WO2003035689A1 - Process for reducing immunogenic reactions using selective th1 cell binding antibodies - Google Patents

Abstract

A process for reducing an undesired immune response in a patient comprising administering to said patient an agent, such as an antibody, against a Th-specific antigen in an amount effective to suppress the ability of Th lymphocytes, especially Th1 celles, to promote such reaction is disclosed. Such immune reactions include autoimmune conditions, graft rejection and the like. Also described is a process for identifying, as well as deleting or eliminating, Th1 cells comprising contacting an antibody specific for homologs of Phan TM4 protein with a sample of cells under conditions promoting binding to the protein wherein said binding indicates the presence of Th1 cells.

Description

PROCESS FOR REDUCING IMMUNOGENIC REACTIONS USING SELECTIVE TH1 CELL BINDING

ANTIBODIES

FIELD OF THE INVENTION

The present invention relates generally to the control of diseases or disorders mediated by the actions of Th1 cells, such control being mediated through purposeful intervention targeted to Th1 cells, such as through their deletion or inactivation, and/or their identification. This invention further relates to antibodies against Th1 cells and to using antibodies specific for antigens present on Th lymphocytes, especially Th1 lymphocytes, for protecting against damage of tissues by the recipient's immune system.

BACKGROUND OF THE INVENTION

Following exposure to foreign antigens, such as that which results from infection or the grafting of foreign tissue, Th cells differentiate into Th1 and Th2 cells with differing functions. Th1 cells produce interferon gamma (IFN-γ) and interleukin 2 (IL-2) (both associated with cell-mediated immune responses). Th1 cells play a role in immune responses commonly involved in protection against pathogens. Th1 cells, however, also have adverse effects in that they play a role in a range of autoimmune diseases (insulin-dependent diabetes, multiple sclerosis, rheumatoid arthritis, lupus erythematosis, scleroderma, psoriasis), in inflammatory bowel disease, in so-called type IV hypersensitivity reactions, and in the rejection of foreign tissue grafts. A surface protein marker for Th1 cells would provide a "target" for specifically identifying such cells within a cell population as well as one for preventing such cells from acting against tissues. In short, such cell-specific "target" antigens would thus act as targets for antibodies and other antigen-specific agents that would reduce the ability of such cells to promote tissue damage, if not eliminate such cells entirely.

An antigen reported to be specific for Th1 cells in mice has been described (see Venkataraman et al, J. Immun. 165:632-636 (2000)). However, while this protein, called Chandra, is reported to be a four-transmembrane domain protein, its surface portions have not been reported. In addition, a human gene corresponding to Chandra is known (US patent No 5,871 ,930),

The published cDNA and predicted protein sequence of Chandra and the mouse TM4 gene are essentially identical.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a process for reducing an immune response by effector cells in a recipient comprising administering to said recipient an agent that reacts with a Th-lymphocyte specific cell surface structure and in an amount effective to decrease the ability of Th lymphocytes to support damage directed to tissues expressing antigens which those T-cell can recognize.

In another aspect, the present invention relates to a process for treating autoimmune diseases (including, but not limited to, insulin-dependent diabetes, multiple sclerosis, rhuematoid arthritis, psoriasis, lupus erythematosis, scleroderma), inflammatory bowel disease, type IV hypersensitivity, and promoting acceptance of tissue transplantation in a transplant recipient, comprising administering to patients an antibody against a Th-specific antigen in an amount effective to treat such disease or disorder or to promote acceptance of a transplant.

In one embodiment of this process, the Th-specific antigen is a Th1 specific antigen, especially where said antigen is a human counterpart of the Chandra protein, dubbed PhanTM4, and most preferably an extracellular domain of PhanTM4, such as the domain loop between the first and second TM (transmembrane) domains (the first external loop region shown in Figure 5) and between the third and fourth TM domains (the third external loop region shown in Figure 5). MS4A6 (human) and MS4A6B (mouse) proteins can likewise be used in such processes.

Still more particularly, the present invention relates to deletion or elimination of Th cells, especially Th1 cells, by use of an agent that binds to an exposed loop portion of a transmembrane protein on Th, especially Th1 , cells and wherein said transmembrane protein is at least 95% identical to the PhanTM4 sequence disclosed herein in SEQ ID NO: 4) or to the MS4A6 (human) and MS4A6B (mouse) protein sequences disclosed herein, such as the loop regions thereof (SEQ ID NO: 13 TO 16).

Still more particularly, the present invention relates to deletion or elimination of human Th1 cells by use of an agent that binds to Th1 cells but does not bind to human T_reg cells, especially wherein said agent binds to an exposed loop portion of a transmembrane protein on Th, especially Th1, cells wherein the exposed domain or domains of said transmembrane protein is at least 95% identical to the exposed domains labeled external loop 1 and/or external loop 3 as depicted in Figure 5. In a preferred embodiment, said agent is an antibody, preferably a monoclonal antibody, most preferably an antibody specific for the PhanTM4 polypeptide disclosed herein, or a protein highly homologous to said protein. Such antibody may also bind to the MS4A6 (human) and MS4A6B (mouse) proteins disclosed herein.

Applicant has found that, with respect to PhanTM4 and MS4A6 (human) and MS4A6B (mouse) proteins, the very short loop between the 1^st and 2^nd TM or transmembrane regions together with the longer loop between the 3^rd and 4^th transmembrane regions are on the outside of the Th1 cell membrane, and are available for cell surface binding with antibodies or other ligands.

In another aspect, the present invention relates to identification and/or detection of Th cells, especially Th1 cells, by use of an agent that binds to an exposed loop portion of a transmembrane protein on Th, especially Th1 , cells and wherein said transmembrane protein is at least 95% identical to the PhanTM4 sequence disclosed herein in SEQ ID NO: 4. In a preferred embodiment, said agent is an antibody, preferably a monoclonal antibody, most preferably an antibody specific for the PhanTM4 polypeptide disclosed herein, or a protein highly homologous to said protein. Useful antibodies also include antibodies against the MS4A6 (human) and MS4A6B (mouse) proteins, especially the loop domains of this protein.

More particularly, the present invention relates to identification and/or detection of human Th1 cells by use of an agent, such as an antibody, that binds to Th1 cells but does not bind to human T_reg cells.

In other embodiments, the present invention relates to processes for reversing tissue damage caused by Th1 (and possibly also Th2 cells). In another aspect, the present invention relates to a process for reducing activity of Th cells in a transplant recipient comprising administering to said recipient an agent that binds to PhanTM4 protein in said recipient, or to the MS4A6 (human) protein, especially the loop domains of these proteins. In particular embodiments thereof, said agent may be an organic chemical capable of binding to said protein and inactivating it or an antibody, such as one that is specific for said protein, preferably an anti-PhanTM4- specific antibody. In other embodiments, said Th cells are preferably Th1 cells, most preferably Th1 cells but not T_reg cells.

