CA2277724C - A composition for inducing humoral anergy to an immunogen - Google Patents

Abstract

Conjugates of stable nonimmunogenic polymers and analogs of immunogens that possess the specific B cell binding ability of the immunogen but lack T cell epitopes and which, when introduced into individuals, induce humoral anergy to the immunogen are disclosed. Accordingly, these conjugates are useful for treating antibody-mediated pathologies that are caused by foreign or self immunogens.

Description

A COMPOSIT::ON FOR INDUCING HUMORAL ANERGY TO AN
IMMUNOGEN
Technical Field This nvent:ion is in the field of immunology and concerns co~aposit:ians and methods for inducing humoral anergy ~:or the purpose of treating antibody-mediated pathologies.
Background of tree Invention In order to survive in a world of pathogenic or potentially pathogenic microorganisms, higher organisms have evolved immune systems which can specifically recognize virtually any foreign substance through its characteristic molecules. This recognition frequently results in the product=ion of specifis proteins called antibodies which bind only to the foreign substance which induced their synthesis, causing the elimination of the invading microorganism. Occasionally an animal's immune system makes ant:ibodieas which recognize some of its own molecules, generating an autoimmune state that may affect the animal's health adversely.
The ir~duction of specific antibodies in response to an i.mmunoc~en involves the interaction of multiple cell types, ~.ncluding thymus-derived lymphocytes (T cells), macraphage;~, and bone marrow-derived lymphocytes (B c:ells).. B cells possess surface immunoglobulin by which they are able to bind immunogens, the first step in their activation and clonal expansion.
The site(s), region,(s) or domains) of the immunogen to which the immunoglobulin binds is called a "B cell epitope". In the second step of B cell activation and expansion, T cells are activated through interaction with the B cell bound-immunogen at a site, region or domain of the immunogen called a "T cell epitope". Once activated, the T cells provide: positive signals) to the B cells bound by the immunogen and they proceed to differentiate and to produce and secrete antibody. Positive signals from the T cell include the secretion of lymphokines, and/or direct contact between the B cells and T cells.
T cell epitopes may be different or more restricted in scope than B cell epitopes. As discussed above, in order for an immunogen to elicit T dependent antibodies, it must have epitopes recognized by both B and T cells.
Past attempts to treat antibody-mediated pathologies have involved both general and specific suppression of the immune response. General suppression has typically employed broad spectrum, nonspecific immunosuppressants such as cyclophosphamide or steroids.
Because these nonspecific drugs suppress many aspects of the immune system, they eliminate its required and beneficial functions as well as the malfunction causing the condition being treated. They are thus used with extreme caution if at all, and subject the patient to risk from secondary infections or other undesirable side effects.
Because of the disadvantages of general immunosuppression, methods for specifically suppressing an immune response to an immunogen without affecting the normal functions of the immune system are highly preferred for treating antibody-mediated pathologies.
The present invention concerns compositions and methods for specifically suppressing the humoral response to immunogens.
Prior att=empts to induce specific immunosuppre~;sion have focused on conjugating haptens and immunogens to nonimmunogenic polymeric carriers.
Benacerraf, R;atz and their colleagues used conjugates of haptens and antigens and copolymers of D-lysine and D-glutamic acid (D-E1~). Their initial studies involved conjugates of the synthetic hapten 2,4-dinitrophenyl (DNP) in guinea pigs and mice and showed the conjugates were capable of inc3ucing humoral unresponsiveness. These initial studies were then extended to conjugates of other haptens and conjugates of immunogens. While the results with haptens were :repeatable, and although their patents (U.S. 4,191,Ei68 and 4,220,565) allege the approach is effective in inducing tolerance to immunogens, subsequent work has shoGrn that conjugates of D_-EK and immunogens do not provide a means for inducing humoral unresponsiveness to the immunogen. For instance, Liu et al., J. Immun.
(1979) 123:25456-2464, report that subsequent studies of those conjugates demonstrate that the conjugates "do not induce unresponsiveness at the level of protein specific B cells." Spmilarly, Butterfield et al., J. Allercry Clin. Immun. (1981) 67:272-278, reported that conjugates of ragweed inamunogen and D-EK actually stimulated both IgE and IgG responses to the immunogen.
This subsequent work and other data dealing with conjugates of nonimmunogenic polymers and immunogens (Saski et al.., Scand. J. Immun. (1982) 16:191-200; Sehon, Prog. Allerm~ (1982) 32:161-202; Wilkinson et al., J.
Immunol._ (987) 139:326-331, and Borel et al., J. Immunol.
Methods (1990) 126:159-168) appear to indicate that the anergy, if any, obtained with such conjugates is due to suppression of T cell activity, not B cell unresponsiveness.

