Note: Descriptions are shown in the official language in which they were submitted.
~VO 95/13288 2 1 7 6 2 0 8 PCT/US9.1/1298~
--1--
SURFACE MEMBRANE PROTEINS AND THEIR EFFECT O~;
IMMUNE RESPONSE
INTRODUCTION
Technical Field
The field of this invention is, ---~ l; r~ of immune response.
Back~round
The immune system is the subject of ever increasing scientific scrutiny. Despitethe enormous interest in the immune system and the contin~o~lcly expanding number of
investigators, both academic and industtial, a complete ulld~,laL~ ding of the system
continues to remain elusive. One of the major blt~Lll~uu6lls was i-IPntifi~tinn of the
interaction between the T-cell receptor and a major l,;~ ;l ,;lity complex antigen.
15 The identification that both class I and class II major l~ ihility complex
antigens have a cleft which binds a small peptide provided a significant key to
",.1~.,~l ,.I;"g~T-cellspecificityandT-cellresttiction.
The llnfil r~tAn~in~ that the MHC served macrophages and B-cells in their role
as antigen presenting cells, where the peptide served to define which T-cells could bind
20 to the MHC-peptide complex still did not explain many other aspects of the T-cell
response. It appeared that the binding of the T-cell receptor to the antigen presenting
cell was not enough by itself to result in T-cell activation. The process of anergy or
Lul~ iul- could not be explained by an activation process solely involving the T-cell
receptor and the MHC antigen. In addition, there was the distinction between helper
25 cells (CD4+) and aulJ~)Icajul/~yLulu~iC cells (CD8+). Some m~hAnicrn was necessary
to associate the helper cells with class Il MHC and the au~ ;aaul/~,yLuLù~-iC cells with
class I MHC. It was ~ . ."lly found that CD4 and CD8 I~uLh,;~ Lul in the MHC
TCR complex by binding a loop of the MHC, enhancing the stability of the complex.
WO 95/13288 2 1 7 6 2 0 8 PCT/IJS9~ 98~ 0
-2-
ln addi~ion, other interactions were uncovered, apparently not directly associated with
the T-cell receptor/MHC complex, where CD28 and B7 were found to bind. Thus,
there appear to be a number of different interaCLions invo~ved with the association of the
antigen presenting cell and the T-cell, which could result in tolerization or acLivaLion of
the T-cel~.
Because of the crucial role that the T-cell plays at the center of a major
component of the immune system, it remains of great importance to be able to
understand how T-cells are selected, activated or tolerized. By lln~Prctl~n~iing the role
thal various participanls play in T-cell activation, there will be ~ " ~ to regulate
the immune system, either enhancing the immune response, where one is dealing with
vaccines, pathogens, neoplasia, or the like, or ~liminiching the immune response,
whereoneisdealingwithA~ yororganla~ alltaLiOII.
A number of il",ll",l.~S"l.~lCS~allL~ have been shown to have specific cellular
proteins as targets. Cyclosponn A binds to cyclophilin, FK506 to FK binding protein,
and d.,~ a~ udlin to both Hsc70 and Hsp70. Hsp 70 has been shown to be a
member of a multigene fan~ily, having at least one constitutively expressed cognate,
Hsc70. The effect of DSG seems to be specific for antigen presenting cells; interfering
with antigen presentation and/or processing. The action of imml~nf cllrpressants is
mediated by their cellular binding proteins, and there is, therefore, interest in
identifying targets and other agents binding to the targets which will provide
modulation of Lhe immune response.
RelGvant T.iter~rllre
Wan er al. (1986) J. Immunol 137:3671-4 describes the Bw4/Bw6 epitopes.
Clayberger, el a~. (1987) Nature 330:763-765 dc.~ sL, - that HLA-A2 peptides canreguiate cytolysis by human allogeneic T IYll.L I~.~Y~t;S. WO93/17699 describes the
activity of peptides from the HLA-A and -B L~l and a2 helices in mf)~ tin~ CTL
activity. HLA-B2702.6U-84 peptide is described therein.
The molecular CllalA~ lr~ I;on of the heai shock protein Hsp 70 is described in
~mmllno~enetics (1990) 32 242-251, and its constitutively expressed homologue inMol. Cell. Bio. (1988) 8:2925-2932.
,1, r~ .;,,.li.,l~ of the i"""",l..s,~ cs~ive effect of d~ y~ ,u~lin (DSG)
may be found in Hoeger, er al. (1994) J. Immungl, 153:3908. The binding of DSG to
Hsp70 and Hsc70 is described in Nadler et al. (1992) Science 258:484 and Nadeau et
al. (1994) BiochPmictrY 33:2561.
~vo 95113288 2 1 7 6 2 0 8 PCTIUS9~11298~
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SUMMARY OF THE INVENTION
Methods are provided for preparing and isolating T-cell proteins which
participate in T-cell activation upon antigen presenting cell - T-cell interaction. The
proteins are .,I.~ i~d by being associated with T cell activation and binding to the
5 HLA-B2702 ctl helix peptide, and are immllnt~ ir~lly cross-reactive with the heat
shock protein cognate, Hsc70. The proteins, fragments thereof and nucleic acids
encoding the proteins and ~ onll(~ o~ are provided, as well as antibodies which
bind thereto. By employing agents which bind to the proteins or inhibit binding of
other agents to the proteins, T~cell activation may be modulated.
DESCRIPTION OF SPECIFIC EMBODIMENTS
Methods and ~,UIIl~o~i~iulls are provided for isolating proteins expressed by T
cells as surface membrane proteins associated with T-cell activation in m~mm~ lnT-cells, which proteins bind to the HLA-B2702 otl helix peptide, nucleic acid encoding
15 such proteins or fragments thereof, and agents for mr)~ in~ the signal
associated with the proteins in the modulation of activation of T-cells
The protein of interest is referred to as p74. It is a surface membrane protein
which is expressed in T cells and a limited number of other cells. It can be readily
isolated by extracting a Iysate of T cells with the peptide of the t~l-helix of ~LA-
20 B2702, namely the RENLRIALRY sequence, peptide extensions thereof, or dimersthereof, particularly palindromic dimers, having the amino acid sequence
YRLAIRLNERRENLRIALRY. The affinity of p74 for the HLA-B2702 palindrome is
at least about 104 M. The extraction may occur with the peptide on a surface, such as
particles, microtiter well walls, etc. or in solution, where the peptide may be labeled
25 with a label which allows for separation of complexes with the peptide. For example,
one may label the amino terminus of the peptide with a ligand, or biotin and precipitate
or sequester complexes with antibody or avidin, respectively. The protein may be freed
from the peptide and ~ ,J as appropriate.
p74 is a protein found in a limited number of cells, particularly activated CTLs,
30 the T cell tumor PEER, and the EBV ~ rull-l.,d cell line JY, in effect, Iymphoid T
and B cells. The p74 protein can be obtained by Iysis with an amphooeric detergent,
such as CHAPS. The Iysate may then be combined with a peptide or protein having an
, Bw4 epitope, for example, HLA-B 2702.84-75-84 palindromic peptide.