In a further aspect, the present invention relates to a process for reducing the proliferation of Th cells in a patient comprising administering to said patient an antibody against a Th-specific antigen present on said Th cells. In a specific embodiment thereof, said antibody is an anti-PhanTM4 antibody.

This invention also relates to treatment with a therapeutic agent that is to treat or prevent a disease wherein administration of the therapeutic agent produces in the host an immune response against the agent. The present invention is applicable to inhibiting, preventing, or ameliorating an immune response against such an agent by administering the above-mentioned antibody (or fragments or derivatives thereof) to a host. Such inhibiting, preventing, or ameliorating an immune response against the agent includes inducing tolerance to the agent. DEFINITIONS

Unless specifically stated otherwise herein, the following terms when used herein have the indicated meanings:

"Th1 cell" refers to a T helper lymphocyte wherein reverse transcriptase- polymerase chain reaction (RT-PCR) data shows that expression of mRNA for the PhanTM4 protein of the invention is high versus the level shown in other lymphocytes, and that after appropriate activation produces interferon-gamma and not interleukin 4.

"Th2 cell" refers to a T helper lymphocyte wherein reverse transcriptase- polymerase chain reaction (RT-PCR) data shows that expression of mRNA for the PhanTM4 protein of the invention is low versus the level shown in Th1 cells, and that after appropriate activation produces interleukin 4 and not interferon-gamma.

"ThO cell" or "naive/ThO" cell refers to a T lymphocyte that, when stimulated, will differentiate into a Th1 or Th2 cell and which, like a Th2 cell, expresses only lower levels of the PhanTM4 protein disclosed herein versus the expression by Th1 cells, and that after appropriate activation produces both interleukin 4 and interferon- gamma.

"Treg cell" refers to a lymphocyte that has the effect of inhibiting other lymphocytes from mounting immune responses do not express the PhanTM4 protein disclosed herein, and that expresses high levels of CD25 and CTLA4 (CD152) compared to ThO, Th1 and Th2 cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows expression of Chandra/TM4 mRNA by quantitative RT-PCR of mouse tissues. Quantitative real time reverse transcriptase polymerase chain reaction (RT-PCR), according to the Applied Biosystems Taqman(TM) methodology, was used to determine the levels of expression of the mouse Chandra/Tm4 mRNA in various tissue samples and cell lines. These data show that the Th1 clone (R2.2), ConA activated spleen cells (that are enriched for ThO and Th1 cells) and normal lymph node cells, express the highest levels of Chandra/TM4 mRNA. Two different Th2 clones, normal resting spleen cells and normal thymocytes were also positive but ranged from 5-20 fold lower than Th1 cells. Treg cells (clone Tr1 D1 ) and a number of normal tissues, including dendritic cells, skin, kidney and brain, had either undetectable Chandra/TM4 mRNA or were more than 100 fold lower than Th1 that is close to the limit of detection in this assay.

Figure 2 shows immunofluorescence analysis of Chandra/TM4 protein expression. The three panels show different immunofluorescence histograms, as measured by FACS analysis, for the expression of Chandra/TM4 protein using a rabbit antibody against the C terminal peptide (NH2-

C)EHQGTNVPGNVYKNHPGEIV(-CONH2). In the top panel the rabbit antibody against Chandra/TM4 has been directly conjugated to the fluorescent dye Alexa488 and used to stain either Th1 (R2.2), Th2 (R2.4) or T_reg (Tr1 D1 ) clones, after fixing and permeabilization (histograms shown in solid colors). When compared to the nonspecific backgrounds (histograms shown as dotted lines) obtained after inhibition with excess peptide, the relative expression of Chandra TM4 protein on each cell type is calculated as Th1=240, Th2=50 and Treg=0 in arbitrary fluorescence units. In the second panel, the unconjugated rabbit anti-Chandra/TM4 was used to stain either live (solid color histogram) or fixed and permeabilized (unfilled histogram), followed by detection with FITC goat anti-rabbit IgG and FACS analysis. This demonstrates that the C terminal peptide sequence of Chandra TM4 is detectable only on the inside of Th1 cells. The third panel shows staining of normal mouse peripheral blood CD4+ T cells with the Alexa488 conjugated anti-Chandra/TM4, in a four color FACS analysis of different subpopulations. The lowest fluorescence, equivalent to background staining (after peptide inhibition, not shown), is seen on the gated Treg population (CD4+, CD25+, CD103+ cells shown in green; median fluorescence = 80), while naive/ThO cells (CD4+, CD25-,CD103- shown in purple; median fluorescence = 108) are weakly positive, while CD4+CD25-CD103+ cells contain the majority of Th1 cells with a strong positive peak (median fluorescence = 285). Figure 3 shows SAGE tag analysis of mRNA expression profiles for Chandra/TM4 and MS4A6B. Serial Analysis of Gene Expression (SAGE) libraries were made for a number of different cell types, as listed down the side of each histogram, using the anchoring enzyme Nla-lll. Each mRNA transcript within the libraries is identified by a unique 10 base pair tag following the most 3' Nla-ll site, and the abundance of each mRNA species is directly related to the frequency of the corresponding tag. The tag for Chandra/TM4 is (CATG)GCAGTGGTTC and the upper histogram shows that the tag and therefore the mRNA is most abundant in Th1 cells, and is also present at a lower level in CD4+CD25+ (comprised of memory cells, Th2 cells and Treg cells) as well as CD4+CD25- (comprised of naive/ThO and Th1 cells). All three Treg libraries are negative for the Chandra/TM4 tag, as are the non-T cell derived libraries. The Th2 library fails to show the Chandra/TM4 tag probably because it contains only approx. 10,000 tags, which would be insufficient to detect the tag if the abundance is 10-20 fold lower than the equivalent Th1 library. The lower panel shows the SAGE profile of another member of the TM4 family (MS4A6B) that has the tag (CATG)TTGTTGGTTC, and is similarly restricted to Th1 clone and the CD4+CD25- (naive/ThO and Th1 ) ceils.

Figure 4 shows an alignment of Th1 expressed TM4 family sequences to predict antibody sites for MS4A6. This figure shows an alignment of mouse Chandra/TM4 and human PHANTM4 together with the mouse MS4A6B and the human MS4A6. The predicted external immunogenic loops between TM3 and TM4 are from the amino acids numbered 141 through 184 and between TM1 and TM2 from amino acids numbered 68 through 85 (please note that as the loops are different sizes there are dashes inserted to indicate where gaps have been inserted to keep the optimal alignment).