Several other references deal with conjugates of nonimmunogenic po:Lymers and DNA. See U.S. 4,191,668;
U.S. 4,650,625; J. C:Lin. Invest. (1988) 82:1901-1907. -As. a whole, these references indicate that these DNA conjugates may suppress the production of antibodies to this lupus autoimmunogen. It should be noted in this regard that DNA, like haptens, does not possess T cell epitopes.
In su.m, applicants believe the prior art shows that antibody ~~roduct:ion to conjugates of nonimmunogenic stable polymers and haptens or DNA, neither of which have T cell epitopes~, may provide B cell unresponsiveness.
Applicants also believe that conjugates of immunogens do not provide B cell unresponsiveness but may activate T cells to directly suppress the immune response.
Disclosure of the Invention The present: invention resides in the discovery that the failure of t:he prior conjugates of nonimmunogenic stable polymers and immunogens to induce B
cell anergy (unresponsiveness) was due to the fact that the immunogens contained both B and T cell epitopes and that if the latter were eliminated, the conjugate would be effective for inducing B cell anergy.

I II I

According to an aspect of the invention, there is provided a conjugate for inducing specific B cell anergy to an immunogen implicated in an antibody-mediated pathology in an individual suffering from said pathology comprising a nonimmunogenic carrier and an analog of the immunogen wherein (a) the analog binds specifically to surface antibody on B cells to which the immunogen binds specifically and (b) the conjugate lacks T cell epitopes capable of activating T
cells in said individual.
The analog may be selected from the group consisting of peptides, polypeptides, proteins, glycoproteins, lipoproteins, carbohydrates, lipids, and polysaccharides.
The immunogen may be an external immunogen, for example, a biological drug, allergen or a D immunogen associated with Rh hemolytic disease.
The immunogen may be a self-immunogen, for example a self immunogen that is associated with thyroiditis, diabetes, stroke, male infertility, myasthenia gravis or rheumatic fever.
The immunogen and the analog may be of the same chemical class, for example, polypetides or may be of different chemical classes.
The carrier is a polymer, for example, a copolymer of D-lysine and D-glutamic acid, polyethylene glycol or triethylene glycol.
The carrier may have three to eight attachment sites.
The antibody-mediated pathology may be an autoimmune disorder.
According to another aspect of the invention, there is provided a pharmaceutical composition for treating an antibody-mediated pathology comprising a therapeutically effective amount of the conjugate described above combined with a pharmaceutically acceptable carrier.
According to another aspect of the invention, there is provided the use of an effective amount of the above-described composition for inducing specific B
cell anergy to a T cell-dependent immunogen in an individual.

