CA2205138A1

CA2205138A1 - Human antibodies to t-cell receptor peptides and methods for their preparation

Info

Publication number: CA2205138A1
Application number: CA002205138A
Authority: CA
Inventors: Douglas F. Lake; William J. Landsperger; John J. Marchalonis
Original assignee: Individual
Current assignee: Baxter International Inc; University of Arizona
Priority date: 1994-11-16
Filing date: 1995-11-15
Publication date: 1996-05-23
Also published as: EP0792294A1; AU4161196A; JPH10509948A; WO1996015153A1; AU705010B2

Abstract

Human antibody preparations enriched for antibodies that bind to human T-cell receptor peptide or conformational determinants are disclosed. Also disclosed are methods for making such antibody preparations from human plasma Cohn fractions by immunoaffinity purification using either recombinant Tcr protein or Tcr variable region peptides. Purification of Tcr antibodies from human or animal antibody mixtures is accomplished by immunoaffinity procedures using recombinant human T-cell receptor proteins. The affinity purified antibodies can bind to intact membrane-bound Tcr using fluorescence activated cell sorting (FACS) and can inhibit T-cell proliferation following stimulation by mitogen. Affinity purified human polyclonal antibodies can inhibit lytic activity of natural killer cells on a monoclonal T-cell line. The antibody preparations can be used for diagnosis, monitoring and therapy of various autoimmune-related syndromes.

Description

CA 0220~138 1997-0~-12 .

HUM~N ANTIBODIES TO T-CELL RECEPTOR PEPTIDES
A~3D METHODS FOR THEIR PREPARATION
This application is a continuation-in-part of U.S.
Serial Number 08/3~1,157, filed November 16, 1994.
=~ ~ BACKGROt~ND OF THE INVENTION
1. Field of the-Invention =~
The present invention relates to antibody preparations that are enriched for antibodie~ 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 ~eptides and conf ormational determinants . The invention f urther relates to ce.rtain methods for~ making such antibody preparations. These ~antibody preparations are useful for diagnosing imr:~une system disorders, such as autoimmune diseases and graft versus host disease ( "GVHD" ) . They also have potential therapeutic value in treating these diseases.

2. Descri~tion of the Back~round 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 autni ~ related syndromes, including classic 25 ~ autoimmune diseases and GVHD. In recent years, it has -CA 0220~138 1997-0~-12 3 PCT/llS95/14869 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-hematolo~ic autoimmune diseases as well as; ,,1- tologic and other diseases with immunopathologic features. ~
Patients suffering from hoth chronic_autoimmune disease and acute GVHD have been f ound to respond to IVIG
treatment regimens. t'lini~ ll~L~.JV~ 'S have been attributed (at least in part) to certain Ant;horl;,~c r~nt~;n~l in these preparations.
T-cells have on their surface.=a T-cell receptor ~"Tcr") 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 maj or histocompatibility complex ( "MHC" ) . The polypeptide chains for the antigen-binding portion of the Tcr are encoded by four different gene loci, designated 0~ and ~. A given T-cell will express either an ~/,3 or a ~i/ y receptor.
The Tcr' s of the ~reat majority of peripheral T-cells are composed of polypeptide products :of the o~ gene~
loci .
The genes encoding the Tcr ~are similar to those which encode antibody. They consist of m~tiple~V, D
and ;r segments which become recombined during T-cell development to produce functional VD~ or Y~
genes. The genes encode the N-terminal variable ~V) domains of the Tcr. The human genome: contains approximately l00 V~ genes and between 50 and l0C V~3 genes .
Kawasaki ' s disease and multiple sclerosis ~ "MS " ) are two examples of ~ltO; f~ diseases in which T-CA 0220~138 1997-0~-12 Wo 96/lSI53 PCrlUS9SI14869 cells are clearly implicated. For example, patients with acute~Kawasaki's disease demonstrate significantly elevated levels of circulating T-cells bearing the products V~2 and V~8.1 genes, indicative of specific up-regulation. The use of IVIG results in significant clinical i~ )LUV~ ' and a return' to near-normal levels of T-ce'll''subsets. Serum from patie~ts suffering from MS have been found to possess elevated levels of T-cells reactive with myelin base proteins, and these T-cells tend to express products of the Tcr V,B 5 . 2 and 6.1 genes rather than the entire pool of Tcr V~3 genes.
Xuman Tcr peptides have been used as immunogens to produce animal antisera . Schluter, S . F . and Marchalonis, J.J., Proc. Natl. Acad Sci. ~SA, 83 :1872-76 (ls86) . 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.
SUMM~RY OF TXE I~VEI~'C~
The present invention provides a '._~a~ :Dody preparation which is enriched fcA- a~ e~ : hind to T cell receptor variable region pe~::3es a-.-conformational - determinants. Also disclose3 :s a method of using such antibody preparation for the 25 diagnosis o~r treatment of an aut~ disease or condition or GVXD which is associated with an elevation of T-lymphocytes bearing a particular Tcr peptide or conformational determinant.
The invention further provides methods for making 30 ~ =antibody preparations enriched for antlbodies with binding specif icities to these Tcr autoantigenic variable region epitopes. In one embodiment, the method involves ~ in~, under antibody-antigen CA 0220~i138 1997-0~i-12 binding conditions, (i) a Cohn human plasma fraction which cnntAinq 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 i~nt;hn~ies from the solid snpport under conditions which break the antibody-antigen bonds, thereby forming an antibody preparation which is enriched fQr antibodies that bind to a human Tcr peptide sequence.
In another-embodiment, the method involves, ` inin~, under antibody-antigen binding conditions ( i ) a human or animal antibody mixture which l-nnt~ine3 ;lntihn~ii~c:
that bind to a human Tcr peptide or conformational determinant and (ii) a soIid support to which a ~
recombinant human Tcr -protein has been i h; 1 i zed;
separating unbound proteins from the solid support; and eluting bound antibodies from the solid su~port under conditions which break the antibody-antigen bonds, thereby forming an antihody preparation which is enriched for antibodies that bind to a human Tcr peptide or c:onformational 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 tQ Tcr protein on the surfaces of T-lymphocytes. The antibody preparations of the present invention provide more potent and selective therapeutic agents.

