AU705010B2 - Human antibodies to t-cell receptor peptides and methods for their preparation - Google Patents

Description

WO 96/15153 PCT/US95/14869 HUMAN ANTIBODIES TO T-CELL RECEPTOR

PEPTIDEP

AND METHODS FOR THEIR PREPARATION This application is a continuation-in-part of U.S.

Serial Number 08/341,157, filed November 16, 1994.

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to antibody preparations that are enriched for antibodies capable of binding to certain T-lymphocytes. More particularly, the invention relates to human antibody preparations that are enriched for antibodies which bind to human T-cell receptor variable region peptides and conformational determinants. The invention further relates to certain methods for making such antibody preparations. These antibody preparations are useful for diagnosing immune system disorders, such as autoimmune diseases and graft versus host disease They also have potential therapeutic value in treating these diseases.

2. Description of the Background Art T-lymphocytes (also referred to herein as T-cells) are involved in cell-mediated immunity. T-cells have been implicated in various immune system disorders, such as autoimmune related syndromes, including classic autoimmune diseases and GVHD. In recent years, it has WO 96/15153 S96/1153 PCT/US95/14869 2 been observed that high dosage administration of intravenous immunoglobulins ("IVIG") has profound effects on a wide variety of immune system-related diseases. These diseases include non-hematologic autoimmune diseases as well as immunohematologic and other diseases with immunopathologic features.

Patients suffering from both chronic autoimmune disease and acute GVHD have been found to respond to IVIG treatment regimens. Clinical improvements have been attributed (at least in part) to certain antibodies contained in these preparations.

T-cells have on their surface a T-cell receptor which is responsible for the immunological specificity of the cells. The Tcr is associated with polypeptides which form the CD3 complex. The Tcr recognizes processed antigen associated with a molecule which is a product of the major histocompatibility complex The polypeptide chains for the antigen-binding portion of the Tcr are encoded by four different gene loci, designated a, C, and A given T-cell will express either an cr a r c tor.

The Tcr's of the great majority cf per r T-cells are composed of polypeptide products ct tne j gene loci.

The genes encoding the Tcr are similar to those which encode antibody. They consist of multiple V, D and J segments which become recombined during T-cell development to produce functional VDJ or VJ genes. The genes encode the N-terminal variable (V) domains of the Tcr. The human genome contains approximately 100 Va genes and between 50 and 100 VP genes.

Kawasaki's disease and multiple sclerosis are two examples of autoimmune diseases in which T- WO 96/15153 PCT/US95/14869 3 cells are clearly implicated. For example, patients with acute Kawasaki's disease demonstrate significantly elevated levels of circulating T-cells bearing the products V32 and V/8.1 genes, indicative of specific up-regulation. The use of IVIG results in significant clinical improvement and a return to near-normal levels of T-cell subsets. Serum from patients suffering from MS have been found to possess elevated levels of Tcells reactive with myelin base proteins, and these Tcells tend to express products of the Tcr V/ 5.2 and 6.1 genes rather than the entire pool of Tcr V3 genes.

Human Tcr peptides have been used as immunogens to produce animal antisera. Schluter, S.F. and Marchalonis, Proc. Natl. Acad. Sci. USA, 83:1872- 76 (1986). A need exists for human antibodies to human Tcr peptide sequences for use in diagnosing certain immune system disorders and for therapeutic use in the treatment of such disorders.

SUMMARY OF THE INVENTION The present invention provides a human antibody preparation which is enriched for antibodies that bind to T cell receptor variable region peptides and conformational determinants. Also disclosed is a method of using such antibody preparation for the diagnosis or treatment of an autoimmune disease or condition or GVHD which is associated with an elevation of T-lymphocytes bearing a particular Tcr peptide or conformational determinant.