i ii i - Sa -According to another aspect of the invention, there is provided the use of a therapeutically effective amount of the above-described composition for treating an individual for an antibody-mediated pathology in which undesired antibodies are produced in response to a T cell-dependent immunogen.
Brief Description of the Drawings Figure 1 graphically illustrates the detection of B cell epitopes in immunized CAF1 mice as described in Example 1.
Figure 2, similarly, illustrates the detection of T cell epitopes as described in Example 1.
Figure 3 illustrates the suppression of antibodies to peptide "L-53" as described in Example 1.
Figures 4 and 5 are graphs of the results described in Example 4.
Modes for Carryina Out the Invention As used herein the term "B cell anergy" intends unresponsiveness of those B cells requiring T cell help to produce and secrete antibody and includes, without limitation, clonal deletion of immature and/or mature B cells and/or the inability of B cells to produce antibody. "Unresponsiveness" means a therapeutically effective reduction in the humoral response to an immunogen. Quantitatively the reduction (as measured by reduction in antibody production) is at least 50%, preferably at least 75%, and most preferably 100%.
"A:ntibody" means those antibodies which are T cell dependent .
As used herein the term "immunogen" means a chemical entity that elicits a humoral immune response when injected into an animal. Immunogens have both B
cell epitopes and T cell epitopes.
As used herein "individual" denotes a member of the mammalian species and includes humans, primates, domestic animals such as cattle and sheep, sports animals such as horses, anal pets such as dogs and cats.
The terms "analog" of an immunogen intends a molecule that (a) binds specifically to an antibody to which the immunoge,n binds specifically and (b) lacks T
cell epitopes. Although the analog will normally be a fragment or derivative of the immunogen and thus be of the same chemical class as the immunogen (e.g., the immunogen is a polypeptide and the analog is a polypeptide), chemical similarity is not essential.
Accordingly, the analog may be of a different chemical class than t:he immunogen (e.g., the immunogen is a carbohydrate and the analog is a polypeptide) as long as it has the functional characteristics (a) and (b) above.
The analog may be a protein, carbohydrate, lipid, lipoprotein, glycoprotein, lipopolysaccharide or other biochemical entity. Further, the chemical structure of neither the immunogen nor the analog need be defined for the purposes of this invention.
"Nonimmu.nogenic" is used to describe the carrier polymer means that the carrier polymer elicits substantially no immune response when it is administered by itself to an individual.
Immunoge:ns that are involved in antibody mediated pathologies may be external (foreign to the individual) immunogens such as biological drugs, allergens, idiopathic contrast media, and the like or self-immunogens (autoimmunogens) such as those associated with thyroiditis (t:hyroglobulin), stroke (cardiolipin), male infertility (cc-sperm), myasthenia gravis (acetylcholine receptor), rheumatic fever (carbohydrate complex), and. Rh hemolytic disease (D immunogen).
Analogs t:o such immunogens may be identified by screening candidate molecules to determine whether they (a) bind specifica7Lly to serum antibodies to the immunogen and. (b) .Lack T cell epitopes. Specific binding to serum antibodies may be determined using conventional immunoassays and the presence or absence of T cell epitopes may be determined by conventional T cell activation assays. In this regard an analog Which "binds specifically"' to serum antibodies to the immunogen exhibits a reasonable affinity thereto. The presence or absence of T cell epitopes may be determined using the tritiated th~znidine incorporation assay described in the examples. Analogs that fail to induce statistically significant incorporation of thymidine above background are deemed to lack T cell epitopes. It will be appreciated that the quantitative amount of thymidine incorporation may ~~ary with the immunogen. Typically a stimulation index below about 2-3, more usually about 1-2, is indicative of a lack of T cell epitopes.
A normal first step in identifying useful analogs is to prepare a panel or library of candidates to screen. For instance, in the case of protein or peptide analogs, libraries may be made by synthetic or recombinant techniques such as those described by Geysen et al. in ~itheti~~ Peptides as Antigens; Ciba Symposium (1986) 119:1'_!1-149; Devlin et al., Science (1990) 249:404-406; Scott et al., Science (1990) 249:386-390;
and Cwirla et. al., PNAS USA (1990) 87:6378-6382. In one _g_ synthetic technique, peptides of about 5 to 30 amino acids are synthesized in such a manner that each peptide overlaps the next <ind all linear epitopes are represented. This is accomplished by overlapping both the carboxyl and amino termini by one less residue than that expected for a B cell epitope. For example, if the assumed minimum requirement for a B cell epitope is six amino acids, then each peptide must overlap the neighboring peptides by five amino acids. In this embodiment, each peptide is then screened against antisera produced against the native immunogen, either by - immunization of animals or from patients, to identify the presence of B cell epitopes. Those molecules with antibody binding activity are then screened for the presence of ~~ cell epitopes as described in the examples.
The molecules lacking T cell epitopes are useful as analogs in the invention.
If the T cell epitope(s) of an immunogen are known or can be identified, random screening of candidate analogs is not necessary. In such instances, the T cell epitope(s) may be altered (e. g., by chemical derivatization, or elimination of one or more components of the epito~~e) to render them inoperative or be eliminated completely, such as, for instance, in the case of peptides, by synthetic or recombinant procedures.
The analogs are coupled to a nonimmunogenic polymeric carrier to prepare the conjugates of the invention. l?referred polymeric carriers are biologically stable, i.e., they exhibit an in vivo excretion half-life of days to months, and are preferably composed of a synthetic single chain of defined composition. They will normally have a molecular weight in the range of about 5,000 to abort 200,000, preferably 5,000 to 30,000.
Examples of ouch polymers are polyethylene glycol, poly-D-lysine, polyvinyl alcohol, polyvinyl pyrrolidone, _g-immunoglobulins, and "D-EK", a copolymer of D-glutamic acid and D-lysine. :Particularly preferred carrier polymers are D--EKs having a molecular weight of about 5,000 to about 30,000, and an E:K (D-glutamic acid:D-lysine) mole ratio o:f approximately 60:40, Conjugation of the analog to the carrier polymer may be effected in any number of ways, typically involving one or more crosslinking agents and functional groups on the analog and carrier.
PolyF~eptid~a analogs will contain amino acid sidechain groups such as amino, carbonyl, or sulfhydryl groups that will serape as sites for coupling the analog to the carrier. Res:idues.that have such functional groups may be aidded ito the analog if the analog does not already contain same. Such residues may be incorporated by solid phase synthesis techniques or recombinant techniques, both of which are well known in the peptide synthesis arts. In the case of carbohydrate or lipid analogs, functional amino and sulfhydryl groups may be incorporated therein by conventional chemistry. For instance, primary amino groups may be incorporated by reaction with eahylendiamine in the presence of sodium cyanoborohydridle and sulfhydryls may be introduced by reaction of cys;teamine dihydrochloride followed by reduction with a standard disulfide reducing agent. In a similar fashion the carrier may also be derivatized to contain functional groups if it does not already possess appropriate functional groups. With specific reference to conjugating peptide analogs and D-EK or other proteinaceous carriers; coupling is preferably carried out using a het.erobil:unctional crosslinker, such as sulfosuccinimid.yl(4-iodoacetyl) aminobenzoate, which links the a amino group on the D-lysine residues of D-EK