Figure 5 shows an alignment of the TM4/CD20 family with the sequence of human PhanTM4 (SEQ ID NO: 4) indicated thereon. The domain structure, showing the indicated internal and external loop regions, are indicated. DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to a process for reducing an immune response by effector cells in a recipient comprising administering to said recipient an agent that reacts with a Th-lymphocyte specific cell surface structure and in an amount effective to decrease the ability of Th lymphocytes to support such immune response.

Such immune response may include the reaction of a graft recipient to grafted tissue, especially where said donor is allogeneic. Such an immune response may also be autologous where an autoimmune disease is involved, such as insulin- dependent diabetes, multiple sclerosis, rhuematoid arthritis, psoriasis, lupus erythematosis, and scleroderma.

The present invention also provides a process for treating autoimmune disease. A number of autoimmune diseases are known which are at least partly mediated by the actions of Th1 lymphocytes and by utilizing an antibody that is specific for such cells, as where the antibody is reactive with the PhanTM4 determinants disclosed herein, said cells can be removed or their actions ameliorated so that the autoimmune condition is reduced without the need for impairing the efficiency of the immune system as a whole. Thus, the present invention provides processes for the inactivation of selected portions of the immune systems, and reduction of selected immune responses, without the need to render the recipient immunologically incompetent, as may result where more indiscriminant immunosuppressive agents are used.

The present invention also relates to the field of promoting transplantation of organs, tissues and cells, and to the deletion and/or identification of Th1 cells. The invention further relates to antibodies against Th1 cells and to promoting acceptance of allogeneic or xenogeneic grafts, using antibodies specific for antigens present on Th lymphocytes, especially Th1 lymphocytes, thereby promoting acceptance of such grafts by the recipient's immune system. In one aspect, the present invention relates to a process for promoting graft acceptance in a graft recipient in need of said graft comprising administering to said recipient an agent that binds to Th-specific cell surface structures and in an amount effective to decrease the ability of Th lymphocytes to support rejection of said transplanted tissue. In one embodiment, such graft is of tissue to replace that injured due to autoimmune disease or other cause, such as where grafted pancreatic tissue is to be used, such as where the graft is allogeneic or xenogeneic, or where tissue is grafted from one part of the body to another in the same recipient where the tissue to be replaced has been compromised due to autoimmune disease.

In accordance with this process, the Th-specific cell surface structure is present on human Th1 cells but not on human T_reg cells, most preferably present on Th1 cells and on no other cells in the organism.

In a particular embodiment of this process, the Th-specific cell surface structure comprises an amino acid sequence at least 95% identical to an external domain of PhanTM4 (SEQ ID NO: 4), preferably wherein said Th-specific cell surface structure comprises an amino acid sequence at least 98% identical to an external domain of PhanTM4 (SEQ ID NO: 4), and most preferably wherein the Th-specific cell surface structure comprises the amino acid sequence of an external domain of PhanTM4 (SEQ ID NO: 4). In especially preferred embodiments, said external domain is external loop domain 1 of PhanTM4 or external loop domain 3 of PhanTM4 as shown for the sequences depicted in Figure 5. Also useful for these processes is the MS4A6 (human) protein, especially the loop domains of this protein.

In accordance with the foregoing, the present invention contemplates that said Th-specific cell surface structure may be any Th1 specific antigen, especially where said antigen is a human counterpart of the Chandra protein, such as that herein dubbed PhanTM4, and most preferably an extracellular domain of PhanTM4, such as the domain loop between the first and second TM (transmembrane) domains (the first external loop region shown in Figure 5) and between the third and fourth TM domains (the third external loop region shown in Figure 5). Also useful for these processes are the MS4A6 (human) and MS4A6B (mouse) proteins, especially the loop domains of these proteins (SEQ ID NO: 13 to 16).

Antibodies useful in practicing the invention may be either polyclonal or monoclonal antibodies, preferably monoclonal, wherein said antibody is specific for an antigenic determinant comprising an amino acid sequence at least 95% identical to an external loop domain of SEQ ID NO: 4, most preferably wherein said antibody is specific for an antigenic determinant comprising an amino acid sequence at least 98% identical to an external loop domain of SEQ ID NO: 4, and most preferably wherein said antibody is specific for an antigenic determinant comprising the amino acid sequence of an external loop domain of SEQ ID NO: 4. In particularly preferred embodiments, said external loop domain is external loop 1 of PhanTM4 or external loop 3 of PhanTM4 (as shown in Figure 5). Also useful for these processes are antibodies that bind to the MS4A6 (human) protein, especially binding to the loop domains of this protein.

The human equivalent of the mouse Chandra protein already known in the literature is the TM4 protein hDCME31 P whose sequence as listed in GenBank is similar to, but shorter than, the sequence called IGERB in US patent No 5,871 ,930 of Bandman et al. The present invention thus discloses the cloned and sequenced cDNA for human PHA activated T cells, dubbed "PhanTM4" (sequence shown in SEQ ID NO: 3) and deduced amino acid sequence shown in SEQ ID NO: 4. The mouse cDNA is SEQ ID NO: 1 and amino acid sequence is SEQ ID NO: 2.

The TM4 family of sequences has gone from 4 known members (FcERbeta, CD20, MS4A3 and MS4A4) at the end of the year 2000 to either 11 or 16 members described in more recent publications (Ishibasi et al, Gene 264:87-93 (2001) and Liang and Tedder, Genomics 72:119-127 (2001)). The PhanTM4 sequence of the present invention does not agree entirely with known sequences (for example, PhanTM4 differs at its N-terminus with the Bandman IGERB sequences and the latter is longer than PhanTM4). As disclosed herein, the correct start of the expressed protein for the human PhanTM4 is:

MTTMQGMEQAMPGAGPGVPQLGN... (SEQ ID NO: 5)

which may be processed or cleaved at the first glycine and modified with a myristoyl group that is commonly seen in signaling proteins and may target them to lipid rafts is:

Myristoyl-GMEQAMPGAGPGVPQLGN... (SEQ ID NO: 6)

This would match the situation predicted for the mouse Chandra/TM4 which starts:

MQGQEQTTMAVVPGVAVPSKN... (SEQ ID NO: 7)

that would be post translationally modified to:

Myristoyl-GQEQTTMAVVPGVAVPSKN... (SEQ ID NO: 8)

Thus, in one aspect, the present invention discloses the use of antibodies to the external TM3-TM4 loops of both the mouse Chandra/TM4 and human PHANTM4/MS4A4 proteins. The genes for these loops have been cloned into appropriate expression vectors and used to prepare transfectants in both mouse and rat T cell lines for the preparation of polyclonal (rat) and monoclonal antibodies. The amino acid sequences of the mouse and human proteins in this region that are useful as targets for making antibodies are especially the 15-mer sequences from within the loop regions between the TM3 and TM4 transmembrane sequences and also the shorter external loop between TM1 and TM2. Also disclosed are the MS4A6 (human) and MS4A6B (mouse) proteins, especially the loop domains of these proteins (SEQ ID NO: 13 to 16). The mouse Chandra/TM4 sequence between TM3 and TM4 is:

VGSQFPFRYNYTITKG (SEQ ID NO: 9)

This is just long enough to be immunogenic, and it should also have an N- linked glycosylation extending above the membrane thereby aiding immunogenicity when using transfectants as immunogens. Of course, all such protein segments can now be readily prepared synthetically by chemical methods known in the art.

