEPGNY QCKTRGSSLS DAVHVEFSPD WLILQALHPV FEGDNVILRC QGKDNKNTHQ KVYYKDGKQL PNSYNLEKIT

VNSVSRDNSK YHCTAYRKFY ILDIEVTSKP LNIQVQELFL HPVLRASSST

PIEGSPMTLT CETQLSPQRP DVQLQFSLFR DSQTLGLGWS RSPRLQIPAM

WTEDSGSYWC EVETVTHSIK KRSLRSQIRV QRVPVSNVNL EIRPTGGQLI

EGENMVLICS VAQGSGTVTF SWHKEGRVRS LGRKTQRSLL AELHVLTVKE SDAGRYYCAA DNVHSPILST WIRVTVRIPV SHPVLTFRAP RAHTVVGDLL

ELHCESLRGS PPILYRFYHE DVTLGNSSAP SGGGASFNLS LTAEHSGNYS

CDADNGLGAQ HSHGVSLRVT VPVSRPVLTL RAPGAQAVVG DLLELHCESL

RGSFPILYWF YHEDDTLGNI SAHSGGGASF NLSLTTEHSG NYSCEADNGL

GAQHSKVVTL NVTGTSRNRT GLTAAGITGL VLSILVLAAA AALLHYARAR RKPGGLSATG TSSHSPSECQ EPSSSRPSRI DPQEPTHSKP LAPMELEPMY

SNVNPGDSNP IYSQIWSIQH TKENSANCPM MHQEHEELTV LYSELKKTHP

DDSAGEASSR GRAHEEDDEE NYENVPRVLL ASDH (SEQ ID NO: 1)

Monoclonal antibodies are disclosed herein that bind IRT A5, such as antibodies that specifically bind the extracellular domain of IRTA5. In one example, IRTA5 has an amino acid sequence set forth as: MLPRLLLLIC APLCEPAELF LIASPSHPTE GSPVTLTCKM PFLQSSDAQF QFCFFRDTRA LGPGWSSSPK LQIAAMWKED TGSYWCEAQT MASKVLRSRR SQINVHRVPV ADVSLETQPP GGQVMEGDRL VLICSVAMGT GDITFLWYKG

AVGLNLQSKT QRSLTAEYEI PSVRESDAEQ YYCVAENGYG PSPSGLVSIT

VRIPVSRPIL MLRAPRAQAA VEDVLELHCE ALRGSPPILY WFYHEDITLG SRSAPSGGGA SFNLSLTEEH SGNYSCEANN GLGAQRSEAV TLNFTVPTGA

RSNHLTSGVI EGLLSTLGPA TVALLFCYGL KRKIGRRSAR DPLRSLPSPL

PQEFTYLNSP TPGQLQPIYE NVNVVSGDEV YSLAYYNQPE QESVAAETLG

THMEDKVSLD IYSRLRKANI TDVDYEDAM (SEQ ID NO: 2) .

The amino acid sequences of the IRTA proteins are well known in the art, and are provided in published PCT Application No. WO 01/39490, which is incorporated herein by reference. In one example, an antibody that specifically binds IRT A3 does not specifically bind IRTAl, IRT A2, IRT A4, IRT A5 and/or IRTA6. Thus, in one embodiment, the antibodies disclosed herein can be used to differentiate the expression of IRT A3 from IRTAl , IRT A2, IRT A4, IRT A5 and/or IRT A6. In another example, an antibody that specifically binds IRT A5 does not specifically bind IRTAl, IRTA2, IRT A3, IRT A4 and/or IRT A6. Thus in another embodiment, the antibodies disclosed herein can be used to differentiate the expression of IRTA5 from IRTAl, IRT A2, IRT A3, IRTA4 and/or IRT A6. Exemplary amino acid sequences of IRTAl, IRT A2, IRT A4, and IRT A6 are given below. IRTAl :

MLLWASLLAF APVCGQSAAA HKPVISVHPP WTTFFKGERV TLTCNGFQFY ATEKTTWYHR HYWGEKLTLT PGNTLEVRES GLYRCQARGS PRSNPVRLLF SSDSLILQAP YSVFEGDTLV LRCHRRRKEK LTAVKYTWNG NILSISNKSW DLLIPQASSN NNGNYRCIGY GDENDVFRSN FKIIKIQELF PHPELKATDS

QPTEGNSVNL SCETQLPPER SDTPLHFNFF RDGEVILSDW STYPELQLPT

VWRENSGSYW CGAETVRGNI HKHSPSLQIH VQRIPVSGVL LETQPSGGQA

VEGEMLVLVC SVAEGTGDTT FSWHREDMQE SLGRKTQRSL RAELELPAIR

QSHAGGYYCT ADNSYGPVQS MVLNVTVRET PGNRDGLVAA GATGGLLSAL

LLAVALLFHC WRRRKSGVGF LGDETRLPPA PGPGESSHSI CPAQVELQSL YVDVHPKKGD LVYSEIQTTQ LGEEEEANTS RTLLEDKDVS VVYSEVKTQH

PDNSAGKISS KDEES (SEQ ID NO: 3)

IRTA2:

MLLWVILLVL APVSGQFART PRPIIFLQPP WTTVFQGERV TLTCKGFRFY SPQKTKWYHR YLGKEILRET PDNILEVQES GEYRCQAQGS PLSSPVHLDF

SSASLILQAP LSVFEGDSVV LRCRAKAEVT LNNTIYKNDN VLAFLNKRTD

FHIPHACLKD NGAYRCTGYK ESCCPVSSNT VKIQVQEPFT RPVLRASSFQ

PISGNPVTLT CETQLSLERS DVPLRFRFFR DDQTLGLGWS LSPNFQITAM

WSKDSGFYWC KAATMPHSVI SDSPRSWIQV QIPASHPVLT LSPEKALNFE GTKVTLHCET QEDSLRTLYR FYHEGVPLRH KSVRCERGAS ISFSLTTENS

GNYYCTADNG LGAKPSKAVS LSVTVPVSHP VLNLSSPEDL IFEGAKVTLH

CEAQRGSLPI LYQFHHEDAA LERRSANSAG GVAISFSLTA EHSGNYYCTA

DNGFGPQRSK AVSLSITVPV SHPVLTLSSA EALTFEGATV TLHCEVQRGS

PQILYQFYHE DMPLWSSSTP SVGRVSFSFS LTEGHSGNYY CTADNGFGPQ RSEVVSLFVT VPVSRPILTL RVPRAQAVVG DLLELHCEAP RGSPPILYWF

YHEDVTLGSS SAPSGGEASF NLSLTAEHSG NYSCEANNGL VAQHSDTISL

SVIVPVSRPI LTFRAPRAQA VVGDLLELHC EALRGSSPIL YWFYHEDVTL

GKISAPSGGG ASFNLSLTTE HSGIYSCEAD NGLEAQRSEM VTLKVAGEWA LPTSSTSEN(SEQ ID NO: 4)

IRTA4:

MLLWSLLVIF DAVTEQADSL TLVAPSSVFE GDSIVLKCQG EQNWKIQKMA

YHKDNKELSV FKKFSDFLIQ SAVLSDSGNY FCSTKGQLFL WDKTSNIVKI

KVQELFQRPV LTASSFQPIE GGPVSLKCET RLSPQRLDVQ LQFCFFRENQ VLGSGWSSSP ELQISAVWSE DTGSYWCKAE TVTHRIRKQS LQSQIHVQRI

PISNVSLEIR APGGQVTEGQ KLILLCSVAG GTGNVTFSWY REATGTSMGK

KTQRSLSAEL EIPAVKESDA GKYYCRADNG HVPIQSKVVN IPVRIPVSRP

VLTLRSPGAQ AAVGDLLELH CEALRGSPPI LYQFYHEDVT LGNSSAPSGG

GASFNLSLTA EHSGNYSCEA NNGLGAQCSE AVPVSISGPD GYRRDLMTAG VLWGLFGVLG FTGVALLLYA LFHKISGESS ATNEPRGASR PNPQEFTYSS

PTPDMEELQP VYVNVGSVDV DVVYSQVWSM QQPESSANIR TLLENKDSQV IYSSVKKS (SEQ ID NO: 5)

IRTA6:

MLLWTAVLLF VPCVGKTVWL YLQAWPNPVF EGDALTLRCQ GWKNTPLSQV

KFYRDGKFLH FSKENQTLSM GAATVQSRGQ YSCSGQVMYI PQTFTQTSET

AMVQVQELFP PPVLSAIPSP EPREGSLVTL RCQTKLHPLR SALRLLFSFH KDGHTLQDRG PHPELCIPGA KEGDSGLYWC EVAPEGGQVQ KQSPQLEVRV

QAPVSRPVLT LHHGPADPAV GDMVQLLCEA QRGSPPILYS FYLDEKIVGN

HSAPCGGTTS LLFPVKSEQD AGNYSCEAEN SVSRERSEPK KLSLKGSQVL FTPASNWLVP WLPASLLGLM VIAAALLVYV RSWRKAGPLP SQIPPTAPGG EQCPLYANVH HQKGKDEGVV YSVVHRTSKR SEARSAEFTV GRKDSSIICA EVRCLQPSEV SSTEVNMRSR TLQEPLSDCE EVLC (SEQ ID NO: 6)

Disclosed herein are the amino acid sequences of murine monoclonal antibodies that specifically bind IRT A5 (anti-IRTA5 antibodies). These monoclonal antibodies each include a variable heavy (V _R ) and a variable light (V _L ) chain and specifically bind IRT A5. For example, the antibodies can specifically bind IRT A5 with an affinity constant of at least 10 ⁷ M ^"1 , such as at least 10 ⁸ M ^"1 , at least 5 X 10 ⁸ M ^"1 , or at least 10 ⁹ M ^"1 .

In one embodiment, the anti-IRTA5 antibody is E3, and includes a V _R polypeptide including an amino acid sequence set forth as EVQLQESGPS LVKPSQTLSL TCSVTGDSIT SGϊWTWIRKF PGNKLEYMGγ ISYSGSTYYN

PSLKSRI S I T RDTSKNQYYL QLNSVTTEDT ATYYCARSYY TGAfDSWGQGT SVTVS S ( SEQ I D NO : 7 ) and a V _L polypeptide including an amino acid sequence set forth as DIVLTQS PASL AVSLGQRAT I S CRASESVDS YGNSFMIMYQ QKPGQPPRLL I FRASNLESG I PARFS GS GS RT DFTLT INP VEADDVATYY CQQSNEDPYT FGGGTKLE I K ( SEQ I D NO : 8 ) . The amino acid sequences of the CDRs from the E3 antibody are shown as bolded italics.

In another embodiment, the anti-IRTA5 antibody is E9, and includes a V _H polypeptide including an amino acid set forth as EVKLEESGGG LVQPGGSMKL SCAASGFTFG DAWMDWVRQS PEKGLEWVAE IRSKANNHAT YYAESVKGRF TI SRDDSKSS VYLQMNSLRV EDTGI YYCTG GNYWFVYWGQ GTLVTVSA ( SEQ ID NO : 9 ) and a V _L polypeptide including an amino acid sequence set forth as DIVMTQSHKF MS T SVGDRVS ITCKASβDVG SIVAW YQQN P GQSPKLLIYW ASTRHTGVPD RFTGSGSGTD FILTI SNVQS EDLADYFCββ rSSYPFTFGS GTKLEIK ( SEQ ID NO : 10 ) . The amino acid sequences of the CDRs from the E9 antibody are shown as bolded italics. The amino acid sequences of the V _H and V _L of E3 and E9 are also set forth in Fig. 17. Functional fragments of the E3 and E9 antibodies and humanized forms of the E3 and E9 antibodies are also disclosed (see below).

In another embodiment, the anti-IRTA5 antibody is E3, which is produced by a hybridoma deposited in accordance with the Budapest Treaty July 11 , 2006 as American Type Culture Collection (ATCC) Deposit No. PTA-7723, or is a humanized form thereof or functional fragment thereof. Hybridoma cells and their progeny that secrete the monoclonal antibodies E3 and E9 are also disclosed.

In several embodiments, the antibody includes a V _H polypeptide having an amino acid sequence at least about 90% identical, such as at least about 95% identical, at least about 98% identical, at least about 99% identical or even 100% identical, to the amino acid sequence set forth as SEQ ID NO: 7 and a V _L polypeptide having an amino acid sequence at least about 90% identical, such as at least about 95% identical, at least about 98% identical, at least about 99% identical or even 100% identical, to the amino acid sequence set forth as SEQ ID NO: 8. In another embodiment, the monoclonal antibody includes a V _H polypeptide having an amino acid sequence at least about 90% identical, such as at least about 95% identical, at least about 98% identical, at least about 99% identical or even 100% identical, to the amino acid sequence set forth as SEQ ID NO: 9 and a V _L polypeptide having an amino acid sequence at least about 90% identical, such as at least about 95% identical, at least about 98% identical, at least about 99% identical or even 100% identical, to the amino acid sequence set forth as set forth a SEQ ID NO: 10.

Also disclosed herein is the murine monoclonal antibody H5 that specifically binds IRT A3 (anti-IRTA3 antibody). The anti-IRTA3 antibody H5 is produced by a hybridoma that was deposited in accordance with the Budapest Treaty on July 11, 2006 as American Type Culture Collection (ATCC) Deposit No. PTA-7724, or is a humanized form thereof or a functional fragment thereof.