to a sulfhydryl side chain from an amino terminal cysteine residue on the peptide to be coupled. This method is preferably carried out such that an average of 3 to 5 analog molecules are coupled to each D-EK molecule and the average mo:Lecular weight of the D-EK prior to coupling is ~~, 000 1~a 30, 00o daltons.
The conjugates will normally be formulated for administration by :injection (e. g., intraperitoneally, intramuscularly, etc.). Accordingly, they will typically be combined urith pharmaceutically acceptable carriers such as saline, Ringer's solution, dextrose solution, and the like. The conjugate will normally constitute about 0.01% to 10% by weight of the formulation. The conjugate is administered to an individual in a "therapeutically effective amount", i.e., an amount sufficient to produce B cell anerg~~ to the involved immunogen and effect prophylaxis, improvement or elimination of the antibody-mediated condition being addressed. The particular dosage regims:n, i.~e., dose, timing and repetition, will depend on ths~ particular individual and that individual's medical history. lKormally, a dose of about 10 ~cg to 1 mg conjugate/kg body weight will be given, daily for three consecutive days. Other appropriate dosing schedules would be 3 doses per week, or one dose per week.
Repetitive administrations, normally timed according to B
cell turnover rates, may be required to achieve and/or maintain a state of humoral anergy. Such repetitive administrations will typically involve treatments of up to 1 mg/kg oj° body weight every 30 to 60 days, or sooner, if an increa:~e in antibody titer is detected.
Alternativel~r, sustained continuous release formulations of the conjugates 'may be indicated for some pathologies.
Various formulations and devices for achieving sustained release are known in the art.

