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IMMUNOLOGICAL TOLERANCE TO HIV EPITOPES
Statement of Rights to Inventions Made Under Federally Sponsored Research
This invention was funded in part by a grant from the National Institute of
Allergy and Infectious Diseases, National Institutes of Health, which provides
to the
United States goverrunent certain rights in this invention.
BACKGROUND OF THE INVENT10N
Field of the Invention
The invention in the fields of immunology, molecular biology and medicine
relates to compositions, primarily fusion immunoglobulins, and methods useful
for
inducing a state of irnmunological tolerance to selected epitopes of human
immunodeficiency virus (HIV) gp120 or target epitopes assocaited with other
diseases. Administration of these composition will induce and maintain
tolerance to
the epitopes in a subject infected with (or at high risk for) HIV, or in whom
an
immune response to a different target epitope is deleterious. Prevention of
antibody
responses to the selected HIV epitopes promotes survival of the host immune
system
and contributes to treatment of HIV disease. The compositions are also useful
as
adjuncts to HIV or other virus vaccines in modulating the immune response to
maximize induction of protective anti-viral T cell immunity.
Description of the Background Art
Immunological tolerance (hereinafter "tolerance"), the basis of the lack of
reactivity of the immune system to self components, can also be induced
artificially
by a wide variety of manipulations. Hence, an animals can be rendered tolerant
to
antigens which are foreign. Autoimmunity is thought to result in part from the
breakdown of tolerance to previously tolerated antigens.
A variety of experimental procedures are known for inducing antigen-specific
tolerance in neonates and adults (Billingham, R.E. et al. (1953) Nature
172:603-606;
Chiller, J.M. et al. (1970) Proc. Natl. Acad. Sci. USA. 65:551-556; Borel, Y.
et al.
(1973) Science 182:76-7$). In the immunocompetent adult, tolerance induction
has
been generally more; difficult. Tolerance to foreign transplantation antigens
or viral
CTL epitopes, for e:Kample, was most effective in models where hematopoietic
or
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lymphoid ablation was followed by reconstitution with antigen-expressing bone
marrow (BM)-derived antigen-presenting cells (APC) (Ildstad, S.T. et al. (
1984)
Nature 307:168-170; Cobbold, S.P. et al. (1984) Nature 312:548-551; Roberts,
J.L. et
al. (1990) J. Exp. Med. 171:935-940; Oehen, S.V., et al. (1994) Cell. Immunol.
158:342-352; Nemazee, D. et al. (1989) Proc. Natl. Acad. Sci. USA 86:8039-
8043).
For autoimmune diseases, studies have focused on the acquired induction of
tolerance
to autoantigens to prevent and/or ameliorate disease. For example, in marine
models
of multiple sclerosis or diabetes, prevention of disease has been accomplished
with
intrathymic, oral, or intravenous administration of , high doses of target
autoantigens
(Tisch, R. et al. (1993) Nature 366:72-75; Higgins, P.J. et al. (1988)
J.Immunol.
140:440-445; Critchfield, J.M., et al. (1994) Science 263:1139-1143).
One well-known way to induce tolerance is by attaching the antigenic
determinant or epitope to be tolerated to isologous or heterologous
immunoglobulin
(Ig) molecules, primarily of the IgG isotype. Such molecules are termed
"tolerogenic
carriers" or "tolerogens" (Scott, D.W. (1979) Immunol. Rev. 43:241 ). Igs of
different
origin may vary in their persistence in an animal after administration andlor
in the
mechanism by which they induce tolerance. However, IgG earners have been by
far
the most efficacious inducers in adult animals of tolerance to haptens,
nucleosides and
peptides (Borel, Y. (1980) Immunol. Rev. 50:71; Scott, D.W. (1976)
Celllmmunol.
22:311 ). These earners owe their superior tolerogenicity to their persistence
in vivo
and the ability of an epitope chemically attached to the IgG molecule to
crosslink
membrane IgM (mIgM) on the surface of B lymphocytes with surface Fc receptors.
However, chemical coupling of epitopes to IgG carriers can be limi'ed by the
availability of free reactive amino groups, structural change of the epitope
as a result
of the coupling reaction, and the uncontrolled targeting of the added
determinant to
different portions of the IgG.
Protein engineering strategies have been used to create molecules containing
heterologous epitopes for the amplification of specific immune responses. For
example, heterologous oligopeptide epitopes of immunological interest have
been
inserted in-frame into bacterial flagellin (Newton, S. et al, (1989) Science
244:70-72;
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Jennings et al., (1989) Protein Eng. 2:365), influenza virus nucleoprotein
(Chimini,
G. et al. (1989) J. E.xp. Med 169:97-302), hepatitis B surface antigen
(Rutgers et al.,
(1988) BiolTechnology 6:1065) and in the complementarity determining regions
(CDR) of immunoglobulins (Billetta, R. et al., (1991) Proc. Natl. Acad. Sci.
USA
88:4713-4717; Zaneati et al. (1992) Nature, 355:476; Zanetti et al.
W090/090804);
Zaghouani, H.et al. (1993) Science 259:224-227; Zaghouani, H. et al., (1993)
Int.
Rev. Immunol. 10:265-278; Zaghouani, H. et al. (1995) Proc. Natl. Acad. Sci.
USA
92: 631-635 ).
Attempts have been made to test the ability of such a recombinant protein to
induce an enhanced immune response to the heterologous oligopeptide. A peptide
immunoglobulin fu;>ion Ig protein or referred to herein as a "fusion Ig" or
"flg" has
been used to induce immunity. For example, a flg was made which expressed in
the
CDR3 of its VH region the repetitive tetrapeptide Asn-Ala-Asn-Pro (SEQ ID NO:1
),
designated (NANP),~ (in single letter amino acid code), of the
circumsporozoite
1 S protein of Plasmodium falciparum, an etiologic agent of malaria (Billetta
et al.,
supra). A monoclonal antibody (mAb) specific for (NANP)~ which was made
against
P. falciparum bound to the above flg and was blocked by a synthetic (NANP)3
peptide. Immunization of rabbits and mice with the engineered fig in adjuvant
elicited antibodies to the (NANP)3 synthetic peptide and to P. falciparum
parasite.
Such antibodies efficiently inhibited the invasion of cultured liver cells by
P.
falciparum. Thus, immunity to malaria was induced in the absence of the
parasite
using antibody V regions engineered to mimic the parasite's molecular
structure. The
authors suggested that antibody (idiotype) mimicry of an exogenous antigen is
possible and may only require a discrete stretch of identity for successful
mimicry.
An alternate and simpler explanation of these results by the present inventors
is that
this material, when administered in adjuvant, simply acted as an immunogenic
hapten-
earner conjugate. C. Bona et al., (1994) Cell Mol. Biol. 40 Suppl 1:21-30
expressed
viral epitopes on Ig molecules by replacing the D segment of a V~y gene with a
B cell
epitope from the V3-loop of HIV-1 envelope glycoprotein gp120, a cytotoxic T
lymphocyte (CTL)-epitope from influenza virus nucleoprotein or a T helper
epitope
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from influenza hemagglutinin. The T cell-targeted peptides in the form of flg
molecules produced by cells transfected with chimeric V genes, activated
specific T
cells. The authors speculated about possible practical applications for Ig
molecules
bearing foreign epitopes for the development of prophylactic and
immunotherapeutic
reagents.
It is noteworthy that Zanetti et al. (supra) and Bona et al. (supra) produced
chimeric Ig molecules (which are figs as the term is used herein) for the
purpose of
immunization (vaccination), not tolerization. Although W090/09084 casually
proferred a speculative notion, lacking any particularity or evidence, that
this type of
construct could be used for tolerization, the authors provided no scientific
basis for
such a utility. In fact, the way in which their exogenous epitope was inserted
into the
Ig framework region resulted only in immunogenic, not toierogenic, constructs.
The
Zanetti et al. reference therefore lacks any proof that its inventors were in
possession
of a tolerogenic fIg and provides no enabling support for ~ tolerogenic
molecule or
preparation. Hence, the induction and maintenance of tolerance to
oligopeptides
presented to the immune system via engineered Ig proteins has not been
demonstrated
prior to the invention as described herein. In particular, the art has not
seen the use of
cells (expressing such fIg molecules) as agents of epitope-specific tolerance
induction
or maintenance. The present invention is the first discovery of tolerogenic
cellular
engineering to achieve a meaningful effect with therapeutic utility.
In summary, the art recognizes that recombinant fusion proteins, including flg
proteins, may be useful as immunogens to induce immune responses to the
heterologous oligopeptide. However, there remains a recognized need to develop
general and specific methods of inducing stable, long-lasting tolerance to any
of a
number of epitopes of clinical significance in a subject. Also needed are
vectors that
can introduce the target epitope to which tolerance is desired into a host
cell or whole
animal, such that the epitope (a) induces tolerance and (b) persists in vivo
so that it
maintains the tolerant state. It is essential that any tolerization protocol
include a
means to maintain the specific state of tolerance. Maintenance of tolerance is
understood to require the persistence of the tolerogenic epitope in vivo
(Smith, R.T.
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( 1961 ) Adv. Immunol. 1:67; Golub, E.S. et al. ( 1967) J. Immunol. 99:6;
Ramsdell, F.
et al. (1992) Science 257:1130-1134).
The present inventors were the first to discover am approach that not only
could induce tolerance to an oligopeptide presented to the immune system in
the form
5 of a recombinant f(g protein but also could maintain a tolerant state in the
subject.
See also Scott and Zambidis, co-pending, commonly assigned application
U.S.S.N.
08/195,874 (allowed) and W095/21926, which applications are hereby expressly
incorporated by reference in their entirety.
One of the present inventors' central hypotheses for explaining the signalling
process in tolerance is that crosslinking with anti-~t chain antibodies
provides "signal
1" to B cells, which, in the absence of T cell help (signal 2), leads to
anergy. At high
concentrations of anti-p, extensive crosslinking of IgM leads to a significant
level of
B-cell apoptosis because a greater proportion of the B cells are forced to
exit the Go
phase and enter the cell cycle. This effect can be mimicked by multivalent
antigen in
specific B cells (Carsetti, R. et al., (1993) Eur. J. Immunol. 23:168). A
unifying
explanation for various experimental results is that that multiple
crosslinking events
are necessary for the: inductian of apoptosis. (See, also, Warner, G. et al. (
1991 ) Cell.
Immunol., 138:404; Scott, D.W. et al. (1987) Immunol. Today, 8:105; Ales-
Martinez,
J.-E. et al. (1992) Se~m. in Immunol. 4:195; Scott et al. (1996) Intern.
Immunol.
9:1375-1385).
The Immune Response to HIV gp120 and its Role in AIDS
The immune response to HIV has been studied extensively. Early studies
suggested a role for neutralizing antibodies in protection or containment of
HIV
infection. This is particularly true in the case of simian immunodeficiency
virus
- 25 (SIV), a relative of HIV, where a cloned virus could be employed (Burns,
D. et al.
(1993) J. Virol. 67:4104). Neutralizing antibodies to the epitopes of the
envelope
glycoprotein gp120, especially the V3 loop, have been described in infected
individuals. However, more recent evidence suggests that the antibody response
to
HIV may not be protective and may, in fact, contribute to the progression of
disease
(Ftist, G. et al. (199:5) Immunol. Today, 16:167; Wang, S. et al. (1994)
Virology
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199:247; Banda, N. et al. (1992) J. Exp. Med. 176:1099). Thus, while antibody
responses against various epitopes of HIV clearly occur, the effectiveness and
therapeutic significance of these responses is unclear.
The CD4 molecule on T lymphocyte serves as receptor for major
histocompatibility complex (MHC) class II antigens and is referred to as
"coreceptor"
because its engagement synergizes with engagement of the T cell receptor for
antigen
(TCR) in activating the cells. When CD4 molecules were engaged by antibody
independently of the TCR (in murine studies), the T cells were induced to
undergo
apoptosis (Wang, Z.Q. et al. (1994) Eur. J. Immunol. 24:1549-1552). Thus,
besides
functioning as a coreceptor with the TCR, CD4 has a function of its own in
facilitating the induction of apoptosis. CD4 also serves as a cellular binding
site or
receptor for the HIV gp 120. In transgenic mice expressing a human CD4
transgene,
appropriate crosslinking of gp120 caused massive deletion of HIV-reactive T
cells in
vivo (Wang, Z.Q. et al. (1994) Europ. J. Immunol. 24:1553-1557). If T cells in
which
CD4 is engaged by anti-CD4 antibody administration are capable of expressing
functional Fas protein on their surface, they degrade their DNA and
disintegrate
rapidly.
Antibodies to gp120 can lead to enhancement of HIV entry into non-T cells
via Fc receptors (Homsy, J. et al. (1989) Science 244:1357, supra). Uptake of
complexes between HIV and anti gp120 antibody by cells of the immune system,
particularly monocytes, can result in establishment of a latent, subclinical
infection
and a virus reservoir susceptible to later activation(Kliks, S.C., (1993)
Proc. Natl.
Acad. Sci. USA 90:11518)). HIV-infected patient sera frequently contain
antibodies
against a peptide of the gp120 C5 region which cross-react with HLA-C
monomorphic determinants (DeSantis, C. et al. (1993) J. Infec. Dis. 168:1396;
Palker,
T.J. et al. (1987) Proc. Nat'I Acad. Sci. USA 84:2479). Though apparently not
causing autoimmune damage, the antibodies are an example of non-protective
antibodies produced during HIV disease. In an equine retroviral disease model,
an
antibody response to a variant virus (EIAV) may end in more extensive disease
(Cook, R. et al. (1995) J. Virology 69:1493). Moreover, production of non-
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neutralizing anti-HfV antibodies (specific for "irrelevant" or "ineffective"
epitopes)
may pre-empt the formation of antibodies to important, neutralizing epitopes.
In AIDS, shifts in cytokines from those produced by TH2 cells to cytokines
made by TH1 cells were observed (Clerici et al. (1994) Proc. Natl. Acad. Sci.
USA
91:11811; Gougeon, M-L. et al. (1993) Science. 268:1269; Ameisen, J-C. (1992)
Immunol. Todav. 13:388). While controversial (Fauci, A. (1993) Science
262:1011),
such shifts may contribute both to apoptosis and to hypergammaglobulinemia.
The
findings discussed above have led those developing the next generation of
vaccines to
re-direct the immune response in patients and to devise T-cell peptides that
serve as
stimulatory ("vaccine") and target epitopes for cytotoxic T lymphocytes (Salk,
J. et ul.
(1993) Science 260:1270; Cease, K.B et al. (1994) Ann. Rev. Immunology.
1?:923.
Evidence obtained in the last few years suggests that HIV may subvert the
immune response through the interaction of viral gp120 with the CD4 receptor
on T
cells. Observations from Finkel's laboratory (Finkel et al., supra; Banda et
al., supra)
and of Newell et al. ( 1990) Nature 347:286), indicate that crosslinking of
CD4 on the
T cell surface may prime T cells for apoptosis, perhaps via the upregulation
of the Fas
molecule, CD95 (Oyaizu, N. et al. ( 1994) Blood 84:2622; Desbarats, J. et al.
( 1996)
Proc. Natl. Acad. Sci. USA 93:11014-11018. Even picomolar concentrations of
gp 120 could prime 'C cells for such activation-induced death.
Apoptosis in. normal, non-infected ("bystander") CD4+ T cells may be
programmed by ( 1 ) allowing gp 120 proteins to bind to CD4 via their natural
affinity,
and then (2) adding anti-gp 120 antibodies to bind and crosslink the gp 120-
CD4
complexes (Finkel eat al., supra; Banda et al., supra). When such programmed
or
"primed" cells are biggered through their TCR, apoptosis follows. This is
reminiscent of the increased rate of apoptosis observed in vitro in T cells
from HIV-
infected subjects (Gougeon, M-L. et al. (1993) Science. 268:1269; Ameisen,
supra)
and provides one explanation for CD4+ T cell depletion in AIDS: According to
this
view, concurrent ini:ection by other organisms or any antigenic challenge for
that
matter would trigger the death of those T cells which bear a TCR recognizing
these
antigens and in which the CD4 molecules have been crosslinked via gp120 and
anti-
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gp120. Such a mechanism may also explain (1) the enhancement of infection
brought
about by certain anti-HIV antibodies, and (2) the paradox that HIV appears to
cause
AIDS after the onset of antiviral immunity.
Ex vivo analysis of CD4+ T cells (without prior culture) indicates that the
increased apoptosis representes a process ongoing in vivo. Varying numbers of
CD41
as well as CD8+ cells in lymph nodes (LN) of HIV infected subjects are
undergoing
apoptosis. In infected adults and children, a fall in the CD4/CD8 ratio
correlates with
increasing apoptosis of CD4+ cells which correlates with CD4 depletion and
disease
severity. In infected humans and monkeys, most apoptosis in the LNs was
occurring
in "bystander" (uninfected) cells. Among infected cells, those expressing
lower levels
of the HIV p24 protein showed higher levels of apoptosis than cells expressing
higher
levels of p24. Thus, the majority of apoptosis appears to takes place in HIV- -
cells,
and the majority of apoptotic cells are HIV- or HIVE°"".
The mechanism for such T cell apoptosis has been suggested by Pahwa and
colleagues (Oyaizu, N. et al. (1993) Blood 82:3392-3400) who examined
apoptosis as
a mechanism for CD4+ T cell depletion in HIV-1 infection. They showed that
( 1 ) patient blood mononuclear cells underwent marked spontaneous apoptosis;
(2) stimulation of patient and normal T cells resulted in increased apoptosis;
and
(3) cross-linking of CD4 molecules was sufficient to induce apoptosis in CD4+
T cells
if cross-linking was performed in unfractionated blood mononuclear cells {but
not in
purified T cells). The accelerated cell death through apoptosis was concluded
to play
an important role in the pathogenesis of HIV=1 infection, and crosslinking of
CD4 in
vivo contributed to this mechanism. Cross-linking of CD4 molecules, induced
either
by anti-CD4 monoclonal antibody (mAb) or by HIV-1 envelope protein gp160
(which
includes gp 120) upregulates Fas mRNA and Fas antigen expression in normal
lymphocytes (Oyaizu et al. (1994) surpra). Upregulation of Fas antigen closely
correlated with apoptotic cell death. CD4 cross-linking resulted in the
induction of
interferon-y (IFNy) and tumor necrosis factor-a (TNF-a) in blood cells, both
of which
cytokines contributed to Fas upregulation. Anti-IFN-y and anti-TNF-a
antibodies
blocked crosslinking-induced Fas upregulation and lymphocyte apoptosis. Hence,
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aberrant cytokine secretion induced by the crosslinking of CD4 and the
consequent
upregulation of Fas antigen were concluded to play a critical role in
triggering
peripheral T cell apoptosis and thereby contribute to HIV disease
pathogenesis.
Cross-linking of the: CD3 molecule (present on all T cells) caused an increase
in the
Fas ligand. This combination of increased expression of Fas and Fas ligand led
to
apoptosis.
Based on thE; foregoing, the present inventors have concluded that the
antibody
response to gp120 in an infected subject is an important pathway leading to
AIDS
progression due to the pathogenetic component of CD4' T cell depletion through
bystander apoptosis as described above. Therefore, they have developed novel
compositions and methods based on their general, flexible approach to the
induction
and maintenance of epitope-specific tolerance to eliminate virus-specific
immune
responsiveness. In lparticular T helper cell and/or antibody responsiveness to
one or
more epitopes of viral gp120 is prevented or inhibited through the induction
and
1 S maintenance of immune tolerance in T helper cells, B cells or both that
are specific for
one or a number of ;selected HIV gp120 epitopes.
Furthermore, the present inventors have extended this approach to the
induction of tolerance to any antigen, be it an autoantigen, an antigen of a
microorganism or a tumor antigen, against which an undesired antibody response
or T
helper cell response occurs in a disease setting and is pathogenic or
otherwise
deleterious to the host.
Citation of t:he above documents is not intended as an admission that any of
the foregoing is pertinent prior art. All statements as to the date or
representation as
to the contents of these documents is based on the information available to
the
applicant and does not constitute any admission as to the correctness of the
dates or
contents of these documents.
SUMMARY OF THE INVENTION
The present inventors have devised novel fusion proteins and DNA constructs
coding therefor. The fusion protein includes a desired peptide epitope or
several
epitopes, toward which immune tolerance is to be established, inserted in
particular
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sites of the immunoglobulin {"Ig") heavy ("H") chain. This product is termed a
"fusion immunoglobulin" and is abbreviated "fIg" herein. A preferred flg
includes an
epitope or epitopes of HIV-1, most preferably from the gp120 glycoprotein of
HIV-1.
To make this construct, DNA encoding the targeted epitope or epitopes is
inserted "in
5 frame" within a DNA construct encoding the Ig heavy (H) chain. If two or
more
targeted epitopes are included, they exist as contiguous or non-contiguous
sequences
in the protein from which they are derived, and may be either linear or
conformational
cpitopes.
This fusion protein construct is then transfected into a cell line, preferably
a
10 myeloma or other line of B lymphocyte lineage (such as a human cell line
transformed
by Epstein-Barr virus) that produces Ig light (L) chains but that cannot
produce H
chains due either to a spontaneous or induced mutation. When the transfected
Ig H
chains are synthesized, they combine naturally with the host cell's Ig L
chains to form
complete immunoglobulin molecules (HzLz) which are secreted. This resultant Ig
1 S fusion protein contains the desired target epitope (or epitopes)
preferably in its N-
terminal region and functions as a tolerogen for both B cells and T cells and
induces
tolerance in vivo. Transgenic mice producing such a fusion protein are highly
tolerant
immunologically to the epitopes included in the flg. The present inventors
have
found that Ig fusion proteins such as these can be presented to the immune
system in a
tolerogenic fashion, either as an flg preparation or in the form of transgenic
hemopoietic precursor cells or B cells expressing the flg, to induce both B
and T cell
tolerance to the targeted HIV-1 gp120 epitope..
The present inventors have conceived of an approach that is useful in
producing improved and effective immunity against a virus, in particular,
human
immunodeficiency virus (HIV-1, HIV-2) by inducing tolerance to selected
nonprotective viral epitopes as discussed above. Thought a peptide comprising
the
desired epitopes can be chemically attached to an autologous Ig carrier for
tolerance
induction, the present inventors have created a fIg comprising one or more
peptide
epitopes and the Ig H chain using recombinant methods as described herein.