The invention further provides methods for making antibody preparations enriched for antibodies with binding specificities to these Tcr autoantigenic variable region epitopes. In one embodiment, the method involves combining, under antibody-antigen WO 96/15153 PCT/US9/14869 4 binding conditions, a Cohn human plasma fraction which contains antibodies that bind to a human Tcr peptide sequence and (ii) a solid support to which Tcr peptide has been immobilized; separating unbound proteins from the solid support; and eluting bound antibodies from the solid support under conditions which break the antibody-antigen bonds, thereby forming an antibody preparation which is enriched for antibodies that bind to a human Tcr peptide sequence.

In another embodiment, the method involves combining, under antibody-antigen binding conditions a human or animal antibody mixture which contains antibodies that bind to a human Tcr peptide or conformational determinant and (ii) a solid support to which a recombinant human Tcr protein has been immobilized; separating unbound proteins from the solid support; and eluting bound antibodies from the solid support under conditions which break the antibody-antigen bonds, thereby forming an antibody preparation which is enriched for antibodies that bind to a human Tcr peptide or conformational determinant.

The novel antibody preparations of this invention have utility in diagnosing or monitoring the progress of human immune system disorders, such as autoimmune diseases and GVHD. These antibody preparations further have value as human therapeutic agents. As a result of the work reported herein, it is believed that IVIG preparations are effective in treating immune disorders because they contain autoantibodies which bind to Tcr protein on the surfaces of T-lymphocytes. The antibody preparations of the present invention provide more potent and selective therapeutic agents.

WO 96/15153 PCT/US95/1486 9 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a fluorescent activated cell sorter (FACS) scan of Jurkat cells treated with antibody free reagents (negative control).

Figure 2 is a FACS scan of Jurkat cells treated with rabbit anti-scTcr antiserum (positive control).

Figure 3 is a FACS scan of Jurkat cells treated with unpurified

IVIG.

Figure 4 is a FACS scan of Jurkat cells treated with IVIG purified by immunoaffinity chromatography using immobilized scTcr.

Figure 5 represents ELISA results showing the reactivity of Cohn Fraction I III with scTcr and various Tcr peptides.

Figure 6 represents ELISA results showing the reactivities of sera from patients with rheumatoid arthritis with scTcr.

Figure 7 represents ELISA results showing the reactivities of sera from patients with SLE with scTcr.

Figure 8 is a graph illustrating the results of cell proliferation assays comparing the inhibitory effects (shown as percent inhibition) of increasing concentrations of purified anti-03 antibodies and a commercial intravenous immunoglobulin

(IVIG).

Figure 9 is an overview depiction of the experimental design of an experiment to study the effects of purified antibodies in modulating T cell activity via interaction with the TcR.

Figure 10 is a graph illustrating the inhibition of lymphocyte killing of tumor targets by anti-TcR antibodies purified from a commercial intravenous immunoglobulin

(IVIG).

WO 96/15153 WO96/15153 PCT/US95/14 8 6 9 6 DETAILED

DESCRIPTION

The novel antibody preparations of this invention are advantageously prepared from human antibody mixtures which contain antibodies having the desired reactivities. Healthy humans, as well as those suffering from autoimmune diseases and conditions, such as systemic lupus erythematosis and rheumatoid arthritis, and autoimmune related syndromes, such as GVHD, contain autoantibodies directed against peptide and conformational determinants occurring in Tcr proteins. See, Marchalonis et al., Proc. Natl. Acad.

Sci., 89:3325-3329 (1992) and Marchalonis et al., Gerontology, 39:65-79 (1993). Thus, pooled serum from healthy individuals or those suffering from autoimmune diseases may be used as sources for the antibody preparations of this invention. Commercially available plasma products or fractions also advantageously may be used as the source for these antibody preparations.

For example, immune serum globulin products and commercial IVIG preparations such as Gammagard®, available from Baxter Healthcare International, and Sandoglobulin, available from Sandoz Pharmaceuticals, can be used as a starting material. Various Cohn plasma fractions (Cohn et al., J. Am. Chem. Soc., 68:459 (1946)) have been found to contain autoantibodies directed to Tcr peptide sequences. Cohn Fractions I and III, which are currently discarded, as well as Cohn Fraction II, can be used as starting materials for the production of the antibody preparations of this invention.