By preparing an affinity column, having the peptide covalently bound to appropnate
35 particles, the Iysate may be passed through the column, followed by elution with Iysis
buffer or other convenient buffer. Effective elution can be achieved using relatively
Wo 95113288 2 1 7 6 2 0 8 PCT/USg~/1298S O
-4-
high pH, conveniently in the range of about 11-1~. See, for example, (Ey (1978)
Imm~mochemistr,v 15:429-436). Alternatively, a different detergent may be used as
described by Mescher er al. (1983) Meth. Enz~mol. 92:86-109. Other separation
techniques employing the peptides bound to a support may also be employed, such as
S panning, magnetic beads, or the like. Usually, after each contacting of the peptide witn
the p74 sample, the supernatant will be removed and the solid support washed to
remove non-specifically bound proteins and other co~ Washes may be any
convenient buffer solution, varying in salt content. The washing should not adversely
affect the complex between the peptide and p74. In this way successive enrichments
may be employed to provide for a protein .,v.lll,o~;lio.~ having at least about 50 weight
% of total protein as p74 protein, preferably at least about 75 weight %, more
preferably at least about 90 weight %, up to 100 % pure.
As an alternative purification scheme, the imml-noln~ir~l cross-reactivity of p74
with the heat shock protein cognate, Hsc70, may be exploited. Antibodies directed
against Hsc70 react with p74. Various affinity pl-rifi~ Ati~n methods are known in the
art. Antibodies may be bound to beads or another insoluble support, and combinedwith the cellular Iysate. The bound protein is washed free of the unbound Iysate. The
bound protein is eluted from the antibody by a number of methods, e.g. heat, salt
gradients, mild detergents, elc.
Instead of affinity Clll~lllAll~A~ Y~ gel clc~ u~l-v-csis of the Iysate may be
performed, where the Bw4 epitope containing peptide, suitably labeled, may be used to
identify the presence of p74. The Bw4 peptide may be labeled with fluorescer,
rA~ icotope, or the like, non-specifically bound peptide washed away, and the
appropriate band developed.
Instead of binding on a solid or in a gel, binding can be ~rcr,mrlich~ in
solution, using a suitably labeled soluble form of the peptide, e.g. N-terminal
bivLillyld~ivll, e~c. By incubating an appropriate cell with the peptide or dimeric form
thereof, pa~ Luly palindromic form, at a mildly elevated t~ diUlC, usually about25 - 40-C. for sufficient time for complexes to form, one obtains specific binding of the
peptide to cell surface proteins. The complexed cells may then be Iysed as described
above. After Iysis, the complexes may be isolated by means of the label, e.~g. biotin,
isolated by means of avidin bound to a solid surface. Alternatively, the label-
1 ,., y binding member complex may be preformed and used to extract specific
protein binding to the peptide as described above. The samples may then be washed
extensively, boiled in SDS and separated using SDS-PAGE gel ele~,~lu~llvlc~;s.
~vo gs/13288 2 1 7 6 2 0 8 PCTiUSg~/12985
-5-
Pnor ~o the affin;ty purificat;on or gel electrophores;s, one may enrich for th~desired p74 protein by using the peptide sequence of the B7.84-75-84 palindrome,YGRLNRLSERRESLRNLRGY, to remove any proteins which bind to that sequence,
as distinct from the B270~.84-75-84 palindrome sequence to which the p74 binds. In
this manner, p74 may be greatly enriched and obtained cllhst~nti~lly free of other
proteins.
The subject proteins may be obtained from any m:~mm~ n species, such as
rodent, bovine, canine, primate, particularly human, or the like.
Using the proteins individually in a sample of at least about 80 weight % punty,based on protein, the sequence of the proteins may be readily determined in accordance
with conventional ways. The protein purified as described above may be sequencedusing ~,u~ -Liollal sequence equipment. The purified protein may be cleaved, using
mild proteolytic flr ~ u ;~ ," . cyanogen bromide, or other mode of cleavage, to provide
fragments under about 20 amino acids. The fragments may then be fr:~rtif~n~t~d e.8.
reverse phase-HPLC or preparative SDS-PAGE and electroelution, and the fragmentssequenced. The sequences of ~he fragments may then be used to deduce t~le ~equence
of the respective protein.
By employing conventional technifll-f-s, degenerate probes may be produced
from the known amino acid sequence of the proteins. Using DNA probes of at leastabout 15 nllrlr~r>tiflrs. usually of at least about 20 nllr1f-r tiflr s, one may screen a cDNA
library of cells known to express the proteins, particularly T-cells. Those cDNAS
which bind to the probes may then be isolated and sequenced. Where the sequence
encodes the ~lu~lidt~ peptide sequence, if the isolated cDNA is not a complete
cDNA, the isolated cDNA may be used as a probe to isolate a complete cDNA which
encodes the entire protein. If desired, the cDNA gene may then be used to isolate the
genomic gene in accordance with conventional te~hni~l~lec (See, for example,
Molecular Cloning: A Laboratory Manual, 2nd ed., J. Sambrook, E. F. Fritsch, T.
Maniatis, CSHL, Cold Spring Harbor, NY, 1989).
The gene or fragments thereof, usually of at least about 25 nucleotides,
preferably of at least about 30 nucleondes and not more than about 60 ~llrlf oti~lrC, may
be used for probing cells to determine whether a subject protein ;s expressed, when in
the cell cycle the protein is expressed, and at what level of dirr~ ialion it may be
expressed, and quantitate the amount of message RNA during activation or deactivation
of T-cells.