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The invention specifically involves:
1. engineered synthesis of peptide immunoglobulin fusion proteins that are
highly tolerogenic.
2. cells transduced with DNA encoding such an engineered flg to induce, but
more importantly, to maintain, a state of tolerance to the peptide epitopes.
The invention provides polynucleotides encoding the flg in the form of
recombinant DNA molecules in vehicles such as plasmid and retroviral vectors,
capable of expression in a desired eukaryotic host cell as disclosed herein.
The
invention also provides hosts transfected or transduced with the flg
constructs which
are capable of producing in culture or in vivo the flg molecules and secreting
them or
displaying them on the cell surface.
This invention is useful for the treatment of any disease in which immunologic
reactions are pathologic. The best-known examples are in infectious and
autoimmune
diseases. In many types of infections, where the host response to the organism
1 S damages the host. :For example in certain arenavirus infections (for
example,
lymphocyte choriomeningitis virus infection), the T cell response is
responsible for as
much or more pathology than the virus itself. Antibody responses and the
interaction
of the antibodies with complement is responsible for the hemorrhagic shock
syndrome
elicited by flaviviruses (in particular) dengue virus or arenaviruses, such as
Junin
virus which causes Argentinean hemorrhagic fever. In both the latter cases, an
efficient immune response leads to disaster for the host. Other examples of
diseases
for which the present invention can be used include viral diseases wherein
virus-
antibody complexes damage the host. For example, infants congenitally infected
with
cytomegalovirus have such circulating complexes that are deposited in the
kidney.
Patients with hepatitis B virus infection have circulating complexes that
result in
arthritis and glomevrulonephritis. Antibodies generated against a virus can
also act as
autoantibodies directed against normal tissues, even tissues not infected
directly by
the virus. An example of this is the polyendocrinopathies that develop in
newborn
animals infected with reovirus type 1 in whom antibodies against antigens in
pancreatic islets, th:e anterior pituitary and the gastric mucosa have been
observed.
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Such examples abound in the art and may be found in any comprehensive treatise
on
microbiology or infectious diseases.
More classical autoimmune diseases are also treatable by the present invention
as either cell-mediated or antibody responses to organ-specific antigens or
common or
cross-reactive antigens are the known pathogenic agents. Specific tolerance
induced
by an fIg of this invention is a promising therapeutic approach to the
treatment of
many types of autoimmune disease.
The invention provides an individual flg H chain or flg H chain dimers. Also
provided by the invention is an flg molecule comprising (i) two different H
chains,
one of which is a fusion protein having one or more HIV gp 120 epitopes
included in
the V region, preferably at the N-terminus of a framework region, most
preferably of
the first framework region, and (ii) native L chains. Preferably, both H
chains of the
flg molecule are the fused H chains.
Specifically, the present invention is directed to a fusion immunoglobulin
(flg)
1 S heavy (H) chain protein comprising a mammalian, preferably human, Ig H
chain
fused in frame after the leader in its N-terminal region to one or more HIV
gp120
epitopes, wherein the flg H chain is tolerogenic in a host with respect to the
gp120
epitopes. The tolerogenic epitope(s) is or are fused to the variable region of
the Ig H
chain, preferably at the N terminus of a framework region of the variable
region.
Most preferably the HIV gp120 epitope or epitopes are fused to the N-terminal
amino
acid residue of the mammalian Ig H chain such that all amino acids encoding
the
gp 120 epitope or epitopes are N-terminal to the Ig-encoding amino acids.
Also provided is an intact fIg protein comprising two Ig H chains and two Ig L
chains, wherein at least one of the H chains is the fIg H chain described
above.
Preferably, both of the H chains are the above flg H chains. A preferred Ig is
one
which fixes complement and has a longer serum half life. Thus, in a preferred
embodiment, the fIg H chain is an Ig y chain, more preferably an Ig y,, yz or
y3 chain.
Most preferably, the Ig is human IgG and preferred fIg isotypes are IgG,,
IgG2.and
IgG,.
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In one embodiment of the above flg, the one or more gp120 epitopes
comprises a full length gp120 protein In other embodiments, the gp120 epitopes
are
one or more peptides selected from the group consisting of the C1 region, the
V3 loop
and the CS region.
In yet other preferred embodiments, the gp120 epitope is a B cell epitope
comprising a peptide selected from the group consisting of:
VPVWKEATTTLFC.ASDAKAY (SEQ ID N0:2), EVHNVWATHACVPTD (SEQ ID
N0:3), YDTEVHNV'WA (SEQ ID N0:4), PQEWLVNVT (SEQ ID NO:S),
PQEVVLVNVTENFIDMWKNDM (SEQ ID N0:6), PNNNTRKSIR (SEQ ID N0:7),
NNNTRKRIRIQRGP'GR (SEQ ID N0:8), RKSIR (SEQ ID N0:9), IQRGPGRAFV (SEQ ID
NO:10), GRAFVTIG;ICI (SEQ ID NO:11 ), PGRAFY (SEQ ID N0:12),
NTRKSIRIQRGPGRAFVTIG (SEQ ID N0:13),
PNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHC (SEQ ID N0:14), NNTRKSIR1QRG
(SEQ ID NO:15), NKRKRIHIGPGRAFYTTKNIIGTIC (SEQ ID N0:16),
1 S RKSIRIQRGPGRAFV (SEQ ID N0:17), IRIQRGPGR (SEQ ID N0:18),
KRIRIQRGPGRAFVTIG (SEQ ID N0:19), QRGPGRAF (SEQ ID N0:20), RGPGRAFV
(SEQ ID N0:21 ), RKRIHIGPGRAFYTT (SEQ ID N0:22), RGPGRAFWIG (SEQ ID
N0:23), SISGPGRAI~YTG (SEQ ID N0:24), KRIHI (SEQ ID N0:25), KRIHIGP (SEQ ID
N0:26), IHIGPGR {SEQ ID N0:27), HIGPGR (SEQ ID N0:28), HIGPGRA (SEQ ID
N0:29), HIGP (SEQ ID N0:30), RIHIGPGRAFYTTG (SEQ ID N0:31), RIQRGPGRAF
(SEQ ID N0:32), IQRGPGRAFV (SEQ ID NO:10), IQRGPGRAF (SEQ ID N0:33),
IRIQRGPGRAFVTI (SEQ ID N0:34), RGPGRAFVTIGKIG (SEQ ID N0:35), QRGPGRA
(SEQ ID N0:36), IX:~GPGRA (SEQ ID N0:37), IGPGR (SEQ ID N0:38), GPGR (SEQ ID
N0:39), GPXR (SEQ ID N0:40), GPGRAF (SEQ ID N0:41 ), RIHIG (SEQ ID N0:42),
HIGPGRAF (SEQ ID N0:43), GRAF (SEQ ID N0:44), GGGDMRDNWRSELYKYKVVK
(SEQ ID N0:45), K~i'KVVKIEPLGVAPTKAKRR (SEQ ID N0:46), LGVAPTKAKR (SEQ
ID N0:47), GGDMRDNWRSELYKYKVVKI (SEQ ID N0:48), IEPLGVAPTK (SEQ ID
N0:49), RRWQRE (SEQ ID NO:50), PTKAKRR (SEQ ID NO:51) and WQREKR (SEQ
ID N0:52).
In yet other preferred embodiments, the gp120 epitope is a T helper cell
epitope comprising a peptide selected from the group consisting of
EQLWVTVYYGVP'V (SEQ ID N0:53), VWGVPVWKEA (SEQ ID N0:54),
CA 02279492 1999-07-29
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14
GVPVWKEATTLFC (SEQ ID NO:55), AHKVWATHACV (SEQ ID N0:56),
NVWATHACVPTD (SEQ ID N0:57), CVPTNPVPQEW (SEQ ID N0:58),
VEQMHEDIISLW (SEQ ID N0:59), EQMHEDIISLWDQ (SEQ ID N0:60),
EQMHEDIISLWDQSL (SEQ ID N0:61), HEDIISLWDQSLK (SEQ ID N0:62),
VTVYYGVPVWKEATTTLFC (SEQ ID N0:63), WLVNVTENFNM (SEQ ID N0:64),
SLKPCVKLTPLCY (SEQ ID N0:65), CTRPNNNTRKSIRIQRGPG(Y) (SEQ ID N0:66),
NTRKSIRIQRGPGR (SEQ ID N0:67), EQRGPGRAFVTIGKI (SEQ ID N0:68),
RIQRGPGRAFVTIGK (SEQ ID N0:69), RIHIGPGRAFYTTKN (SEQ ID N0:70),
GRAFVTIGKIGNMRQ (SEQ ID N0:71 ), QRGPGRAFVTIGKIGNMRQAI-I (SEQ ID
N0:72), VGKAMYAPPISGQIR (SEQ ID N0:73), GNSNNESEIFRPGGG (SEQ ID N0:74),
FRPGGGDMRDNWRSEL (SEQ ID N0:75), DMRDNWRSELYKYKV (SEQ ID N0:76),
RDNWRSELYKYKVVK (SEQ ID N0:77), CKYKVVKIEPLGVAPT (SEQ ID N0:78),
YKYKVVKIEPLGVAP (SEQ ID N0:79), KVVKIEPLGVAPTKAICRRVVQREKRC (SEQ
ID N0:80), ITLPCRIKQIINMWQEVGKAMYAPPISGQIRC (SEQ ID N0:81 ), and
ELYKYKVVKIEPLGVAPTKAKRRVVQREKR. (SEQ ID N0:82)
The present invention is further directed to a DNA molecule comprising a
nucleotide sequence encoding any fusion Ig H chain as described above.
Also provided is an expression vector useful for producing the above fusion Ig
product and for inducing and maintaining immunological tolerance to one or
more
epitopes of HIV gp120 protein in a subject, preferably a human. The vector
preferably comprises (a) a DNA molecule as above, operably linked to (b)
transcriptional and translational control regions operable in a hematopoietic
cell or
lymphoid cell of the subject. The transcriptio~al and translational control
regions
provide for constitutive expression of the DNA sequence in a lymphoid cell or
a
hematopoietic cell. A preferred vector is a retroviral vector. A naked DNA
vector
may also be used.
The present invention also provides a hemopoietic or lymphoid cell
transformed by a vector as above, which cell stably expresses the fIg protein.
Stable expression is expression which is not transient, and persists for weeks
or even months, preferably for the in vivo lifespan of the cell in which the
fIg
is expressed. Such a cell is preferably a human bone marrow cell, a resting B
CA 02279492 1999-07-29
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lymphocyte or an activated B lymphocyte which has been activated by a
mitogen or other polyclonal B cell activator.
Also included is a method for producing the fusion Ig of the invetnion
by culturing the above transformed cell. For in vitro production of the flg,
5 however, any cell type may be used which can express an Ig H chain gene as
well as the DNA encoding the fIg and secrete it into the culture medium.
The present invention includes a pharmaceutical composition
comprising:
(a) a tolerogenic; amount of a fusion Ig molecule having a fusion Ig H chain
as
10 described above; and
(b) a pharmaceutically acceptable earner or excipient for parenteral
administration.
Preferably, in the pharmaceutical composition, the fIg is an isologous IgG
molecule.
Also provided herein is a method for immunologically tolerizing a subject to
15 one or more HIV gp120 epitopes comprising administering to the subject an
effective
amount of a fusion l:g pharmaceutical composition as described above.
A method for immunologically tolerizing a subject to one or more HIV gp120
epitopes comprising; introducing into the subject an effective amount of
transformed
cells as described above, thereby tolerizing the subject.
In another embodiment, the invention is directed to a method for
immunologically tolerizing a subject to one or more HIV gp120 epitopes
comprising
introducing into the subject an effective amount of transformed cells as
above, thereby
tolerizing the subject. Prior to introducing the transformed hemopoietic cells
into the
subject, the subject ,may be treated to diminish the host's hemopoietic cells,
although
this may not be necessary in a patient with AIDS. Tolerance may also be
achieved by
a combination of treatment with transformed cells and a pharmaceutical
composition
comprising fIg as dE;scribed above.
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16
This invention is also directed to a method of (i) inducing and (ii)
maintaining
immunological tolerance to an epitope or epitopes ofHIV gp120 protein in a
subject,
comprising:
(a) administering to the subject an effective amount of a fIg pharmaceutical
S composition as described above to induce the tolerance to the epitope or -
epitopes; and
(b) administering to the subject an effective amount of transformed
hemopoietic
or lymphoid cells as described above to maintain the tolerance to the epitope
or epitopes,
thereby inducing and maintaining the tolerance. However, tolerance is also
induced
and maintained by means of administering the transformed hemopoetic or
lymphoid
cells without resorting to the flg itself. Thus, expression of the flg by the
transformed
cells is sufficient to accomplish the induction and the maintaining functions.
Also included is a method for identifying whether a candidate HIV gp120
epitope or epitopes are tolerogenic in a first subject when presented to the
subject in a
fusion Ig molecule, comprising the steps of
(a) producing a expression vector as above, wherein the gp120 epitope or
epitopes
in the fusion Ig are the candidate epitopes;
(b) stably transforming a population of autologous or matched allogeneic cells
of
the subject with the vector;
(c) introducing the transformed cells into the subject; and
(d) determining whether the subject is tolerant to the candidate epitope or
epitopes
by
(i) immunizing the subject with the candidate epitope or epitopes in
immunogenic form and measuring the immune response in vivo or in
vitro, and
(ii) comparing the response to an immune response in a second control
subject similarly immunized which has not been treated with the
transformed cells,
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17
such that, if the first subject is tolerant, the candidate epitope or epitopes
in the fusion
immunoglobulin construct is identified as being tolerogenic. In the above
method, the
subjects are preferably humans and the transformed cells are human cells.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 (sheets 1/1 through 1/3) shows the amino acid sequence of HIV-1
gp120 (mature protein). The boldface sequence, SEQ ID N0:83, (with position
numbers in the rif;ht margin) is the consensus sequence of the protein from
subtype B
virus (the prevalent HIV-1 subtype in the United States). The following
characters are
used in the consensus sequence:
( 1 ) single letter code; UPPER CASE letter indicates that the amino acid
residue in
that position is conserved for all known viral isolates of subtype B;
(2) lower case letter indicates the amino acid residue is conserved in more
than 50%
of known isolates;
(3) a "?" indicates lack of consensus at that position (no single residue is
found in the
majority of isolates).
The concensus sequence is read left to right. Shown vertically below each
position in
the consensus sequence (where appropriate) are alternative amino residues that
have
been identified at that position in mutants or variants of subtype B. Residues
which
happen to be adjacent to one another below the consensus sequence line are NOT
to
be read left to right as they do NOT represent adjacent residues in an actual
gp120
sequence. (Note: all the variant residues below the consensus sequence line
are
UPPER CASE for clarity only.) All of the sequence information in Fig. 1 (and
Fig. 2)
was obtained from The Human Retroviruses and AIDS Genetic Sequence 1995
Compendium, published by the Los Alamos National Laboratory: Theoretical
Biology
and Biophysics C~ivision, Los Alamos, NM.
Figure 2 (sheets 2/1 - 2/4) shows the aligned consensus sequences for the
major subtypes or "clades" of HIV-1 as published in The Human Retroviruses and
AIDS Genetic Sequence 1995 Compendium (see Figure 1 ). HIV subtypes are
defined
and distinguished based on their nucleotide (and not amino acid) sequences.
Certain
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18
"signature sequences" are characteristic of a subtype, for example, the GPGR
consensus sequence at the tip of the V3 loop of the subtype B that appears as
a GPGQ
consensus for most other subtypes. The letter/symbol conventions are the same
as
those used in the consensus sequence in Figure 1. In addition, the presence of
a ' :" at
a position indicates that most isolates (and hence, by definition, the
consensus
sequence for the subtype) lack an amino acid at that position. All sequences
are
shown relative to the consensus A sequence (SEQ ID N0:84), and a "-"indicates
the
same residue as in subtype A at that position. The gp120 sequences in Figure 2
(SEQ
ID NOS:84-104) include the signal peptide (N terminal to the mature protein
and
indicated above the CONSENSUS-A sequence). The mature protein begins to the
right of the "/" Other landmarks indicated include the V3 neutralization loop,
the C
terminus of gp120 (indicated by a "/" on sheet 2/4) and the N-terminal segment
(about
16 residues) of the HIV-1 gp41 protein.
Figure 3 (sheets 3/1 and 3/2) shows the aligned amino acid sequences of
gp 120 (including the signal sequence) from several strains or isolates of HIV-
1. The
top line of each grouping (in boldface) is the subtype B consensus sequence
(SEQ ID
NO:105; also appearing in Figures 1 and 2). The footnotes describing each
variant or
isolate and the markings used in Figure 3 are as follows:
(1) The first 27 - 30 amino acids left of the "/P' mark comprise the signal
sequence of gp120.
The mature gp 120 protein begins to the right of the "/P'. In general, a space
appears after
each 10 residues. To preserve alignment, spaces have sometimes been omitted
and for the
consensus sequence, additional residues have been placed above the main
sequence line.
(2) CON-B is the consensus sequence for gp120 of subtype B (SEQ ID NO:105).
UPPER or
lower case letters are as described for Figs. 1 and 2. The presence of single
letter amino
acid codes or "?" above the consensus sequence line indicates the existence of
additional
residues in some subtype B isolates at approximately the positions indicated.
In some
locations, arrows appear in the sequence line as place indicators for such
additional residues.
Each arrow is _not intended to correspond to a single residue and points to
the known residues
(usually "?") that may occupy that region in various isolates..
(3) BH10 isolate (SEQ ID N0:106): Ratner, L. et al. Nature 313:277-284(1985)
(Genbank
SWISS PROT Accession No. P03375)
(4) LAV-BRU isolate (SEQ ID N0:107): Wain-Hobson, S. et al., Cell 40:9-
17(1985) (Genbank
SWISS PROT Accession No. P03377 )
(5) ARV2/SF2 isolate (SEQ ID N0:108): Sanchez-Pescador, R., et al., Science
227:484-
492(1985) (Genbank SWISS PROT Accession No. P03378)
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19
(6) MN isolate (SEQ ID N0:109): Gurgo, C. et al., Virology 164:531-536(1988)
(Genbank
SWISS PROT Accession No. P05877)
(7) 92US712.4 isolate (SEQ ID NO:110). This sample was part of a set of
sequences generated
through the N1AID/NIH DAIDS HIV variation program. The virus was derived from
an
S asymptomatic individual from Baltimore, thought to be infected by parenteral
i.v, drug user
contact. The env aequence clustered with HIV-1 B subtype sequences. Gao, F. et
aL, J. Virol.
70:165I-1667 (1996) (Genbank SWISS PROT Accession Number U08449). This
sequence
was randomly chosen as a subtype B isolate for illustrative purposes and for
comparison
with the more common variant sequences.
Figures 4A and 4B illustrate a preferred engineering strategy for inserting a
foreign epitope at the N-terminus of an IgG y chain. Figure 4A depicts the
incorporation of an oligonucleotide (SEQ ID NO:111 ) encoding the ~, phage C 1
repressor peptide 12-26 (SEQ ID N0:112) as described in Examples. This flg was
expressed in marine J558 myeloma cells. The present invention introduces an
1 S oligonucleotide or polynucleotide encoding one or more native or synthetic
gp120
peptide epitopes into an Ig H chain, preferably a human y chain (Figure 4B).
Figures SA a:nd SB show strategy for the construction, expression, and epitope
recognition of a fusion Ig gene by inserting a foreign epitope into a VH gene.
Fig. SA
presents a scheme for constructing the fIg. A modified 12-26 nucleotide
sequence
was ligated into a P:ctI site of a 1.3-kb marine VH (LVDJ) chain fragment. The
PstI
site appears at the coding sequence of the fifth amino acid of the FRl;
therefore, a
repeat of the first five FR1 amino acids was designed to follow the coding
sequence of
the 15 a~rnino acids of 12-26, so as not to perturb proper framework region
folding
after insertion. SDS/10% polyacrylamide gel'electrophoresis of purified H-
chain
transfected immunoglobulins demonstrated proper assembly of H chains with L
chains. Fig. SB is a gel pattern showing recognition of epitopes by
immunoblotting.
Purified control IgG (P6) of 12-26-IgG (Q3) samples were electrophoresed on
SDS/10% polyacryl~amide gels, transferred onto nitrocellulose, and probed with
anti-
mouse IgGl (left lanes) or with biotinylated anti-12-26 mAb B3.11 (right
lanes) plus
AP-conjugated secondary reagents.
Figure 6 shows in vivo effects of 12-26-IgG pretreatment on peptide-specific
humoral immune responses. Male BALB/c mice were injected i.v. with a single 1-
mg
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dose of deaggregated protein G-purified P6 ( 1 ) or Q3 ( / ) IgG. Mice were
immunized and total or isotypic anti-peptide IgG titers were quantitated by
ELISA 8
days after a secondary antigenic boost. Isotyped anti-peptide titrations (IgG,
and
IgGZb) represent mean absorption values from assays of three individual mice
in each
S group. 0 , Preimmunization sera
Figure 7 shows in vivo effects of 12-26-IgG pretreatment on peptide-specific
cellular immune responses. Tertiary cytokine (IL-2 and IL-4) responses of
enriched
splenic T cells (3 x 1 O6 cells per ml) from mice displaying tolerized humoral
immune
secondary responses are shown. IL-2 and IL-4 production in supernatants was
1 U determined in triplicate by CTLL and CT.4S assay, respectively. "Medium
only"
backgrounds were subtracted; these values ranged from 1 to 4 units/ml in all
assays.
Figure 8 shows structure and genomic Southern blotting of transgenic mice
expressing 12-26-IgG specifically in the B-lymphocyte lineage. A murine
IgG,° I~
chain construct containing endogenous immunoglobulin promoter and enhancer
(EH)
15 sequences was modified to express 12-26 peptide and a repeat of perturbed
framework
region sequence (FR1) at the N-terminus. Fertilized embryos were injected with
this
linearized construct and transgenic mice were generated via standard
procedures.