The novel antibody preparations are conveniently obtained by immunoaffinity purification using immobilized Tcr peptides or recombinant human Tcr proteins. The latter may be preferred because of their WO 96/15153 PCTfUS95/14869 7 ability to bind to antibodies through both peptide and conformational determinants. Tcr peptides are immobilized by bonding them to a conventional solid support, such as agarose beads, using well known conventional methods. See, Marchalonis et al., 1992, supra. Various peptides corresponding to sequences of the Tcr antigen binding region can be used in the immunoaffinity purification procedure. These peptides are preferably encoded by Va and/or V/ genes, but may also be encoded by V6 and/or Vy genes. Tcr peptides chosen represent regions of the Tcr found to be autoimmunogenic as manifested by production of IgG and IgM autoantibodies. Marchalonis et al., 1992, supra.

Recombinant Tcr proteins contain these sequences. Lake et al., Biochem. Biophys. Res. Comm., 201 (301): 1502- 109 (1994).

The Tcr peptides used for the antibody purifications may be chemically synthesized using conventional peptide synthesis techniques. Recombinant human Tcr proteins may be made by various recombinant DNA procedures, by cDNA cloning techniques using messenger RNA obtained from commercially-available T-cell lines, or from T-cells cultured from normal blood or blood from patients suffering from autoimmune diseases. Genes from the Va, VO, V6 and Vy loci have been sequenced (Toyonaga, B. And Mak, Ann. Rev.

Immunol., 5, 585-620 (1987)), and these sequences may be utilized for designing PCR primers and probes for amplifying and identifying cDNA clones.

As indicated above, particular autoimmune diseases have been associated with elevated levels of circulating T-cells bearing the products of specific Tcr genes. As used herein, a Tcr protein, peptide or conformational determinant is said to be associated WO 96/15153 WO 915153 PCT/US95/1486 9 8 with a particular immune system disorder or condition when T-cells bearing that determinant are elevated in patients having the disorder or condition. Thus, Tcells bearing the products of the V32 and V08.1 genes are said to be associated with Kawasaki's disease, because those T-cells are elevated in patients having that disease. Similarly, T-cells bearing the products of V/5.2 and V36.1 genes are associated with MS. The analysis of Tcr variable regions associated with immune system disorders is progressing at a rapid pace.

Upon identification of Tcr variable region genes associated with a particular disease, proteins containing those sequences can be produced by cDNA cloning procedures using published sequence information. Antibody preparations enriched for antibodies which bind to a desired Tcr peptide or conformational determinant may then be prepared by the immunoaffinity procedures described herein.

In one aspect, the method of the present invention utilizes a recombinant single chain Tcr protein (scTcr) for the immunoaffinity purification of antibodies from IVIG and from Cohn Fractions I and III and from Cohn Fraction II. Construction of the scTcr was based upon complete Va and V3 regions of the Jurkat T-cell line.

The Jurkat cell line is a human monoclonal CD4*, helper a/0' leukemia T-cell line, which is available from the American Type Culture Collection, Rockville, Maryland, USA, under Accession No. ATCC 152-T1B. This scTcr molecule contains the Va and V9 gene products joined by a linker peptide. The construction of the scTcr from the Jurkat T-cell line using cDNA cloning techniques is described by Lake et al., 1994, supra, incorporated herein by reference. The Lake et al. publication WO 96/15153 WO 96/15153 PCT/US95/148 6 9 9 describes the expression of the scTcr peptide from E.

coli strain BL21(DE3) transformed with plasmid PET21d.

Recombinant human Tcr proteins may also be used to .prepare animal antisera enriched for antibodies which bind to human Tcr or conformational determinants. The animal antisera may be prepared by immunizing animals with Tcr peptides, recombinant human Tcr proteins or human T-cells. The animal antibody preparations so produced have utility as diagnostic reagents.