CA 0220~138 1997-0~-12 WO 96/lS153 PCT/[TS9Sl14869 S ~=
BRIEF DESCRIPTION OF T~IE 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 ~rkat cells treated with unpurif ied IVIG . - -Figure 4 is a FACS scan of Jurkat cells treated with IVIG purified by; Int-=ffinity chromatography using i h; 1 i 7ed 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 -f om patients with rheumatoid arthritis with scTcr. : ~ ~
Figure ~ represents ELISA results showing the reactivities of sera from patients with SLE with scTcr.
Figure 8 is a graph illustr.ating the results of cell proliferation assays comparing the inhibitory effects ~(sEown as percent inhibition) o_ increasing concentrations of purified a~ti-~3 ~ntiho~ies and a commercial intravenous immunoglohulin ( IVIG) .
Figure--~ is an overview depiction of the experimental design of an experiment to study the effects of purified ~ntihr~rlies in modulating T cell activity via i~teraction with the TcR.
Figure 10 is a graph illustrating the inhibition 3 0 o_ lymphocyte killing of tumor targets by anti-TcR
antibodies purified from a commercial irltravenous immunoglobulin ( IVIG) .

CA 0220~138 1997-0~-12 DETAILED DESCRIPTION
The novel antibody preparations of this inventlon are advantageously prepared from human antibody mixtures which contain antibodies having the desired reactivities. ~Iealthy humans, as well as those suffering from ~7~;mm1ln~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,, Gerontoloqv, 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. ~" -rc-i~lly 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 f~mm~d"', 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., 3. Pm. 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 3 0 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 prefbrred because of their CA 0220~il38 l997-0~i-l2 WO 96/lSlS3 PCI/US951l4869 ability to bind to i7nt; ho~ q 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., lg92, supra. Various peptides corrF-~p~n~l;ng to sequences of the Tcr~ antigen binding region can be used in the ~ffinity puriication procedure. These peptides are pref erably encoded by V~ and/or Vl~ genes I but may also be encoded by V~ and/or V~ 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. I~ake et al., Biochem. Bio~hYs. Res Comm., 201 (301): 1502-109 (1994) .
The Tcr peptides used f ar the antibody:
purifications may be chemically synthesized using conventional peptide synthesis techniques. Recombinant human Tcr proteins may be made by various r,o~: ; ni9nt DNA procedures, e . g., by cDNA cloning techniques using messenger RNA obtained from commercially-available T-cl-l 1 1 inl-~, or from T-cells cultured from normal blood or blood from patients suffering from autoimmune 2~ diseases. Genes from the V~, V~, V~ and V~ loci have been sequenced (Toyonaga, B. And Mak, T.W., Ann. Rev.
Immunol., 5, 585-620 (198~) ), and these sequences may be utilized for designing PCR primers and probes for amplifying and identifying cDNA clones.
3 0 As indicated above, particular autoimmulle diseases have been associated with elevated levels of circulating T-cells bearing the products of specific Tcr genes.~ ~As use:d herein, a Tcr protein, peptide or conformational determinant is sa:d to be associated CA 0220~138 1997-0~-12 with a particular immune sy6tem disorder or condition when T-cells bearing that determinant are eIevated in patients having the disorder or condition. Thus, T-cells bearing the product6 of the V~2 and V,B8.1 genes 5 are said to be associated with Kawa6aki' 6 di6ease, becau6e tho6e T-cell6 are ~ elevated in patients having that disease. Similarly, T-cells bearing the products of V~5 . 2 and V~6 . l genes are associated with MS . The analysis of Tcr variable regions associated with immune lO sy6tem di60rder6 i6 progre6sing at a rapid pace.
Upon identification of Tcr variable region genes associated with a particular disease, proteins cnnt~;n;n~ those sequences can be produced by cDNA
cloning procedures using pllhl i F:h~.l sequence 15 in~orm-ation. Alltibody preparations enriched for antibodies which bind to a desired Tcr peptide or conformational determinant may then be prepared by the immunoaff inity procedures de6cribed herein .
In one a6pect, the method of- the crese~ ..Yention 2~ utilizes a recombinant single chair. Tc- ~~^~e:-. ~ scTcr) for the i ~ffinity purificat~ a~.~ ~ 5 irom IVIG and from Cohn FractI=ons I a-.i ~~ ~-.~ '--~ _-.n Fraction II. Construction of the s~ se~ upon complete V(Y and V,B regions of the Juri~a~ ~-ce` ' 1 lne .
25 The Jurkat cell line is a~ human monoclonal CDY-, helper leukemia T-cell line, which ig available from the American Type Culture Collection, Rockville, Maryland, USA, under Accession Mo. :ATCC 152-Tl~. This scTcr molecule contains the V~Y and V,B gene products j oined by 3~ a linker peptide. The construction of the scTcr from the Jurkat T-cell line using cDNA cloning techniques i6 de6cribed by Lake et al., 1994, 6ul~ra, inco,rporated herein by reference. The Lake et al. publication CA 0220~138 1997-0~-12 WO 96/lSl53 PCT/US9S/14869 describes the expression of the scTcr peptide from E.
coli strain B~21(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 detormlnAntq. The animal antisera may be prepared by i ;7;n~ 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 ^ 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 immunochemical detection techniques may be used f or detecting the interaction of the antibodies and T-lymphocytes For example! EBISA
and flow cytometry using a fluorescent activated cell sorter ("FACS"), as well as other conv,-n~irm~l immunochemical procedures, may be used for the detection o~ the antibody-T-ce~L 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 :accorda~ce with this invention, it has been shown that these immunoglobulin . ~