Genomic DNA from tail biopsies was digested with BamHI and EcoRI to release a
1.3
kb V,_, fragment, fractionated on 0.8% agarose/TBE, and transferred onto nylon
20 membranes via alkaline Southern transfer . Southern blots were probed with
random-
primed 32P-labeled DNA sequence containing 3 tandem repeats of 12-26
nucleotide
sequence. Densitometry studies using known amounts of purified, linearized
transgene DNA was used to estimate that there are 2-3 integrated copies in
Lines 5
and 17.
Figures 9A and 9B show profound peptide-specific cellular and humoral
immune tolerance in 12-26-IgG-expressing transgenic mouse lines. Fig. 9A
presents
titers of total anti-peptide IgG (open symbols), or IgG, isotype (closed
symbols) for
Line 5 transgenic mice measured after peptide immunizations and secondary
boosts.
Fig. 9B presents splenic T cell cytokine responses from tolerant Line 5
trangenic (Tg)
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21
mice determined by CTLL assay. Error bars signify the standard error of the
mean for
3-4 mice per group.
Figure 10 shows profound peptide-specific cellular and humoral immune
tolerance in transgenic bone marrow chimeras expressing 12-26-IgG. Chimeras
were
S prepared with 1:1 mixtures of Line 17 Tg and non-transgenic (NTg) bone
marrow (/).
Antibody responses. to peptide are shown. Anti-HEL specificity controls showed
no
differences between groups. Nonirradiated mice injected with saline (0)
displayed
immune responses similar to control chimeras reconstituted with 100% NTg bone
marrow (0). Error bars signify standard error of the mean of 2-3 mice per
group.
Figure 11 stows the induction of peptide-specific humoral immune tolerance
in normal immunocompetent adults by intravenous injection of various
preparations
of 12-26-IgG-expressing lymphoid tissues. Normal, nonirradiated BALB/c males
were injected iv with 4x10'sex-matched splenocytes, Percoll°°-
gradient-purified (60-
70% fraction) resting B cells, 48-hour activated LPS blasts, or crude
unfractionated
bone marrow cells lsom Line 17 transgenic mice. Recipients were rested for 7-
10
days before immunization with 50 pg peptide in CFA (SC base of tail). Mice
were
boosted with an additional 50 p.g in saline 2 weeks later and serum antibody
titers
determined 8 days later.
Figures 12A. and 12B present an analysis of B-cell tolerance induction in
tolerized transgenic or normal adult subjects. Fig. 12A: Nontransgenic ( 0 ),
Line S
transgenic ( O ), or line 17 transgenic ( / ) mice were immunized
intraperitoneally with
50 p,g 12-26-HEL conjugate in CFA, and boosted with the same in saline 2 weeks
later. Anti-peptide and anti-HEL (all >105, not shown) titers were determined
by
ELISA as described in the text. Fig. 12B: Serum titers from adoptively
transferred
recipients boosted vvith 50 p,g 12-26-HEL conjugate in IFA were similarly
determined: BALB/c recipients were irradiated with 400 rads, and injected with
5 x
10' splenocytes from Line 17 Tg-tolerized donors (closed circles, various
sources of
lymphoid tissue) or non-transgenic injected, non-tolerized donors (open
diamonds).
Splenic donors had been previously primed and boosted with 12-26 peptide and
HEL
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WO 98136087 PCT/US98/02766
22
(at different subcutaneous locations), and had previously displayed tolerance
(experiment from Figure 11 ).
Figures 13A. l3B,and 13C summarize studies showing the induction of
tolerance in previously-primed adult recipients by either resting, B cells, B
cell blasts
S or chemically fixed B cells. BALB/c mice were immunized SC with 50 pg 12-26
peptide in CFA 1-2 weeks before iv injection of4x107 Line 17 transgenic (!) or
nontransgenic control ( 0 ) purified resting B cells (Fig. 13A), LPS-activated
B cell
blasts (Fig. 13B), or carbodiimide-fixed B cells (Fig. 13C). The mice were
challenged
IP with SO ug soluble peptide 1-2 weeks following tolerizing injections, and
antibody
1 U titers (ELISA) determined 8 days later. The graphs show peptide-specific
total 1gG or
two IgG isotypes (IgG, and IgG~b),
Figures 14 and 15 show B cell expression, epitope recognition, and direct
antigenic presentation of retrovirally-synthesized peptide-IgG. Fig. 14 shows
the
structure and proviral integration of marine Moloney leukemia retroviral
construct
15 MBAE.BAK. Ten pg of genomic DNA from transduced, 6418-resistant (+) or
control (-) A20 cells was digested with Sac I, fractionated on 0.8% agarose,
Southern-
blotted, and probed with 32P-labeled DNA probe containing three tandem copies
of
12-26 sequence. Sac I digestion releases an ~5.1 kb proviral genome. Fig. 15
shows
tissue expression of 12-26 mRNA in long-term (~3 months) recipients of gene-
20 transferred ( f ) or mock-transduced ( - ) BM progenitors. RNA from bone
marrow
(B), thymus (T), or spleen (S) was assayed by 12-26 sequence RT-PCR (25). One-
tenth of each PCR reaction (except for A20 controls: 1/100th) was Southern-
blotted
and probed with a non-complementary 32P-labeled 12-26-specific
oligonucleotide.
Figure 16 shows the induction of peptide-specific cellular immune tolerance in
25 adult bone marrow chimeras infused with peptide-Ig-expressing progenitor
cells.
BALB/c mice were sublethally irradiated (600 rads) and injected iv with 1-
2x106
gene-modified or mock-transduced BM. Recipients were immunized with peptide in
CFA 2 months post-infusion and draining LN cells were restimulated in vitro
with
dilutions of synthetic peptide and 25-50 p,g/ml purified protein derivative
(PPD,
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23
Connaught) in RPMI 1640 with 0.5% heat-inactivated autologous serum.
Stimulation
indices (SI) represent ratios of proliferation to medium alone backgrounds
(5,609~2,271 cpm)., IL-2 and IFN-y were quantitated by CTLL and ELISA assays,
respectively (Gilbert, K.M., et al. (1994) J. Exp. Med. 179:249-258).
Additional
experiments also revealed a diminution of peptide-specific IL-4 release in LN
of
tolerized recipients. Error bars signify standard error of the mean for 3
individual
mice per group. This experiment was done at least twice with 3-4 mice per
group
with similar results..
Figures 17A,, 17B and 17C show the induction of peptide-specific humoral
immune tolerance i;n adult bone marrow chimeras infused with peptide-Ig-
expressing
progenitor cells. B.ALB/c mice were sublethally irradiated with either (A) 2U0
rads
(Fig. 17A) , or 600 rads (Fig. 17B,C) and infused with 1-2x106 gene-
transferred
(triangles) or mock-transduced (circles) BM cells. Mice were primed and
boosted for
humoral responses .either (Fig. 17A) one month, or (Fig. 17B,C) 2 months post-
1 S infusion with synthetic 12-26 peptide, and HEL as a specificity control.
Non-
manipulated, immunized BALB/c always produced titers similar to recipients
infused
with mock-transduc:ed BM cells (Fig. 17A, diamonds). Both total peptide-
specific
IgG (open symbols), or the main isotype IgGl (closed symbols) were diminished
in
all experiments. Normal recipients in Fig. 19B and 19C received either S-FU-
pretreated normal EtA,LB/c BM or SCIDBALB/c BM cells. Flow cytometric analysis
at the one month sacrifice time (of mice from Fig. 17A) revealed comparable
levels of
CD4+ and Ig+ splenocytes in normal BALB/e reconstituted with either normal or
SCID BM: (CD4+: 18-25%; Ig+: 40-65%). All experiments were done at least twice
with 3-5 mice per group with similar results.
Figures 18-'L 9 show peripheral tolerance induction in immunocompetent adults
with gene-transferred peripheral B cells expressing engineered peptide-Ig.
Figure 18
shows humoral and cellular tolerance induction. Unirradiated mice were
injected with
>1x107 LPS blasts co-cultured with retrovirus-producing F6P (+) or mock-
transducing yr-2 (-). One week later, mice were primed and boosted for humoral
responses, and sacrificed 3 months later for analysis of splenic memory T cell
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24
responses. Cytokine release in individual splenic cultures was determined at
24 hours
(IL-2) or 48 hours (IL-4); medium alone background values were less than 1-2
U/ml
and were subtracted for clarity (~U/ml). Figure 19 shows persistence of gene-
transferred B cells. Hybridomas generated from spleens of tolerized mice by
PEG
fusion of A20 cells with LPS-activated splenocytes (48 hours, 50 ~g/ml LPS).
Hybridomas were selected in 1 mg/ml 6418 and tested for their ability to
activate T-
cell hybrid 9C127 as above. Eight representative A20 hybridomas from each
recipient (Mice #1-3) are shown.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The nucleotide and amino acid sequences of most known isolates of HIV-1 are
published in "The Human Retroviruses and AIDS Genetic Sequence 1995
Compendium" (hereinafter, "Compendium"), as well as earlier editions of the
Compendium). This document is available in paper or electronic form from its
publisher, the Los Alamos National Laboratory: Theoretical Biology and
Biophysics
Division, Los Alamos, NM. The complete nucleotide and amino acid sequences of
individual HIV isolates as well as consensus sequences for recognized HIV-1
subtypes or Glades (A, B, C, D, E, F, G, O U, CPZ) have been published in this
database for a number of years. The HIY Molecular Immunology Database 1995,
Editors: B. Korber et al., Los Alamos National Laboratory: Theoretical Biology
and
Biophysics, Los Alamos, NM, 1995, (referred to herein as the "HIVMID")
provides
T-cell epitope maps, alignments, and annotation (for T helpe epitopes and for
CTL
epitopes) , as well as a summary and map of linear B cell epitopes and
monoclonal
antibodies recognizing such epitopes. This application incorporates by
reference the
latest Compendium and HIVMID, but is also intended to include updates
containing
sequences of additional viral isolates as they are added and published. The
compendium (database) and HIVMID are publicly available on the World Wide Web
at the address "http://hiv-web.lanl.gov".
In particular, the HIV-1 env gene (which encodes the gp160 precursor protein
of both gp120 and gp41 envelope proteins) consensus nucleotide sequences for
10
CA 02279492 1999-07-29
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viral subtypes appear at pages I-A-358 to I-A-364 of the 1995 Compendium (NOV
1995). The env nucleotide sequences of individual viral isolates, grouped by
subtypes
and appearing along; with the consensus sequence for the subtype, are at pages
I-A-
174 to I-A-357 of the 1995 compendium.
Crosslinkinl; of CD4 molecules on human T cells either by (a) HN-1 virions
bound to CD4 via viral gp120, or (b) anti-gp120 antibodies crosslinking of
soluble
gp 120 bound to CD4, primes or programs the T cells for apoptosis, as
described
herein. Thus, an infected subject's antibody response to HIV-1, particularly
to one or
more epitopes of gp~120, contributes to the pathogenetic process by targeting
10 bystander T cells to self destruct. The inventors have discovered an
approach to
modulate these responses by inducing selective immunological tolerance either
at the
level of B cells, T helper cells or both, resulting in diminished antibody
responses to
one or more gp120 epitopes. Inducing and maintaining such tolerance in a
subject
provides a therapeutic approach for treating HIV infection. Furthermore, such
15 tolerance induction to selected epitopes can be used as part of a
therapeutic or
prophylactic vaccine approach. Thus, an improved HIV vaccine may include in
addition to an HN immunogenic preparation, a flg in accordance with this
invention
to reduce or prevent undesired antibodies.
The term "tolerant" or "tolerance" as used herein is defined functionally in
20 terms of the immune response to an immunogenic challenge with an antigen. A
subject is tolerant if his response to an immunogenic challenge is reduced by
at least
about 50%, more preferably at least about 80% relative to a non-tolerant
control
subject.
Tolerance may be manifest by reduced reactivity in vivo such as antibody
25 formation or in vitn~, for example, by reduced lymphocyte proliferation.
A "toleroge;n" is a form of antigen which, when it encounters the immune
system, induces a state of immunological tolerance or hyporesponsiveness or
anergy
in the host. Such a state is tested by subsequent immunization or challenge of
lymphocytes in vitro with the specific antigen in immunogenic form.
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26
The term "immunogenic" with reference to an antigen or epitope is also a
functional term which is dependent on the nature, form, dose and route of
administration of the antigen (epitope) such that it has immunogenic
properties, i.e., it
induces immune reactivity resulting in antibodies or cellular immunity. Thus
the
same molecule, e.g., a protein, can be imrnunogenic or tolerogenic depending
on it
form (e.g., aggregated or deaggregated) dose or route of administration, all
of which is
well-known in the art. As described herein, antigens, including low molecular
weight
haptens, can be rendered non-immunogenic and even tolerogenic by coupling them
to
homologous immunoglobulin molecules. In fact, a key observation underlying
this
invention is that such "coupling" can be achieved by recombinant techniques in
the
form of a flg wherein a peptide epitope (or "antigenic determinant" or minimal
antigenic structure) for which tolerance is desired is made part of the fIg
using
methods described herein.
Because certain tolerogenic flg preparations, or cells expressing such an flg,
can induce hyporesponsiveness in an already primed or immunized subject (see
below), the present invention is useful as a therapeutic tolerogen, to curtail
an ongoing
immune response to a selected gp120 epitope or epitopes during the course of
HIV
disease. In fact, that may be the more significant clinical utility of this
invention.
Alternatively, the fIg tolerogen is to modulate the response to an HIV vaccine
such
that the subject immunized with the vaccine and treated with the tolerogen
responds to
particular desired viral epitopes (expressed by the vaccine) and is prevented
(or
suppressed) in his response to other selected epitopes (expressed by the
tolerogen)
By judicious selection of these HIV epitopes, most preferably gp120 epitopes,
it is
possible to render a host selectively tolerant at the level of T helper cells,
B cells, or
both. In the case of HIV infection, this would inhibit or prevent the
production of
antibodies that are of no benefit (e.g., non-neutralizing), and more
importantly, are
harmful via mechanisms such as bystander apoptosis or enhancing antibodies
which
promote infection of host cells. On its face, it might appear counterintuitive
to inhibit
an immune response to a virus which one wishes to eradicate. However, given
the
differences between epitopes of HIV recognized by antibodies and by cytotoxic
T
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27
lymphocytes (CTL;) (see HIVMID). B cell and/or T helper cell tolerance to one
or
more (even all) epitopes of gp120 molecule may still permit an effective CTL
response against other (non-tolerizing) gp120 epitopes or, importantly, other
non-
envelope HIV proteins which are known to be immunogenic. HIV T helper epitopes
and CTL epitopes have been described in a number of publication, for example,
Berzofsky, J.A. (1S~95) Ann N YAcad Sci, 754:161-168; Meister GE et al. (1995)
Vaccine, 13:581-591; Cease KB et al. (1994) Annu Rev Immunol, 12:923-989;
Shirai
M; et al. ( 1994) J Immunol, 152:549-556; Ahlers JD et al. ( 1993) J Immunol,
1 SO
5647-5665; Berzofsky JA, ( 1991 ) Biotechnol Ther, 2:123-135; De Groot AS, et
al.
(1991 ) Jlnfect Dis.,164:1058-1065; Berzofsky JA et al. ( 1991 ) J Clin
Invest,
88:876-884; Clerici M et al. (1991) EurJImmunol,11:1345-1349 Palker TJ et al.
{1989) Jlmmunol, 142:3612-3619, which references are hereby incorporated by
reference.
The present inventors have developed a flexible fusion protein approach for
induction of unresponsiveness to defined B-cell and T-cell epitopes in vivo
and in
vitro. See, for example, Scott and Zambidis, co-pending application U.S.S.N.
08/195,874, PCT Publication WO 95/21926 and Zambidis, E.T. et al., (1996)
Proc.
Natl. Acad. Sci. US'A 93:5019-5024, which references are hereby incorporated
by
reference in their e~atirety. As described herein, this approach originally
set forth for
other antigens, is adapted for the production of compositions and methods
useful for
inducing unresponsiveness to one or more HIV gp120 epitopes.
Epitope-specific tolerance is used to ablate undesired antibody responses
while
maintaining protective CTL responses. Hence, by inducing B cell tolerance and
T
helper cell tolerance to all gp120 epitopes, either by use of a flg into which
a complete
gp120 sequence or one or more partial gp120 sequences have been inserted, or
by
using a mixture of fusion Ig's each including a subset of gp120 epitopes, anti-
gp120
antibody responsiveness can be prevented or diminished. Because the CD8 arm of
the
immune response is not affected, protective antiviral cell-mediated immunity,
in
particular CTL responses to HIV epitopes, remains intact.
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IgG-gp120 peptide fusion proteins are effective tolerogens which modulate
anti-gp120 responses. Furthermore, human hematopoietic precursor cells,
whether
from BM or other tissues, and their progeny B cells which express the IgG-
gp120
peptide fusion proteins are themselves tolerogenic agents which deliver or
present on
their surface the selected HIV peptides in tolerogenic form for induction
and/or
maintenance of the tolerant state. Thus, in one embodiment, the ongoing
maintenance
of tolerance is achieved by first transfecting bone marrow (BM) cells or
peripheral
hematopoietic stem cells from any tissue (for example, CD34' peripheral blood
stem
cells in the human) with a DNA vector which includes a DNA sequence encoding a
IgG-gp120 fusion protein of the present invention. In another embodiment, the
tolerogen is presented expressed in a myeloid cell (as determined in studies
using
SCID mouse BM). The B cell expressing the tolerogenic flg may be a resting B
cell,
an activated B cell or B cell blast, or a transformed B cell (e.g., leukemia
or
lymphoma) which has been appropriately attenuated to ablate its oncogenic
potential
for use in human subjects. Long-lasting, even permanent tolerance can be
induced by
grafting transfected BM stem cells or peripheral stem cells. This approach is
described
in more detail in Example IV.
Tolerogen Presentation
B cells are known to be capable of inducing tolerance by presentation of
appropriate surface molecules in a tolerogenic fashion (Eynon, E.E. et al.
(1992) J.
Exp. Med. 175:131, using human IgM and IgD; Fuchs, E. et al. (1992) Science
258:1156, for the H-Y antigen).
The present inventors discovered that resting B cells expressing a fIg, after
injection into a recipient subject, induce tolerance for natural epitope
included in the
flg, such as the phage 7~ 12-26 epitope. Larger blast cells induced by
stimulating such
B cells with bacterial lipopolysaccharide (LPS) (termed "LPS blasts") also
tolerize for
this peptide. Activated B cells are better tolerogenic vehicles in primed
recipients
than resting B cells. This is in contrast to the observations of Yuschenkoff
et al.
(supra) who found that activated B cells from mice tzansgenic for and
expressing
human p, chains lost the ability to tolerize. Transgenic lymphoma cells
activated in
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29
this way did not induce tolerance but rather appeared to induce an immune
response
for the same epitope.
In marine studies, splenic B cells stimulated with LPS are infected with a
retrovirus construct containing the desired epitope. In a model system, the 12-
26 IgG
flg has been used successfully in this way. Such LPS blasts are tolerogenic
for that
epitope. Hybridom~as produced from the splenic B cells expressing the flg also
express the flg transgene. When transgenic BM expressing 12-26 flg or normal
(control) BM is injected into recipient mice irradiated with 2008, and the
animals are
immunized with the: peptide in immunogenic form (in adjuvant), the following
results
have been obtained:
( 1 ) T cells in recipients of transgenic BM are tolerant, measured by T cell
proliferation and production of cytokines (IL2, IL4, IFN-y,. etc.).
(2) recipients are tolerant as far as making IgG antibodies to the peptide.
Tolerance to a desired HIV gp120 peptide epitope included in an flg construct
is achieved using as a source of B cells expressing the flg on their surface
any
population of lymphocytes known to contain B cells or to differentiate into B
cells.
This may include au unfractionated population, a cell preparation enriched in
B cells
or their precursors, or a purified B cell population. Any conventional method
for
enriching or purifying B cells may be employed. Examples of tissue sources for
B
cells include BM, spleen, LN, peripheral blood or lymph. B cells may be
resting or
preferably are activated, for example, LPS blasts.
As described in the Examples, when normal marine spleen cells were first
stimulated by LPS followed by infection with a retroviral vector carrying the
flg
transgene and then infused into normal recipients, followed by immunization,
the
following results wc;re obtained: The T cell responsiveness to the 12-26
peptide
showed decreased IlL-2 and IL-4 production. Animals had a decreased antibody
response to the peptide. The effect on the antibody response can be explained
by the
T helper cell compartment being tolerant, the B cells being tolerant, or more
likely,
both. Table I summarizes results in a primed subject:
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TABLE I
Recipient Outcome Measure Small B CellsLPS BlastsFixed B
Cells'
Normal IgG Titers ~"~ ,~~, NDz
Primed' IgGI Response - ~.~. partial ~.
not tolerogenic
' Fixed cells are treated with a carbodiimide.
2 ND=not determined
' Recipients injected 10 days earlier with 12-26 in complete Freund's
adjuvant.
5 Without wishing to be bound by any mechanism, the present inventors
propose two possible mechanisms to explain these results:
( 1 ) Antigen presentation without "signal 2" (R. Schwartz ( 1989) Cell 5
7:1073-
1081 ) results in anergy.
(2) LPS blasts may induced "propriocidal" cell death..
10 Lenardo and colleagues (Boehme S.A. et al. (1993) Eur. J. Immunol.
23:1552-1560; Boehme S.A. et al. (1993) Leukemia 7 (Suppl 2):S45-S49;
Critchfield,
J.M. et al. (1995) Cell. Immunol. 160:71-78; Pelfrey, C.M. et al. (1995) J.
Immunol.
154:6191-6202) found that stimulated T cells (or T cell hybridomas) produced
IL-2
but also underwent suicide termed "propriocidal death." This response is
thought to
15 be important for regulating an ongoing immune response wherein suicide of
responding cells serves to bring the response to a timely termination.