The novel purified antibodies of this invention may be of any isotype, and those purified from Cohn Fractions I and III are primarily of either IgG or IgM isotype.

The antibody preparations of this invention may be used for diagnosing or monitoring the progress of immune system disorders or conditions, including autoimmune diseases and GVHD. The novel antibody preparations are used diagnostically by determining the extent to which they bind to T-lymphocytes obtained from human subjects. Various immunochemica: detection techniques may be used for detecting tne .ctra-tion of the antibodies and T-lymphocytes. For exa-r..

ELISA

and flow cytometry using a fluorescent act::.ate cell sorter as well as other conventiona immunochemical procedures, may be used for the detection of the antibody-T-cell interactions.

In addition to their utility for the diagnosis of immune system disorders and conditions, the antibody preparations of this invention have potential therapeutic value. As indicated above, it is known that commercially available immune serum globulin preparations can be used for treating autoimmune diseases and GVHD. In accordance with this invention, it has been shown that these immunoglobulin WO 96/15153 PCTIUS95/1 4869 preparations contain antibodies to Tcr peptide sequences and conformational determinants. The antibody preparations of this invention are enriched for antibodies to Tcr peptide and conformational determinants up to about 1,000 times over the levels in unprocessed IVIG. Dosages of IVIG used in the treatment of GVHD and autoimmune diseases range from 100 mg to 5 g of IVIG/kg body weight. Based upon in vitro and in vivo studies (mouse model), effective therapeutic doses of affinity purified antibodies are within the range of about 0.1 mg to about 100 mg/kg of body weight. Such doses can be administered by any suitable methods, with intravenous administration being preferred. These antibody preparations are expected to have numerous advantages over currently-available

IVIG

preparations in the treatment of these diseases. These advantages arise from the higher potencies and greater selectivities of the antibody preparations of the present invention. Therefore, lower dosages and thus, lower protein loads on the patient can be realized.

This invention is further illustrated by the following examples, which are not intended to be limiting.

EXAMPLE I Purification of Antibodies from Cohn Fraction I and III Cohn Fraction I and III was obtained from the Hyland Division of Baxter Healthcare International, Duarte, California, U.S.A. The plasma fraction was centrifuged and dialyzed and then filtered through 0.45 pm filter to remove insoluble Celite and particulates.

The resulting solution was subjected to immunoaffinity purification essentially as described by Marchalonis et WO 96/15153 PCT/US95/1 4 8 69 11 al., 1992 supra. The immunoaffinity column was prepared as follows: BL21 (DE3) E. coli cells were purchased from Novagen. PET21d plasmid which contained recombinant scTcr gene was used to transform the BL21 (DE3) E. coli so that the E. coli would produce the recombinant scTcr protein. The recombinant protein (1.1 milligrams) was dissolved in 10 ml of 0.1 M sodium carbonate (pH and incubated with 0.75 g of activated CH Sepharose-4B (Pharmacia Fine Chemicals, Piscataway, NJ, at room temperature for 2 hours. The Sepharose was then washed with 40 ml of phosphate-buffered saline (50 mM NaCl/150 mM sodium phosphate, pH treated with ml of 1M ethanolamine (pH 8.0) for 1 hour to block unreacted sites, washed with 100 ml of TBS and packed into a 10 cm x 1 cm column.

To remove nonspecific (sticky) antibodies, all samples were first applied to a column packed with ovalbumin immobilized on CH-Sepharose at a flow rate of -1.2 ml/min. The ovalbumin column previously had been equilibrated with TBS. The effluent from the ovalbumin column was applied directly to the immunoaffinity column (also previously equilibrated with TBS). After washing with 10 bed volumes of TBS, the bound antibodies were eluted with 150 mM glycine-HCl, pH collected in 2 ml fractions and immediately neutralized with 3M Tris-NaOH at pH EXAMPLE