The gene may be introduced into an expression vector for expression in a wide
variety of hosts, both prokaryotic and eukaryotic. A large number of promoters are
Wo 95/13288 2 1 7 6 2 0 8 PCT/US9~11298~ ~
-6-
commercial~y available today which are included upstream (in the direction of
Ll.~ JLiull) of a polylinker for insertion of a gene to be under the transcnptional and
rranslational control of a transcriptional and translational initiation regulatory region
functional in an expression host. Besides the L ~ and ~ l initiation
5 regulatory region, the expression vector may include dUWI~LI~ I from the polylinker,
a trRncrrir~ nRl and translational t~rrninRtinn regulatory region, functional in the
expression host. Thus, the gene may be inserted into the vector after lil~ iull of
the vector with a restriction enzyme which cuts at a convenient site in the polylinker. In
addition to the L~ 1 and trRn~tRtirmRl regulatory regions, the expression vector1û may also include a gene which allows for selection of hosts comprising the expression
vector. For the most part, these genes will provide for antibiotic resistance, but may
also serve to ~--",1 1 "...1l an 21U~U~lU~ host, or other mode of selection. The vector
may also include an origin for episomal Ill~ or the vector may allow for
integration into the expression host. The vector may be introduced into the expression
host by any convenient means, including el~ lu~ul~Liull, fusion, calcium ~ iUi~l.t~
DNA, transfection, etc., the particular mode employed not being cntical tû this
invention and depending upon the choice of expression host.
In addition to using the expression construct for production of the subject
proteins, the expression construct can also be used in the rll~ri~lRtil.n of T-cell
activation. These constructs may be used, for example, in c(-".l~ studies.
For example, cells lacking one or both of the subject proteins, as well as other surface
member proteins associated with T-cell activation, may be modified so as to express
one or both of the subject proteins. The effect of p74 binding on various pathways in
the cell may then be studied. These studies may also examine the effect of mutations on
the activity of p74. In addition, one may choose to use the cytoplasmic portion of p74
and bind it to a different extracellular portion, so as to provide a fused protein, which
will transduce the signal when a ligand or other binding molecule for the -YtrRrt lllllAr
portion binds to the t ~ctrRr~ lRr portion. For example, one may use various receptors
with known ligands, such as the EGF receptor, IL2 receptor, insulin receptor, CD4, or
the like, where binding of the protein, particularly ~ lu~ hil~g on the surface, will
result in signal trRnC~llrriOn Thus, one may use a natural ligand or antibodies to the
receptor, so as to provide for the signal Llrll~ l A soluble form of p74 may be
uul~LIu~ by deletion of the ~ lle region. The soluble protein is used as a
competitor for the native protein, thereby modulating the cellular immune response.
The expression constructs may also be used to produce fusion proteins, where
p74 may be fused to a marker, such as 13-gal, CAT, ~acZ, and the like. The fusion
~WossJL3288 2 1 7 6208 PCT/USg~/12985
-7 -
proteins ma~ serve as markers for translational e~pression, for production of
antibodies, in assays, or the like. The fusion proteins may serve to aid in the
of a source of p14, where p74 0r truncated portion thereof may be joined to
a sequence which allows for ,UUI ;r.~ ,.. in the Qiagen process or other process.
p74 binds to HLA-B 2702.75-84, particularly as the palindromic dimer. and not
to HLA-B7 75-84 or its palindromic dimer. It is associated with the process of T-cell
activation. Binding to p74 can result in inhibition of cytolysis by CTL as well as CTL
dirr~,.c-~Li~-Lion. By employing agents which bind to the p74 of CTL, the immuneresponse can be significantly inhibited. Alternatively, by inhibiting binding agents,
either naturally occurring or synthetic to p74, CTL activity may be mAin~inf~rl, other
aspects necessary for CTL activity being present. For inhibition of CTL activity.
peptides having the Bw4 epitope may be employed in an amount sufrlcient to inhibit
differentiation or cytolysis of target ce~ls. Alternatively, antibodies may be employed
which bind to p74, where the antibody may provide for activation or inhibition.
Depending upon the antibody, it may inhibit binding of an agent to p74 allowing for
acti tion of the CTL, it may provide for activation of the CTL in conjunction wit}l
other;,,~ A~ llC of surface member proteins or other signals being transduced into the
cell, or it may i",l, .l)....l. ,~lly inactivate the CTL through the binding of p74. Whole
antibodies need not be used, binding fragments thereof, such as Fab, F(ab')2, Fv, or
20 the like, or other binding entities, either naturally occumng or synthetic.
p74 when bound to the B2702 peptide results in a large calcium influx in
.."",."~r""". ~1 T cells. The magnitude and kinetics are essentially the same as that
induced by anti-CD3 ant~odies, supporting the conclusion that the rise in intracellular
calcium is involved in the dCVCI~ L of anergy. This response appears to be specific
to the B2702.75-84 peptide.
Soluble p74 may be prepared by truncating the respective gene and removing
the ~ e sequence and the intracellular sequence. The truncation can be
achieved in A~ e with conventional methods, using restriction enzymes, primer
repair, exonucleases, or synthesis of the extracellular portion of the protein. The
soluble p74 may be used for a variety of purposes. The soluble p74 may be used in
diagnostic assays to determine the presence and amount of p74 in a sample, may be
used for screening ~,ull~poulld~ which bind to p74 in accordance with conventional
binding assays, for prophylactic or therapeutic purposes (i.e. to inhibit binding of
agents to the native protein, as well as in research to elucidate the, 'f ~ 1 l ' ' ;`" ' of T cell
activation.
WO95/13288 2 1 7 6 2 0 8 PCT/US9.1/1298S O
-8-
Soluble p74 may be used for identifying APCs which bind to p74 as well as
agents which interfere with such binding. Using labeled p74, cells binding ~o p74 can
be readily detected by magnetic separation, nuulc~cll~c~ etc. Soluble p74 may also be
used to modulate CTL response in vivo or in vitro in a mixture of T cells and antigen
S presenting cells, such as Illa.,luplla~ and B cells.
Antibodies to the subject proteins may be prepared in aucul-lall-,c with
conventional methods. p74, native or soluble form, may be used as an ;IIIIIIUI10~
being injected intravascularly,; " l, AI,r,; 1~ ,. ,. Ally,; " ~ Iy, ~1 l1). I l l A l~r ~ y~ or the
like normally in conjunction with an adjuvant, into an a~J~uulvlidlc mammal, e.g.