Anti-~T helper cell treatments may be administered tc>gether with the conjugates. Such treatments usually employ agents that suppress T cells such as steroic;s or c:yclosporin.
The following examples are intended to further illustrate the invention and its uniqueness. These examples are nat internded to limit the scope of the invention in any manner.
Example 1 B Cell A.neray to the Acetylcholine Receptor Preparation of Peptides and D-EK/Peptide Conjugates:
The a-subunit of the acetylcholine receptor of Torpedo californicus is described by Stroud, R.M., and Finer-Moore, J., Ann. Rev. Cell Biol. (1985) 1:317:351, and Sumikawa, K., et al., Nucl. Acids Res. (1982) 10:5809-22. The peptide defined by residues 47-127 of that a-subunit is called the major immunogenic region (MIR).. .
Two peptidea, L-42 and L-53, corresponding to residues 61-77 and 11.2-127 of that a-subunit, were synthesized using conventional solid-phase methods and purified to homogeneity by HPLC. An amino terminal cysteine was added to each sequence for the purpose of attachment of the peptide to D-EK via a thio ether linkage.
Each peptide (40 mg) was dissolved in 0.1M
sodium borate buffer, pH 9Ø The solution was reacted with citraconic anhydride (400 ~,L) at room temperature;
the pH was maintained. above 7.0 by addition of 1M NaOH.
The solution was then. made 20 mM in dithiothreitol and was warmed at 37°C for 20 minutes to reduce the peptide.
The mixture was quickly desalted over G-10 Sephadex*-*Trademark columns which. were equilibrated with O.1M sodium borate, pH 7Ø
D-E;K (200 mg, weight average mw = 10, 000 -30,000) was dissolved in 2.0 mL of O.1M sodium borate.
Sulfosuccinim.idyl (4-iodoacetyl) aminobenzene (SSIAB, mg, Pierce. Chemical) was added to the mixture and the mixture was reacted for 90 minutes at room temperature in the dark. Th.e mixture was then desalted over a 10 mL
G-25 column, equilibrated with O.1M sodium borate, 10 pH 7Ø
The: desalted SSIAB-D-EK was mixed with the reduced and desalted peptide and reacted overnight. The resulting conjugates was placed in dialysis tubing with a 14 Kd cutoff and was dialyzed against 5% acetic acid to remove citrac:onyl groups. The dialysis buffer was changed to ph,osphai:e-buffered saline and the dialysis continued.
Detection of B cell epitopes:
CAf1 mice were immunized (day 0) intraperitone:ally (i.p.) with 50 ~g of recombinant torpedo MIR absorbed onto alum plus B. pertussis vaccine (Iverson, G.M., (1986) Handbook of Experimental ImmunoloaY, V'ol. 2" p. 67, D.M. Weir ed., Blackwell Scientific Puiblicai=ions, Palo Alto, CA). The mice received a booster injection of the same protein in saline, IP, on day 21 and were bled from the tail vein on day 28. Sera. from these mice (anti-MIR sera) were used to screen peptides L-42 and L-53 for the presence of B
cell epitopes;, as i~ollows. The sera were added to microtiter wells coated with 10 ~.g/ml of the indicated peptide conjugates.. The plates were incubated at 37°C
for one hour, washead 3 times, 100 ~,1 of alkaline phosphatase-conjugated goat antimouse antibody was added, incubated at 37°C for one hour, washed 3 times, and 100 ~,1 of developer (substrate) was added to each well.

The plates were incubated at room temperature for 30 minutes and the amount of color in each well was determined in a Ti.tertek~ Multiskan. Results are illustrated graphically in Figure 1. The curve labelled "L42 or L53, NMS" contains the values obtained using normal mouse serum (NMS) instead of the anti-MIR sera on plates coated with either L42 or L53. As shown in Figure 1, both peptides reacted specifically with antibodies from the immunized mice indicating the presence of B cell epitopes on both peptides.
Detection of T cell epitopes:
T cell activation was assayed by the general procedure of Bradley, M.L., (1980) in Mishell and Shigii, eds., Selected Methods in Cellular Immunoloay (W. H.
Freeman and Co., ~~an Francisco, CA), p. 164. CAF1 mice were immunized on the footpad with 50 ~g MIR in Complete Freund's Adjuvant (CFA) on day 0. On day 7 the popliteal lymph nodes were removed and placed in culture in microtiter plates using 5 x 105 cells per well. The peptides or peptide-DEK conjugate were added to the cultures, and on day 4, 1 ~Ci of tritiated thymidine was added to each well. to measure proliferation of T cells.
The cultures were harvested on day 5 with a Skatron~ cell harvester. The amount of incorporated 3H-thymidine was determined in a Beckman L6800~ liquid scintillation counter. The stimulation index was calculated by dividing the CPM incorporated with peptide by the CPM
incorporated from cultures without any peptide. A
stimulation index > 1 was indicative of the presence of a T cell epitope on the peptide added to the well. As shown in Figure 2, L-42 but not L-53 possessed T cell epitopes in this assay.