The mouse Chandra/TM4 sequence between TM1 and TM2 is:

TTLFSELPTSVMLM (SEQ ID NO: 10)

although this loop in the mouse may be too short to be of immunogenic utility and probably does not extend sufficiently outside of the membrane.

The human PHANTM4 sequence between TM3 and TM4 is:

FYSFHHPYCNYYGNSNNCHGTMSILMG (SEQ ID NO: 11 )

This is the longest external loop. Unlike the equivalent Chandra TM3 to TM4 loop it does not contain a consensus N-linked glycosylation site, although this does not rule out glycosylation.

The human PHANTM4 sequence between TM1 and TM2 is: CMASNTYGSNPISVHIG (SEQ ID NO: 12)

Of course, the processes of the invention also readily make use of longer sequences that comprise the disclosed loop regions and well as segments of these loop regions that are shorter than those provided herein yet bind to antibodies useful in the processes of the invention. In sum, the specific sequences disclosed herein for loop regions and polypeptides are not to be taken as limiting the bounds and breadth of the invention but the processes disclosed herein can make use of variants of these sequences, including active fragments thereof.

As shown in Figure 1 , the mouse Chandra/TM4 mRNA is expressed in Th1 more than Th2 while T_reg cells are negative for this antigen. Figure 2 shows that the mouse Chandra/TM4 protein (as detected by an antibody to the C terminal peptide) is expressed at high levels in Th1, at lower levels in Th2, ThO and naive T cells while being negative on T_reg cells. This also confirms the orientation of the molecule in the membrane with the C terminus only detectable inside the cell (and thus the external loops are the TM1 - TM2 (TM = transmenbrane) and the TM3-TM4 stretches.

The different levels of mRNA are depicted in Figure 3 (as measured by SAGE) and identifies a SAGE tag for a different TM4 protein with similar specificity (i.e., for Th1 but not for T_reg cells). Figure 4 shows an alignment used as the basis for the determination of the immunogenic external loop sequences disclosed herein for both the mouse MS4A6B (GenBank Accession No. AF237909) and the equivalent human MS4A6 (GenBank Accession No. XM_015538) proteins. The sequences are as follows:

for the mouse MS4A6B TM3-TM4 loop:

GLHPASEQCLQSKELRPTEYHYYQFLDRNECFAAKSVLAG

(SEQ ID NO: 13)

and for the human MS4A6 TM3-TM4 loop:

TLNPASLQCELDKNNIPTRSYVSYFYHDSLYTTDCYTAKASLAG

(SEQ ID NO: 14)

and for the mouse MS4A6B TM1-TM2 loop:

SVPSNLHFTSVFSVLLKSG (SEQ ID NO: 15) and for the human MS4A6 TM1-TM2 loop:

SASFSPNFTQVTTSTLLNSA (SEQ ID NO: 16)

It should be noted that these TM3-TM4 loops (SEQ ID NO: 13 and 14) are considerably longer than the Chandra TM4 or PhanTM4 equivalents, thereby making them more immunogenic and thus more useful targets. Thus, in accordance with the processes of the invention, these sequences (SEQ ID NO: 13 to 16), as well as active fragments thereof and polypeptides incorporating these sequences, may be employed as a target in any of the processes disclosed herein for reducing an immune response, including all assays, as well as all therapeutic and/or prophylactic treatments. Consequently, all processes disclosed herein utilizing PhanTM4 protein and fragments, such as loop regions, thereof can also use MS4A6 (human) and MS4A6B (mouse) proteins and loop domains as well.

More particularly, the present invention relates to deletion or elimination of Th cells, especially Th1 cells, by use of an agent that binds to an exposed loop portion of a transmembrane protein on Th, especially Th1 , cells and wherein said transmembrane protein is at least 95% identical to the PhanTM4 sequence disclosed herein (SEQ ID NO: 4) or to MS4A6 disclosed herein.

In particular embodiments, the present invention relates to deletion or elimination of human Th1 cells, or reduction of their activity, or decrease in their proliferation, by use of an agent that binds to Th1 cells but does not bind to human T_reg cells.

As disclosed herein, such agent preferably binds to an exposed loop portion of a transmembrane protein on Th, especially Th1 , cells wherein the exposed domain or domains of said transmembrane protein is at least 95% identical to the exposed domains labeled external loop 1 and external loop 3 as depicted in Figure 5 for PhanTM4, preferably at least 98% identical to such loop domains and most preferably comprise the amino acid sequences of these loop domains. In preferred embodiments of this process, the Th cells are Th1 cells and the agent is an antibody. In still other preferred embodiments, such agent, including antibodies, especially monoclonal antibodies, bind to Th1 cells but not to human T_reg cells. Other useful agents are those that bind to the MS4A6 (human) protein, especially the loop domains of this protein (SEQ ID NO: 13 to 16).

In another aspect, the present invention relates to identification and/or detection of Th cells, especially Th1 cells, by use of an agent that binds to an exposed loop portion of a transmembrane protein on Th, especially Th1 , cells and wherein said transmembrane protein comprises external loop domains at least 95% identical in amino acid sequence to the PhanTM4 sequence disclosed herein in SEQ ID NO: 4, preferably at least 98% identical thereto, and most preferably comprise the sequence of at least one of said external loop domains. Also useful for these processes are the MS4A6 (human) protein, especially the loop domains of this protein.

In particular embodiments, said agent, preferably an antibody as disclosed elsewhere herein for use with the process of the invention, binds to Th1 cells but does not bind to human T_reg cells.