10 mouse, rat, guinea pig, or the like. Usually, one or more booster injections will be
employed to enhance the specificity of the antisera. The immunized host may then be
sacrificed, the spleen isolated and ~ o~yLcs immortali~ed by any convenient means,
e.g. fusion with a hybridoma. The hybndomas may then be grown using limiting
dilution and screened for binding characteristics with p74, as appropnate. Hybridomas
15 which show binding affinity for p74, may then be expanded and the ~ulJ~,IIlàLallL used
for isolation of mnn-lrlonAI an~ibodies, or the hybridomas used for the preparation of
ascites fluid as a source of mr~norlnlnsl antibodies. The llyblidulllds may then be
further subcloned and screened to identify antibodies which have a desired binding
affinity. These antibodies may be then be further screened with T-cells having p74
20 under conditions where the physiological effect of the antibodies can be determined.
For procedures for preparing m~-nr,rl--nsl antibodies, see Antibodies: A Laboratory
Manual, Eds., Ed Harlow and David Lane, Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY, 1988.
For use as ~JIu,ull~la~,LiC or therapeutic reagents in a host, the antibodies may be
25 modified by replacing constant regions and framework regions of the antibody with
sequences utilized by the host to be treated. Techniques for isolating the variable
rcgions of the heavy and light chains and fusing them to variable regions native to the
host, as well as :,ub~liLuLiull of framework regions native to the host, have been
described in numerous publications. See, for example, EPA 173,494 and
30 WO92/16562.
The subject therapeutic agents may be prepared as formulations in
p~ 1y acceptable media, for example saline, PBS, in glucose, generally at a
pharmacologically effective dose, the conr~n~rsAtinn~ of which will be determined
empirically in accordance with .UII V'~,llLi(Jlldl procedures for the particular purpose. The
35 filrrn-llAAtirm~ may include ~srt~ riri~Al agents, stabilizers, buffers or the like. The
amount ddlllillisL~Icd to the host will vary depending on what is being a.llllilli,u,lcd, the
~NO 9sll3288 2 1 7 6 2 0 8 PCTIUS9-1112985
purpose of the adn1lnistration, such as prophylaxis or therapy, whether inhibition or
activation is desired, the state of the host, the manner of a.l..~ Li~,--, and the like. In
order to enhance the half life of the subject peptide agents, the peptides may be
r~i, introduced into the lumen of liposomes, prepared as a colloid, modified
5 by binding to stable peptides, such as the constant region of IgG, or polyalkyleneoxy
groups, or other conventional techniques.
The role of p74 in the mixed Iylll~llo~ , reaction, B cell activation, and other
cellular or compound i., l r, ,~ may be investigated. For example, in culture, the p74
or biologically active fragment thereof may be added to the culture medium in from
about I to 500, Lg per I x 105 cells, where inhibition of T cell activation is studied. In
addition, p74 may be used for screening novel compounds for agonist or antagonist
activity to CTL activation. Thus, one can combine the protein bound to a peptide or
other agent which binds to the protein, where the peptide or other agent is labeled, so as
to be detectable. By carrying out a ~mr~titi-)n between the complex involving p74 and
15 the labeled agent with the compound being analyzed, and fh~tl~rminin~ the rate of release
of the labeled agen~ from p7~, one can measure the affinity of the candidate compollnd
for p74. Alternative~y, one may use CTLs where the peptide or other agent competes
for binding to the protein with the candidate compound and measure the effect of the
candidate compound on CTL ~lirr~ ion or cytolytic activity as compared to a
20 standard, where the candidate compound is not present.
The following examples are offered by way of illustration and not by way of
limitation.
EXPERIMENTAL
Effect of a Varietv of HLA Per)tide,s of Amino Acids 60-84 and HLA- B
2702/0~.145-169 on Lvsis
The following peptides were :,y--ll-.,;,i~.
HLA-B2702.60-84 WDRETQICKAKAQTDRE~LR~LRY
HLA-B2705.60-84 WDRETQICKAKAQTDREDLRTLLRY
HLA-Bw46.60-84 WDRETQKYKRQAQTDRVSLRNLRGY
HLA-Bw62.60-84 WDRETQISKTNTQTYRESLRNLRGY
HLA-A2. 1.60-84 WDGETRKVKAHSQTHRVDLGTLRGY
HLA-B2702/05.145-169 RKWEAARVAEQLRAYLEGECVEWLR
HLAB38.6084 WDRNTQICKTNTQTYRENLRL~LRY
WO 95113288 2 1 7 6 2 0 8 PCT/US9~/1298~ 0
-10-
The effect of the above sequences on Iysis of long-term CTL speciflc for
HLA-A2, -B2705, -Bw46, -Bw62, and -Cw4 was rlr-lr-lTninr-rl, including CTL specific
for HLA-B27 and the HLA-Cw4. None of the peptides inhibited or enhanced Iysis
with the exception of the B2702.60 84 peptide. This peptide blocked Iysis by all CTL,
5 regardless of their HLA specificity. This effect was due to interaction with the CTL
and not the target cell as shown by pre-treatment ~
These peptides were tested for effects on the dirrclclllia~iul~ of CTL from CTL
precursors in limiting dilution assay. The procedure was modified from Skinner and
Marbrook (J. Exp. Med. 143:1562; 1976) as follows: PBL from normal HLA-typed
10 donors were punfied over Ficoll-Hypaque and co-cultured in round bottom microtiter
wells with irradiaoed (10,000 R) EBV ~al~rullll~d B-lymphoblasts expressing the
HLA allele of interest. Responder PBL were added at 3000, 600û, lOOûO and 30000
cells per well while stimulators were added at 6000 cells per well. 20-4 replicates were
setupforeach..,"...,~ ;r.)lofrespondercellsinRPMI-1640medium~u~ l.t~"1
15 with lû% fetal bovine serum plus L-glutamine. Plates were incubated for six days in â
5% C02/95% air humidifled incubator at which time the contents of each well were
mixed by pipetting five times with a multi-channel pipette. Fifty microliter aliquots
were transferred to the V-bottom microtiter wells to which 1000 5ICr labeled targets of
known HLA type were then added. Lysis was determined in a four-hour cyLulu~icily20 assay. Wells were designated positive if specific Iysis was >10%. CTL precursor
frequency was determined by linear regression analysis using a computer program.The B2702.60-84, Bw46.60-84 and Bw62.60-84 peptides all blocked the
d;rrclcll~;ali~ll of CTL, whereas the other peptides had no effect.