II

Flow Cytometry Jurkat cells obtained from ATCC were used to demonstrate cell surface binding of anti-Tcr antibodies. Cells were cultured in RPMI 1640 medium supplemented with 5% fetal calf serum at 370C in a WO 96/15153 PCT/US95/148 6 9 12 air 5% CO 2 atmosphere. Cells were harvested during exponential growth and separated from the culture supernatant by centrifugation. Cells (1 x 106) were resuspended in phosphate buffered saline and combined with 0.5 ml of 10 Jg/ml of affinity purified antibodies from an IVIG preparation designated Gammagard® and available from Hyland Division of Baxter Healthcare International, which was purified by the procedure of Example I. An equal number of cells were treated with 0.5 ml of 1:2000 dilution of serum from a rabbit immunized with scTcr (positive control). Cells treated only with buffers (no antibodies) were used as negative controls.

Primary antibody was detected by incubation on ice for one hour with a 1:5000 dilution of goat (Fab') 2 anti-human IgG antibody) coupled to fluorescein isothiocyanate Cells were washed free of antibody with PBS and analyzed on a Becton Dickinson FACscan flow cytometer.

The results are shown in Figures 1-4. Figure 1 represents the instrument output for the negative control, and shows a reactivity of 4.39% positive.

Figure 2 represents the positive control and shows a reactivity of 95.44% positive. Figures 3 and 4 represent unpurified IVIG and immunoaffinity purified IVIG respectively. The unpurified antibody mixture had a reactivity of 3.43% positive and the purified antibody preparation had a reactivity of 91.47% positive.

EXAMPLE

III

The reactivities of affinity purified antibodies in Cohn Fractions I and III with various human Tcr peptides and with the recombinant Jurkat scTcr WO 96/15153 WO15153 PCT/US95/14869 13 described above were analyzed by ELISA as follows: Hexadecapeptide antigens 03, 08, and 017 corresponded to the first complementarity determining region, the third framework region and the constant region of the YT35 (Jurkat T cell myeloma cell line) 3-chain respectively. Peptide antigens or the scTcr were dissolved in 0.2 M carbonate buffer, pH 9.6. These solutions (100 pg/ml) were added to wells of a microtiter plate and dried overnight at 37C. Wells were blocked with phosphate-buffered saline, pH 7.4, containing 0.005% Tween 20 (PBST) and 1% gelatin Cohn Fraction I III was reacted with the antigen for one hour followed by washing 4 times with PBST.

Peroxidase conjugated rabbit antibody to human IgM or IgG was used as a developing reagent at a dilution of 1:1000 for the anti-IgM and 2:4000 for the anti-IgG.

Conjugate incubations were also for 1 hour. After five washes with PBST, 0.03% substrate (2,2-azino-bis-(3ethylbenzthioazoline-6-sulfonic acid) in 0.1 M citrate buffer, pH 4.0 and 0.01% hydrogen peroxide were added. Color development was read at 405 mm on a Titertek Multiscan (Flow Labs) after 60 minutes incubation at room temperature. The results are shown in Figure 5. Immunoreactivity was greatest for the 33 peptide followed by scTcr, 08 and 017 in that order.

The 33 and 08 peptides were contained in the scTcr, while 117 was not.

EXAMPLE IV The reactivities of antibodies in sera from four patients with rheumatoid arthritis and eight patients with systemic lupus erythematosis with scTcr were analyzed by ELISA using essentially the procedures described in Example III. The results are WO 96115153 PCT/US95/1486 9 14 shown in Figures 6 and 7 respectively. Significant reactivities with the recombinant scTcr were observed in these patient's sera.

EXAMPLE V Antibodies which bind to a human Tcr protein, peptide or conformational determinant were affinity purified as in Example I but from a commercial intravenous immunoglobulin (IVIG, Gammagard®) using column chromatography in which peptide 33, corresponding to the first complementarity determining region of a human T cell line (also obtained from 3 chain, was immobilized on CH-Sepharose.