Evaluation of Potentially Tolerogenic gp 120 Epitopes for Use in fIg
Human y globulin (HGG) (American Red Cross), a model tolerogenic carrier,
is used as a carrier in these evaluations of a given peptide ("PEP")
con:esponding to
20 one or a combination of epitopes of gp120. MBS (m-maleimodobenzoyl-N-
hydroxysuccinimide ester) is a preferred coupling agent because of ease of use
and
thiol-cleavability (i.e., to prepare control peptide-conjugates, as well as to
determine
conjugation ratios). A known antigen (hapten) may be used as a specificity
control
for tolerance, e.g., FITC-coupled HGG. In a standard protocol based upon long-
term
25 experience in the present inventors' laboratory (Scott, D.W. et al. (1979)
Immunol.
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31
Rev. 43:241; Warnc;r, G.L. et al. (1991) J. Immunol. 146:2185), murine spleen
cells
are cultured for 24 lhours with increasing concentrations of PEP-HGG, FITC-HGG
or
anti-~. (positive control for tolerance); these cells are washed and then
challenged with
LPS in microculture for 4 days. ELISAs for IgM and IgG anti-PEP, anti-gp 120,
anti-
s HGG and anti-FIT(: are then performed by standard methodology. This protocol
allows for polyclonal stimulation that elicits measurable responses to all of
these
epitopes Once established, the evaluation can be performed in PEP-primed
subjects to
verify that tolerance: induction can be achieved in secondary B cells (Linton
PJ, et al.
( 1991 ) J. Immunol. 146:4099).
It is also helpful to perform dose response studies using PEP-HGG conjugates,
as well as free peptide, administered intravenously. For example, groups of 4-
5 mice
are injected intravenously with 0.1, 0.3 or I mg of PEP alone, PEP-HGG, or
FITC-
HGG as a specificity control. Four to seven days later, mice are challenged
with
gp120 in complete iFreund's adjuvant (CFA). Mice are bled on day -7 (before
tolerance) and at 1 f and 20 days after challenge; mice can then be boosted on
day 20
and bled 7 days late-r to evaluate secondary IgG responsiveness. Heterologous
IgG's
are known to be tolerogenic in vivo at <10-a M (~0.1-1 mg/mouse). Peptides for
inducing T cell tolerance are commonly administered at higher concentrations
(approximately 10-'M).
It may also be advantageous to establish epitope density requirements for
tolerance. Typically, hapten-protein ratios of 5-10 are used with Ig
conjugates. It
would be desirable to control coupling reactions to achieve molar ratios
(PEP:HGG)
of 2,4,8, and 16. Because the MBS cross-linker is cleavable, it is possible to
quantitate ratios and create peptide-linker only controls. Primed recipients
may
require tolerogens with a higher epitope density. In the fIg embodiment,
higher
epitope density is translated into inclusion of more copies of the DNA
encoding the
epitope, for example 2-10 copies, in the fIg DNA construct if this is required
to
overcome a state of preexisting immunity in the subject.
Achievement of tolerance using the above chemical-coupling approaches
along with determinations of optimal dose-response relationships and epitope
CA 02279492 1999-07-29
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32
densities will provide the basis and indicate the efficacy of the epitopes to
be inserted
into the recombinant fIg fusion proteins for use as tolerogens.
Choice of HIV gp120 Epitopes for B Cell and T Helper Cell Tolerance Induction
The tolerogenic IgG-gp-120 peptide fusion proteins may include one or more
peptides of gp120, including the full-length gp120 protein. If more than one
peptide
epitope is present, the different peptides may be arranged in the fusion
protein in the
same order and in contiguous form as they are in the native gp 120 protein.
Alternatively, the peptides may be "reshuffled" in the fusion protein.
Furthermore,
one or more of the gp120 peptides may be present in the fusion protein in two
or more
copies, either alone or with another gp120 peptide. In a preferred embodiment,
the
one or more epitopes selected for use in the tolerogenic fIg is a linear
epitope.
However, as conformational epitopes become better defined, it will be possible
to
construct a flg having one or more epitopes which, in combination, yield the
conformational determinant in the expressed flg.
It is advantageous to use the largest fragment of the native gp 120 protein
that
(a) can be fused with the Ig H chain while maintaining the required tertiary
structure
of the Ig portion of the fusion protein for tolerogenic activity and (b) can
be
accommodated by the vector used to transfer the fIg-encoding DNA. The
advantage
lies in the fact that the appropriate epitopes of such a flg are selected by
the host MHC
proteins (of antigen-presenting cells or, in this case, tolerogen-presenting
cells) for
presentation and tolerance induction. In humans, this would obviate the need
to select
a priori those epitopes of gp120 which would interact with the HLA-DR
molecules of
a given subject to yield an active tolerogen for that subject. As more
information
relating various HL-A types with HIV gp 120 epitopes becomes available, it
will
become easier to tailor smaller tolerogenic epitopes for a given subject.
Approaches
to accomplish this for T helper cells epitopes are already available through
various
computer based algorithms which are discussed in much more detail below.
Expression of the epitope on the fIg can be tested using a conventional
immunoassay with an antibody specific for the epitope (if it is a B cell
epitope) or
with lymphocyte proliferation or cytokine secretion assay (for a T helper cell
epitope).
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33
Antibodies recognizing such epitopes are available, and T cells can be
prepared in
vitro or long-term T cells lines of the appropriate specificity are available
or can be
prepared using conventional methods. The Compendium, and in particular the
HIVMm lists antibodies specific for each of the epitopes of Table II, for
example.
The antibodies may be rodent mAbs, human polyclonal or mAbs or hybrid
antibodies
generated from such human or rodent mAbs. Alternatively or additionally, the
soluble
flg can be administered in adjuvant to a host and tested for generation of
peptide-
specific T-cell responses in vivo, due to processing and presentation by
endogenous
APC, even in the context of an Ig scaffold (see Examples).
A gp120 epitope of the present invention, in particular a linear or
"sequential"
epitope, is preferably one comprising a "natural" sequence, defined as the
sequence as
it occurs in a consensus gp120 sequence of a particular HIV subtype or a
naturally
occurring mutant thereof which has been isolated and characterized.
However, the; epitope sequence may also be a variant of a natural sequence
defined here as a sequence in which one or more amino acid residues has been
replaced by a different residue, including substitutions not known to occur in
natural
viral isolates. The only condition is that the variant sequence maintain the
secondary
and tertiary structure. needed to create the desired the tolerogenic epitope
when
expressed in a fIg protein either in solution or on a cell surface. Hence, it
is preferred
that any variant maintain (a) the structure of the peptide backbone in the
area of the
substitution, for example, as a sheet or helical conformation, (b) the charge
or
hydrophobicity of the molecule at the substitution site, or (c) the bulk of
the side
chain.
For a detailed description of protein chemistry and structure, see Schulz,
G.E.
et al., Principles of Protein Structure, Springer-Verlag, New York, 1978, and
Creighton, T.E., Proteins: Structure and Molecular Properties, W.H. Freeman &
Co.,
San Francisco, 1983, which are hereby incorporated by reference. The types of
substitutions which may be made in the gp 120 protein or peptide molecule of
the
present invention may be based on analysis of the frequencies of amino acid
changes
between a homologous protein of different species (e.g., Table I-2 of Schulz
et al.
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34
(supra) and Figure 3-9 of Creighton (supra). Base on such analysis,
conservative
substitutions are defined as exchanges within one of the following five
groups:
I . Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr (Pro,
Gly);
2. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gln;
3. Polar, positively charged residues: His, Arg, Lys;
4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys); and
S. Large aromatic residues: Phe, Tyr, Trp.
The three amino acid residues in parentheses above have special roles in
protein architecture. Gly is the only residue lacking a side chain and imparts
flexibility to the chain. Pro, because of its unusual geometry, tightly
constrains the
chain. Cys participates in disulfide bond formation which is important in
protein
folding. Tyr, because of its hydrogen bonding potential, has some kinship with
Ser,
Thr, etc.
Most deletions and insertions, and substitutions according to the present
invention are those which do not produce radical changes in the structural or
immunological characteristics of the gp120 protein or peptide molecule when
expressed as part of a flg. However, when it is difficult to predict the exact
effect of
the substitution, deletion, or insertion in advance of doing so, one skilled
in the art
will appreciate that the effect will be evaluated by routine screening assays.
For
example, a variant typically is made by chemical synthesis or site-specific
mutagenesis of the peptide-encoding nucleic acid, expression of the variant
nucleic
acid in an flg construct in recombinant cell culture, and, optionally,
purification from
the cell culture, for example, by immunoaffinity chromatography using an
immobilized antibody specific for the natural (non-variant) epitope. The
presence of
the desired epitope can be readily ascertained by one skilled in the art using
an
antibody, for example in an immunoassay using a mAb the binding of which
defines
the epitope. A standard assay, for example immunofluorescence of flow
cytometry
may be used to detect the variant epitope on the surface of a cell.
Alternatively, the
presence of the desired epitope can be detected using a cellular assay, for
example an
assay which measures the stimulation of T lymphocytes to proliferate or to
secrete
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cytokines. Such assays are well known in the art and are described in detail
in the
Examples below.
In preferred embodiments, a tolerogenic gp I20-IgG fusion Ig protein
modulates the responsiveness ofB and/or T cells to non-neutralizing gp120
epitopes,
5 for example in the C:1 region or in the CS region which contains HLA-cross-
reactive
CS epitopes.
The CI region of gp120 is noteworthy for its dominance in being a target for
immune reactivity. The V3 loop, in particular the V3 region of the loop (see
Tables II
and III), is noteworthy as a target for neutralizing antibodies. The CS region
is
I O noteworthy for its cross reactivity with HL-A molecules and its
stimulation of
autoimmunity. Hence one or more epitopes of one of more of these regions would
be
useful as tolerogenic epitopes. In particular any gp120 epitope which
stimulates
autoimmunity or against which an autoimmune host response is directed
(irrespective
of mechanism of induction) is a preferred epitope for use in a tolerogenic fIg
of this
I S invention.
Because the host immune system will process any administered fIg for
presentation to T lymphocytes in conjunction with the host's MHC
glycoproteins on the; antigen-presenting cells ("APC")(or more appropriately,
"tolerogen-presenting" cells), it is preferably to include a peptide of
sufficient
20 length for binding to host MHC molecules and subsequent presentation. As
shown below, in particular for T helper cell epitopes, the gp120 peptide may
be as short as about 6 amino acids. Generally larger peptides are preferred,
including those with more than one gp120 epitope in the flg. For example,
about 10-20 amino acids, preferably about 10-40 amino acids, more preferably
25 about 10-60 amino acids are included in the flg. This will allow the host
cells
to select the epitopes appropriate for the particular MHC type. Alternatively,
as discussed below, T helper epitopes may be identified and selected using
various published computer-based algorithms. It is preferable, though not
required, to exclude cysteine from the tolerogenic fIg because of the
30 constraints this amino acid imposes on uncontrolled secondary structure.
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The present inventors have developed a model system which utilizes mice
made transgenic for human CD4 which is used to screen fIg constructs for their
efficacy and utility in humans. Administration to these mice of gp120 and anti-
gp120
or HIV virions or crosslinked gp120 leads to sensitization for subsequent
apoptosis.
Once the animals have been primed in this way, apoptosis occurs
"spontaneously" in
response to environmental exposure to antigens which engage the TCR and
trigger the
apoptotic process. Specific antigens including peptides with defined epitopes
are
administered to more precisely activate T cell apoptosis. This can be
evaluated by
testing the animals for epitope-specific T cell unresponsiveness or
hyporesponsiveness. The induction of B-cell tolerance and T helper cell
tolerance to
selected gp120 epitopes can readily be tested in this model for its effect on
the
pathway of T-cell apoptosis.
The present inventors have utilized a peptide that contains both a T-cell and
a
B-cell epitope, created a fusion protein of this peptide with an IgG molecule
serving
as a "carrier" and have used it to induce epitope-specific T-cell and B-cell
tolerance
(see Examples). This success indicates the utility of inducing epitope-
specific B-cell
tolerance to HIV peptides. Such tolerance is exploited to counteract the non-
protective or even harmful antibody responses to certain gp 120 peptides
(Homsy, J. et
al. (1989) Science 244:1357; Finkel, T. et al. (1994) Curr. Opin. Immunol.
6:605;
Fiist, G. et al. ( 1995) Immunol. Today, I b:167), as described below.
In the C 1 region of gp 120, both T-cell and B-cell epitopes have been shown
to
be immunodominant (Abacioglu, Y. et al. (1994) AIDS Res. Xuman Retrovirus.
10:371). Within C1, these investigators defined boundaries, termed Cla and
Clb.
The Cla peptide epitope FNMWKND (corresponding to residues 63-69 of Figure 1,
SEQ ID N0:83) can be detected with monoclonal antibody B10. A preferred
peptide
has the structural motif similar to that used earlier to form a 14-mer with a
C-terminal
cysteine for coupling ease using MBS (m-maleimodobenzoyl-N-hydroxysuccinimide
ester) AAAFNMWKNDGGGC (SEQ ID N0:113). This peptide can be chemically
conjugated to HGG for evaluation in vivo and in vitro for tolerogenicity.
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37
Table IV, .above provides amino acids sequences of T helper cell epitopes of
gp120 that have been identified using either human or murine test systems and
have
been entered in the HIVMID published on the Los Alamos National Laboratory
World Wide Web Site. Preferred fTg constructs include one or more of the
epitopes
presented in Table: IV linked to the N-terminus of an Ig H chain as was
described
above for B cell epitopes (e.g., those in Table III). However, the present
inventors do
not intend to be limited by this listing of sequences which are specifically
based on
the amino acids sequences of HIV subtype B viruses. The art permits
identification of
other epitopic sequences derived from other HIV subtypes (discussed above) as
well
as viral isolates or "quasi species" thereof.
TABLE II
Defined Regions Of g p120 For Use As Tolerogenic Epitopes
Region of gp1:20 Residues (approximate)'
C1 1-95
C1 (subregion) 60-90
V 1 loop 101-127
V2 loop 144-166
C2 168-269-
C2 223-238
V3 loop 266-301
V3 region 274-288
C3 298-360
C3(subregion) 331-348
V4 loop 355-388
C4 383-427
VS 428-442
CS 443-481 (C-terminus)
' See Figure 1 for consensus
amino acid sequence of HIV-1
subtype B gp 120
(and selected individual
amino acid substitution
variants in individual viral
isolated.
See Figure 2 for consensus amino acid sequence of other HIV-1 subtypes
CA 02279492 1999-07-29
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38
TABLE III
HIV gp120 B CeII Epitopes Identified by mAbs
Region or Virus Amino Acid SEQ
Positions Strain* Residues ID
S _NO:
30-51 LAI ATEKLWVTVWGVPVWKEATTT 114
31-50 LAI TEKLWTVWGVPVWKEATT 115
31-50 LAI GVPVWKEATT 116
42-61 LAI VPVWKEATTTLFCASDAKAY 2
64-78 IIIB EVHNVWATHACVPTD 3
51-70 LAI YDTEVHNVWA 4
81-90 LAI PQEVVLVNVT
81-100 LAI PQEVVLVNVTENFDMWKIVDM 6
89-103 IIIB PNPQEVVLVWTENF 117
91-100 LAI ENFDM WKNDM 1
I
8
93-96/94-97 LAI/BH10 FNMW 119
94-99 BH10 FNMWKN 120
91-100 LAI ENFDM WKNDM 118
101-110 LAI VEQMHEDIIS 121
101-120 LAI VEQMHEDIISLWDQSLKPCV 122
311-321 HXB10 EQMHEDIISLWDQSLKPCVK 123
101-120 LAI LWDQSLKPCV 124
102-121 LAI EQMHEDIISLWDQSLKPCVK 123
114-123 IIIB MHEDIISLWD 125
122-141 LAI LTPLCVSLKCTDLKNDTNTN I26
162-169 HXB2 STSIRGKV 127
162-171 V2 BH10 STSIRGKVQ 128
170-180 BH10 QKEYAFFYKLD 129
or IIIB
172-191 HXB2 EYAFFYKLDIIPIDNDTTSY 130
162-181 BH10 STSIRGKVQKEYAFFYKLDI 131
172-181 HXB 2 EYAFFYKLDI 132
221-220 LAI EPIPIHYCAPA 133
21 I -230 LAI EPIPIHYCAPAGFAILKCNN 134
222-231 LAI GFAILKCNNK 135
242-261 LAI RPVVSTQLLL 136
252-271 LAI RPVVSTQLLLNGSLAEEEW 137
257-262 BH10 TQLLLN 138
257-263 BH10 TQLLLNG 139
262-281 LAI NGSLAEEEVVIRSVNFTDNA 140
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Table III, cont.
Region or Virus Amino Acid
SEQ
Positions Strain* Residues
ID
_NO: --
261-280 LAI VIRSVNFTDN 141
299-304 IIlB 1NCTRP
142
299-304 IIIB SVEINCTRPNNNTRKSI 143
299-308 IIIB PNIVNTRKSIR 7
300-315 HXB 10 NNNTRKRIRIQRGPGR g
304-308 IIIB RKSIR
309-318/329-338
IIIB IQRGPGRAFV / AHCNISRAKW 144
314-323/494-503
infec GRAFVTIGKI / LGVAPTKAKR 145
1 S 316-322 infec PGRAFY
12
302-321 BH10 NTRKSIRIQRGPGRAFVTIG 13
306-338 BH10 PNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHC
14
307-318 IIIB NNTRKSIRIQRG I
S
308-313 MN NKRICRIHIGPGRAFYTTKNIIGTIC 16
308-313 MN V3 tip
304-318 LAI RKSIRIQRGPGRAFV 17
299-304 IIIB IRIQRGPGR 1
g
299-304 IIIB KRIRIQRGPGRAFVTIG 19
308-328 BRU
20
V3 BRU RGPGRAFV 21
V3 MN RKRIHIGPGRAFYTT 22
V3 infec -I----G--FY-T 146
311-321 HXB10 RGPGRAFVTIG 23
V3 infec SISGPGRAFYTG 24
V3 MN KRIHI 25
V3 infec IXIGPGR 147
V3 MN KRIHIGP 26
V3 MN IHIGPGR 2~
(orinfec)
V3 MN HIGPGR 28
V3 infec HIGPGRA 29
V3 infec RKRIHIGPGRAFYTT 22
V3 ? HIGP 30
311-324 MN RIHIGPGRAFYTTG 31
312-318 MN IXIGPGR 147
307-316 IIIB RIQRGPGRAF 32
307-316 IIIB IQRGPGRAFV 10
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Table III, cont.
Region or Virus Amino Acid SEQ
Positions Strain* Residues I
D
_NO: _
S 308-316 IIIB IQRGPGRAF 33
V3 IIIB IRIQRGPGRAFVTI 34
V3 316-330 HXB2 RGPGRAFVTIGKIG 35
V3 ? QRGPGRA 36
V3 IIIB IXXGPGRA 37
10 V3 infec IGPGR 3g
V3 MN GPGR 39
V3 MN GPXR 40
308-313 MN GPGRAF 41
V3 MN RIHIG 42
15 V3 MN HIGPGRAF 43
V3 IIIB GRAF 44
V3 IIIB RAF 148
361-380 LAI IFKQSSGGDPEIVTHSFNCGG 149
362-381 LAI FKQSSGGDPEIVTHSFNCGGE 150
20 380-393 LAI GEFFYCNSTQLFNS 151
C3 HIV2ROD HYQ [core] I52
C3 HIV2ROD RNISFKA 153
C3 HIV2ROD APGK[core] 154
395-400 BH10 WFNSTW 155
25 423-437 IIIB IINMWQKVGKAMYAP 156
429-443 EVGKAMYAPPISGQI 157
429-438 BRU EVGKAMYAPP 158
429-438 BRU GKAMYAPPIS 159
CD4 bs* IIIB AMYAPPI 160
30 CD4 bs IIIB AMYAPPISGQ 161
425-441 IIIB NMWQEVGKAMYAPPISG 162
412-453 MN GKAMYAPPIS 159
451-470 LAI SNNESEIFRL 163
471-490 LAI GGGDMRDNWRSELYKYKVVK 45
35 490-508 IIIB KYKVVKIEPLGVAPTKAKRR 46
314-323 and 494-503 GRAFVTIGKI and LGVAPTKAKR 11
and 47
472-491 LAI GGDMRDNWRSELYKYKVVKI 48
491-S00 - LAI IEPLGVAPTK 49
40 503-509 infec RRVVQRE 50
C terminus infec PTKAKRR S
1
C terminus infec VVQREKR 52
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TABLE IV
HIV gp120
T Helper
Cell
Epitopes
Virus SEQ
Position No. Strain Amino Acids _Rec. In
No.
39-51 EQLWVTVYYGVPV 1 53
45-55 VYYGVPVWKEA 1 54
48-61 GVPV WKEATTLFC 1 55
72-82 AHKV WATHACV 1 56
74-85 LAI NVWATHACVPTD 2 57
81-92 CVPTNPVPQEVV ~ Sg
108-119 LAI VEQMHEDIISLW 2 59
109-121 EQMHEDIISLWDQ ~ 60
109-123 IIIB EQMHEDIISLWDQSL 3 61
112-124 IIIB, HEDIISLWDQSLK 3-9 62
BH10
115-126 LAI IISLWDQSLKPC 2 164
204-216 SVITQACSKVSFE 1 165
215-228 FEPIPIHYCAFPGF 1 166
233-244 LAI AGFAILKCNNKT 2 167
269-283 IIIB EVVIRSANFTDNAKT l0 168
B10
274-288 IIIB SANFTDNAKTIIVQL 10 169
:B 10
296-312 LAI IIVQLNQSVE 2 I70
292-300 SF2 NESVAINCT 11 171
MN ESVQIN 12 172
303-321 IIIB CTRPNNNTRKSIRIQRGPG(Y) ~3 66
307-322 IIIB NTRKSIRIQRGPGR 14 67
309-323 IIIB EQRGPGRAFVTIGKI 10 68
:B 10
315-329 IIIB RIQRGPGRAFVTIGK 6-8,15,669
MN analogRIHIGPGRAFYTTKN 16 70
314-328 IIIB GRAFVTIGKIGNMRQ 10 71
:810
324-338 IIIB FVTIGKIGNMRQAHC 3 173
327-341 HXB2 RQAHCNISRAKWNNT 17 174
342-356 IIIB RAKWNNTLKQICSKL 3 175
346-359 QIVKKLREQFGNNK 18 176
364-378 IIIB SSGGKPEIVTHSFNC 10 177
:810
368-377 LAI NKTIIFKQSS 2 178
369-383 IIIB PEIVTHSFNCGGEFF l0 179
:B10
394-408 IIIB TWFNSTWSTKGSNNT 10 180
:810
399-413 IIIB TWSTKGSNNTEGSDT 10 181
:B 10
410-429 PV22 GSDTITLPCRIKQFINMWQE 19,20 182
424-438 IIIB INMWQEVGKAMYAPP 10 183
:B10
428-443 IIIB KQIINMWQEVGKAMYA 3-8,12 ,13,16,21,22
:B 10 184
432-446 IIIB NMWQEVGKAMYAPPI 3 185
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Table IV. cont.