25 Effect of Peptides C~JIIC~IJOIId~ to HLA Re~ions on CTL Precursor Frequencv as
Determined by Limitin~ Dilution Analysis
Peptide l/CTL Precursor Frequency
B2705.60-84 1 64,245
B2702.60-84 349.99
B38.60-84 334,937
A2. 160-84 164,245
Bio46.60-84: : 995,400
Bio62.60-84 995,400
B27.145-169 164,245
WO 95/13288 2 1 7 6 2 0 8 PCTIUS9 J/12985
PBL from a normal donor (HLA-A3; B-7, 38: Cw4; DR4,6) were cultured wi~l
ty (HLA-A2; B7; DR4,6) or HOM2 (HLA-A3; B27) in the presence of 10-100 ~Lg/ml
peptide. After 6 days, Iysis was tested on 5ICr-labeled CIR cells expressing either
HLA-A2.5 or HLA-B2705. Results are shown for HLA-A2 specific Iysis but similar
5 results were obtained for HLA-B27 specific Iysis.
'rhe effect was not allele specific since the lirrL~ id~iOI. of CTL specific for a
number of different HLA molecules was inhibited. None of the peptides affected
Class Il restricted responses, including mixed Iy.~ L~y~e responses and mitogen
induced ~ILlir~l~Liull.
PBL from normal donors were cultured at sx105 cells/round bottom microtiter
well in RPMI-1640 supplemented with 10% fetal bovine serum and L-glutamine.
Cultures were ~ with either 5x lO3 irridiated (10,000 R) pBV l, ... ,~r~ ", .~ ~B Iymphoblasts or 10 ~g/ml phyt~hpm~e~ tinin P (PHA-P). Cells were incubated at
37-C for 3 days for PHA-P and 5 days for alloantigen at which point 3H-thymidine15 was added (2 lali/well). After 16 hours wells were harvested and 3H-thymidine incorporation deter~nined by srilltill~tir)n counter.
EffectofTruncatedSe~uencesonL-rsisandDirr~li..,li~l,."~
Since the B2702.60-84 and B2705.60-84 peptides differed by only 3 amino
acids, additional peptides were prepared to investigate the effect of these differences.
Three addihonal peptides were ~y~ l.,s;~d~
HLA-B2702.75-84 RENLRIALRY
HLA-B2705.75-84 REDLRTLLRY
HLA-B2702/05.60-69 WDRETQICKA
The peptide l,L~ ;~LII~dillg to residue 60-69 of HLA-B2702/05 had no effect on
the assays described above. The peptide LollL~vlldillg to residue 75-84 of
HLA-B2702 blocked all Class I specific CTL responses, whereas the peptide
~, ." ~ e to the same region of HLA-B27o5 did not~
To determine which residue(s) mediated the inhibitory effects, 3 more peptides
were synthesized in which single amino acid changes were introduced at residues 77,
80 and 81 to convert the B2702 sequence into the B2705 sequence at that position. The
B2702.75-84(D) and B2702.75-84(L) peptides still blocked Iysis by existing CTL and
;rr~ of pre-CTL while the B2702.75-84(T) peptide had no inhibitory activity.
Thus, the isoleucine at position 80 is required for inhibition.
HLA-B2702.75-84(D) REDLRIALRY
HLA-B2702.75-84(T) RENLRIALRY
Wo 95/13288 2 1 7 6 2 0 ~ PCT/US9~/12985 o
-12-
HLA-B~70~.75-84(L~ RENLRLLEY
It was also found by the following assay that B2702.60-84, B38.60-84 and
B2702.75-84 when pre-bound to plastic caused cells to bind. None of the other
peptides were found to have this effect. However, when the B2702.60-84 peptide was
5 conjugated to bovine serum albumin or to beads via the cysteine at residue 67, the
blocking effect and the ability to bind cells to plastics were lost.
The plastic binding procedure was as follows: peptide (100 ~Lg/ml) was
dissolved in PBS and 50 111 was added to round bottom microtiter wells or 5-10 1ll to
petri dishes. After 60 minutes at ~7 C or overnight at 4', the solution was removed and
the plates washed twice in RPMI-1640 s~rrl~-m~ nt~ with 10% fetal bovine serum.
Cells were added and incubated at 4- for 30 minutes. Binding to petri dishes wasdetermined by inspecting the dishes under a Illil,lU~l_U~lC following gentle agitation.
Binding to microtiter wells was determined after centrifugation at 500 rpm for 3minutes. Cells which did not bind formed a small pellet at the bottom of the well
15 whereas cells that did bind did not form a pellet.
Binding occurred equally well at 4, 25, or 37' and was not dependent on
exogenously added divalent cations since binding was observed in medium containing
EDTA. However, if cells were preincubated with 1% NaN3 or fixed with
~,a,Arulllldldcll~de, no binding was observed, indicating that viable cells and most likely
20 generation of ATP were required.
Isolation and CI.aIA~ ~r~ Of p74
The amino terminal amino groups of the B2702.60-84, B2702.84-75-84,
B2702.84-79/79-84, B2702.84-75T17S-84T, B7.60-84, and B7.84-75nS-84 peptides
25 were conjugated to biotin-(CH)12-for use with strepavidin-agarose (SAA) to isolate the
peptide receptor from 35S-methionine and cysteine labeled cells.
HLA-B2702.60-84 WDRETQICKAKAQTDRENLRIALRY
B2702 84-75-84 Palilld.u~ ; YRLAIRLNERRENLRIALRY
B2702 84-79-84 Palindrome YRLAIRRIALRY
30 B2702 84-75TnS-84T Palindrome YRLAIRLNETRENLRIALRT
B7.60-84 WDRETQICKAKAQTDRESLRNLRGY
B7.84-75nS-84 Palindrome YGRLNRLSERRESLRNLRGY
Two different protocols were used. In the first, the biotinylated peptide was
complexed to the SAA and allowed to bind to labeled cells at 4 C for 30 minutes. The
35 cells were washed free of excess complex and Iysed by addition of CHAPS containing
Iysis buffer. This method ~lcrtlc~ lly ~.c,,i~ ..t., material from the cell surface. In
~VO 95/13288 2 1 7 6 2 0 8 PCT/US9.1/12985~
Ihe second protocol cell ~ysates were prepared in CHAPS ~ysis buffer and the ~ysate
was incubatcd with biotiny~ated peptide/SAA comp~exes for 30 minutes at 4-C fol~owed
by extensive washing. This method preferential~y precipitates inn ArP~ r material
For both methods proteins wcre separated by SDS-PAGE and visua~iæd by
nu J~ . The B2702.84-75nS-84 peptide bnngs down 2 bands of 70 and 74 kD
whi~e the c~osely re~ated B7.84-75J7S-84 peptide does not. The 70 and 74 kD bands
were a~so broughl down with the B2702.60-84 and B2702.84-79/7g-84 peptides but
not with the B7.60-84 peptide. These two bands cou'd be ~ iL. t~ d from the surface
or Iysate of CTL ~ines and periphcral blood Iymphocytes but on~y from the Iysate of
most other cel~s tested. Substitution of either iso~eucine with threonine ~ed to a
signif1cant decrease in the intensity of both bands and the doub~e threonine substituted
peptide B2702.84-75TnS-84T did not precipitate either band.