CA 0220~138 1997-0~-12 ~VO 96115153 PCrllJS95114869 preparations contain ~n~;hnflies to Tcr peptide sequences and conformational determinants. The antibody preparations of this invention are enriched for antibodies to Tcr peptide a~d conformational determinants up to about l,000 time~s over the levels in unprocessed IVIG. Dosages of= IVIG used in the treatment of GV~ and autoilhmune diseases range from lO0 mg to 5 g of~IVIG/kg body weight. Based upo~ in vitro and in vivo studies~ (mouse model), effective therapeutic doses of affinity purified antlbodies are within the range of about 0 . l mg to about lO0 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 $urrently-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 reali~ed.
This invention is further illustrated by the ~orlowing examples, which are not_intended to be_ limiting .
EXAMPJ~E 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 llm f ilter to remove insoluble C~llte ~ and particulates .
The resulting solution was subjected to i ~;3ffinity purif ication essentially~as described by Marchalonis et CA 0220~138 1997-0~-12 al., 1992 su~ra. The; ffin;ty column was - prepared as f ollows ~
BL21 (DE3 ) E. coli eells were purehased from Novagen . PET21d plasmid which contained re~ ; n~nt 5 scTcr gene was used to transform the BL21 (DE3) E. eoli so that the E. coli would produee the reeombinant scTcr protein. The recombinant protein (1.1~ milligramsl was dissolved in 10 ml of 0.1 M sodium carbonate (pH 8.0) and incubated with 0 . 75 g of activated CH Sepharose-4B
(Pharmacia Fine Chemicals, Piscataway, NJ, U.S.A. ) 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 8 . 0), treated with 40 ml of 1M e~hanolamine (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) antibQdies, all samples were first applied tq a column packed with ovalbumin immobillzed on CH-Sepharose at a flow rate of ~1.2 ml/min. The ovalbumin column preYiously had been equilibrated with TBS. The effluent from the ovalbumin column was applied directly to the i~--ln~ffinity column (also previously equilibrated with TBS) . After washing with 10 bed volumes of TBS, the bound 25 .: :antibqdies were eluted with 150 mM glycine-HCl, pH 2.0, collected in 2 ml fractions and i ~ ly neutralized with 3M Tris-~aOH at pH 9Ø
EXAMPLE I I
Flow CYtomet ry 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 59; fetal calf serum at 37C in a 959 air - 5~6 COz atmosphere. Cells were harvested during exponential growth and separated from the culture supernatant by centrifugation. Cells (I x 106) were resuspended in phosphate buffered sallne ("PBS") and combined with 0.5 ml of lQ ~Lg/ml of affinity puri~ied antibodies from an IVIG preparation designated rd~ and available from Hyland Division of ~Baxter Healthcare International, which was purified by the procedure of Example I. ~n equal number of cells were treated with 0.5 ml of 1:2000 ~ t;-~n 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 (2 antibody) coupled to, fluorescein isothiocyanate ("FITC") . Cells were washed free of 2 antibody with PBS and analyzed on a Becton Dickinson FACscan f low cytometer .
The results are shown in Figures 1-4 Figure l represents the instrument output for the negative control, and shows a reactivity of 4.399~ llositive.
Figure 2 represents the positive control and shows a reactivity of 95 . 4496 positive. Figures 3 and 4 represent unpurified IVIG~ and ~ ffinity purified IVIG respectively. The unpurified antibody mixture had a reactivity of 3 . 439; positive and the puri~ied antibody preparation had a reactivity o~ 91.47 positive .
