WO2008087219A1 - Compositions and methods for regulating t cell activity - Google Patents

Abstract

The present invention relates to antibodies, antibody fragments, and derivatives thereof that specifically bind to Vα7.2-Jα33 T cell receptor polypeptides present on the surface of Mucosal Associated Invariant T (MAIT) cells. The invention also relates to cells producing such antibodies; methods of making such antibodies; fragments, variants, and derivatives of the antibodies; pharmaceutical compositions comprising the same; methods of using the antibodies to purify MAIT cells and/or for diagnostic or therapeutic purposes in subjects.

Description

COMPOSITIONS AND METHODS FOR REGULATING T CELL ACTIVITY

FELD OF THE INVENTION

The present invention relates to antibodies, antibody fragments, and derivatives thereof that specifically bind to T cell receptors present on the surface of a subpopulation of T cells called MAIT, or Mucosal Associated Invariant T Cells. The invention also relates to cells producing such antibodies; methods of making such antibodies; fragments, variants, and derivatives of the antibodies; pharmaceutical compositions comprising the same; methods of using the antibodies to purify MAIT cells; and the use of such antibodies and compositions for diagnostic or therapeutic purposes in subjects.

BACKGROUND

B and T lymphocytes display a wide repertoire of antigen receptors that are produced by the random recombination of V, (D) and J segments. Two T lymphocyte subsets have been described that display conserved T cell receptors (TCR) with limited diversity. The NK-T cells, which are found in the thymus, liver, spleen and bone marrow, use an invariant α chain (hAV24AJ18 in humans, mAV14AJ18 in mice; see, e.g., Lantz et al. (1994) J. Exp. Med. 180:1097-1106; Dallabona et al. (1994) J. Exp. Med. 180:1171-1176; Bendelac A et al. 1997. Annu. Rev. Immunol. 15:535-62), a CDR3 of constant length, and a limited repertoire of Vβ segments. The second type of invariant T cells are called MAIT, or Mucosal Associated Invariant T cells (Tilloy et al. (1999) J. Exp. Med. 189:1907-1921; Treiner et al. (2003) Nature 422:164-169). MAIT cells are present in mucosal tissues such as the gut lamina propria and the lungs, and use Vα7.2-Joc33 in humans and the homologous Vαl9-Joc33 segments in mice and cattle (Tilloy et al., J. Exp. Med., 1999, 1907-1921). MAIT cells use a limited repertoire of Vβ segments: Vβl3 and Vβ2 in humans (Vβ6 and Vβ8 in mice), without restriction in the usage of Jβ segments or in the length/composition of CDR3β. MAIT cells are mostly CD4-/CD8- (double negative, or DN) or CD8αα, but rarely CD4 and never CD8αβ. They seem to be the result of oligoclonal expansions (Tilloy et al., J. Exp. Med., 1999, 1907-1921), and represent 10 to 15% of the DN lymphocytes in human peripheral blood.

As with NK T cells, MAIT cell populations require the expression of their selecting element on hematopoietic derived cells (Treiner et al. (2003) Nature 422:164-169). MAIT cells are restricted by the non-classical MHC class I molecule MRl (Treiner et al. (2003) Nature 422:164-169). It has also been shown that the expansion of MAIT cells in the gut lamina propria requires the presence of commensal flora as well as that of B cells expressing MRl (Treiner et al. (2003) Nature 422:164-169).

One of the main methods of defense against gut pathogens is the secretion of high quantities of IgA in the intestinal fluids. There are high numbers of IgA secreting cells in both Peyer's Patches (PP) and Intestinal Lamina Propria (LP) that depend upon the presence of microbial flora and are largely T cell dependent. Previous studies have shown that transgenic overproduction of MAIT cells induces the activation of LP B cells associated with higher levels of IgA in the intestinal fluids (Treiner et al. (2003) Nature 422:164-169). Indeed, it appears that MAIT cells are involved in the regulation of lamina propria B cell functions through the recognition of MRl through the secretion of lymphokines such as IFN-γ and TGFβl.

Until now, the detection and analysis of MAIT cells has been based solely on quantitative PCR techniques. These techniques, however, are limited by poor precision and are difficult to perform. There is thus a great need in the art for new tools to specifically recognize MAIT cells in humans, which would be useful, inter alia, for recognizing, purifying, enriching, or modulating the activity of these cells, in vivo or in vitro. The present invention addresses these and other needs.

SUMMARY OF THE INVENTION

The present invention provides novel compositions and methods involving antibodies and antibody fragments and derivatives that allow specific binding to human MAIT cells, in vivo or in vitro. Such compositions and methods are useful for a multitude of applications, including purifying human MAIT cells, specifically labeling them in vitro or in vivo, e.g. for diagnostic purposes, and modulating their activity, e.g., for the treatment or prevention of infections or of mucosal immune disorders.

Accordingly, in one aspect, the present invention provides an isolated antigen binding agent that specifically binds an epitope present on a Vα7.2-Joc33 polypeptide. Preferably the agent does not substantially bind an epitope present on a mouse Vαl4 or Vαl9, or human Vα2, Vαl 1 or Voc24 polypeptide. The antigen binding agent may advantageously be an antibody or other peptide or polypeptide-based agent; such agents are generally known and can be prepared by providing a test agent obtained using any suitable method and testing the agent as to whether it binds to an epitope present on a Vα7.2-Joc33 polypeptide. The latter testing step can be advantageously performed using e.g. the 3C10 or 1A6 antibodies and the methods described herein. While the present application will refer to antibodies that bind a Vα7.2-Joc33 polypeptide, it will be appreciated that the term antibody can be replaced by the term antigen binding agent. In one embodiment, the antibody or agent binds to substantially the same epitope as monoclonal antibody 3C10 (also referred to as 3C10A8) or 1A6. In another embodiment, the antibody is a monoclonal antibody or a fragment or derivative of a monoclonal antibody. In a particularly preferred embodiment, the antibody is the monoclonal antibody is 3C10 or 1A6 or a fragment or derivative thereof. In another embodiment, the Vα7.2-Joc33 polypeptide is a human Vα7.2-Joc33 polypeptide. In another embodiment, the antibody or agent specifically binds to human or non- human primate MAIT cells. In another embodiment, the antibody or agent increases the activity of human or non-human primate MAIT cells. In another embodiment, the antibody or agent decreases the activity of human or non-human primate MAIT cells. In one embodiment, the antibody or agent leads to the elimination or killing of MAIT cells, either directly (e.g. an antibody comprising a cytotoxic moiety) or indirectly (e.g. an antibody capable of inducing ADCC or CDC toward a MAIT cell to which it is bound.

In further embodiments, the invention encompasses a cell producing any of the antibodies of the invention. In one embodiment, the cell is a recombinant host cell made to express the antibody or other antigen binding agent of the invention (e.g. a cell transformed with a DNA encoding the antibody or agent). In one embodiment, the cell is the hybridoma having CNCM registration number 1-3693. The hybridoma producing antibody 3C10A8 was deposited on November 15th, 2006 at the Collection Nationale de Culture de Microorganismes, Institute Pasteur, 25, Rue du Docteur Roux, F-75725 Paris, France, under reference identification number 3C10A8 and registration number CNCM 1-3693.

In another embodiment, the antibody is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, F (ab') 2, Fv, diabodies, single-chain antibody fragments, and multispecifϊc antibodies comprising multiple different antibody fragments. In another embodiment, the antibody is a humanized antibody or a chimeric antibody. In another embodiment, the antibody is conjugated or covalently bound to a toxin, or radio-isotopes, a detectable moiety, or a solid support.

In another aspect, the present invention provides a hybridoma comprising: a) a B cell from a non-human mammalian host that has been immunized with an antigen that comprises an epitope present on a Vα7-Joc33 polypeptide, fused to b) an immortalized cell, wherein the hybridoma produces a monoclonal antibody that specifically binds to the epitope.

In one embodiment, the monoclonal antibody is capable of specifically binding to or modulating the activity of MAIT Cells. In one embodiment, the antibody is an agonist antibody. Preferably an agonist antibody stimulates the activity of MAIT cells; optionally said agonist antibody increases the production of cytokines (e.g. ILlO, RANTES, TNF and/or an interferon) by MAIT cells. In a particularly preferred embodiment, the hybridoma produces a monoclonal antibody that binds to substantially the same epitope as monoclonal antibody 3ClO or 1A6. In another embodiment, the hybridoma produces a monoclonal antibody 3C10 or 1A6. In another embodiment, the antibody is an antibody produced by the hybridoma having CNCM registration number 1-3693.

In another aspect, the present invention provides a method of producing an antibody that specifically binds MAIT Cells, the method comprising the steps of : a) immunizing a non- human mammal with an immunogen comprising a Vα7-Joc33 polypeptide; and b) preparing antibodies from said immunized animal that bind said Vα7-Joc33 polypeptide.

In one embodiment, the antibody prepared in step (b) is a monoclonal antibody. In another embodiment, the non-human animal is a rabbit or a mouse. In another embodiment, the method further comprises the steps: c) selecting antibodies of (b) that are capable of modulating the activity of MAIT cells. In another embodiment, the antibodies selected in step (c) cause an increase or decrease in MAIT cell activity of at least about 10%, 20%, 30%, 40%, 50%, or more. In another embodiment, the polypeptide is a human polypeptide.

In another aspect, the present invention provides a pharmaceutical composition comprising an antibody that specifically binds a Vα7-Joc33 polypeptide, wherein the antibody is present in an amount effective to detectably modulate MAIT cell activity in a patient or in a biological sample comprising MAIT cells; and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the composition further comprises a therapeutic agent selected from an immunomodulatory agent, a hormonal agent, a chemotherapeutic agent, an anti-angiogenic agent, an apoptotic agent, a second antibody that binds to and inhibits an MAIT receptor, an anti- infective agent, a targeting agent, an anti- inflammation drug, a steroid, an immune system suppressor, an antibiotic, an anti-diarrheal drug, and an adjunct compound. In another embodiment, the antibody increases the activity of human MAIT cells. In another embodiment, the antibody decreases the activity of human MAIT cells. In another embodiment, the Vα7.2- Joc33 polypeptide is a human Vα7.2-Joc33 polypeptide.

In another aspect, the present invention provides a composition comprising an antibody that specifically binds a Vα7.2-Joc33 polypeptide, wherein the antibody is capable of modulating MAIT cell activity, and wherein said antibody is incorporated into a liposome.

The present invention also provides kits comprising any of the herein-described antibodies or compositions, optionally also including instructions for their use, e.g., based on the present methods.

The invention also provides methods of regulating human MAIT cell activity in vitro, ex vivo, or in vivo, comprising contacting human MAIT cells with an effective amount of an anti-

Voc7.2-Jα33 antibody of the invention, a fragment of such an antibody, a derivative of either thereof, or a pharmaceutical composition comprising at least one of any thereof. Preferred methods comprise the administration of an effective amount of a pharmaceutical compositions of this invention and are directed at increasing or decreasing the activity of human MAIT cells, most preferably ex vivo or in vivo, in a subject having a cancer, an infectious disease, an immune disease involving the mucosa, Crohn's Disease, ulcerative colitis, irritable bowel disease, or Celiac disease.

In another aspect, the present invention provides a method of modulating MAIT cell activity in a patient in need thereof, comprising the step of administering to the patient any of the anti- Voc7.2-Jα33 antibodies or pharmaceutical compositions of the invention. In one embodiment, the composition enhances the activity of the MAIT cells. In one embodiment, the composition induces the proliferation of the MAIT cells. In one embodiment, the composition induces the production of TNF, RANTES, and/or IL- 10 by MAIT cells, or the expression of CD69 on MAIT cells. In another embodiment, the composition inhibits the activity of the MAIT cells. In one embodiment, the composition leads to the depletion of MAIT cells.

In one embodiment, the patient is suffering from a mucosal immunological disorder. In another embodiment, the disorder is selected from the group consisting of cancer, infection, Crohn's disease, ulcerative colitis, irritable bowel disease, and Celiac disease.

In another embodiment, the method further comprises the step of administering to the patient an appropriate additional therapeutic agent selected from the group consisting of an immunomodulatory agent, a hormonal agent, a chemotherapeutic agent, an anti- angiogenic agent, an apoptotic agent, a second antibody that binds to and modulates a receptor present on MAIT cells, an anti- infective agent, a targeting agent, an anti- inflammation drug, a steroid, an immune system suppressor, an antibiotic, an anti- diarrheal drug, and an adjunct compound.In another aspect, the present invention provides a method of detecting the presence of MAIT cells in a biological sample or a patient, the method comprising the steps of a) contacting the biological sample or patient with any of the anti-Voc7.2-Joc33 antibodies of the invention, under conditions that would allow binding of the antibodies to Vα7.2-Joc33 epitopes present in the sample or patient; and b) detecting the presence of the bound antibody in the biological sample or patient.

In one embodiment, the antibody is conjugated or covalently bound to a detectable moiety.

In another aspect, the present invention provides a method of purifying MAIT cells from a biological sample, the method comprising the steps of: a) contacting the sample with any of the anti-Voc7.2-Joc33 antibodies of the invention under conditions that allow the cells to bind to the antibody, wherein the antibody is conjugated or covalently bound to a solid support; and b) eluting the bound cells from the solid support-bound antibody. In another, related, aspect, the present invention provides a method of purifying MAIT cells from a biological sample, the method comprising: a) providing a fluorescently-labeled anti-Voc7.2-Joc33 antibody of the present invention and a biological sample, and b) using the labeled antibody to purify cells displaying Vα7.2-Joc33 from the biological sample by FACS sorting.

In one embodiment of either of these aspects, the biological sample is a blood sample. In another embodiment, the biological sample is a tissue biopsy comprising mucosal tissue. In another embodiment, the mucosal tissue is gut, gut lamina propria, or lung. In another embodiment, the biological sample is taken from a patient, and the cells are purified for diagnostic purposes. In another embodiment, the biological sample is taken from a healthy individual.

In another aspect, the invention provides a method for identifying an antibody that is efficacious in the treatment of a mucosal immunological disorder by administering an anti-Voc7.2-Joc33 antibody of the invention to a non-human primate model of the disorder and assessing the ability of the antibody to prevent or ameliorate the disorder, or a symptom thereof, or to modulate the activity of MAIT cells in the primate.

In another aspect, the present invention provides that the anti-Voc7.2-Joc33 agents or antibodies of the invention can be used in any of the methods for the detection, depletion, purification or modulation of activity of MAIT cells, in vitro or in vivo.

The invention also provides several additional markers of MAIT cells that can be used separately or in combination with each other, and/or separately or in combination with the anti- Voc7.2-Jα33 agents or antibodies of the invention, in any methods of detection, depletion, purification or modulation of the activity of MAIT cells, in vitro or in vivo. While the markers (other than the anti-Voc7.2-Joc33 agents) are not individually specific for MAIT cells, agents that detect each marker can nevertheless be useful in methods to detection, depletion, purification or modulation of the activity of MAIT cells, generally in combination with a second agent specific for a second marker. The following markers were positive on both flow cytometry and affymetrix data: 2B4 (CD244), CD161, CD26, CD28, CD94, CD96, IL12Rb (CD212), IL18Ra (CD218b), and NKG2D (CD314). These human polypeptide are well known in the art and their nucleic acid and amino acid sequences, methods of preparing them, as well as antibody reagents that recognize them are available commercially or can be readily prepared. CD antigen designation are well known, for example the full list of CD molecules may be accessed through the 8th HLDA Workshop (HLDA8) website fwww.hlda8.org). in Science (2006) 313(5792), or at Expasy.org (see http://www.expasy.org/cgi-bin/lists7cdlist.txt), the disclosures of which are incorporaed herein by reference. The following additional markers were positive in flow cytometry and not negative on micro-arrays: CD122, CD127, CD27 and CD45RO. Agents that specifically bind such polypeptide markers are therefore optimally used in combination; preferred methods for detecting, depleting, purifying, or modulating the activity of MAIT cells, in vitro or in vivo, comprise the use of two, three, four, five or more agents selective for a polypeptides selected from the group consisting of 2B4, CDl 61, CD26, CD28, CD94, CD96, IL12Rb, ILl 8Ra, and NKG2D; optionally the group also includes N CD122, CD127, CD27 and CD45RO. Preferably, the methods further comprise the combined use of an agent selective for a Voc7.2-Jα33 polypeptide. The agents which recognize the aforementioned additional markers are preferably antibodies and can generally be used in the same way as anti-Vα7.2-Joc33 polypeptide described herein to detect, deplete, purify or modulate the activity of MAIT cells.