Following elution, the antibodies were immediately neutralized with NaOH-glycine. The antibodies then were used in inhibition of phytohaemagglutinin

(PHA)

stimulated T cell proliferation. The T cell proliferation assay is a standard assay and was performed essentially as in Current Protocols in Immunology (Coligan, J.E. et al.; 19940 Series Ed.

Richard Coico) vol. I, Section 7.10 (John Wiley and Sons, Inc.) as follows. Serial dilutions of antibodies were pre-incubated at 37 0 C with normal peripheral blood lymphocytes (PBL) at a concentration of 10 s cells/well in a 96 well microtiter plate for 30 minutes. 5 gg of PHA were added to the PBL-antibody mixture and the cells were cultured for 72 hours at 37 0 C. Sixteen hours prior to harvest, one microcurie of tritiated thymidine was added to each well. At the end of the 72 hour incubation, the nuclei were harvested on a cell harvester and counted in a liquid scintillation counter. Percent inhibition by the anti-Tcr antibodies was calculated by the following formula: WO 96/15153 WO15153 PCT/US95/14869 1-[(sample-background)/(max. proliferationbackground)] X 100%.

The results are shown in Figure 8. Although unprocessed IVIG showed only non-specific background inhibition up to 2500 pg/ml under defined experimental conditions, the affinity purified antibody-induced inhibition increased steadily to 87% as the antibody concentration increased from 3.1 to 25.0 g/ml. These data demonstrate that antibodies with T cell activity can be specifically purified from commercial

IVIG.

EXAMPLE VI The possible effects of affinity purified antibodies in modulation of T cell activity were studied via interaction with the Tcr in a "proof of principle" model in C57/BL mice. An overview of the experiment is shown in Figure 9. A well-developed sponge model of concomitant tumor immunity was used.

The concepts of concomitant tumor immunity are reviewed in Gorelik, E, Adv. Cancer Res. 39:71 (1983), and the complete details of the gelatin sponge model used are described in Akporiaye, E.T. et al., Cancer Res.

58:1153 (1988), and in Akporiaye, E.T. and K.

Muthulakshmi, Cancer Immunol. Immunother. 29:199 (1989). In this model, an animal harboring a primary EMT6 mammary tumor is challenged with a secondary tumor implant through a pre-implanted gelatin sponge. During the manifestation of concomitant tumor immunity, the second tumor is rejected and the effector cells can be recovered from the sponge and their tumoricidal activity studied in vitro. These cytotoxic tumor rejecting T cells (TRLs) predominantly express V91 and V08 Tcrs. The aim of this experiment was to test the effect of anti-V91 and anti-V8 antibodies affinity WO 96/15153 14869 16 purified from IVIG to modulate in vitro the tumoricidal activity of the isolated TRLs.

C57/BL mice were injected on day 0 with Balb/c mouse mammary derived EMT6 tumor cells. On day 8, the mice were surgically implanted with a gelatin sponge and the incisions were allowed to heal. On day fresh EMT6 cells were injected into the gelatin sponges. On day 17, the sponges were removed, digested with gelatinase and the TRLs, which had infiltrated in order to reject the tumor cells, were recovered.

Antibodies which bind to a human Tcr protein, peptide or conformational determinant were affinity purified from IVIG (Gammagard®) using column chromatography with peptides immobilized on CH- Sepharose. The peptides used corresponded to the CDR1 region of Balb/c mouse Vl1 (EQHLGHNAMY) and V?8 (NQTNNHNNMY) Tcr chains. The purification procedure was as described in Example 1. Final preparations of affinity purified antibody were extensively dalvzed against

PBS.

Affinity-purified antibodies prepare= each peptide in separate purificaticn ste-s -umn flow through IVIG were evaluated ir ar :i :rchromium release assay. The chromium release assay was performed essentially as described in Akporiaye,

E.T.

and K. Muthulakshni, Cancer Immunol. Immunother. 29:199 (1989) with the following modifications. Fifty Al of the TRLs obtained above (1.5 X 10 5 cells per well) were added to appropriate wells of a 96 well culture plate.