In a particular embodiment of such a process, the present invention relates to a process for identifying Th1 cells comprising contacting an antibody specific for a protein at least 95% identical, preferably 98% identical, most preferably the protein with the amino acid sequence depicted in SEQ ID NO: 4 as the PhanTM4 protein, especially where said antibody is specific for an exposed loop region of said protein, most especially the external loop regions depicted in Figure 5, with a sample of cells under conditions promoting binding of said antibody to said protein and detecting the binding of said antibody to said protein thereby identifying Th1 cells as cells to which said antibody is bound. Such antibodies may advantageously incorporate a label for ease of measurement and said label may include chromatic labels, such as fluorescent labels, or a radiolabel. Also useful for these processes are the MS4A6 (human) and MS4A6B (mouse) proteins, especially the loop domains of these proteins (SEQ ID NO: 13 to 16).

Such processes readily lend themselves to use in diagnostic procedures founded on distinguishing Th1 cells from T_reg cells. In one such embodiment, identification of Th1 cells, or absence of the antigen(s) disclosed herein, serves as an endpoint in various types of therapy, such as tolerance therapies disclosed herein, particularly autoimmunity, transplantation, specific antigen acceptance or determining lack of response against therapeutic agents that generate an immune reaction. In a particular embodiment of such a process, one monitors the overall ratio of PhanTM4- and/or MS4A6-positive cells (effector cells) to CD4-positive, PhanTM4-negative cells (Tr_eg cells) during, before or after treatment with an antigenically active agent, such as a therapeutic agent that elicits an immune response. In another embodiment, one monitors the ratio of Th1 cells (PhanTM4-and/or MS4A6-positive cells) to CD4 negative cells that are antigen specific, for example, by combining PhanTM4 and/or MS4A6B staining with fluorescent MHC tetramer-antigen peptide techniques (well known in the art), or following a brief antigen specific stimulation in vitro.

The present invention also relates to a process for reducing the activity of Th cells in a transplant recipient comprising administering to said recipient an agent that reduces the activity of the PhanTM4 protein and/or the MS4A6 (human) protein, in said recipient. Such reduction in activity may be accomplished by deleting or eliminating the Th cells, especially Th1 cells, using said agent.

The present invention also relates to processes for reducing the effects of autoimmune diseases where said diseases are mediated or influenced by the actions or presence of Th1 cells. This is accomplished by reducing the activity of Th cells, especially Th1 cells, in a patient afflicted with an autoimmune disease, such as insulin-dependent diabetes, multiple sclerosis, rhuematoid arthritis, psoriasis, lupus erythematosis, scleroderma, comprising administering to said patient/recipient an agent, such as an antibody, that reduces the presence of the PhanTM4 and/or MS4A6 protein in said recipient. Such reduction in activity may be accomplished by deleting or eliminating the Th cells, especially Th1 cells, using said agent. In particular embodiments, said agent includes an antibody, either polyclonal or monoclonal, preferably monoclonal, as already disclosed herein for use in the processes of the invention.

The present invention further relates to a process for reducing the proliferation of Th cells in a patient comprising administering to said patient an agent that acts against the PhanTM4 and/or MS4A6B protein, preferably wherein the Th cells are TH1 cells, and preferably wherein said agent is an antibody as disclosed elsewhere herein for use with the processes of the invention.

In accordance with the present invention, there are provided polyclonal rabbit antibodies against the PhanTM4 C-terminal peptide, or the MS4A6 (human) protein, especially the loop domains of this protein, to stain a range of normal T cells and T cell clones (after fixation and permeabilisation), and confirming that the protein is highly expressed on activated Th1 cells, is positive at a lower level on naϊve T cells and Th2 cells, and is negative on T_reg clones and natural T_reg populations, using multicolor staining with a range of different antigens. These are useful in detecting the presence of Th1 cells in a population.

In one embodiment, such antibody is a monoclonal antibody (MAb) that reacts with a cell surface structure present on Th cells, especially Th1 cells, wherein said structure is at least 95% identical to the PhanTM4 polypeptide or MS4A6 polypeptide sequence disclosed herein, preferably at least 95% identical to said sequence, most preferably where said structure is the PhanTM4 sequence disclosed herein, especially where said structure is at least 95% identical to an exposed loop domain of the PhanTM4 sequence disclosed herein, preferably at least 98% identical to said exposed loop domain, and most preferably wherein said structure is an exposed loop domain of the PhanTM4 polypeptide sequence disclosed herein, including the MS4A6 (human) and MS4A6B (mouse) loop domains of these proteins (SEQ ID NO: 13 to 16). As already disclosed, the present invention further relates to processes for promoting tissue grafts wherein the donor of said tissue is allogeneic or xenogeneic to the recipient. The process of the invention may also be employed to treat autoimmune diseases wherein Th1 (possibly also Th2 cells) are responsible for adverse immunological reactions. In accordance therewith, said agent may be administered to the graft recipient prior to or contemporaneously with, as well as after, said engraftment or transplantation.

In one such embodiment, the graft is one used to replace tissue injured due to autoimmune disease, or other causes, and wherein the processes of the present invention have the effect of reducing the autoimmune condition by reducing the activity of Th cells, preferably Th1 cells, most preferably Th1 cells and not T_reg cells, thereby selectively reducing the effectiveness of the recipient's immune system in supporting the autoimmune process while at the same time not compromising the recipient's immune system as a whole. This selective inhibition of targeted portions of the recipient's immune system can be readily used against any disease that involves an immune reaction provided that said reaction is mediated by the actions of Th1 cells. Such diseases can include the autoimmune diseases and graft rejections already elucidated herein, as well as different forms of inflammation (for example, inflammatory bowel disease and so-called type IV (Gell and Coombs classification) hypersensitivity reactions and the like).

The present invention further relates to antibodies and other therapeutic ligands made against the human sequences contained within the external loops of the Th1-specific protein disclosed herein, and in particular the longer loop between the 3^rd and 4^th transmembrane regions of the molecule. Such antibodies or ligands find utility either to kill or inhibit the function of Th1 cells while allowing preferential T_reg cell development for immunological tolerance and concomitant portion of transplantation graft acceptance.

In accordance with the invention disclosed herein, such killing could either be through the natural killing mechanisms of antibodies (e.g., complement, cell mediated killing) or by attachment of toxins or prodrugs (e.g., ricin) or by the induction of signalling for apoptosis (as seen with antibodies against CD20, a different molecule of the same family found on B cells and B cell tumours). Inhibition of function results from the function of PhanTM4 (and similar proteins) in Th1 cells in accordance with the observation that expression increases markedly with activation, making activated Th1 cells more sensitive to killing or inhibition. Th2 and ThO cells express lower levels of mouse Chandra/TM4, making Th2 and ThO effector cells targets as well.

