CA1301064C - Immunomodulating compositions and their use - Google Patents

Abstract

IMMUNOMODULATING COMPOSITIONS
AND THEIR USE

ABSTRACT OF THE DISCLOSURE
Novel methods or compositions are provided for modulating the immune system, so as to be able to se-lectively stimulate or inactivate lymphocytes in rela-tion to a particular transplantation antigen context.
Particularly, mixtures may be employed associated with the more common transplantation antigens of a host pop-ulation. In this manner, a large number of people can be treated, for example, by immunization, stimulation of particular T-cells or B-cells in relation to a path-ogenic invasion or other aberrant state, e.g. neopla-sia, treatment of autoimmine diseases, and the like.
Particularly, the compositions may involve an oligopep-tide involving as a first region a consensus sequence and an epitope or the first region may be joined to a second region comprising an antibody target sequence which is capable of competing with an epitopic site of an antigen of interest.

Description

13(1~4 IMMUNOMODULATING COMPOSITIONS
AND THEIR USE

Methods and compositions in immunology, where the S immune system may be activated or deactivated in relation to particular transplantation antigens and T-cells. The methods and compositions involve vaccination, organ transplants, autoimmune diseases, pathogenic infections, as well as other health status situations which involve the immune system.

Vertebrates have developed a sophisticated system to protect themselves against a wide variety of hazards, neoplasia by viruses and various microorganisms, such as bacteria and fungii, gene-tic diseases, neoplasia, and the effect of a variety of toxins. The system has evolved based on the ability to recognize self as distinct from non-self.
broad panoply of defense mechanisms are involved, including phagocytosis, lysis, such as complement mediated or perforin mediated, killer T-cells, such as cytotoxic-T-lymphocytes, natural killer cells, antibody dependent cyto-13~

toxic cells, and the like. Various types of cells havedifferent mechani~ms whereby the invader or endogenous diseased cell may be eliminated.
A key to the immune defensive mechanism is the T-cell. T-cells have been found to be restricted in that they respond to an antigen in relation to one or a few specific transplantation antigens associated with their natural host. In vitro, T-cells from one haplo-type host respond to an antigen in relation to a trans-plantation antigen of a different haplotype host. TheT-cell receptor repertoire appears to be narrower than the ~-cell immunoglobulin repertoire. In addition, rather than directly binding to the antigen, the T-cell receptor appears to require concomitant binding to an antigenic epitope and a transplantation antigen.
The transplantation antigens are divided into two classes, Class I and Class II, where the former class o~ antigens is relatively ubiquitous on host cells, while the latter class is relatively limited to lymphocytes, macrophages, and dendritic cells. Differ-ent T-cells appear to be activated in relation to one or the other class of transplantation antigen. In the main, the nature of the activity of a T-cell will vary with the class of the transplantation antigen to which it is complementarY-In effect it appears that a T-cell clone recognizes a specific antigen in conjunction with a specific transplantation antigen allele. Furthermore, variation in the antigen sequence, affects the nature o~ the response when the T-cell, antigen, and antigen presenting cell are brought together in culture.
Depending upon the nature of the change, all three possibilities are encountered, namely, no change, increased stimulation or decreased stimulation.
In view of the above described events, it would be of substantial interest to be able to modify the immune response in vivo and in vitro, where one ~i 13~

could provide stimulation or inactivation o~ a partic-ular immune response. In this manner, the natural response to a particular event could be modulated, either by activating particular lymphocytes to enhance the protective response or by deactivating particular lymphocytes to diminish or prevent an immune response.
By employing the immune system a~ the source o~ protec-tion, a more holistic approach to medicine and disease can be achieved.

Relevant Literature Review articles that demonstrate the extraor-dinary progress and rate at which an understanding o~
T-cell restriction and the immune system has occurred is shown by Berzof~ky, The Year in Immunology (1986) 2:28-38; Schwartz, Ann. Rev. Immunol. (1985) 3:237-261;
Shastri et al., J. Exp. Med. ~1986) 164 :882-896. See also Shastri _ al. ibid (1985) 162:332-345; Unanue and_ _ Allen, Science (1987) 236:551 -557; Guillet et al., 20 Science (1987) 235:865-870 (which discloses a portion of the work described in the subject application).
Re~erence~ of interest concerned with peptide analogs and an understanding of the interaction between Ia gene products, antigenic fragments, and T-cell 25 receptors, may be found in DeLisi and Berzofsky, Proc.
Natl. Acad. Sci. USA (1985) 82:7048-7052; Watts et al., Proc. Natl. Acad. Sci. USA ( 1985) 82: 5480-5484;
Schwartz et al., J. Immunol. ( 1985) 135 :2598-2607;
Babbitt et~al., Nature (1985) 317:359-361; Gammon et 30 _., Nature (1986) 319:413-415; Babbitt et al., Proc,_ _ Natl. Acad. Sci. USA (1986) 83:4509-4513; Watts et al., Nature (1 986) 320 : 179-181; Finnegan et al., J. Exp.
Med. (1986) 164:897-910; Lechler et al., J. Exp. Med.
(1986) 163:678-696; Ashwell and Schwartz, Nature (1986) 35 320:176-179; Townsend et al., Cell (1986) 44:959-968;
Shimonkevitz et al., J. Immunol. (1984) 133:2067-2074;
Berkower et al., J. Immunol. ( 1986) 136 :2498-2503.

13~tl(~4 Ba~tin et al., J. Exp. Med. (1987) 165:1508-1523; and Hirayama et al., Nature (1987) 327:426-430.
Other references of intere~t include Rock and Benacerraf, J. Exp. Med. (1984) 160:1824-1829; Rock and 5 Benacerraf, ibid ( 1983) 157:1618-1634; Rao et al., Cell (1984) 36:889-895; Berkower et al., J. Immunol. (1984) 132:1320-1378; A~hwell et al., ibid (1986) 136:389-395;
Mengle-Gaw and McDevitt, Proc. Natl. Acad._Sci. USA
(1983) 80:762-765; Werdelin, J. Immunol. (1982) 129:
10 1883-1891; Choi et al., Science (1983) 222:283-286;
Lakey et al., Eur. J. Immunol. (1986) 16:721-727; Marx, Science (1987) 235:843-844; Kaiser and Kezdy, Science, (1984) 223 :249-254.
See also U.S. Patent Nos. 4,599,230 and 15 4,469,677. Other patent~ of intere~t include U.S.
Patent Nos. 4,473,555; 4,478,823; and 4,565,696.

Methods and compo3itions are provided compris-ing oligopeptides ha~ing defined contiguous and/or non-contiguous amino acid ~equences for enhanced affinity of immunogens restricted by one or more transplantation antigens. Oligopeptides are prepared which can be used to modulate an immune response when a ly~phocytic sys-25 tem is contacted with one or more antigen~. The compo-sitions employed may have a single or mixture of oligo-peptides, 90 that a single compostion may be used with one or more cellular ~ystems having a plurallty of hap-lotypes. A method for defining the enhanced affinity 30 amino acid ~equence is also provided.

In the drawings:
Figure 1 is a schematic representation of the internal complementarity, consisting of an external 35 receptor and an internal ligand, which is associated with domains of the transplantation antigen.

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Figure 2 is a graphic representation of the inhibition of activity of T-cell hybridoma 7B7.3 by related peptides. Activity was measured in the pre-sence of various concentrations of P15-26, in medium (RPMI 1640~ alone (---) or in the presence of P12-24 at 20 ~M ( o - o ) or at 60 ~m (o-o). A20 B-cell lymphoma (5x104 cells/well) was used as antigen-presenting cells. After 24 hours of culture, 50 ~l supernatant were harvested and assayed for IL-2 concentration by following incorporation of [3H] thymidine into the IL-2 dependent CTL-1 cell line (104 cells~well). The values represent the arithmetic mean of triplicate samples taken from the experiments carried out ab initio.
Figure 3 is a graphic representation of the inhibition of activity of T-cell hybridoma 7B7.3 by chicken ovalbumin (Ova) or Staphylococcal nuclease (Nase). Activity wa~ measured in the presence of various concentration~ of P15-26, in (RPMI 1640) medium alone (---) or in the presence of 30 ~M of Ova (P324-336) (~ -c~) or 60 ~M of Nase (P61-80) (o-o). The values represent the arithmetic means of triplicate values as explained in Figure 1.
Figure 4 is a graphic representation o~ the inhibition of activity of DO-11.10 T-cell hybridomas, when cultured with various concentrations of (P324-336) ovalbumin and A20 presenting cells in medium alone ~ ), in the presence of influenza hemagglutinin P111-120 ( - ) or P12-26 (o-o) or influen2a hemag-glutinin site 2 (P126-138) (---), each at 50 ~M. The values shown represent the arithmetic mean in tripli-cate samples as explained in Figure 1.
Figure 5 shows the amino acid ~equences of peptides restricted by the I-Ed Class II molecule.
Myoglobin (P135-147) i5 recognized by I-Ed re~tricted T-cell clones. Nuclease from Stephyloccoccus aureus (P66-78) i9 recognized by H-2d restricted T-cell - clones. cI protein from lambda repressor (P12-24) 13~6~

I-EBd sequence (69-81) I-EBk sequence (69-81).
Hemagglutinin from influenza virus (P111-120) is recog-nized by I-Ed restricted T-cell hybridomas. I-EBd sequence (28-36). I-EBk sequence (28-35).
Numbers in parenthesis represent the amino acid residue positions in either the immunosen or the Ia molecule. Leu in parenthesis means that leucine and phenylalanine at the first position of peptide 111 to 120 from hemagglutinin are equivalent ~or stimulation of the hemagglutinin-specific I-Ed-restricted T-cell clone. The underlined residues show identity between the antigens and I-EBd protein regions.
Figure 6 shows a comparison of a partial amino acid sequence of ovalbumin and myoglobin with Class II
antigen amino acid sequences. Ovalbumin (P326-339) is recognized by DO-11.10. I-ABb sequence (42-55). Myo-globin (P102-118). Numbers in parenthesis represent the amino acid residue position in the native molecule.
Figure 7 ~how~ the amino acid ~equences of 5 peptides restricted by the I-Ad Clas~ II molecule: myo-globin (P106-118); ovalbumin (P323-336); lambda repres-sor cI (P12-26); nuclease from Staphylococcus aureus (P66-80) and ragweed allergen (P54-65). Number~ in parenthe~i~ represent the amino acid residue positions in the immunogen molecule. A dash repre~ents a dele-tion at that location compared to the peptide above.
Figure 8 shows the amino acid sequences of 4 peptides restricted by the I-Ek Class II molecule: moth cytochrome (P95-103); nuclease from Staphylococcus aureus (P89-97); cI protein from lambda repre~sor tP18--26) and hen egg white lysozyme (P88-96). Numbers in parenthesis represent the amino acid residue positions in the immunogen molecule.