The invention therefore provides a method of purifying MAIT cells from a biological sample, said method comprising the steps of : a) contacting said sample with an agent that binds a human Vα7.2-Joc33 polypeptide and/or an agent that binds a polypeptide selected from the group consisting of 2B4, CD161, CD26, CD28, CD94, CD96, ILl 2Rb, ILl 8Ra, NKG2D, CD 122, CD 127, CD27 and CD45RO, under conditions that allow said cells to bind to said agent, optionally wherein said agent is conjugated or covalently bound to a solid support; and b) selecting cells bound to said agent, optionally eluting said bound cells from said agent conjugated or covalently bound to a solid support. Also provided is a method of profiling a cell population (e.g. a MAIT cell population), or a method of identifying a MAIT cell, comprising a step of detecting in said cell population the presence or absence of Vα7.2-Joc33 polypeptide. The method may further comprising detecting in said cell population the presence or absence of a polypeptide selected from the group consisting of: 2B4, CDl 61, CD26, CD28, CD94, CD96, ILl 2Rb, ILl 8Ra, NKG2D, CD122, CD127, CD27 and CD45RO. The determination that the aforementioned polypeptide(s) is present will identify the cell as a MAIT cell or candidate MAIT cell.

The invention also provides a population of cells obtained according to any of the methods described herein. In one aspect, the invention provides an isolated population of MAIT cells, wherein said cell population being characterized by purity of at least 50%, 75%, 80%, 90%, 95%, 98% or 99% of Vα7.2-Joc33 polypeptide-expressing cells. In another aspect, the invention provides an isolated population of MAIT cells, optionally wherein the population of cells is selected with regard to a Vα7.2-Joc33 polypeptide, optionally using an anti- a Vα7.2-Joc33 antibody of the invention, wherein said cell population being characterized by purity of at least 50%, 75%, 80%, 90%, 95%, 98% or 99% of Vα7.2-Joc33 polypeptide-expressing cells, optionally cells bound by an agent specific for a Vα7.2-Joc33 polypeptide. Purity is preferably assessed by cell counting method more preferably by a FACS method. The present invention is preferably directed to purity of a cell preparation being a cell count based purity. The cell count based purity indicates the portion of number of pure (e.g. being bound by an agent specific for a Voc7.2-Jα33 polypeptide) from the total number of cells.

The present invention also provides isolated or purified MAIT cell compositions, wherein the MAIT cells are bound by two, three, four, five or more agents selective for a polypeptide selected from the group consisting of 2B4, CD161, CD26, CD28, CD94, CD96, ILl 2Rb, ILl 8Ra, and NKG2D; optionally the group also includes CD122, CD127, CD27 and CD45RO. Preferably, the MAIT cells are also bound an agent selective for a Vα7.2-Joc33 polypeptide.

These and additional advantageous aspects and features of the invention may be further described elsewhere herein.

BRIEF DESCRIPTION OF THE DRAWINGS

3C10A8 is a subclone of 3C10 and the name 3C10 or 3C10A8 will be used interchangeably herein.

Figure 1 shows the method by which mouse anti-Vα7.2-Joc33 antibodies were obtained.

Figure 2 shows the strategy for the primary screening. The example shown was not selected for further study as it was positive also on B cells and no staining was observed on DN CD3 T cells.

Figure 3 shows the results of a FACS analysis carried out during the secondary screening allowing the identification of four types of reactivity by studying four different patients and antibodies to CD4, CD3, CD8 and the supernatant. One hybridoma of each category of reactivity was selected for further study. The results for one of the hybridoma are shown.

Figure 4 shows the Multicolor FACS strategy used to characterize the supernatant reactivity .

Figure 5 shows the strategy of FACS sorting experiment using a labeling with anti- CD3, TCRγδ, CD4, CD8, and the supernatant allowing the sorting of cells expressing the epitope recognized by the SN among the CD4, CD8 and DN cells The RNA is then extracted and retro transcribed, and the results are shown in Figure 6.

Figure 6 shows the results of quantitative RT PCR in positive or negative 3C10 fractions from DN, CD4 or CD8 TCRαβ T cells allowing the demonstration of the anti-Voc7.2 specificity of this supernatant. Figure 7 shows the results of a FACS analysis to examine the proportion of Vβ2, 8 and 13 among the different SN+ fractions. It is shown that the DN recognized by the anti-Voc7.2 antibody candidates have an increased proportion of Vβ2 cells in accordance with the results found previously in Tilloy et al, 1999, JEM.

Figure 8 shows 3C10 binding to the following human Vα7.2 transfectants: 58α-β-, 3Cl 1 (huVα7.2+mVβl3), αDPβDP (mVα8+mVβl3), ICElO (huVα7.2+ mVβ8.2) and αDPβDP (mVα4+mVβ8.2). We observed that antibody 3C10 recognizes transfectants 3Cl 1 and ICElO which both express the Vα7.2 chain.

Figure 9 shows multicolor FACS analysis on gated CD4, CD8 and DN TCRαβ T cells with 3C10 on one hand and anti-Voc2/Val2/Voc24 on the other hand to show that the 3C10 antibody is not an anti-Vα framework antibody. This experiment revealed that 3C10 did not recognize Vα2, 12 nor Vα24. Together with the other data this demonstrates that 3C10 is specific for

Vα7.2.

Figure 10 shows the analysis of Va repertoire after sorting CD4, CD8 or DN TCRαβ T cells into 3C10 positive or negative fractions. Binding of antibody 3C10 to the different fractions was followed by quantitative RT-PCR using a battery of Va primers to determine the identity of the Va chain expressed by the T cells. It is shown that Vα7.2 is highly enriched in the 3C10 positive fractions without much other Va remaining. Panel 1OA displays the results on CD8 and CD4 fractions. Panel 1OB displays the results on DN T cells.

Figure 11 shows the principle of the sequence analysis of Vα7 and Vαl 1 amplicons derived from 3Cl 0+ DN TCRαβ T cells demonstrating that indeed 3C10 is specific for Vα7 segments as the sequencing found that the putative Vαl 1 amplicons were in fact Vα7 in the 3Cl 0+ fraction. These results indicate that the Vαl 1 primer is cross-reactive with Vα7 segment.

Figure 12 shows that 3C10 has an agonist effect on Ly T CD4-, CD8- cells and on CD3+, γδ-, CD4-, CD8- cells; anti-CD28 antibody used in combination, as demonstrated by a specific dilution of CFSE in the 3C10+ fraction after stimulation by 3C10 and anti-CD28 coated to plastic.

Figure 13 shows the characterization of the cytokine secretion pattern of 3C10 positive and negative DN, CD4, CD8 T cells after stimulation by anti-CD3 and anti-CD28 antibody.

Figure 14 shows the amino acid sequence of human Vα7.2-Jα33 polypeptide as well as mouse and cattle equivalents. Figure 15 shows example of surface staining on frozen section of human thymus.

Figure 16 shows the nucleotide and polypeptide sequence of the heavy chain variable regions of the 3C10 and 1A6 monoclonal antibodies, with the CDR regions indicated. Also indicated are the differences between the two antibodies.

Figure 17 shows the nucleotide and polypeptide sequence of the light chain variable regions of the 3C10 and 1A6 monoclonal antibodies, with the CDR regions indicated. Also indicated are the differences between the two antibodies.

DETAILED DESCRIPTION OF THE INVENTION Introduction

The present invention provides novel methods for producing and using antibodies and other compounds suitable for the treatment of mucosal immune disorders such as autoimmune and inflammatory disorders. Antibodies, antibody derivatives, antibody fragments, and hybridomas are encompassed, as are methods of producing the same and methods of treating patients using the antibodies and compounds.

The present invention is based, in part, on the discovery that antibodies can be generated that specifically and efficaciously bind to MAIT cells, particularly Vα7.2-Joc33 TCR alpha chains present on MAIT cells, and furthermore than such antibodies can be obtained that are MAIT cell agonists. In addition to antibodies, antibody fragments, antibody derivatives, hybridomas, and methods of making each of these, the present invention also provides methods of purifying MAIT cells, as well as methods of treating and diagnosing MAIT cell-related disorders. Such methods involve first, optionally detecting the prevalence of cells expressing the Vα7.2-Joc33 polypeptide, and then, if appropriate, administering one or more present antibodies that can modulate the activity of or otherwise target the Vα7.2-Joc33 polypeptide. In this way, the function of the cells can be either enhanced when antibodies are used that stimulate the Vα7.2- Jα33 -comprising TCR, or inhibited when antibodies are used that block activation of the receptor; alternatively, derivatized antibodies can be used to kill the cells by virtue of a toxic moiety conjugated to the antibody.

Definitions

As used herein, "T" cells refers to a sub-population of lymphocytes that mature in the thymus, and which display, among other molecules, T cell receptors on their surface. T cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including the TCR, CD4 or CD8, the ability of certain T cells to kill tumor or infected cells, the ability of certain T cells to activate other cells of the immune system, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify T cells, using methods well known in the art.

The term "MAIT" cells, or "Mucosal- Associated Invariant T cells" refers to a population of T cells present in mammals, preferably humans, that display an invariant TCR alpha chain comprising Vα7.2-Joc33 (in humans), a CDR3 of constant length, and a limited number of Vβ segments together with an activated phenotype (CD44) (see, e.g., Lantz and Bendelac. 1994. J. Exp Med. 180:1097-106; Tilloy et al., J. Exp. Med., 1999, 1907-1921; Treiner et al. (2003) Nature 422:164-169, the entire disclosures of each of which are herein incorporated by reference). MAIT cells are generally CD4⁺ or CD47CD8^' (DN) or CD8αα in humans, and are restricted by the non-classical MHC class I molecule MRl. In terms of localization, MAIT cells are generally absent from the thymus, liver, spleen and bone marrow, but are abundant in the gut lamina-propria (LP), the mesenteric lymph nodes (MLN), and in other mucosal tissues, such as the lungs. For the purposes of the present invention, any T cells that express the invariant Voc7.2-Jα33 alpha TCR chain are considered to be MAIT cells. Preferably, the alpha chain is associated with an invariant CDR3 and with either Vβ2 or Vβl3. Also preferably, the MAIT cells are present in a mucosal tissue, such as the gut — or more specifically, the gut lamina propria — the mesenteric lymph nodes, or the gut.

Voc7.2-Jα33 is a T cell receptor expressed by MAIT cells. As used herein, "Vα7.2-Joc33" inlcudes any variant, derivative, or isoform of therearranged Vα7.2-Joc33 gene or encoded protein. The amino acid sequence of human and mouse Vα7.2-Joc33 are described in Tilloy et al. (1999) J. Exp. Med. 189(12): 1907-1921, the disclosure of which is incorporated herein by reference. Sequences of human and mouse are also shown in Figure 14, reproduced from Tilloy et al. and in SEQ ID NOS 17, 18 and 19 for human, mouse and cattle amino acid sequences, respectively. There is some variability at the junction between the Vα7.2 and Jα33 segments with variability of the sequence. The consensus sequence starting at amino acid position 89 to position 93 in the human sequence in Figure 14 is therefore CAXXD, wherein X is any amino acid.

Also encompassed are any nucleic acid or protein sequences sharing one or more biological properties or functions with wild type, full length Vα7.2-Joc33, and sharing at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or higher nucleotide or amino acid identity.

"Autoimmune" disorders include any disorder, condition, or disease in which the immune system mounts a reaction against self cells or tissues, due to a breakdown in the ability to distinguish self from non-self or otherwise. Examples of autoimmune disorders include, but are not limited to, Celiac disease, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, Grave's disease, polymyositis, Guillain Barre, Wegener's granulomatosus, polyarteritis nodosa, polymyalgia rheumatica, temporal arteritis, Bechet's disease, Churg-Strauss syndrome, Takayasu's arteritis, and others. Autoimmune disorders can involve any component of the immune system, and can target any cell or tissue type in the body.

"Inflammatory diseases" refer to any disorder, condition, or disease characterized or caused by excessive or uncontrolled inflammation, or any aspect of inflammation such as redness, swelling, heat, pain, etc. Inflammatory diseases include, but are not limited to, irritable bowel disease, Crohn's disease, ulcerative colitis, allergies, including allergic rhinitis/sinusitis, skin allergies such as urticaria/hives, angioedema, atopic dermatitis, food allergies, drug allergies, insect allergies, and rare allergic disorders such as mastocytosisasthma, asthma, arthritis, including osteoarthritis, rheumatoid arthritis, and spondyloarthropathies, gastrointestinal inflammation, neuroinflammatory disorders, and autoimmune disorders.

The terms "decreasing," "inhibiting," and "downregulating," with respect to Vα7.2-Joc33- expressing cells means a process, method, or compound that can slow down, reduce, reverse, or in any way negatively affect the activity or number of Vα7.2-Joc33 receptors or of cells expressing Vα7.2-Joc33. These terms can refer, e.g., to compounds such as antibodies that inhibit the stimulation of Vα7.2-Joc33 by a ligand, e.g., MRl or MRl -associated ligand, that act antagonistically in the absence of a ligand to decrease the activity of the receptor, that decrease the expression level of the receptor, that block Voc7.2-Joc33-triggered signaling or gene expression, or that block any other activity, e.g., proliferation, cytokine production, or B cell stimulation, of the Voc7.2-Joc33-displaying. In a preferred embodiment, the inhibiting compound or method blocks the binding of Vα7.2-Joc33 by a ligand, e.g. by binding to Vα7.2-Joc33 on the cell surface and preventing ligand access. The number of Vα7.2-Joc33 receptor molecules, or the number or activity of Vα7.2-Jα33-presenting cells, can be measured in any of a number of ways, as described elsewhere in the present specification.

The term "antibody," as used herein, refers to polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chains, antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. Several of these are further divided into subclasses or isotypes, such as IgGl, IgG2, IgG3, IgG4, and the like. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The terms variable light chain (V_L) and variable heavy chain (V_H) refer to these light and heavy chains respectively. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are termed "alpha," "delta," "epsilon," "gamma" and "mu," respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. IgG and/or IgM are the preferred classes of antibodies employed in this invention, with IgG being particularly preferred, because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting. Preferably the antibody of this invention is a monoclonal antibody. Particularly preferred are humanized, chimeric, human, or otherwise-human-suitable antibodies. "Antibodies" also includes any fragment or derivative of any of the herein described antibodies.

The term "specifically binds to" means that an antibody can bind preferably in a competitive binding assay to the binding partner, e.g. Vα7.2-Joc33, as assessed using either recombinant forms of the proteins, epitopes therein, or native proteins present on the surface of isolated T or MAIT or other target cells. Competitive binding assays and other methods for determining specific binding are further described below and are well known in the art.

When an antibody or agent is said to "compete" or "bind to substantially the same epitope" as a particular monoclonal antibody (e. g. 3C10, 1A6), it means that the antibody or agent competes with the monoclonal antibody in a binding assay using either recombinant Vα7.2-Joc33 molecules or surface expressed Vα7.2-Joc33 molecules. For example, if a test antibody or agent reduces the binding of 3C10 or 1A6 to a Vα7.2-Joc33 polypeptide in a binding assay, the antibody or agent is said to "compete" with 3C10 or 1A6, respectively.

By "immunogenic fragment," it is herein meant any polypeptidic or peptidic fragment that is capable of eliciting an immune response such as (i) the generation of antibodies binding said fragment and/or binding any form of the molecule comprising said fragment, including the membrane-bound receptor and mutants derived therefrom, (ii) the stimulation of a T-cell response involving T-cells reacting to the bi-molecular complex comprising any MHC molecule and a peptide derived from said fragment, (iii) the binding of transfected vehicles such as bacteriophages or bacteria expressing genes encoding mammalian immunoglobulins.

Alternatively, an immunogenic fragment also refers to any construction capable of eliciting an immune response as defined above, such as a peptidic fragment conjugated to a carrier protein by covalent coupling, a chimeric recombinant polypeptide construct comprising said peptidic fragment in its amino acid sequence, and specifically includes cells transfected with a cDNA of which sequence comprises a portion encoding said fragment. "Toxic" or "cytotoxic" peptides or small molecules encompass any compound that can slow down, halt, or reverse the proliferation of cells, decrease their activity in any detectable way, or directly or indirectly kill them. Preferably, toxic or cytotoxic compounds work by directly killing the cells, by provoking apoptosis or otherwise. As used herein, a toxic "peptide" can include any peptide, polypeptide, or derivative of such, including peptide- or polypeptide- derivatives with unnatural amino acids or modified linkages. A toxic "small molecule" can includes any toxic compound or element, preferably with a size of less than 10 kD, 5 kD, 1 kD, 750 D, 600 D, 500 D, 400 D, 300 D, or smaller.