Virus SEQ
Positions Strain Amino Acids _Ref. ID
NO.
437-451 IIIB VGKAMYAPPISGQIR 3 73
S 459-473 IIIB B10 GNSNNESEIFRPGGG Io 74
466-481 FRPGGGDMRDNWRSEL 18 75
474-488 IIIB B10 DMRDNWRSELYKYI~V 10 76
483-497 IIIB RDNVVRSELYKYKVVK 3 77
C492-506 IIIB CKYKVVKIEPLGVAPT 3 78
484-498 IIIB B10 YKYKVVKIEPLGVAP l0 79
494-518 IIIB KVVKIEPLGVAPTKAKRRVVQREKRC 14
80
References: 1. K.J. Sastry et al., AIDS, 1991 5:699-707; 2. R.D. Schncr et
al., J. Immunol. 1989
142:1166-1176; 3. P.M. Hale et al., Int'1. Immunology, 1989 1:4:409-415; 4.
K.B. Cease et al., Proc.
1 S Natl. Acad. Sci. USA, 1987 84:4249-4253; 5. J.A. Bercofsky et al., Nature,
1988 334:706-708; 6. M.
Clerici et al., Nature, 1989 339:383-385; 7. M. Clerici et al., J. lmmunol.,
1991 146:2214-2219; 8.
M. Clerici et al., Eur. J. Immunol., 1991 21:1345-1349; 9. A. Hosmalin et al.,
J. Irnmunol., 1991
146:1667-1673; 10. B. Wahren et al., Vaccines, 1989 89:89-93; 11. P. Botarelli
et al., J. Immunol.,
1991 147:3128-3132; 12. F.D.M. Veronese et al., J. Mol. Biol., 1994 243:167-
172; 13. T. J. Palker et
al., J. Immunol., 1989 142:3612-3619; 14. G. Goodman-Snitkoff et al., Vaccine,
1990 8:257-262;
15. H. Takahashi et al., J. Exp. Med., 1990 171:571-576; 16. M. Clerici et
al., J. Inf. Dis., 1992
165:1012-1019; 17. A. P. Warren et al., AIDSRes. Hum. Retrovir., 1995 8:559-
564; 18. J. Krowka
et al., J. Immunol., 1990 144:2535-2540; 19. K. M. Callahan et al., J
Immunol., 1990 144:3341-
3346; 20. M. Polydefkis et al., J. Exp. Med., 1990 171:875-887; 21. B.F.
Haynes et al., J. Exp. Med.,
1993 177:717-727; 22. D. M. Klinman et al., AIDSRes. Hum. Retrovir., 1995 1 I
:97-105
Preferred approaches to identification and selection of T cell epitopes, in
particular T helper cell epitopes, for inclusion in a flg as described herein,
utilize
computer-based algorithms. Several computer-driven algorithms have been
devised
in the art which exploit the alphabetic representation of amino acid sequence
information to search for T cell epitopes by searching the amino acid sequence
of a
given protein for characteristics believed to be common to immunogenic
peptides, and
thereby locating regions that are likely to induce cellular immune response in
vitro.
With the rapid expansion of sequence data on geographic subtypes (clades) of
HIV
and individual HIV quasi-species, the application of these algorithms to HIV
proteins
can significantly reduce the number of regions which would require in vitro
testing for
the desired property (generally immunogenicity) although as envisioned by the
present inventors, the desired property is tolerogenicity when presented to
the immune
system as an flg. Computer-driven algorithms can identify regions of HIV
proteins
CA 02279492 1999-07-29
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43
that contain epitopes and are less variable among geographic HIV isolates;
alternatively, computer-driven algorithms can rapidly identify regions of each
geographic isolate':. more variable proteins that should be included in a
multi-Glade
tolerogenic ftg.
Well-known and conventional ways to identify T cell epitopes within protein
antigens, which may be used for the present invention, employ a variety of
methods,
including the use of whole and fragmented native or recombinant antigenic
protein,
and the "overlapping peptide" method. This approach involves synthesis of
overlapping peptidc;s which span the entire sequence of a given protein
antigen, in the
present case, gp120. These overlapping peptides are then tested for their
capacity to
stimulate the relevant T cell responses in vitro, for example T cell
proliferative
responses (Vordermeier, H.M. et al. (1993) Immunology 80:6-12; Ashbridge, K.R.
et
al. (1992) J. Immur~ol. 148:2248-2255). While the overlapping peptide method
is
thorough, it is both cost- and labor-intensive.
The computer based algorithm methods minimize the cost and labor of the
overlapping peptide; method and avoid the potential omission of sites between
overlapping fragments. Such computer-based algorithms designed to predict T
cell
epitopes from the amino acid sequences of proteins include AMPHI. AMPHI
searches a protein's primary structure for peptides with a high probability of
folding as
amphipathic structures (Margalit, H. et al. (1987) J. Immunol. 138:2213-2229;
Cornette, J.L. et al. In: The Amphipathic Helix (Ed. Epand, R.M.), CRC Press,
Boca
Raton, 1993). Seventy percent of published epitopes were found to contain
sequences
that would have been predicted by AMPHI (Margalit et al., supra; Spouge, J.L.
et al.
(1987) J. Immunol. 138:204-212). The number of known T cell epitopes has
quadrupled since the design of AMPHI, and of these, 65% are amphipathic, such
that
the correlation rem~~ins highly significant (Cornette et al., supra). Other
epitope
prediction algorithms which analyze protein sequences for specific secondary
structural or sequer.~ce characteristics (Stifle, C.J. et al. (1987) Mol.
Immunol.
24:1021-1027; Rotlhbard, J.B. et al. (1988) EMBO J. 7:93-100; Salomon, M. et
al.,
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(1993) Vaccine 11:1067-1073) generally search for a spacing of hydrophobic
residues
similar to that searched for by the AMPHI algorithm.
DeGroot and colleagues (Meister, G.E. et al. (1995) Vaccine, 13581-591)
developed two computer-based algorithms for T cell epitope prediction, OptiMer
and
EpiMer, which incorporate current knowledge of MHC-binding motifs. OptiMer
locates amphipathic segments of protein antigens with a high density of MHC-
binding
motifs. EpiMer identifies peptides with a high density of MHC-binding motifs
alone.
These algorithms exploit the tendency for MHC-binding motifs to cluster within
short
segments of each protein. Epitopes predicted by these algorithms contain
motifs
corresponding to many different MHC alleles, and may contain both class I and
class
II motifs, features thought to be ideal for the peptide components of
synthetic subunit
vaccines. Use of these two algorithms provide sensitive and efficient means
for the
prediction of promiscuous T cell epitopes that may be used to development
preparations such as epitope-specific vaccines, or, for the present
application, specific
tolerogenic epitopes to be used in an flg.
OptiMer examines known amino acid sequences of proteins and generates a
list of peptides that contain these motifs; the algorithm then identifies
peptides that
would be amphipathic if folded as a helix or twisted as a beta-strand, using
the
AMPHI algorithm. These potentially amphipathic peptides are compared to the
list of
MHC-binding motif matches. OptiMer extends the predicted amphipathic peptides,
to
maximize the density of MHC-binding motif matches per length of protein
region.
The EpiMer algorithm searches protein amino acids sequences for MHC-
binding motif matches, generating a list of matches for each protein. The
relative
density of these motif matches is determined along the length of the antigen,
resulting
in the generation of a motif density histogram. Finally, the algorithm
identifies
protein regions in this histogram with a motif match density above an
algorithm-
defined cutoff density value, and produces a list of subsequences representing
these
clustered, or motif rich regions. The regions selected by EpiMer may be more
likely
to act as mufti-determinant binding peptides than randomly chosen peptides
from the
same antigen, due to their concentration of MHC-binding motif matches.
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OptiMer and EpiMer, have been used to predict putative epitopes in five
Mycobacterium tuberculosis (Mtb) protein antigens (14 kDa, 16 kDa, 19 kDa, 38
kDa,
and 65 kDa) and three human immunodeficiency virus (HIV) protein antigens
(nef,
gp160 which is the precursor of gp120 and gp4l, and reverse transcriptase
{RT). To
5 evaluate the new algorithms' predictive power, Meister et al. compared
OptiMer- and
EpiMer-predicted epitopes, AMPHI-predicted epitopes, and peptides that would
have
been synthesized using the "overlapping peptide" method, to a selection of
published
T cell epitopes for the above proteins. These algorithms were used to predict
T cell
epitopes from within the published sequences of three HIV protein antigens.
Epitopes
10 published for the HIV protein antigens nef and gp160 were almost
exclusively class I
MHC-restricted, while epitopes published for RT were both class I- and class
II-
restricted.
A version o:f either OptiMer or EpiMer based on the list of class I-restricted
MHC-binding motifs was used to predict putative epitopes for nef and gp160,
while
15 versions of both algorithms based on the combined list of class I- and
class II-
restricted motifs were employed to predict putative epitopes for the HIV
protein
antigen RT. In all, 29 putative epitopes were generated by the class I-
specific version
of OptiMer (totaling 661 amino acids in length); 30 putative epitopes were
generated
by EpiMer, totaling 614 amino acids in length. AMPHI generated 36 putative
epitopes
20 (totaling 666 amino acid residues), and 104 peptides (totaling over two
thousand
residues in length) would have been required by the overlapping peptide
method. For
these two HIV protein antigens, the class I-restricted implementations of both
OptiMer and EpiMer identified published epitopes with an efficiency comparable
to
that of AMPHI, and greater than that of the overlapping peptide method.
EpiMer's
25 sensitivity per amino acid exceeds that of either OptiMer or AMPHI. For RT,
the
combined class I/class II implementation of OptiMer generated 18 putative
epitopes
(totaling 422 amino acids); the same implementation of EpiMer generated 22
putative
epitopes (totaling 361 amino acids in length). These values compare with 23
putative
epitopes generated by AMPHI (totaling 433 amino acids) and 55 peptides
(totaling
30 over one thousand residues) required by the overlapping peptide method.
OptiMer
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and EpiMer predict published T cell epitopes for the HIV protein RT with both
efficiency and sensitivity comparable to that of the AMPHI algorithm. EpiMer
again
attains the highest sensitivity per amino acid of these three algorithms. In a
recent
comparison of EpiMer predictions to published HIV protein T cell epitopes, the
EpiMer algorithm was shown to be 2.4-fold more sensitive (per amino acid
residue)
than the overlapping peptide method for detecting published T cell epitopes
for four
HIV proteins, gp160, nef, tat, and gag. In contrast, AMPHI was somewhat less
sensitive ( 1.6-fold) (Roberts, C.G.P. et al. ( 1996) AIDS Res. Human
Retrovir. 12:593-
607). A summary of comparisons of the overlapping peptide method with the
AMPHI and EpiMer prediction method is provided by Roberts et al. (supra).
The above approach to HIV epitopes has been embodied in an algorithm
recently named EpiMatrix/H1V which predicts the sequences most likely to bind
to
MHC molecules when given a number of primary HIV protein sequences and which
was developed by A.S. De Groot at Brown University and implemented for the
Internet by AVX Design Inc., Providence, Rhode Island. Both a website and an
online tool, EpiMatrix is located on the Internet at
http://www.epimatrix.com/hiv as of
November 1, 1996. Use of this algorithm in accordance with the present
invention
allows selection of peptides that are highly likely to bind to a particular
subject's
MHC, thereby enabling identification of T-helper epitopes (as well as
cytotoxic T-cell
epitopes for vaccine development). The EpiMatrix algorithm yields a score for
each
peptide in a 10-mer frame. Scoring is a quantitative estimate of the
likelihood
(relative to other sequences) that a peptide will bind to a given HLA
molecule. Two
scoring methods are used: single-allele predictions score for specific HLA
alleles and
clustered predictions score peptides by the prevalence of MHC alleles in
selected
populations Matrices for all of the major (greater than 10% population
prevalence)
MHC alleles representing world populations are included in the algorithm (B.M.
Jesdale et al., Vaccines '97, Cold Spring Harbor Laboratory Press). EpiMatrix
reduces the total number of regions of HIV proteins to be evaluated in vitro,
permitting more rapid identification of desired epitopes. (See, also
AIDSWEEKLY
Plus, 18 November 1996 issue).
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Additional MHC binding motif based algorithms have been described by K.C.
Parker et al. (J. Immunol. (1994) 152:163-175) and Y. Altuvia et al. (1995) J.
Mol.
Biol. 249:244-250). In these algorithms, binding to a given MHC molecule is
predicted by a linear function of the residues at each position, based on
empirically
S defined parameters, and in the case of Altuvia et al., known
crystallographic structures
are also taken into consideration. J. Hammer et al. (J. Exp. Med. (1994)
180:2353-
2358) described a tE:chnique known as "peptide side chain scanning" which is
used to
predict binding peptides for an MHC allele.
The EpiMer/EpiMatrix algorithm predicted putative T cell epitopes from protein
sequences for HIV-1 nef, gp160, gag p55, and tat that required fewer peptides
and
therefore fewer amino acid residues to be synthesized than either AMPHI-
predicted
peptides or overlapping peptides. For the four HIV-1 proteins, EpiMer
predicted 43
peptide epitopes, AMPHI predicted 68 peptides , and the overlapping peptide
method
(20 amino acid long peptides overlapping by 10 amino acids) would have
required
161 peptides. Details (amino acid start and stop, number of MHC binding
motifs) of
the predicted proteins are available36. Regions of HIV proteins that contain
as many
as 20 to 30 MHC binding motifs can be identified using this algorithm.
The various known methods for epitope prediction are not mutually exclusive.
As the contributions of side chains and tertiary peptide structure to peptide-
MHC
binding are better quantified, the development of a computer algorithm that
predicts T
cell epitopes based on a matrix of side chain information such as one
described by J.
Hammer (1995) Curr. Opin. Immunol. 7:263-269) will become available. The
identification of novel structural features which are able to independently
predict
peptide recognition and their subsequent synthesis into a combined algorithm
with
statistically verifiable predictive capacity, allows a dramatic reduction in
the time and
effort required to s3mthesize and test potential T cell antigenic sites for
HIV proteins,
by allowing the prediction of sites with a high concentration of antigenic
features.
HIV protein regions that contain multiple overlapping class-II restricted
epitopes, also knov~m as "mufti-determinant" or mufti-determinant peptides,
have been
identified in mice and humans. Such regions might be important to include in
the
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synthesis of an fIg having multiple tolerogenic T helper cell epitopes as
described
herein. This is particularly useful if a multi-determinant T cell epitope is
involved in
stimulating antibody responses (i.e., to B cell epitopes).
Table V, below presents a list of epitopes of gp120 (and several N-terminal
epitopes of gp41) which were identified using EpiMer (Roberts et al., supra).
These
sequences are from the BH10 strain of HIV-1. The amino acid sequence of this
HIV
strain was obtained from the SWISS-PROT protein sequence data bank , Accession
No. P03375 (EMBL Data Library, Heidelberg, Germany). The residue numbers
shown in Table V are from this sequence bank. Those residues beyond amino acid
511 are part of gp4l, not gp120. In a preferred embodiment, the present
invention
provides a tolerogenic fIg H chain or intact fIg molecule which includes at
the N
terminus of the H chain one or more of the HIV peptide epitopes listed in
Table V.
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TABLE V
T Cell Epitopes of Hiv gp120 Identified by Epimer Algorithm
AMINO AMINO
ACIDS SEQUENCE ACIDS SEQUENCE
19 - TMLLGNILMICSATEKL 168 -
34 185 KVQKEYAFFYKLDIIPID
(SEQ ID NO:186) (SEQ ID
N0:187)
20 - MLLCiMLMIC 168 - KVQKEYAFF
28 176
21 - LLGMLMICS 169 - VQKEYAFFY
29 177
21 - LLGMLMICSA 169 - VQKEYAFFYK
30 178
22 - LGMLMICS 171 - KEYAFFYKL
29 179
22 - LGMLMICSA 173 - YAFFYKLDI
30 181
24 - MLMIICSATE 173 - YAFFYKLDII
32 182
24 - MLMIfCSATEK 174 - AFFYKLDI
33 I 81
25 - LMICSATEK 174 - AFFYKLDII
33 182
26 - MICS.ATEKL 175 - FFYKLDII
34 182
175 - FFYKLDIIP
183
36 - 175 - FFYKLDIIPI
54 184
VTVYYGVPVWKEATTTLFC
(SEQ 176 - FYKLDIIPI
ID 184
N0:63)
36 - VTVYYGVPV
44
36 - VTVYYGVPVW 198 - TSVITQACPKVSFEPIP
45 214
37 - TVY~'GVPVWK (SEQ ID N0:188)
46
38 - VYYCiVPVWK 198 - TSVITQACPK
46 207
39 - YYGVPVWK 199 - SVITQACPK
46 207
42 - VPVV~KEATTT 199 - SVITQACPKV
51 208
44 - VWK1EATTTL 200 - VITQACPKV
52 208
44 - VWKIEATTTLF 202 - TQACPKVSF
53 210
45 - WKEATTTLF 204 - ACPKVSFEPI
53 213
45 - WKEATTTLFC 205 - CPKVSFEPI
54 213
84 - WLVNVTENFNM 249 - HGIRPWSTQLLL
95 261
(SEQ ID N0:64) (SEQ ID NO:189)
.
85 - VLVNVTENF 249 - HGIRPVVS
93 256
85 - VLVNVTENFN 251 - IRPVVSTQL
94 259
87 - VNV7.'ENFNM 251 - IRPVVSTQLL
95 260
252 - RPVVSTQLL
260
115 SLKPCVKLTPLCY
-127
(SEQ 1D N0:65) 284 - IIVQLNQSVEINC
296
116 LKPCVKLTP (SEQ ID N0:190)
- 124
116 LKPCVKLTPL 284 - IIVQLNQSV
- 125 292
117 KPCV'KLTPL 285 - IVQLNQSVE
- 125 293
119 CVKI,TPLCV 286 - VQLNQSVEI
- 127 294
286 - VQLNQSVEIN
295
288 - LNQSVEINC
296
SUBSTITUTE SHEET (RULE 26)
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TABLE V, cont.
AMINO AMINO
ACIDS SEQUENCE ACIDS SEQUENCE
310 QRGPGRAFVTIGKIGN 482 - 527 ELYKYKWKIEPLGVA
- 330
MRQAH (SEQ ID N0:191 PTKAKRRWQREKRAV
)
312 GPGRAFVTI GIGALFLGFLGAAG
- 320
314 GRAFVTIGK (SEQ ID N0:194)
- 322
31 S RAFVTIGK 482 - 490 ELYKYKVVK
- 322
31 S RAFVTIGKI 483 - 491 LYKYKVVKI
- 323
317 FVTIGKIGN 483 - 492 LYKYKVVKIE
- 325
318 VTIGKIGNM 486 - 494 YKVVKIEPL
- 326
318 VTIGKIGNMR 486 - 495 YKVVKIEPLG
- 327
319 TIGKIGNMR 488 - 496 VVKIEPLGV
- 327
320 IGKIGNMRQ 488 - 497 VVKIEPLGVA
- 328
320 IGKIGNMRQA 489 - 497 VKIEPLGVA
- 329
491 - 499 IEPLGVAPT
351 EQFGNNKTIIFKQ 493 - 502 PLGVAPTKAK
- 363
(SEQ ID N0:192) 494 - 502 LGVAPTKAK
351 EQFGNNKTI 495 - 503 GVAPTKAKR
- 359
353 FGNNKTIIF 495 - 504 GVAPTKAKRR
- 361
353 FGNNKTIIFK 496 - 504 VAPTKAKRR
- 362
496 - SOS VAPTKAKRRV
381 EFFYCNSTQLFN 497 - 505 APTKAKRRV
- 392
(SEQ ID N0:193) 500 - 508 KAKRRVVQR
382 FFYCNSTQL 503 - 511 RRVVQREKR
- 390
382 FFYCNSTQLF 505 - 513 VVQREKR/AV'
- 391
383 FYCNSTQLF 506 - 514 VQREKR/AVG
- 391
383 FYCNSTQLFN 506 - 515 VQREKR/AVGI
- 392
414 ITLPCRIKQIINMWQEV 507 - 515 QREKR/AVGI
- 445
GKAMYAPPISGQIRC 510 - 518 KR/AVGIGAL
(SEQ ID N0:81 ) , 511 - R/AVGIGALF
519
414 ITLPCRIKQ
- 422
414 ITLPCRIKQI
- 423
416 LPCRIKQII
- 424
416 LPCRIKQIINM
- 426
418 CRIKQIINM
- 426
420 IKQIINMWQ
- 428
420 IKQIINMWQ
- 428
420 IKQIINMWQE
- 429
424 INMWQEVGKA
- 433
426 MWQEVGKAMY * "~" follows the C terminal
- 435 residue of
427 WQEVGKAMY gp120
- 435
428 QEVGKAMYA
- 436
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Application of the above approach to general HIV tolarogenic preparations
may be restricted by t:he amount of sequence variation in individual quasi-
species,
HIV strains, and HIV subtypes as well as by the MHC background of the subject
population. For example, the region of amino acids at about 130 to 160
(depending
S upon which subtype or isolate), has a great deal of inter-strain variation
and may
therefore best be avoided in designing a tolerogenic fIg which has the
broadest range
of applicability across virus variants and subtypes. HIV peptide epitopes
which
contain multiple MHC binding motifs, either conserved across HIV strains or
derived
from several different HIV strains, may be ideal candidates for targeting for
T helper
cell-directed tolerance induction, as it is assumed that the tolerogen will be
presented
in vivo by host MHC molecules. Thus, epitopes with multiple MHC binding motifs
or
having an MHC binding motif present in the highest frequency in the subject
population (race, ethnic group, e. ) would be preferably selected for
inclusion in a
tolerogenic flg. The EpiMer algorithm is particularly well suited for
identifying and
selecting such epitopes.