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Novel oligopeptide~ are provided, as well as their u~e in modulating the immune response of a lym-phocyte ~ystem to an epitope, normally a~sociated with a hapten or anti8en. The oligopeptides are deqigned so a~ to have a greater affinity for one or more trans-plantation antigen a~ compared to an immunogen of intere~t, immunologically cros~-reactive with the epi-tope. The oligopeptide ~equence may be a fragment of a naturally occurring polypeptide, a9 a fragment or as a part of the whole polypeptide, or a synthetic polypep-tide prepared by recombinant techniques or chemical synthesi~.
In referring to an epitope, the epitope will be the basic element or smallest unit of recognition by a receptor, particularly immunoglobulin~ and T-cell receptors, where the sequence may be contiguous or non-contiguous, and the amino acids essential to the recep-tor recognition may be contiguous and/or non-contiguous 20 in the sequence.
The polypeptide reaBents employed in the sub-~ect invention will have from one to two region3. A
first region will compri~e the oligopeptide~ having the amino acid ~equence which provides for enhanced binding 25 to target transplantation antigen(s). The amino acid - sequence which binds to the tran~plantation antigen will be referred to as the "agretope. n The agretope i3 a single unit Or recognition which specirically bind~
to one or a limited number of tran~plantation antigens.
30 The agretope i~ defined by a set of agretopio amino acid residues, with the individual amino acid residue~
frequently separated. The amino acids interspersed between the agretopic re~idue~ may provide the epitopic recognition by themselveq or in con~unction with one or 35 more of the agretopic reqidues. An amino acid sequence may have one or more different units of recognition, ~o that in referring to an agretope being present on the , ~

13U~ 4 oligopeptide, it should be understood that there may be a plurality of sets of agretopic amino acid units, where even the same set may vary in its binding affin-ity in a few cases depending upon the adjacent amino acids. The interspersed amino acids which separate the agretope amino acids are selected as to which T-cell receptors bind to the complex of the agretope-con-taining-oligopeptide and the transplantation antigen.
The interspersed amino acids are referred to as the "epitope," (includes épitopes, that is the amino acids may define a plurality of recognition sites) but in view of the nature of the subject invention, will be referred to as the "first epitope." This interspersed sequence defining the epitope may be selected to acti-vate a particular subset(s) of T-cells or to prevent activation of a particular subset(s) of T-cells, depending upon whether one wishes to enhance or dimin-ish the immune response to a particular immunogen. The oligopeptide including the agretope(s) and first epi-tope(s) may be ~oined to an antibody target sequencewhich may be considered as a ~econd epitope, which may or may not bind the same T-cell receptor(s) bound by the first epitope. However, by virtue of the enhanced affinity of the agretope for the target transplantation antigen(s), an enhanced immune response will be obtained to the antibody target sequence. 8y employing various compositions, modified as indicated above, the immune response o~ a lymphocyte system may be modu-lated, by being deactivated or activated toward one or more particular immunogens.
The immune response which is modulated i~ pre-dicated upon a ternary complex, involving a cell having a transplantation antigen, a T-cell receptor restricted by the transplantation antigen, and a polypeptide which specifically binds to the combination of transplanta-tion antigen and T-cell receptor. The transplantation antigens may be divided into two classes, Class I and 13(~ 4 Class II. Class I is relatively ubiquitous, found on most cells of a mammalian host, and has a polymorphic ~-chain and a conserved ~-chain. By contrast, Class II
transplantation antigens are restricted to relatively few sets of cells, primarily lymphocytes macrophages, and dendritic cells, and comprise polymorphic - and ~-chains. In the case of Class I transplantation anti-gens, one is primarily concerned with cellular aber-rations or diseased states, such as viral infection, mycoplasma infection, neoplasia, or the like, or with organ transplants. The T-cells involved will normally cause the destruction of the aberrant cell.
By contrast, Class II transplantation antigens are concerned with activation of the cellular immune system, resulting in the expansion of cells involved with protection of the host against aberrant physio-logical states. The aberrant physiological ~tates may be involved with pathogenic invasions, including vi-ruses, bacteria, fungii, protista, toxins, o~ the like.
In addition, the Class II cells may be involved with various autoimmune diseases, such as rheumatoid arth-ritis, systemic lupus erythematosus, diabetes, etc., as well as those cellular aberrations or diseased states associated with Class I transplantation antigens.
Class I transplantation antigens are also in-volved in rejection of organ transplants. The immune system is able to recognize the foreign nature of the transplantation antigens present on the tran~planted organ and attack the organ. In this situation, one does not wish to protect the host from the foreign cells, but rather diminish the subset of the immune system which is specific for attacking the organ transplant.
In many situations, there will also be inter-est in controlling lymphocyte response in vitro. Thesesituations may involve sp~cifically destroying cells infected with mycoplasma or virus, removing a particu-13(i`1(~64 1 o lar subset of cells from a mixture of neoplastic andnormal cells, expanding a particular subset or subsets of cells in the mixture, providing for conditioned medium for production of various lymphokines, such as 5 IL-2, or the like. The in vitro system may involve whole blood, plasma, serum, cellular fractions, normal or immortalized cells, etc.
The manner in which the oligopeptide is defined is in relation to the transplantation antigen 10 sequence, the polymorphic region, and the immunogens restricted by the transplantation antigen. The se-quence of the transplantation antigen m~y be defined by comparing homology between the immunodominant sequences of antigens restricted by the transplantation antigen, 5 where included in the comparison may or may not be the polymorphic region of the transplantation antigen.
(For a di~cussion of the immunodominant sequence see, for example, Berzofsky, (1986) supra, and references cited therein.) Transplantation antigens have polymorphic regions, where the individual alleles are associated with specific hosts. For the most part, the host will be diploid and heterozygous, ~o that each host will have two haplotypes, meaning that there will be two different copies of a particular transplantation anti-gen type from the ~ame locus, unless the host is homo-zygous at that particular locus.
The homology of the immunodominant sequences may be compared using the FASTP algorithim, although 3 any other algorithim which allows for comparison of homology may be employed. For a description of FASTP, see Lipman and Pearson, Science (1985) 227:1435-1441.
The immunodominant sequences should have about 30~
homology or greater, with up to a total of 20%, u~ually - 35 not more than about 15% based on the number of amino acids in the sequence of deletions or insertions to provide for the desired homology~ That is one counts ~a~

the non-conservative substitutions first, then dele-tions and insertions to provide the best homology.
Desirably, the homology will involve identity, rather than conservative substitutions. Usually, there will 5 be not more than a total of 2 amino acids involved in insertions or deletions, and usually not more than about 1 insertion or deletion, usually deletion.
The following table indicates conservative substitutions, where any amino acid on the same line 10 may be substituted for any other amino acid on the same line.

Amino Acids Aliphatic non-polar G, A, (P) V, L, I
polar neutral C, M, S, T
N, Q
acidic D, E
basic K, L, (H) aromatic F, W, Y, (H) Proline (P) may be considered equivalent, but 3 ~ill normally not be substituted for the other amino acids on the same line. Similarly, Histidine (H) may be substituted for the other amino acids on the same line, but will not normally be considered an equiva-lent. In addition, in some instances, the acidic, ba~ic, and polar amic amino acids (N, Q) may be substi-tuted one for the other in determining homology.

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Normally, at least 2 immunodominant sequences will be involved in the determination of a consensus sequence for an agretope, more usually 3 and not more than about 8 should suf~ice, usually 3 to 6 sequences 5 will suffice. The immunodcminant sequences may be identified in a number of ways. Particularly, the protein restricted by the transplantation antigen may be divided into a number of sequences, which may ~e synthesized and then used in an assay, where cells containing the particular transplantation antigen and T-cells restricted by the transplantation antigen and specific for the specific antigen are combined. (See Experimental section for assay.) By determining the level of secretion of IL-2 (interleukin-2), one can define the sequence which is immunodominant. Usually there will be a ~ingle immunodominant sequence, al-though in some antigens, there may be more than 1.
However, where there i~ more than 1, both the sequences may be used in defining a consensus sequence for optimizing binding affinlty to the particular trans-plantation antiBen.
Usually, there will be not more than 7 amino acids involved with the agretope, more usually not more than 5 amino acids defining the agretope, normally at least 2 amino acid~, more usually at least 3 amino acids. The amino acids may be in tandem or-separated by from 1 to 4 amino acid~, usually 2 to 4 amino acids.
By comparing the homologies, particularly identities, desirably at lea~t 2 identities, more desirably at 3 least 3 identities, and the spacing of the consensus sequence, usually differing by not more than 1 amino acid, one can define a sequence which will have a higher affinity for the transplantation antigen than one or more, usually all of the antigens employed for the determination of the consensus ~equence. Thus, the amino acids having homology will usually be 1-4, 4-8, or 6-12 amino acids apart, usually 2-3 or 4-9 amino 13(~ 4 acids apart, between any 2 amino acids, although tandem homologie~ may be encountered. Once the consensus sequence is defined, one may then scan any antigen restricted by the particular transplantation antigen to define the immunodominant region containing the agre-tope and the first epitope. The information concerning the sequence may then be used for immunomodulation of a lymphocytic sy~tem.
The oligopeptide comprising the agretope and epitope (oligo-a-e) will normally differ from a sequence present in the polymorphic region of the transplantation antigen to which it binds. It is found that there is a sequence in the polymorphic region which serves as an internal ligand to a receptor of the transplantation antiBen. Thu~, this region will have some homology to the immunodominant regions restricted by the transplantation antigen in many cases. However, the internal ligand will normally differ significantly from the consensus sequence, although sharing some moderate homology with the con~ensus sequence. By employing an oligo-a-e having the identical sequence to the internal ligand sequence of the transplantation antigen, one can substantially inhibit any T-cell response. The effect is as if the oligo-a-e is recog-nized as self, and either the ternary complex does notform or, if formed, the T-cell is not activated.
A number of antigens have been described in the literature, where particular sequences have been described as antibody producing and useful in produc-tion of antibodies for protection of hosts against par-ticular pathogens or toxins. These compositions will for most part be less than about 5 kD, usually less than about 3 kD, so that they would normally be con-~idered haptenic. These sequence~ may be used as the antibody target sequence and be joined to an oligo-a-e. The re~ulting molecule will have two domains to form a novel polypeptide where each domain 13(}~ 4 will be fewer than about 70g of the total molecule, usually the oligo-a-e agretope-containing domain being from about 30 to 70% and the antibody target sequence domain being from about lO to 60g.
For the most part, the oligo-a-e agretope-containing domain will vary from about 8 to 30 amino acids, more usually from about 8 to 20 amino acids, while the antibody target sequence domain will vary from about S to 25 amino acids, more usually from about 5 to 20 amino acids.
The agretope-containing domain may be selected in a variety of ways. One may use a universal oliso-a-e, where the first epitope may be also selected in a variety of ways. One may select the first epitope arbitrarily, insuring that it is not identical with the transplantation polymorphic region in a majority of the host~ with which it will be used. Alternatively, it may be selected ~o a~ to have an epitopic site asso-ciated with an innocuous epitope, such as already indicated, e.g. tetanus toxoid, keyhole limpet hemocyanin, bovine gamma-globulin, BCG, human gamma-globulin, or portions of cell endogenous proteins, etc.
Finally, it may have an epitope of interest which shares some relationship with the purpose for which the antibody target sequence is being employed, e.g. the fir~t epitope may be an epitope different from the antibody target sequence but present on the ~ame antigen or a different antigen associated with the antigen of`the antibody target ~equence. For example, in the case of a virus, the f$r~t epitope and antibody target sequence could be present on the same or differ-ent capsid proteins, envelope proteins, etc. Particular antibody target sequences of interest include neutra-lizing antibody target sequences (epitopes) of patho-genic microorganisms, e.g. bacteria and viruses.

13(~1~6~

The oligopeptides employed in this inventionwhich define immunodominant regions and are not joined to heterologous sequences (that is sequences other than the natural sequence to which they are normally joined), will not include cytochrome c, ovalbumin, myo-globin, nuclease from Staphylococcal aureus, lysozyme, repetitive sequences of 1, 2, and 3 amino acids, influenza hemagglutinin, naturally occurring hormones of fewer than about 20 amino acids, such as oxytocin, bradykinin and angiotensin, herpes glycoprotein d, insulin, particularly bovine insulin B-chain, rat myelin basic protein, ragweed allergen Ra3, human ACh receptor-Y, VP1 foot-mouth virus, angiotensin 2, fibrinopeptide B-14, minimum stimulatory polymer, HLA
CW3, myelin basic protein, particularly rat and guinea pig, rabies glycoprotein, flu matrix protein, tubercu-losis 65 kd protein, or other digopeptide prepared prior to the effective filing date of the subject application.
As already indicated, the agretope will normally have greater binding affinity to the trans-plantation antigen than the antigen of interest, so that the oligo-a-e may succes~fully compete with the antigen of interest in forming the ternary complex.
The antigen of interest is the antigen(s) to which the immune system is being modulated. The oligo-a-e sequence may be the total sequence of a molecule, or only a portion of a much larger peptide. The oligo-a-e may be ~oined in a variety of ways to the second epitope.
For _ vivo purposes, there will be a target antigen of interest to which the host may be or is exposed. For example, where a close match has been made between a donor and recipient of an organ trans-plant, there may be only a few antigenic sequences orepitopes present on the organ which could result in a strong immune response. In accordance with the subject ~;~QlCP64 invention, one would block the T-cells which respond to the immunodominant resions of such antigens to diminish the rejection response by the host. In another situa-tion, where an individual would be entering an area in which a parasite is prevalent, the individual would be vaccinated with an oligopeptide having one or more antibody target sequence(s) cross-reactive with the parasitic antigen(s) to provide cellular and humoral protection. These and many other situations will be served by the subject invention. In preparing oligopeptides for the different situations, various strategies will be employed.
One composition would have the antigen of interest mutated at the immunodominant site to enhance the affinity of the antigen of interest for one or more transplantation antigens present in the host. Particu-larly, with B-cells and Class II transplantation anti-gèns, the various epitopes of an immunogen of interest wlll bind to the surface lmmunoglobulin of the B-cells present in the lymphocyte system, whereby the immuno-dominant sequence will then bind to the transplantation - antigen and be pre~ented to the T-cell recognizing the particular immunodominant sequence. Because of the higher binding affinity, an enhanced immune response would be achieved. By employing the mutated immunogen, a plurality of B-cells may be activated in con~unction with T-cells having a T-cell receptor which recognizes the particular immunodominant region.
Alternatively, one may qolely use the mutated immunodominant region, whereby only a few B-cells will b~ activated which bind to the immunodominant region in conjunction with the particular T-cell. Another alter-native is to select one or more epitopic sites present on the antigen to which the immunodominant region may be joined, either directly or through a bridge, usually having fewer than about 50 amino acids, more usually having fewer than about 30 amino acids. In thi~

~3Ul.l;~

manner, a ~ingle fused protein may be obtained, where the immunodominant region is fused to one or more epitopic sites of interest.
Rather than having a fused protein, one may join the immunodominant region by linking the immuno-dominant region to a peptide of interest. The linkage may take a variety of forms, the particular manner of linkage not being critical. Thus, one can provide for a cysteine to be present at a terminus or other site of the olisopeptide, where the antigen of interest may be functionalized with a maleimide group. By combining the cysteine modified immunodominant sequence with the functionalized antigen, a thioethyl linkage may be achieved. If appropriate, a carboxyl group present on the immunodominant sequence may be activated, using carbodiimide, or forming an active ester, e.g. p-nitro-phenyl ester, where any available amino groups present on the oligopeptide are blocked. After reacting the oligopeptide with the antigen of interest to form a peptide bond, the blocking groups may be removed.
Other techniques may also be employed, as described by Fieser and Fieser, Reagents for Organic Synthesis, Vol.
3, Wiley-Interscience, NY, 1972.
The immunodominant region may be modified, not only as to the agretope, but also as to the first epi-tope to provide the oligo-a-e. Thus, one can modify the T-cell receptor which will recognize the immuno-dominant region, so that the immunodominant region may be recognized by more than one T-cell, or by different T-cells from the T-cell which recognize the wild-type immunodominant region. Particularly, where the immuno-dominant region is joined to an antibody target se-quence, one may wish to modify the agretope, so as to either minimize any T-cell response or provide a T-cell response to a substantially immunologically unreactive epitope.