As already stated, a preferred embodiment of the present invention is the use of antibodies to selectively eliminate and/or identify Th1 cells as a means, for example, of promoting graft acceptance in a graft recipient in need of such graft or transplant.

The antibodies cited for use in the processes of the present invention include humanized antibodies that bind to the same epitopes (i.e., the external loop domains of PhanTM4 (and like proteins, such as the MS4A6 (human) and MS4A6B (mouse) proteins) as well as humanized antibodies that have the same CDRs (complementarity determining regions) as antibodies that bind to these same loop domains but which have a different humanized framework and/or a different human constant region, wherein the term "framework" refers to the amino acid sequences that are part of the heavy and light chain variable regions of the antibody but not within the CDR regions. Also contemplated for use in the processes of the present invention are humanized antibodies that bind to this same epitope (one or more of the external loop domains of PhanTM4) and/or MS4A6 but wherein one or more amino acids of one or more of the CDRs have been changed (preferably but not necessarily by a conservative amino acid substitution, where one or more amino acid residues are replaced by a different amino acid that is of the same chemical character, such as where one hydrophobic residue is replaced by another) and in which the framework may remain the same or have a different humanized framework or in which one or more of the amino acids of the framework region have been changed and/or in which the constant region may be the same as or different from that of an antibody that binds one or more of the native external domains of PhanTM4 and/or MS4A6. Thus, once an antibody binding to, and presumably specific for, one or more of the external domains of PhanTM4 and/or MS4A6 has been produced, such as by monoclonal antibody technology, the present invention specifically contemplates that such antibody may be changed, as where portions of its amino acid sequence are changed, to produce a more specific or selective antibody for use in the processes of the invention. By way of non-limiting example, an antibody could be initially generated from a mouse or from murine hybridoma cells and then selectively humanized for administration to a human graft recipient or used to identify human Th1 cells by the processes disclosed herein.

The antibodies contemplated for use in the processes of the invention include rat, murine, porcine, bovine, human, chimeric, humanized antibodies, or fragments or derivatives thereof.

The processes disclosed according to the present invention not only promote graft acceptance where such acceptance would otherwise be negated by the actions of Th1 cells in the recipient but also are useful in ameliorating any undesired immune responses having as their mechanism the actions of Th1 cells. As used herein, the term "immune response(s)" is intended to mean immune responses dependent upon Th1 cell activation and proliferation which includes both cellular effects and T cell dependent antibodies which may be elicited in response to, by way of example and not limitation: (i) grafts, (ii) graft versus host disease, (iii) auto-antigens resulting in autoimmune diseases and (iv) therapeutically administered biologic and gene therapy products that elicit undesirable immune responses.

The term "fragment" as used herein means a portion of an antibody, by way of example such portions of antibodies shall include but not be limited to CDR, Fab, or such other portions, which bind to the external loop domains of PhanTM4 or MS4A6 as disclosed herein.

Thus, the present invention also relates to antibodies that can react (i.e., bind to) PhanTM4 or MS4A6 protein as disclosed herein, preferably where such antibodies bind to an external loop domain of PhanTM4 or MS4A6 protein, most preferably where said domain is either the external loop 1 or external loop 3 domains (as depicted in Figure 5). The term "antibody" as used herein includes polyclonal and monoclonal antibodies as well as antibody fragments and derivatives, as well as antibodies prepared by recombinant techniques, such as chimeric or humanized antibodies, single chain or bispecific antibodies which bind to the external loop domains of PhanTM4 or MS4A6 as disclosed herein. The term "molecules" includes by way of example and not limitation, peptides, oligonucleotides or other such compounds derived from any source which mimic the antibody or binds to the same epitope or a portion thereof as the antibody fragment or derivative thereof. Any of these are specifically contemplated for use in the processes of the present invention.

In one embodiment, the antibody is one that binds to one or more external loop domains of PhanTM4 or MS4A6 and which is a humanized antibody that includes modified constant regions of a human antibody, and light and heavy chain framework and CDR regions, in which the framework regions of the light and heavy chain variable regions correspond to the framework regions of the light and heavy chain variable region of a human antibody, and the CDRs derived from a mouse monoclonal antibody that binds to one or more external loop domains of PhanTM4 as disclosed herein.

The preparation of humanized antibody suitable for the purposes of the present invention are no doubt apparent to those skilled in the art from the teachings herein. For example, such an antibody may be prepared by recombinant techniques known to those skilled in the art.

The antibodies of the present invention may be used to inhibit an immune response in an animal by administering the antibody (or fragment thereof) in an amount effective to inhibit such immune response as disclosed elsewhere herein.

It should be borne in mind that none of the processes disclosed herein are contemplated to necessarily act alone but the agents, such as antibodies, disclosed for use in the processes of the invention may certainly be employed along with other agents known to facilitate the desired result. Thus, by way of non-limiting example, the antibodies that binds to one or more of the external loop domains of PhanTM4 or MS4A6 as disclosed herein may be administered to a graft recipient along with one or more other therapeutic, such as immunosuppressive, agents as part of a larger therapeutic scheme and use of such other agents in no way detracts from the effectiveness of antibodies described herein.

Also in accordance with the processes of the present invention, the antibody specific for TH1 cells, as disclosed herein, may be administered prior to, in combination with, or subsequent to administration of a therapeutic agent or of a graft or both. The method of administration is dependent on a variety of factors, including, but not limited to, the specific indication, specific therapeutic agent and optimal dosing schedule. If administered prior to the therapeutic agent and/or graft, the antibody is administered from about 1 hour to about 10 days prior to the administration of the therapeutic agent and/or graft, preferably from about 1 hour to about 24 hours prior to the administration of the therapeutic agent and/or graft. If administered after the administration of the therapeutic agent, the antibody is administered from about 1 hour to about 10 days after the administration of the therapeutic agent, preferably from about 1 hour to about 24 hours after the administration of the therapeutic agent and/or graft. Each of these may be used to treat host versus graft disease.

For example, in some cases, treatment with a therapeutic agent includes an immune response against the therapeutic agent. As representative examples of such therapeutic agents there may be mentioned monoclonal antibodies such as ReoPro and OKT3, enzymes for replacement therapy such as, but not limited to, glucocerebrosidase for Gaucher's disease and clotting factors such as Factor VIII, and products of gene therapy and gene therapy delivery vehicles such as adenovirus derived vectors.