Preparation of Recombinant flg and its Transfer
The present invention provides polynucleotides encoding the fIg in the form of
recombinant DNA molecules in vehicles such as plasmid and retroviral vectors,
capable of expression in a desired eukaryotic host cell as disclosed herein.
The
invention also provides hosts transfected or transduced with the fIg
constructs which
are capable of producing in culture or in vivo the fIg molecules and secreting
them or
displaying them on the cell surface.
A preferred engineering strategy for inserting a foreign epitope at the N-
terminus of an IgG y chain is shown in Figure 4A and 4B. Figure 4A depicts the
incorporation of an oligonucleotide, in this example encoding the ~, phage C 1
repressor peptide 12-26. However, the present invention exploits the same
general
scheme wherein a native or synthetic gp120 peptide epitope is inserted in
place of the
12-26 peptide. This :is illustrated in Figure 4B. Any Ig gene construct may be
used
for insertion of the to~lerogenic epitope or epitopes. A preferred Ig gene
encodes
human Ig, more preferably an Ig comprising a human y chain.
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The general principles of recombinant DNA technology are utilized, as
described for example, in Sambrook, J. et al., Molecular Cloning: A Laboratory
Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989;
Ausubel, F.M. et al. Current Protocols in Molecular Biology, Voi. 2, Wiley-
Interscience, New York, 1987; Lewin, B.M., Genes IV, Oxford University Press,
Oxford, (1990); Watson, J.D. et al., Recombinant DNA, Second Edition.
Scientific
American Books, New York, 1992, which references are hereby incorporated by
reference in their entirety.
The DNA construct encodes an individual flg H chain, although the protein
products of this invention include both the fIg H chain and a complete
assembled Ig
molecules comprising the flg H chain having one or more HIV gp120 epitopes in
combination with a native human Ig L chain. The flg may also comprise two
different
H chains, one of which is a fusion protein having one or more HIV gp120
epitopes
added to or included in the V region.
Genetic sequences, especially cDNA sequences, encoding either a complete
flg H chains, the fIg V regions or a human Ig C region of any Ig isotype, most
preferably, an IgG isotype (i.e., a human Cy chain) are also provided herein.
The invention also provides a genetic sequence, especially a cDNA sequence
encoding an Ig V region fusion protein in which the V region encoding DNA has
been
combined in frame with one or more HIV gp120 epitopes. Though, genomic DNA
sequences may also be used, cDNA sequences are particularly preferred.
One non-limiting approach to producing the flg comprises the steps of
1. Selection of one or more gp120 epitopes as described below for which
tolerance is
desired;
2. Preparation of DNA encoding the epitope or epitopes selected above; this
can be
done by isolating HIV RNA and cloning an preparing cDNA corresponding to all
or part of gp120, by isolating and cloning DNA from HIV-infected cell, or if
the
DNA is sufficiently short, synthesizing an oligonucleotide having the desired
coding sequence. The latter synthetic approach permits construction of
artificial
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combinations of two or more gp120 epitopes or which are not contiguous in the
native protein.
3. Introduction of appropriate restriction enzyme recognition sites in the gp
120 DNA
to permit ligation to Ig H chain encoding DNA, preferably human y chain-
encoding DNA; this can be done by PCR, site-directed mutagenesis or
synthetically;
4. Selection of endogenous restriction sites of the Ig-encoding DNA or
modification
of the DNA as above to introduce restriction sites corresponding to those in
the
gp 120 DNA such that they can form cohesive ends and be ligated
5. Ligation of the gI>120 DNA to the Ig H chain DNA using conventional
methods.
6. Expression and production of the fIg H chains or intact Ig molecules (H~Lz)
in a
selected host, preferably human lymphoid or hematopoietic cells.
Oligonucleotides which can be used as primers for introducing useful
restriction sites into the gp120 and human Ig DNA for subsequent linkage are
1 S well known in the art. See, for example, Sambrook et al., supra.
In an alternate: embodiment, rather than using DNA encoding an entire Ig H
chain, the gp120 DN,A is linked to an Ig V gene cassette. Because the antibody
specificity of the fIg :is not important, any V region DNA can be selected. A
preferred
V gene is one which encodes a protein which, after fusion of a gp120 epitope
or
epitopes, even a full llength gp 120 protein, still maintains its ability to
fold properly in
an full Ig molecule (I-izLz).
The variable !;V) domain of an Ig chain includes hypervariable (HV) regions
which are also knov~m as complementarily-determining regions (CDRs) because
they
are important in "determining" the structure of the antibody combining site
that is
complementary the epitope bound. Each H and L chain V region has three HVs or
CDRs. The segments on either side of each HV region which are relatively
invariant
are termed "framework regions" (FRs). Thus, the order of these regions in a V
domain (from the N=terminus)is as follows: FRl-HV 1-FR2-HV2-FR3-HV3-FR4.
For example, the three HV regions are roughly from residues 28-35, 49-59 and
92-
103, respectively.
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The framework regions form the ~i sheets that provide th structural framework
of the domain, with the HV sequences corresponding to three loops at one edge
o each
sheet that are juxtaposed in the folded protein. The HV loops from the VH and
VL
domains are brought together, creating a single HV site at the tip of the Fab
fragment
which forms the antigen binding site. (See, for example, Janeway, C.A., Jr. et
al.,
IMMUNOBIOLOGY, 2n ed., Garland Publishing Inc., New York, 1996, chapter 3).
The first framework region (FR1 ) is the most N-terminal of the V region.
Eisen, H.N., GENERAL IMMUNOLOGY, (J. Lippincott Co., Philadelphia, 1990) at
pages 57-59, in particular Figure 14-19 at page 58, shows the amino acid
sequences of
the first framework region of 5 different human H chains. The first framework
region
includes the 30 N-terminal amino acids at which point the HV 1 region follows.
A
framework region of nine different human x L chains belonging to three
different
groups VKI, VKII and VKIII are shown in this textbook figure.. Again, the FRs
are
about 30 residues, with a number of positions in each group serving as
"framework
residues" which serve to characterize each VK group. In the present invention,
the
heterologous epitope of the fIg is preferably inserted immediately N terminal
to the
first framework region. In other embodiments, it may be fused "deeper" into
the Ig
sequence within the V region.
A spacer comprising between about 1 and 10 amino acids, preferably about 3-
5 residues, can be present between the C terminal residue of the heterologous
epitope(s), preferably a gp 120 epitope(s) and the N terminal residue of the
Ig V
region, provided that the protein can fold properly to present the gp120
epitope while
maintaining its tolerogenic properties. In a preferred arrangement, as
exemplified
below, a repeat of the 5 N-terminal amino acids of the Ig H chain is inserted
N-
terminal from the added the gp120 peptide (or peptides) such that this
pentapeptide
sequence is repeated on either end of the inserted gp120 sequence. If more
than one
gp120 peptide is included, a spacer as described herein may be linked to one
or more
of the added gp 120 peptides. A major purpose of the spacer is to permit
unimpeded
folding and proteolytic processing of the flg as if it were an normal Ig
protein. This
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assures proper surface expression of the flg and association with MHC proteins
on the
surface of a tolerogerl-presenting cell.
Advantage can be taken of the natural antigen-binding property of the V
region, as is described below for the model marine constructs wherein the V
region
5 was specific for the 1'JIP hapten. A complete Ig H chain is constructed by
combining
the now altered V gene construct containing additional gp120 DNA with a C gene
construct encoding a desired human C region. preferably a human Cy protein.
The
most preferred C region would be that encoding the y3 isotype.
Ig H chain (o:r VH) cDNA vectors are typically prepared from human cells and
10 modified by site-directed mutagenesis to place a restriction site at the
position in the
human sequence in vrhich the gp 120 DNA is to be grafted. Preferably this is
5' to the
nucleotide encoding the N-terminus of the Ig H chain or the VH protein.
Two coding DNA sequences are said to be "operably linked" if the linkage
results in a continuously translatable sequence without alteration or
interruption of the
I S triplet reading frame.. A DNA coding sequence is operably linked to a gene
expression element i:f the linkage results in the proper function of that gene
expression
element to result in expression of the coding sequence.
Expression vehicles include plasmids or other vectors, such as retroviral
vectors. A preferred vehicle carries a functionally complete human VH and CH
having
20 appropriate restriction sites engineered so that any gp 120-encoding
nucleotide
sequence with approvpriate cohesive ends can be conveniently ligated thereto.
These
vehicles can be used as intermediates for propagation of DNA encoding any
desired H
chain (VHC,.~ ready to receive a gp120 DNA sequence, and for the expression of
the
complete flg (gp 120-V"CH).
25 Preferred hosts are mammalian cells, most preferably human cells, grown in
vitro for prolonged periods, or taken from a host, cultured in vitro for
purposes of
transfection and then reintroduced into the host. Mammalian cells provide
post-translational modifications to the Ig protein molecules including leader
peptide
removal, folding and assembly of H and L chains, glycosylation of the protein
chains
30 and secretion of the complete functional fIg protein. Mammalian cells which
may be
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useful as hosts for the production of flg proteins include cells of lymphoid
origin,
such as the hybridoma Sp2/0-Agl4 (ATCC CRL 1581) or the myeloma P3X63Ag8
(ATCC TIB 9), also abbreviated as P3, and its derivatives. A preferred marine
cell
line for expressing the flg of this invention is J558L. Any cell line which
allows for
efficient expression and secretion of the fIg constructs of the present
invention and
which promotes proper folding of the flg is preferred. Known human lymphoid or
hematopoietic cell lines may be used, including B lymphoblastoid lines,
lymphomas,
hybridomas or heterohybridomas. Examples of cell lines and approaches for
expression of recombinant or chimeric or hybrid or modified Ig genes are
described in
Shin, S.U. et al., (1993) Int. Rev. Immunol. 10:177-186; Wright, A. et al.,
(1992) Crit.
Rev. Immunol. 12:125-168; Shin, S.U. et al. (1992) Immunol. Rev. 130:87-107;
Morrison, S.L., (1992) Annu. Rev. Immunol. 10:239-265; Morrison, S.L. et al.,
(1989)
Adv. Immunol. =14:65-92; Weidle et al., (1987) Gene .51:21; Whittle et al..
(1987)
Protein Engineering 1:499; Morrison, S.L., (1985) Science 229:1202-1207;
Morrison
S.L. et al., ( 1984) Annu. Rev. Immunol. 2:239-256, all of which references
are
incorporated by reference in their entirety. In a preferred embodiment, human
hematopoietic cells obtained from the intended recipient or those
histocompatible
with the recipient are transfected with the fIg DNA construct.
Many vector systems are available for the expression of cloned Ig H and L
chain genes in mammalian cells (see Glover, D.M., ed.(1985) DNA Cloning, Vol.
II,
pp143-238, IRL Press). Different approaches can be followed to obtain complete
HzLZ antibodies. It is possible to co-express H and L chains in the same cells
to
achieve intracellular association and linkage of H and L chains into complete
tetrameric HZLz antibodies. The co-expression can occur by using either the
same or
different plasmids in the same host. Genes for both H and L chains can be
placed into
the same plasmid, which is then transfected into cells, thereby selecting
directly for
cells that express both chains. Alternatively, cells may be transfected first
with a
plasmid encoding one chain, for example the L chain, followed by transfection
of the
resulting cell line with an H chain plasmid containing a second selectable
marker.
Cell lines producing HzL, molecules via either route could be transfected with
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plasmids encoding additional copies of H, L, or H plus L chains in conjunction
with
additional selectable markers to generate cell lines with enhanced properties,
such as
higher production of assembled H,L, antibody molecules or enhanced stability
of the
transfected cell lines.
S One particular strategy for inserting an HIV peptide sequence at or near the
N-
terminus of an Ig H chain is related to that described in Hebell, T. et al. (
1991 )
Science 254:102-105 and Ballard, D.W. et al. (1993) Proc. Natl Acad Sci. USA
83:9626-9630. A first plasmid is constructed which preferably includes .a full
genomic sequence of the Ig H chain and selectable markers, for example,
neomycin
and or/ampicillin resistance genes. The source DNA encoding the HIV gp120
epitope
or epitopes PCR is amplified to create the DNA encoding the desired single or
multiple epitopes. Appropriate restriction sites are included on the primers
so that the
epitope-encoding DrdA can be spliced into the Ig gene-containing vector. The
gp120
epitope sequence is subcloned into a site, preferably the VH site of the first
plasmid.
Recombinant clones are analyzed for proper orientation and polymerase induced
errors by double stranded DNA sequencing methods (e.g., Sequenase~ kit from
U.S.
Biochemical).
The promoter sequences useful for the DNA constructs of the of the present
invention are any promoters which allow efficient expression of the flg DNA of
the
invention in a target cell of choice, for example a hematopoietic progenitor
cell or a
lymphoid cells, more preferably a B cell. Preferred promoters are the
promoters of
the Ig gene into which the foreign epitope-encoding DNA is being inserted.
However,
other known promoters of either eukaryotic or viral origin may be used.
Suitable
promoters are inducible or repressible or, more preferably, constitutive.
Examples of
useful eukaryotic/viral promoters include the promoter of the mouse
metallothionein I
gene (Hamer, D., et al. (1982) J. Mol. Appl. Gen. 1:273-288); the TK promoter
of
Herpes virus (McKnight, S. (1982) Cell 31:355-365); the SV40 early promoter
(Benoist, C., et al. ('1981 ) Nature 290:304-310); and the yeast gal4 gene
promoter
(Johnston, S.A., et ail. (1982) Proc. Natl. Acad. Sci. (USA) 79:6971-6975;
Silver, P.A.,
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et al. (1984) Proc. Natl. Acad. Sci. (USA) 81:5951-5955). Strong promoters are
most
preferred.
The flg construct into which the gp120 epitope(s) has been inserted is
introduced ("gene transfer") into the appropriate target cells by conventional
methods,
e.g., direct physical transfer of plasmid DNA, or preferably, by virus-
mediated
transfer, for example using a retroviral vector, as discussed below.
A number of means for transferring genes are known in the art and may be
used herein, including, for example, electroporation and lipofection. A
preferred, and
reiativeiy efficient means for achieving transfer of genes is by retrovirus-
mediated
gene transfer (Gilboa, E. (1987) Bio-Essavs 5:252-258; Williams, D.A. et al.
(1984)
Nature 310:476-480; Weiss, R.A. et al., RNA Tumor Viruses, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York, 1985). One class of
retroviruses,
recombinant amphotropic retroviruses have been used as vectors for the
transfer of
genes into human cells (Cone, R.D. et al. ( 1984) Proc. Natl. Acad Sci. USA
81:6349-
6353; Danos, O. et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464.
When the targets for gene therapy are bone marrow or blood stem cells, for
example, it may be advantageous to manipulate the cells in vitro with
cytokines and
then to infect them with the vector bearing the flg gene (Wilson, J.M. et al.
(1990)
Proc. Natl. Acad. Sci. USA 87:8437-8441 ). Recombinant amphotropic
retroviruses
have been recognized as useful vectors for transferring genes efficiently into
human
cells, for example to correct enzyme deficiencies (Cone, R.D. et al. ( 1984)
Proc. Natl.
Acad. Sci. USA 81:6349-6353; Danos, O. et al, ( 1988) Proc. Natl. Acad Sci.
USA
85:6460-6464). For safety reasons, it is important that a retroviral vector
used for
gene therapy be capable of infecting only desired cells and not cause
generalized
infection of cells throughout the body of the individual being treated. In the
past, this
has generally been accomplished by using helper-defective virus preparations,
or
mutants lacking the yr packaging sequence, etc.
Another viral vector system useful for this invention is the recombinant adeno
associated viral (AAV) transduction system (Lebkowski, J.S., et al. (1988)
Mol. Cell.
Biol. 8:3988-3996). AAV DNA integrates into cellular DNA as one to several
tandem
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copies joined to cellular DNA through inverted terminal repeats {ITRs) of the
viral
DNA. (Kotin, R.M., et al. (1990) Proc. Natl. Acad Sci. USA 87:2211-2215). The
transgene DNA size limitation and packaging properties are the same as with
any
other DNA viral vectors. AAV is a linear single stranded DNA parvovirus, and
requires co-infection by a second unrelated virus in order to achieve
productive
infection. AAV carrica two sets of functional genes: rep genes, which are
necessary
for viral replication, and structural capsid protein genes (Hermonat, P.L., et
al. ( 1984)
J. Y'irol. 51:329-339). The rep and capsid genes of AAV can be replaced by a
desired
DNA fragment to generate AAV plasmid DNA. Transcomplementation of rep and
capsid genes are requiired to create a recombinant virus stock. Upon
transduction
using such virus stock., one recombinant virus uncoats in the nucleus and
integrates
into the host genome by its molecular ends.
Liposomes may be used to encapsulate and deliver a variety of materials to
cells, including nucleic acids and viral particles (Falter, D.V. et al. (1984)
J. Virol.
49:269-272). Preformed liposomes that contain synthetic cationic lipids form
stable
complexes with polymionic DNA (Felgner, P.L., et al. (1987) Proc. Natl. Acad.
Sci.
USA 84:7413-7417). Cationic liposomes, iiposomes comprising some cationic
lipid,
that contained a membrane fusion-promoting lipid dioctadecyldimethyl-ammonium-
bromide (DDAB) effiiciently transfer heterologous genes into eukaryotic cells
(Rose,
J.K., et al. (1991) Bio~techniques 10:520-525). Cationic liposomes can mediate
high
level cellular expressiion of transgenes, or mRNA, by delivering them into
cultured
cell lines (Malone, R.., et al. (1989) Proc. Natl..Acad. Sci. USA
86:60776081).
Ecotropic and amphotropic packaged retroviral vectors infect cultured cells in
the presence of cationic liposomes, such as Lipofectin (BRL, Gaithersburg,
MD), and
in the absence of specific receptors (Innes, C.L. et al. ( 1990) J. Virol.
64:957-961 ).
Physical means well-known in the art can be used for direct gene transfer,
including administration of plasmid DNA (Wolff et al., 1990, supra) and
particle-
bombardment mediated gene transfer, originally described in the transformation
of
plant tissue (Klein, T'.M. et gl. (1987) Nature 327:70; Christou, P. et al.
(1990) Trends
Biotechnol. 6:145) but also applicable to mammalian tissues in vivo, ex vivo
or in vitro
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(Yang, N.-S., et al. (1990) Proc. Natl. Acad. Sci. USA 87:9568; Williams, R.S.
et al.
( 1991 ) Proc. Natl. Acad. Sci. USA 88:2726; Zelenin, A. V. et al. ( 1991 )
FEBS Lett.
280:94; Zelenin, A.V. et al. ( 1989) FEBS Lett. 244:65; Johnston, S.A. et al.
( I 991 ) In
Vitro Cell. Dev. Biol. 27:11 ). Furthermore, electroporation, a well-known
means to
S transfer genes into cell in vitro, can be used to transfer DNA molecules
according to
the present invention to tissues in vivo (Titomirov, A.V. et al. (1991)
Biochim.
Biophys. Acta 1088:131 ).
Gene transfer can also be achieved using "carrier mediated gene transfer" (Wu,
C.H. et al. ( 1989) J. Biol. Chem. 264:16985; Wu, G.Y. et al. ( 1988) J. Biol.
Chem.
10 263:14621; Soriano. P. et al. (1983) Proc. Natl. Acad Sci. USA 80:7128:
Wang, C-Y.
et al. (1982) Proc. Natl. Acad. Sci. USA 8-1:7851; Wilson, J.M. et al. (1992)
J. Biol.
Chem. 267:963). Preferred carriers are targeted liposomes (Nicolau, C. et al.
(1983)
Proc. Natl. Acad Sci. USA 80:1068; Soriano et al., supra) such as
immunoliposomes,
which can incorporate acylated monoclonal antibodies into the lipid bilayer
(Wang et
1 S al., supra}, or polycations such as asialoglycoprotein/polylysine (Wu et
al., 1989,
supra).
In general, improved efficiency of gene transfer is attained by the use of
promoter enhancer elements in the plasmid DNA constructs (Philip, R., et al.
(1993)
J. Biol. Chem. 268:16087-16090).
20 The disclosure provided herein focuses on the gp 120-derived amino acid
sequence that is present in the final flg product. The nucleotide sequences
encoding
the desired peptide epitopes are not specifically listed here but are evident
to those
skilled in the art. First, the full native sequences for HIV gpo 120
(consensus for each
subtype as well as individual viral isolates reported to date) are provided in
the
25 Compendium cited above. One skilled in the art will know how to utilize
alternate
coding sequences for expressing the desired native or synthetic gp 120
peptides which
are to be included in the tolerogenic flg. Any nucleotide sequence which
encodes a
chosen peptide epitope or series of epitopes may be used. Distinct gp120
epitopes
may be combined in any order or combination provided that the coding nucleic
acids
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provide an in-frame sequence both with respect to the gp 120 epitopes and with
respect
to the Ig H gene utilized to construct the flg.
Uses of the Invention
Treatment of an individual infected with HIV using the tolerogenic fIg of this
invention comprises ~parenterally administering a single or multiple doses of
the flg to
a subject, preferably .a human. The fIg is preferably an isologous Ig, that
is, of the
same species as the subject. A most preferred fIg is fusion IgG molecule. An
effective tolerogenic dose is a function of the size and number of particular
HIV
gp120 epitopes included in a particular flg construct, the patient and his
clinical
status, and can vary from about 0.01 mg/kg body weight to about 1 g/kg body
weight.
A subject can be given this amount in a single dose or in multiple repeated
doses.
Doses of hematopoietic cells or B cells expressing the fIg are preferably
administered at a dose between about 106 and 10'° cells on one or
several occasions.
The route of administration may include intravenous (iv) , subcutaneous (SC),
intramuscular, intrapulmonary, intraperitoneal or other known routes. The
preferred
route for administratiion of flg proteins or cells for tolerogenesis is by iv
injection.
The fIg of this invention may be advantageously utilized in combination with
other therapeutic agents useful in the treatment or prevention of HIV disease,
including prophylactic or therapeutic vaccine preparations, antiviral
chemotherapeutic
agents, immune response modulators including cytokines and hematopoietic
growth
factors, protective antibody reagents, etc.