Hybridoma cells and their progeny that secrete the monoclonal antibody H5 are also encompassed by this disclosure. The H5 monoclonal antibody includes a variable heavy (V _H ) and a variable light (V _L ) chain and specifically binds IRT A3. For example, the H5 antibody can specifically bind IRT A3 with an affinity constant of at least 10 ⁷ M ^"1 , such as at least 10 ⁸ M ^"1 , at least 5 X 10 ⁸ M ^"1 , or at least 10 ⁹ M ^"1 . The production of chimeric antibodies, which include a framework region from one antibody and the CDRs from a different antibody, is well known in the art. Thus humanized antibodies that specifically bind IRT A3 or IRT A5 are disclosed. In some embodiments, a humanized antibody that specifically binds IRTA3 is a humanized form of the H5 monoclonal antibody of a functional fragment thereof. In some embodiments, a humanized antibody that specifically binds IRTA5 is a humanized form of the E3 or E9 monoclonal antibody of a functional fragment thereof. The antibody or antibody fragment can be a humanized immunoglobulin having complementarity determining regions (CDRs) from a donor monoclonal antibody that binds IRT A5 (such as E3 or E9, see Table 1) or an antibody that binds IRT A3 (such as those from H5, produced by the above-described hybridoma deposited with ATCC) and immunoglobulin and heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light chain frameworks. Generally, the humanized immunoglobulin specifically binds to IRT A3 or specifically binds to IRT A5 with an affinity constant of at least 10 ⁷ M ^"1 , such as at least 10 ⁸ M ^"1 at least 5 X 10 ⁸ M ^"1 or at least 10 ⁹ M ^"1 . The location of the CDRs for the E3 and E9 are set forth in Table 1 below: Table 1

Humanized monoclonal antibodies can be produced by transferring donor CDRs from heavy and light variable chains of the donor mouse immunoglobulin (such as H5, E3, or E9) into a human variable domain, and then substituting human residues in the framework regions when required to retain affinity. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of the constant regions of the donor antibody. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321 :522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al, Proc. Nat'lAcad. Sd. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev. Biotech.12:437, 1992; and Singer et al., J. /mmw/rø/.150:2844, 1993. The antibody may be of any isotype, but in several embodiments the antibody is an IgG, including but not limited to, IgG ₁ , IgG ₂ , IgG ₃ and IgG ₄ . In one embodiment, the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Thus, the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 75%, at least about 85%, at least about 95%, or at least about 99% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. The sequences of the heavy and light chain frameworks are known in the art. Human framework regions, and mutations that can be made in a

humanized antibody framework regions, are known in the art (see, for example, in U.S. Patent No. 5,585,089, which is incorporated herein by reference).

Exemplary human antibodies LEN and 21/28 CL are of use in providing framework regions. Exemplary light chain frameworks of human MAb LEN have the following sequences:

FRl : DIVMTQS PDSLAVSLGERATINC (SEQ ID NO: 11)

FR2: WYQQKPGQPPLLIY (SEQ ID NO: 12)

FR3: GVPDRPFGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 13)

FR4: FGQGQTKLEIK (SEQ ID NO: 14) Exemplary heavy chain frameworks of human MAb 21/28' CL have the following sequences:

FRl : QVQLVQSGAEVKKPQASVKVSCKASQYTFT (SEQ ID NO: 15)

FR2: WVRQAPGQRLEWMG (SEQ ID NO: 16)

FR3: RVTITRDTSASTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 17) FR4: WGQGTLVTVSS (SEQ ID NO: 18).

Antibodies, such as murine monoclonal antibodies, chimeric antibodies, and humanized antibodies, include full length molecules as well as fragments thereof, such as Fab, F(ab') ₂ , and Fv which include a heavy chain and light chain variable region and are capable of binding the epitopic determinant. In some embodiments, the antibodies have V _L and V _H regions having the amino acid sequences shown above (for example, see SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10 or the sequences for V _L and V _H regions for antibodies H5, see above). Fv antibodies are typically about 25 kDa and contain a complete antigen-binding site with three CDRs per each heavy chain and each light chain. To produce these antibodies, the V _H and the V _L can be expressed from two individual nucleic acid constructs in a host cell. If the V _H and the V _L are expressed non-contiguously, the chains of the Fv antibody are typically held together by noncovalent interactions. However, these chains tend to dissociate upon dilution, so methods have been developed to crosslink the chains through glutaraldehyde, intermolecular disulfides, or a peptide linker. Thus, in one example, the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chain variable region and the light chain variable region are chemically linked by disulfide bonds.

In an additional example, the Fv fragments comprise V _H and V _L chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the V _H and V _L domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are known in the art (see Whitlow et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al., Science 242:423, 1988; U.S. Patent No. 4,946,778; and Pack et al., Bio/Technology 11 :1271, 1993).

Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5 S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Patent No. 4,036,945 and U.S. Patent No. 4,331,647, and references contained therein; Nisonhoff et al., Arch. Biochem. Biophys.%9:230, 1960; Porter, Biochem. J. 73: 119, 1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4). Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

One of skill will realize that conservative variants of the antibodies can be produced. Such conservative variants employed in antibody fragments, such as dsFv fragments or in scFv fragments, will retain critical amino acid residues necessary for correct folding and stabilizing between the V _H and the V _L regions, and will retain the

charge characteristics of the residues in order to preserve the low pi and low toxicity of the molecules. Amino acid substitutions (such as at most one, at most two, at most three, at most four, or at most five amino acid substitutions) can be made in the V _H and the V _L regions to increase yield. Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Thus, one of skill in the art can readily review SEQ ID NOs: 7-10, or the sequences of the H5 antibody, locate one or more of the amino acids in the brief table above, identify a conservative substitution, and produce the conservative variant using well- known molecular biology techniques. Generally, conservative variants will bind the target antigen with an equal to or greater efficiency than the parent monoclonal antibody. Effector molecules, such as therapeutic, diagnostic, or detection moieties can be linked to an antibody that specifically binds IRT A3 or to an antibody that specifically binds IRT A5, using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used. The procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector. Polypeptides typically contain a variety of functional groups (such as carboxylic acid (COOH), free amine (-NH ₂ ) or sulfhydryl (-SH) groups) which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to join the antibody to the effector molecule. The linker is

capable of forming covalent bonds to both the antibody and to the effector molecule. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the effector molecule from the antibody when the immunoconjugate has reached its target site. Therefore, in these circumstances, immunoconjugates will comprise linkages that are cleavable in the vicinity of the target site. Cleavage of the linker to release the effector molecule from the antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site. When the target site is a tumor, a linker which is cleavable under conditions present at the tumor site (for example when exposed to tumor-associated enzymes or acidic pH) may be used.

In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, label (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide. In some examples a label is a detectable label such as a fluorophore (for example FTIC, PE and the like), an enzyme (for example HRP), a radiolabel, or a nanoparticle (for example a gold particle or a semiconductor nanocrystal, such as a quantum dot (QDOT®)). Therapeutic agents include various drugs such as vinblastine, daunomycin and the like, and effector molecules such as cytotoxins such as native or modified Pseudomonas exotoxin or Diphtheria toxin, encapsulating agents, (such as, liposomes) which themselves contain pharmacological compositions, target moieties and ligands. The choice of a particular therapeutic agent depends on the particular target molecule or cell and the biological effect desired to be evoked. Thus, for example, the therapeutic agent may be an effector molecule that is cytotoxic which is used to bring about the death of a particular target cell. Conversely, where it is

merely desired to invoke a non-lethal biological response, a therapeutic agent can be conjugated to a non-lethal pharmacological agent or a liposome containing a non- lethal pharmacological agent.

Immunotoxins are chimeric molecules (such as a recombinant immunotoxins) in which a cell targeting moiety is fused to a toxin (see for example Pastan et al, Science, 254: 1173-1177, 1991). If the cell targeting moiety is an antibody, the molecule is termed a recombinant immunotoxin. The toxin moiety is typically genetically altered so that it cannot bind to the toxin receptor present on most normal cells. Thus, immunotoxins, such as recombinant immunotoxins, selectively kill cells which are recognized by the antigen binding domain.

Toxins can be employed with an antibody that specifically binds IRT A3 or an antibody that specifically binds IRT A5 and fragments of these antibodies, such as a svFv or a dsFv, to yield chimeric molecules, which are of use as immunotoxins. Exemplary toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and subunits thereof, ribotoxin, ribonuclease, saporin, and calicheamicin, as well as botulinum toxins A through F. These toxins are well known in the art and many are readily available from commercial sources (for example, Sigma Chemical Company, St. Louis, MO).

Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins is mutated to reduce or to eliminate nonspecific toxicity. A mutant known as CRM 107, which has full enzymatic activity but markedly reduced non-specific toxicity, has been known since the 1970's (Laird and Groman, J. Virol. 19:220, 1976), and has been used in human clinical trials. See, U.S. Patent No. 5,792,458 and U.S. Patent No. 5,208,021. As used herein, the term "diphtheria toxin" refers as appropriate to native diphtheria toxin or to diphtheria toxin that retains enzymatic activity but which has been modified to reduce non-specific toxicity.

Ricin is the lectin RCA60 from Ricinus communis (Castor bean). The term "ricin" also references toxic variants thereof. For example, see, U.S. Patent No. 5,079,163 and U.S. Patent No. 4,689,401. Ricinus communis agglutinin (RCA) occurs in two forms designated RCA ₆ O and RCA ₁₂ O according to their molecular weights of approximately 65 and 120 kD, respectively (Nicholson & Blaustein, J.

Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing cells. The B chain binds ricin to cell-surface galactose residues and facilitates transport of the A chain into the cytosol (Olsnes et al, Nature 249:627-631, 1974 and U.S. Patent No. 3,060,165). Ribonucleases have also been conjugated to targeting molecules for use as immunotoxins (see Suzuki et al, Nat. Biotech. 17:265-70, 1999). Exemplary ribotoxins such as α-sarcin and restrictocin are discussed in, for example, Rathore et al, Gene 190:31-5, 1997; and Goyal and Batra, Biochem 345 Pt 2:247-54, 2000. Calicheamicins were first isolated from Micromonospora echinospora and are members of the enediyne antitumor antibiotic family that cause double strand breaks in DNA that lead to apoptosis (see, for example, Lee et al, J. Antibiot 42:1070-87. 1989). The drug is the toxic moiety of an immunotoxin in clinical trials (see, for example, Gillespie et al, Ann Oncol 11 :735-41, 2000).

Abrin includes toxic lectins from Abrus precatorius . The toxic principles, abrin a, b, c, and d, have a molecular weight of from about 63 and 67 kD and are composed of two disulfide-linked polypeptide chains A and B. The A chain inhibits protein synthesis; the B chain (abrin-b) binds to D-galactose residues (see, Funatsu et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978). In one embodiment, the toxin is Pseudomonas exotoxin (PE). Native

Pseudomonas exotoxin A ("PE") is an extremely active monomeric protein (molecular weight 66 kD), secreted by Pseudomonas aeruginosa, which inhibits protein synthesis in eukaryotic cells. The native PE sequence and the sequence of modified PE are provided in U.S. Patent No. 5,602,095, incorporated herein by reference. In one embodiment, native PE has a sequence set forth as:

AEEAFDLWNE CAKACVLDLK DGVRSSRMSV DPAIADTNGQ GVLHYSMVLE GGNDALKLAI DNALSITSDG LTIRLEGGVE PNKPVRYSYT RQARGSWSLN WLVPIGHEKP SNIKVFIHEL NAGNQLSHMS PIYTIEMGDE LLAKLARDAT FFVRAHESNE MQPTLAISHA GVSVVMAQTQ PRREKRWSEW ASGKVLCLLD PLDGVYNYLA QQRCNLDDTW EGKIYRVLAG NPAKHDLDIK PTVISHRLHF PEGGSLAALT AHQACHLPLE TFTRHRQPRG WEQLEQCGYP VQRLVALYLA

ARLSWNQVDQ VIRNALASPG SGGDLGEAIR EQPEQARLAL TLAAAESERF VRQGTGNDEA GAANADVVSL TCPVAAGECA GPADSGDALL ERNYPTGAEF LGDGGDVSFS TRGTQNWTVE RLLQAHRQLE ERGYVFVGYH GTFLEAAQSI VFGGVRARSQ DLDAIWRGFY IAGDPALAYG YAQDQEPDAR GRIRNGALLR VYVPRSSLPG FYRTSLTLAA PEAAGEVERL IGHPLPLRLD AITGPEEEGG RLETILGWPL AERTVVIPSA IPTDPRNVGG DLDPSSIPDK EQAISALPDY ASQPGKPPRE DLK (SEQ ID NO: 19) .

The method of action of PE is inactivation of the ADP-ribosylation of elongation factor 2 (EF-2). The exotoxin contains three structural domains that act in concert to cause cytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding. Domain II (amino acids 253-364) is responsible for translocation into the cytosol and domain III (amino acids 400-613) mediates ADP ribosylation of elongation factor 2. The function of domain Ib (amino acids 365-399) remains undefined, although a large part of it, amino acids 365-380, can be deleted without loss of cytotoxicity. See Siegall et ah, J. Biol. Chem. 264:14256-14261, 1989.