The 70 and 74 kD bands were subsequently shown to be reactive with
antibodies specific for members of the heat shock protein family. Biotin-conjugated
B2702.84-75/75-84 or B7.84-75/75-84 peptides were used to precipitate proteins from
CTL Following separation by SDS-PAGE proteins were electrophoreticall~
transferred to a ny~on membrane and the Westem b~ot was probed with a m-7m-~Ir-nAI
antibody specific for Hsp70. It is seen that the antibody reacts specifica~y with the p74
protein.
Isoe~ectric focusing on the p74 protein as we~ as on the Hsc70 after
JiLdLiOl~ with either peptide or anti-Hsc70 mAb shows an identica~ molecu~ar
weight and isoelectric points. The B2702 peptide a~so binds to the heat inducible fomm
commonly referred to as Hsp70 although the binding to the Hsc70 is much more
VUIIC~:l. Both forms are expressed on the cell surface and are upregulated
following treatment of cells at 43- for one hour. Expression of the Hsc70 protein
corre~ates with peptide induccd ca~cium increase and the induction of unresponsiveness.
Binding of the B2702 peptide to ~ r -" "~ rl T cel~s was found to result in a
large calcium infLux having a magnitude and kinetics l - 1~ O that induced by
anti-CD3 antibodies in support of the peptide binding to a surface membrane protein
and being involvcd in the dcvcl~--- .-L of anergy. Other Class I HLA ccl helix derived
peptides did not induce this response under ~ conditions.
The correlation between the peptide induced increase in calcium and
iL lLiull of the 70 and 74 kD bands from the surface for a number of different cel~
types is shown in Tab~e 1. The largest increase in intrace~ular calcium is observed in
thehumanCTL PBL Jurkat andratsp~eence~s. WiththeexceptionofJurkat these
Wo 95/13288 2 1 7 6 2 0 8 PCT/US9~/12985 0
-14-
cells also express the highest levels of the 70 and 74 kD bands. Six ~Lg of p70 and p74
have been purified.
Table 1 Effect of B2702.84-75-84 peptide on intr~rPlllllrr calcium and correlation with
~ J;Ldliull of 70 ar d 74 kD bands fronr the cell surface.
CELL CALClUM[nM] 70 kD BAND 74 kD BAND
C~L - CD8+ T cells 500 +++ +++
PBL- Human 250 ++ +++
Jurkat - al3 T cell tumor 400 - +
HUT-78 - a~ T cell tumor 50 - +/-
Peer - ~3 T cell tumor None
HSB - al3 T cell tumor None
NK clones - Human 200 ND ND
JY - B Iy~ Jhul,ldst None - +/-
MS - Burkitt's 100 - +
Daudi - ~2m-Burkitt's 75 - +
K562 - Pre-erythrocyte None
HEL - Pre-erythrocyte None
Rat Iy.. l~Jl,~yLc~ -ACI spleen 200 - +++
An increase in ;"l. ,.r~ Ca2+ induced by peptide was observed in only some
of the cell lines tested. The greatest changes in in~r~rrll~ r Ca2+ were ,~ l inPBL, CTL lines, and the human T cell tumor Jurkat. A smaller but significant increase
in intrAr~ r Ca2+ was induced by the inverted repeat B2702.84-75~75-84 peptide in
two Burkitt's Iymphoma cell lines, Daudi and MS. The B2702.84-75/75-84 peptide
did not cause a Ca2+ flux in the human T cell lines Peer, HUT-78, HSB; human
Epstein Barr virus tr~ncforrnrr1 B cell lines, including JY and 721.221; the natural killer
cell line YT2C2; or several pre-erythrocytic cell lines, including K562 and HEL.Neither the B2702.75-84 peptide nor the inverted repeat B7.84-75/75-84 peptide
initiated Ca2+ Illubili~Liull in any of the cells tested.
The threonine substituted peptides, which failed to cause IIIIIC:~)Ull~ l in T
cells, were also unable to promote Ca2+ mobilization. The maximum intr~r~ r
Ca2+ level induced by the B2702.84-75/75-84T and B2702.84-75T/75-84 peptides
was only 10-30% of that achieved with the lln~llh~ti~l~l~l B2702.84-75n5-84 peptide.
The double substituted B2702.84-75T/75-84T peptide did not initiate any Ca2+ flux.
These results suggest that the B2702 peptides cause an increase in intr~r~ lAr Ca2+
_ _ _ ,, ,
~!0 9sl~3288 2 1 7 6 2 0 8 PCT/US9 ~1298~i
that results in T cell unresponsiveness. The B270~.84-75T/75-84T pept;de, which did
inhibit any T cell responses, did not cause a rise in inrr~rP~ r Ca2+.
Mobili~ation of intracellular Ca2+ is mediated by at least two major
mf ch~nicmc IP3 is one messenger molecule that links surface receptor activation to
S the release of Ca2+ from internal stores by binding to a receptor on the Pnrir~rl~cmir
reticulum. The level of IP3 in cells is elevated following the activation of either G
protein linked receptors or by tyrosine kinase linked receptors. The other major"~ ,- is Ca2+ induced Ca2+ release (CICR). It has recently been rl~mr~nctr~tPd
that a molecule called cyclic adenosine .l,l.l,b,l.l, . ribose (cADPR), a metabolite of
10 .l;~ padenine~ lrlF~ll;flF-(NAD+)~isthemessenger~ Jb~l~;L/lcforregulating
this channel through the ryanodine receptor. Using the T cell line Jurkat, we observed
that stim~ tirm with anti-TCR mAb produced a substantial increase in IP3 whereastreatment with the B2702.84-75n5-84 peptide had no effect on levels of IP3. The
peptide induced increase in intracellular Ca2+ is likely to be mediated through the
15 ryanodine receptor.