A "human-suitable" antibody refers to any antibody, derivatized antibody, or antibody fragment that can be safely used in humans for, e.g. the therapeutic methods described herein. Human- suitable antibodies include all types of humanized, chimeric, or fully human antibodies, or any antibodies in which at least a portion of the antibodies is derived from humans or otherwise modified so as to avoid the immune response that is generally provoked when native non- human antibodies are used.

For the purposes of the present invention, a "humanized" or "human" antibody refers to an antibody in which the constant and variable framework region of one or more human immunoglobulins is fused with the binding region, e.g. the CDR, of an animal immunoglobulin. Such antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding regions are derived, but to avoid an immune reaction against the non- human antibody. Such antibodies can be obtained from transgenic mice or other animals that have been "engineered" to produce specific human antibodies in response to antigenic challenge (see, e.g., Green et al. (1994) Nature Genet 7:13; Lonberg et al. (1994) Nature 368:856; Taylor et al. (1994) Int Immun 6:579, the entire teachings of which are herein incorporated by reference). A fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art (see, e.g., McCafferty et al. (1990) Nature 348:552-553). Human antibodies may also be generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incorporated in their entirety by reference).

A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity. The terms "isolated", "purified" or "biologically pure" refer to material that is substantially or essentially free from components which normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

The term "biological sample" as used herein includes but is not limited to a biological fluid (for example serum, lymph, blood), cell sample, or tissue sample (for example bone marrow or tissue biopsy including mucosal tissue such as from the gut, gut lamina propria, or lungs).

The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

Within the context of this invention, the term antibody that "binds" a common determinant designates an antibody that binds said determinant with specificity and/or affinity.

Producing Anti-Vα7.2-Jα33 Antibodies

The antibodies of this invention specifically bind to Vα7.2-Joc33 polypeptides, e.g., Vα7.2-Joc33 polypeptides on the surface of human MAIT cells. The ability of the antibodies to bind Vα7.2- Joc33 polypeptides makes them useful for numerous applications, e.g., purifying human or other primate MAIT cells, or specifically labeling human or other primate MAIT cells in vitro, in vivo, or ex vivo. The ability to specifically purify and label MAIT cells is useful for, inter alia, diagnostic purposes. In certain embodiments, the antibodies also modulate the activity of the TCRs comprising the Vα7.2-Joc33 polypeptides, and thereby modulate the activity of the MAIT cells. Depending on the antibody (or fragment or derivative thereof) that is used, such modulating antibodies can either increase or decrease the activity of the MAIT cells. . It will be appreciated that certain antibodies (e.g. 3C10, 1A6) are capable of increasing the activity of MAIT cells, preferably comprising a constant region capable of crosslinking receptors (e.g. human IgGl subtype). Such antibodies can readily be modified so as to have properties of decreasing MAIT function, for example in a format that does not crosslink (e.g. fragments, human IgG4). Antibodies may also bind MAIT cells and result in their depletion, e.g. by including a toxic moiety or by mediating depletion of the antibody-bound MAIT by cellular effectors.

As such, the present antibodies are useful for, inter alia, treating or preventing immune conditions involving the mucosa, e.g., conditions resulting from an increase or decrease in the number or activity of MAIT cells, or conditions that can be prevented or ameliorated by increasing or decreasing the number or activity of MAIT cells.

In a preferred embodiment, the invention provides an antibody that binds the human Vα7.2- Joc33 TCR alpha chain, modulates the activity of MAITs, and competes with monoclonal antibody 3C10 or 1A6 for binding to human Vα7.2-Joc33. Optionally, said antibody is a chimeric, human, or humanized antibody. Depending on the antibodies or particular derivative or fragment used, the antibodies of the invention can either increase or decrease the activity of MAIT cells.

In an advantageous aspect, the invention provides an antibody that competes with monoclonal antibody 3C10 or 1A6 and recognizes, binds to, or has immunospecificity for substantially or essentially the same, or the same, epitope or "epitopic site" on a Vα7.2-Joc33 molecule as the monoclonal antibody 3C10 or 1A6. In other embodiments, the antibody competes with a monoclonal antibody 3C10 or 1A6. In other embodiments, the monoclonal antibody consists or, or is a derivative or fragment of, an antibody 3C10 or 1A6.

It will be appreciated that the present antibodies can recognize and be raised against any part of the Voc7.2-Jα33 (or, e.g., Vα7.2-Jα33/Vβ2 or Vα7.2-Jα33/Vβl3) polypeptide. For example, Voc7, Voc7.2, Joc33, fragments thereof, or any combination of any of these polypeptides or fragments, can be used as immunogens to raise antibodies, and the antibodies of the invention can recognize epitopes at any location within the Vα7.2-Joc33 (or, e.g., Vα7.2-Jα33/Vβ2 or Vα7.2-Jα33/Vβl3) polypeptide. Preferably, the recognized epitopes are present on the cell surface, i.e. they are accessible to antibodies present outside of the cell. As discussed elsewhere in the specification, antibodies recognizing distinct epitopes within Va7.2-Ja33 can be used in combination, e.g. to bind to Vα7.2-Joc33 polypeptides with maximum efficacy and breadth among different individuals.

The antibodies of this invention may be produced by a variety of techniques known in the art. Typically, they are produced by immunization of a non-human animal, preferably a mouse, with an immunogen comprising a Vα7.2-Joc33 polypeptide, preferably a human Vα7.2-Jα33 polypeptide. The Vα7.2-Joc33 polypeptide may comprise the full length sequence of a human

Voc7.2-Jα33 polypeptide, or a fragment or derivative thereof, typically an immunogenic fragment, i.e. , a portion of the polypeptide comprising an epitope exposed on the surface of cells expressing a Vα7.2 receptor, preferably MAIT cells. Such fragments typically contain at least about 7 consecutive amino acids of the mature polypeptide sequence, even more preferably at least about 10 consecutive amino acids thereof. Fragments typically are essentially derived from the extra-cellular domain of the receptor. In a preferred embodiment, the immunogen comprises a wild-type human Vα7.2 Jα33 polypeptide in a lipid membrane, typically at the surface of a cell. In a specific embodiment, the immunogen comprises intact MAIT cells, particularly intact human MAIT cells, optionally treated or lysed. In another preferred embodiment, the polypeptide is a recombinant Vα7.2Jα33/Vβl3 polypeptide.

The step of immunizing a non-human mammal with an antigen may be carried out in any manner well known in the art for stimulating the production of antibodies in a mouse (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988), the entire disclosure of which is herein incorporated by reference). The immunogen is suspended or dissolved in a buffer, optionally with an adjuvant, such as complete Freund's adjuvant. Methods for determining the amount of immunogen, types of buffers and amounts of adjuvant are well known to those of skill in the art and are not limiting in any way on the present invention. These parameters may be different for different immunogens, but are easily elucidated.

Similarly, the location and frequency of immunization sufficient to stimulate the production of antibodies is also well known in the art. In a typical immunization protocol, the non-human animals are injected intraperitoneally with antigen on day 1 and again about a week later. This is followed by recall injections of the antigen around day 20, optionally with adjuvant such as incomplete Freund's adjuvant. The recall injections are performed intravenously and may be repeated for several consecutive days. This is followed by a booster injection at day 40, either intravenously or intraperitoneally, typically without adjuvant. This protocol results in the production of antigen-specific antibody-producing B cells after about 40 days. Other protocols may also be utilized as long as they result in the production of B cells expressing an antibody directed to the antigen used in immunization.

For polyclonal antibody preparation, serum is obtained from an immunized non-human animal and the antibodies present therein isolated by well-known techniques. The serum may be affinity purified using any of the immunogens set forth above linked to a solid support so as to obtain antibodies that react with Vα7.2 Jα33 receptors.

In an alternate embodiment, lymphocytes from an unimmunized non-human mammal are isolated, grown in vitro, and then exposed to the immunogen in cell culture. The lymphocytes are then harvested and the fusion step described below is carried out.

For monoclonal antibodies, the next step is the isolation of splenocytes from the immunized non-human mammal and the subsequent fusion of those splenocytes with an immortalized cell in order to form an antibody-producing hybridoma. The isolation of splenocytes from a non- human mammal is well-known in the art and typically involves removing the spleen from an anesthetized non-human mammal, cutting it into small pieces and squeezing the splenocytes from the splenic capsule and through a nylon mesh of a cell strainer into an appropriate buffer so as to produce a single cell suspension. The cells are washed, centrifuged and resuspended in a buffer that lyses any red blood cells. The solution is again centrifuged and remaining lymphocytes in the pellet are finally resuspended in fresh buffer.

Once isolated and present in single cell suspension, the lymphocytes can be fused to an immortal cell line. This is typically a mouse myeloma cell line, although many other immortal cell lines useful for creating hybridomas are known in the art. Preferred murine myeloma lines include, but are not limited to, those derived from MOPC-21 and MPC-11 mouse tumors available from the SaIk Institute Cell Distribution Center, San Diego, U. S. A. , X63 Ag8653 and SP-2 cells available from the American Type Culture Collection, Rockville, Maryland U. S. A. The fusion is effected using polyethylene glycol or the like. The resulting hybridomas are then grown in selective media that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT- deficient cells.

Hybridomas are typically grown on a feeder layer of macrophages. The macrophages are preferably from littermates of the non-human mammal used to isolate splenocytes and are typically primed with incomplete Freund's adjuvant or the like several days before plating the hybridomas. Fusion methods are described in Goding, "Monoclonal Antibodies: Principles and Practice," pp. 59-103 (Academic Press, 1986), the disclosure of which is herein incorporated by reference.

The cells are allowed to grow in the selection media for sufficient time for colony formation and antibody production. This is usually between about 7 and about 14 days.

The hybridoma colonies are then assayed for the production of antibodies that specifically bind to Voc7.2-Jα33 receptor gene products. The assay is typically a colorimetric ELISA-type assay, although any assay may be employed that can be adapted to the wells that the hybridomas are grown in. Other assays include and radioimmunoassay. The wells positive for the desired antibody production are examined to determine if one or more distinct colonies are present. If more than one colony is present, the cells may be re-cloned and grown to ensure that only a single cell has given rise to the colony producing the desired antibody.

Hybridomas that are confirmed to produce a monoclonal antibody of this invention can be grown up in larger amounts in an appropriate medium, such as DMEM or RPMI- 1640. Alternatively, the hybridoma cells can be grown in vivo as ascites tumors in an animal.

After sufficient growth to produce the desired monoclonal antibody, the growth media containing monoclonal antibody (or the ascites fluid) is separated away from the cells and the monoclonal antibody present therein is purified. Purification is typically achieved by gel electrophoresis, dialysis, chromatography using protein A or protein G-Sepharose, or an anti- mouse Ig linked to a solid support such as agarose or Sepharose beads (all described, for example, in the Antibody Purification Handbook, Biosciences, publication No. 18-1037-46, Edition AC, the disclosure of which is hereby incorporated by reference). The bound antibody is typically eluted from protein A/protein G columns by using low pH buffers (glycine or acetate buffers of pH 3.0 or less) with immediate neutralization of antibody-containing fractions. These fractions are pooled, dialyzed, and concentrated as needed.

Positive wells with a single apparent colony are typically re-cloned and re-assayed to insure only one monoclonal antibody is being detected and produced.

Antibodies may also be produced by selection of combinatorial libraries of immunoglobulins, as disclosed for instance in (Ward et al. Nature, 341 (1989) p. 544, the entire disclosure of which is herein incorporated by reference).

The identification of one or more antibodies that bind(s) to substantially or essentially the same epitope as the anti-Voc7.2-Joc33 monoclonal antibodies described herein can be readily determined using any one of variety of immunological screening assays in which antibody competition can be assessed. A number of such assays are routinely practiced and well known in the art (see, e. g. U. S. Pat. No. 5,660, 827, issued Aug. 26, 1997, which is specifically incorporated herein by reference). It will be understood that actually determining the epitope to which an antibody described herein binds is not in any way required to identify an antibody that binds to the same or substantially the same epitope as the monoclonal antibody described herein.

For example, where the test antibodies to be examined are obtained from different source animals, or are even of a different Ig isotype, a simple competition assay may be employed in which the control (3C10 or 1A6, for example) and test antibodies are admixed (or pre-adsorbed) and applied to a sample containing Vα7.2-Joc33 polypeptides. Protocols based upon Western blotting and the use of BIACORE analysis are suitable for use in such simple competition studies.

In certain embodiments, one pre-mixes the control antibodies (3C10 or 1A6, for example) with varying amounts of the test antibodies (e.g., about 1 :10 or about 1 :100) for a period of time prior to applying to the Vα7.2-Joc33 antigen sample. In other embodiments, the control and varying amounts of test antibodies can simply be admixed during exposure to the Vα7.2-Joc33 antigen sample. As long as one can distinguish bound from free antibodies (e. g. by using separation or washing techniques to eliminate unbound antibodies) and 3C10 or 1A6 from the test antibodies (e. g. , by using species-specific or isotype-specific secondary antibodies or by specifically labeling 3C10 or lAόwith a detectable label) one can determine if the test antibodies reduce the binding of 3C10 or 1A6 to the two different antigens, indicating that the test antibody recognizes substantially the same epitope as 3C10 or 1A6. The binding of the (labeled) control antibodies in the absence of a completely irrelevant antibody can serve as the control high value. The control low value can be obtained by incubating the labeled (3C10 or 1A6) antibodies with unlabelled antibodies of exactly the same type (3C10 or 1A6), where competition would occur and reduce binding of the labeled antibodies. In a test assay, a significant reduction in labeled antibody reactivity in the presence of a test antibody is indicative of a test antibody that recognizes substantially the same epitope, i. e., one that "cross- reacts" with the labeled (3C10 or 1A6) antibody. Any test antibody that reduces the binding of 3C10 to Voc7.2-Jα33 antigens by at least about 50%, such as at least about 60%, or more preferably at least about 70% (e. g., about 65-100%), at any ratio of 3C10 or lA6:test antibody between about 1 :10 and about 1 : 100 is considered to be an antibody that binds to substantially the same epitope or determinant as 3C10 or 1A6. Preferably, such test antibody will reduce the binding of 3Cl 0 or 1 A6 to the Vα7.2-Joc33 antigen by at least about 90% (e.g., about 95%).

Competition can be assessed by, for example, a flow cytometry test. In such a test, cells bearing a given Vα7.2-Joc33 polypeptide can be incubated first with 3C10 or 1A6, for example, and then with the test antibody labeled with a fluorochrome or biotin. The antibody is said to compete with 3C10 or 1A6 if the binding obtained upon preincubation with a saturating amount of 3C10 or 1A6 is about 80%, preferably about 50%, about 40% or less (e.g., about 30%) of the binding (as measured by mean of fluorescence) obtained by the antibody without preincubation with 3C10 or 1A6. Alternatively, an antibody is said to compete with 3C10 if the binding obtained with a labeled 3C10 or 1A6 antibody (by a fluorochrome or biotin) on cells preincubated with a saturating amount of test antibody is about 80%, preferably about 50%, about 40%, or less (e. g., about 30%) of the binding obtained without preincubation with the antibody. A simple competition assay in which a test antibody is pre-adsorbed and applied at saturating concentration to a surface onto which a Vα7.2-Joc33 antigen is immobilized may also be employed. The surface in the simple competition assay is preferably a BIACORE chip (or other media suitable for surface plasmon resonance analysis). The control antibody (e.g., 3C10 or 1A6) is then brought into contact with the surface at a Vα7.2-Joc33 -saturating concentration and the Vα7.2-Joc33 and surface binding of the control antibody is measured. This binding of the control antibody is compared with the binding of the control antibody to the Vα7.2-Joc33- containing surface in the absence of test antibody. In a test assay, a significant reduction in binding of the Voc7.2-Joc33-containing surface by the control antibody in the presence of a test antibody indicates that the test antibody recognizes substantially the same epitope as the control antibody such that the test antibody "cross-reacts" with the control antibody. Any test antibody that reduces the binding of control (such as 3C10 or 1A6) antibody to a Vα7.2-Joc33 antigen by at least about 30% or more, preferably about 40%, can be considered to be an antibody that binds to substantially the same epitope or determinant as a control (e.g., 3C10 or 1A6). Preferably, such a test antibody will reduce the binding of the control antibody (e.g., 3C10 or 1A6) to the Voc7.2-Jα33 antigen by at least about 50% (e. g. , at least about 60%, at least about 70%, or more). It will be appreciated that the order of control and test antibodies can be reversed: that is, the control antibody can be first bound to the surface and the test antibody is brought into contact with the surface thereafter in a competition assay. Preferably, the antibody having higher affinity for the Vα7.2-Joc33 antigen is bound to the surface first, as it will be expected that the decrease in binding seen for the second antibody (assuming the antibodies are cross-reacting) will be of greater magnitude. Further examples of such assays are provided in, e. g., Saunal and (1995) J. Immunol. Methods 183: 33-41, the disclosure of which is incorporated herein by reference.