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1~
In this situation, the first epitope may be modified so as to mimic the epitope of a vaccine, such as tetanu~ toxide or other relatively innocuous antigen such as previously described. Alternatively, one may modify the first epitope to a sequence to which there is no T-cell receptor which is activated. This may be considered to be a "hole in the repetoire," where a particular epitope finds no homologous T-cell receptor to bind. One might anticipate that those epitopes which mimic self-epitopes would provide this property, as well as other epitopes which will be discovered to specific haplotypes. Depending upon the desired result, various combinations of agretopes, first epi-topes and antibody target sequences can be employed, to activate or inactivate particular subsets of B-cells and/or T-cells. Thus, one can selectively choose a single subset of T- or B-cells or groups of subsets of either or both T- or B-cells to modulate toward a ~pecific purpose.
For example, if one wished to inactivate a specific subset of B-cells specific for a particular epitope, one would prepare a polypeptide having two regions. One region, the antibody target sequence~
would involve an amino acid sequence which mimics the 25 epitope of interest. The other region would be an oligo-a-e which would not provide for T-cell receptor binding, for example, an identical sequence to a se-~uence of the polymorphic region of the transplantation antigen(s) of the host of interest. In this situation, 30 the resulting polypeptide would be bound by those B-cell~ which recognize the second epitope and be presented to the T-cell receptor under conditions which would block activation. Furthermore, the antibody target sequence would be selected so as to minimize the 35 affinity of the epitope to the transplantation antigen.
In this way, the T-cell will not be activated by the binding of the polypeptide to the transplantation anti-i3t}1~

gen so that those B-cells which recognize the polypep-tide will remain unstimulated by T-cells. If one wished to block any T-cell stimulation of B-cells a T-cell non-activating oligo-a-e could be used.
Where one wishes to stimulate a particular subset of r-cells, one would employ an agretope specif-ic for a Class II transplantation antigen. In this situation, the first epitope would be chosen to be directed to the particular subset of T-cells of inter-est. In this manner, one could build up a T-cell popu-lation directed to a particular antigen, for example, as in the case of a neoplastic condition, in cases of autoimmune diseases, or the like.
For stimulating both B- and T-cells, one could employ an oligo-a-e with an epitope specific for the antigen of interest. In addition, one could employ the oligo-a-e joined to other epitopes of the same or asso-ciated antigens, ~o that a plurality of B-cells would be ~timulated, where the different subsets of B-cells were speci~ic for different epitopic sites. This situ-ation would have application in a number of different situations. In ~ddition, one could prepare an oligo-peptide or protein having a plurality of oligo-a-e's, so that the same molecule would be effective for a plurality of transplantation antigens.
For example, in preparing a vaccine, one could provide for epitopes of different ~trains for a partic-ular pathogen, e.g. virus or bacterium, so that B-cells recognizing all of the different ~trains would be ~tim-ulated at the same time, by virtue of a single mole-cule. One could also provide for epitopes of different molecules, where one wi~hes to vaccinate a host against a plurality of organisms, for example the TORCH serie~
for pregnant women (Toxoplasmosis, Rubella, Cytomegalo-viru~, and Herpes Simplex virus). Therefore, particu-larly in those situations where a battery of antigens are involved, the subject methodology allows for a 13(; 1~

single molecule to provide for immunization against a plurality of epitopes associated with different organisms and for a plurality of transplantation antigens to be bound.
Usually, the number of different antibody target sequences will range from 0 to 20, more usually from about 0 to 10, conveniently from about 1 to 6.
Each of the antibody target sequences will have at least about 5, usually 8 amino acids and not more than about 30 amino acids, more usually not more than about 20 amino acids. Depending upon the nature of the indi-vidual epitopes, the epitopes may be joined head-to-tail or may be separated by bridging groups of from about 1 to 30 amino acids, usually from about 1 to 20 amino acids. The bridging groups may be any convenient sequence, but will usually be subject to selection so as to avoid interference with the proper binding of the epitopic sequence~ and to avoid creating unde~irable immune re~pon~es to epitope~ which could be detrimental to the host.
The compositions of this invention may be a single polypeptide or a mixture of polypeptides, usu-ally a mixture of polypeptide~. Generally, the number of polypeptides will not exceed about 20, more usually not exceed about 12, and preferably not exceed about 8, more preferably not exceed about 6. The mixtures will usually be directed to those tran3plantation antigens which are most frequently found in the population of interest. That is, particular tran~plantation antigens 3 may be more frequent in certain population group~, for example, different species, such as humans, other pri-mates, domestic animal~, lab animals, etc. such as ovine, porcine, equine, avian, etc. By properly se-lecting agretope3 which bind to the most common trans-plantation antigens, either or both Class I or ClassII, there is the opportunity to minimize the total number of different polypeptide~ involved. Further-13~ 4 more, in ~any situations, different transplantationantigens may share particular amino acids in the con-sensus sequence, so that higher or equivalent affinity as to the antigen(s) of interest, may be achieved with a number o~ transplantation antigens with a single olLgo-a-e or consensus sequence.
For the most part, since most hosts will be heterozygous, if one wishes to involve more than one transplantation antigen allele, it will be desirable to have at least two molecules of different consensus sequences unless the two transplantation antigens have substantially similar consensus sequences, and up to 6 consensus sequences in humans.
The subject compositions may be formulated in a variety of ways for administation to a host or for use in vitro. They may be formulated in any convenient physiologically acceptable medium for administation to a host. These media include water, saline, phosphate buffered saline, oil emulsions, etc. In some instances it may be desirable to formulate the subject peptides as tablets, microcapsules, e.g. slow release, lipo-somes, gels, powders, precipitates, e.g. alum, or the like. In some situations, it may be desirable to pro-vide for continuous infusion into the host, by employ-ing convenient delivery systems, such as catheters,constant diffusion membranes, pumps, or the like.
These formulations and techniques are well known in the ; literature. Administration may be by injection, for example, intravascular, peritoneally, subcutaneously, subtopically, intradermal patches, etc.
The amount of the subject compositions will vary widely depending upon the particular purpose, the manner of administration, the nature of the host, the duration of the treatment, the frequency of repetitive treatment, and the like. Thus, for the most part, with each composition, the amount used will be determined empirically. However, some general considerations can 13(~ti~

be made concerned with the administration of oligo-peptides to a host. To that extent, the oligopeptides will generally range from about 0.01 to 10 ~g~kg of host, where concentrations will generally range from about 10 ~g/ml-lmg/ml. Other additives may be included in the formulations, such as stabilizers, antibiotics, excipients, adjuvants, precipitates for adsorption, slow release additives, etc.
The subject polypeptides may be prepared by any convenient means. Usually, either chemical synthe-sis will be employed or recombinant techniques. For the most part, the polypeptides employed in this inven-tion will have fewer than 200 amino acids, more usually fewer than 150 amino acids, preferably fewer than about 100 amino acids, and more preferably fewer than about 75 amino acids, generally ranging from about eight to 60 amino acids. However, as previously indicated, a wild-type or naturally occurring protein may be employed where the immunodominant regionts) have been mutagenized to change the transplantation antigen affinity.
Based on known techniques, one can synthesize genes encoding the subject compositions. Techniques for synthesizing oligodeoxynucleotide ~ingle ~trands are well established and strand~ may be obtalned of 200 bases or more. By appropriately overlapping strands, large synthetic sequences can be prepared. Alterna-tively, where one has mutated the immunodominant sequence of an available antigen, various techniques 3 are available for precisely introducing the mutation, such as _ vitro mutagenesis, restriction and insertion of a synthetic sequence, or the like. The particular manner in which the mutation is carried out is not critical to this invention.
Once the gene has been obtained it may be used in accordance with conventional techniques for expression. A significant number of expression vectors 13(~1Q6~

are available commercially or in the literature, which may be used with advantage in the subject invention.
The host transformed with the vectors may provide for stable extrachromosomal maintenance or integration into the host genome. Convenient expression hosts include E. coli, B. subtilis, B. licheniformis, Saccharomyces, e.g. cerevisiae, Kluyveromyces, e.g. lactis. Of primary interest will be mircoorganisms, particularly bacteria and fungii, e.g. yeast.
If desired, the constructs can be prepared in conjunction with known signal leaders for secretion.
Signal leaders include the ~-factor o~ yeast, ~-amylase, penicillinase, surface membrane proteins, etc.
The signal leader with its processing signal may be joined at the 5' terminus of the gene, so as to provide for a fused precursor, which upon maturation loses the signal leader and processing signal.
The human transplantation antigens of interest as being the ones being generally encountered include for Class II: see Figueroa and Klein, Immunology Today (1986) 7:78-81, and references cited therein; for Class I: see Klein and Figueroa ibid (1986) 7:41-44, and references cited therein. The human Class II antigens are divided into DP, DQ and DR, where the mouse H-LA
(I-A) corresponds to HLA-DQ and the mouse H-LE (I-E) corresponds to DR. The different transplantation antigens may play different roles. As reported by Hirayama et al., supra, and references cited therein, suggest that suppressor T-cells (CD8+) are restricted 3 by DQ while helper T-cells (CD4') are restricted by DR. Using the relationship, either helper or suppressor cells may be stimulated to provide for immunomodulation of the immune response of a host to an epitope of an antigen.
The first epitopes and antibody target sequences of interest will include sur~ace membrane proteins, envelope proteins, capsid proteins, 13~1Q6~

oncogenes, transplantation antigens, toxins, allergens, carbohydrates, polysaccharides, etc. Antigens asso-ciated with viruses will include such viruses as Herpes, Hepatitus, HIV, influenza, FeLV, Rhinoviruses, and other pathogenic viruses. Antigens associated with unicellular pathogens will include such organisms as Plasmodium, Hemophilus, E. coli, Salmonella, Tripanosomes, Pseudomones, and Toxoplasmosis and other pathogenic bacteria and parasites. Antigens associated with oncogenes will include such oncogenes as human fas, mye, abl, ras, etc. Antigens associated with toxins will include such toxins as aflatoxin, diptheria, botulins, etc. A list of pathogens of interest may be foun in U.S. Patent No. 3,996,345.
A large number of epitopes have been defined for a wide variety of organisms of interest. Of par-ticular interest are those epitopes to which neutra-lizing antibodies are directed. ~isclo~ures of such epitope~ are in many of the references cited in the Relevant Literature section.
The various epitopes may be joined to sequences having the consensus sequence for binding to the polymorphic regions of the transplantation anti-gens. Of particular interest are the regions involved with the Class II polymorphic domains ~-2 and ~-3 variable region~. In these domains, amino acids 20 to 40, and 65 to 85, more particularly 2 to 30 and 68 to 78 are of interest. Thus, the compositions which are prepared in accordance with this invention will be 3 varied from these regions by having at least one muta-tion which enhances the affinity of the sequences for the transplantation antigen by including an amino acid associated with the agretope as defined by the consensus sequence.
Illustrative of vaccines is a vaccine involving an antibody target sequence for Plasmodium parasites, particularly falciparum. An antibody target 13~0~g sequence is prepared havins at least three repeatingunits of the tetramer (N-A-N-P)n, where n is at least 3 and usually not more than 12. This oligomer may be covalently bonded or fused to one or more oligo-a-e's 5 having specific affinity for particularly host haplotypes. (For a description of the Plasmodium epitope, see Lavale, et al., Sclence (1985) 228:1436-1440; Miller et al., Science ( 1986) 234: 1349-1356. The oligo-a-e(s) may be selected to have an innocuous first epitope and at least three, preferably at least four members of the consensus sequence as to each haplotype.
The same strategy employed for preparing a malovia vaccine may be extended to other parasites and pathogens.
The sequences of the polymorphic regions of the Class I and Class II antigens which have been sequenced are as follows:

26 ~3Q:lQ~i4 i~ d c~ ~ J ,~
_ z ~ ~ 1-- Z Z - 2 ~ J ~ J -- ~ ~ ~ J ~ ~ J
r: C: Q~ Q Q' ir ~ Q ~IQ~ Q i~ ~ ,- O
~r Q 0~ Q 15: Q 4 ~r r~ n- Q- Q~ Q X cr I
J Q ~-L ~ ;r~ z ~ r,r~ C~ Z V~ n Z Z i'~ O Z C~ Z V~ Z Z Q Q Q~, _ Q = ir~ _ Q Q ~ Q , Q~
QL ' v ,~ ~ ~ ~^ ~ ~ ~ i~ ~ ~ h; ~ j ~ > _ ~ _, _ ~
~ =; h~ Q' Q Q' !'~ Q' Q' Q' ~ I Q' Q' Q' C: ~ Q- Q~ Q~ Q~ Q~ c~ Q ~ 3 3 3 3 3 3 - 3 ~ ~ ~ 3 '3: ~ 3 3 3 3 3 3 3~1 ro O ._~~ 3 -- rJl ~ U~ 3 Vl Vl U'1 h. ' 1-- ~-- 1-- ~-- ~ 1-- ~ ~ )-- U) ;~' ~ ~ ~` ~ ~ ~ ~ ~ ~ Y Y _~
O c~ cr cJ c~r C~ O O O O c!r O O O C!r V or O ~wr c~r O ~ w r~
Z C~ Z Z C21 Z i~ I ~ Z ~ ;~1 ~ 0 I O O O O C ~ ~ r ~ o r~ ~ r ~ i~ J t.:~ O C~ J J O ~ ~ 1 Y ~ .Y :y y `~ Y ~t Y Y r-- 1-- Y Y--Y y y y y y ~ y : J ~ ~ ~ _J _ y Q ~ y ~ ~ Q ~ ~ ,_ y z y ,j~ _ y y Q, cS eS c~
O O O O O ~ O O O O J i~J U: Q- O 3 O C J O O .~ Q ~ h~ `~ Q Q~ ~ Q' Q : ~ Q ;~1~ Q Q- C~
E ,., o z z ". ~ --hJ ~J I~ J 1~ 4J IAJ W Z Z hJ Z hJ O O --~
Q Q- h-- Q h~0 O ~ Q. Q~ C~ C~ O ~ w i~ w w ~
~o 33 ~ 3 ~ 3 3 ~ 3 3: 3 3 3 ~ ~ 3 3 ~ 3 ~ ~ 0 ~ ~:1 ~ W w w ~ -hJ W w ~JL~J ~ ~1 1.~ hJ 1~ W ~ Y w h~ w w C5 O ~ O CJ V O w O i' S O cC ~ S q O CL ~ h. a ~ Q ~ ~ t~ ~ O_ h. ~L n h. ~. Q l:L ~ h.
J l.J h~ o ~C~ ~ O ~ O O ~ ) LLI Q 3 2 1 ~ 3 3 ~ 3 3 3 2 3 3 1 3 3 3 3 ~ _ 3i ul C~ ~ L" I,~J L~ 1.~ hl L~ J J w h~ h~ h~ Y hJ L.J ~ ~ Y ~' Y y y Y Y Y ~C Y Y ~' Y -- -Y Y y Y y O O o O O V O O OO O O O O O o Q' Q' O O O/ I Q I -r ~ h-' Q' C~ -h- ~ n I ~ ~ Q: n~ C: ~i ~hJ W IJ 1.~ ~ h~ h~ L.J hJ l.. J hJ h~ I.J h~ IJ hJ i~.l ~ ~ rw Q' Y ~1: Y Y ~: Q. Q. C~: ~ ~ Q~ C: ~C Cl: `~ y o O y O O ~y 9~ _ 2 2 3c ~ 2 ~ ~ 3 3 3 3 3 3 2 3 3 3 ~
CL Q~ C~ C ~ ~ ~ h. Q Q CL Q Q Q _~ O O O ~ o O O O q EQ~ Q Ct~ Q. ~ Q. a~ Q' Q' D~ ~ h~
V 11 ~ ~ ~ ~ ~ o ~ ~L J J D Q h. I:L ~ Cl_ (~ ~L O ~ ~ s l-- ~: , ~ S ~ s _ _ ~_ s E s ~ E ~ I~ s EQ~ w L~ J Y X /~J hJ l~ l h~ L.J LJ h~ w hJ, a a o o a a a a C~ a CJ C~ a C~ ~ a ~ a a ~ C~
~ v~~ h ~ ~- ~ 2 ~ ~ w O hJ O _ cr Cl: CC c~ ~S C et Ct ~ cr C C C C c~ C S ~ C c~
~ Q~ Q~ V Q~Ir Q h-- ~ ~ Q Q' ~ Q Q' Q - a: C ~1: ~ Ct e c ~ c c ~ c ct ~
V aJ rC Z Z Z Z Z Z z Z ~ _ ~ V ~ ~n l/~ Ul ~ V~ V~ U7 v7 3c ~ 3i ;c 3i _ 3i 3i ;C 3 3 3 3 ~ ~ 3ii 3 3C 31 ~ v ~ CJ ~~ a ~ l~1 V W L~ e C ~ CC ~: ~ c cr ~ r ~ ~ ~ ~ ~ ~ j n in Vl ~r ~ r U~ ~ _n in ~j Ql Q~l ee~e~scel:~l ~eeeee3~ YYYYYYY YYYYYQ~Q QQ~n Q,~
~ L~ V U~O 4 Q C~ ,' J J ~ J I J J J J ~ ~ _ _ J I _ _ _ _ J _ J
O ~ v; _ O O ~w~ 4 Cl 4 0 4 o O O ~ o Q C~ w o W o O _ D V ~ ~o 4 0 4 4 0 0 4 l Z Q ~ O ~ ~ L~J .,~1 hJ w hJ hl hJ LJ l~J J w L.l w L.J h~
D ~ Q a.~ L-- 4 4 4 1~ ~ ~ ~ 4~' L~ 4 L~ L. ~i Z Z Z z Z z z z z z z z~ 2_z Z z Z z -- Q vl Q ~ 2~ 1~ h-- Q 0: t~ Q: ~ h-- C:~ I ~ J J ~ ~ J _ ~ _ ~ _~
Z~0 ~ 10 ~i4 1-- L~ 4 4 4 4 ~ 4~ 1., h. ~ h. 11~ 4 _ -- _ _ _ -- _ _ ~ _ _ ~ ~ _ ~ _ _ _ _ _--~ o ~ DL~` a a w ~ "~`~ o a a ~ o o o a o o o o o C5 ~ ~ ~ _ ~~c E c 'r~ r ~ ~ y ~ y r ~ r ~ ~ ~ r r r - ~ ~ ~ - ~ ~ a ~ o c~ ~ o O o c~ O O o o 2 o o O o ~ d ,~o o O O O _0 0O ~,~ O ~ O O ,,~ c~ O O 0 ~1 ~w Q- r_ ~W r~ W ~w Q~ r. r~ r~
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;~ ~ ~ ~ ~, , - . ~ 4 r , 4 = = ~ r -- ~ T T ~ rJ~
~ h~e~r~or~nh~ ~ eeeeee~ er~nrJ~--r~ ~aoooo~-ooOoooOoooooOo~r;~
~j l_ r r_ r~ _ r~ -- r ~ _ --~ ~ ~ ~ _ ~ ~ A_ - 4 r r L~ 1, 1~ ~
~ rY r~ r~ '3 t~ _ 3 Ir r~ r~ I ~ n- Q: ~I r~ rJ r" rJ r,~ rJ ro r~ r~ r~ e e o r~ r~ r~ o O r~ ~ t.a ~7 9 ~ i~ 0 ~ r~ r,~ r~ ri r~ ,~ r", rl r~ r rl r~ r r~ ~ r r~ r~ ~ r~ rr r~ r~ ra~ r,~ ~ r,~ r,~ r~ 1~ r~
~ ~ 4~ r~l r~ 1~1 r~ l,J : J ~ J ~ ~ I ~ _ r~ _ J ~
~ r, ~ r,~ r.~ ~ r~ ~ r, ~ r ~ rJ ~:~ rl ` J ~ J J J J J ~ r~ r ~ J r~ 1~ b. r r1~ I r~ J r~-:
!J ~ ~ e r r ~ r r ~ r ~ r ~~ o o o o o o o r o r r ~ n ~
E~ rn ,~ ,~ rn rn rn rn ~n rn ~n rn ~n rn ~n rn ~n ~n rn rni '-~'rn ~ ~, ~ ' o .~ ~ ~ ~ ~ ~ ~ r r ~ r r r ~ U r 4 4 ~ 4 1~ 4 r~ 41~ ~ 4 4 4 4 4 1_ 4 ~ r . ~ s _ s ~ = s S s s s . s . 2 2 2 ~ ~- r2 ~ - ~ ~ 2 1 ~ Q'0:~ rY~l3r~ ~I Q~ Q'4Q'r.~ QQQ'Q'Q''" OOOOOOOOOOOi_OOO aoa-pr;~
~J ~ J~J ~^, E~'~J E J ~'J J _ :~ S s-- E ~ S ~ _ ~ J, J ~
n ~n r~ rJ~ `~n rn ~n ~n r~~n r~J7` rn ~n r~ ~n r~ ~n r~ r~ ~n ~n r~ ~ .~
~ T T T I X S T T T T T ~ T I T T 1 I I I I T ~C I I I I T T T ~ T I T Zf V~ CJ^ ~Q Q Q--rl' Q ~n Q o v~ ~n ~n rn rn rn rn rn rn rn rn ~n rn c 'rn rn rn rn ~n U~ ~ ~n rn ~n ~ ~ n U~ ~n ~n ~n rn rn r r u~ aJ C ~ r~ ~ ~ r~ r,~ r~.~ r., ~ 1~ Q~ r~ ~ r.,~ . . ~ ~ r~ ~ r~ ~ 1 r,_7 t~ ~ rJ rJ ~J ~ ~J r~ r~ r~
_ O
r~lJ ~ ~ r~
r~ C

~ ~ C ~ a ~ ~ ~ ~ ~ ~ r~r Cr ~ ~ ~ ~ r U ~ r _ ~ ~ , ~
Yi~ ~J I I I I I I I I I I I I I I I I I I T T r~ r~- ~ I I 1 i T I I I I I I I I I I I :i: ~ I r~- r 27 13(J~ti4 , .. .` ; ... . . . .. ; ~