The term "Pseudomonas exotoxin" ("PE") as used herein refers as appropriate to a full-length native (naturally occurring) PE or to a PE that has been modified. Such modifications may include, but are not limited to, elimination of domain Ia, various amino acid deletions in domains Ib, II and III, single amino acid substitutions and the addition of one or more sequences at the carboxyl terminus, such as KDEL (SEQ ID NO: 20) and REDL (SEQ ID NO: 21), see Siegall et al, supra. In several examples, the cytotoxic fragment of PE retains at least 50%, preferably 75%, more preferably at least 90%, and most preferably 95% of the cytotoxicity of native PE. In one embodiment, the cytotoxic fragment is more toxic than native PE.

Thus, the PE used in the immunotoxins disclosed herein includes the native sequence, cytotoxic fragments of the native sequence, and conservatively modified variants of native PE and its cytotoxic fragments. Cytotoxic fragments of PE include those which are cytotoxic with or without subsequent proteolytic or other processing in the target cell (for example as a protein or pre-protein). Cytotoxic fragments of PE known in the art include PE40, PE38, and PE35.

In several embodiments, the PE has been modified to reduce or eliminate non-specific cell binding, typically by deleting domain Ia, as taught in U.S. Patent No. 4,892,827, although this can also be achieved, for example, by mutating certain residues of domain Ia. U.S. Patent No. 5,512,658, for instance, discloses that a mutated PE in which Domain Ia is present but in which the basic residues of domain Ia at positions 57, 246, 247, and 249 are replaced with acidic residues (glutamic acid, or "E") exhibits greatly diminished non-specific cytotoxicity. This mutant form of PE is sometimes referred to as PE4E.

PE40 is a truncated derivative of PE (see, Pai et al., Proc. Natl Acad. Sci. USA 88:3358-62, 1991; and Kondo et al., J. Biol. Chem. 263:9470-9475, 1988).

PE35 is a 35 kD carboxyl-terminal fragment of PE in which amino acid residues 1- 279 have been deleted and the molecule commences with a met at position 280 followed by amino acids 281-364 and 381-613 of native PE. PE35 and PE40 are disclosed, for example, in U.S. Patent No. 5,602,095 and U.S. Patent No. 4,892,827. In some embodiments, the cytotoxic fragment PE38 is employed. PE38 is a truncated PE pro-protein composed of amino acids 253-364 and 381-613 of SEQ ID NO: 19 which is activated to its cytotoxic form upon processing within a cell (see for example, U.S. Patent No. 5,608,039, and Pastan et al., Biochim. Biophys. Acta 1333:C1-C6, 1997). While in some embodiments, the PE is PE4E, PE40, or PE38, any form of

PE in which non-specific cytotoxicity has been eliminated or reduced to levels in which significant toxicity to non-targeted cells does not occur can be used in the immunotoxins disclosed herein so long as it remains capable of translocation and EF-2 ribosylation in a targeted cell. Conservatively modified variants of PE or cytotoxic fragments thereof have at least 80% sequence similarity, preferably at least 85% sequence similarity, more preferably at least 90% sequence similarity, and most preferably at least 95% sequence similarity at the amino acid level, with the PE of interest, such as PE38. Nucleic acids encoding the amino acid sequences of the IRT A3 and IRT A5 antibodies and immunotoxins are provided herein. For example, an exemplary nucleic acid sequence encoding the E3 V _H is provided as

GAGGTGCAGC TTCAGGAGTC AGGACCTAGC CTCGTGAAAC CTTCTCAGAC TCTGTCCCTC ACCTGTTCTG TCACTGGCGA CTCCATCACC AGTGGTTACT

GGACCTGGAT CCGGAAATTC CCAGGGAATA AACTTGAGTA CATGGGGTAC ATAAGCTACA GTGGTAGCAC TTACTACAAT CCATCTCTCA AAAGTCGAAT CTCCATCACT CGAGACACAT CCAAGAACCA GTACTACCTG CAGTTGAATT CTGTGACTAC TGAGGACACA GCCACATATT ACTGTGCAAG ATCCTATTAC TATGGTATGG ACTCCTGGGG TCAAGGAACC TCAGTCACCG TCTCCTCA (SEQ ID NO: 22 ), an exemplary nucleic acid sequence encoding E3 V _L is provided as

GACATTGTGC TGACCCAATC TCCAGCTTCT TTGGCTGTGT CTCTAGGGCA GAGGGCCACC ATATCCTGCA GAGCCAGTGA AAGTGTTGAT AGTTATGGCA ATAGTTTTAT GCACTGGTAC CAGCAGAAAC CAGGACAGCC ACCCAGACTC CTCATCTTTC GTGCATCCAA CCTAGAATCT GGGATCCCTG CCAGGTTCAG TGGCAGTGGG TCTAGGACAG ACTTCACCCT CACCATTAAT CCTGTGGAGG CTGATGATGT TGCAACCTAT TACTGTCAGC AAAGTAATGA GGATCCGTAC ACGTTCGGAG GGGGGACCAA GCTGGAAATA AAA (SEQ ID NO: 23), an exemplary nucleic acid sequence encoding the E9 V _H is provided as

GAAGTGAAGC TTGAGGAGTC TGGAGGAGGC TTGGTGCAAC CTGGAGGATC CATGAAACTC TCTTGTGCTG CCTCTGGATT CACTTTTGGT GACGCCTGGA TGGACTGGGT CCGCCAGTCT CCAGAGAAGG GGCTTGAGTG GGTTGCTGAA ATTAGAAGCA AAGCTAATAA TCATGCAACA TACTATGCTG AGTCTGTGAA AGGGAGGTTC ACCATTTCAA GAGATGATTC CAAAAGTAGT GTCTACCTGC AAATGAACAG CTTAAGAGTT GAAGACACTG GCATTTATTA CTGTACGGGG GGTAACTACT GGTTTGTTTA CTGGGGCCAA GGGACTCTGG TCACTGTCTC TGCA (SEQ ID NO: 24), and an exemplary nucleic acid sequence encoding E9 V _L is provided as

GACATTGTGA TGACCCAGTC TCACAAATTC ATGTCCACAT CTGTAGGAGA

CAGGGTCAGC ATCACCTGCA AGGCCAGTCA GGATGTGGGT AGTACTGTAG

CCTGGTATCA ACAGAATCCA GGACAATCTC CTAAACTACT GATTTACTGG

GCATCCACCC GGCACACTGG AGTCCCTGAT CGCTTCACAG GCAGTGGGTC TGGGACAGAT TTCATTCTCA CCATTAGTAA TGTGCAGTCT GAAGACTTGG

CAGATTATTT CTGTCAGCAA TATAGCAGTT ATCCATTCAC GTTCGGCTCG GGGACAAAGT TGGAAATAAA A (SEQ ID NO: 25). Other nucleic acids encoding antibodies including one ormore CDRs from E3 or E9 can readily be produced by one of skill in the art, using the amino acid sequences provided herein, and the genetic code. Nucleic acids encoding the amino acid sequence of the antibody produced by H5 can be readily determined by one of skill in the art. In addition, one of skill can readily construct a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same effector molecule ("EM") or antibody sequence or nucleic acid sequences that differ due to the degeneracy of the genetic code. Thus, nucleic acids encoding antibodies, conjugates and fusion proteins are provided herein.

Nucleic acid sequences encoding the antibodies and/or immunotoxins can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al. , Meth. Enzymol. 68:109- 151, 1979; the diethylphosphoramidite method of Beaucage et al, Tetra. Lett. 22:1859-1862, 1981; the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra. Letts. 22(20): 1859-1862, 1981, using an automated synthesizer as described in, for example, Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method of U.S. Patent No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill would recognize that while chemical synthesis of DNA is generally limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.

Exemplary nucleic acids encoding sequences encoding an antibody that specifically binds IRT A3 or an antibody that specifically binds IRT A5, such as an immunotoxin, can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook et al., supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, MO), R&D SYSTEMS® (Minneapolis, MN), Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), ChemGenes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemika-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), INVITROGEN™ (San Diego, CA), and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to one of skill.

Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill.

In one example, an antibody of use is prepared by inserting into a vector the cDNA which encodes a variable region from an antibody that specifically binds IRT A3 or an antibody that specifically binds IRT A5. In some examples the cDNA which encodes a variable region from an antibody that specifically binds IRT A3 or an antibody that specifically binds IRT A5 is inserted into a vector which comprises the cDNA encoding an effector molecule (EM). The insertion is made so that the variable region and the EM are read in frame so that one continuous polypeptide is produced. Thus, the encoded polypeptide contains a functional Fv region and a functional EM region. In one embodiment, cDNA encoding a cytotoxin is ligated to a scFv so that the cytotoxin is located at the carboxyl terminus of the scFv. In one example, cDNA encoding a Pseudomonas exotoxin ("PE"), mutated to eliminate or to reduce non-specific binding, is ligated to a scFv so that the toxin is located at the amino terminus of the scFv. In another example, PE38 is located at the amino terminus of the scFv. In a further example, cDNA encoding a cytotoxin is ligated to a heavy chain variable region of an antibody that specifically binds IRT A3 or an antibody that specifically binds IRT A5, so that the cytotoxin is located at the carboxyl terminus of the heavy chain variable region. The heavy chain- variable region can subsequently be ligated to a light chain variable region of the antibody that specifically binds IRT A3 or an antibody that specifically binds IRT A5 using disulfide bonds. In a yet another example, cDNA encoding a cytotoxin is ligated to a light chain variable region of an antibody that specifically binds IRT A3 or an antibody that specifically binds IRT A5, so that the cytotoxin is located at the carboxyl terminus of the light chain variable region. The light chain- variable region can subsequently be ligated to a heavy chain variable region of the antibody that specifically binds IRT A3 or the antibody that specifically binds IRT A5 using disulfide bonds.

Once the nucleic acids encoding the immunotoxin, antibody, or fragment thereof are isolated and cloned, the protein can be expressed in a recombinantly engineered host cell such as bacteria, plant, yeast, insect and mammalian cells, for example by expressing in vivo by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art. Alternatively the DNA sequences encoding the immunotoxin, antibody, or fragment thereof can be expressed in vitro.

Polynucleotide sequences encoding the immunotoxin, antibody, or fragment thereof, can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.

The polynucleotide sequences encoding the immunotoxin, antibody, or fragment thereof can be inserted into an expression vector including, but not limited to a plasmid, virus or other vehicle that can be manipulated to allow insertion or incorporation of sequences and can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art.

Transformation of a host cell with recombinant DNA may be carried out by conventional techniques known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl ₂ method using procedures well known in the art. Alternatively, MgCl ₂ or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of trans fection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with polynucleotide sequences encoding the immunotoxin, antibody, or fragment thereof, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). One of skill in the art can readily use an expression systems such as plasmids and vectors of use in producing proteins in cells including higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.

Isolation and purification of recombinantly expressed polypeptide (such as an immunotoxin, antibody or fragment thereof) can be carried out by conventional means including preparative chromatography and immunological separations. Once expressed, the recombinantly expressed polypeptide can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, R. Scopes, Protein Purification, Springer-Verlag, N. Y., 1982). Substantially pure compositions of at least about 90 to 95% homogeneity are disclosed herein, and 98 to 99% or more homogeneity can be used for pharmaceutical purposes. Once purified, partially or to homogeneity as desired, if to be used therapeutically, the polypeptides should be substantially free of endotoxin. Methods for expression of single chain antibodies and/or refolding to an appropriate active form, including single chain antibodies, from bacteria such as E. coli have been described and are well-known and are applicable to the antibodies disclosed herein. See, Buchner et al., Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991; Huse et al., Science 246:1275, 1989 and Ward et al., Nature 341 :544, 1989, all incorporated by reference herein.

Often, functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization using strong denaturants,

and subsequent refolding. During the solubilization step, as is well known in the art, a reducing agent must be present to separate disulfide bonds. An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of the disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena et al., Biochemistry 9: 5015-5021, 1970, incorporated by reference herein, and especially as described by Buchner et al., supra.

Renaturation can be accomplished by dilution (for example, 100-fold) of the denatured and reduced protein into refolding buffer. An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, the heavy and light chain regions are separately solubilized and reduced and then combined in the refolding solution. An exemplary yield is obtained when these two proteins are mixed in a molar ratio such that a 5 fold molar excess of one protein over the other is not exceeded. Excess oxidized glutathione or other oxidizing low molecular weight compounds can be added to the refolding solution after the redox-shuffling is completed.

In addition to recombinant methods, the immunotoxins, EM, and antibodies disclosed herein can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of the polypeptides of less than about 50 amino acids in length can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany & Merrifϊeld, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifϊeld et al., J. Am. Chem. Soc. 85:2149-2156, 1963, and Stewart et al, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, 111., 1984. Proteins of greater length may be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxyl terminal end (for example, by the use of the coupling reagent N, N'-dicycylohexylcarbodiimide) are well known in the art.

Detection Methods and Kits

A method is also provided herein for detecting IRT A3 or for detecting IRT A5 in a biological sample. The method includes contacting the sample with one or more of an antibody that specifically binds IRT A3 or an antibody that specifically binds IRTA5 to form an antibody-IRTA3 or an antibody-IRTA5 complex. The presence or absence of the complex is detected. In one example, the method detects the presence of soluble IRT A3 or soluble IRT A5. In several examples the antibody is E3, E9, or H5 or a humanized form thereof or a functional fragment thereof. The sample can be any sample, including, but not limited to, tissue from biopsies, autopsies and pathology specimens. Biological samples also include sections of tissues, such as frozen sections taken for histological purposes. Biological samples further include body fluids, such as blood, serum, spinal fluid or urine. A biological sample is typically obtained from a mammal, such as a rat, mouse, cow, dog, guinea pig, rabbit, or primate. In one embodiment, the primate is macaque, chimpanzee, or a human. Thus is some examples a subject, such as a human subject is selected a biological sample from that subject is tested for the presence of IRT A3 and/or IRT A5 using the disclosed antibodies or fragments thereof. In some examples, the method is a method of diagnosing or confirming a diagnosis of a B cell malignancy.