Next, 50 1p aliquots (30 Ag of antibody per well) were added and the plates incubated for eight hours at 37 0

C.

The degree of cell lysis was determined by measuring the amount of chromium released in the supernatant using a gamma counter.

WO 96/15153 PCT/US95/1 4869 17 Figure 10 shows that anti-V)l and anti-V/8 independently gave 80% and 50% inhibition of T cell activity, respectively. Added together, they yielded inhibition. Column flow through as a control showed no inhibition. These data demonstrate that antibodies with specific reactivities are able to inhibit the lytic activity of cytotoxic T-cells via reactivity and modulation of the T-cell receptor. In addition, these data, as a proof of principle, clearly demonstrate the validity of the hypothesis for the ultimate clinical efficacy in the treatment of certain autoimmune disorders.

Claims (30)

1. A human antibody preparation which is enriched for antibodies which bind to conformational epitopes of a recombinant human Tcr protein.

2. The human antibody preparation of claim 1 which is enriched for antibodies which bind to conformational epitopes of a recombinant human Tcr protein encoded by a Va gene or for antibodies which bind to conformational epitopes of a recombinant human Tcr protein encoded by a Vp gene.

3. The human antibody preparation of claim 1 which is enriched for antibodies which bind to conformational epitopes of a recombinant human Tcr protein encoded by a V8 gene or for antibodies which bind to conformational epitopes of a recombinant human Tcr protein encoded by a Vy gene. 0:

4. The human antibody preparation of claim 1 which is enriched for antibodies which bind to conformational epitopes of a recombinant human Tcr peptide or of a conformational determinant thereof which is associated with an autoimmune disease or condition. The human antibody preparation of claim 4, wherein the autoimmune disease is multiple sclerosis, systemic lupus erythematosis, Kawasaki's disease or rheumatoid arthritis. 9/3/99

6. The human antibody preparation of claim 1 which is enriched for antibodies which bind to conformational epitopes of a recombinant human Tcr peptide or of a conformational determinant thereof which is associated with graft versus host disease (GVHD).

7. The human antibody preparation of claim 1 which is enriched for antibodies which bind to conformational epitopes of Va and Vp peptide sequences of the Jurkat T-cell line, ATCC No. 152-T1B.

8. The antibody preparation of claim 1 which is enriched for antibodies which bind to conformational epitopes of a single-chain Tcr protein derived from the Va and Vp gene sequences of a human T cell.

9. A method for making an antibody preparation which is enriched for antibodies that bind to conformational epitopes of a recombinant human Tcr peptide sequence, which comprises: combining, under antibody-antigen binding conditions, a human plasma Cohn fraction which contains antibodies that bind to a human Tcr peptide or to a conformational determinant thereof, and (ii) a solid support to which conformational epitopes of a recombinant human Tcr peptide have been immobilized; separating unbound proteins from the solid support; eluting bound antibodies from the solid support under conditions which break the antibody-antigen bonds, thereby forming an antibody preparation which is enriched for antibodies that bind to conformational epitopes of a recombinant human Tcr peptide sequence. K 9/3/99 The method of claim 9, wherein the human plasma Cohn fraction employed in step is Cohn Fraction I or III.

11. The method of claim 9, wherein the human plasma Cohn fraction employed in step is Cohn Fractions I and III.

12. The method of claim 9, wherein the human plasma Cohn fraction employed in step is Cohn Fraction II.

13. A method for making an antibody preparation which is enriched for antibodies that bind to conformational epitopes of a recombinant human Tcr peptide or of a conformational determinant thereof, which comprises: combining, under antibody-antigen binding conditions, an antibody mixture which contains antibodies that bind to human Tcr peptide or to a conformational determinant thereof and (ii) a solid support to which conformational epitopes of a recombinant human Tcr protein have been immobilized; separating unbound proteins from the solid support; eluting bound antibodies from the solid support under 0l.• conditions which break the antibody-antigen bonds, thereby forming an antibody preparation which is enriched for antibodies that bind to conformational epitopes of a recombinant human Tcr peptide or of a conformational determinant thereof.