Having now ;generally described the invention, the same will be more readily
understood through reference to the following examples which are provided by
way of
illustration, and are not intended to be limiting of the present invention,
unless
specified.
EXAMPLE I
Epitope-Specific Tolerance Induction by Gene Transfer of an Engineered Peptide
Immunoglbulin Fusion Protein
In this study.,. the present inventors took advantage of the IgG molecule as a
tolerogenic carrier, and created an engineered tolerogen with a grafted
epitope at the
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N-terminus of an IgG heavy chain. This engineered IgG was recognized by the
immune system in a tolerogenic manner. The model epitope chosen for this
initial
analysis is the well-characterized class-II MHC-restricted peptide sequence
from the
cI ~, repressor protein (pl-102), residues 12-26. This peptide contains both a
B- and T-
cell epitope. and is the immunodominant determinant in H-2d mice immunized
with
the entire protein (26-30). It was thus possible to measure tolerance
induction to a
single determinant at both the B-cell and T-cell levels. Furthermore,
tolerogenic Ig-
peptide constructs could be expressed in adoptively-transferred hematopoietic
tissue
for the permanent modulation of epitope-specific immune responses in mature
adults.
I O These studies show that the model flg, 12-26-IgG, is an efficient
tolerogen in adult
animals and serves as the basis for expansion of this approach to other
epitopes of
clinical utility as described below.
MATERIALS AND METHODS
Mice. Male and female BALB/cByJ (H-2°) and CAF 1 (H-2°'a) mice
were obtained
15 from the Jackson Laboratory (Bar Harbor, ME) and were used at 6-10 weeks of
age.
Medium: RPMI 1640 medium (GIBCO-BRL, Gaithersburg, MD) was supplemented
with 5% FCS (Hyclone, Logan, UT), 2-ME, L-glutarnine, penicillin,
streptomycin,
MEM nonessential amino acids, and sodium pyruvate.
Antibodies: Hybridoma B3.11, which produces a monoclonal IgG, specific for the
20 12-26 peptide was a kind gift of Drs. Tom Briner and Malcolm Gefter
(Immulogic,
Waltham, MA). B3.11 was affinity purified with goat anti-mouse IgG sepharose
columns and biotinylated, or used as a neat culture supernatant. All alkaline-
phosphatase (AP)-conjugated reagents were purchased from Southern
Biotechnology
Assoc. (Birmingham, AL).
25 Synthetic peptide: The 12-26 15-mer LEDARRLKAIYEKKK (SEQ ID N0:112) was
prepared with a solid-phase method and purified to >92% homogeneity using
standard
HPLC methods. Peptide was conjugated to bovine albumin serum (BSA) rabbit
gamma globulin (RGG), or keyhole limpet hemocyanin (KLH) as described (Roy, S.
et al. (1989) Science. 244:575-575).
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Oligonucleotides: The following complementary synthetic oligonucleotides
encoding
the 12-26 sequence were designed with BamHI/CIaI restriction ends,
phosphorylated
with T4 kinase and ATP, and cloned into the hypervariable region of flagellin
construct pPX 1647:
S DWS1: (SEQ ID N0:195)
5'-CGA TCT GGA GGA CGC GCG GCG GCT GAA GGC GAT ATA CGA GAA GAA GAA GG-3'
DWS2: (SEQ ID N0:196)
5'-GAT CCC TTC TTC TfC TCG TA T ATC GCC TTC AGC CGC CGC GCG TCC TCC AGA T-3'
PCR primers were also designed to amplify a modified 12-26 sequence from the
chimeric 12-26-flagc:llin construct. This sequence includes 5' FR1 VH sequence
and
PstI restriction sites at each flanking ends:
Ig-one: 5'-TGATCTACTGCAGCTGGAGGACGCGCGGCG G-3' (SEQ ID N0:197)
Ig-two:
5'- CGACCTCCTGCAGTTGGACCTGCTTCTTCTTCTCGTATAT-3' SEQ ID N0:198)
ELISA: To determine the specificity of binding of our peptide-specific mAb
B3.11 to
12-26-fusion proteins, competitive inhibition ELISA's were conducted as
follows:
biotinylated B3.11 vvas incubated 1:1 (vol/vol) with decreasing amounts of
inhibitor
in ELISA binding buffer (0.25% BSA, 0.05% Tween 20 in saline). Mixtures were
then incubated on peptide-coated (10 ltg/ml) ELISA plates (Immulon 4
Dynatech),
and subsequently streptavidin-AP was added as a secondary reagent. Percent
inhibition of binding (A4os) was calculated as: [(average binding of antibody
alone
minus average binding of antibody incubated in presence of inhibitor)/average
binding
of antibody alone] x 100. ELISA determinations of serum peptide-specific IgG
responses were donE; by coating ELISA plates with 50 pg/ml synthetic peptide.
Antigen-coated plates were blocked with 1% gelatin/0.05% Tween 20 buffer, and
duplicate serial dilutions of serum were incubated and probed with goat anti-
mouse
IgG isotype-specific: secondary reagents. Titers are expressed as the
geometric mean
of the reciprocal dilution required to bring A49o readings to prebleed levels
or <0.08
O.D.
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Protein Engineering Design:
Preparation of murine H chain IgGI construct encoding the 12-26 sequence at
the N-
terminus
Our strategy for inserting a foreign peptide sequence at the N-terminus of an
IgG H chain is similar to what has been described (Hebell, T. et al. ( 1991 )
Science
254:102-105; Dal Porto, J. et al. {1993) Proc. Natl. Acad. Sci. USA 90:6671-
6675).
Plasmid pSNR (Ballard, D.W. et al. (1986) Proc. Natl. Acad. Sci. USA.
83:9626-9630), which contains neo and amp resistance genes, as well as the
full
genomic sequence for a IgG,b H chain specific for the NP hapten, was obtained
from
Dr. Douglas Fearon (Cambridge University) and modified. A modified 12-26
sequence was created via PCR amplification of this sequence from the chimeric
flagellin construct A29 (described in W095/21926) utilizing PCR primers "Ig-
one"
and "Ig-two". The modified 12-26 sequence was subcloned into the VH site of
pSNR
and recombinant clones were analyzed for proper orientation and Taq polymerase
mutational errors by double-stranded DNA sequencing methods (USB Sequenase 2.0
kit).
Expression purification, and quantitation of transfected IgG:
Construct pQ3.EZ1 (Q3), as well as the control pSNR IgG, construct (P6)
were electroporated into J558L myeloma cells (which produce only a ~, light
chain) as
described by Hebell et al., supra, Ballard et al., supra, and Dal Porto et
al., supra.
Stably transfected clones were isolated in 1 mg/ml 6418 (GIBCO-BRL),
subcloned,
and transfected IgG's from selected clones were purified from bulk
supernatants or
ascites with anti-mouse IgG-Sepharose or protein G columns. Since the original
H
chain binds with high affinity to the NIP (5-iodo-4-hydroxy-3-
nitrophenylacetyl)
hapten, purified or serum transfectoma IgG was quantitated using a modified
NIP-
gelatin binding ELISA , using anti-mouse IgG,-AP as a secondary reagent.
In vitro and in vivo tolerance induction and immunization protocols:
Peptide-specific tolerance induction in adult recipients was accomplished by
intravenous ("iv") injection (in the lateral tail vein) of either 1 mg
purified,
deaggregated, chimeric (Q3) or control IgG (P6) diluted in saline, or by 3
repeated
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injections of mitomycin C-treated (50 p.g/ml, SIGMA) P6- or Q3-secreting
transfectomas. For measurement of humoral immune responses 10 days following
iv
tolerization, animals were immunized subcutaneously ("SC") at the base of the
tail
and intraperitoneally ("ip"), with 50 ~tg synthetic 12-26 peptide and 25 pg
hen egg
5 lysozyme (HEL, SIGMA) emulsified in Freund's complete adjuvant (CFA, SIGMA).
Mice received an additional antigenic boost of 50 Itg peptide and 25 ~tg HEL
injected
ip in saline 2 weeks after initial priming. Mice were bled to assess serum
anti-peptide
antibody responses !3 days after this boost. Splenic memory T cell
("tertiary")
responses were analyzed in culture 8 weeks following secondary boosts. Splenic
T
10 cells were enriched by panning on anti-Ig coated plates, and restimulated
(3x106/ml)
with dilutions of peptide and irradiated splenic APC (2500 rads, 106/ml). For
analysis
of secondary (LN) responses following iv tolerization, animals were immunized
in
hind footpads with c'.0 Itg peptide emulsified in CFA, and draining popliteal
LNs were
harvested 9 days latf:r and restimulated in culture with dilutions of peptide
and 50
15 ~g/ml purified protein derivative (PPD, Connaught, Swiftwater, PA). IL-2
and IL-4
secreted into the medium were determined from culture supernatants at 24 and
48
hours, respectively, in LN or splenic T-cell cultures using recombinant
cytokines as
standards.
In vitro B-cell tolerance induction experiments were done on enriched splenic
20 B cells essentially as described by before (Waldschmidt, T.J. et al. (1983)
J. Immunol.
131:2204-2209; Phillips, N.E. et al., (1983) J,. Immunol. 130:602-606; Warner,
G.L. et
al., ( 1991 ) J. Immuraol. 146:2185-2191 ). Supernatants from 3-4 day cultures
were
assayed for IgM production by ELISA by coating wells with peptide- or FITC-BSA
conjugates, and probing with goat anti-mouse IgM-AP. Results represent
experiments
25 repeated 2-3 times; :individual points represent the arithmetic mean of
triplicate or
quadruplicate values with standard deviations generally less than 15% (omitted
for
clarity).
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RESULTS
Preparation and characterization of a marine IgG, self carrier containing the
~, cI
repressor 12-26 peptide at the V,, N-terminus
The 12-26-IgG construct was prepared by modifying plasmid pSNR, which
S contains the genomic sequence encoding a marine IgG,b H chain. Isologous
IgG, was
chosen because of its documented activity as a tolerogenic "carner" of potency
equal
to IgG,, and greater than other Ig isotypes or other serum proteins. We chose
to insert
a foreign epitope at the N-terminus of the VH region (Figure SA), because
insertions at
this location have been shown not to alter normal immunoglobulin folding and
structure (Hebell et al., supra; DaPorta et al., supra). Analysis of
transfected, purified
chimeric 12-26-IgG (Q3) or control pSNR IgG (P6) by SDS-PAGE showed that H
chains can successfully pair with J558L light chains (L). The chimeric H chain
(Q3)
containing the additional 12-26 sequence was about 1.8 kDa larger than the
control
IgG (P6).
Purified transfected IgGs expressed the 12-26 epitope as shown by western
blotting and ELISA utilizing peptide-specific mAb B3.11 (Figure SB).
Furthermore,
in competitive inhibition ELISA, chimeric 12-26-IgG effectively competed with
free
synthetic peptide or a chemical conjugate of 12-26 with rabbit IgG for binding
to mAb
B3.11. These results suggest that the inserted peptide is recognized
efficiently by
epitope-specific antibodiesB cells on the exterior surface of the recombinant
IgG,
without significantly perturbing H chain tertiary structure.
Additionally, the recombinant 12-26-IgG chimera is immunogenic and capable
of priming 12-26-specific T and B cells in vivo. Mice immunized with Q3
emulsified
in CFA were able to prime 12-26-specific T cells comparable to the response
elicited
with synthetic peptide. In vitro restimulation of LN cultures with synthetic
peptide
resulted in T-cell proliferation as well as IL-2 and IL-4 production in
peptide- and
Q3-primed, but not P6-primed LN cells. Immunization also led to a high serum
anti-
12-26 IgG antibody titer detectable by peptide-specific ELISA. 12-26-IgG
stimulated
IL-2 production in an I-Ad-restricted 12-26 specific T-cell hybridoma (9C
127). These
results suggest that the confirmation of the inserted foreign epitope is not
only
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recognized by specific antibodies, but the peptide (or one like it ) can also
be
processed and presented to T cells in a physiologically relevant manner by APC
even
in the context of a self IgG scaffold.
In vivo induction of peptide-specific immune self tolerance with soluble
purified
engineered 12-26-IgCi.
To test the efficacy of genetically engineered tolerogens, we analyzed both
humoral and cellular immune responsiveness after iv administration of high
doses of
soluble, deaggregatecl 12-26-IgG fusion Ig. Mice were injected with 1 mg of
either
chimeric Q3 or control P6 immunoglobulins, and challenged 10 days later with a
mixture of 12-26 peptide and HEL (as a specificity control) emulsified 1:1 in
CFA.
Secondary humoral immune responses were analyzed one week after an additional
boost. Figure 6 shows that mice receiving pretreatments of Q3, but not control
P6,
were dramatically unresponsive to peptide challenge as assessed by ELISA of
anti-
peptide IgG, whereas control anti-HEL antibody titers were unaffected.
Although the
predominant Ig isotype in this anti-peptide response in Balb/c mice is IgG,,
antibodies
of all isotypes including IgG2b were consistently diminished by the
tolerogenic
treatment with 12-26-IgG (Figure 6).
To test the potential of inducing unresponsiveness with peptide-Ig-transfected
cells as a model for gene-therapy-based tolerogenesis, Balb/c mice received 3
(consecutive weekly iv injections of transfectomas secreting Q3 (or P6 control
IgG);
the cells had first been treated with mitomycin C. This protocol resulted in
transient
appearance in serum ~of the transfected IgG's at levels reaching at least 10-
500 ng/ml
(assessed by NIP-gelatin ELISA). This type of treatment resulted in diminution
of
peptide-specific hum~oral immune responses as well as reduction of LN cell
proliferative responses.
Since unresponsiveness as measured by serum antibodies may results from
tolerance of B-cells, 'r-cells or both, the cellular basis of the observed
tolerance was
analyzed by measuring T-helper (Th) cytokine responses 8 weeks after
immunogenic
challenge. Restimulation of splenic memory T cells (Figure 7) revealed that
both
Thl-type (IL-2) and 'fh2-type (IL-4) responses were absent in tolerized mice,
a result
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consistent with the observed lack of anti-peptide IgGZb and IgG,
antibodies(Figure 6),
which are dependent on these Th cell subsets. The T cell response to peptide
was
diminished in 12-26-IgG pretreated animals when measured as short-term LN
restimulation assays . Mice toierized with 1 mg of 12-26-IgG 10 days previous
to
peptide challenge had reduced LN IL-2 responses, but unaffected recall
proliferative
responses to the antigen PPD compared to control P6-injected animals. These
results
indicate efficient induction of Th cell tolerance to the immunodominant
peptide which
results in an inability to prime any subset of T cell response to the peptide.
Thus, a foreign immunogenic peptide genetically engrafted into an Ig scaffold
can be very efficiently presented to the immune system in a tolerogenic manner
when
administered by the appropriate route and method. Thus pretreatment with
peptide-Ig
chimeras delivered either as single high doses or via slow release by
transfected
autologous B cells have utility in achieving efficient epitope-specific
manipulation of
undesired T-cell responses.
Analysis of a novel 12-26-flagellin immunogen for testing the efficacy of 12-
26-IgG
on B-cell tolerance.
To test the efficacy of the flg 12-26-IgG as a B-cell tolerogen, it was
necessary
to challenge B cells with an immunogenic, T-independent form of the 12-26
epitope.
Since polymerized flagellin is a well-characterized T-independent antigen, we
constructed a 12-26 flagellin fusion protein with a strategy previously
described
(Newton et al., supra). Western blotting and ELISA analyses of purified WT
(pPX)
and 12-26-flagellin (A29) showed that although flagellin epitopes are readily
expressed in both recombinant flagellins, the inserted epitope was detectable
only in
chimeric flagellin A29. Polymerized 12-26-flagellin stimulated splenic B cells
to
secrete anti-12-26 IgM antibodies. The stimulatory effect was comparable to
that of
the polyclonal B cell mitogen, bacterial lipopolysaccharide (LPS). A
concentration of
0.1 pg/ml was found to be minimally mitogenic (as assessed by anti-fluorescein
[FITC] IgM ELISA's) and used for subsequent experiments. These results broaden
the context in which the inserted epitope can be recognized: IgG and the
polymerized
flagellin molecule. In the latter context, the epitope readily stimulate B
cells to
produce epitope-specific IgM antibodies.
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We also tested the ability of 12-26-IgG to induce specific B-cell
unresponsiveness. Enriched B cell populations were incubated in vitro with
various
doses of Q3 or P6 control IgG's, washed, and then cultured with either
mitogenic LPS
or 12-26-flagellin. Alternatively, BALB/c mice were injected iv with 1 mg of
each
S protein, and splenic lB cells were harvested and challenged in vitro 10 days
later.
Supernatants from 3 day cultures were assayed for 12-26-specific IgM antibody
or
anti-FITC antibody ~~s a specificity control. Pretreatment with Q3, but not
P6, either
in vitro or in vivo. markedly suppressed the anti-12-26 IgM response, whereas
anti-
FITC control IgM responses were unaffected. Thus, in addition to inducing
potent Th
tolerance, the flg construct is independently can induce epitope-specific
unresponsiveness in B cells. The magnitude of B cell tolerance was more modest
in
vivo than T cell tolerance, possibly reflecting either a requirement for
higher epitope
valency (the fIg provides only a bivalent epitope, one on each arm of the H
chain), or
a higher dose requirement. A reduction of antibody responsiveness of similar
magnitude was observed after adoptive transfer of in vivo-tolerized B cells,
admixed
with nontolerized naive T cells, into secondary immunodeficient recipients
which
were then challenged with the peptide in CFA.
DISCUSSION
The development and maintenance of the unresponsive state in newly
emerging lymphocy~~es is a lifelong process requiring the persistence of
antigen.
Exposure of mature B and T cells to antigen in an adult immune system may lead
to
either activation or tolerance depending on the route and method of exposure,
as well
as the availability of costimulatory signals from specialized APC. Since a
major goal
in clinical therapy in a variety of conditions (e.g., infection, autoimmunity,
allergy,
transplantation) is tt»e induction of specific immune unresponsiveness in
adult mature
lymphocytes, a varieay of approaches have exploited these pathways of
exposure. Of
these approaches, experimental tolerance induction with gamma-globulin
carriers has
been most extensively described. IV administration of soluble, deaggregated
IgG's in
the absence of adjuvants, induces both antigen-specific B-cell and T-cell
tolerance
even in the absence of a thymic environment. Mechanisms of specific cional
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anergy/inactivation and deletion have been implicated in this type of
experimental
model.
In the foregoing studies, the present inventors described for the first time,
the
tolerogenic capability of an engineered self IgG expressing a model class II
MHC-
restricted immunodominant peptide. This novel epitope, deliberately expressed
at the
N-terminus of an IgG heavy chain construct, was tolerogenic in vivo and in
vitro.
Conceptually similar approaches have been utilized to express immunogenic
(rather
than tolerogenic) malarial or viral peptides in the CDR3 loop of Ig H chains
for the
induction of enhanced anti-peptide immune responses, as described above.
10 As with other such epitopes, the 12-26-IgG protein could act as an
efficient
immunogen when administered in an immunogenic manner (i.e., emulsified in
CFA).
Zaghouani et al., 1993, supra showed that T-cell activation (for a class II-
restricted
epitope) was enhanced 100-1000 fold when the epitope was part of an Ig-
chimera,
presented in vitro by stimulatory dendritic cells as APC. The present results
similarly
15 show that an approximately 100-fold lower molar quantity of 12-26-IgG (as
compared
to free peptide) stimulated similar numbers of peptide-specific LN T cells
from
immunized mice.
The increased efficacy of the flg's of the present invention, both as immune
activators and as tolerance inducers, may indicate that common pathways are
utilized.
20 The increased efficacy may directly result from (a) an increased half life
and (b) an
Fc-receptor mediated uptake of the "carrier" portion of the Ig molecule
(Stockinger,
B. (1992) Eur. J. Immunol. 22:1271-1278) leading to improved presentation of
the
grafted foreign epitope(s). In the absence of adjuvants (which act in part by
mobilizing APC having e~cient costimulatory capability), high doses of
soluble,
25 deaggregated serum protein may be preferentially taken up by "non-
professional"
APC, such as resting B cells, via the process of Fc receptor-mediated
endocytosis or
phagocytosis, and subsequently presented by these "non-professional" APC
(Parker,
D.C. et al. ( 1991 ) FASEB J. 5:2777-2784; Eynon, E.E. et al. ( 1992) J. Exp.
Med
175:I3I-138; Fuchs, E.J. et al. (1992) Science 258:1156-1159). Furthermore,
IgG
30 carriers can induce efficient B-cell unresponsiveness by mechanisms
involving the
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crosslinking of surface IgM to Fc receptors. One or more of the above
mechanisms
may be responsible for the enhanced tolerogenic efficiency of Ig Garners. In
contrast,
the mere iv injection of soluble, deaggregated peptides can suffice to induce
effective
Th cell tolerance (Scherer, M.T. et al. ( 1989) Cold Spring Harbor Symp.
Quant. Biol.
54:497-504), but is insufficient to induce specific B cell unresponsiveness.
The present inventors findings are summarized as follows. A fIg, specifically
the 12-26-IgG fusion protein, can present an epitope in a tolerogenic fashion
and
induce both B- and T-cell tolerance. A convenient property of this epitope
allows
simultaneous study of both cellular and humoral immune responses to a single
immunodominant peptide. The 12-26 peptide can induce a vigorous antibody
response which is predominantly of the IgG, isotype, and can prime Th cells of
both
the Thl and Th2 phenotype. Tolerance induction with 12-26-IgG was globally
effective in suppressing every type of immune response which can be elicited
by this
immunodominant peptide.
The inventors have therefore provided a powerful approach to determining the
efficacy of inducing specific unresponsiveness to a defined antigens,
particularly
peptide antigens, for the modulation of undesired immune responses. The
present
approach has advantages of that inserting heterologous epitopes into the H
chain
CDR3 because the N-terminus insertion does not restrict the size of the
epitope or
epitopes fused to the; tolerogenic IgG Garner. Therefore, not only short
peptides, but
also larger, more complex foreign antigens may be fused in an fIg construct
for
tolerogenic presentation.