In one embodiment, a kit is provided for detecting IRT A3 or for detecting IRT A5 in a biological sample, such as a blood, serum, or plasma sample. Kits for detecting a polypeptide will typically comprise an antibody that specifically binds IRT A3 or an antibody that specifically binds IRT A5, as disclosed herein. In some embodiments, an antibody fragment, such as an Fv fragment is included in the kit. For in vivo uses, the antibody can be a scFv fragment. In a further embodiment, the antibody is labeled (such as with a fluorescent, radioactive, or an enzymatic label).

In one embodiment, a kit includes instructional materials disclosing means of use of an antibody that specifically binds IRT A3 (such as but not limited to H5) or an antibody that specifically binds IRT A5 (such as but not limited to E3 or E9). The instructional materials may be written, in an electronic form (for example computer

diskette or compact disk) or may be visual (such as video files). The kits may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like). The kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.

Methods of determining the presence or absence of a cell surface marker are well known in the art. For example, the antibodies can be conjugated to other compounds including, but not limited to, enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorochromes, metal compounds, radioactive compounds or drugs. The antibodies can also be utilized in immunoassays such as but not limited to radioimmunoassays (RIAs), enzyme linked immunosorbant assays (ELISA), or immunohistochemical assays.

In one embodiment, the diagnostic kit comprises an immunoassay. Although the details of the immunoassays may vary with the particular format employed, the method for detecting IRT A3 or for detecting IRTA5 in a biological sample generally includes the steps of contacting the biological sample with an antibody which specifically reacts, under immunologically reactive conditions, to an IRT A3 polypeptide or contacting the biological sample with an antibody which specifically reacts, under immunologically reactive conditions, to an IRT A5 polypeptide. The antibody is allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

The antibodies disclosed herein can also be used for fluorescence activated cell sorting (FACS). A FACS employs a plurality of color channels, low angle and obtuse light-scattering detection channels, and impedance channels, among other more sophisticated levels of detection, to separate or sort cells (see U.S. Patent No. 5, 061,620). Fluorescence activated cell sorting (FACS) can be used to sort cells that are IRTA3 or IRT A5 positive, by contacting the cells with an appropriately labeled antibody, such as an anti-IRTA3 antibody or an anti-IRTA5 antibody (for

example the H5, E3 or E9 antibody functional fragment thereof or humanized form thereof). The techniques can also include contacting the cells with a first antibody that binds IRT A3 or IRT A5 and then contacting the cells with a second labeled antibody that specifically binds the first antibody. However, other techniques of differing efficacy may be employed to purify and isolate desired populations of cells. The separation techniques employed should maximize the retention of viability of the fraction of the cells to be collected. The particular technique employed will, of course, depend upon the efficiency of separation, cytotoxicity of the method, the ease and speed of separation, and what equipment and/or technical skill is required.

Additional separation procedures may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents, either joined to a monoclonal antibody or used in conjunction with complement, and "panning," which utilizes a monoclonal antibody attached to a solid matrix, or another convenient technique. Antibodies attached to magnetic beads and other solid matrices, such as agarose beads, polystyrene beads, hollow fiber membranes and plastic Petri dishes, allow for direct separation. Cells that are bound by the antibody can be removed from the cell suspension by simply physically separating the solid support from the cell suspension. The exact conditions and duration of incubation of the cells with the solid phase-linked antibodies will depend upon several factors specific to the system employed. The selection of appropriate conditions, however, is well known in the art.

Unbound cells then can be eluted or washed away with physiologic buffer after sufficient time has been allowed for the cells expressing a marker of interest (for example IRTA3, or IRTA5) to bind to the solid-phase linked antibodies. The bound cells are then separated from the solid phase by any appropriate method, depending mainly upon the nature of the solid phase and the antibody employed, and quantified using methods well known in the art. In one specific, non-limiting example, bound cells separated from the solid phase are quantified by FACS. Antibodies may be conjugated to biotin, which then can be removed with avidin or streptavidin bound to a support, or fluorochromes, which can be used with FACS to enable cell separation and quantitation, as known in the art.

An anti-IRTA3 antibody, such as the H5 antibody disclosed herein, can be used to separate and/or detect the presence of IRT A3 expressing T cells. It is disclosed herein that IRT A3 is expressed on peripheral CD4 ⁺ T cells (such as CD4+CD25+FoxP3+ regulatory T cells, or CD4+CD25+CD127 ^low FoxP3+ regulatory T cells) and CD8 ⁺ T cells in human blood. In addition, it is disclosed herein that IRT A3 is expressed on a subset of CD4+CD25+FoxP3+ cells and/or CD4+CD25+CD127 ^low FoxP3+ cells. Thus, an antibody that specifically binds IRT A3 can be used to detect and/or isolate these subsets of T cells, such as regulatory T cells that express IRT A3. It is disclosed herein that Treg cells expressing IRT A3 (IRT A3+ Tregs) do not proliferate in response to stimulation with exogenous IL-2. Thus, the disclosed IRT A3 antibodies can be used to detect and/or isolate Treg cells that do not proliferate in response to exogenous IL-2. In some examples, the IRT A3 antibodies are used to detect the presence of Treg cells that do not proliferate in response to exogenous IL-2 in a subject, for example using a sample obtained from a subject. Thus, in some embodiments, the method includes selecting a subject with a disorder that can be treated with exogenous IL-2. Thus, the method can include detecting IRTA3+ T cells to determine if the subject would benefit from IL-2 therapy.

In some examples, an antibody that that specifically binds IRT A3 is used to detect and/or isolate T cells. These methods include contacting a sample comprising T cells with an anti-IRTA3 antibody detecting binding of the antibody T cells. For example, the method can include contacting the sample with a first antibody that specifically binds IRT A3 and contacting the cells with another antibody that specifically binds T cells, such as an antibody that specifically binds CD4, CD8, CD25, CD 127 or CD3 expressing T cells. Multiple antibodies can be used in these methods, so that one or more of CD4, CD8, CD25, CD127, and/or CD3 are detected. The method optionally can include measuring expression of FoxP3, or performing a bioassay for Treg cell activity. Methods for assaying Treg activity are known in the art, and include bioassays (see PCT Publication No. 2006/012641, incorporated herein by reference). Methods for detecting FoxP3 are known in the art (see, for example, PCT Publication No. 2006/012641 and the description below).

Methods of Monitoring Therapeutic Response

FoxP3 levels differ between subjects with an autoimmune disease and healthy controls (including, the FoxP3 gene, transcript and/or protein). Accordingly FoxP3 expression can be used monitor the efficacy of autoimmune disease therapies. Disclosed herein are methods for assessing the efficacy of a therapy in a subject with an autoimmune disease. These methods include administering a therapy to a subject, isolating IRT A3+ T cells from a biological sample containing T cells obtained from the subject and determining the expression of Foxp3 in the IRT A3+ T cells. The expression of FoxP3, and/or the amount of IRTA3+ cells is compared to a control. An increase in the expression of FoxP3 and/or an increase in the number or IRT A3+ cells, or both, indicates that the therapy is effective for the treatment of the autoimmune disease.

In some embodiments, the T cells express CD3 (are CD3+). In additional embodiments, the T cells express CD4 (CD4+), and/or CD 127(CD 127+ and/or CD127 ^low ), and/or CD25 (CD25+). In some embodiments, the control is a standard value indicative of a basal level of Fox3p. In other embodiments, the control is the expression of FoxP3 in IRT A3+ CD3+ cells, and/or the number of IRTA3+ T cells, prior to the onset of the therapy. If the number of IRTA3+ T cells is increased relative to the control, the therapy is considered effective. However, if the number of IRTA3+ T cells in decreased or remains constant relative to the control the therapy is not considered effective. In additional embodiments, if FoxP3 expression is increased relative to the control, the therapy is considered effective (and thus could be continued). However, if FoxP3 expression is decreased relative to the control, then the therapy is not considered effective (and thus could be discontinued).

In certain method embodiments, an expression level (transcript or protein) and/or activity (protein) of FoxP3 is different with respect to a control. A variety of controls can be used. In some instances, a control is the expression and/or activity of FoxP3 in IRT A3+ T cells isolated from a different subject not treated with the therapeutic agent. In other examples, a control is an average (or "normal-range") value for the expression and/or activity of FoxP3 in subjects, which normal-range value has been determined from population studies. In particular applications, such

as some methods for determining the efficacy of an autoimmune disease therapy, a control also can be, for example, the expression and/or activity of FoxP3 in IRT A3+ T cells from the same subject prior to onset of the therapy, and/or after some period of time following (or during) the therapy. Alternatively, the efficacy of an autoimmune disease therapy can be determined by comparing the expression and/or activity of FoxP3 in a test subject, who is receiving therapy, as compared to a second subject suffering from an autoimmune disease, who is receiving a placebo. In this latter situation, it is expected that the expression levels and/or activities of FoxP3 in the treated subject would diverge from those of a placebo-treated subject, with such expression levels and/or activities in an effectively treated subject approaching corresponding values observed in a healthy control subject.

In some disclosed methods, an expression level and/or activity of FoxP3 (for example, gene, transcript or protein) can differ from a reference expression level and/or activity by at least 10%; for example, by at least about 15%, at least about 25%, at least about 40%, at least about 50%, at least about 60%, at least about 95%, or at least about 90%.

Biological samples containing IRTA3+ T cells can be obtained from normal, healthy subjects or from subjects who are predisposed to or who are suffering from any one of a variety of autoimmune diseases such as, but not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosis, type I diabetes mellitus, Crohn's disease; myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, ankylosing spondylitis, and psoriasis. The disclosed methods contemplate as a subject any living organism capable of experiencing an autoimmune disease, including veterinary subjects (such as, felines, canines, rodents (for example, mice and rats), equines, bovines, ovines, and the like) and human subjects (including, adults, adolescents, and children).

The expression level and/or protein activity of FoxP3 can be detected by any method, including the expression of a transcript from, and/or expression or activity of a polypeptide encoded by, the FoxP3 gene. In particular examples, the expression of FoxP3 is determined by measuring mRNA levels (for example using a gene array, RT-PCR, quantitative PCR, in situ hybridization, Northern blot, or other method(s) commonly known in the art). In other examples, the expression of FoxP3 is

determined by measuring the level or activity of FoxP3 protein (for example, using an antibody array, immunofluorescence, Western blot, radioimmunoassay, sandwich immunoassays (including ELISA), Western blot, affinity chromatography (affinity ligand bound to a solid phase), in situ detection with labeled antibodies, or any of a number of functional assays described herein).

In some disclosed methods, the upregulation or downregulation of FoxP3 can be detected, leading to a relative increase or decrease, respectively, in corresponding transcript and/or protein levels. In other disclosed methods, an increase or decrease in an activity of FoxP3 protein relative to a control can be determined. Particular methods involve detecting a downregulation (and/or decrease in an activity) of FoxP3.

In the methods disclosed herein, FoxP3 expression levels are measured. A variety of methods can be used to detect and quantify FoxP3 expression. In some embodiments, FoxP3 mRNA is measured. FoxP3 mRNA can be measured by any method known to one of skill in the art. For example, polymerase chain reaction (PCR) can be used. Briefly, total RNA is extracted from IRTA3+ CD3+ cells by any one of a variety of methods well known to those of ordinary skill in the art. Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, Greene Publ. Assoc, and Wiley-Intersciences, 1992) provide descriptions of methods for RNA isolation. The extracted RNA is then used as a template for performing reverse RT- PCR amplification of FoxP3 cDNA. FoxP3 -specific primers for the PCR reaction can be obtained, for example, from Applied Biosystems (Foster City, CA). Methods and conditions for PCR are described in Kawasaki et al, (In PCR Protocols, A Guide to Methods and Applications, Innis et al. (eds.), 21-27, Academic Press, Inc., San Diego, California, 1990). In other examples, Northern blotting or RNA dot blots can also be used to detect FoxP3 mRNA.

An additional method for measuring FoxP3 expression levels utilizes measurements of FoxP3 protein. Antibodies to FoxP3 have been described (see for example, PCT Publication No. WO 02/090500 A2, which is incorporated herein by reference). These antibodies can be used in methods such as immunoassays (for

example RIAs and ELISAs), immunohistochemistry, and Western blotting to assess the expression of FoxP3.

Briefly, for Western blotting, total cellular protein is extracted from T cells and electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel. The proteins are then transferred to a membrane (for example, nitrocellulose or PVDF) by Western blotting, and an anti-FOXP3 antibody (for example, a rabbit anti-human FoxP3 antibody) preparation is incubated with the membrane. After washing the membrane to remove non-specifϊcally bound antibodies, the presence of specifically bound antibodies is detected by the use of (by way of example) an anti-rabbit antibody conjugated to an enzyme such as alkaline phosphatase. Application of an alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results in the production of a dense blue compound by immuno localized alkaline phosphatase.