3 0 - EXAMP~E I I I
The reactivities of ~affinity purified antibodies in Cohn Fractions I and TII with various human Tcr peptides and with the recombinant ~Jurkat scTcr -CA 0220~138 1997-0~-12 described above were analyzed by ELISA afi follows:
~T~Y~ reptide antigens ~3, ,~8, and ~7 corr.-qrnn-lF-fl to the firfit complementarity - determining region, the - third ~ Lh region and the confitant region of the YT35 (Jurkat T cell myeloma cell line) ~-chain refipectively. Peptide antigenfi or the ficTcr :were dififiolved in 0 . 2 M carbonate buf f er, pH 9 . 6 . ~ Thefie fiolutionfi (100 I~g/ml) were=added to wellfi of a microtiter plate and dried overnight at 37C. Wellfi were blocked with phofiphate-buffered fialine, p~ 7.4, cnnt;~in;ng 0 . 005% Tween 20 (PBST) and l96 gelatin (w/v) .
Cohn Fraction I + III wafi ~reacted with the antigen for one hour followed by wafihing 4 tlmefi with PBST.
Peroxldafie - conjugated rabbit ant`ibody to human IgM or IgG wafi ufied as a developing reagent at a dilution of l:lO00 for the anti-I=gM and 2:4000 for~the anti-IgG.
Conjugate ~incubations =were also for l hour . Af ter five washes with PBST, 0.03~6 fiubstrate (2,2-azino-bis- ~3-ethylbenzthioazoline-6-sulfonic acid) in ~ ate buffer, p~ 4 . O and 0 . 0l~6 hydrogen percx ~ were added. =Color~deveIopment was read a~ .. a Titertek Multiscan (Flow Labs~ a :e~
incubation at room temperature. T-~e -''5'__'5 a-e shown in Figure 5. Immunoréactivity wafi grea~es~ '.,- the ~3 peptide followed by ficTcr, ,B8 and ~17 in that order.
The ~3 and ~8 peptidefi were c-nn~;~in~i1 in- the scTcr, while ~17 was not.
EXAMPLE IV
The reactlvltlefi of antibodlefi in fiera from four patlentfi wlth rheumatoid arthrltls ( "RA" ) and eight patlents with fiyfitemic lu~ufi erythematosis ("SLE") wlth scTcr~were analyzed by ELISA using essentially the procedures descrlbed in Example III The ~esultfi are =
CA 02205138 1997-0~-12 WO 96/15153 PC'r/US95/14869 showrl in Figures 6 and 7 respectively . Signif icant reacti~ities with the :recrrnhin~nt scTcr were observed in these patient' s sera.
EXAMPLE V
5 - ~ntihn~ies 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, t~ ~rd'~)) using column chromatography in which peptide ,l~3, 10 corrl~prnflinr to the first complementarity determining region of a human T cell line (also obtained from YT35?) ~ chain, was i 1-i1 i7ed on CX-Sepharose.
Following elution, the antibodies were immediately neutrali~ed with NaOX-glycine. The antibodies then 15 were used in inhibition o~ phytohaemagglutinin~(PH~) stimulated T cell proliferation. The T cell proliferation assay is a ~standard assay and was performed essentially as-in~ Current ~rotocols in Irnmunology (Coligan, J.E. et al.; 19940 Series Ed.
20 Richard Coico) vol. I, Section 7.10 (John Wiley and Sons, Inc. ) as follows . Serial dilutions o~ antibodies were pre-incubated at 37C with normal peripheral blood lymphocytes (PBL) at a rrnr~ntration of l05 cells/well in a 96 well microtiter plate for 30 minutes. 5 ~g of 25 PX~ were added to the PBL-antibody mixture and the cells were cultured for ~72 hours at 37C. Sixteen hours prior~to harYest, one microcurie o~ tritia~ted thymidine was: added to .each well . At the end of the 72 hour inrl1h~tion, the nuclei were haryested on a~cell 30 harvester and rounted in a liquid crintill~tion counter. Percent inhibition by the anti-Tcr antibodies was calculated by the following formula:

CA 0220~il38 l997-0~i-l2 Wo 96115153 _ : PCTIUS95/14869 1- L ~sample-background) / (max. ~ proliferation-- background) ] X 1 0 0 ~ .
The results are shown in Figure 8 Although - unprocessed IVIG ~showed only non-specific background 5 inhibition up to 2500 ~g/ml under-defined experimental conditions, the affinity purified antibody-induced inhibition increased steadily to 87~6 as the antibody rnnr,~ntration increased from 3 1 to 25 . O llg/ml . These data demonstrate that antibodies with T cell activity can be specifically purified from commercial IVIG.
EXAMP~E VI
The possible effects of affinity purified antibodies i~ modulation of T cell activity were studied via interaction with the ~Tcr in a "proof of principle" model in C57/B~ mice An overview of the experiment is shown in Figure 9 A we~ll-developed sponge model of concomitant tumor immunity was used The concepts of concomitant tumor immunity are reviewed in Gorel~k, E, Adv. Cancer ~es. 39:71 (1983), and the complete details of the gelatin= s~onge model used are descrihed in Akooriaye, E T et al , Cancer Res.
58:1153 (1988), and in Akporiaye, E T and K
thlll ~kqhm;, 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-im~lanted gelatin sponge. During the manifestation of concomitant tumor immunity, the second tumor is rejected ana the effector cells can be recovered from the sponge and their tumoricidal activity studied in vitro These cytotoxic tumor rejecting T cells (TR~s~ pr,-~lnm~n~ntly express V,(~1 and V~8 Tcrs The aim of this experiment was to test the effect of anti-V~1 and anti-V~8 antibodies affinity CA 0220~138 1997-0~-12 16 - ~
purified from IVIG to modulate i~ 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 5 ~ ~ mice were surgically implanted with a gelatin sponge and the incisions were allowed to heal. O~ day 1o, fresh EMT6 cells were injected into the gelatin ~
sponges. On day 17, the sponges were removed, digested with gelatinase and the TRLs, which had inf iltrated in order to re~ect the tumor cells, were recQvered.~
;Z~nt; ho~i es which bind to a human Tcr protein, peptide or conformational determinant were affinity purified from IVIG (Gammagard~3~ using column chromatography with peptides immobilized on CH- ~
Sepharose. The peptides used corresponded to the CDR1 region of Balb/c mouse V~1 (EQHLGHNAMY) and V,~38 (Ns~rlNN~lNNMy) Tcr chains . ~The purification procedure was as described in Example 1.- Final preparations of af f inity purif ied antibody were extensive~y dialyzed against PBS. ~ ~
Affinity-purifiea antibodies prepared using ~each peptide in separate~ purification steps plus the column f low through IVIG were evaluated in an in vi tro chromium release assay. The chromium release assay was performed essentially as descrihed in Akporiaye, ~E.T.
and K. Muthulakshni, eancer Il2lrnunol. Immunotller. ~29:199 (1989~ with the following modifications. Fifty 1ll of the TBLs obtained a~o~e_~1.5 ~ 105_cel1s per well) were _ added to apFropriate welIs of a 96 well culture~ plate.
~Next, 50 ~Ll- aliquots (30 ~Lg of antibody per welI) were added and the plates i~cubated for eight hours at 37C.
The degree of cell lysis was determined ~by measuring the amount of ohromium released in the supernatant using a gamma counter.