C C ~ ;!_ i` OL ~ C C ~ C C G C ~ _ t' C - ~ C " '~
a a a a a a q a .s a ct a a ~ ~a ~ ~ --~--J----J -I--J J--_ _ _ _ _ _ _. _ _ _ _ ~,; ~ ~ ~, , r. ~, ~ 3 3 3 3 3: ~ -C 3 -C 3 ~ r ~ 3 3 3 3 3 3 3 3 ~r ~ ~
v~ v~ v~ v V) u- vl ~- ~7 I I I = I = Y- = ~ I I I -r I = _ = I = = = r = = = =
~ C Z X r 1.
U~ Vl Vl V) V/ Jl (~ V ~7 Vl VJ V- Vl V~ V7 V~ V- Vl V Vl V~ V~ I ;~ ;" ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~' ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~' r C. X C Q ~ C ~ C or r- tr, tr 1~: C ~J Y X X ~ Y Y Y ~C Y Y Y ~ Y ~C
YXYY~:XXYXXXXY"1_~YXYXXwO~
onc~ooGc~aooooot~
J ~ -' J -- -- J J -- J ~ ;, J J ~_ `~
_ _ _ , _ _ ~ _ , J~
Z ~ C:~ t~ O ^ . 0__0 0 C. t~ ~ C ~ ~ C '` --_ J _ _ _ J J J J J , J _ _ ~ , J, J _ , J O ~ ) C Q O C~ O 1.~ 0 0 ~ O ~ L.. l ~ W ~.~1 1.1 ,~ C C 1~ ;C ~ C ~ C ~ C ~ c~ ~ C C i~ c~ a I I I T I I _ I T I I T I I ~C ~ ct ~; 5 C~ ~:t C~ V~ V~ IJ- V~ J V~ _~l U- V! ~ ~r U- V~ V~
/~ Y Y Y X X Y X Y X X X X X X X ~ X Y ~' i Y X ~' ^ D. O_ tL r ~L C G O_ O_ G CL C ~ tL C C C G G ~ _ ^ G C _ J ~ J ~ ~:~ O ~ t,~ O Q t_. O ~ J
a ~ s a ~s a a ~ a ~ ~, cc a a r ct a ~: cc ~S -- ~ ~ ~ ~ c 4 51. t~
~ _ _ J _I J _ _ J _~ _ _ _ J _ _ _ _l _ _ _l _ _ ~ _ tC 'Z ~ ~ ct Ct ~r _'--t ~ = T T T
_~ J ~~ _ J ~ J J J ~
w ~ _~ V ~ C~ ~ w w w (~ ~ T I _ :~: 2 .~ ~ Y X Y Y ~c X Y X --OOOoOOC10 OOC~tJC~0 OOO~OOOVt30 ~ '~ ~ ~ ~ ~' '~ ~ ~ 1~ ~~ '-- ~-- ~ ~ -- ~' ~-- -- -- -- -- -- -- ~ -C ~ ¢.~ ~5~0_ 5~ ~ U~ V~ V~ V~ V~ Vl V~ VJ u7 V~ V) V~ V~ VJ ,,~
Q--f~.w ~QQ,w w ~ ~ w w W l~;W ~ I I ~ _, ~ = _ ~ T T ~ ~ -- T I ~ T -- = = _ = T _ _ w~L~ ~O~OCC~OOOOOOOOOO--OOOOOOC~
v~ _ V) ~ vi ~tt v~ IJ') vl v- v~ vl V~ q ~
a _ ~- ~5 CS CC ~C ~C ~C ~C C L~ ^.~ ^r .. = ~ _-J J J J ~ J J _ I_ J _ ~ Ct ~ L ~_ ~L ~L C t~
O O O O O O O (~ I 9~; O t~:.X ~.~ ~a;~O I ~ ~ ~ ~ J ~ J ~ ~ ~ J ~ ~ ~ J ~ ~ ~~ ~ --~ ~ ~ ~ ~ ~ --~,o,~ ~ ~ ~ ~ C :L ~ .. ~ n. ~ ~ O^ ~ kJ.. hl.bl.~.~L~ ~,tL 1.. w .. , w .. w hJ W ~ W W W W ~_ ~ W ~.J W _ _. ~. _ '_ ~ _ ~ J _ _ J _ _ J ~ _ J J J J ~ J ~ ~ J , __~n ~n vn vn n vn vn v n vl v v v, n 'f - ~
D ~ 3 ~ ~: 3 ~ '~ 3 '~ 3 3 3 ~ :t I 3 3 ~ 3 3: 3 C ~r 3s /'n~ ~ O ~_ U (:~ U (~ U U 1~ J U L:~ C ~:1 O U
o '7 -- ------------~~~~ --~5,_:~: cr~ _ _~s___,--__ --,----_------____ E ~ X W W W I~ W ~ ~ W I.J W ~ W ~
Y ~c Y x x x Y Y x Y x Y Y Y V~ Y Y Y y Y ~ Y Y ~n vn vn vn vn vn vn vn - ` vn m vn vn vn vn c ._ c c .^L t~ c ~
V YYxxYxYx _ _--yYxxYYYYx~ ,~YxxxxYxxY ~ S~ qYxYxx,q_w c~ o o o c~ o o o ~ o ~ ~,~ ~ ~gx o o ~ U U O ~ U L1 C~ C~
SoJ____J~~~~~~~~ _ ~ ___~ ZZZZZZ~Z~Z;~ !~Z~Z
O O O O O O O O O O O O O O O O O O O ,la;P ~ a O 2~ CC 3~ C C G :1: G _ G ~ ~
:~ :. :. _. ~ ~ :~ ~ ~ ~ ~ ~ ~ ~ ~ ~, ~ ~ ~ ~ ~ ~ ~ ~ r J ~ ~ ~ ~ ~ J J ~ ~ J J ~ ~ J ~ -- ~ -- ~ _ _ _ _ J
K~ ~ T ~ V~ 3 3 3 3 ~ ~' 3 3 ~ 3 3: 3 ~ 3 3 ~ 3 3 3: 3 3 3 3: 3 3 ~ w w 1,1 w l,~ J w w I~J w w L~J w w w 1,~ V L.i ~ Z Z Z ;C ~ ~ Z Z ~ ~ ;~ ~ ~ ~ ~ a~ ;~ ~ ~ ~ Z ;C ~ Z ~1 OO O O O Cl O O O O O O O O O O O w O O O O ~,O I~J, O ~_~ ~ _,~ ~ ~ ~ ~ ~ ~ ~ ~^ ~ ~ ~ ~ r ,_,_ J
C~ O O O O O w O O O O O w O O O C C~ O O O~ w O O ~L C C C C C C lL C C G C ~ ~ C C C G C C ~ G C C C ~
o ~ .J~ ~ ~ G C O_ G tL ~ C G 11 1 1 0_ '` ~L C C '` ~ 1 ~ 1~ 1~ I:L C CL t~.
;~ ~L.~L.~WWWWWWL.J~OOOOOOOL~
r~,~ ~in~ -e~ ~3jZ2,,Z,~Z,ZZz~,.zcæ.~æ~YYY~xxYY;C~
O O O C O O O O C~ O O O O C~ O O O /:~ O Cl O O ~ O O O
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y __u7 v~ ~ m v~ v7_vn v~ ~n vn vn ~ n o ~ ~ ~ u ~
Q O ~ O ~ C:l ~ X ~ ~ _ J J J J J _ J J J _ _ _ J J _ J _ J J J _~ J _ ~~L^. ~ ~ u~ h~ -- z ~ ~--~ ~ z z ~ z ~ ~ ~ z ;~ ~ z ~ c ~ ~ :~ z 2uaoc7oooo3oaoooJJJJJJJooo ,~A ^----c ~_~__~__~La~____~
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D ~: L y ~5~ ^ `~ Y `~ Y X Y X Y ~ Y Y Y Y Y Y _y ~ y y y T ~ r Ul = = ~ ~ = I ~ T 1 2 = 3: T j I~ ~ _ _ ~ J J ~ J ~
~ Q & r~ ~ C~ ~ O CJ C C~ ~ Q ~ ~ w ~ ~ ~ I w Q ~ ~:a ~ ~ ~ ~, . ~, ~, I ~ ~ ;. ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . ~ ~ ;a c ~ ~ I Y Y~ C ~ > ~ ~ -~ 2. > ~
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13~ 6~

~9 A domain of particular interest i~ the Class II third variable region, more particularly the amino acids 68 to 80. In this region, the polymorphic amino acids are L-E-D*-A*-R-A-S*-V-D-T-Y*-C-R, where the polymorphic sites have an *, so that the amino acid at that site may vary conservatively or non-conservatively.
The following examples are offered by way of illustration and not be way of limitation.
First, observations will be given, followed by the experimental protocols for obtaining the observations.

EXPERIMENTAL
T-cell hybridomas were obtained as described in Guillet et al., Science (1985) 235:865-870; and Lechler et al., J. Exp. Med. (1986) 163:678-696 (particularly reference~ cited therein). The procedure 20 for determining inhibition or activation i~ described in Guillet et al., supra.

Inhibition of antigen-~pecific T-cell activation by non-stimulatory peptide analogs Inhibition of antigen-specific T-cell activation by non-stimulatory peptide analogs was assessed, using the T-cell hybridoma 7B7.3 and the T-cell hybridoma 8I.
7B7.3 was derived from a Balb/c mouse 3 immunized with the immunodominant peptide derived from bacteriophage lambda repressor, cI. It is stimulated with the peptide P15-26, which contains amino acid residues 15-26 of cI, in the context of I-Ad. The T-cell hybridoma 8I is derived from the A/J strain and 35 recognizes the same peptide (P15-26), but in the context of I-Ek.

13~ 64 3o Neither 7B7.~ nor 8I can be stimulated by homolo30us peptide P12-24 (which contains residues 12-24 of cI). Other T-cells, however, derived from Balb/c mice, can recognize P12-24 in the context of I-Ad.

5 This suggests that P12-24 can bind to the I-Ad molecule but presumably cannot stimulate 7B7.3 because it lacks a specific T-cell interaction residue (an epitope). It was found that P15-26 dependent 7B7.3 activation would be inhibited when the peptida P12-24 was also included in the culture. See Figure 2.
In the case of 7B7. 3, P12-24 inhibited activation by P15-26 in a dose-dependent manner; the potency of the inhibition depended on the concentration of the inhibitor. P12-24 changed the apparent affinity 15 of 7B7.3 for P15-26 (See Example II). The inhibitory effect was reversed by increasing the concentration of P15-26 in culture.
In contrast, the same peptide (P12-24) had no statistically significant effect on 8I activation, as demonstrated by IL-2 response of the hybridoma.
Inhibition of repressor-specific T-cell activation by other I-Ad-restricted peptides The competitive ability of peptides derived from two other antigen systems to inhibit P15-26 25 dependent activation of 7B7. 3 was determined. For this purpose, peptides derived from Staphylococcus nuclease (Nase), residues 61-80 (P61-80) and from ovalbumin (Ova), residues 324-336 (P324-336) were used to inhibit cI P15-26 dependent activation of 7B7.3. Each is I-Ad-30 restricted and immunodominant for it~ respective anti-- gen. This work is described in detail in Example III.
Results showed that 7B7.3 activation is inhi-bited, in a dose-dependent manner, by these peptides.
Specificity is ~een in that the same peptides are with-35 out significant effect on the I-Ek restricted P12-26 ~pecific T-cell, 8I.

l3ula~4 Inhibition of ovalbumin-specific T-cell activation by other I-Ad-restricted peptides The T-cell hybridoma D0-11.10, is ovalbumin specific and I-Ad-restricted. It responds to the peptide P323-339 derived from ovalbumin. It responds less to the truncated analog P324-336. P324-336 peptide was used as stimulator. The lambda repressor cI peptide P12-26 and a peptide influenza hemagglutinin site 2 (I-Ad-restricted) were used as potential inhibitors. Neither of these can stimulate D0-11.10 on its own. As shown in Figure 4, these non-stimulatory peptides act as inhibitors for ovalbumin-specific T-cell activation. The I-Ed restricted influenza hemagglutinin-derived peptide P111-120 was used as a control and had no effect on ovalbumin-specific T-cell activation.
Results of this work demonstrated competitive inhibition of T-cell activation by unrelated peptides restricted by the same Class II molecule (I-Ad).
Binding of repressor peptide P12-26 to Class II molecules in vitro The lambda repressor (cI) P12-26 peptide was labelled with 125I and tested for its ability to bind to various Class II molecules. It was observed that the peptide could bind to Class II molecules isolated from the d and k haplotypes, as shown in Table 1 (Example V). The I-Ad and I-Ek molecules are restricting elements for the peptide, but the I-Ak and I-Ed molecùles are not.
3 The P12-26 peptide binds most tightly to the I-Ed molecule, despite the fact that this molecule was never observed to act as a restricting element for P12-26-specific T-cells derived from Balb/c mice.
To determine whether the binding of P12-26 is specific for the I-Ed molecule, its ability to compete for binding with a myoglobin-derived peptide, known to be restricted by (and to bind to) I-Ed was examined.

13('~ ;4 The binding is specific as shown in Table 2 (Example V).
P12-26 also competes with other immunodominant peptides for bindins to their respective Class II mole-cules. Competition, however, is not observed for thelysozyme-derived peptide restricted by I-Ak, a Class II
molecule which is not bound by P12-26 (see Table 1 of Example V). In fact, P12-26 binds best to the I-Ed molecule, as shown by its relative binding ability as 10 well as its relative competitive ability.
In view of this unexpected result, the presence of T-cells restricted by I-Ed in the Balb/c mouse immunized with the NH2-terminal domain of cI was re-examined.
There appears to be an absence of T-cells in cI immune mice able to recognize P12-26 in the context of the I-Ed molecule; there is apparently a hole in the repertoire with respect to P12-26 and the I-Ed molecule.