Pharmaceutical Compositions and Therapeutic Methods

Compositions are provided that include one or more of the antibodies, immunotoxins, or fragments thereof that are disclosed herein (such as an antibody that specifically binds IRT A3 or an antibody that specifically bind IRT A5 or a humanized form thereof or a functional fragment thereof). In some examples the immunotoxin, antibody, or fragment thereof is formulated with a carrier. In several examples the antibody is E3, E9, or H5 or a humanized form thereof or a functional fragment thereof. The antibodies of use in the methods provided can include any of the antibodies described above. Exemplary antibodies include, but are not limited to, an E9, E3 (produced by a hybridoma or progeny thereof deposited as ATCC Deposit No. PTA-7723) or H5 antibody (produced by a hybridoma or progeny thereof deposited as ATCC Deposit No. PTA-7724) or a humanized form thereof or a functional fragment thereof. The antibodies of use in the methods disclosed herein also can include an antibody that includes both a light and heavy chain with CDRl, CDR2 and CDR3, wherein H-CDRl comprises the amino acid sequence set forth as amino acids 31 to 35 of SEQ ID NO: 7, H-CDR2 comprises the amino acid sequence set forth as amino acids 50 to 65 of SEQ ID NO: 7, and H-CDR3 comprises the amino acid sequence set forth as amino acids 98 to 105 SEQ ID NO:

7, and wherein L-CDRl comprises the amino acid sequence set forth as amino acids 24 to 38 of SEQ ID NO: 8, L-CDR2 comprises the amino acid sequence set forth as amino acids 50 to 56 of SEQ ID NO: 8, and L-CDR3 comprises the amino acid sequence set forth as amino acids 93 to 101 of SEQ ID NO: 8, wherein the antibody specifically binds IRT A5. The antibodies of use in the methods disclosed herein also can include an antibody that includes both a light and heavy chain with CDRl, CDR2 and CDR3, wherein H-CDRl comprises the amino acid sequence set forth as amino acids 31 to 35 of SEQ ID NO: 9, H-CDR2 comprises the amino acid sequence set forth as amino acids 50 to 68 of SEQ ID NO: 9, and H-CDR3 comprises the amino acid sequence set forth as amino acids 101 to 107 of SEQ ID NO: 9 and wherein L-CDRl comprises the amino acid sequence set forth as amino acids 24 to 34 of SEQ ID NO: 10, L-CDR2 comprises the amino acid sequence set forth as amino acids 50 to 56 of SEQ ID NO: 10, and L-CDR3 comprises the amino acid sequence set forth as amino acids 89 to 97 of SEQ ID NO: 10. One or more of these antibodies may be used in the methods disclosed herein.

The compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating physician to achieve the desired purposes. The antibody, immunotoxin, or fragment thereof can be formulated for systemic or local (such as intra-tumor) administration. In one example, the antibody, immunotoxin, or fragment thereof that specifically binds IRT A3 or the antibody, immunotoxin, or fragment thereof that specifically binds IRT A5 is formulated for parenteral administration, such as intravenous administration. Any of the antibodies disclosed herein, variants thereof (such as immunotoxins) and humanized forms thereof can be used in these methods. The compositions for administration can include a solution of the antibody, immunotoxin, or fragment thereof that specifically binds IRT A3 or the antibody, immunotoxin, or fragment thereof that specifically bind IRT A5 dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as

required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of antibody in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject's needs.

A typical pharmaceutical composition for intravenous administration includes about 0.1 to 10 mg of antibody per subject per day. Dosages from 0.1 up to about 100 mg per subject per day may be used, particularly if the agent is administered to a secluded site and not into the circulatory or lymph system, such as into a body cavity or into a lumen of an organ. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995).

Antibody, immunotoxin, or fragment thereof may be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The solution is then added to an infusion bag containing 0.9% Sodium Chloride, USP, and typically administered at a dosage of from 0.5 to 15 mg/kg of body weight. Considerable experience is available in the art in the administration of antibody drugs, which have been marketed in the U.S. since the approval of RITUXAN® in 1997. Drugs containing an antibody, immunotoxin, or fragment thereof can be administered by slow infusion, rather than in an intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level. For example, an initial loading dose of 4 mg/kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30 minute period if the previous dose was well tolerated. The antibody, immunotoxin, or fragment thereof that specifically binds

IRTA3 or the antibody, immunotoxin, or fragment thereof that specifically bind IRT A5 can be administered to slow or inhibit the growth of cells, such as tumor

cells. In one embodiment, a method is provided for inhibiting the growth of a B cell of a B cell leukemia or lymphoma. The method typically includes contacting the cell with an effective amount of at least one antibody disclosed herein conjugated to an effector molecule, such as the immunotoxins disclosed herein. Methods for measuring cell growth are well known in the art (see, for example, Lewis et al. (1996) Cancer Res. 56: 1457-65; Tian et al. (2001) Nutrition and Cancer 40: 180- 184). The method may be performed in vivo or in vitro.

A method is also provided for treating B cell leukemia or lymphoma in a subject, including administering to the subject a therapeutically effective amount of at least one antibody, immunotoxin, or fragment thereof disclosed herein conjugated to an effector molecule. In theses applications a subject with a tumor, such as a B cell malignancy (for example a lymphoma or leukemia) is selected for treatment with a composition that contains an at least one antibody, immunotoxin, or fragment thereof disclosed herein conjugated to an effector molecule. Suitable subjects include those with B cell malignancies such as precursor B cell lymphoblastic leukemia and lymphoma B cell chronic lymphocytic leukemia, small lymphocytic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B cell lymphoma, nodal marginal zone lymphoma, extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue (MALT) type, Hairy cell leukemia, plasma cell myeloma/plasmacytoma follicular lymphoma, mantle cell lymphoma, diffuse large cell B cell lymphoma, and Burkitt's lymphoma/Burkitt's cell leukemia. Thus, suitable subjects include subjects that have precursor B cell lymphoblastic leukemia and lymphoma B cell chronic lymphocytic leukemia, small lymphocytic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B cell lymphoma, nodal marginal zone lymphoma, extranodal marginal zone B cell lymphoma of mucosa- associated lymphoid tissue (MALT) type, Hairy cell leukemia, plasma cell myeloma/plasmacytoma follicular lymphoma, mantle cell lymphoma, diffuse large cell B cell lymphoma, or Burkitt's lymphoma/Burkitt's cell leukemia. A therapeutically effective amount of an antibody-effector molecule conjugate, such as an immunotoxin, is administered to a subject in an amount sufficient to inhibit growth of cells expressing IRT A3 or IRT A5. Amounts effective

for this use will depend upon the severity of the disease and the general state of the patient's health. A therapeutically effective amount of the antibody, immunotoxin, or fragment thereof is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. These compositions can be administered in conjunction with another chemotherapeutic agent, either simultaneously or sequentially.

Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of at least one of the antibodies disclosed herein, such as an immunotoxin, to effectively treat the patient. The dosage can be administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy. In one example, a dose of the antibody, such as an immunotoxin, is infused for thirty minutes every other day. In this example, about one to about ten doses can be administered, such as three or six doses can be administered every other day. In a further example, a continuous infusion is administered for about five to about ten days. The subject can be treated with the immunotoxin at regular intervals, such as monthly, until a desired therapeutic result is achieved. Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.

Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, A.J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995) incorporated herein by reference. Particulate systems include microspheres, microp articles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μm so that only nanoparticles are administered intravenously. Microp articles are typically around 100 μm in diameter

and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992) both of which are incorporated herein by reference.

Polymers can be used for ion-controlled release of the antibody compositions disclosed herein. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer, polaxamer 407, exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has been shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid- capsulated drug (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Patent No. 5,055,303; U.S. Patent No. 5,188,837; U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; U.S. Patent No. 4,957,735; U.S. Patent No. 5,019,369; U.S. Patent No. 5,055,303; U.S. Patent No. 5,514,670; U.S. Patent No. 5,413,797; U.S. Patent No. 5,268,164; U.S. Patent No. 5,004,697; U.S. Patent No. 4,902,505; U.S. Patent No. 5,506,206; U.S. Patent No. 5,271,961; U.S. Patent No. 5,254,342 and U.S. Patent No. 5,534,496).

EXAMPLES

Example 1

Methods

This example describes exemplary methods used in the following examples.

Preparation of recombinant IRTA 1-6 proteins as the Fc-fus ion proteins:

Extracellular domains of IRTA 1-6 were genetically fused with human IgGl Fc in pcDNA3 vector plasmid (INVITROGEN™, Carlsbad, CA). The FcγRI-binding site of the human Fc had been altered by a mutation to abolish binding to FcγRI. Plasmids were transfected into 293T cells to secrete the Fc- fusion proteins in the supernatants. The transfection and the purification of the Fc- fusion proteins by a protein A column was carried out as described by Ise et al. (Ise et al, Clin. Cancer Res. l l(l):87-96, 2005). The concentration of the Fc- fusion proteins was measured by a human IgG-specific sandwich ELISA. The fusion constructs of IRTAl, IRTA2, IRTA3, IRTA4, IRTA5, IRTA6 and human IgGl used for the generation of antibodies specific for IRTAl, IRT A2, IRT A3, IRT A4, IRT A5.

Transfection of ^' 293T cells: Typical experiments used 5 x 10 ⁵ of 293 T cells seeded in a 10-cm dish (BD

Biosciences, Bedford, MA) for 24 hours prior to the transfection. Four μg of plasmid DNA were transfected by LIPOFECTAMINE™ and Plus reagent (INVITROGEN™) according to the manufacturer's instruction. After a 48-72 hour transfection, the cells transfected with the plasmids encoding IRTAs were used for fluorescence activated cell sorter (FACS) analysis, cell-enzyme-linked immunosorbent assay (cell-ELISA), Western blot or immunofluorescence. 293T cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

ELISA Assay to Determine Antibody Cross-reactivity:

In a typical ELISA for the screening of hybridomas, microtiter plates (MaxiSorp; Nalgene Rochester, NY) were coated with 100 ng/50 ul/well of goat

anti-human IgG in phosphate buffered saline (PBS) for 2 hours at room temperature. Next 100 ng/50 ul/well of each IRTA-Fc in blocking buffer (25% DMEM, 5% fetal bovine serum (FBS), 25 mM HEPES, 0.5% bovine serum albumin (BSA), 0.1% sodium azide in PBS) was added to each well and incubated for 1 hour at room temperature. After washing with PBS containing 0.05% TWEEN™ 20, 50 ul of the hybridoma supernatants were added and incubated for 2 hours at room temperature. After washing, the bound MAbs were detected by a 24 hour incubation with horse radish peroxidase (HRP)-labeled goat anti-mouse IgG followed by a tethamethylbenzidine (TMB) substrate kit.

Cell culture of human cell lines:

Seven human cell lines were obtained. They were Daudi (Burkitt's lymphoma American Type Culture Collection (ATCC) number CCL-213); NAMALWA (Burkitt's lymphoma ATCC number CRL-1432); Raji (Burkitt's lymphoma ATCC number CCL-86); Jurkat (T cell lymphoma); SU-DHL-5 (B cell non-Hodgkin's lymphoma (B-NHL, diffuse large cell noncleaved cell type)); BL2 (Burkitt's lymphoma); and L540 (Hodgkin's disease).

Daudi cells were grown in RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS). NAMALWA, Raji, Jurkat and SU-DHL-5 cells were grown in RPMI 1640 medium supplemented with 10% FBS. BL2; and L540 cells were grown in Isocove's modified Dulbecco's medium (IMDM) supplemented with 10% FBS.

Human peripheral blood lymphocytes (PBMC) and the cell culture: Buffy coats of healthy donors were obtained from the department of transfusion medicine, National Institutes of Health (NIH, Bethesda, MD), according to a protocol approved by NIH. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation over Ficoll/Hypaque (GE bioscience, Freiburg, Germany). The PBMCs were freshly used or kept in culture up to 40 hours in (IMDM supplemented with 10%FBS at 10 ⁶ cells/ml before the FACS analysis. In some examples, the isolated PBMCs were stimulated by anti-CD3 antibody (clone UCHTl, BD bioscience, San Jose, CA) and anti-CD28 antibody

(clone CD28.2, BD) and time-course samples were analyzed. In another examples, PBMCs were treated an anti-CD25 immunotoxin, LMB-2, at 20 ng/ml for 5 days to specifically deplete CD25 ⁺ cells. LMB-2 is a recombinant fusion protein of an anti- CD25 single-chain Fv fragment to a truncated form of the bacterial Pseudomonas exotoxin A (Kreitman et al, Blood, 83(2):426-434, 1994). In other examples, fractions of cells sorted by FACS (10 ⁴ cells/ml) were treated with recombinant IL-2 (10 ng/ml, Abeam, Cambridge, MA) for 5 days. To monitor the cell growth, the glucose consumption by the cells was determined by measuring glucose level of the culture media using Autokit Glucose CII (Wako chemicals, Richmond, VA). In some T cell activation examples, the freshly isolated PBMCs (5 x 10 ⁵ cells) were seeded in 24- well culture plate (1 ml/well) and stimulated by adding 2 x 10 ⁶ of DYNABEADS® goat anti-mouse- IgG (INVITROGEN™ 110-33) precoated with anti-CD3 antibody (clone UCHTl, BD bioscience, 2 μg/10 ⁷ beads) and anti-CD28 antibody (clone CD28.2, BD bioscience, 2 μg/10 ⁷ beads).