Induction of B Cell Anergy to L-53 by L-53/D-EK Conjugate:
CAF1 mice. were immunized with 50 ~g of MIR, i.p., absorbed onto alum plus B. pertussis vaccine on day 0. On days 21,. 22 and 23 the mice (6 mice per group) received 10 or 100 ug of either L-42-D-EK conjugate or L-53-D-EK con.jugate. One group received only saline. On day 28 all mice received a booster injection of MIR in saline and on. day 35 all mice were bled and assayed for the presence of antibodies to L-42 and L-53 in their sera, using an ELISA assay as described above with respect to Figure .1. The results for antibodies to L42 are shown in Figure. 3A and for antibodies to L53 are shown in Figure 3B" The L-53 conjugate suppressed antibody formation to L-53 but not to L-42. The L-42 conjugate did. not suppress the antibody response to either L-42 or L-53 but rather may have increased antibody production to L-42. The antibody titers are expressed as a percent of a standard sera. The P values were determined by a standard t test comparing each dose to the saline: control.
Examgle 2 Failure o7: Ovalbumin-D-EK Coniuc~ate to Induce B Cell Anerqy to Ovalbumin This example is further evidence that conjugates of immunogens and D-EK do not induce B cell anergy.
Synthesis of Ovalbumin-D-EK Conjugate:
Chicken egg ovalbumin (50 mg) was dissolved in 5 mL of O.1M sodium borate buffer, pH 9.0, containing 10 mM EDTA. After the addition of 3.0 mg of 2-iminothiolane (Traut's reagent), the mixture was reacted for 2.5 hours at room temperature. D-EK (54 mg), dissolved in 0.5M
sodium borate:, pH X3.0, at a concentration of 100 mg/mL, was reacted wit;! SSIAB (18 mg; Pierce Chemical) for 2.5 hours in the dark, at room temperature. The two reaction mixtures described above were desalted separately on G-25 columns (Pha:rmacia; 10 mL column volume, equilibrated with O.1M sodium borate, pH 9.0) and the excluded fractions were ~~ombined and reacted for 16 hours at 4°C, in the dark. The reaction product was fractionated by *..
gel filtration aver Sephacryl ~-200 (490 mL, Pharmacia) columns, equilibrated with 0.2M ammonium bicarbonate.
l0 Fractions containing conjugate, as assessed by polyacrylamide ~~el electrophoresis, in the presence of sodium dodecyl aulfate (SDS-PAGE), were pooled and dried under vacuum. 'rhe dried material was reacted with 0.8 mL
of citraconic anhydride, maintaining the pH between 7 and 9 by the addition of 1M NaOH, in order to efficiently separate conjugated ovalbumin from unreacted protein.
The citraconylal~ed conjugate was rechromatographed over S-200, and fracl=ions containing high molecular weight material (> 80,000 daltons), as assessed SDS-PAGE, were used for biolog:ecal studies. , ChickESn _ovalbumin, when conjugated to D-EK, does not induce B cell anergy in mice immunized to chicken ovalbumin:
FemalEa CAF1 mice were primed with chicken ovalbumin (ova; 100 ~C~g/mouse, i.p.) precipitated on alum, with B. gertussis vaccine added as an adjuvant. Sixteen weeks later, the. mice were divided into two groups of six mice each. One group (control) was treated with saline, and the second croup was injected with a conjugate of ova and D-EK (ova-D--EK; 200 ~Cg/mouse/day, i.p.). The mice were dosed on three successive days. One week after the first dose, the mice .in both groups were boosted, i.p., with ova in saline (100 ~g/mouse). One week later, the mice were bled f=rom a tail vein. The plasma was harvested and a~~sayed f.or the amount of anti-ova *Trademark antibodies b~T an ELISA assay. As shown in Table 1, the ova-D-EK con;jugate did not suppress the anti-ova response.
Table 1 Percent ~f Anti-Ova Group Treatment Standard Serum ~ S.D.
1 saline 70.7 ~ 36 2 ova-D-EK 160.2 ~ 167 1 The amount of anti-ova antibody was determined in an ELISA, measured against a standard pool of sera obtained from CAF1 mice immunized and boosted with ova. The values shown are the mean and standard deviation for the six mice in each group.
Example 3 lFailure of MIR-D-EK Conjuq"ate to Induce B Cell Anercty to MIR
This example is still further evidence that conjugates o:E immunogens and D-EK do not induce B cell anergy.
Synthesis of MIR-D-EK Conjugate:
MII~ was modified on its carboxyl-terminus to include a sequence of 8-amino acids (Arg-Ser-Lys-Ser-Lys-Ser-Lys-Cys (SEQ. ID NO.: 1)). The amino-terminus was extended by one amino acid, proline. Purified modified MIR (250 mg) was reduced with 100 mM
dithiothreit«1 and was desalted over Sephadex G-25 (Pharmacia), equilibrated with 0.1 M sodium borate buffer, pH 9.0, containing 10 mM EDTA. D-EK (400 mg) was reacted with SSIAB (29 mg) as in the previous examples.
The product Haas desalted over G-25. The excluded volumes from the modified MIR and D-EK G-25 column runs were combined and reactE=_d at 4°C for 16 hours, in the dark.
Excess SSIAB group, were quenched with 2-mercaptoethanol, and the reaction mixture was concentrated to 20 mL over a PM-10 membrane (Am:icon Corporation). The mixture was treated with 1.0 mL of citraconic anhydride and chromatographed over S-300 (Pharmacia; 1.8 L), equilibrated with !5% ammonium hydroxide. Fractions containing two or more modified MIR groups per D-EK, as assessed by f~DS-PA~sE, were pooled and used for biological studies.
MIR-D-EK conjugate contains T cell epitopes in rats immunized witlh MIR from the same species:
T cell activation was assayed by the general procedure of Bradley, supra. Female Lewis rats were immunized in the footpad with MIR (50 fig) in complete Freund's adjuvant (CFA) on day 0. On day 7, the popliteal lymph nodes were removed and placed in culture in microtitei plates using 5~105 cells per well. MIR-D-EK was added,, and, after four days of culture, the wells were pulsed with t:ritiated thymidine (1-uCi) to measure proliferation of T cells. The cultures were collected after 5 days of culture with a Skatron"' cell harvester.
The amount oi° incorporated 3H-thymidine was determined by scintillation spectrometry. The stimulation index was calculated bit dividing the counts incorporated in the absence of the conjugate. A stimulation index of greater than 1 Was considered indicative of the presence of a T
cell epitope on the added conjugate. The stimulation index was 4 or _greater at all concentrations of MIR-D-EK
tested (10 ~cc~/mL to 400 mg/mL). This proves that T cells from MIR-immunized rats recognize T cell epitopes on the MIR-D-EK con:jugate :in this assay.