Preferably, monoclonal antibodies that recognize a Vα7.2-Joc33 epitope will react with an epitope that is present on a substantial percentage of or even all MAIT cells, but will not significantly react with other cells, i.e., immune or non-immune cells that do not express Vα7.2 Joc33. In preferred embodiments, the antibodies will bind to MAIT cells from an individual or individuals with a mucosal immune disorder, i.e. an individual that is a candidate for treatment with one of the herein-described methods using an anti-Voc7.2-Joc33 antibody of the invention. Accordingly, once an antibody that specifically recognizes Vα7.2 Jα33 on MAIT cells, preferably human MAIT cells, it can be tested for its ability to bind to MAIT cells taken from a patient with a mucosal immune disorder such as cancer, infection, irritable bowel disease, Crohn's disease, ulcerative colitis, or Celiac disease. In particular, prior to treating a patient with one of the present antibodies, it will be beneficial to test the ability of the antibody to bind MAIT cells taken from the patient, e.g. in a blood sample or mucosal tissue biopsy, to maximize the likelihood that the therapy will be beneficial in the patient.

In one embodiment, the antibodies of the invention are validated in an immunoassay to test their ability to bind to Voc7-Joc33-expressing cells, e.g. MAIT cells. For example, peripheral blood lymphocytes (PBLs) are taken from a plurality of patients, and MAIT cells are enriched from the PBLs, e.g., by flow cytometry using relevant antibodies (see, e.g., Examples and Tilloy et al. (1999) J. Exp. Med 189:1907-1921; Treiner et al. (2003) Nature 422:164-169). The ability of a given antibody to bind to the cells is then assessed using standard methods well known to those in the art. Antibodies that are found to bind to a substantial proportion (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80% or more) of cells known to express Vα7.2-Joc33, e.g. MAIT cells, from a significant percentage of individuals or patients (e.g., 5%, 10%, 20%, 30%, 40%, 50% or more) are suitable for use in the present invention, both for diagnostic purposes to determine the presence or level of MAIT cells in a patient or for use in the herein-described therapeutic methods, e.g., for use to increase or decrease MAIT cell number or activity. To assess the binding of the antibodies to the cells, the antibodies can either be directly or indirectly labeled. When indirectly labeled, a secondary, labeled antibody is typically added. The binding of the antibodies to the cells can then be detected using, e.g., cytofluorometric analysis (e.g. FACScan). Such method are well known to those of skill in the art.

While described in the context of 3C10 for the purposes of exemplification, it will be appreciated that the herein-described immunological screening assays and other assays can also be used to identify antibodies that compete with other anti-Vα7.2-Joc33 antibodies (e.g. 1A6), and other antibodies described herein or obtained according to the teachings of the present specification.

Determination of whether an antibody binds within one of the epitope regions defined above can be carried out in ways known to the person skilled in the art. As one example of such mapping/characterization methods, an epitope region for an anti-Voc7.2-Joc33 antibody may be determined by epitope "foot-printing" using chemical modification of the exposed amines/carboxyls in the Vα7.2-Joc33 protein. One specific example of such a foot-printing technique is the use of HXMS (hydrogen- deuterium exchange detected by mass spectrometry) wherein a hydrogen/deuterium exchange of receptor and ligand protein amide protons, binding, and back exchange occurs, wherein the backbone amide groups participating in protein binding are protected from back exchange and therefore will remain deuterated. Relevant regions can be identified at this point by peptic proteolysis, fast microbore high-performance liquid chromatography separation, and/or electrospray ionization mass spectrometry. See, e. g. , Ehring H, Analytical Biochemistry, Vol. 267 (2) pp. 252-259 (1999) Engen, J. R. and Smith, D. L. (2001) Anal. Chem. 73,256A-265A. Another example of a suitable epitope identification technique is nuclear magnetic resonance epitope mapping (NMR), where typically the position of the signals in two-dimensional NMR spectra of the free antigen and the antigen complexed with the antigen binding peptide, such as an antibody, are compared. The antigen typically is selectively isotopically labeled with 15N so that only signals corresponding to the antigen and no signals from the antigen binding peptide are seen in the NMR-spectrum. Antigen signals originating from amino acids involved in the interaction with the antigen binding peptide typically will shift position in the spectrum of the complex compared to the spectrum of the free antigen, and the amino acids involved in the binding can be identified that way. See, e. g. , Ernst Schering Res Found Workshop. 2004; (44): 149-67; Huang et Journal of Molecular Biology, Vol. 281 (1) pp. 61-67 (1998); and Saito and Patterson, Methods. 1996 Jun; 9 (3): 516-24.

Epitope mapping/characterization also can be performed using mass spectrometry methods. See, e.g., Downward, J Mass Spectrom. 2000 Apr; 35 (4): 493-503 and Kiselar and Downard, Anal Chem. 1999 May 1; 71 (9): 1792-801. Protease digestion techniques also can be useful in the context of epitope mapping and identification. Antigenic determinant-relevant regions/sequences can be determined by protease digestion, e. g. by using trypsin in a ratio of about 1 :50 to Vα7.2-Joc33 or o/n digestion at and pH 7-8, followed by mass spectrometry (MS) analysis for peptide identification. The peptides protected from trypsin cleavage by the anti- Voc7.2-Jα33 binder can subsequently be identified by comparison of samples subjected to trypsin digestion and samples incubated with antibody and then subjected to digestion by e. g. trypsin (thereby revealing a footprint for the binder). Other enzymes like chymotrypsin, pepsin, etc. also or alternatively can be used in similar epitope characterization methods. Moreover, enzymatic digestion can provide a quick method for analyzing whether a potential antigenic determinant sequence is within a region of the Vα7.2-Joc33 polypeptide that is not surface exposed and, accordingly, most likely not relevant in terms of immunogenicity/antigenicity. See, e. g., Manca, Ann 1st Super Sanita. 1991; 27 : 15-9 for a discussion of similar techniques.

The binding of the antibodies to MAIT or other Voc7.2-Joc33-expressing cells can also be assessed in non-human primates, e.g. cynomolgus monkeys. The invention therefore provides an antibody, as well as fragments and derivatives thereof, wherein said antibody, fragment or derivative specifically binds to Vα7.2-Joc33 receptors at the surface of human MAIT cells, and which furthermore binds to MAIT cells from cynomolgus monkeys. The invention also provides a method of testing the toxicity of an antibody or fragments or derivatives thereof, or their ability to modulate MAIT cell activity, wherein said antibody, fragment or derivative specifically binds to Vα7.2-Joc33 polypeptides at the surface of human MAIT cells, wherein the method comprises testing the antibody in a cynomolgus monkey. In certain embodiments, the non-human primate is a model for a mucosal immune disorder such as the MAIT cell-related disorders addressed herein. Upon immunization and production of antibodies in a vertebrate or cell, particular selection steps may be performed to isolate antibodies as claimed. In this regard, in a specific embodiment, the invention also relates to methods of producing such antibodies, comprising: (a) immunizing a non-human mammal with an immunogen comprising a Vα7.2-Joc33 polypeptide; and (b) preparing antibodies from said immunized animal, wherein said antibodies bind said Voc7.2-Jα33 polypeptide. In one embodiment, the method further comprises step (c), selecting antibodies of (b) that are capable of modulating Vα7.2-Joc33 mediated MAIT cell activity. Methods of accessing MAIT cell activity are described below and are known in the art (see, e.g., U.S. Patent Application No. 20030215808; Kawachi et al. (2006) J Immunol. 176(3):1618- 27; Huang et al. (2005) J Biol Chem. 280(22):21183-93; Treiner et al. (2005) Microbes Infect. 7(3):552-9; Treiner et al. (2003) Nature 422(6928): 164-9; the entire disclosures of which are herein incorporated by reference).

In preferred embodiments, the antibodies prepared according to the present methods are monoclonal antibodies. In preferred embodiments, the non-human animal used to produce antibodies according to the methods of the invention is a mammal, such as a rodent, bovine, porcine, horse, rabbit, goat, or sheep.

According to an alternate embodiment, the DNA encoding an antibody that binds an epitope present on Vα7.2-Joc33 polypeptides is isolated from the hybridoma of this invention and placed in an appropriate expression vector for transfection into an appropriate host. The host is then used for the recombinant production of the antibody, or variants thereof, such as a humanized version of that monoclonal antibody, active fragments of the antibody, or chimeric antibodies comprising the antigen recognition portion of the antibody.

DNA encoding the monoclonal antibodies of the invention, e.g., antibody 3C10 or 1A6, can be readily isolated and sequenced using conventional procedures (e. g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. As described elsewhere in the present specification, such DNA sequences can be modified for any of a large number of purposes, e.g., for humanizing antibodies, producing fragments or derivatives, or for modifying the sequence of the antibody, e.g., in the antigen binding site in order to optimize the binding specificity of the antibody.

Recombinant expression in bacteria of DNA encoding the antibody is well known in the art (see, for example, Skerra et al. , Curr. Opinion in Immunol., 5, pp. 256 (1993); and Pluckthun, Immunol. 130, pp. 151 (1992). The invention therefore provides a host cell capable of expressing an antibody that binds an epitope present on Vα7.2-Joc33 polypeptides, including but not limited to a recombinant host cell which has been transformed with a nucleic acid encoding an antibody that binds an epitope present on a Vα7.2-Joc33 polypeptide.

Antibody 3C10 or its subclone 3C10A8

Nucleic acid and amino acid sequences from variable regions of the heavy and light chains for antibody 3C10 are listed in SEQ ID NOS 1 to 4, respectively. In a specific embodiment, the antibody binds essentially the same epitope or determinant as one of monoclonal antibodies 3C10. In one preferred embodiment, the monoclonal antibody comprises the Fab or F(ab')₂ portion of 3C10. According to another preferred embodiment, the monoclonal antibody comprises the three CDRs of the variable heavy chain region of 3C10 (CDRl = amino acids 26 to 33 of SEQ ID NO: 2; CDR2 = amino acids 51 to 58 of SEQ ID NO: 2; CDR3 = amino acids 97-110 of SEQ ID NO: 2). Also provided is a monoclonal antibody that comprises the variable heavy chain region of 3C10 (3Cl OVH; SEQ ID NO: 2). According to another preferred embodiment, the monoclonal antibody comprises the three CDRs of the variable light chain region of 3C10 (CDRl = amino acids 27 to 32 of SEQ ID NO: 4; CDR2 = amino acids 50 to 52 of SEQ ID NO: 4; CDR3 = amino acids 89-97 of SEQ ID NO: 4). Also provided is a monoclonal antibody that comprises the variable light chain region of 3C10 (3 Cl OVK; SEQ ID NO: 4). In another preferred embodiment, the antibody comprises a heavy chain comprising one, two or three of the CDRs of the variable heavy chain region of 3C10, said CDRs comprising an amino acid sequence selected from the group consisting of: GFNIKDTH (SEQ ID NO: 5; CDRl); TDPASGDT (SEQ ID NO: 6; CDR2); and AHYYRDDVNYAMDY (SEQ ID NO: 7; CDR3), or any sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 contiguous amino acids thereof (to the extent such sequence is consistent with the length of the SEQ ID), or any sequence which is at least 50%, 60%, 70%, 80% or 90% identical thereto over the length of of the aforementioned CDR sequence. In another preferred embodiment, the antibody comprises a light chain comprising one, two or three of the CDRs of the variable light chain region of 3C10, said CDRs comprising an amino acid sequence selected from the group consisting of: QNVGSN (SEQ ID NO: 8; CDRl); SSS (SEQ ID NO: 9; CDR2); and QQYNTYPYT (SEQ ID NO: 10; CDR3), or any sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 contiguous amino acids thereof (to the extent such sequence is consistent with the length of the SEQ ID), or any sequence which is at least 50%, 60%, 70%, 80% or 90% identical thereto over the length of the sequence of the aforementioned CDR sequence. Optionally any one or more of said light or heavy chain CDRs may contain one, two, three, four or five amino acid modifications (e.g. substitutions, insertions or deletions). In another preferred embodiment the antibody is 3C10. The hybridoma producing antibody 3C10 was deposited on November 15th, 2006 at the Collection Nationale de Culture de Microorganismes, Institute Pasteur, 25, Rue du Docteur

Roux, F-75725 Paris, France, under reference identification number 3C10A8 and registration number CNCM 1-3693.

Antibody 1A6

Nucleic acid and amino acid sequences from variable regions of the heavy and light chains for antibody 1A6 are listed in SEQ ID NOS 11 to 14, respectively. In a specific embodiment, the antibody binds essentially the same epitope or determinant as one of monoclonal antibodies 1A6. In one preferred embodiment, the monoclonal antibody comprises the Fab or F(ab')₂ portion of 1A6. According to another preferred embodiment, the monoclonal antibody comprises the three CDRs of the variable heavy chain region of 1A6 (CDRl = amino acids 26 to 33 of SEQ ID NO: 12; CDR2 = amino acids 51 to 58 of SEQ ID NO: 12; CDR3 = amino acids 97-110 of SEQ ID NO: 12). Also provided is a monoclonal antibody that comprises the variable heavy chain region of 1A6 (1A6VH; SEQ ID NO: 12). According to another preferred embodiment, the monoclonal antibody comprises the three CDRs of the variable light chain region of 1A6 (CDRl = amino acids 27 to 32 of SEQ ID NO: 14; CDR2 = amino acids 50 to 52 of SEQ ID NO: 14; CDR3 = amino acids 89-97 of SEQ ID NO: 14). Also provided is a monoclonal antibody that comprises the variable light chain region of 1A6 (1 A6VK; SEQ ID NO: 14). In another preferred embodiment, the antibody comprises a heavy chain comprising one, two or three of the CDRs of the variable heavy chain region of 1A6, said CDRs comprising an amino acid sequence selected from the group consisting of: GFNIKDTH (SEQ ID NO: 5; CDRl); TDPASGDI (SEQ ID NO: 15; CDR2); and AHYYRDDVNYAMDY (SEQ ID NO: 7; CDR3) , or any sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 contiguous amino acids thereof (to the extent such sequence is consistent with the length of the SEQ ID), or any sequence which is at least 50%, 60%, 70%, 80% or 90% identical thereto over the length of the aforementioned CDR sequence. In another preferred embodiment, the antibody comprises a light chain comprising one, two or three of the CDRs of the variable light chain region of 1A6, said CDRs comprising an amino acid sequence selected from the group consisting of: QNVGTN (SEQ ID NO: 16; CDRl); SSS (SEQ ID NO: 9; CDR2); and QQYNTYPYT (SEQ ID NO: 10; CDR3), or any sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 contiguous amino acids thereof (to the extent such sequence is consistent with the length of the SEQ ID), or any sequence which is at least 50%, 60%, 70%, 80% or 90% identical thereto over the length of the aforementioned CDR sequence. Optionally any one or more of said light or heavy chain CDRs may contain one, two, three, four or five amino acid modifications (e.g. substitutions, insertions or deletions). In another preferred embodiment the antibody is 1 A6.

Both activating, inhibitory and depleting monoclonal anti-Voc7.2-Joc33 antibodies against will generally be modified so as to make them suitable for therapeutic use in humans. For example, they may be humanized, chimerized, or selected from a library of human antibodies using methods well known in the art. Such human-suitable antibodies can be used directly in the present therapeutic methods, or can be further derivatized into cytotoxic antibodies, as described infra, for use in the methods.

In one preferred embodiment, the DNA of a hybridoma producing an antibody of this invention, e.g. an an antibody that binds to substantially the same epitope as 3C10 or other antibody of the invention, can be modified prior to insertion into an expression vector, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous non-human sequences (e.g., Morrison et al. (1984) PNAS 81 :6851), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide. In that manner, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity of the original antibody. Typically, such non- immunoglobulin polypeptides are substituted for the constant domains of an antibody of the invention.

In a preferred embodiment, the antibody comprises the variable heavy chain region of 3C10 (SEQ ID NO:2), or one, two or three CDRs comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 5, 6 and 7, fused to a human heavy chain constant region, or the variable heavy chain region of 1A6 (SEQ ID NO:12), or one, two or three CDRs comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 5, 15 and 7, fused to a human heavy chain constant region. In one preferred embodiment, the human heavy chain constant region is an IgG4 constant region. In another preferred embodiment, the human heavy chain constant region is an IgGl constant region.