Com etition at the level of the I-a molecule p The immunodominant peptides derived from both Staphylococcal nuclease and ovalbumin are able to inhibit T-cell activation of a cI-specific T-cell hybridoma. The degree of inhibition observed is dependent on the ratio of stimulator to competitor, and, therefore, the inhibition with regard to the activator appears to be competitive in nature.
Similarly I-Ad-restricted peptides are shown to inhibit 3 the activation o~ an ovalbumin-specific T-cell hybridoma.
In order to observe inhibition of the P12-26-specific T-cells 7B7.3, it was necessary to use the weakly stimulatory truncated analog, P15-26, as an activator. Similarly, for the Ovalbumin-specific T-cell, DO-11.10, a poorly stimulating peptide analog was used as activator.

Antigen peptides as analogs of self In light of the fact that the cI peptide binds to both I-Ad and I-Ed and the Nase peptide can appar-ently be restricted by both Class II molecules andpresumably also binds to both, the amino acid sequences of the two peptides were compared to those of other peptides restricted by either I-Ad or I-Ed.
As shown in Figure 5, the I-Ed-restricted, immunodominant peptide derived from sperm whale myoglobin bears a homology to both the cI peptide and the Nase peptide at residues 1, 2, 5, 6, 9, and 13 (see Figure 5).
Given that the cI peptide is homologous to other I-Ed-restricted peptides, it was unclear why there were apparently no T-cells able to recognize it in the context of the I-Ed molecule. To explain this, a comparison was made of the sequence~ of the I-Ed-restricted peptides and the I-Ed molecule itself.
Result~ showed that in the third hypervariable region of the EB chain (residues 69-81), residues 1, 2, 5, and 13 as aligned were homologous to the peptides restricted by the I-Ed molecule (see Figure 5). The cI
peptide is identical to the I-EBd molecule at residues 1, 2, 3, 4, 5, and 11 and homologous at 13. Further-more, comparison of the sequences of the I-EBd molecule with that of the I-EBk molecule in this region, shows that residues 3, 4, 7, and 11 are the only polymorphic ones. The cI peptide i~ identical to the I-Ed molecule at three of these polymorphic residues; the other I-Ed-restricted peptides are not. This identity appears to account for the presumed "hole in the repertoire."
As shown in Figure 5, the I-Ed-restricted immunodominant peptide derived from influenza hemagglutinin, bears little homology to the other peptides described above (except at residues 1 and 2). This peptide however, does bear a striking 13~ 4 ho~ology to the I-EBd ~olecule itself. In this case, the residues of the I-E8d molecule used for comparison are taken from the second hypervariable region of the EB chain. Mengle-Gaw, L. and H. McDevitt, Proceedings of the National Academy of Sciences, USA, (1983) _ :
7621-7625. As aligned, residues 1, 2, 3, 4, 6, and 10 are identical and residues 7 and 9 are homologous. The influenza peptide bears an insertion with respect to the I-EBd molecule at residue 5 and this residue has been shown to be an epitope for T-cell recognition.
Hackett, C et al , Journal of Immunology, (1985) 135:1391-1394.

Summary Thus, the work described above and in the Examples which follow show~ that antigen-specific T-cell activation (i.e., activation by a specific peptide bound by a particular Class II molecule) can be inhibited by a homologou~ peptide if it is presented in the same Class II context as that in which the stimulatory peptide i9 presented. This is true even though the homologous peptide i9 unable to activate the T-cells. However, the same homologous peptide does not inhibit activation of T-cells which recognize the specific peptide bound by a different Class II
molecule.
This supports, in the first case, competition for binding to the Class II antigen and interference by the homologous peptide in T-cell activation is the 3 result of its binding to the Class II molecule (thus preventing the specific peptide from doing so). In the second case, it supports that because there is no competition between the homologous peptide and the ~pecific peptide for binding to a ~econd Class II
molecule, the homologous peptide does not interfere with T-cell activation.

~3~ 64 It has also been demonstrated that only one peptide binding site is present on each Class II mole-cule and that unrelated peptides restricted by a given Class II molecule act as competitive inhibitors of each other's ability to stimulate specific T-cells through competition in binding to the Class II molecule.
Comparison of the amino acid sequences of the following peptides and the I-Ed Class II molecule itself supports the conclusion that selection of an immunodominant peptide within an antigen for T-cell recognition rests on its ability to bind to a transplantation antigen:

1. cI peptide, the immunodominant peptide derived from lambda repressor which is I-Ad and I-Dk restricted but binds most tightly to the I-Ed molecule.

2. Nase peptide, the immunodominant peptide derived from Staphylococcus nuclease, which is I-Ad and I-Ed restricted and presumably binds to both;

3. The immunodominant peptide derived from sperm whale myoglobin, which is I-Ed-restricted; and 4. The immunodominant peptide derived from sperm whale hemagglutinin, which is I-Ed_ 3 restricted.

Comparison of peptides 1-3, summarized in the top ~egment of Figure 5, ~howed homology among three at 6 of the 13 residues (i.e., at 1, 2, 5, 6, 9, and 13).
Such homolozy explains their common restriction and/or ability to bind to the same Class II molecule (I-Ed)-13~1064 Comparison of the sequences of these three I-Ed-restricted peptides with that of the I-Ed molecule itself showed homology between four residues in the peptides and in the third hypervariable region of the IB chain. For example, in the whale myoglobin peptide, the cI peptide and the EB chain, leucine (a non-polar amino acid) occurs at residue 1, glutamic acid at residue 2 and arginine (a basic amino acid) at residue 5; in N~se, the residue at each of these locations was the same or homologous: valine (nonpolar) at residue 1, glutamic acid (same amino acid) at residue 2 and lysine (basic) at residue 5. All three immunodominant peptides were shown to have lysine (a basic amino acid) at residue 13 and the Class II molecule to have arginine (also a basic amlno acid) at that location.
Comparison of the amino acid sequences of these three peptides and the amino acid sequence of the influenza peptide, which i8 also I-Ed-restricted showed little homology. Comparison of the influenza peptide sequence with that of the second hypervariable region of the I-Ed molecule showed striking homology between the two, however; six residues are identical and two homologous.
The results support the conclusion that the basis of selection of an immunodominant peptide within an antigen for T-cell recognition rests upon its ability to bind to a transplantation antigen (e.g., a Class I or a Class II molecule) and that the chemical requirements for such binding rest upon a homology with a segment of the transplantation antigen itself.
These observations support the presence of an internal complementary (ligand-receptor) associated with particular domains (segregated on each chain) of the Class II molecule and that the internal "ligand"
for binding is, in part, encoded by the polymorphic region. The same would be expected to be true for the internal "receptor". The immunodominant peptides would 13(~1~64 then be bound to the Class II molecule, displacing the internal "ligand" and assuming equivalent geometry.
Foreign "ligands" (immunodominant peptides) would then be seen by T-cells as analogs of the internal, self "ligand.~
In the case of the lambda repressor peptide (cI) described above, it appears that the foreign ligand is indistinguishable (at the polymorphic sites) from the self Class II molecule and the "hole in the repertoire" is brought about by self tolerance. This idea of recognition as self, resulting in what appears to be a "hole in the repertoire," is not new. Burnet, F. M., Cambridge University Press (1959); Herne, N., European Journal of Immunology, (t971) 1:1-5. However, the data presented herein provides the first molecular evidence demonstrating that the notion has a physical basis.
It is reasonable to expect that alloreactivity is a result of T-cell recognition of the internal "ligand" o~ a foreign Class II molecule. In the limit, if the internal "ligand" were to be composed of a number of polymorphic residues and the histotopic residues (sites of T-cell binding to the Class II
molecule) were not, then a given T-cell could not distinguish between a foreign "ligand" bound to a self Class II molecule and a foreign Class II molecule bound with its own internal "ligand." If there were places of identity between the two ligands, alloreactivity would result. Given the results above (i.e., that 3 there is homology between all "ligands"), foreign internal "ligands" could be readily considered as analogs of self and therefore chemically equivalent to self plus X. Each polymorphic residue of the foreign "ligand" would represent, in principle, a different foreign antigen in the context of self. Hence a large percentage of T-cells would be able to respond to a single allo Cla~s II molecule. Ashwell, J. et al., Journal of Immunology, (1986) 136:389-395.

13G~ 4 The sequence for the I-Ad-restricted, immunodominant peptide of ovalbumin (described herein) is shown in Figure 6. It is aligned with a polymorphic resion of (residues 42-55) of the AB chain. Choi, E.
et al., Science, (1983) 221:283. It bears an identity at residues 12, 17, 19, and 20 as aligned. The single residue that is polymorphic in this region is residue 12, which is histidine in the I-ABb molecule (and ovalbumin) and tyrosine in the I-ABd molecule (the restricting element), as well as in the I-ABk molecule.
The ovalbumin-specific T-cell hybridoma, DO-11.10 which recognizes ovalbumin in the context of I-Ad, shows an alloreaction with I-ABb but not with I-ABk. Further-more, this region was shown by Germain and colleagues to control the alloreaction of DO-11.10. This histidine residue was shown by McConnell and colleagues to be essential for recognition of another ovalbumin-specific T-cell. Lechler, R. et al., Journal of Experimental Mediclne, (1986) 163:678-696; Watts, T. et al., Proceedings of the National Academy of Sciences, USA, (1985) 82:5480.
This indicates that the DO-11.10 cell cannot distinguish self plu9 X (where X is the ovalbumin peptide) and allo (where allo is I-ABb).
The ovalbumin peptide described here appears to contain two regions of "permis~ive" residues (allowing it to bind to Class II molecules). One region involves positions 17 to 20 (T-cells like DO-11.10 require this region). The peptide deleted for 3 these residues binds to I-A, as shown by the fact that it can stimulate T-cells like Do-54.8. Shimonkevitz, R. et al., Journal of Immunology (1984) 133:2167;
Watts, T., et al., Proceedings of the National Academy of Sciences, USA, (1985) 82:5480. Recent evidence indicates that residues in the region preceding position 12 also form a "permissive" framework for I-Ad binding. Cease, K. B. et al., Journal of Experimental Medicine, (1986) 164:1779. T-cells recognizing a .
peptide derived from sperm whale myoglobin in the context of I-Ad also show an alloreaction with I-Ab.
Berkower, I. et al., Journal of Immunology, (1986) 5 164:1779. Myoglobin shows a homology with the I-Ab molecule at positions 2, 5, 7, 8, and 12 (Figure 6).
Here, too, it appears that the histidine residue at position 12 is responsible for the similarity between myoglobin in the context of self, I-Ad, and allo, I-Ab.
An obvious homology motif may not always be ~ound among peptides restricted by the same Class II
molecule. Motifs associated with each ligand-like domain will be found (Figures 5 and 6). Figure 7 presents a compilation o~ the data which indicates that 15 there are three motifs associated with ligands for the I-Ad molecule. The alignments in Figure 8 indicate the possibility of two motifs for the I-Ek molecule.

EXAMPLE I Preparation of T-cell Hybridomas and 20 Antigen-Presenting Cells The I-Ad-I-Ek positive A20.2J Balb/c B
lymphoma line which presents antigen in an MHC
restricted fa~hion to T-cells was a gift from Drs. J.
Kappler and P. Marrack. D0-11.10 is an ovalbumin I-Ad 25 restricted T-cell hybridoma. Shimonkevitz, R. et al., Journal of Immunology, (1984) 133:2067-2074. TA.3 is an antigen-presenting cell line obtained by fusing lipopolysaccharide stimulated B-cells from (Balb/c x A/J)F1 donors with cell~ from the M12.4.1 Balb/c B
30 lymphoma cell line (a gift from Dr. L. Glimcher). The phenotype of TA3 B-cell-B lymphoma hybridomas, I-Ak/d-I-Ek/d, make this cell line able to present antigen to either H2d or H2k restricted Th (T-helper lymphocytes) cell hybridomas.
Culture Condition~. T-cell hybridomas as well a~ antigen presenting cells were maintained in large wells (24 well plates, Costar no. 3424) in RPMI 1640 13(~06~

medium supplemented with 2 x 10 3 M glutamine, 5 x 10 5 M 2-mercaptoethanol, 100 ~/ml penicillin, 100 ~g/ml streptomycin and 10% fetal bovine serum (Grand Island Biological Company, no. 200.5140). The cell lines were duplicated every two days by serial dilution and expanded in T25 and T75 flasks (Falcon) with, respectively, 5 ml and 50 ml of medium be~ore they were used in the work described below.
Peptides. All peptides were synthesized by the solid phase method of Merrifield as previously described. Merrifield, R., Journal of the American Chemical Society, (1963) 85:2149-2154. The amino acid composition and the sequence analysis of the synthesized peptides correspond to the expected compositions. The purity of the peptides as determined by sequence and/or HPLC is 93-94%.