MIR-D-EK does not induce B cell anergy in rats immunized with MIR:
Female Lewis rats were primed with MIR (100 ~Cg/rat) in CfA. S.ix months later, the rats were divided into three groups of three rats each. One group was treated with saline (control) and the other two groups were treated with 1KIR-D-EK (100 ~Cg/rat, i.p.) on three successive days. ,after one week, the rats in the control group and one, group that had been treated with MIR-D-EK
were boosted with :recombinant MIR (1000 ~,g/rat, i.p.) in saline. One week later, all three groups of rats were bled from then tail vein. The plasma was harvested and assayed for i:he amount of anti-MIR antibodies by an ELISA
assay. TablEa 2 below reports the data from those assays.
Table 2 Group Treai:ment MIR Boost ~g/ml anti-MIR2 P vs.
(mean ~ S.D.) Group 1 1 Saline Yes 130.5 ~ 74.7 2 MIR-D-EK Yes 85.5 t 31.1 0.195 3 MIR-D-EK No 230.6 ~ 31 0.049 2 The conceni~ration of anti-MIR antibodies was determined in an ELISA measured against a standard pool of rat anti-MIR sera. The values shown are the mean and standard deviation of the three rats in each group.
P values ware determined by a Standard t test. Group 2 is not significantly different from Group 1. Group 3 (the non-b«osted group) is significantly higher than 3 0 Group 1.
As shown in Table 2, the data on Group 1 animals (saline control) indicate that MIR itself is an immunogen. 'rhe data for the Group 2 and 3 animals indicate that the M:IR-D-EK conjugate did not affect the anti-MIR response. In fact, MIR-D-EK boosted the anti-MIR response in Group 3.
Example 4 Tests with ConiuQate of L-42 and KLH
These tempts, taken together with the results of Example 1 show that: the moiety conjugated to D-EK will cause anergy in B cells recognizing that moiety if the moiety either does not contain a T cell epitope or is not recognized by T ce7~.ls.
Synthesis of L42 peptide-KLH conjugate:
Reduced h-42 (see Example 1) was conjugated to keyhole limpet hemocyanin (KLH) using thioether chemistry similar to that de:~cribed above with respect to D-EK.
L-42 laci;s a T cell epitope(s) in mice immunized with L-4:?-KLH:
Activation of T cells by peptides was measured by the general procedure of Bradley, sutra. Female CAF1 mice were ima:unized in the footpad with L-42 peptide conjugated KI~H (L-~~2-KLH; 50 fig) in CFA on day 0. On day 7, the poplit:eal lymph nodes were removed and placed in culture in mi.crotii~er plates, at a cell density of 5'10 5 cells/well. Peptides were added, and, after four days of culture, the wells were pulsed with 1 ~Ci of tritiated thymidine to measure proliferation of T cells. The cultures were: collEacted after 5 days of culture with a SkatronT" cell. harvEaster. The amount of incorporated 3H-thymidine wa~~ determined by scintillation spectrometry.
The stimulation index was calculated by dividing the counts incorF~orated in the absence of peptide. An index of greater than 1 :is indicative of the presence of a T
cell epitope on the added peptide.
The: data in Figure 4 demonstrate that the L-42 did not stimulate the growth of T cells taken from L-42 KLH-immunized mice, and therefore did not contain an epitope(s) recognized by T-cells induced by immunization with L-42-KLF~.
L-~E2-D-E:K conjugate induces a B cell anergy in mice immunizs~d to :L-42-KLH:
CA3?1 mice were primed with 100 ~g/mouse of L-42-KLH on alum plus B. pertussis vaccine as an adjuvant.
Three weeks :Later, the mice were divided into groups of six mice eactl. One group was treated by i.p. injections on three successive days with saline (control); the other groups were :similarly treated with L-42-KLH (50 ~g/mouse, i.p.), and, after a wait of one week, they were bled from the tail vein. The plasma was harvested and assayed for the amount o~° anti-L-42 and anti-KLH antibodies by ELISA
assays. Data are expressed as a percent of a standard serum. An a:~terisk indicates that a data point was significantllt different from the control as determined by a standard t test.
The= data in Figure 5 demonstrate that the L-42 response, bui~ not the anti-KLH response, was suppressed in this assay by the L-42-D-EK conjugate. Thus, the studies summarized :in Example 1 and these data demonstrate i~he L-42-D-EK induces B cell anergy when the mice are immunized in a manner that does not induce the proliferation of T cell clones that recognize the L-42 peptide. On the other hand, L-42-D-EK did not induce B cell anerg!~ in animals that were immunized with an immunogen (M:IR) which induced T cells that recognized the L-42 peptide.
Modifications of the above-described modes for carrying out the invention that are obvious to those of ordinary sk ill in the fields of immunology, chemistry, medicine and related arts are untended to be within the scope of the following claims.