14. The method of claim 13, wherein the antibody mixture employed in step is a human antibody mixture. 9/3/99 The method of claim 13, wherein the antibody mixture employed in step is an animal antibody mixture.

16. The method of claim 14, wherein the antibody mixture is a human plasma Cohn fraction.

17. The method of claim 16, wherein the antibody mixture is Cohn Fraction I and III.

18. The method of claim 16, wherein the antibody mixture is Cohn Fraction II. i

19. The method of claim 14, wherein the antibody mixture employed in step is a human IVIG preparation.

20. The method of claim 13, wherein the immobilized conformational 0:00 epitopes of the recombinant human Tcr protein employed in step is a conformational epitope of a protein encoded by a region of a Va, VP, VS or Vy gene.

21. The method of claim 13, wherein the immobilized conformational epitopes of the recombinant human Tcr protein employed in step is a conformational epitope of a single-chain Tcr protein derived from the Va and VP gene sequences of a human T Cell.

22. A method of diagnosing a human for an autoimmune disease or Scondition or GVHD, which comprises: 9/3/99 identifying a Tcr peptide or conformational determinant thereof that is elevated in humans having said autoimmune disease or condition or GVHD; combining T-lymphocytes from said human with a human antibody preparation which is enriched for antibodies which bind to conformational epitopes of said Tcr peptide or of said conformational determinant; determining the extent to which antibodies in said antibody preparation bind to the said T-lymphocytes.

23. The method of claim 22, wherein the autoimmune disease is multiple sclerosis, systemic lupus erythematosis, Kawasaki's disease or rheumatoid arthritis. 0•

24. The method of claim 22, wherein the said Tcr protein or said conformational determinant is encoded by a region of a Va or V8 gene. S The method of claim 22, wherein the said Tcr peptide or said conformational determinant is encoded by a region of a VP or Vy gene. O S O

26. The method of claim 22, wherein step is accomplished by means S* of an ELISA procedure.

27. The method of claim 22, wherein step is accomplished by fluorescent flow cytometry. 9/3/99

28. A method of treating a patient suffering from an autoimmune disease or condition or graft versus host disease (GVHD) which comprises administering to the patient a human antibody preparation which is enriched for antibodies which bind to conformational epitopes of a recombinant human Tcr peptide or of a conformational determinant thereof that is present in elevated levels of circulating T cells in patients having said autoimmune disease or GVHD, wherein said preparation comprises a sufficient amount of said antibodies to bind to said conformational epitopes.

29. The method of claim 28, wherein the antibodies are administered in a dosage range of about 0.1 to about 100 mg/kg body weight.

30. The method of claim 28, wherein the antibody preparation is enriched for antibodies which bind to conformational epitopes of a recombinant human Tcr protein encoded by a Va gene or for antibodies which bind to conformational epitopes of a recombinant human Tcr protein encoded by a VP gene. «*01 o*

31. The method of claim 28, wherein the antibody preparation is enriched for antibodies which bind to conformational epitopes of a recombinant human Tcr protein encoded by a V6 gene or for antibodies which bind to conformational epitopes of a recombinant human Tcr protein encoded by a Vy gene.

32. The method of claim 28, wherein the disease is an autoimmune disease. 9/3/99

33. The method of claim 32, wherein the autoimmune disease comprises Kawasaki's disease, multiple sclerosis, rheumatoid arthritis or systemic lupus erythematosis.

34. The method of claim 28, wherein the disease is GVHD. Dated this 9th day of March 1999. BAXTER INTERNATIONAL INC and ARIZONA BOARD OF REGENTS, UNIVERSITY OF ARIZONA By their Patent Attorneys PETER MAXWELL ASSOCIATES 4 4 0 *.0 8