FACS analysis:

Typically 10 ⁶ cells in 200 μl of FACS buffer (phospho buffered saline (PBS) containing 5% FBS and 0.1% sodium azide) were stained with a panel of conjugated antibodies for FACS analysis. In some examples, cells were incubated with a cocktail of optimal dilution of each MAb. Typically 106 cells were incubated in 200μl of the antibody cocktail in FACS staining buffer (PBS containing 5% FBS and 0.1% sodium azide) for 1 hour at 4°C. To stain intracellular Foxp3 or CTLA4 proteins, cells post-stained by other surface markers were fixed and permeabilized by Fixation/Permeabilization solution (eBioscience), and then stained with the MAbs against Foxp3 or CTLA4. Flow cytometry data was acquired on

FACSCALIBUR® or on LSR II flow cytometer (BD Biosciences). At least, 50,000 and 300,000 events were collected for three or less colors and more than 4 colors analysis, respectively. All the data was further analyzed by Flow Jo software (Tree Star Inc.). The gating strategies including lymphocyte gating, CD3-SSC gate, and CD4-SSC gate are described in figure legends. To determine positive and negative events for each marker, all reagents except for the one of interest (fluorescence minus one, or "FMO" controls) were used to identify expressing cells in the fully

stained sample. Unless indicated otherwise, positive thresholds for each marker was determined so that fewer than 0.2% of cells in the counter FMO control were included in the positive ranges. Cell sorting experiments were performed on FACSARIA® or FACSVANTAGE® cell sorter (BD Biosciences). The following antibodies were used: the anti-IRTA3, H5, disclosed herein was conjugated to R-phycoerythrin (PE) (custom labeled by INVITROGEN®). MAbs against IRTA5 (E3 and E9, disclosed herein), IRTA4 (B24), IRTAl (Al), and IRTA2 (F56) were prepared and labeled with PE (INVITROGEN®). The following antibodies were purchased: fluorescein isothiocyanate (FITC)-labeled antibodies, anti-Foxp3 (PCHlOl, eBioscience 11-4776), anti-CD16 (3G8, BD

555406), anti- CD19 (HIB19, eBioscience 11-0199), anti-CD25 (BC96, eBioscience 21-0259), anti-CD38 (HIT2, BD 555459), anti-CD45RA (HIlOO, eBioscience 11- 0458), anti-CD56 (MEM188, eBioscience 11-0569), anti-CD69 (FN50, eBioscience 11-0699), anti-CD103 (B-Ly7, eBioscience 11-1038); PE-labeled antibodies, IgG2b- isotype control (27-35, BD 555743); peridinin chlorophyll-alpha protein (PerCP)- Cy5.5-labeled antibodies, anti-CD3 (SP34-2, BD 552852), anti-CD4 (SK3, BD 341654), anti-CD5 (L17F12, BD 341089), anti-CD8, (SKl, BD 341051), anti-CD19 (SJ25C1, BD 340951), anti-CD20 (L27, BD 340955); PE-Cy7-labeled antibodies, anti-CD4 (RPA-T4, eBioscience 25-0049), anti-CD25 (BC96, eBioscience 25- 0259), anti-CD62L (DREG-56, eBioscience 25-0629); allophycocyanin (APC)- labeled antibodies, anti- AITR (eBioAITR, eBioscience 17-5875), anti-CD 19 (HIB 19, BD 555415), anti-CD25 (BC96, eBioscience, 17-0259), anti-CD45RA (HIlOO, eBioscience, 17-0458), anti-CD45RO (UCHLl, eBioscience 17-0457), anti- CD56 (B159, BD 555518), anti-CD69 (FN50, eBioscience 17-0699), anti-CD127 (eBioRDR5, eBioscience 11-1278), anti-HLA-DR (LN3, eBioscience 17-9956), anti- CTLA4(CD152) (BN13, BD, 555855); APC-Cy7-labeled antibodies, anti-CD3 antibody (SK7, BD 557832), anti-CD62L (SREG56, eBioscience 10-0629); Pacific blue (PB)-labeled antibodies, anti-Foxp3 (PCHlOl, eBioscience 57-4776), anti-CD8 (OKT8, eBioscience 57-0086), anti-CD19 (HIB19, eBioscience 57-0199), anti- CD127 (eBioRDR5, eBioscience 57-1278); AmCyan-labeled anti-CD4 (SK3, BD 339187). Individual staining combinations included: IRTA3-PE/CD3-PerCP-Cy5.5, IRTA3- PE/CD19-PerCP-Cy5.5, CD56-FITC/IRTA3-PE, IRTA3-PE/CD4-PerCP-

Cy5.5, IRTA3- PE/CD8-PerCP-Cy5.5 in Fig. 3; IRTA3-PE/CD3-PerCP- Cy5.5/CD4-PE-Cy7/CD8- PB/CD45RA-APC/CD62L-APC-Cy7, IRTA3-PE/CD3- PerCP-Cy5.5/CD8-PB/CD4- AmCyan/CD45RA-APC/CD62L-APC-Cy7 in Fig. 4A: IRTA3-PE/CD3-PerCP-Cy5.5/CD4- PE-Cy7/CD8-PB/CD45RO-APC, IRT A3- PE/CD3-PerCP-Cy5.5/CD8-PB/CD4- AmCyan/CD45RO-APC, in Fig. 4B; IRTA3- PE/CD3-PerCP-Cy5.5/CD4-PE-Cy7/CD8- PB/CD25-APC, CD25-FITC/IRTA3- PE/CD4-PE-Cy7/CD8-PB/CD3-APC-Cy7 in Fig. 4C; CD69-FITC/IRTA3-PE/CD4- PE-Cy7/CD8-PB/CD25-APC/CD3-APC-Cy7, CD25- FITC/IRTA3-PE/CD4-PE- Cy7/CD8-PB/CD69-APC/CD3-APC-Cy7 in Fig. 5; CD127- FITC/IRTA3-PE/CD4- PerCP-Cy5.5/Foxp3-PB/CD25-APC in Figs. 6 and 7; CD25- FITC/IRTA3-PE/CD4- PerCP-Cy5.5/Foxp3-PB/CTLA4-APC, CD25-FITC/IRTA3-PE/CD4- PerCP- Cy5.5/Foxp3-PB/AITR-APC, CD25-FITC/IRTA3-PE/CD4-PerCP-Cy5.5/Foxp3- PB/HLA-DR-APC, CD38-FITC/IRTA3-PE/CD4-PerCP-Cy5.5/Foxp3-PB/CD25- APC, CD 103- FITC/IRTA3-PE/CD4-PerCP-Cy5.5/Foxp3-PB/CD25-APC in Fig. 13; CD127-FITC/IRTA3- PE/CD4-PerCP-Cy5.5/CD25-APC for the cell sorting in Figs. 9, 10, and 11; CD127- FITC/IRTA3-PE/CD4-PerCP-Cy5.5/Foxp3-PB/CD25- APC, CD103-FITC/IRTA3-PE/CD4- PerCP-Cy5.5/Foxp3-PB/CTLA4-APC in Fig. 11.

In vitro suppression assays:

To measure the suppressor activity of the sorted population of cells, the isolated cells (candidates of Treg cells) were mixed with responder CD4+CD25- CD127hi cells (Teff cells) that had also been sorted from the autologous PBMCs. The responder cells were labeled with 2 μM of CFSE (INVITROGEN®) in IMDM supplemented with 1% FBS by alO-min incubation at room temperature.

Suppression assays were performed by coculturing of 4,000 CFSE-labeled Teff cells with different numbers of the sorted cells (typically 4,000, 2000, 1000 and 250) in 200 μl of IMDM containing 5% human AB serum (Bioreclamation Inc.) in roundbottom 96-well microtiter plates. The cells were stimulated by adding 25,000/well of anti- CD3/CD28 beads that had been prepared as described above. The beads to cell ratio for the optimal cell activation was predetermined in titration experiments. After 80hours or 96hours of incubation, 100 μl of supernatant was

removed from each well for cytokine assays. The cells were harvested in 0.3 ml of FACS buffer containing 7-amino-actinomycin D (0.5 μg/ml, BD bioscience) for viability staining and 20,000 of FITC-conjugated beads (Bangs Laboratories Inc., #512) were used as the standard for cell counting. CFSE dilution by Teff cell dividing was analyzed in lived cell population by LSR II flow cytometer and Flow Jo software.

In vitro cell proliferation assays:

The isolated cells by sorting were labeled with CFSE. The labeled cells (5,000) were incubated with or without different numbers of anti-CD3/CD28 beads (25,000, 50,000, 100,000) in 200 μl of IMDM containing 5% human AB serum in round-bottom 96-well microtiter plates. In some cultures, exogenous recombinant human IL-2 (BIOSOURCE®/INVITROGEN®) was added at 10 ng/ml.

Induction ofFoxp3+ cells from Teff cells in vitro by treatment of TGF -β:

Sorted CD4+CD25-CD127 ¹¹¹ cells (5 x 105) were cultured in 1 ml of IMDM containing 5% human AB serum in 24-well culture plates for 5 days with or without

10 ng/ml of recombinant human TGF-β (eBioscience) or anti-CD3/CD28 beads

(106/ml) or IL-2 (lOng/ml).

Cytokine assay:

Cytokine production in the culture supernatants of the suppressor and proliferation assays were measured using human Thl/Th2 cytokine cytometric bead array kit (BD bioscience) according to the manufacturer's instruction with a minor modification. The human cytokines measured were IL-2, IL-4, IL-5, IL-10 and

TNF-α.

Reverse transcriptase polymerase chain reaction (RT-PCR):

The mRNA expression of IRT A3 or control genes in the sorted cell fractions was examined by a RT-PCR method. Total RNA was prepared from each fraction of cells (10 ⁶ cells) using TRIZOL® LS reagent (INVITROGEN®). cDNA was made by SUPERSCRIPT™ III Reverse Transcriptase (INVITROGEN®) with

random hexamer priming according to the manufacturer's instruction. PCRs were performed using the cDNAs and 3 sets of primers for IRT A3, CD3, and β-actin with 20-28 cycles of amplification (95 ⁰ C, 15 sec; 55 ⁰ C, 30 sec; 72 ⁰ C, 1 min). The primer sequences were as follows: IRTA3-F, 5 '-CAGCACGTGGATTCGAGTCAC-S' (SEQ ID NO: 26); IRTA3-R, 5'-CAGATCTGGGAATAAATCGGGTTG- 3'(SEQ ID NO: 27); CD3-F, 5'-TTTGGGGGCAAGATGGTAATG-SXSEQ ID NO: 28); CD3-R, 5'-GGGGTAGCAGACATAATAACCAC-3'(SEQ ID NO: 29); β-actin-F, 5'-AGCCCTTCAGACTCGGACTC-3'(SEQ ID NO: 30); β-actin-R: 5'- TGGGGCAGCCTAAATCTT-3'(SEQ ID NO: 31).

Example 2

Generation of anti-IRTA3 monoclonal antibody This example describes exemplary methods for the production of anti- IRT A3 antibodies. DNA-immunization method followed by a boost immunization with

IRT A3 -trans fected 293T cells as described by Nagata et al. (Nagata et al, J Immunol Methods 280(l-2):59-72, 2003) was used to produce the anti-IRTA3 antibodies. In brief, IRTA3 full length cDNA in pcDNA3 was subcutaneously injected in a 6-week-old female Balb/c mouse. The mouse was boosted with 293T cells transfected with the same plasmid, and 3 -days later the spleen cells were fused with SP2/0-neo myeloma cells. SP2/0-neo myeloma cells were maintained in IMDM supplemented with 15% FBS. The hybridomas were screened for secretion of specific MAbs in an ELISA using IRTA3-Fc antigen. After multiple rounds of cell cloning by limiting dilution, the established hybridoma was grown to a high density to harvest the MAbs in the culture supernatants. The crossreactivity of the MAb with other IRTA proteins were tested both in ELISA using other IRTAs-Fc antigens and in fluorescence-activated cell sorter (FACS) analysis using 293T cells transfected with each IRTA-encoding plasmid . The isotype of the MAbs was determined by mouse MAb isotyping reagents (ISO2; Sigma-Aldrich, St. Louis, MO). Immunoglobin (Ig) concentrations in the culture supernatants were determined by a sandwich ELISA as described in Nagata et al, Hybridoma 10(3):369-378, 1991. Purification of MAbs by protein A was carried

out as described by Nagata et al. (Nagata et ah, J Immunol Methods 280(l-2):59- 72, 2003). H5 MAb reacted with IRT A3 -Fc fusion protein in ELISA but did not react with FCRLl, 2, 4, 5, 6-Fc fusion proteins in the same assay; H5 MAb bound to the surface of IRTA3-transfected 293 T cells in a flow cytometry but did not bind to IRTAl, 2, 4, 5, 6-transfected cells (Fig. 12A and 12B). H5 MAb is specific to IRT A3 and not cross reactive with other IRTA proteins. H5 MAb also bound to endogenous FCRL3 protein on several human B cell lines in which IRT A3 mRNA was expressed (Fig. 12C). H5 MAb did not bind to recombinant IRT A3 proteins in Western blotting, indicating that H5 MAb recognizes a conformational epitope whose structure is denatured by SDS treatment.