Claims (18)

1. A conjugate for inducing specific B cell anergy to an immunogen implicated in an antibody-mediated pathology in an individual suffering from said pathology comprising a nonimmunogenic carrier and an analog of the immunogen wherein (a) the analog binds specifically to surface antibody on B cells to which the immunogen binds specifically and (b) the conjugate lacks T
cell epitopes capable of activating T cells in said individual.

2. The conjugate according to claim 1 wherein the analog is selected from the group consisting of peptides, polypeptides, proteins, glycoproteins, lipoproteins, carbohydrates, lipids, and polysaccharides.

3. The conjugate according to claim 1 or 2 wherein the immunogen is an external immunogen.

4. The conjugate according to claim 3 wherein the external immunogen is a biological drug, allergen or a D immunogen associated with Rh hemolytic disease.

5. The conjugate according to claim 1 or 2 wherein the immunogen is a self-immunogen.

6. The conjugate according to claim 5 wherein the self immunogen is that associated with thyroiditis, diabetes, stroke, male infertility, myasthenia gravis or rheumatic fever.

7. The conjugate according to any one of claims 1 to 6 wherein the immunogen and the analog are same chemical class.

8. The conjugate of claim 7 wherein the immunogen and the analog are polypeptides.

9. The conjugate according to any one of claims 1 to 6 wherein the immunogen and the analog are of different chemical classes.

10. The conjugate according to any one of claims 1 to 9 wherein the carrier is a polymer.

11. The conjugate according to claim 10 wherein the polymer is a copolymer of D-lysine and D-glutamic acid.

12. The conjugate of claim 10 wherein the polymer is polyethylene glycol.

13. The conjugate of claim 10 wherein the polymer is triethylene glycol.

14. The conjugate according to any one of claims 1 to 13 wherein the carrier has three to eight attachment sites.

15. The conjugate according to any one of claims 1 to 14 wherein the antibody-mediated pathology is an autoimmune disorder.

16. A pharmaceutical composition for treating an antibody-mediated pathology comprising a therapeutically effective amount of the conjugate according to any one of claims 1 to 14 combined with a pharmaceutically acceptable carrier.

17. Use of an effective amount of the composition of claim 16 for inducing specific B cell anergy to a T cell-dependent immunogen in an individual.

18. Use of a therapeutically effective amount of the composition of claim 16 for treating an individual for an antibody-mediated pathology in which undesired antibodies are produced in response to a T cell-dependent immunogen.