In accordance with an aspect of the present invention, an antibody as hereinabove described (or fragment of such antibody) is administered to a patient that is to be treated with such therapeutic agent, with the antibody (or fragment) being administered in an amount effective to inhibit the immune response against the therapeutic agent. The antibody may be administered prior to, in combination with, or subsequent to administration of the therapeutic agent. The method of administration is dependent on a variety of factors, including, but not limited to, the specific indication, specific therapeutic agent and optimal dosing schedule. If administered prior to the administration of the therapeutic agent, the antibody is administered from about 1 hour to about 10 days prior to the administration of the therapeutic agent, preferably from about 1 hour to about 24 hours prior to the administration of the therapeutic agent. If administered after the administration of the therapeutic agent, the antibody is administered from about 1 hour to about 10 days after the administration of the therapeutic agent, preferably from about 1 hour to about 24 hours after the administration of the therapeutic agent.

The amount of antibody administered, the dosing schedule and the number of times that the antibody is administered is dependent upon the therapeutic agent and the regimen used for treating a patient with the therapeutic agent. The antibody may also be administered to a graft prior to transplant as a means of preventing, or at least ameliorating, graft versus host disease.

In general, the antibody may be used in an amount from 0.1 milligram to 3 grams per dose. The dose utilized may depend to some extent on whether the recipient of a graft is being treated or the graft itself is being treated. Either treatment can serve to promote graft acceptance.

For prevention or lessening of potential graft versus host disease (GVHD) an antibody or active fragment thereof or molecule of the type hereinabove described may be administered ex Vo in accordance with the present invention to decrease the density of PhanTM4 or MS4A6 expression on the cell surface and/or modify signal transduction, thus reducing the functionality of Th1 lymphocytes and/or the number of Th1 cells of the donor tissue. Thus, by way of example and not limitation, in an ex vivo procedure, such antibodies or fragments or derivatives thereof or molecules would be infused into donor bone marrow prior to transplantation to prevent the onset of graft versus host disease upon transplantation. Such adverse immunological response can also result where the graft is autologous to the recipient but would be rejected due to autoimmune reactions by the host so that transplantation of healthy graft tissue would be futile due to the ability of the host to specifically reject such tissues. In one embodiment of the processes of the invention, a patient afflicted with an autoimmune condition could have otherwise healthy tissue removed while it is still viable and functional and then be treated with an agent as disclosed herein, such as an antibody reactive with the PhanTM4 or MS4A6 protein of the invention, which treatment serves to reduce the numbers and/or activity or effectiveness of Th1 cells and thus their ability to compromise the physiological functions of the host tissues. Using the processes disclosed herein for detection of Th1 cells, such as with an anti-PhanTM4 or anti-MS4A6 antibody of the invention, when the numbers and/or activity of such cells have been reduced to safe levels, the healthy tissues can be regrafted back into the patient with reduced ability of the patient's immune system to further injure such tissues.

Of course, in the same way, autoimmune diseases, quite apart from any involvement of grafts or transplants, can be combated in a general way by administering agents as disclosed herein, which agents have the effect of reducing the numbers and/or activity of Th cells, preferably Th1 cells, most preferably Th1 cells and not T_reg cells. Such selective reduction of the actions of a specialized component of the immune system thus allows a systemic treatment of autoimmune diseases while at the same time avoiding general compromising of the immune system as a whole. Such procedures are useful against all manner of autoimmune diseases, including many types of inflammation.

The agents, such as antibodies and active fragments thereof, useful in the processes of the invention are generally administered in a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, and saline. A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ. current edition).

The antibodies contemplated for use in the processes of the invention may be administered in vivo intravenously, subcutaneously, or by intramuscular administration, etc., to a patient, or receiving a therapy in which neutralizing immune responses are generated in the patient.

As hereinabove indicated, the antibodies disclosed for use in the present invention are administered in vivo in an amount effective to inhibit an immune response against an antigen(s). The term "an effective amount" for purposes of this invention shall mean that amount of antibody capable of producing the desired effect. In general, such antibody is administered in an amount of at least 0.1 milligram per dose. It is to be understood that lower amounts could be used. In addition after the initial treatment, the hereinabove described amounts may be reduced for subsequent treatments, if any. Thus the scope of the invention is not limited by such amounts.

The processes disclosed herein are intended to induce acceptance of a graft of foreign tissue, which may thus be allogeneic, and thus induce acceptance of a foreign antigen or where the graft is autologous but the diagnosis of an autoimmune conditions makes such grafting of minimal value. The term "acceptance", as used herein, means that a T-cell non-response (i.e., a Th1 cell non-response or reduced response) persists against an antigen after stopping the antibody treatment, even in the case of challenge. If needed, however, booster or reinforcing doses of the antibody may be given in order to maintain such acceptance.

As already stated hereinabove, the processes of the present invention for inhibiting the actions of Th1 cells may be employed alone or in combination with other processes, drugs or compounds for inhibiting the activation of Th1 cells or inhibiting graft rejection or graft versus host disease or in treating various autoimmune diseases. Examples may include drugs such as rapamycin and cyclosporine A, or other immunomodulatory compounds including monoclonal antibodies directed against co-stimulatory molecules such as CD2, CD8 and CD28, as well as monoclonal antibodies directed against adhesion molecules. #868185

SEQUENCE LISTING

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<120> Process for Reducing Immunogenic Reactions Using Selective THl Cell Binding Antibodies

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Claims

WHAT IS CLAIMED IS:

1. A process for reducing an immune response by effector cells in a recipient comprising administering to said recipient an agent that reacts with a Th-lymphocyte specific cell surface structure and in an amount effective to decrease the ability of Th lymphocytes to support an immune response.

2. The process of claim 1 wherein said Th-specific cell surface structure is present on human Th1 cells but not on human T_reg cells.

3. The process of claim 1 wherein said Th-specific cell surface structure comprises an amino acid sequence at least 95% identical to an external domain of PhanTM4 (SEQ ID NO: 4) or MS4A6.

4. The process of claim 3 wherein said Th-specific cell surface structure comprises an amino acid sequence at least 98% identical to an external domain of PhanTM4 (SEQ ID NO: 4).

5. The process of claim 3 wherein said Th-specific cell surface structure comprises the amino acid sequence of an external domain of PhanTM4 (SEQ ID NO: 4).

6. The process of claim 5 wherein said external domain is external loop domain TM1 - TM2 of PhanTM4.

7. The process of claim 5 wherein said external domain is external loop domain TM3 - TM4 of PhanTM4.

8. The process of claim 1 wherein said agent is an antibody.

9. The process of claim 8 wherein said antibody is a monoclonal antibody.

10. The process of claim 9 wherein said monoclonal antibody is specific for an antigenic determinant comprising an amino acid sequence at least 95% identical to an external loop domain of SEQ ID NO: 4.

11. The process of claim 9 wherein said monoclonal antibody is specific for an antigenic determinant comprising an amino acid sequence at least 98% identical to an external loop domain of SEQ ID NO: 4.