EXAMPLE II Inhibition of Antigen~Specific T-cell Activation by Non-Stimulatory Peptide Analogs The T-cell hybridoma 7B7.3 was derived from a Balb/c mouse immunized with lambda repres~or. It can be stimulated with the peptide P15-26 (residues 15-26 of the immunogen) in the context of I-Ad. The T-cell hybridoma 8I, derived from the A/J strain, recognizes the same peptide but in the context of I-Ek. Neither T-cell can be stimulated with a homologous peptide analog P12-24 (residues 12-24 of the immunogen). Other T-cells, however, derived from Balb/c can recognize P12-24 in the context of I-Ad.
3 This suggests that P12-24 can bind to the I-Ad molecule but presumably cannot stimulate 7B7.3 because it lacks a specific T-cell interaction residue (epi-toQes). The activation of T-cell hybridomas was measured by their ability to secrete IL-2 (T-cell hybridomas t5 x 104) that were incubated with 5 x 104 antigen-presenting cells in wells of a 96-well tissue Costar plate (no. 3596) in 200 ~l RPMI complete medium 13~ 4 containing the approoriate antigen concentrations).
After 24 hrs of culture, supernatant (50 ~l) was removed and then assayed for IL-2 content by its ability to maintain the growth of the IL-2 depenOent CTL-L cell~ Ci of 3H tritiated thymidine was added per well. Six hours later the cells were harvested by an automatic cell harvester (Skartron Inc., Sterling, VA) and thymidine incorporation measured by scintillation counting.
Since both peptides share 11 of 13 residues, inhibition of P15-26-dependent 7B7.3 activation ws observed when the peptide P12-24 was also included in the cultures. See Figure 2.
The activity of T-cell hybridoma 7B7.3 was measured in the pre~ence of various concentrations of P15-26, either in RPMI 1640 with 10 percent fetal calf serum alone or in the presence of P12-24 20 ~M or P12-24 60 ~M. A20 B-cell lymphomas (5 x 104 cells/well) were used as antigen presenting cells. After 24 hrs of culture, supernatant (50 ~l) was harvested and then assayed for IL-2 concentration by following incorpora-tion of 3H thymidine into the IL-2 dependent CTL-L cell line (104 cells/well). The indicated values in Figure 2 represent the arithmetic mean of triplicate samples.
In the case of 7B7.3, P12-24 was found to inhibit activation by P15-26 in a dose dependent manner. The potency of the inhibition depend~ on the inhibitor concentration. As shown in Figure 3, P12-24 changed the apparent affinity of P15-26 to 7B7.3: the 50% stimulation point of the P15-26 antigen dose response curve iJ at a concentr~tion of 20.7 i 1.2 ~M
when P15-26 is cultured with 7B7.3 alone. When 7B7.3 is co-cultured with P15 26 and P12-24, the 50 percent stimulation point is 29.8 i 2.8 ~M and 70.3 ~M when P12-24 is at 20 ~M and 60 ~M, respectively. The inhibitory effect can be reversed by increasing the concentration of P15-26 in culture. The sample 13~`~0~i4 peptide, P12-24, had no ef~ect on the IL-2 rsponse of hybridoma 8I, a P12-26 responsive, I-Ek-restricted T-cell hybridoma. It has also been demonstrated that the residues of P12-26 required for interaction with I-Ad and I-Ek are different. The absence of inhibition of the I-Ek-re3tricted T-cells by P12-24 may therefore be due to the inability of that peptide to bind to the I-Ek molecules.

EXAMPLE III Inhibition of Repres30r-Specific T-cell Activation by Other I-Ad-Restricted Peptides The following work was carried out to examine the competitive ability of peptides derived from Staphylococcus nuclease (Nase), residues 61-80 (P61-80); and from ovalbumin (Ova), residues 324-336 (P324-336) to inhibit P15-26-dependent activation of the hybridoma 7B7.3. Each of these peptide~ has been shown in their respective cases to be I-Ad-restrLcted and to be immunodominant for their respective antigen3.
Finnegan, A. et al.. Journal of Experimental Medicine (1986) 164:897-910; Shimonkevitz, R. et al., Journal of Immunology (1984). 5 x 104 TA3 cell3 were added to each well. Variou3 concentration~ of P15-26 were added with either medium (RPMI 1640 with 10 percent fetal calf 3erum) alone or 30 ~M or Ova (P324-36) or 60 ~M of Nase (P62-80). A20 B-cell lymphoma (5 x 104 cell3/well) wa~ u3ed as antigen presenting cell3.
7B7.3 T-cell hybridoma3 (5 x 104) were then added and cultured for 24 hr3. The level of T-cell ~timulation 3 wa~ assayed by determining the IL-2 concentration a~
indicated in Example II. As 3hown in Figure 3, these peptides inhibit 7B7.3 activation in a dose-dependent manner. The degree of inhibition i5 independent upon the ratio of activator to inhibitor in each ca3e.
8I (I-Ek-restricted) T-cell hybridomas and TA3 - B presenting cell3 (5 x 104) were cocultured with various concentration of P15-26 and either medium alone ~3~

or 60 ~M of Ova (P324-336) or 60 ~M Nase (P61-80) as described above. Speci~icity is seen in that the same peptides are without effect on the I-Ek-restricted-P15-26-specific T-cell, 8I. In addition, no other peptides representative of the remainder of the Nase sequence were able to inhibit 7B7.3 activation.

EXAMPLE IV Inhibition of Ovalbumin-Specific T-cell Activation by Other I-Ad-Restricted Peptides The T-cell hybridoma DO-11.10 is ovalbumin-specific, and I-Ad-restricted. It responds to the peptide P323-33g derived from ovalbumin. It responds less well to the truncated analog P324-336, which was used as a stimulator. As inhibitors, the lambda repressor peptide P12-26 and a peptide influenza hemagglutinin site 2 (P126-138) derived from influenza hemagglutinin (I-Ad-restricted) were used. Neither of these peptLde~ can stimulate DO-11.10 on its own. DO-11.10 was cultured with various concentrations of Ova (P324-336) and A20 presenting cells, either in medium alone or in the presence of influenza hemagglutinin P111-120 or P12-26 or influenza hemagglutinin site`2 (P126-138), each at 50 ~M. After 24 hrs of culture, stimulation was determined by quantitating the amount of IL-2 released. In Figure 4, the values represent the arthmetic mean of triplicate samples. Conditions were as described in Example III.
As shown in Figure 4, the~e non-stimulatory peptides act a~ inhibitors for ovalbumin-specific T-cell activation. The influenza hemaggiutinin-deriYed peptide P111-120, which is I-Ed-restricted, serYed as a control; it had no effect on ovalbumin-specific T-cell activation.

13~ 6~

EXAMPLE V Bindinq of Repressor Peptide P12-26 to Class II Molecules In Vitro To gain further insight into the mechanism of competitive inhibition observed between peptides restricted by the same Class II molecule, a study of peptide binding to Class II molecules in v tro was conducted. Results are shown in Table 1.
The peptide P1?-26 was modified by the addition of a tyrosine residue to the N-terminus to serve as an acceptor for the 125I.
Ia-molecules were purified from Nonidet* P-40 (NP-40) lysates of A20 tH2d) or AKTB-lb (H2k) cells by affinity chromatography using the following monoclonal antibodies:
MK-D6 (I-Ad-specific), 10-3-6 (I-Ak-specific) or 14-4-4 (I-Ed/k-specific) coupled to Sepharose 4B beads (Pharmacia Fine Chemicals, Sweden).
A gel filtration assay was u~ed for determining the degree of association between immunogenic peptides and Ia.
The assay is described in: Buus, S. t al., Proceedinq of the National Academy of Sciences, USA (1986) 83:3968.
Briefly, 40 ~mole of purified Ia in 1 percent NP-40/PBS was mixed with 0.2 ~mole of 125I-labeled peptide (approximately 200,000 cpm for each experiment) and incubated for 48 hours at room temperature to allow for formation of the Ia-peptide complex. The Ia-peptide complexes were separated from free peptide by gel filtration and the percentage of peptide bound to Ia was calculated as the ratio of the 125I-labeled peptide in the void volume to the total 125I-labeled peptide recovered.

* Trade Mark .. ,. ~, ~3(~

Table 1~ Binding of 125I cI P12-26 Analog to Class II
Molecules Class II Antigen Percent of Peptide Bound _Ad 1.6 + 0.8 (n-5) _Ed 8.9 + 2.2 (n-7) _Ak 0.3 + 0.5 (n=5) I_Ek 2.3 + 1.7 (n=5) Level of binding is significantly different from the other ~lass II molecules at the greater than 99 percent confidence level.

As shown in Table 1, the peptide P12-26 is able to bind to both molecules as expected. (The modified form of P12-26 used for binding is as active for T-cell activation as P12-26.) Specificity is apparently shown by the inability of P12-26 to bind to the I-Ak molecule; the latter does not show acitivity in vivo for restriction of this peptide. The peptide P12-26, however, binds exceedingly well to the I-Ed molecule. No T-cells from Balb/c immune mice were found to be restricted by this molecule.
To determine if the binding of P12-26 is non-specific for the I-Ed molecule, examination was carried out of its ability to compete for binding with a myoglobin derived peptide, shown to be restricted by (and to bind to) I-Ed. Results are shown in Table 2.
For the inhibition assay a dose-range of unlabeled cI P12-26 peptide was added to the incubation nlixture of Ia and 125I-labeled peptide (600, 120, 24, ad 48 llM). The degree of association between Ia and labeled peptide was determined by gel f`iltration as described in Table 1. The percentage of peptide bound in the absence of inhibitors is 10.6 percent for ovalbumin (323 to 339)/I-Ad; 6.5 percent for 13V~

myoglobulin (132 to 153)/I-Ed; 21.5 percent for lysozyme (46 to 61)/I-Ak; 2.5 percent for cytochrome C
(88 to 104)/I-Ek. The concentration of inhibitory peptide required to obtain a 50 percent inhibition of binding wa~ calculated. Each experiment was repeated three times.

Table 2. Capacity of Lambda-Repressor Peptide P12-26 to Inhibit Binding of Isotopically Labeled Peptides to Various Class II

Concentration (~M) of P12-26 Required 125I LabeledClass II for 50 percent PeptideMolecule Bound Inhibition Ovalbumin I-Ad 300 (P323-339) Myoglobin I-Ed 4 (P132-153) Lysozyme (Hen) I-Ak greater than (P46-61) 2,500' Cytochrome c I-Ek 300 (pigeon) (P88-104) Numbers in parenthesis refer to the amino acid residue positions in the parent molecule.

No inhibition of binding was detected at 2500 ~M of P12-26.
P12-26 also competes with other immunodominant peptides for binding to their respective Clas~ II

13(Jl~

molecules. Competition, however, is not observed for the lysozyme-derived pepti~e, restricted by I-Ak~ a Class II molecule not bound by P12-25 (see Table 1).
It is noteworthy that in fact, P12-26 binds best to the 5 I-Ed molecule, as shown by its relative binding ability and its relative competitive ability.
In view of the result, indicating that P12-26 binds specifically to the I-Ed molecule, the question as to whether or not there were T-cells restricted by 10 I-Ed in the Balb/c mouse immunized with the NH2-terminal domain of cI was re-examined.
Of more than 300 hybrids specific for P12-26 recovered from 15 cI immune mice, none were shown to be restricted by the I-Ed molecule. That is, they could 15 be stimulated by antigen in the presence of L cells expressing I-Ad, but could not be shown to be stimulated by antigen in the presence of L cells expressing I-Ed.
In contrast, of 80 hybrids specific for 20 myoglobin (P135-147), 78 were shown to be restricted to I-Ed as as~ayed on L cells expressing I-Ed.
Furthermore, unfractionated lymph node-derived T-cells from P12-26 immune mice showed significant proliferation (68,000 cpm thymidine incorporation) when 25 cultured in a standard lymph node proliferation assay as described by Shastri et al. with 10 llM P12-26 (Shastri et al., Journal of Experimental Medicine, (1986) 1_:882). However, the lymph node cells failed to show significant proliferation (1250 cpm) above 30 culture~ with no antigen added when the monoclonal antibody, MKD6, specific for the I-Ad molecule, was added to identical cultures with 10 ~M P12-26. The proliferation of these same cultures was not inhibited (75,000 cpm) by a monoclonal antibody, 34-1-4S, which 35 is specific for I-Ed and which inhibits the stimulation of a hemocyanin specific, I-Ed resticted T-cell hybridoma.