CA 0220~138 1997-0~-12 Wo 96/151S3 PCI/US95114869 Figure lO shows that anti-V~l and anti-V,B8 independer~tly gave 8096 and 50% inhibition of T cell activity, respectively. Added together, they yielded - 90~ + inhibition. Column fIow through as a control showed no inhibition. These data demonstrate that anti~1odies with specific reactivities are able to inhi~it: 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 ef f icacy in the treatment of certain autoimmune disorders.

Claims

WHAT IS CLAIMED IS:

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

2. The human antibody preparation of claim 1 which is enriched for antibodies which bind to a recombinant human Tcr protein encoded by a V.alpha. gene or antibodies which bind to a recombinant human Tcr protein encoded by a V.beta. gene.

3. The human antibody preparation of claim 1 which is enriched for antibodies which bind to a recombinant human Tcr protein encoded by a V.delta. gene or antibodies which bind to a recombinant human Tcr protein encoded by a V.gamma. gene.

4. The human antibody preparation of claim 1 which is enriched for antibodies to a Tcr peptide or conformational determinant which is associated with an autoimmune disease or condition.

5. The human antibody preparation of claim 4, wherein the autoimmune disease is multiple sclerosis, systemic lupus erythrematosis, Kawasaki's disease or rheumatoid arthritis.

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

7. The antibody preparation of claim 1, which is enriched for antibodies which bind to V.alpha. and V.beta. peptide sequences of the Jurkat T-cell line, ATCC No. 152-TlB.

8. The antibody preparation of claim 1, which is enriched for antibodies which bind to a single-chain Tcr protein derived from the V.alpha. and V.beta. gene sequences of a human T cell.

9. A method for making an antibody preparation which is enriched for antibodies that bind to a human Tcr peptide sequence, which comprises (a) combining, under antibody-antigen binding conditions, (i) a human plasma Cohn fraction which contains antibodies that bind to a human Tcr peptide or conformational determinant, and (ii) a solid support to which Tcr peptide has been immobilized;
(b) separating unbound proteins from the solid support;
(c) 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 or conformational determinant.

10. The method of claim 9, wherein the human plasma Cohn fraction employed in step (a) is Cohn Fraction I or III.

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

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

13. A method for making an antibody preparation which is enriched for antibodies that bind to a human Tcr peptide or conformational determinant, which comprises:
(a) combining, under antibody-antigen binding conditions, (i) an 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;
(b) separating unbound proteins from the solid support;
(c) 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.

14. The method of claim 13, wherein the antibody mixture employed in step (a) is a human antibody mixture.

15. The method of claim 13, wherein the antibody mixture employed in step (a) 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.

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

20. The method of claim 13, wherein the immobilized recombinant human Tcr protein employed in step (a) is a protein encoded by a region of a V.alpha., V.beta., V.delta. or V.gamma. gene.

21. The method of claim 20, wherein the recombinant human Tcr protein is a single-chain Tcr protein derived from the V.alpha. and V.beta. gene sequences of a human T cell.

22. A method of diagnosing a human for an autoimmune disease or condition or GVHD which comprises (a) identifying a Tcr peptide or conformational determinant that is elevated in humans having said autoimmune disease or condition or GVHD;
(b) combining T-lymphocytes from said human with a human antibody preparation which is enriched for antibodies which bind to said Tcr peptide or conformational determinant;
(c) 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.

24. The method of claim 22, wherein the Tcr protein or conformational determinant is encoded by a region of a V.alpha. or V.delta. gene,

25. The method of claim 22, wherein the Tcr peptide or conformational determinant is encoded by a region of a V.beta. or .gamma. gene.

26. The method of claim 22, wherein step (c) is accomplished by means of an ELISA procedure.

27. The method of claim 22, wherein step (c) is accomplished by fluorescent flow cytometry.

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 a Tcr peptide or conformational determinant 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 peptide or conformational determinant.

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 a recombinant human Tcr protein encoded by a V.alpha. gene or antibodies which bind to a recombinant human Tcr protein encoded by a V.beta. gene.

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

32. The method of claim 28, wherein the disease is an autoimmune disease.

33. The method of claim 30, 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.