In another preferred embodiment, the antibody comprises the variable light chain region of 3C10 (SEQ ID NO:4), or one, two or three CDRs comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 8, 9 or 10, fused to a human light chain constant region, or the antibody comprises the variable light chain region of 1A6 (SEQ ID NO: 14), or one, two or three CDRs comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 16, 9 and 10, fused to a human light chain constant region. More preferred is an antibody that comprises the variable light chain region of 3C10 or 1A6 fused to the human kappa (k3) light chain constant region.

Even more preferred is an antibody comprising both (a) 3Cl OVK, or one, two or three CDRs comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 8, 9 or 10, fused to a human light chain constant region and (b) 3Cl OVH, or one, two or three CDRs comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 5, 6 or 7, fused to a human heavy chain constant region. In another embodiment, the invention provides an antibody comprising both (a) 1A6VK, or one, two or three CDRs comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 5, 15 and 7, fused to a human light chain constant region and (b) 1A6VH, or one, two or three CDRs comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 16, 9 and 10, fused to a human heavy chain constant region. In one example, the light chain constant region is a kappa (k3) constant region and the heavy chain constant region is selected from IgG4 or IgGl (e.g. IgGlm(-l, -2, -3)).

Fragments and Derivatives of the present Monoclonal Antibodies

Fragments and derivatives of antibodies of this invention (which are encompassed by the term "antibody" or "antibodies" as used in this application, unless otherwise stated or clearly contradicted by context), preferably a 3C10 or 1A6-Iike antibody, can be produced by techniques that are known in the art. "Fragments" comprise a portion of the intact antibody, generally the antigen binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F (ab') 2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single chain polypeptide"), including without limitation (1) single-chain Fv molecules (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific antibodies formed from antibody fragments.

Fragments of the present antibodies can be obtained using standard methods. For instance, Fab or F (ab') 2 fragments may be produced by protease digestion of the isolated antibodies, according to conventional techniques. It will be appreciated that immunoreactive fragments can be modified using known methods, for example to slow clearance in vivo and obtain a more desirable pharmacokinetic profile the fragment may be modified with polyethylene glycol (PEG). Methods for coupling and site-specifically conjugating PEG to a Fab' fragment are described in, for example, Leong et al, 16 (3): 106-119 (2001) and Delgado et al, Br. J. Cancer 73 (2): 175- 182 (1996), the disclosures of which are incorporated herein by reference.

Alternatively, the DNA of a hybridoma producing an antibody of this invention, preferably a 3C10 or 1A6-Iike antibody, may be modified so as to encode a fragment of this invention. The modified DNA is then inserted into an expression vector and used to transform or transfect an appropriate cell, which then expresses the desired fragment. In certain embodiments, the DNA of a hybridoma producing an antibody of this invention, preferably a 3C10 or 1A6-Iike antibody, can be modified prior to insertion into an expression vector, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous non-human sequences (e. g. , Morrison et al., Proc. Natl. Acad. Sci. U. S. pp. 6851 (1984) ), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide. In that manner, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity of the original antibody. Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody of the invention.

In one particularly preferred embodiment, the antibodies of this invention are humanized. "Humanized" forms of antibodies according to this invention are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') ₂, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from the murine or other non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary- determining region (CDR) of the recipient are replaced by residues from a CDR of the original antibody (donor antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody. In some instances, Fv framework residues of the human immunoglobulin may be replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in either the recipient antibody or in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of the original antibody and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. For further details see Jones et al. (1986) Nature 321: 522; Reichmann et al. (1988) Nature 332: 323; Verhoeyen et al. (1988) Science 239:1534 (1988); Presta (1992) Curr. Op. Struct. Biol. 2:593; each of which is herein incorporated by reference in its entirety.

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called "best- fit" method, the sequence of the variable domain of an antibody of this invention is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the mouse is then accepted as the human framework (FR) for the humanized antibody (Sims et al. (1993) J. Immun., 151 :2296; Chothia and Lesk (1987) J. MoI. Biol. 196:901). Another method uses a particular framework from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al. (1992) PNAS 89:4285; Presta et al. (1993) J. Immunol. 51 :1993)).

It is further important that antibodies be humanized while retaining their high affinity for Voc7.2-Jα33, preferably human and non-human primate Vα7.2-Joc33, and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

In one example, the invention provides human or humanized activating anti- Vα7.2-Joc33 antibodies having a half- life of at least 5, 6, 8, 9, 10, 15 or 20 days, and do not substantially bind human FcgammaRIIIa (CD 16) (e.g. via their constant region). For example an antibody having a constant region of the IgG4 type, or a F(ab')2 fragment will typically have low or no CD 16 binding). In another example, the invention provides human or humanized activating anti- Voc7.2-Jα33 antibodies having a half-life of at least 5, 6, 8, 9, 10, 15 or 20 days, and does bind human FcgammaRIIIa (CD16). More preferably, the activating anti-Voc7.2-Joc33 antibody is a humanized antibody and completely competes with 3C10 or 1A6 antibody for binding to human Voc7.2-Jα33. For the purpose of illustration with preferred antibodies suitable for use according to the methods herein, a 3C10 or 1A6 antibody can be used to prepare a humanized antibody. Preferred humanized antibodies according to the invention comprise a human framework, at least one CDR from a non-human antibody, and in which any constant region present is substantially identical to a human immunoglobulin constant region, e.g., at least about 60-90%, preferably at least 95% identical. Hence, all parts of a humanized antibody, except possibly the CDR's, are substantially identical to corresponding parts of one or more native human antibody sequences. In some instances, the humanized antibody, in addition to CDRs from a non-human antibody, would include additional non-human residues in the human framework region.

The design of humanized antibodies can be carried out as follows. When an amino acid falls under the following categories, the framework amino acid of a human antibody to be used

(acceptor antibody) is replaced by a framework amino acid from a CDR-providing non-human antibody (donor antibody): (a) the amino acid in the human framework region of the acceptor antibody is unusual for human antibody at that position, whereas the corresponding amino acid in the donor antibody is typical for human antibody in that position; (b) the position of the amino acid is immediately adjacent to one of the CDR's; or (c) the amino acid is capable of interacting with the CDR's in a tertiary structure antibody model (see, C. Queen et al. Proc. Natl. Acad. Sci. USA 86, 10029 (1989), and Co et al., Proc. Natl. Acad. Sci. USA 88, 2869 (1991) the disclosures of which are incorporated herein by reference).

For further detailed description of the production of humanized antibody, See Queen et al., op. cit. and Co et al, op. cit. and U.S. Pat. Nos. 5,585,089; 5,693,762, 5,693,761, and 5,530,101, the disclosures of which are incorporated herein by reference. Usually, the CDR regions in humanized antibodies are substantially identical, and more usually, identical to the corresponding CDR regions in the mouse antibody from which they were derived. Although not usually desirable, it is sometimes possible to make one or more conservative amino acid substitutions of CDR residues without appreciably affecting the binding affinity of the resulting humanized antibody. Occasionally, substitutions of CDR regions can enhance binding affinity. Other than for the specific amino acid substitutions discussed above, the framework regions of humanized antibodies are usually substantially identical, and more usually, identical to the framework regions of the human antibodies from which they were derived. Of course, many of the amino acids in the framework region make little or no direct contribution to the specificity or affinity of an antibody. Thus, many individual conservative substitutions of framework residues can be tolerated without appreciable change of the specificity or affinity of the resulting humanized antibody. The antigen binding region of the humanized antibody (the non- human portion) can be derived from an antibody of nonhuman origin, referred to as a donor antibody, having specificity for Vα7.2-Joc33 . For example, a suitable antigen binding region can be derived from a 3C10 or 1A6 monoclonal antibody. Other sources include Vα7.2-Joc33 - specific antibodies obtained from nonhuman sources, such as rodent (e.g., mouse and rat), rabbit, pig, goat or non-human primate (e.g., monkey) or camelid animals (e.g., camels and llamas). Additionally, other polyclonal or monoclonal antibodies, such as antibodies which bind to the same or similar epitope as a 3C10 or 1A6 antibody, can be made (e.g., Kohler et al., Nature, 256:495-497 (1975); Harlow et al., 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor, N. Y.); and Current Protocols in Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel et al., Eds. (John Wiley & Sons: New York, N. Y.), Chapter 11 (1991)).

In one embodiment, the humanized antibody having binding specificity for human Vα7.2-Joc33 comprises at least one CDR of nonhuman origin. For example, a humanized antibody having a binding specificity for human Vα7.2-Joc33 comprises a heavy chain and a light chain. The light chain can comprise a CDR derived from an antibody of nonhuman origin which binds Vα7.2- Joc33 and a FR derived from a light chain of human origin. For example, the light chain can comprise CDRl , CDR2 and/or CDR3 which have the amino acid sequence similar or substantially the same as that of the respective CDR of a 3C10 or lAόantibody such that the antibody specifically binds to the human Vα7.2-Joc33. The heavy chain can comprise a CDR derived from an antibody of nonhuman origin which binds Vα7.2-Joc33 and a FR derived from a heavy chain of human origin. For example, the heavy chain can comprise CDRl, CDR2 and CDR3 which have the amino acid sequence set forth below or an amino acid similar or substantially the same as that of the respective CDR of the 3C10 or 1A6 antibody such that the antibody specifically binds to the human Vα7.2-Joc33.

An embodiment of the invention is a humanized antibody which specifically binds to human Voc7.2-Jα33 and comprises a humanized light chain comprising three light chain CDRs from a 3C10 or 1A6 antibody and a light chain variable region framework sequence from a human antibody light chain. The invention further comprises a humanized heavy chain that comprises three heavy chain CDRs from a 3C10 or 1A6 antibody and a heavy chain variable region framework sequence from a human antibody heavy chain.

The portion of the humanized antibody or antibody chain which is of human origin (the human portion) can be derived from any suitable human antibody or antibody chain. For example, a human constant region or portion thereof, if present, can be derived from the kappa or lambda light chains, and/or the gamma (eg, gammal, gamma2, gamma3, gamma4), μ, alpha (eg, alphal, alpha2), delta or epsilon heavy chains of human antibodies, including allelic variants. A particular constant region, such as IgG2b or IgG4, variants or portions thereof can be selected to tailor effector function. The latter constant regions, or portions therefore are particularly preferred in that they do not substantially bind FcgammaIIIa receptor on NK cells (CD 16) and therefore do not substantially induce ADCC mediated lysis of NK effectors to which the anti- Voc7.2-Jα33 antibodies of the invention are bound. For example, a mutated constant region, also referred to as a "variant," can be incorporated into a fusion protein to minimize binding to Fc receptors and/or ability to fix complement (see e.g., Winter et al., U.S. Pat. No. 5,648,260; Morrison et al., WO 89/07142; Morgan et al., WO 94/29351). In addition, a mutated IgG2 Fc domain can be created that reduces the mitogenic response, as compared to natural Fc regions (see e.g., Tso et al., U.S. Pat. No. 5,834,597, the teachings of which are incorporated by reference herein in their entirety). If present, human FRs are preferably derived from a human antibody variable region having sequence similarity to the analogous or equivalent region of the antigen binding region donor. Other sources of FRs for portions of human origin of a humanized antibody include human variable consensus sequences (See, Kettleborough, C. A. et al., Protein Engineering 4:773-783 (1991); Queen et al., U.S. Pat. Nos: 5,585,089, 5,693,762 and 5,693,761, the teachings all of which are incorporated by reference herein in their entirety). For example, the sequence of the antibody or variable region used to obtain the nonhuman portion can be compared to human sequences as described in Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, U.S. Government Printing Office (1991). In a preferred embodiment, the FRs of a humanized antibody chain are derived from a human variable region having at least about 60% overall sequence identity, and preferably at least about 80% overall sequence identity, with the variable region of the nonhuman donor (e.g., 3C10 or 1A6 antibody).

The phrase "substantially identical," in context of two nucleic acids or polypeptides (e.g., DNAs encoding a humanized antibody or the amino acid sequence of the humanized antibody) refers to two or more sequences or subsequences that have at least about 80%, most preferably 90- 95% or higher nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using the following sequence comparison method and/or by visual inspection. Such "substantially identical" sequences are typically considered to be homologous. Preferably, the "substantial identity" exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues, or over the full length of the two sequences to be compared. As described below, any two antibody sequences can only be aligned in one way, by using the numbering scheme in Kabat. Therefore, for antibodies, percent identity has a unique and well-defined meaning.

Amino acids from the variable regions of the mature heavy and light chains of antibodies are designated Hx and Lx respectively, where x is a number designating the position of an amino acid according to the scheme of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991). Kabat lists many amino acid sequences for antibodies for each subgroup, and lists the most commonly occurring amino acid for each residue position in that subgroup. Kabat uses a method for assigning a residue number to each amino acid in a listed sequence, and this method for assigning residue numbers has become standard in the field. Kabat's scheme is extendible to other antibodies not included in his compendium by aligning the antibody in question with one of the consensus sequences in Kabat. The use of the Kabat numbering system readily identifies amino acids at equivalent positions in different antibodies. For example, an amino acid at the L50 position of a human antibody occupies the equivalent position to an amino acid position L50 of a mouse antibody. From N-terminal to C-terminal, both light and heavy chain variable regions comprise alternating framework and (CDRs)" FRl , CDRl , FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each region is in accordance with the definitions of Kabat (1987) and (1991), supra and/or Chothia & Lesk, J. MoI. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-

883 (1989).

Binding and/or adhesion assays or other suitable methods can also be used in procedures for the identification and/or isolation of humanized antibodies (e.g., from a library) with the requisite specificity (competition assays for example).

The antibody portions of nonhuman and human origin for use in the invention include light chains, heavy chains and portions of light and heavy chains. These antibody portions can be obtained or derived from antibodies (e.g., by de novo synthesis of a portion), or nucleic acids encoding an antibody or chain thereof having the desired property (e.g., binds Vα7.2-Joc33., sequence similarity, for example with the 3C10 or 1A6 antibody) can be produced and expressed. Humanized antibodies comprising the desired portions (e.g., antigen binding region, CDR, FR, C region) of human and nonhuman origin can be produced using synthetic and/or recombinant nucleic acids to prepare genes (e.g., cDNA) encoding the desired humanized chain. To prepare a portion of a chain, one or more stop codons can be introduced at the desired position. For example, nucleic acid sequences coding for newly designed humanized variable regions can be constructed using PCR mutagenesis methods to alter existing DNA sequences (see e.g., Kamman, M., et al., Nucl. Acids Res. 17:5404 (1989)). PCR primers coding for the new CDRs can be hybridized to a DNA template of a previously humanized variable region which is based on the same, or a very similar, human variable region (Sato, K., et al., Cancer Research 53:851-856 (1993)). If a similar DNA sequence is not available for use as a template, a nucleic acid comprising a sequence encoding a variable region sequence can be constructed from synthetic oligonucleotides (see e.g., Kolbinger, F., Protein Engineering 8:971-980 (1993)). A sequence encoding a signal peptide can also be incorporated into the nucleic acid (e.g., on synthesis, upon insertion into a vector). If the natural signal peptide sequence is unavailable, a signal peptide sequence from another antibody can be used (see, e.g., Kettleborough, C. A.,

Protein Engineering 4:773-783 (1991)). Using these methods, methods described herein or other suitable methods, variants can be readily produced. In one embodiment, cloned variable regions can be mutagenized, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al., U.S. Pat. No. 5,514,548; Hoogengoom et al., WO 93/06213, published Apr. 1, 1993)).

The invention also relates to isolated and/or recombinant (including, e.g., essentially pure) nucleic acids comprising sequences which encode a humanized antibody or humanized antibody light or heavy chain of the present invention.

Another method of making "humanized" monoclonal antibodies is to use a XenoMouse (Abgenix, Fremont, CA) as the mouse used for immunization. A XenoMouse is a murine host according to this invention that has had its immunoglobulin genes replaced by functional human immunoglobulin genes. Thus, antibodies produced by this mouse or in hybridomas made from the B cells of this mouse, are already humanized. The XenoMouse is described in United States Patent No. 6,162, 963, which is herein incorporated in its entirety by reference.

Human antibodies may also be produced according to various other techniques, such as by using, for immunization, other transgenic animals that have been engineered to express a human antibody repertoire (Jakobovitz et Nature 362 (1993) 255), or by selection of antibody repertoires using phage display methods. Such techniques are known to the skilled person and can be implemented starting from monoclonal antibodies as disclosed in the present application.

The antibodies of the present invention, preferably a 3C10 or 1A6-Iike antibody, may also be derivatized to "chimeric" antibodies (immunoglobulins) in which a portion of the heavy light chain is identical with or homologous to corresponding sequences in the original antibody, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (Cabilly et al. , supra; Morrison et al. , Proc. Natl. Acad. Sci. U. S. A., pp. 6851 (1984)).