12. The process of claim 9 wherein said monoclonal antibody is specific for an antigenic determinant comprising the amino acid sequence of an external loop domain of SEQ ID NO: 4.

13. The process of claim 12 wherein said external loop domain is external loop TM1 - TM2 of PhanTM4.

14. The process of claim 12 wherein said external loop domain is external loop TM3 - TM4 of PhanTM4.

15. The process of claim 1 wherein said immune response is host versus graft disease.

16. The process of claim 1 wherein said immune response is a response against a graft.

17. The process of claim 16 wherein the donor of said graft is allogeneic to said recipient.

18. The process of claim 16 wherein said agent is administered prior to transplantation of said graft.

19. The process of claim 16 wherein said agent is administered simultaneously with transplantation of said graft.

20. The process of claim 16 wherein said agent is administered after transplantation of said graft.

21. The process of claim 16 wherein the donor of said graft is xenogeneic to said recipient.

22. The process of claim 1 wherein said immune response is an autoimmune disease.

23. The process of claim 22 wherein said autoimmune disease is a member selected from the group consisting of insulin-dependent diabetes, multiple sclerosis, rheumatoid arthritis, psoriasis, lupus erythematosis, and scleroderma.

24. The process of claim 22 wherein said autoimmune disease is insulin- dependent diabetes.

25. The process of claim 22 wherein said autoimmune disease is rheumatoird arthritis.

26. The process of claim 1 wherein said immune response is an inflammation.

27. The process of claim 26 wherein said inflammation is inflammatory bowel disease.

28. The process of claim 1 wherein said immune response is a type IV hypersensitivity reactions.

29. The process of claim 1 wherein said agent is administered intravenously.

30. The process of claim 1 wherein said agent is administered prior to administering a different immunosuppressive agent.

31. The process of claim 1 wherein said agent is administered after administering a different immunosuppressive agent.

32. The process of claim 1 wherein said agent is administered simultaneously with administering a different immunosuppressive agent.

33. The process of claim 1 wherein said immune response is graft versus host disease.

34. A process for reducing graft versus host disease in a graft recipient comprising administering to a donor an effective amount of the agent of claim 1 prior to transplantating tissue from said donor to said recipient.

35. A process for reducing graft versus host disease in a graft recipient comprising contacting tissue to be grafted with an effective amount of the agent of claim 1 prior to transplanting said graft.

36. A process for reducing an autoimmune response comprising removing tissue from a host, contacting said tissue with an effective amount of an agent of claim 1 and reintroducing said tissue into said host.

37. The process of claim 36 wherein said agent is an antibody.

38. The process of claim 37 wherein said antibody is specific for an external domain of PhanTM4.

39. An antibody specific for an external domain loop of PhanTM4.

40. The antibody of claim 39 wherein said external domain loop is external domain loop TM1 - TM2 of PhanTM4.

41. The antibody of claim 39 wherein said external domain loop is external domain loop TM3 - TM4 of PhanTM4.

42. A process for identifying Th1 cells comprising contacting a Th1 specific antibody with a sample of cells under conditions promoting binding of said antibody to said protein and detecting the binding of said antibody to said protein thereby identifying Th1 cells as cells to which said antibody is bound.

43. The process of claim 42 wherein said antibody is selected from the group consisting of the antibodies of claims 39, 40 and 41.

44. The process of claim 43 wherein said antibody comprises a label.

45. The process of claim 44 wherein said label is a fluorescent label.

46. The process of claim 44 wherein said label is a radiolabel.

47. A process for reducing activity of Th cells in a patient in need of such reducing comprising administering to said recipient an agent that reduces the activity of the PhanTM4 protein in said recipient.

48. The process of claim 47 wherein said Th cells are Th1 cells.

49. The process of claim 48 wherein said agent is an antibody.

50. The process of claim 49 wherein said antibody is selected from the group consisting of the antibodies of claims 39, 40 and 41.

51. The process of claim 47 wherein said patient is a graft recipient.

52. The process of claim 47 wherein said patient is afflicted with an autoimmune disease.

53. A process for reducing the proliferation of Th cells in a patient comprising administering to said patient an agent that acts against the PhanTM4 protein.

54. The process of claim 53 wherein said Th cells are Th1 cells.

55. The process of claim 54 wherein said agent is an antibody.

56. The process of claim 55 wherein said antibody is selected from the group consisting of the antibodies of claims 28, 29 and 30.

57. The process of claim 1 wherein said immune response is an immune response against a therapeutic agent.

58. An antibody specific for MS4A6.

59. An antibody specific for an external domain loop of MS4A6.

60. The antibody of claim 59 wherein said external domain loop is external loop TM1 - TM2 of MS4A6 (SEQ ID NO: 16).

61. The antibody of claim 59 wherein said external domain loop is external loop TM3 - TM4 of MS4A6 (SEQ ID NO: 14).

62. An antibody specific for MS4A6B.

63. An antibody specific for an external domain loop of MS4A6B.

64. The antibody of claim 63 wherein said external domain loop is external loop TM1 - TM2 of MS4A6 (SEQ ID NO: 16).

65. The antibody of claim 63 wherein said external domain loop is external loop TM3 - TM4 of MS4A6 (SEQ ID NO: 14).

66. A process for determining the extent of an immune response by Th1 cells in a patient comprising determining the ratio of effector cells to non-effector cells in said patient wherein said ratio is determined by determining the binding of an antibody selected from the group consisting of the antibodies of claim 39, 40, 41 , 58, 59, 60, 61 , 62, 63, 64, and 65 to cells in a population of blood cells drawn from said patient wherein the level of said binding is proportional to the percentage of TH1 cells in said population and thereby indicates the extent of said immune response.

67. The process of claim 1 wherein said Th-specific cell surface structure comprises an amino acid sequence at least 95% identical to a member selected from the group consisting of MS4A6 and MS4A6B.

68. The process of claim 67 wherein said Th-specific cell surface structure comprises an amino acid sequence at least 95% identical to a member selected from the group consisting of an external domain of MS4A6 and an external domain of MS4A6B.

69. The process of claim 67 wherein said Th-specific cell surface structure comprises an amino acid sequence at least 98% identical to a member selected from the group consisting of an external domain of MS4A6 and an external domain of MS4A6B.

70. The process of claim 69 wherein said external domain is TM1-TM2 of MS4A6.

71. The process of claim 69 wherein said external domain is TM1-TM2 of MS4A6B.

72. The process of claim 69 wherein said external domain is TM3-TM4 of MS4A6.

73. The process of claim 69 wherein said external domain is TM3-TM4 of MS4A6B.