13(~

Thus, there appears to be an absence of T-cells in cI i~mune mice able to recognize P12-26 in the context of the I-Ed molecule; there is app~rently a hole in the repertoire with respect to P12-26 and the I-Ed molecule.
The next study was involved with a synthetic polypeptide for use as a malaria vaccine.

Materials The synthesis and analysis of synthetic peptides has been described previously (Guillet et al., supra). Peptides used in this study were:
P12-26, derived from the cI protein of bacteriophage lambda.
P12-26 LF is a variant of P12-26 in which residues 14 and 15 are replaced by LF.
(NANP)1-12-26, (NANP)2~12-26, and (NANP)3-23-26 are respectively 19, ~3, and 27 residues long. They begin with NANP repeated 1-3 times followed by P12-26.
NANP-50 is a polymer of NANP repeated 50 times.

METHODS

Antibody Production For antibody production, mice were given a primary intraperitoneal and foot pad in~ection of 100 ~g of antigen (NANP)3-12-26) emulsified in complete Freund'~ Adjuvant (CFA) on day 0 followed by a 3 secondary subcutaneous in;ection of 50 ~g of antigen on day 21 with incomplete Freund's Adjuvant (IFA). Sera from a bleed on days 7, 21 (before boost) 28, and 42 were used.

T~cell Proliferation -For T-cell proliferation studies 100 ~g of (NANP)3-12-26 emulsified in CFA was in~ected in the 13U~64 base of the tail and also food pad of mice on day 0.
On day 10, draining nodes were removed. Cells were seeded at 8 x 105/well flat bottom microtiter plates 1/2 volume in 0.1 ml RPMI containing 10% FCS, 5 x 10 5 M2ME, penicillin, streptomycin and antigen. On day 2, 1 ~Ci of 3H thymidine (ICN) was added to triplicate or duplicate cultures. 16 hr later cells were harvested and incorporated 3H was assayed by scintillation counter.
Estimation of Antibody Titer Against NANP~ (NANP)n, where n indicates number of repeats) An ELISA test was used. NANP50 or 12-26-BSA
conjugated (10 ~g/ml, 2 ~g/ml, respectively (25 ~l)) was added to polyvinyl wells for 1 hr at room temperature or overnight at 4C. Wells were then blocked, sera added and a goat anti-mouse IgG coupled to peroxidase was added (1 x 1000 dilution). Finally, substrate (ABTS) was added and absorbance was read at 405 nm. Sera were tested at variou~ dilutions.

RESULTS

(NANP)3-1?-26 Stimulates T-cells Specific for P12-26 Activation of T-cell hybridoma 9H35 and 9C127 was studied. Antigen presenting cells were the B-cell lymphoma A20 and the antigen, either P12-26 or (NANP)3-12-26, wa~ added at various doses (0-50 ~g/ml). IL-2 release was taken as a measure of activation of T-cell~
3 and was assayed on an indicator cell line requiring IL-2 for growth.
The results obtained showed that for two H2d-restricted T-helper cell hybridomas, 9H35 and 9C127, specific for P12-26 (ijolated from cI protein-immunized animals), the peptide (NANP)3-12-26 is able to act as a stimulator in the presence of suitable (A20) antigen presenting cells. The NANP-containing peptide is more l3al~4 active. This result demonstrates that the T-cell stimulatory capacity of an extended Class II-bindins peptide is not gre~tly altered as studied in vitro.

The (NANP)-3-12-26 Peptide can be_Bound by Both Monoclonal Antibodies Specific for_(NANP)50, and One Specific for P12-26 Antibody recognition of (NANP)3-12-26 by monoclonal antibodies was ~tudied. The antigen, bovine serum albumin (BSA) conjugated to P12-26 was adsorbed to microwells and horse radish peroxidase (HRP) labeled antibody ~pecific for P12-26 added. In the absence of (NANP)3~12-26 or in tne presence of P12-26LF, there is no inhibition of monoclonal antibody B3.11 binding.
Inhibition is observed with P12-26, (NANP)3-12-26, and P12-26S (position 18), in the order of decreasing inhibition.
The above study was repeated except the antiBen wa~ radiolabelled (NANP)50 which was bound to microwell~. It wa~ found that P12-26 or (NANP)2-12-26 did not inhibit binding while (NANP)3 and (NANP)3-12-26 did inhibit binding.
The results demonstrate that the monoclonal antibody, B3.11 can be inhibited in its binding to bovine serum albumin coupled with P12-26 by the addition of (NANP)3-12-26. Similarly, a monoclonal antibody specific for (NANP)50, isolated from malarial parasite-immune mice, can be inhibited in its binding by the (NANP)3-12-26 peptide. These results again demonstrate that antibodies specific for the NANP
portion of the synthetic molecule are able to recognize the "hapten" in the presence of an additional Class II-binding peptide.

~3Ul(~6 Mice Unable to Synthesize Antibodies to (NANP)5~ when (NANP)50 or (NANP)3 are Used for Immunization, are able to Synthesize Antibodies to (NANP)5~, (NANP)3, and to Sporozoite~ when (NANP)3-12-26 is used as an Immunogen Pairs of mice were immunized with (NANP)3-12-26, (NANP)50, (NANP)1-12-26, and (NANP)2-12-26. The serum titers were tested by direct binding assays as indicated in the methods section at either seven days after a primary immunization, 20 days after, or ten days after a secondary boost (control sera gave no detectable binding). Sera from mice immunized with (NANP)3-12-26 (10 days after secondary boost) were found to bind directly to circumsporozoite falciparum by conventional radiolabelled anti-mouse globulin RIA.
Lymph node-derived T cell proliferation was observed in mice eight days after immunization with (NANP)3 12-26. T-cell proliferation as assyed by thymidine incorporation was seen with the immunogen, P12-26, (NANP)1-12-26, and (NANP)2-12-26. No proli~eration i8 seen with (NANP)3 or (NANP)50 in these ~ame mice. These results indicate that the presence of Clas~ II molecule binding sequences on the immunogen are su~ficient to impart T-cell stimulatory activity to (NANP)3 which on its own is inactive in these mice.
All T-cell activity is directed to the Class II binding sequence. These results support that the antibody activity induced in mice immunized with (NANP)3-12-26 again~t the (NANP) sequences present on the 12-26 portion of the immunogen, and that such an "activating"
sequence can be added to inactive sequences and generate antibodies against the latter.
In a similar experiment, Balb/c mice immunized with (NANP)3-12-26LF showed T-cell proliferative responses (taken from the spleen 16 day~ post immunization) to the immunogen but not to (NANP)3-12-26 (no more than to P12-26 alone). This result again shows that there is no induction of T-cell reactivity to the (NANP) portion of the immunogen. In this case, as described in the paragraph above, it is clear that (NANP~3-12-26 can bind to Class II molecules and present to T-cells for activation. Mice immunized with the (NANP)3-12-26LF also induce antibodies specific for the (NANP)50 polymer. These antibodies are of the ~ and Y-1 class.
The inability to detect T-cell reactivity to the (NANP) portion of the immunogen is a property of Balb~c mice and not the property of the immunogen.
57B6/H-2b mice which were immunized with (NANP)3-12-26, are able to respond directly to (NANP)3 or (NANP)50, and respond to immunization with (NANP)3 12-26 with T-cell reactivity to the (NANP) portion of the immunogen.
Thus, the utility of this method is shown in a system which has utility for the production of immunity to a human pathogen.
The above result~ demonstrate the power and breadth of the sub~ect invention in modulating the immune system in a wide variety of contexts, both in vitro and in vivo, both as to stimulating B~ and/or T-cells or inactivating B- and/or T-cells. The ~ubject invention provideq for exquisite specificity in select-ing for one or more subsets of lymphocytes in response to a particular event and, furthermore, in relation to the context of the host, that is, the transplantation antigens of the host. By virtue of the ability to mimic a polymorphic region of the transplantation anti-gens of a particular host, the T-cell immune system may be substantially inactivated. By contrast, by varying the consensus sequence for binding to the transplanta-tion antigens of the target host(s) one can activate specifically one or a few subsets of lymphocytes to provide for a stimulated immune system for purpoqes of vaccination, enhanced response to a pathogenic invader, - or other event associated with protection by the immune system. In addition, one can modul~te the autoimmune ~3U~6~

system by inactivating lymphocytes associated with attack on native tissue. Thus, there is an extensive spectrum of uses of the subject invention for enhancing or diminishing particular cells in relation to their function.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifi.cations may be practiced within the scope of the appended claims.

Claims (22)

1. A method for modulating the immune response of a cellular system to a epitope of a first antigen to which said cellular system is immunologic-ally responsive, said cellular system comprising T-cells restricted by a first transplantation antigen and cells comprising said first transplantation antigen, said method comprising:
combining said cellular system with a molecule comprising a first domain of at least an eight amino acid sequence of an immunodominant sequence of said first antigen or a mutated sequence thereof restricted by said transplantation antigen, wherein said mutation results in greater conformity with the consensus sequence of the agretope restricted by said transplan-tation antigen or a sequence consisting essentially of a sequence of a polymorphic region of said transplan-tation antigen;
with the proviso that when said first domain comprises said immunodominant sequence, said domain is joined to a second epitopic site of a molecule other than said first antigen or an epitopic site of said first antigen joined by other than the natural sequence of said first antigen;
whereby the immune response of said cellular system to said epitope is modulated.

2. A method according to Claim 1, wherein said cellular system is whole blood.

3. A method according to Claim 2, wherein said immunodominant sequence is a mutated sequence mutated to conform with the consensus sequence Or the agretope restricted by said transplantation antigen.

4. A method according to Claim 1, wherein said first domain comprises a sequence of a polymorphic region of said transplantation antigen.

5. A method according to Claim 1, wherein said first domain is joined to a second epitopic site.

6. A method for modulating the immune response of a cellular system to an epitope of a first antigen to which said cellular system is immunologic-ally responsive, said cellular system comprising heterozygous T-cells restricted by at least one of first and second transplantation antigens and hetero-zygous cells comprising said first and second trans-plantation antigens, each of said transplantation antigens responding to a consensus sequence, said method comprising:
combining said cellular system with at least two molecules, each molecule comprising a first domain of at least an eight amino acid sequence of an immuno-dominant sequence of said first antigen or a mutated sequence thereof, said mutation resulting in increased conformity with the consensus sequence of the agretope restricted by either said first or second transplanta-tion antigen or a sequence consisting essentially of a polymorphic region of said first or second transplanta-tion antigen;
whereby a first of said two molecules binds to said first transplantation antigen and a second of said two molecules binds to said second transplantation antigen;
whereby the immune response of said cellular system to said epitope is modulated.

7. A method according to Claim 6, wherein said cellular system is whole blood.

8. A method according to Claim 6, wherein said molecules are conjugated to a second epitope different from said first epitope and cross-reactive with a second antigen different from said first antigen.

9. A method according to Claim 8, wherein said second eptiope is an epitope of a pathogen.

10. A method according to Claim 9, wherein said pathogen is a virus.

11. A method according to Claim 9, wherein said pathogen is a parasitic organism.

12. A method according to Claim 11, wherein said parasitic organism is Plasmodium.

13. A method according to Claim 9, wherein 20 said pathogen is a bacterium.

14. A composition comprising two novel molecules for modulating the immune response to a first epitope of a first antigen of lymphocytes having transplantation antigens, each of said molecules characterized by a first domain of at least an eight amino acid sequence of an immunodominant sequence of said first antigen or a mutated sequence thereof restricted by said transplantation antigens, said mutation resulting in increased conformity with a consensus sequence of different agretopes restricted by said transplantation antigens or a sequence consisting essentially of a polymorphic region of said transplantation antigens: wherein each of said molecules specifically binds to different transplantation antigens.

15. A composition according to Claim 14, wherein said molecules are characterized by a mutated sequence of said immunodominant sequence conjugated to a sequence comprising a second epitope different from said first epitope and cross-reactive with a second antigen different from said first antigen.

16. A composition according to Claim 15, wherein each of said molecules is of fewer than 100 amino acids.

17. A composition according to Claim 14, wherein each of said molecules is of fewer than 30 amino acids.

18. A composition according to Claim 14, wherein said molecules are characterized by comprising said consensus sequence.

19. A composition according to Claim 14, wherein said first antigen is an antigen of a pathogen.

20. A composition comprising a mutated sequence of at least eight amino acids of an immunodominant sequence of a first antigen joined to a sequence defining an epitopic site of said first antigen or a different second antigen.

21. A composition according to Claim 20, wherein said epitopic site is of said second antigen and said second antigen is an antigen of a pathogen, an oncogene, a transplantation antigen, or a lipopolysaccharide.

22. A composition comprising a sequence of at least eight amino acids of an immunodominant sequence of a first antigen joined to a sequence defining an epitopic site of a different second antigen.