Other derivatives within the scope of this invention include functionalized antibodies, i. e. , antibodies that are conjugated or covalently bound to a toxin, such as ricin, diphtheria toxin, abrin and Pseudomonas exotoxin; to a detectable moiety, such as a fluorescent moiety, a radioisotope or an imaging agent; or to a solid support, such as agarose beads or the like.

Methods for conjugation or covalent bonding of these other agents to antibodies are well known in the art.

Conjugation to a toxin is useful for targeted killing cells displaying Vα7.2-Joc33 receptors on its cell surface, e.g. MAIT cells. Once the antibody of the invention binds to the cell surface of such cells, it is internalized and the toxin is released inside of the cell, selectively killing that cell. Such methods are useful, e.g., for the treatment of disorders caused by or associated with an increase in MAIT cell activity or number, e.g. cancer, infection, irritable bowel syndrome, Crohn's disease, Celiac disease or ulcerative colitis.

Conjugation to a detectable moiety is useful, inter alia, when an antibody of the invention is used for diagnostic purposes. Such purposes include, but are not limited to, assaying biological samples, e.g., a blood sample or mucosal tissue biopsy, for the presence of MAIT cells, and detecting the presence, level, or activity of MAIT cells in an individual. Such assay and detection methods are also alternate embodiments of the present invention. Such method are useful, e.g., for diagnosing conditions caused by or associated with an increase in MAIT cell activity or number, e.g. cancer, infection, irritable bowel syndrome, Crohn's disease, Celiac disease or ulcerative colitis. Labeled antibodies of the invention can also be used in FACS sorting to purify or isolate MAIT cells from a biological sample.

Conjugation of an antibody of this invention to a solid support is useful as a tool for affinity purification of cells bearing a Vα7.2-Joc33 receptor on their cell surface from a biological sample, such as a blood sample or mucosal tissue biopsy from an individual. This method of purification is another alternate embodiment of the present invention, as is the resulting purified population of cells.

In an alternate embodiment, an antibody that binds an epitope of a Vα7.2-Joc33 polypeptide, wherein said antibody is capable of modulating MAIT cell activity, may be incorporated into liposomes ("immunoliposomes"), alone or together with another substance for targeted delivery to an animal. Such other substances include, but are not limited to, nucleic acids for the delivery of genes for gene therapy or for the delivery of antisense RNA, or siRNA for suppressing a gene in a MAIT cell, or toxins or drugs for the targeted killing of cells.

Purifying MAIT cells using the antibodies of the invention

In certain embodiments, the present antibodies are used to purify MAIT cells from a biological sample. Biological samples can be obtained from a patient, e.g. for diagnostic or ex vivo therapeutic purposes, or from individuals or non-human primates to obtain a source of such cells for research purposes.

MAIT cells can be purified using the present antibodies with any of a number of standard methods. For example, peripheral blood cells can be sorted using a FACS scanner using labeled antibodies specific for Vα7.2-Joc33, e.g., and optionally to other cell surface molecules typically present on MAIT cells, e.g., CD8, CD3, etc.

In addition, the antibodies of the invention can be conjugated or covalently linked to a solid support and used to purify MAIT cells or any cells expressing Vα7.2-Joc33 from a biological sample, e.g., from a blood sample or mucosal tissue biopsy from a patient or other individual. Specifically, the biological sample is placed into contact with the antibodies under conditions that allow cells within the sample to bind to the antibody, and then the cells are eluted from the solid-support-bound antibody.

Regardless of the method used to isolate or purify the MAIT cells, the ability to do so is useful for numerous purposes, e.g. to diagnose an immune disorder by assessing the number or activity or other characteristics of MAIT cells obtained from a patient, or to evaluate the ability of the antibodies of the invention, or fragments or derivatives thereof, to modulate the activity or behavior of MAIT cells of a patient prior, e.g., to one of the herein-described treatments using the antibodies. The ability to isolate or purify MAIT cells is also useful for ex vivo therapies. Further, purified MAIT cells are useful in a research context, e.g., to better characterize the cells and their various properties and behaviors, as well as to identify compounds or methods that can be used to modulate MAIT cell behavior, activity, or proliferation.

Assessing the ability of antibodies to modulate MAIT cell activity

The antibodies of this invention are able to modulate the activity of Vα7.2-Joc33 expressing cells, particularly MAIT cells. For example, certain antibodies can stimulate the Vα7.2-Joc33 receptors and thereby activate MAIT cells and, in turn, enhance an MAIT cell-mediated immune response. Such antibodies are referred to herein interchangeably as "agonist", "activating" or "stimulatory" antibodies. They are useful, e.g., for treating or preventing a condition caused by a decrease in MAIT cell activity or number, or where increased MAIT cell activity can ameliorate, prevent, eliminate, or in any way improve the condition or any symptom thereof. Activating antibodies include, without limitation, for example, antibodies that are bivalent or greater valency, and/or antibodies that bind CD 16 (e.g. via their constant regions). Other antibodies, on the other hand, can inhibit the activation of MAIT cells, e.g. they can block the binding of endogenous ligands such as MRl to the Vα7.2-Joc33 receptors. These antibodies are thus referred to as "neutralizing" or "inhibitory" or "blocking" antibodies. Such antibodies are useful, inter alia, for decreasing MAIT immune cell activity, e.g. for the treatment or prevention of conditions involving excess MAIT cell activity or number, or where decreased MAIT cell activity can ameliorate, prevent, eliminate, or in any way improve the condition or any symptom thereof. Neutralizing antibodies include, without limitation, for example, antibodies that are monovalent (e.g. antibody fragments) and/or antibodies that do not bind CD 16 (e.g. do not bind CD 16 via their constant regions). Preferred antibodies also include anti-Voc7.2-Joc33 antibodies capable of depleting MAIT cells by causing the elimination of MAIT cells in vitro or in vivo. For example, an anti-Voc7.2-Joc33 antibody may mediate killing of MAIT cells (e.g. CDC, ADCC, or by use of a toxic moiety); examples of antibodies that mediated ADCC include antibodies that have a constant region that binds CD16 (e.g. IgGl, IgG3) or other activating Fc receptors.

Any of a large number of assays, including molecular, cell-based, and animal-based models can be used to assess the ability of anti-Voc7.2-Joc33 antibodies to modulate MAIT cell activity. For example, cell-based assays can be used in which cells expressing Vα7.2-Joc33 are exposed to MRl or another Vα7.2 Jα33 ligand (or cells expressing the ligand), and the ability of the antibody or a test compound to disrupt the binding of the ligand or the stimulation of the receptor (as determined, e.g., by examining any of the MAIT cell activities addressed herein) is assessed.

The activity of MAIT cells can also be assessed in the absence of a ligand, by exposing the cells to the antibody itself and assessing its effect on any aspect of the cells' activity or behavior. In such assays, a baseline level of activity (e.g., cytokine production, proliferation, see below) of the MAIT cells is obtained in the absence of a ligand, and the ability of the antibody or compound to alter the baseline activity level is detected. In one such embodiment, a high- throughput screening approach is used to identify compounds capable of affecting the activation of the receptor.

Examples of assays that can be used to assess MAIT cell activity can be found, inter alia, in U.S. Patent Application No. 20030215808; Kawachi et al. (2006) J Immunol. 176(3):1618-27; Huang et al. (2005) J Biol Chem. 280(22):21183-93; Treiner et al. (2005) Microbes Infect. 7(3):552-9; Treiner et al. (2003) Nature 422(6928): 164-9; the entire disclosures of each of which are herein incorporated by reference.

Any suitable physiological change that reflects Vα7.2-Joc33 activity can be used to evaluate test antibodies or antibody derivatives. For example, one can measure a variety of effects, such as changes in gene expression (e.g. CD69), cell growth, cell proliferation, pH, intracellular second messengers, e.g., Ca²⁺, IP3, cGMP, or cAMP, or activity such as ability to activate B cells or cytotoxicity. In one embodiment, the activity of the receptor is assessed by detecting the expression of Vα7.2-Joc33-responsive genes or the production of Vα7.2-Joc33-responsive cytokines or other factors, e.g., ILlO, RANTES, IFN-gamma or TNF-alpha.

In one embodiment, the activity of MAIT of this invention is assessed in a assay in which MAIT cells are incubated in the presence or absence of a test antibody and with B cells, e.g. B cells presenting the MHC class Ib molecule, MRl . Optionally, microbial flora is also present in the incubation. The effect of the presence of the antibody on the properties of the B or T cells, e.g. their proliferation, activity, cytotoxicity, IgA production, or production of cytokines such as ILlO, RANTES, TNF-β or IFN-γ are assessed. See, e.g., U.S. Patent Application No. 20030215808; Kawachi et al. (2006) J Immunol. 176(3):1618-27; Huang et al. (2005) J Biol Chem. 280(22):21183-93; Treiner et al. (2005) Microbes Infect. 7(3):552-9; Treiner et al. (2003) Nature 422(6928):164-9; the entire disclosure of which is herein incorporated by reference.

In another embodiment, the effect of the present antibodies on MAIT cells is assessed in non- human primates in vivo. For example, a pharmaceutical composition comprising an anti-Voc7.2- Joc33 antibody of the present invention is administered to a non-human primate that is either healthy or affected by a mucosal immune condition, and the effect of the administration on, e.g., the number or activity of MAIT cells in the primate, on the IgA production in the gut of the primate, or on the progression of the condition is assessed. Any antibody or antibody derivative or fragment that effects a detectable change in any of these MAIT -related parameters is a candidate for use in the herein-described methods.

In any of the herein-described assays, an increase or decrease of 5%, 10%, 20%, preferably 30%, 40%, 50%, most preferably 60%, 70%, 80%, 90%, 95%, or greater in any detectable measure of Voc7.2-Joc33-stimulated activity in the cells indicates that the test antibody is suitable for use in the present methods.

Compositions and Administration

The present invention also provides pharmaceutical compositions that comprise an antibody, or a fragment and derivative thereof, wherein said antibody, fragment or derivative specifically binds to Vα7.2-Joc33 polypeptides on the surface of cells, and optionally modulates the activity of TCRs comprising the polypeptides and, consequently, the activity or behavior of the cells expressing the polypeptides, e.g., MAIT cells. In certain embodiments, the antibodies stimulate the TCRs and thus enhance the activity or proliferation of the cells. In other embodiments, the antibodies inhibit the TCRs, e.g., by blocking the interaction of an antigen or ligand such as MRl to the receptor, and thus inhibits the proliferation or activation of the cells. The composition further comprises a pharmaceutically acceptable carrier. Such compositions are also referred to as "antibody compositions" of the invention. In one embodiment, antibody compositions of this invention comprise an antibody disclosed in the antibody embodiments above. The antibody 3C10 or 1A6 is included within the scope of antibodies that may be present in the antibody compositions of this invention.

The invention further provides a method of modulating MAIT cell activity in a patient in need thereof, comprising the step of administering to said patient a composition according to the invention. In one embodiment, the MAIT cell activity is enhanced, wherein the patient has a disease or disorder wherein such enhancement may promote, enhance, and/or induce a therapeutic effect (or promotes, enhances, and/or induces such an effect in at least a substantial proportion of patients with the disease or disorder and substantially similar characteristics as the patient, as may determined by, e. g. , clinical trials). In one embodiment, the composition induces proliferation of MAIT cells; in another embodiment, the composition induces the production of cytokines, for example IL-2 and/or IL-10. In another embodiment, the MAIT cell activity is inhibited, wherein the patient has a disease or disorder wherein such inhibition may promote, enhance, and/or induce a therapeutic effect (or promotes, enhances, and/or induces such an effect in at least a substantial proportion of patients with the disease or disorder and substantially similar characteristics as the patient-as may determined by, e. g. , clinical trials). Such treatment methods can be used for a number of mucosal immune disorders, including, but not limited to, cancer, infection (e.g. viral infection), irritable bowel syndrome, Crohn's disease, ulcerative colitis, and Celiac disease.

In some embodiments, prior to the administration of the anti-Voc7.2-Joc33 antibody or composition, the presence of Vα7.2-Joc33 on cells of the patient will be assessed, e.g., to determine the relative level and activity of MAIT cells in the patient as well as to confirm the binding efficacy of the antibodies to the MAIT cells of the patient. This can be accomplished by obtaining a sample of PBLs or cells from the site of the disorder (e.g., from mucosal tissue), and testing e.g., using immunoassays, to determine the relative prominence of markers such as CD4, CD8, etc., as well as Vα7.2-Joc33 on the cells.

In one embodiment, where it is sought to increase the activity of a patient's MAIT cells, a "responder" test can be carried out. In brief, the ability of the anti-Voc7.2-Joc33 antibody or composition to increase the activity of the patient's MAIT cells is assessed, preferably the ability to induce the proliferation, activation, of cytokine expression of the MAIT cells. If the activity of the MAIT cells is increased by the anti-Voc7.2-Joc33 antibody or composition, the patient is determined to be responsive to therapy with an anti-Voc7.2-Joc33 antibody or composition, and optionally the patient is treated with an anti-Voc7.2-Joc33 antibody or composition.

In other embodiments, the method may comprise the additional step of administering to said patient an appropriate additional therapeutic agent selected from an immunomodulatory agent, a hormonal agent, a chemotherapeutic agent, an anti-angiogenic agent, an apoptotic agent, a second antibody that binds to and modulates a Vα7.2 Jα33 receptor, an anti-infective agent, a targeting agent, an anti-inflammation drug, a steroid, an immune system suppressor, an antibiotic, an anti- diarrheal drug, or an adjunct compound. Such additional agents can be administered to said patient as a single dosage form together with said antibody, or as a separate dosage form. The dosage of the antibody (or antibody and the dosage of the additional therapeutic agent collectively) are sufficient to detectably induce, promote, and/or enhance a therapeutic response in the patient. Where administered separately, the antibody, fragment, or derivative and the additional therapeutic agent are desirably administered under conditions (e.g., with respect to timing, number of doses, etc.) that result in a detectable combined therapeutic benefit to the patient.

Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat.

The compositions of this invention may be employed in a method of modulating, e.g. enhancing or inhibiting, the activity of MAIT cells in a patient or a biological sample. This method comprises the step of contacting said composition with said patient or biological sample. Such method will be useful for both diagnostic and therapeutic purposes.

For use in conjunction with a biological sample, the antibody composition can be administered by simply mixing with or applying directly to the sample, depending upon the nature of the sample (fluid or solid). The biological sample may be contacted directly with the antibody in any suitable device (plate, pouch, flask, etc.). For use in conjunction with a patient, the composition must be formulated for administration to the patient.

The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

The compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include, e.g., lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

Alternatively, the compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non- irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used.

The compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents. Several monoclonal antibodies have been shown to be efficient in clinical situations, such as

Rituxan Herceptin (Trastuzumab) or Xolair (Omalizumab), and similar administration regimens (i.e., formulations and/or doses and/or administration protocols) may be used with the antibodies of this invention. Schedules and dosages for administration of the antibody in the pharmaceutical compositions of the present invention can be determined in accordance with known methods for these products, for example using the manufacturers' instructions. For example, an antibody present in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulated for IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection. The pH is adjusted to 6.5. An exemplary suitable dosage range for an antibody in a pharmaceutical composition of this invention may between about 10 mg/m2 and 500 mg/m2. However, it will be appreciated that these schedules are exemplary and that an optimal schedule and regimen can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition that must be determined in clinical trials.

Quantities and schedule of injection of an antibody in a pharmaceutical composition of this invention that saturate MAIT cells for 24 hours, 48 hours, 72 hours, or a week or a month will be determined considering the affinity of the antibody and the its pharmacokinetic parameters.

According to another embodiment, the antibody compositions of this invention may further comprise another therapeutic agent, including agents normally utilized for the particular therapeutic purpose for which the antibody is being administered. The additional therapeutic agent will normally be present in the composition in amounts typically used for that agent in a monotherapy for the particular disease or condition being treated. Such therapeutic agents include, but are not limited to, therapeutic agents used in the treatment of cancers, therapeutic agents used to treat infectious disease, therapeutic agents used in other immunotherapies, cytokines (such as IL-2 or IL- 15), anti-inflammation agents, steroids, immune system suppressors, antibiotics, anti-diarrheal drugs, and other antibodies and fragments thereof.

The antibody compositions and methods of the present invention may be combined with any other methods generally employed in the treatment of the particular disease, such as immune disorders involving the mucosa. So long as a particular therapeutic approach is not known to be detrimental to the patient's condition in itself, and does not significantly counteract the activity of the antibody in a pharmaceutical composition of this invention, its combination with the present invention is contemplated.