Another early event in T cell activation is a change in the patterns of
phosphorylation of many proteins. Therefore, it was determined whether the peptides
that inhibited T cell function affected protein ~ llulyldP~oll or the level of;..., ~, . 11"1~.
calcium. None of the peptides examined thus far has had any effect on the pattern of
20 tyrosine phosphorylation observed by Western blot analysis in whole Iysates from T
cells.
A C~ dli~J-- of the effects of the B2702 84-75-84 palindrome with other
known ;II,Il~ dlll~ f' a novel pattern of activity. The results are
~.~....A.;,..lil~T~ble2
Wo 95113288 2 1 7 6 2 0 8 PCT/US9~/1298~ 0
-16-
Tab~- 2
CSA FK~06 Rapamycin d~u~ya~ udlin B2702
Block T cell +++ +++ +++ +++ +
proliferation
Block Cytotoxiciy - - - - +++
Block Cytokine +++ +++ - ND ND
Block IL-2 +++ ND - ND ND
T"" . ~
Induce Ca++ flux - - - - +++
Block calcineurin +++ +++ - ND ND
Cellular Receptor Cyclophilin FKBP FKBP Hsc70 p74
The data d~ oll~LIaL~s that B2702 haâ a novel pattern of activity, as compared
to the known immunusu~ ;s~ànts~ cyclosporin A, FK~06, rapamycin and
5 deoxyspergualin.
It is evident from the above results, that p74 plays an important role in the
mt~ tirm of CTL activity. By controlling protein interactions resulting in signal
transduction or the absence of signal ~ lc ~ 111, CTL ~irr~ .c~id~iul~ and cytolytic
activity can be modulated.
All ~ c and patent A~ Al;llllC cited in this cre~ rir)n are herein
i~lcullJuldl~;d by reference as if each individual publication or patent application were
specifically and individually indicated to be i~,u~ ,d by reference.
Although the fo}egoing invention has been described in some detail by way of
illustration and example for purposes of clarity of ulld~,laLdlldillg, it will be readily
15 apparent to those of ordinary skill in the art in light of the teachings of this invention
that certain changes and " " K 1; r~ may be made thereto without departing from the
spirit or scope of the appended claims.
~VO 95/13288 2 1 7 6 2 0 8 PCT/US9 ~/12985
--17--
SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) A?PLICANT The Board of Trustees for the Leland Stanford Junior
University
(ii) TITLE OF ~NVENTION SURFACE MEMBR~NE PROTEINS AND THEIR
EFFECT ON IMM[~NE RESPONSE
(iii) NtlMBER OF SEQUENCES 20
(iv) ~U~ UN~4N ~ ADDRESS
A) T~nnVRCCRR FLEHR, HOHBACH, TEST, ALBRITTON ~/ ~ERBERT
B) STREET 4 r ' C~dT LU Center, Suite 3400
C) CITY San Francisco
D) STATE l"Alil'nrrliA
, E ) COUNTRY USA
F) ZIP 94111-4187
(v) COM?UTER READABLE FORM
(A MEDIUM TYPE Floppy disk
(B COMPUTER ~BM PC compatible
(C OPERATING SYSTEM PC-DOS/MS-DOS
(D SOFTt\TARE PatentTn Release #1 0, Version #1 25
(vi) CURRENT A?PLICATION DATA
(A) A?PLICATION N0MBER PCT/USs4/
(B) FILING DATE 10-NûV-1994
( C) CLASSIFICATION
(vii) PRIOR A?PLICATION DATA
(A) A?PLICATION N[~MBER 08/150,493
(B) FILING DATE 10-NOV-1993
(viii) ATTORNEY/AGENT INFORMATION
(A) NAME Rowland, Bertram I
(B) REGISTRATION NtlMBER 20, 015
(C) ~~ /DOCl~ET N~MBER FP-58976/BIR
(ix) TEL~;~VI_ JNl~TION INFORMATION
(A) TELEPHONE (415) 781-1989
(B) TELEFAX (415) 398-3249
(C) TELEX 910 277299
(2) INFORMATION FOR SEQ ID NO 1
( i ) SEQUENCE ~TD ~ A ~
A LENGTH 2 0 amino acids
B TYPE amino acid
C sTR7lT~RnNEcc single
~ D TOPOLOGY linear
(ii) MOLECULE TYPE peptide
(xi) SEQUENCE ~ U~ SEQ ID NO 1
Tyr Arg Leu Ala Ile Arg Leu Asn Glu Arg Arg Glu Asn Leu Arg Ile
Ala Leu Arg Tyr
(2) INFûRMATION FOR SEQ ID NO 2
(i) SEQUENOE r~rTv~ ;1lU j:
(A) LENGTH 20 amino acids
-
WO 95113288 - 2 1 7 6 2 0 8 PCTII~S9~/1298~ 0
-18-
(B) TYPE: amino acid
(C) STR~ nNRcq single
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Tyr Gly Arg Leu ~sn Arg Leu Ser Glu Arg Arg Glu Ser Leu Arg Asn
eu A~g Gly Tyr
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE rp~ DrTR~r.qTIcs
A) LENGTB: 25 amino aclds
B) TYPE: amino acid
C) ST~ r~n~Rqs single
D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE L'~;S~ llUlY: SEQ ID NO:3:
Trp Asp Arg Glu Thr Gln Ile Cys Lys Ala Lys Ala Gln Thr Asp Arg
lD l5
Glu Asn Leu Arg Ile Ala Leu Arg Tyr
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE r~DrTR~TqTICS:
A LENGTB: 20 amino acids
B' TYPE: amino a~id
C sT~D~nRnN~.q~: single
,D TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Tyr Arg Leu Ala Ile Arg Leu Asn Glu Arg Arg Glu A~n Leu Arg Ile
~ 10 15
la Leu Arg Tyr
(2) INFORMATION FOR SEQ ID NO:5:
(i) sEgJENcE rp~ rTRl7rqTIcs
A LENG~: 12 amino acids
B TYPE: amino acid
C sT~Rn~Ecq: single