Example 3 Generation of anti-IRTA5 monoclonal antibodies

This example describes the production of anti-IRTA5 MAbs. The anti-IRTA5 MAbs E3 and E9 were produced and isolated by procedures similar to those described above. Briefly, IRTA5 full length cDNA in pcDNA3 was subcutaneously injected into a Balb/c mouse. The mouse was boosted with 293T cells transfected with the same plasmid, and the spleen cells were fused with SP2/0-neo myeloma cells. The fusion experiments resulted in the production of 9 anti-IRTA5 MAbs. The anti-IRTA5 MAbs E3 and E9 were chosen for further study. The characteristics of the of the anti-IRTA5 MAbs E3 and E9 as well as the anti-IRTA3 MAbs H5 are summarized in Fig. 1. With reference to Fig. 1, an expression plasmid for full-length of each IRTA was used as an immunogen as described in Example 1. All the MAbs possess K light chain. The cross-reactivity with other IRTA family members was examined by ELISA (see Example 4) using each IRTA-Fc fusion protein by FACS using 293T cells transfected with each IRTA expression plasmid. Both assays showed no cross- reactivity of the MAbs.

Example 4 Cross reactivity of anti-IRTA5 and anti-IRTA3 monoclonal antibodies

This example describes the determination of the cross reactivity of anti- IRTA MAbs. Because of the high homology that exists between IRTA family members the cross reactivity of the anti-IRTA5 MAbs and anti-IRTA3 MAbs to other IRTAs at a saturated concentration was performed by ELISA and to native IRTAs exposed on transfected 293T cells. Fig. 15 shows the results of ELISA using IRTA-Fc fusion proteins. Anti-IRTA5 MAbs E3 and E9 only reacted with IRTA5-Fc but did not react with other IRTA-Fcs and CD30-Fc. Anti-IRTA3

MAb H5 only reacted with IRT A3 -Fc but did not react with other IRTA-Fcs and CD30-Fc. As shown in Fig. 16, FACS analysis using 293T cells transfected with each IRTA resulted in the same reactivity as in the ELISA assays (Fig. 15). Thus, the binding of anti-IRTA5 MAbs E3 and E9 was specific to IRT A5 in both assays, and the binding of anti-IRTA3 MAb H5 was specific to IRT A3 in both assays.

Example 5 Expression of IRT A3 and IRT A5 on human cell lines

This example describes the determination of IRT A3 and IRT A5 expression on several human cell lines.

The reactivity of each anti-IRTA3 and anti-IRTA5 MAb with 7 cell lines (Daudi, Raji, Jurkat, NAMALWA, SU-DHL-5, BL2, and L540 cells) was determined (See Fig. 16 and Fig. 1). The anti-IRTA3 MAb H5 reacted with Raji and SU-DHL-5 cells. The anti-IRTA5 MAb E3 and E9 reacted with Daudi, NAMALWA, and BL2 cells. None of the MAbs reacted with Jurkat cells.

Example 6

The anti-IRTA3 MAb specifically reacts with the extracellular domain of

IRTA3 protein This example describes the determination of the specificity of the isolated anti-IRTA3 IgG2b MAb H5.

The specificity of the anti-IRTA3 MAb H5 the cross reactivity of the H5 was determined. Fig. 2A shows the result of ELISA using each IRTA extracellular domain-Fc fusion protein. The MAb H5 bound to an IRT A3 -Fc coated plate in a dose-dependent manner and did not show any reactivity with IRTAl, 2, 4, 5-Fc proteins. Fig. 2B shows a FACS analysis using MAb H5 and 293T cells transfected expression plasmids that express each IRTA (1, 2, 3, 4, and 5). MAb H5 reacted with IRT A3 -transfected cells and did not react with IRTAl, 2, 4, 5-transfected cells as well as non-transfected 293T cells. The surface expression of all the IRTA proteins on the transfected cells was confirmed by FACS using each anti-IRT A MAb. Based on this data it was determined that MAb H5 specifically reacts with the extracellular domain of IRT A3 in both ELISA and FACS. In Western blotting, MAb H5 did not bind to any of the IRTA- Fc fusion proteins or to recombinant IRTA proteins expressed in 293 T cells, indicating that MAb H5 binds to a conformational epitope whose structure is denatured by SDS treatment. The absence of the cross reactivity of MAb H5 to FcγRl (CD64), FcγRIIb (CD32) and FcRIIIa (CD 16) was confirmed in single or double colors FACS staining of HL60 cells, B cells in human blood, and CCRF- SB cells, respectively.

Example 7

IRT A3 is expressed on T cell subsets in human PBMC

This example describes the determination of the set of T cells that express IRT A3.

To examine IRT A3 expression on peripheral lymphoid cells, MAb H5 was used together with other surface markers in multi-color staining of human PBMCs from normal donors. IRT A3 protein expression was observed on B cells (CD 19 ) as well as on natural killer (NK) cells (CD56 ⁺ ). Unexpectedly, it was found that there was significant IRT A3 expression on a subset of T cells (Fig. 3A). The modest level of the FACS signal on B and NK cells is consistent with a previous report describing IRT A3 protein expression on B cells and NK cells (Poison et al, Int. Immunol. 18(9): 1363-1373, 2006). The low IRTA3 signal made it difficult to conclude if all the B cells and NK cells express IRT A3 or if only subsets of these

cells express IRT A3. There was no clear correlation between IRT A3 expression and a memory marker (CD27) expression in the B cell compartment see also Poison et al. {Int. Immunol. 18(9): 1363-1373, 2006). However, the two peaks with different IRT A3 expression levels in T cells indicated the presence of minor IRTA3 ⁺ and IRTA3 ^" cell subsets in the peripheral T cells (Fig. 3A). Expression of IRT A3 protein in T cells has not been described, perhaps because of the small ratio of IRT A3 ⁺ cells in T cells and the modest level of the expression.

Fig. 3B shows two-color staining of human PBMC with the anti-IRTA3 and anti-CD3 (pan T), anti-CD4 (helper T) or anti-CD8 (cytotoxic T) antibodies. In these trials (n=8), T cells (CD3 ⁺ ) represented about 70% of total PBMCs and consisted of helper (CD4 ) and cytotoxic (CD8 ) T cells at roughly a 2:1 ratio, consistent with reference values (Deneys et al., J. Immunol. Methods 253(1 -2):23-

+

36, 2001; Bisset et al., Eur. J. Haematol. 72(3):203-212, 2004). IRTA3 subsets both in CD4 ⁺ and CD8 ⁺ cells was observed, indicating that IRTA3 expression is not linked to those of CD4 and CD8 proteins. As summarized in Fig. 3C, 1.5- 14.3% of CD3 ⁺ cells expressed IRT A3 (n=8) and 0.5-12.9% of CD4 ⁺ cells and 2.6-24.7% of CD8 ⁺ cells expressed IRT A3. The frequencies of IRT A3 expression in CD8 ⁺ cells tended to be higher than in CD4 ⁺ cells, although there is no statistical significance in the difference. In CD3 ⁺ cells, there was no correlation between IRT A3 and CD56 expressions, indicating that IRT A3 is not preferentially expressed in CD3 CD56 ⁺ natural killer T cells.

To confirm IRTA3 expression in IRTA3 ⁺ CD3 ⁺ T cells, PBMCs were separated into 4 fractions (P1-P4) by a cell sorter based on their differential staining for cell surface IRTA3 and CD3 (Fig. 3D). RT-PCR analysis indicated the presence of IRT A3 mRNA in IRTA3 ⁺ cells and CD3 mRNA in CD3 ⁺ cells, all cells expressed for β-actin mRNA. Cloning and sequencing of the fragments amplified by IRT A3 primers confirmed IRT A3 mRNA expression. The complete agreement of the FACS and RT-PCR data demonstrated the presence of CD3 ⁺ cells that express IRTA3 protein.

Example 8 Phenotype of IRTA3-positive T cells

This examples describes the phenotypic analysis of IRT A3 -positive T cells. The phenotype of IRT A3 ⁺ T cell (CD3 ) was further characterized by FACS.

Other surface markers on IRT A3 + T cells were investigated by multi-color flow cytometry. In the analysis, we identified IRTA3+ and IRTA3- cells in CD3+CD4+ or in CD3+CD8+ cell populations by appropriate gating and determined the expression level of different markers including CD45RA, CD62L, CD45RO and CD25. As shown in Figs. 4A and 4D, IRT A3+ cells less frequently expressed naϊve T cell makers, CD45RA and CD62L, than IRT A3- cells in both CD4+ and CD8+ cell fractions. In contrast, IRTA3+ cells more frequently expressed a memory marker, CD45RO, than IRTA3- cells in both CD4+ and CD8+ cell fractions (Figs. 4B and 4D). Thus IRTA3+ T cells tend to show memory phenotype rather than naϊve phenotype as for these markers. CD25 expression on IRT A3+ and IRT A3- cells was also examined. As shown in Fig. 4C and Fig. 4D, 49% of IRTA3+CD4+ cells expressed CD25, whereas only 5% of IRTA3-CD4+ cells expressed CD25. In contrast, there was no difference of CD25 expression frequency between IRTA3+CD8+ and IRTA3-CD8+ cells. Therefore, IRTA3 expression is associated with the constitutive expression of CD25 in CD4+ human peripheral T cells, which is a putative marker set for natural Treg cells. The memory phenotype of CD4+IRTA3+ T cells (Figs. 4A and 4B) also supports the association of IRT A3 expression with nTregs, which generally show memory phenotype.

Example 9

IRTA3 expression is not induced by T cell stimulation in vitro

This example demonstrates that IRT A3 expression is independent of T cell stimulation.

When the T cells are activated in vitro by T cell receptor stimulation (anti- CD3) with coreceptor stimulation (anti-CD28), the T cells start to proliferate and express early activation marker, CD69, followed by a late activation marker, CD25. Because CD25 is also known to be up-regulated upon activation, IRT A3 expression

kinetics on T cells in the course of activation via TCR and co-receptor using anti- CD3/CD28-coated beads was examined. The stimulation induced the transient expression of an early phase activation marker, CD69, followed by the expression of a late activation marker, CD25 in both of CD3+CD4+ and CD3+CD8+ T cells (Fig. 5 A and 5B). However, the frequencies of IRTA3+ population in any cell fraction were consistently low level (<10%) during the time course, indicating that IRT A3 expression was not up-regulated or down-regulated by the T cell activation. Stimulation with mitogens such as phytohaemagglutinin and concanavalin A did not increase the level or frequency of IRT A3 expression. In long term culture of human T cells in the presence of IL-2, the expression of IRT A3 was diminished and finally disappeared after a few weeks of culture. IRT A3 expression was not detected in 3 different tumor infiltrated lymphocyte cultures (CD8+).

Example 10 IRT A3 expression on natural regulatory T cells

This example describes IRT A3 expression in regulatory T cells. The activation trial indicated that IRT A3 ⁺ cells in CD4 ⁺ cells are anergic to T cell receptor stimulation and frequently express CD25. Constitutive expression of CD25 in anergic CD4 ⁺ T cells is a characteristic of regulatory T cells. The regulatory (suppressive) activity can be monitored by proliferation of CD4 ⁺ and CD8 ⁺ effector T cells cocultured with the regulatory T cells enriched in the CD4 CD25 ⁺ peripheral T cell population. To more precisely identify regulatory T cells, the detection of an intracellular transcription factor, forkhead box P3 (Foxp3), is widely used because genetic experiments indicate that the Foxp3 transcription factor is associated with suppressor function of the regulatory T cells.

PBMCs were stained with CD4, CD25, CD 127, and IRT A3 -specific MAbs. After fixation and permeabilization, intracellular Foxp3 was assessed. In the analysis, the stained cells were gated by CD4-side scatter profile followed by three different gating strategies as shown in Fig. 6A. Tregs were identified as Foxp3+, CD25+CD127 ^low and CD25 ^hl cells in P2, P4 and P7 gates, respectively. As the counter populations for comparison, non-Treg cells were identified as Foxp3-, CD25-CD127 ¹ " and CD25- cells in Pl, P3 and P5 gates, respectively. The P6 gate

defined CD25 ^dim cells that contain functional Tregs. To depict the mutual relationships between these gated populations, marker expression and the frequency of each population in CD4+ cells are summarized in Figs. 6B and 6D. The P2 Tregs (Foxp3+) account for 14% of CD4+ cells contained both CD25+CD127 ^low and CD25-CD127 ¹ " cell populations. The Pl non- Tregs (Foxp3-) were always CD25- CD127M cells. In contrast, all P4 Tregs (CD25+CD127 ^low ) were Foxp3+ whereas P3 non-Tregs (CD25-CD127 ¹¹¹ ) were Foxp3-. Therefore, Foxp3+ cells (P2) included CD25+CD127 ^low cells (P4) that account for 6% of CD4+ cells. The P7 gate is part of P4 gate; accordingly P7 Tregs (CD25 ^hl ) were a subset of P4 Tregs (CD25+CD127 ^low ). The Foxp3 expression level of P7 Tregs (CD25 ^hl ) is uniformly high whereas P4 Tregs (CD25+CD1271ow) consist of two populations with different Foxp3 levels in the positive range. In addition, P6 cells (CD25 ^dim ) contained Foxp3 dimly-positive population. Therefore, P7 Tregs (CD25 ¹ ") is a subpopulation with the highest level of Foxp3 expression of P4 Tregs (CD25+CD127 ^low ). These characteristics of human Treg populations defined by the three strategies are well consistent with the data previously reported: intracellular Foxp3+ cells contain nTregs and a subset of conventional T cells and account for 10-15% of peripheral human CD4+ cells; Tregs defined in CD25+CD127 ^low gate comprise 6-8% of CD4+ cells and are all suppressive in the suppressor assay; CD25 ^hl Tregs comprise a part of functional Treg population with the highest level of Foxp3 expression. Fig. 6C shows the IRT A3 expression of these gated cell populations. All the three Treg populations (P2, P4, and P7) contained two distinctive subpopulations that correspond to IRTA3+ and IRTA3- cells. In contrast, the three non- Treg populations (Pl, P3, and P5) were almost all IRT A3- cells. Thus, IRTA3 is preferentially expressed on nTregs in human CD4+ cells. As summarized in Fig. 6E, the ratios of IRTA3+ cells in CD25+CD127 ^low (P4) and in CD25 ^hl (P7) are 40% and 44%, respectively, whereas IRTA3+ cell ratio in Foxp3+ (P2) was 22%.