When one or more additional therapeutic agents are used in combination with an antibody- containing composition of this invention in a therapeutic regimen, there is no requirement for the combined results to be additive of the effects observed when each treatment is conducted separately. Although at least additive effects are generally desirable, any increased effect above one of the single therapies would be of benefit. Also, there is no particular requirement for the combined treatment to exhibit synergistic effects, although this is certainly possible and advantageous.

In connection with solid tumor treatment, the pharmaceutical compositions of the present invention may be used in combination with classical approaches, such as surgery, radiotherapy, chemotherapy, and the like. The invention therefore provides combined therapies in which a pharmaceutical composition of this invention is used simultaneously with, before, or after surgery or radiation treatment; or are administered to patients with, before, or after conventional chemotherapeutic, radiotherapeutic or anti- angiogenic agents, or targeted immunotoxins or coaguligands.

In other aspects, immunomodulatory compounds or regimens may be administered in combination with or as part of the antibody compositions of the present invention. Preferred examples of immunomodulatory compounds include cytokines. Various cytokines may be employed in such combined approaches. Examples of cytokines useful in the combinations contemplated by this invention include IL-2, IL- 3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL- 12, IL-13, IL-15, TGF- beta, GM-CSF, M-CSF, G-CSF, TNF-alpha, TNF-beta, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-alpha, and IFN-beta. Cytokines used in the combination treatment or compositions of this invention are administered according to standard regimens, consistent with clinical indications such as the condition of the patient and relative toxicity of the cytokine.

The present antibodies can also be administered in conjunction with anti- inflammatory agents, such as NSAIDS, aspirin, salsalate, diflunisal, ibuprofen, ketoprofen, nabumetone, piroxicam, naproxen, diclofenac, indomethacin, sulindac, tolmetin, etodolac, ketorolac, oxaprozin, celecoxib, corticosteroids, oral steroids, prednisone, prednisolone, beclomethasone, fluticasone, budesonide, betamethasone, dexamethasone, aclomethasone and clobetasone.

In another embodiment, two or more antibodies of this invention having different cross- reactivities, e.g. antibodies that specifically bind to distinct epitopes within the Vα7.2-Joc33 polypeptide, are combined in a single composition so as to target as many distinct Vα7.2-Joc33 gene products as possible, e.g. to account for diversity in the polypeptides within an individual or in different patients, and to do so as efficaciously as possible. In addition, an antibody composition of this invention may comprise multiple antibodies that recognize a single Vα7.2- Joc33 epitope. Such combinations would again provide wider utility in a therapeutic setting. The invention also provides a method of modulating MAIT cell activity in a patient in need thereof, comprising the step of administering a composition according to this invention to said patient. The method is more specifically directed at increasing MAIT cell activity in patients having a disease in which increased MAIT cell activity is beneficial, or which is caused or characterized by insufficient MAIT cell activity, or, contrarily, at decreasing MAIT cell activity in patients having a disease in which decreased MAIT cell activity is beneficial, or which is caused or characterized by excessive MAIT cell activity.

Diseases and conditions treatable using the present methods include cancer, other proliferative disorders, infectious disease, or immune disorders such as inflammatory diseases and autoimmune diseases.

The antibodies of this invention can be used to treat or prevent infectious diseases, including preferably any infections caused by viruses, bacteria, protozoa, molds or fungi.

The present antibodies can be included in kits, which may contain any number of antibodies and/or other compounds, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any other number of therapeutic antibodies and/or compounds, as well as, in certain embodiments, antibodies or other diagnostic reagents for detecting the presence of Vα7.2-Joc33 on cells. Such diagnostic antibodies will often be labeled, either directly or indirectly (e.g., using secondary antibodies). Therapeutic antibodies can be either modified, e.g. by the addition of a cytotoxic agent, or unmodified, working, e.g., by modulating Vα7.2-Joc33 activation, or by simply binding to target cells and thereby stimulating or inhibiting them, triggering cell death, or marking them for destruction by the immune system. It will be appreciated that this description of the contents of the kits is not limiting in any way. For example, the kit may contain other types of therapeutic compounds as well, such as other anti-inflammatory agents. Preferably, the kits also include instructions for using the antibodies, e.g., detailing the herein-described methods.

Further aspects and advantages of this invention will be disclosed in the following experimental section, which should be regarded as illustrative and not limiting the scope of this application.

EXAMPLES

Example 1. Characterization of MAIT-specific monoclonal antibodies.

Part 1) Production of monoclonal antibodies and initial screening:

To obtain anti-Voc7.2 antibodies, Balb/c mice were immunized with a recombinant Vα7.2- Jα33/Vβl3 protein (Figure 1). Primary screening of PBLs was carried out by flow cytometry with three antibody targets: Goat anti-mouse F(ab')2 Ig-PE fragment; PE-Cy5 anti-human CD3;

FITC anti-human CD4; FITC anti-human CD8 (Figure 2, strategy for primary screening).

Selection of hybridomas of interest:

Antibodies recognizing an antigenic T cell target (i.e. supernatants labeling CD3+ cells, regardless of whether they were CD4+, CD8+ and/or DN), were selected; the main selection criteria was reactivity against CD3 lymphocytes alone, no reactivity on B cells, weak reactivity against CD4 and CD8 cells, and strong reactivity against DN cells. 500 hybridomas were screened and 20 were selected for high reactivity on DN CD3+ T cells and no reactivity on non- T cells. An example of a non-selected hybidoma is shown in Figure 2.

During the secondary screening, labeling experiments were carried out using supernatants from potentially interesting clones. The FACS labeling protocol involved: FITC anti-human CD4; APC anti-human CD3; PE Cy5 anti-human CD8; SN+ Goat anti mouse F (ab')2 IgG PE. In addition, in subsequent experiments, a goat anti-mouse F (ab')2 anti-IgG+M PE was used to prevent the undue elimination of an IgM. In this way, we were able to identify distinct groups of antibodies based on the percentages of CD4, CD8, and DN cells recognized from four donors. An example of such a labeling experiment is presented in Figure 3 (strategy for secondary screen), showing staining obtained with the supernatant of the 3C10 hybridoma during the secondary screen; from left to right, the four panels show (1) gated live cells, with CD4 on the x-axis and CD8b on the y-axis, and in (2)-(4) CD3-APC on the y-axis and in (2) CD4 on the x-axis showing 0.96% CD4-CD3APC cells, (3) CD8 on the x-axis showing 19.19% CD8-CD3APC cells, and (4) DN on the x-axis showing 27.33% DN-CD3APC cells, These experiments allowed us to define four distinct groups of antibodies based on their reactivities.

Conclusion from this initial characterization:

The proportion of labeled CD4 cells was low, and that of DN cells was compatible with what was expected of MAIT cells in this fraction. The proportion of CD8 cells was very high: approximately 10-20 fold higher than the level of MAIT cells among the CD8 cells. This indicates that the different antibodies are likely not clonotypic. This conclusion was made possible using a multicolor FACS strategy (Figure 4) showing PBMCs staining with TCRpanφγ-PC5, CD3-APC, CD4-APCCy7, CD8-ECD, Supernatant-GAM-PE, Vβl3+2-FITC and DAPI. The strategy took into account quantitative aspects (capacity of antibody production as meansured by mean fluorescence, shown in the top and middle row of panels in Figure 4) and qualitative aspects (Vbeta2+13 reactivity on CD4, CD8 and DN cells, shown in the bottom three panels of Figure 4).

In order to better explore the reactivities of the antibodies, we decided to examine the positive fractions and to quantify the Vα7.2-Jα33 rearrangements within the different fractions.

We selected four supernatants, one from each group. These supernatants, each having a different profile, including the supernatant designated 3C10 were judged as interesting for further study, and were subcloned.

Figure 5 shows the strategy of FACS sorting experiment using a labeling with anti-CD3, TCRγδ, CD4, CD8, and the supernatant allowing the sorting of cells expressing the epitope recognized by the SN among the CD4, CD8 and DN cells; Figure 5 shows an example for sorting of 0.5*10-6 cells on Vbetal3+Vbeta2 on the y-axis and either CD4, CD8 or DN (panels from left to right) on the x-axis, with the bracketed section on the left of each panel showing the negative fraction and the bracketed section on the right of each panel showing the positive fraction. The RNA is then extracted and retro transcribed.

Recovery of cellular fractions (from 15,000 to 500,000 cells, depending on the sample and the supernatant). Resuspension in 200μl RNAbIe. Storage at -20⁰C. RNA extraction. Reverse transcription. Quantitative real-time PCR on the cDNAs using specific primers for huCαH and huVα 7.2 / huJα33, and Taqman probes specific for primer-amplified regions (Vα7.2 or Ca).

Using this method, we were able to quantify the proportion of Vα7.2-Jα33 TCR among the αβ LT in each of the sorted fractions (as a function of the fluorescence kinetics corresponding to the amplification of the Vα7.2-Jα33 fragment relative to that of the Ca fragment). The results were interpreted by calculating the ratios of the Ct Vα7.2Jα33 to Ca (positivation cycles).

Figure 6 shows the results in fractions 3C10 allowing the demonstrations of the anti-Voc7.2 specificity of this supernatant. In each of the three panels in Figure 6 corresponding to the 3C10 fractions that were CD4+, CD8b+ or DN, respectively, results show that the 3C10A8+ fraction corresponded to Vα7.2 cells as assessed by quantitative RT-PCR. Results are also summarized in Table 1 below.

Table 1 : Quantitative RT-PCT

Remarks: Not all of the fractions could be sorted for all supernatants due to insufficient quantities of supernatant. The fractions were split into two immediately following sorting (as long as sufficient cells were available) in order to have duplicates for the RNA extraction step. The DN fractions were the most interesting, because MAITs are significantly more abundant among this population.

We observed an enrichment of Vα7.2-Jα33 T cells in the positive fractions for all six supernatants. Nevertheless, certain of the supernatants were particularly interesting: they both enriched the positive fractions by a factor of 100 to 200 relative to the total fractions (for DNs), and the negative fractions were almost completely devoid of Vα7.2-Jα33 T cells. For antibody 3C10, enrichment of Vα7.2-Jα33 T cells was by a factor of between 20 and 25,000. Four supernatants were retained at this stage.

Since all of the supernatants enriched the Vα7.2-Jα33 fractions by a significant amount, we investigated the possibility (as we not working under a hood), that the antibodies used in the staining experiments were contaminated in some way (the Vα7.2-Jα33 plasmids had been previously used in the same lab, although in a different room).

We thus decided to re-sort the CD4, CD8 and DN fractions for SN+ and SN- under sterile conditions (staining under a hood with new antibodies) for the four retained supernatants. Further, we decided to alter the staining in an attempt to exclude γδ T cells, which make up a substantial proportion of DN T cells in the blood.

Antibodies used: FITC anti-human CD4, APC anti-human CD3, PE Cy5 anti-human TCR pan γδ, PE-TR anti-human CD8β, and SN+ goat anti-mouse F (ab')2 IgG+M-PE;DAPI was used as well; methods: 7 color cytometry (FACS Aria, BD).

Remarks: for one clone, the fraction sorted for CD4 SN+ contained very few cells (<1000) and PCR yielded no product. Nevertheless, for the three other re-sorted supernatants we observed a significant enrichment of Vα7.2-Jα33 T cells in the SN + fractions (factor of 100 for two subclones (one of which is 3Cl 0), and a factor of 3 for another subclone) As a further confirmation, the PCR products for Vα7.2-Jα33 and Vα7.2-Cα were sequenced to confirm the presence of the canonical MAIT CDR3α sequence. For the selected fractions sorted for the supernatants (i.e. SN+/SN-), total cDNA was obtained and amplified using primers for Vα7.2-Cα and Vα7.2-Jα33 (positive control). The samples were purified and sequenced. From this it was observed that that the CD4, CD8 and DN cell populations recognized only by two antibodies (including 3C10) which associate preferentially with Vα7.2-Jα33.

To ensure the specificity of the monoclonal antibodies, several additional experiments were carried out.

Confirmation Tests: Tests on the CD4, CD8 and DN fractions for 3C10. We used quantitative PCR to determine the proportion of T cells expressing the different Vβ fragments. We specifically wanted to know whether there was an enrichment of segments Vβ2 and Vβl3 (which are preferentially expressed by MAITs) in the fractions, with a concomitant global depletion of other Vβs.

As with our earlier results, we observed that the frequency of segments Vβ2 and V βl3 were higher in the 3Cl 0+ fractions than in the 3Cl 0- fractions. We also observed a depletion of certain other fragments (Vβ5, Vβ8, Vβl2...).

FACS with double staining for Vβ2 and SN: anti-Vβ2 FITC; SN + Gam PE (IgG+M); anti-CD3 APC; anti-TCR panγδ PC5; anti-CD4 APC Cy7; anti-CD8β PE TR. 7 color cytometry (FACS Aria, BD).

The goal of the following experiment was to determine the proportion of Vβ2 + cells among the SN+ fractions, as well as the proportion of SN+ cells among the Vβ2+ fractions. A sample staining is shown in Figure 7.

Results: For DN cells: Depending on the supernatant, excluding the γδ T cells between 5 to 35% (for 3Cl 0) of the DN cells were SN+. More than 80% of the Vβ2+ DN cells were stained by the different antibodies, versus only 30%-60% for the Vβ2- DN cells. 12% to 15% of the SN+ DN cells were Vβ2+, versus less than 3% of the SN- DN cells.

For CD8β cells: Around 5% of the CD8β cells were SN+ (regardless of the SN). We also observed a greater percentage of SN+ cells among the Vβ2+ than among the Vβ2- cells. Similarly, the percentage of Vβ2+ cells was higher among the SN+ than among the SN- cells.

For CD4 cells: For the CD4 fraction, which were only very poorly stained by the supernatants (around 1% of the CD4 cells were SN+), we did not observe a significant difference for Vβ2.

We thus observed an association of Vβ2 and Vβl3 which are predominant but not exclusive, and represent: 30%a 40% of DN, 10 a 15% of CD4 and CD8 recognized by the antibody. Other

Vβ tested such as 17, 1, 5.1,11, etc. are present in similar proportions as Vβ8. We note that the different subclones behaved similarly. These results lend further support to the notion that these antibodies allow an enrichment of sorted fractions containing MAITs. 3C10 being the most promising for recognition of Vα7.2-Jα33. These hybridomas were subcloned again to obtain stable clones, which allowed us to produce ascites and purify a large quantity of antibodies as well as to obtain cell lines. Antibodies were biotinylated for use in FACS and immunohistochemistry.

Example 2. Characterization of antibodies 3C10 and 1A6

In order to further characterize the antibodies, we first tested 3C10 binding on a human Vα7.2 transfectant using FACS analysis. Figure 8 shows the results on the following transfectants tested: 58α-β-, 3Cl 1 (huVα7.2+mVβl3), αDPβDP (mVα8+mVβl3), ICElO (huVα7.2+ mVβ8.2) and αDPβDP (mVα4+mVβ8.2). We observed that antibody 3C10 recognizes transfectants 3Cl 1 and ICElO which both express the Vα7.2 chain. Figure 8, top left panel shows binding of control IgGl antibody, and the binding to transfected cells is similar and not significant cell transfected with each of 58α-β-, 3Cl 1, and αDPβDP; top right panel shows binding of control IgGl antibody, and the binding to transfected cells is similar and not significant cell transfected with each of 58α-β-, ICElO and αDPβDP. Figure 8, bottom left panel tests binding of antibody 3C10A8 (2ug/ml) to each of 58α-β-, 3Cl 1, and αDPβDP, demonstrating strong binding (thick curve, indicated by arrow) to only 3Cl 1

(huVα7.2+mVβl3). Figure 8, bottom left panel tests binding of antibody 3C10A8 (2ug/ml) to each of 58α-β-,lCE10 and αDPβDP, demonstrating strong binding (thick curve, indicated by arrow) to only ICElO (huVα7.2+ mVβ8.2). Thus, 3C10A8 is able to recognize huVα7.2 TCR.

Antibody 3C10 was then tested to assess its specifity for different Va segments. Figure 9 shows multicolor FACS analysis on gated CD4, CD8 and DN TCRαβ T cells with 3C10 on one hand and anti-Vα2/Vαl2/Vθc24 on the other hand to show that the 3C10 antibody is not an anti-Vα framework antibody. This experiment revealed that 3C10 did not recognize Vα2, 12 nor Vα24.