1 D TOPOLQGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Tyr ~rg Leu Ala Ile Arg Arg Ile Ala Leu Arg Tyr
~VO 95/13288 2 1 7 6 2 0 8 PCT/US94/1298~
--19-
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE r'BZ~PrTT;'RTCTICS:
(A) LENGTH: 20 amino acid6
(B) TYPE: amino acid
(C) 5T~NDRn~Rqq 6ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Tyr Arg Leu Ala Ile Arg Leu A6n Glu Thr Arg Glu A6n Leu Arg Ile
5 10 15
la Leu Arg Thr
(2) INFORMATION FOR SEQ ID NO:7:
( i ) SEQUENCE r~TP~ p ~ LW
A) LENGTH: 25 amino acids
B) TYPE: amino acid
C) ST~ ~CC: 6ingle
'D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Trp A6p Arg Glu Thr Gln Ile Cy6 Lys Ala Ly6 Ala Gln Thr A6p Arg
Glu Ser Leu Arg A6n Leu Arg Gly Tyr
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQ-~ENCE rup~ T7~TcTIcs
AI LENGTH: 20 amino acids
B TYPE: amino acid
C ST~ nN~qq ~ingle
D I TOPOLOGY: lir~ear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE l~ ~LlUN: SEQ ID NO: 8:
Tyr Gly Arg Leu A6n Arg Leu Ser Glu Arg Arg Glu Ser Leu Arg Asn
eu Arg Gly Tyr
(2) INFORMATION FOR SEQ ID NO:g:
(i) SEQUENCE ~P~Pr~TF~TCTICS:
(A I LENGTH: 10 amino acid6
(B TYPB: amino acid
(C sT~ n~c~: single
(Dl TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE J~;S~l:Clrll~?N: SEQ ID NO:g:
WO 95113288 2 1 7 6 2 0 8 PCT/US9~1112985 0
-20 -
Ary Glu Asp Leu Arg Ile Ala Leu Arg Tyr
~2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE r~T~TPrT~TCTIC5:
~A LENGT~ 10 amino acids
B TYPE: amino acid
C ST~Nn~n~ECC: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQIJENCE ~ b~ lUN: SEQ ID NO:10:
Arg Glu Asn Leu ~ Thr Ala Leu Arg Tyr
rg 10
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENOE rT~ rT~TqTICS:
A) LENGTH: 10 amino acids
B) TYPE: amino acid
C) ST~NnRnl~T~cc: ~ingle
D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Arg Glu Asn Leu Arg Ile Leu Leu Glu Tyr
(2) INFORMATION FOR SEQ ID NO:12:
~i) SEQ-JENCE rF~ rT~Z~TqTICS:
A LENGTH: 10 amino acids
B TYPE: amino i~cid
C sT~ nNRqc: ~ingle
ID. TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Arg Glu Asn Leu Arg Ile Ala Leu Arg Tyr
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE r~ rT~TcTIcs
(~ LENGTE: 10 amino acids
(B TYPE: amino acid
(C ST~ ~TlnNF~c single
(D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
rg Glu Asp Leu Arg Thr Leu Leu Arg Tyr
5 10
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE r~T~rT~7TCTICS:
(A) LENGTH: 10 amino acids
~WO 95/13288 2 1 7 6 2 0 8 PCT/US9~/12985
--21--
(B) TYPE: amino acid
(C) sT~zlNn~nN~c.~: single
(D) TOPOLOGY: linear ~-:
(ii) MOLBCULE TYPE: peptide
(xi) SEQUENCE ~ ~lrLlUN: SEQ ID NO:14:
Trp Asp Arg Glu Thr Gln Ile Cys Lys Ala
(2) INFORMATION POR SEQ ID NO:15:
(i) SEQUENCE r~7\DprT~TcTIcs:
(A LENGTH: 25 Lmino acids
(B, TYPE: amino acid
(C sTRLNn~nN~ cc ~ingle
(D TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Trp Asp Arg Glu Thr Gln Ile Cys Lys Ala Lys Ala Gln Thr Asp Ar
Glu Acp Leu Arg T}Lr Leu Leu~Arg Tyr 15
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE rT~p~prTR~TcTIcs:
Al LENGTH: 25 amino acids
B TYPE: amino acid
C sTRpl~Tnli:nN~!cs: ~ingle
I D TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi ) SEQUENCE DESCRIPTION: SEQ ID NQ :16:
Trp Asp Arg Glu Thr Gln Lys Tyr Ly~ Arg Gln Ala Gln Thr Asp Arg
Val Ser Leu Arg Asn Leu Arg Gly Tyr
(2) INFORMATION' FOR SEQ ID NO:17:
(i) SEQUENCE rrPl~prT~TcTIcs~
A) LENGTH: 25 amino acids
B) TYPE: amino acid
C) sT~Nn~nN~:.C: single
ID) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE l,l~ llUN: SEQ ID NO:17:
Trp Asp Arg Glu Thr Gln Ile Ser Ly~ Thr Asn Thr Gln Thr Tyr Arg
lu Ser Leu Arg Asn Leu Arg Gly Tyr
20 25
(2) LNr1 --TrN FOR SEQ ID NO:18:
(i) SEQ~ENCE rFrP~P~ 5:
WO95113288 21 7 6 2 0 8 PCTIUS9~/1298: 0
(A) LENGT~: 25 amino acids
(B) TYPE: amino acid
(C) sTl7~Mn~nN~cq: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Trp A~p Gly Glu Thr Arg Lys Val Lys Ala His Ser Gln Thr His Arg
5 l0 15
Val A6p Leu Gly Thr Leu Arg Gly Tyr
(2) ~NFORMATION FOR SEQ ID NO:l9:
( i ) SEQUENCE r~
A) I.ENGTH: 25 amino acids
B) TYPE: amino acid
C) ~ bb: single
D) TOPOLOGY- liDear
(ii) MOLECI~LE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:
Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu
Glu Gly Glu Cys Val Glu Trp Leu Ar
(2) INFO~MATION FOR SEQ m NO:20:
(i) SEQ-~ENCE r~T3~'T~.~rCTICS:
A LENGTH: 25 amino acids
B TYPE: amino acid
C 1 ~ ~ single
~ D' TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQ~ENCE L~ ~: SEQ ID NO: 20:
Trp Asp ~rg Asn Thr Gln Ile Cys Lys Thr Asn Thr Gln Thr Tyr Arg
5 10 15
Glu Asn Leu Arg Ile Ala Leu Arg Tyr