Example 11 IRT A3 expresses on a subset of CD25+CD1271owCD4+ regulatory T cells

This example describes IRT A3 expression in a subset of regulatory T cells.

Because of the importance of CD25+CD127 ^low phenotype in Treg suppressor function, the relationships of the levels between IRT A3, CD 127 and CD25 on CD4+Foxp3+ cells was precisely analyzed (Fig. 8). The PBMCs were stained with the same panel of MAbs used in experiments performed above, and then IRT A3+ and IRTA3- cells in Foxp3+CD4+ cell compartment were gated for the analysis. The gated cells with different levels of IRT A3 expression were separately evaluated for CD25 and CD 127 expression. Consistent with the analysis using whole CD4+ cells (Fig. 6C), IRTA3+Foxp3+cells showed a CD25+CD127 ^low phenotype, although IRTA3-Foxp3+cells contained both CD25-CD127 ^hl cells and CD25+CD127 ^low cells (Fig. 8A). As summarized in Fig. 8B, 70% of

IRTA3+Foxp3+ cells fell into CD25+CD127 ^low Treg gate and only 23% of IRTA3+Foxp3+ cells were classified into CD25-CD127 ¹ " non-Treg phenotype. In contrast, only 27% of IRTA3-Foxp3+ cells were CD25+CD127 ^low Treg cells and 68% of IRTA3-Foxp3+ cells were CD25 -CD 127 ^hl non-Treg cells. Therefore, IRT A3 expression on Foxp3+ cells is associated with the CD25+CD127 ^low functional Treg phenotype. Fig. 8B also shows that the ratios of CD25 ¹ " cells in IRTA3+Foxp3+ and IRTA3-Foxp3+ cell compartments were 30% and 10%, respectively, which closely correlate with the CD25+CD127 ^low cell ratios in IRTA3+Foxp3+ and IRTA3-Foxp3+ cells. This indicates a similar level and frequency of IRT A3 expression on both CD25 ¹ " cells and CD25+CD127 ^low cells regardless of the different distribution of CD25 and Foxp3 expression. Therefore, Tregs with a CD25+CD127 ^low phenotype can be divided into two groups by IRT A3 expression. The association of IRT A3 expression with other markers was also examined (Fig. 6). Cells expressing CTLA-4, GITR, and HLA-DR were enriched in the Treg population and there was no significant difference in these markers expression between IRT A3+ and IRT A3- Tregs. There also was no association of IRT A3 expression with CD30 or with CD 103 expression, which subdivide CD25 ^lu CD4+ cells into two relatively even subpopulations.

Example 12

Both IRTA3+ and IRTA3- Treg cells (CD25+CD1271ow) suppress T cell proliferation in vitro.

This example describes the determination of suppressor activity of IRTA3+ positive Treg cells on T cell proliferation

CD25+CD127 ^low Tregs can be divided into two groups by IRT A3 expression. CD25+CD127 ^low Treg cells were previously considered as a single population. The regulatory (suppressor) activity of IRTA3+ and IRT A3- subsets of CD25+CD127 ^low cells on the proliferation of non-Treg cells was examined. According to the gates shown in Fig. 9A, CD25-CD127 ¹¹¹ (Pl) and CD25+CD127 ^low cells (P2) were isolated from CD4+ cells for samples PBMCs by a cell sorter. The CD25-CD127 ¹ " (Pl) cells were used as responder cells (T effector cells, Teffs) in the following proliferation assay to test candidate Treg cells isolated from autologous PBMCs. IRTA3- (P3) and IRTA3+ (P4) cells in the CD25+CD127 ^low cell compartment were also isolated using the second gating (Fig. 9A). The post sorting panels confirm that these isolated cell populations fell into the same gates established for their sorting. The isolated IRTA3- (P3) and IRTA3+ (P4) cells cannot be distinguished in their CD25 vs. CD127 profiles, which is consistent with the data shown in Figs. 6 and 8. The responder Teff cells (Pl) were labeled with carboxy fluoroscein succinimidyl ester (CFSE) and added to plates with or without anti-CD3/CD28 beads.

The sorted cells (Pl, P2, P3 and P4) were added back to the labeled Teffs at ratio of 1 :2. Fig. 9B shows CFSE levels of the responder Teffs after 80 hours of culture. Without the beads stimulation, all the Teffs showed a single peak with the initial level of CFSE reflecting that CFSE had not been diluted by cell division. Stimulation of the Teffs with anti- CD3/CD28 antibodies induced cell division evidenced by the stepwise reduction in levels of CFSE in the offspring of the Teffs. Adding non-labeled Teffs (Pl) to the labeled Teffs did not block the proliferation of labeled cells. In contrast, the addition of total CD25+CD127 ^low cells (P2), IRTA3- CD25+CD127 ^low cells (P3) or IRTA3+ CD25+CD127 ^low (P4) remarkably suppressed the proliferation of the labeled Teffs. A time course experiment (Fig. 9C) and an experiment using various addback ratios (Fig. 9D) indicated that the

IRTA3- (P3) and IRTA3+ (P4) cells of the CD25+CD127 ^low population manifested similar levels of the suppressive activity to the total (P2) CD25+CD127 ^low cells. All the three populations similarly suppressed IL-2, IL-4, IL-5 and TNF-α production by the Teffs. IL-IO, was present at trace level in the growth-suppressed cultures, which confirmed that CD25-CD127 ^hl cells do not contain a significant number of adaptive Tregs. When CD25 ¹ " gating was used to identify nTregs, their IRTA3 expression was also independent on the suppressor activity.

Example 13 IRT A3 expression on nTreg cells correlates with the cell hypoproliferative property

A characteristic of nTregs is their relative inability to proliferate in vitro in response to TCR stimulation. Treg cells do not produce IL-2 and require exogenous IL-2 in addition to TCR stimulation for proliferation. IL-2 is produced by non-Treg cells in vivo and shows an essential role in Treg development and/or peripheral homeostasis. Therefore, proliferation of the sorted IRTA3+ and IRTA3- cells was examined with or without exogenous IL-2. Cells were sorted according to the gates in Fig. 9A and labeled with CFSE. Fig. 1OA shows cell division after 80 hours of culture as monitored by the dilution of intracellular CFSE. The labeled non-Treg CD25-CD127 ¹ " cells (Pl) proliferated when stimulated with anti-CD3/CD28 beads. In contrast, IRTA3- (P3), IRTA3+ (P4) or the total (P2) of CD25+CD127 ^low cells showed no proliferation in response to such stimulation. Increasing the ratio of the stimulating beads to cells did not induce proliferation of any of the three Treg populations. CD25-CD127 ¹ " Teff cells showed a similar saturating level of cell proliferation. Fig. 1OB shows the effects of exogenous IL-2 on proliferation. The anti-CD3/CD28 beads alone or IL-2 alone showed no increase in stimulated cell proliferation. In a contrast, when cells were stimulated both with anti-CD3/CD28 beads and IL-2, the IRTA3- (P3) population of the CD25+CD127 ^low cells regained the proliferation activity but the IRT A3+ (P4) population of the CD25+CD127 ^low cells did not. The total (P2) of the CD25+CD127 ^low cells showed moderate proliferation that approximately corresponded to the 2:3 ratio of IRTA3+ and IRTA3- populations in the CD25+CD127 ^low cells (Fig. 6E). Fig.lOC shows the

growth curves of the different cell populations in the presence of both of anti- CD3/CD28 beads and IL-2. Nonparallel growths of IRTA3+ and IRT A3- nTreg cells indicated that the difference of susceptibility to IL-2 between these two populations was maintained during the 5 -day culture period. These results demonstrate that IRT A3 expression on nTreg cells correlates with the hypoproliferation state in the presence of IL-2. As shown in Fig. 1OD, both IRT A3+ and IRTA3- Treg cells did not produce significant amounts of IL-2, IL-4, IL-5, IL- 10 or TNF-α upon anti-CD3/CD28 stimulation. In addition, the supplement of exogenous IL-2 also did not induce secretion of IL-4, IL-5 and TNF-α either from IRT A3+ and IRT A3- Tregs. However, IL-IO production from IRT A3- nTregs was induced by exogenous IL-2 whereas IRT A3+ nTregs didn't produce IL-10 under the same conditions. This confirms that IRT A3- Tregs in are not responsive to exogenous IL-2.

Example 14

No IRTA3 expression on induced Foxp3+ cells from CD25-CD127 ^hi non-Treg cells by TGF- β treatment in vitro.

Various ex vivo generation and expansion methods of Tregs have been actively studied partly as the clinical potential of Treg cells to control immune response in patients. TGF-β treatment of peripheral CD4+CD25- cells is one of the most extensively characterized methods to obtain the induced Treg cells (iTregs). TCR stimulation in addition to TGF-β treatment is required for induction of iTregs. IL-2 contributes to the generation of iTregs in this condition. For these reasons, non-Treg CD25-CD127 ¹¹¹ cells (Pl in Fig. 9A) were stimulated with anti-CD3/CD28 beads, TGF- β, IL-2 and their combinations for 4 days. Expression of various makers including IRT A3, Foxp3, CTLA-4, CD25 and CD 127 in CD4+ cells was monitored by multicolor cytometry analysis. As shown in the first line of panels in Fig. 12, CD25-CD127 ^hl cells maintained the same CD25- and CD127 ^hl phenotype during the incubation without any stimulation; Foxp3 as well as CD25 or CTLA-4 was not induced. In contrast, anti-CD3/CD28 beads activated CD25- CD127 ^hl cells to grow and become granular cells with higher side scatters and markedly induced CD25 and CTLA-4 expression. Distinct from these markers, Foxp3 was only

slightly induced by the activation. TGF-β treatment alone did not change phenotype of CD25-CD127 ¹¹¹ cells. However, a combination of TGF-β with anti CD3/CD28 beads converted a significant fraction of the stimulated cells to Foxp3 -positive cells. The induced Foxp3+ cells showed a marker profile similar to nTreg in PBMCs, such as high levels of CD25 and CTLA-4 and lower levels of CD127. The induced Foxp3+ cells, however, did not express IRT A3. Addition of exogenous IL-2 showed no effects on IRTA3 expression.

IRT A3 protein expresses on 40% of CD4+CD25+CD127 ^low Foxp3+ natural regulatory T cells in human peripheral blood. The IRT A3 expression was associated with CD25+CD 127 ^low phenotype rather than with Foxp3+ or with CD25 ¹ " phenotype, indicating that functional human nTregs denoted by CD4+CD25+CD127 ^low is a cell population that can be divided into two subgroups based on different levels of IRT A3 expression. One established property for nTregs is hyporesponsiveness (anergy) to TCR and coreceptor stimulation, which can be cancelled by addition of exogenous IL-2. The nTregs propagated by IL-2 temporarily lose their suppressive activity but spontaneously regain the suppressive activity once IL-2 is removed.

IRTA3- nTregs and not IRTA3+ nTregs are responsible for the IL-2- dependent proliferation, indicating that nTreg expansion and maintenance by IL-2 is associated with IRT A3- nTreg subpopulation. The generation of two nTreg populations with and without IRT A3 could be sensibly controlled in vivo to regulate nTreg expansion locally and timely. Other markers specific to IRT A3- nTregs or IRT A3+ nTregs (Fig. 13) have not been detected. High levels of Foxp3 are observed in IRTA3- nTregs and in IRTA3+ nTregs. Foxp3 plays a large part for conferring the regulatory function on Tregs by activating or repressing hundreds of genes by forming transcription complexes with other factors. The presence of IRT A3- and IRT A3+ subpopulations of Foxp3+ nTregs and their association with different response to IL-2 imply that nTregs maintenance by IL-2 in vivo is controlled in a Foxp3-independent manner. It is also questioned how IRTA3- nTregs and IRT A3+ nTregs differently respond to IL-2. The difference in IL-IO production after IL-2 stimulation indicates that IL-2 induces different sequential events between in IRTA3- nTregs and in IRTA3+ nTregs to express the

dissimilarity. The addition of the anti-IRTA3 MAb in the culture induced no detectable changes in marker phenotype of IRTA3+ nTregs. As disclosed herein, the finding that IRT A3 + nTregs is less reactive to IL-2 than IRT A3- nTreg indicates that the induced IRTA3 expression on nTregs changes the IL-2-dependent survival of nTregs in the periphery, thereby breaking the peripheral balance between nTregs and self-reactive cells toward autoimmunity.

While this disclosure has been described with an emphasis upon particular embodiments, it will be obvious to those of ordinary skill in the art that variations of the particular embodiments may be used, and it is intended that the disclosure may be practiced otherwise than as specifically described herein. Features, characteristics, compounds, chemical moieties, or examples described in conjunction with a particular aspect, embodiment, or example of the invention are to be understood to be applicable to any other aspect, embodiment, or example of the invention. Accordingly, this disclosure includes all modifications encompassed within the spirit and scope of the disclosure as defined by the following claims.