Cytometry is insufficient for determining the specificity of Vα7.2 due to lack of available anti Va. A Q-PCR method was therefore used to study the Va repertoire. Binding of antibody 3C10 to CD4, CD8 and DN fractions was followed by quantitative PCR using a battery of Va primers to determine the identity of the Va chain expressed by the T cells. This experiment revealed that 3C10 did not recognize Vαl4 nor Vα24. Thus, 3C10 is specific for Vα7.2. Figure 1OA and 1OB shows the results. Figure 1OA, left top, left middle and left bottom panels show the study of the Valpha repertoire for the total CD8 population, the 3C10A8 positive CD8 population, and the 3C10A8 positive CD8 population, respectively. Figure 1OA, top right, middle right and bottom right panels show the study of the Valpha repertoire for the total CD4 population, the 3C10A8 positive CD4 population, and the 3C10A4 positive CD8 population, respectively. Figure 1OB, top, middle and bottom panels show the study of the Valpha repertoire for the total DN population, the 3C10A8 positive DN population, and the 3C10A4 positive DN population, respectively.

Antibody 3C10 was next tested for specific reactivity with the Vα7.2 chain. The Vαl 1 chain is highly similar to the Vα7.2 chain and could present an epitope that could be recognized by an anti- Vα7.2 antibody. Binding of antibody 3C10 to DN fractions was followed by quantitative PCR using a battery of Vα7.2 and Vαl 1 primers to determine the identify of the Va chain expressed by the T cells. Figure 11 shows the results; the top panel shows the repertoire of

Valpha segments in total DN TCR alpha-beta T cells, the middle panel shows the repertoire of Valpha segments in 3C101A8 positive DN TCR alpha-beta T cells, the bottom panel shows the repertoire of Valpha segments in 3C101A8 negative DN TCR alpha-beta T cells. It appeared at first that 3C10 regonized the Valpha7 chain and the Valphal 1 chain. Due to the sequence similarity between Valpha7 and 11 chains, PCT primers amplified both, and amplicons were sequenced to confirm the identity of the amplified Va sequence. This amplicon sequencing revealed that 3C10 did not recognize Vαl 1.

Finally, antibody 3C10 was tested for its ability to act either as an agonist or antagonist. Figures 12 show that 3C10 has an agonist effect on Ly T CD4-, CD8- cells and on CD3+, γδ-, CD4-, CD8- cells; anti-CD28 antibody used in combination). Figure 12 shows a large increase in specific 3C10 positive cell proliferation by a dilution of CFSE. Figure 13 shows the characterization of the cytokine secretion pattern of 3C10 positive and negative T cells after stimulation by anti-CD3 and anti-CD28 antibody and demonstrates a large increase in production of TNF, RANTES , IL-10 and a very little production of IL-2.

Figure 15 shows example of surface staining on frozen section of human thymus. The 3C10 antibody stains huValpha7.2 chain transfected-58alpha-beta cells as well as cells in the frozen section of human thymus. The antibody can therefore be useful to identify MAIT cells in diagnostic (e.g. immunohistochemistry) protocols.

Two antibodies were sequenced: the 3C10 and 1A6. Figure 16 shows the nucleotide and polypeptide sequence of the variable regions of the heavy chains of the 3C10 and 1A6 monoclonal antibody. The DNA sequences encoding 3C10 and 1A6 heavy chain variable region sequence are also listed in SEQ ID NOS 1 and 11, respectively, and amino acid sequences for the 3C10 and 1A6 heavy chain variable region sequence are also listed in SEQ ID NOS 2 and 12, respectively. The 3C10 and 1A6 antibodies differ at amino acid position 58 in the heavy chain of SEQ ID NO 2 and 12, where 3C10 has a threonine at this position and 1A6 an isoleucine. Figure 17 shows the nucleotide and polypeptide sequence of the variable regions of the lightchains of the 3C10 and 1A6 monoclonal antibody. The DNA sequences encoding 3C10 and 1A6 light chain variable region sequence are also listed in SEQ ID NOS 3 and 13, respectively, and amino acid sequences for the 3C10 and 1A6 light chain variable region sequence are also listed in SEQ ID NOS 4 and 14, respectively. The 3C10 and 1A6 antibodies differ at amino acid positions 26 (3C10 has an arginine and 1A6 has a serine), 31 (3C10 has a serine and 1A6 has a threonine), 52 (3C10 and 1A6 have a serine, but different codon usage) and 107 (3C10 has a arginine and 1A6 has a lysine) in the light chain of SEQ ID NO 4 and 14, where 3C10 has a threonine at this position and 1A6 an isoleucine. For each sequence, the CDR sequences are indicated by underlining, and sequence differences between 3C10 and 1A6 are shown.

The 3C10 and 1A6 antibodies were compared for binding. The ClO and 1A6 Mab recognize the same epitope. PBL were purified by Ficoll gradient density from normal volunteers and then incubated with unlabelled 1A6 or 3C10 supernatant washed and then with a PE-conjugated goat anti-mouse antibody. The cells were then extensively washed. The free antibody sites were saturated with mouse serum. The cells were then incubated with the following antibody mixture CD19-PCy5/CD3-FITC/CD4-APC/CD8β-PETR/3C10-Biot, washed and revealed with a PE- Cy7 conjugated streptavidin. It was observed that 1) all the cells recognized by one of the antibody is stained by the other, and2) increasing the concentration of one of the two antibodies decreases the staining of the other.

Conclusion. The results obtained with double staining for Vβ2/SN clearly show that the antibodies recognize a TCR-linked epitope, and probably an alpha chain. Most interesting is antibody 3C10 specifically recognizes Vα7.2, and do not recognize Vαl 1, Vαl4 nor Vα24. Moreover this antibody is able to act as an agonist, and Vα7.2-Jα33 are able to produce large amount of TNF, RANTES and IL-10.

In a further experiment, membrane phenotype of MAIT cells was studied, using the newly obtained Vα7.2 specific antibodies. A summary of the studies carried out on MAIT cells is displayed in Table 2, below. 3C10 DN human T cells were stained with the indicated antibodies or alternatively sorted by FACS. Their transcription pattern was studied using the Affymetrix Ul 33 micro-array and compared to mainstream memory (CD45RO+) T cells sorted from the same 3 donors. The following markers were positive on both flow cytometry and Affymetrix data: 2B4, CD161, CD26, CD28, CD94, CD96, ILl 2Rb, ILl 8Ra, and NKG2D. The following additional markers were positive in flow cytometry and not negative on micro-arrays: CD 122, CD127, CD27 and CD45RO. Table 2

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e. g. , all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate).

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated.

The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability and/or enforceability of such patent documents, The description herein of any aspect or embodiment of the invention using terms such as reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of," "consists essentially of or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e. g. , a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

This invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum extent permitted by applicable law.

All publications and patent applications cited in this specification are herein incorporated by reference in their entireties as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. An isolated antibody that specifically binds an epitope present on a Vα7.2-Joc33 polypeptide.

2. The antibody of claim 1, wherein said polypeptide is Vα7.2-Jα33/Vβl3.

3. The antibody of claim 1, wherein said polypeptide is a human polypeptide.

4. The antibody of any one of the above claims, wherein said antibody or a fragment thereof is further characterized by not specifically binding to a Vαl 9 polypeptide.

5. The antibody of any one of the above claims, wherein said antibody or a fragment thereof is further characterized by not specifically binding to a Vαl 1 polypeptide.

6. The antibody of any one of the above claims, wherein said antibody or a fragment thereof is further characterized by not specifically binding to a Vα24 polypeptide.

7. The antibody of claim 1, wherein said antibody binds to substantially the same epitope as a monoclonal antibody 3C10.

8. The antibody of claim 7, wherein said monoclonal antibody is 3C10.

9. The antibody of any one of the above claims, wherein said monoclonal antibody is an agonist of a cell that expresses a Vα7.2-Joc33 polypeptide.

10. The antibody of any one of the above claims, wherein said antibody is a monoclonal antibody or a fragment or derivative of a monoclonal antibody.

11. The monoclonal antibody or fragment thereof according to claim 1 , wherein the antibody or fragment has a CDR comprising an amino acid sequence of any one of SEQ

ID NOS 5 to 7 or any one of SEQ ID NO 5, 15 and 7.

12. The monoclonal antibody or fragment thereof according to claim 1, wherein the antibody or fragment has a CDR comprising an amino acid sequence of any one of SEQ ID NOS: 8 to 10 or any one of SEQ ID NOS: 16, 9 and 10.

13. The antibody of claim 8, wherein said monoclonal antibody is 3C10, or a fragment or derivative thereof.

14. The antibody of claim 9, wherein said cell is a human MAIT cell.

15. The antibody of any one of the above claims, wherein said antibody is an antibody fragment selected from Fab, Fab', Fab'-SH, F (ab') 2, Fv, diabodies, single-chain antibody fragment, or a multispecific antibody comprising multiple different antibody fragments.

16. The antibody of any one of the above claims, wherein said antibody is a humanized antibody or a chimeric antibody.

17. The antibody of any one of the above claims, wherein said antibody is conjugated or covalently bound to a toxin, a detectable moiety, or a solid support.

18. The antibody of claim 1, wherein said antibody specifically binds to human MAIT cells.

19. The monoclonal antibody according to claim 1, wherein said antibody comprises:

(a) a heavy chain comprising the amino acid sequence of SEQ ID NO:2 fused to a human IgGl chain constant region; and a light chain comprising the amino acid sequence of SEQ ID NO:4 fused to human kappa chain constant region; or (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 12 fused to a human IgGl chain constant region; and a light chain comprising the amino acid sequence of SEQ ID NO: 14 fused to human kappa chain constant region.

20. The antibody of any one of the above claims, wherein said antibody increases the activity of said human MAIT cells.

21. The antibody of any one of the above claims, wherein said antibody causes the elimination of human MAIT cells.

22. The antibody of any one of the above claims, wherein said antibody specifically binds to, increases the activity of, or decreases the activity of, or causes the elimination of non-human primate MAIT cells.

23. A hybridoma comprising: a) a B cell from a non-human mammalian host that has been immunized with an antigen that comprises an epitope present on a Vα7.2-Joc33 polypeptide, fused to b) an immortalized cell, wherein said hybridoma produces a monoclonal antibody that specifically binds to said epitope.

24. The hybridoma of claim 23, wherein said polypeptide is Vα7.2-Jα33/Vβl3.

25. The hybridoma of claim 23, wherein said polypeptide is a human polypeptide.

26. The hybridoma of claim 23, wherein said monoclonal antibody specifically binds to, increases the activity of, or decreases the activity of, human MAIT cells.

27. The hybridoma of claim 23, wherein said hybridoma produces a monoclonal antibody that binds to substantially the same epitope as monoclonal antibody 3C10 or 1A6.

28. The hybridoma of claim 23, wherein said monoclonal antibody is 3C10.

29. The hybridoma of claim 23, wherein said monoclonal antibody is 1A6.

30. The hybridoma of claim 23 to 29, wherein said monoclonal antibody is an agonist of a cell which expresses a Vα7.2-Joc33 polypeptide.

31. The hybridoma of claim 27, wherein said monoclonal antibody is 3C10.

32. The hybridoma of claim 27, wherein said hybridoma is the cell deposited at CNCM having registration no. 1-3693

33. A method of producing an antibody that specifically binds MAIT cells, said method comprising the steps of: a) immunizing a non-human mammal with an immunogen comprising a Vα7.2-Joc33 polypeptide; and b) preparing antibodies from said immunized animal that specifically bind to said Vα7.2-Joc33 polypeptide.

34. The method of claim 33, wherein said polypeptide is a Vα7.2-Jα33/Vβl3 polypeptide.

35. The method of claim 33, wherein said polypeptide is a human polypeptide.

36. The method of claim 33, wherein said MAIT cells are human MAIT cells.

37. The method of claim 33, wherein the antibodies prepared in step (b) are monoclonal antibodies.

38. The method of claim 33, further comprising the step: c) selecting antibodies of (b) that are capable of modulating the activity of MAIT cells.

39. The method of claim 38, wherein the antibodies selected in step (c) cause an at least about 10%, 20%, 30%, 40%, 50%, or greater increase or decrease in MAIT cell activity.

40. The method of claim 37, further comprising the step of making fragments or derivatives of the selected monoclonal antibodies.

41. The method of claim 40, wherein said fragments or derivatives are selected from the group consisting of Fab, Fab', Fab'-SH, F (ab') 2, Fv, diabodies, single-chain antibody fragment, multispecifϊc antibodies comprising multiple different antibody fragments, humanized antibodies, and chimeric antibodies.

42. The method of claim 40, wherein said monoclonal antibodies are derivatized to make them humanized, human, or chimeric.

43. A pharmaceutical composition comprising an antibody that specifically binds a human

Voc7.2-Jα33 polypeptide, wherein said antibody is present in an amount effective to detectably modulate MAIT cell activity in a patient or in a biological sample comprising MAIT cells; and a pharmaceutically acceptable carrier or excipient.

44. The composition of claim 43, further comprising a therapeutic agent selected from an immunomodulatory agent, a hormonal agent, a chemotherapeutic agent, an anti- angiogenic agent, an apoptotic agent, a second antibody that binds to and inhibits an MAIT receptor, an anti-infective agent, a targeting agent, an anti-inflammation agent, a steroid, an immune system suppressor, an antibiotic, an anti-diarrheal drug, and an adjunct compound.

45. The composition of claim 43, wherein said antibody increases the activity of human

MAIT cells.

46. The composition of claim 43, wherein said antibody decreases the activity of human MAIT cells.

47. A method of modulating MAIT cell activity in a patient in need thereof, comprising the step of administering to said patient a composition of claim 43.

48. The method of claim 47, wherein said patient is suffering from an immune disorder involving the mucosa.

49. The method of claim 47, wherein said disorder is selected from the group consisting of cancer, infection, viral infection, irritable bowel syndrome, ulcerative colitis, and Celiac disease.

50. The method of claim 47, wherein said MAIT cell activity is increased in the patient.

51. The method of claim 47, wherein said MAIT cell activity is decreased in the patient.

52. The method of claim 47, comprising the additional step of administering to said patient an appropriate additional therapeutic agent selected from an immunomodulatory agent, a hormonal agent, a chemotherapeutic agent, an anti- angiogenic agent, an apoptotic agent, a second antibody that binds to and inhibits an MAIT receptor, an anti-infective agent, a targeting agent, an anti- inflammation agent, a steroid, an immune system suppressor, an antibiotic, an anti-diarrheal drug, and an adjunct compound.

53. A method of detecting the presence of MAIT cells in a biological sample or an individual, said method comprising the steps of a) contacting said biological sample or individual with the antibody of claim 1 ; and b) detecting the presence of said antibody in said biological sample or individual.

54. The method of claim 53, wherein said antibody is conjugated or covalently bound to a detectable moiety.

55. A method of purifying MAIT cells from a biological sample, said method comprising the steps of : a) contacting said sample with the antibody of claim 1 under conditions that allow said cells to bind to said antibody, optionally wherein said antibody is conjugated or covalently bound to a solid support; and b) selecting cells bound to said antibody, optionally eluting said bound cells from said antibody conjugated or covalently bound to a solid support.

56. A method of purifying MAIT cells from a biological sample, said method comprising the steps of : a) contacting said sample with an agent that binds a human Vα7.2-Joc33 polypeptide and/or an agent that binds a polypeptide selected from the group consisting of 2B4, CD161, CD26, CD28, CD94, CD96, IL12Rb, IL18Ra, NKG2D, CD122, CD 127, CD27 and CD45RO, under conditions that allow said cells to bind to said agent, optionally wherein said agent is conjugated or covalently bound to a solid support; and b) selecting cells bound to said agent, optionally eluting said bound cells from said agent conjugated or covalently bound to a solid support.

57. A method of profiling a MAIT cell population, or a method of identifying a MAIT cell, comprising a step of detecting in said cell population the presence or absence of a polypeptide selected from the group consisting of: 2B4, CDl 61, CD26, CD28, CD94, CD96, IL12Rb, IL18Ra, NKG2D, CD122, CD127, CD27 and CD45RO.

58. A method of profiling a cell population comprising a step of detecting in said cell population the presence or absence of a Vα7.2-Joc33 polypeptide, optionally wherein the presence of a Vα7.2-Joc33 polypeptide designates the cell as a MAIT cell.

59. The method of claim 57, further comprising detecting in said cell population the presence or absence of a polypeptide selected from the group consisting of: 2B4, CD161, CD26, CD28, CD94, CD96, IL12Rb, IL18Ra, NKG2D, CD122, CD127, CD27 and CD45RO.

60. A population of cells obtained according to the method of claims 55 or 56.

61. An isolated population of MAIT cells, wherein said cell population is characterized by purity of at least 90 % of Vα7.2-Joc33 polypeptide-expressing cells