Finally, because this approach provides what may be envisioned as a
genetically transmissible "hapten-carrier" complex, these tolerogenic figs
when
expressed as a transgene-transferred into hematopoietic tissues or cells, can
be used to
both induce and maintain tolerance for the long term. Such studies are
reported in
Example III, below. Recipients of BM stem cells which have been transduced
with a
retroviral vector for the long-term expression of flg cDNA constructs. The
application of most immediate interest for the present invention is the use of
this
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approach to block and ineffective and potentially harmful antibody responses
which
occur during HIV infection (Clerici, M. et al., (1993) Immunol. Today 14:107-
110).
EXAMPLE II
Tolerance to HIV gp120 Epitopes from the CS Region: Detection
The study described above using a phage ~, epitope was extended to two gp 120
epitopes:
(1) a CS peptide KYKVVKIEPLGVAPTKAKRRVVQREKR (SEQ ID
N0:199)
positions 485-511 of gp120 from the BH10 strain (see Figure 3) which is cross-
reactive with HLA-C monomorphic determinants (DeSantis, C. et al. (1993) J.
Infectious Dis. 168:1396; Palker, T.J. et al. ( 1987) Proc. Nat'1 Acad. Sci.
USA
84:2479; and
(2) a C1 immunodominant peptide (such as residues 90-120 of the BH10 isolate,
above, that contains distinct B- and T-cell epitopes (Abacioglu et al.
(supra).
Many antibody responses to HIV can be non-protective, and can enhance viral
uptake by monocytes or promote T-cell apoptosis (Finkel et al., supra; Banda
et al.,
supra; Kliks, S.C. et al. (1996) Proc. Nat'1 Acad. Sci. USA 90:11518.
Initially, the
inventors focused on defining peptides smaller than the original 35-mer in CS
defined
by Beretta and colleagues (DeSantis, C. et al. (1993) J. Infectious Dis.
168:1396.
Using mAbs (Robinson, W.E. Jr. et al. (1990) Proc. Nat. Acad. Sci. USA
87:3185,
peptides were examined that contain either the KYK or KAKRR (SEQ ID
N0:200)motifs that have been defined for HLA cross-reactivity. At least two 15-
18-
mers were identified by an ELISA inhibition assay.
Results discussed in Example I, above, indicated that, in contrast to free
peptide, ~. 12-26-IgG was tolerogenic for B cells as a bivalent molecule,
although the
mechanism of this unresponsiveness was not determined.
Design of shortened peptides containing major CS epitopes:
As a model epitope, the inventors initially chose the C-terminal peptide
KYKVVKIEPLGVAPTKAKRRVVQREKR (SEQ ID N0:199)
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(residues 485-51 in the BH10 variant (Figure 3) and which ; corresponds
approximately to positions 455-481 of the consensus sequence in Figure 1 ).
This is
in the conserved CS C-terminal region of gp120. This peptide contains the B-
cell
epitope consisting of the KYK--------KAKRR (SEQ ID N0:200)motifs that are
recognized by the M:38 marine mAb (DeSantis et al., supra; Palker et al.,
supra). The
epitope recognized b;y M38 has been noted to be KYKVVKEIPLGVAPTKAKRR of
SEQ ID N0:199. M.Ab M38 also binds to the C-terminus of gp120, in a gp41
binding
region. M38 also reacts with a common motif in the HLA-C heavy chain al region
(KYKRQAQADRVI~ILRKLR: SEQ ID N0:201 ) that is mimicked in this CS peptide.
HIV-infected individuals have HLA class I-gp120 cross-reactive antibodies.
The inventors first established that a 35-mer containing this M38-defined
epitope was tolerogenic in vivo when chemically coupled to heterologous rabbit
IgG.
Since the CS peptide was relatively large and not readily available. Shorter
peptides
containing the KYK and KAKRR sequences with different spacer residues and with
a
C-terminal cysteine :for more controlled coupling to IgG carriers can be
designed.
Although the residuea between the two M38 epitopes (IEPLGVAPT; SEQ ID
N0:202) are not recognized in seropositive individuals (Scott, D.W. et al.
(1993) Adv.
in Molec. and Cell. i'mmunol. l:l 19, it was important to determine the
contribution of
these amino acids to the epitope conformation. The mAbs described in Robinson
et
al., supra, were used to analyze reactivity to these new peptides as well as
the
requirement for the intervening sequences. The peptides designed were:
1. AAKYKGGGGGKAKRRGGC (SEQ ID N0:203)
2. AAKYKGGGPTKAKRRGGC (SEQ ID N0:204)
3. AAKYKGVAPTKAKRRGGC (SEQ ID N0:205)
Control peptides (for example, available from the National Institute of
Allergy
and Infectious Dise~~ses) encompassed the KYK, KAKRR (SEQ ID N0:200) motifs
or the entire IEPTGVAPTKAKRR (SEQ ID N0:206)sequence recognized by the
human mAbs. Using a competitive ELISA assay, the inventors found that peptide
#3,
above ("P3"), was similar in activity to the full sequence and that peptides
containing
only the KYK motif were noninhibitory, as expected, with these human anti-CS
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mAbs. Treatment with P3-HGG induced unresponsiveness for an anti-P3 response
(and an anti-HGG response). These results suggest that VAPT motif, while not
immunoreactive, contributes to the structural integrity of this epitope.
Peptide 3 (P3)
contains both motifs that have been reported to be recognized by M38 and are
important in anti-HLA-C recognition, for which tolerance induction is one
goal.
Importantly, these results mean that shorter peptides still express B-cell
epitopes.
It is expected that anti-C1 and V3 loop antibody responses would not be
affected by the above peptides because the stimulus is a polyclonal mitogen.
It is
expected that anti-HLA crossreactivity will be eliminated if KYK-specific
unresponsiveness is induced. In order to achieve tolerance to C1 region or V3
loop
epitopes, the present invention would require that the fIg include one or more
epitopes
from these regions.
EXAMPLE III
Restin and Activated B Lymphocytes Expressing flg are Tolerogenic Vehicles
Since antigen-presenting B-lymphocytes are known to either augment or
downregulate T-cell dependent immunity , it should be possible to modulate the
immune response to a selected antigen (such as an autoantigen, a viral antigen
or a
tumor antigen) via gene-transfer of exogenous genes and constitutive
expression in
vivo by autologous APC. Such an approach would be advantageous for the
induction
of unresponsiveness, since tolerance to foreign antigens could be maintained
indefinitely in vivo, especially if gene-transfer into long-lived lymphoid
progenitors is
achieved.
Previous models have led to apparently divergent results and have shown that
B cells can be either essential (Ron, Y. et al. (1981 ) Eur. J. Immunol.
11:964-968;
Janeway, C.J. et al. (1987) J. Immunol. 138:1051-1055; Constant, S. et al.
(1995) J.
Immunol. 155:3734-3741; Morns, S.C. et al. (1994) J. Immunol. 152:3777-3785)
or
nonessential (Sunshine, G.H. et al. (1991) J. Exp. Med. 174:1653-1656;
Ronchese, F.
et al. (1993) J. Exp. Med. 177:679-690; Epstein, M.M et al. (1995) J. Exp.
Med.
182:915-922) for T cell priming, and can be critical for either activating or
tolerizing
(Eynon, E.E. et al. (1991) Transplant. Proc. 23:729-730; Eynon, E.E. et al.
(1992) J.
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Exp. Med. 175:131-138; Fuchs, E.J. et al. (1992) Science. 258:1156-1159;
Buhlmann,
J.E. et al. (1995) Immunity. 2:645-653) naive T cells, and even previously
activated T-
cell clones (Gilbert, K.M. et al. (1994) J. Exp. Med. 179:249-258). The state
of
activation of the collaborating B cells and T cells as well as the antigen-
specificity for
5 the interactions have appeared to be important for the different outcomes.
To analyze the ability of antigen-presenting B cell to serve as a modulator of
the immune response, the present inventors generated a unique transgenic mouse
system (see Example; I) in which a foreign class II-restricted immunodominant
epitope
is expressed as a self antigen specifically in the B cell compartment. The
foreign
10 epitope, residues 12-26 from ~, cl repressor protein was grafted in-frame
at the N-
terminus of a murine~ IgG, heavy chain and is made endogenously as a transgene
in
the B-lymphocyte lineage. The tolerogenic capabilities of this soluble
engineered
immunoglobulin in immunocompetent adult mice is described above. The present
study describes the tolerogenic nature of transgenic hematopoietic tissue
expressing
15 such a fIg molecule. This approach takes advantage of the efficiency of the
immunoglobulin secretory and endocytic pathways to synthesize and present an
exogenous "neo" self peptide, and provides a model for inducing peripheral
tolerance
to undesirable humoral and cellular immune responses using gene therapy
strategies.
I. Materials and Methods
20 Mice and Reagents. Male and female B6D2 (H-2~'d) and BALB/cByJ (H-2d)
mice were purchased from the Jackson Laboratories (Bar Harbor, ME) at 3-8
weeks of
age, and housed in pathogen-free, microisolater cages. RPMI 1640 medium (GIBCO-
BRL, Gaithersburg, MD) was supplemented with either heat-inactivated S% FCS
(Hyclone, Logan, U'T), or heat-inactivated 0.5% autologous mouse serum
(Jackson
25 Immunochemicals), 2-ME, L-glutamine, penicillin, streptomycin, MEM
nonessential
amino acids, and sodium pyruvate. Hybridoma B3.11, which produces an IgG,
specific for the 12-26 peptide was from Drs. T. Briner and M.Gefter (Immulogic
Corp., Waltham, MA), and was originally derived by fusion with splenocytes
from
peptide-immunized BALB/c mice. Monoclonal antibody (mAb) B3.11 was affinity
30 purified from bulk-cultured supernatants with goat anti-mouse IgG sepharose
columns
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and biotinylated. All alkaline-phosphatase (AP)-conjugated secondary reagents
were
purchased from Southern Biotechnology Assoc. (Birmingham, AL). The 12-26 15-
mer LEDARRLKAIYEKKK (SEQ ID N0:112), or an N-terminal cysteine-modified
16-mer was prepared with a solid-phase method and purified to >92-95%
homogeneity using standard HPLC methods. The cysteine-modified 12-26 peptide
was covalently conjugated to hen egg white lysozyme (HEL) with Sulfo-MBS
(Pierce,
Rockford, Illinois), a sulfhydryl-specific crosslinking reagent.
Generation of Transgenic (T~ Mice. The preparation of a chimeric marine
IgG,b H chain construct, specific for the NP hapten and engineered to express
the 12-
26 peptide at the N-terminus, is described in detail above. The entire ~10 kb
genomic
construct containing the original endogenous immunoglobulin promoter,
enhancer,
and polyadenylation sequences was shuttled into pBluescript KS+/- (Stratagene,
La
Jolla, CA) and excised as a Xhol/NotI fragment. The linearized transgene was
purified over a continuous 10-40% (wt/vol) sucrose gradient and dialyzed
against 5
mM Tris-HCI/0.15 mM EDTA (ph 7.5). Tg mice were derived by pronuclear
injection of fertilized B6D2 eggs, and implantation into pseudopregnant
females as
described by Hogan et al. (Hogan, B. et al. ( 1986) Manipulating the Mouse
Embryo: A
Laboratory Manual. Cold Spring Harbor Lab. Press, Plainview, N.Y. pp. 81-141,
incorporated by reference). Three original Tg founders were identif ed by
genomic
Southern blotting of tail DNA with a'ZP-labeled probe containing 3 cloned,
tandem
copies of the 12-26 cDNA sequence. Two of these founders (Line 5 and Line 17)
were selected for further analysis, bred onto the BALB/c background for at
least 5-10
generations, and confirmed for H-2d homozygosity via RFLP Southern blot
analysis
before use in BALB/c adoptive transfer experiments. Lines 5 and 17 were also
rederived by Cesarean section (Taconic Labs) and thereafter housed in
sterilized
microisolater units at the Holland Laboratory to ensure healthy microorganism-
free
strains of Tg mice. Tg offspring obtained via BALB/c coatings were, in
general,
heterozygous for their transgene and distinguished from their nontransgenic
(NTg)
littermates by either 12-26 sequence Southern blotting of tail DNA, or serum
NIP-
binding IgG, ELISA.
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Preparation of Bone Marrow Chimeras. Eight week-old BALB/c recipients
were sublethally irradiated (650 rads) with a "'Cs source and injected iv with
10' cells
consisting of a 1:1 pooled mixture of non-Tg/Line 17 Tg bone marrow (BM) cells
that
had been depleted o1"erythrocytes. Control mice were injected with NTg
iittermate
BM cells(after 650 rads) or saline only (with no irradiation). All Tg/NTg
donor BM
was completely sex-matched and syngeneic with BALB/c recipients. Adoptively-
transferred mice were rested for 7-8 weeks before immunization studies.
Preparation of Lymphoid Cells for Tolerance Induction. 12-26 peptide-
specific tolerance induction in normal adult (6-10 week old) BALB/c mice was
accomplished by iv injection of Line 17 Tg hematopoietic tissue. Unconditioned
recipients were generally injected with 2-4 x 10' cells from preparations of
purified
resting B cells, LPS-activated B cell blasts, unfractionated splenocytes, or
crude BM
cells from Tg or control NTg donors. Ten days following such injections,
recipients
were immunized with antigens SC as described below.
Bone marrow (from both femurs and tibiae) or spleen tissue was prepared in
serum-free completE; RPMI and depleted of erythrocytes. Splenic B cells were
obtained by depleting splenocytes of T cells by treatment with anti-T cell
cocktail plus
baby rabbit complernent. Resting B cells were harvested by further
fractionation on
Percoll gradients and collecting the 60-70% layers as previously described
{29). For
preparation of activated B cell populations, purified B cells (4x106/ml) were
incubated
for 48 hrs in compleae RPMI (5% FCS) in the presence of 50 p,g/ml LPS (Sigma,
St.
Louis, MO), and washed 3 times before further use. For chemical fixation,
purified B
cells were treated with carbodiimide (ECDI, Sigma) by incubating 108 cells in
0.5 ml
of 75 mM ECDI (in saline) for 1 hour, on ice. All cells were washed
extensively prior
to iv injection.
Measurement of Peptide-Specific Cellular and Humoral Immune Responses.
All protocols are essentially as described previously (Lai, M-Z et al. (1987)
J.
Immunol. 139:3973-3980; Scherer, M.T. et al. (1989) Cold Spring Harbor Symp.
Quant. Biol. 54:497-504; Soloway, P. et al. (1991) J. Exp. Med. 174:847-858).
Ten
days following iv tolerization, animals were immunized to induce cellular or
humoral
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immune responses. For measurement of humoral immunity, animals were injected
SC
at the base of the tail with SO pg synthetic 12-26 peptide emulsified 1:1 in
complete
Freund's adjuvant (CFA). In some experiments, animals were also injected with
20
~tg hen egg lysozyme (HEL) in CFA, intraperitoneally (ip). Two weeks later,
mice
received an additional antigenic boost of 50 pg peptide and 10 pg HEL in
saline,
injected ip. Antibody titers were determined from serum obtained 8 days after
secondary boosts. Splenic memory T cell responses were measured in vitro 6-8
weeks
following these secondary challenges.
For more detailed analysis of cellular immune responses, animals were
immunized SC in the hind footpads with 20-50 ug peptide in CFA, and draining
popliteal and inguinal lymph nodes (LN) were harvested 9 days later. LN cells
were
restimulated in vitro with synthetic peptide or 25-50 p.g/ml purified protein
derivative
(PPD, Connaught, Swiftwater, PA) in complete RPMI with 0.5% heat-inactivated
autologous mouse serum (Jackson Immunochemicals, West Grove PA). On day 3,
1 S cultures were pulsed with 1 ~Ci/well of ['H]thymidine and incubated an
additional 14-
hours, for the determination of proliferative responses. Cells were then
harvested
on glass fiber filters and incorporated'H was detected using a direct beta
counter
(Packard, Matrix 9600). IL-2 and IL-4 cytokine production was quantitated
using
CTLL and CT.4S bioassays, respectively, testing serial dilutions of culture
20 supernatants. Recombinant IL-2 (Genzyme, Cambridge, MA) and IL-4 (from Dr.
William Paul, NIH) were used to generate standard curves. Dilutions of anti-IL-
2
mAb S4B6 and anti-IL-4 mAb 11B11 (ATCC, Rockville, MD.) were included in the
assays to establish cytokine specificity.
For determination of B-cell tolerance induction, Tg mice or adoptively
transferred recipients were immunized with a chemical conjugate of cysteine-
modified
12-26 and HEL ( 12-26-HEL). Tg or NTg control mice were immunized ip with 50
p,g
12-26-HEL emulsified 1:1 in CFA and then boosted with 10 ~g of the same
conjugate
in saline 2 weeks later. Titers of IgG antibodies specific for the peptide- or
HEL were
determined by ELISA 8 days following this boost. Irradiated (400 rad) BALB/c
recipients were adoptively transferred (iv) with 5 x 10' splenocytes from
previously
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tolerized BALB/c, and boosted ip with 100 ~g 12-26-HEL conjugate in incomplete
Freund's adjuvant (IfA). Serum bleeds were collected 8 days following this
boost,
and antibody titers determined by ELISA.
Antigen-Presentation Studies. The ability of Tg B cells to directly present
endogenous 12-26 peptide was assessed with T-cell hybridoma 9C 127 which
recognizes 12-26 peptide in the context of 1 Ad. Tg or control littermate B
cell APC
were purified as described above, and recultured in varying numbers in 200 ltl
microcultures with 1 (1° 9C 127 cells/well in complete RPMI with 5%
FCS.
Supernatants were harvested 48 hours later, and multiple dilutions were
assayed for
IL-2 production as above.
Immunologic Methods. ELISA determinations of serum peptide-specific or
HEL-specific IgG responses were performed by coating plates with 50 pg/ml
synthetic peptide or 5 pg/ml HEL and following standard ELISA protocols.
Briefly,
antigen-coated plates were blocked with 1 % gelatin/0.05% Tween 20 buffer, and
duplicate serial diluti~ons of serum were incubated and probed with goat anti-
mouse
IgG isotype-specific secondary reagents conjugated to alkaline phosphatase.
Titers
are expressed as the geometric mean of the reciprocal dilution required to
bring A49o
readings to prebleed levels or <0.09 O.D.
12-26-IgG H chain protein was detected in serum of Tg mice via its ability to
bind to the NIP (5-iodo-4-hydroxy-3-nitrophenylacetyl) hapten using a modified
NIP-
binding ELISA (Grosschedl, R. et al. (1984) Cell. 38:647-658). Dilutions of
sera
from Tg mice were incubated on ELISA plates coated with NIP-gelatin or NIP-BSA
conjugates ( 10 ltg/ml), and subsequently probed with goat anti-mouse IgG,-AP
as a
secondary reagent. L>etection of the 12-26 epitope in Tg sera could be
demonstrated
by similarly using N1P-sepharose beads (from Dr. T. Imanishi-Kari, Tufts
University)
to immunoprecipitate: 12-26-IgG. Samples were boiled in 2X SDS loading buffer,
electrophoresed on 10% SDS-PAGE, and transferred onto nitrocellulose filter in
a
buffer with 25 mM T'ris, 192 mM glycine, and 20% methanol, pH 9.0 at
20°C
overnight. Blots were blocked in 2% BSA in TBST (50 mM Tris, 200 mM NaCI, pH
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7.5, 0.05% Tween 20), and probed with biotinylated mAb B3.11 (anti-12-26
epitope)
plus streptavidin-AP as a secondary reagent.
FACS analysis. Cells were stained for surface antigens and analyzed by flow
cytometry using standard methods. The following conjugated antibodies were
from
commercial sources and used with appropriate fluorochrome-labeled
isotype/species
matched or secondary reagent controls: RA3-6B2, rat anti-mouse Ly5 (B220)-PE
(Caltag, San Francisco, CA), YTS 191.1, rat anti-mouse L3/T4 (CD4)-PE
(Caltag),
YTS 169.4, rat anti-mouse Ly-2 (CD8)-FITC (Caltag), goat anti-mouse IgGI
(adsorbed)-PE (Caltag), goat anti-mouse IgM (H+L)- FITC (Hyclone). Data was
10 acquired on a Becton Dickinson FACScan and analyzed with LYSIS II software.
II. RESULTS
Generation of Transgenic Mouse Lines Expressing a Novel Peptide-IgG_,
Construct
Specifically in the B-Lymphocyte Compartment
The unique tolerogenic properties of an engineered peptide-IgG fusion protein
15 expressing residues 12-26 of ~, cI repressor protein at the N-terminus of a
marine H
chain specific for the NP hapten is described above. The engrafted epitope is
recognized on the exterior surface of assembled IgG by a peptide-specific mAb
(B3.11). More importantly, soluble fusion protein administered in adjuvant can
efficiently generate peptide-specific T-cell responses in vivo, suggesting
that 12-26 (or
20 an extremely similar peptide) is processed and presented by endogenous APC,
even in
the context of an Ig scaffold. To further characterize the potential for
expressing
tolerogenic IgG fusion proteins in vivo, we generated Tg mice expressing the
engineered genomic (rearranged) H chain construct driven by its endogenous
immunoglobulin promoter/enhancer sequences (Example I). Tg founders possessing
25 2-3 integrated copies were identified via Southern blotting of genomic tail
biopsy
DNA using a cDNA probe containing 12-26 sequence (Figure 8). Two lines (5 and
17) were bred onto the BALB/c (H-2d) strain and further analyzed for
expression of
engineered IgG. Unlike Tg mice expressing rearranged IgM constructs, both
lines had
no apparent suppression of endogenous Ig rearrangements and expressed amounts
of
30 surface IgM, serum levels of IgM and IgG, as well as B220, CD4, and CD8
markers
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that were comparable to NTg littermates. These results are in agreement with
previous observations for IgG H chain Tg experiments (Storb, U. (1987) Ann.
Rev.
Immunol. .5:151 -174; Yamamura, K. et al. (1986) Proc. Natl. Acad. Sci. USA.
83:2152-2156.; Tsao, B.P. et al. (1992) J. Immunol. 149:350-358; Radic, M.Z.
et al.
(1995)J.Immunol.1.55:3213-3222).
Serum expression of the NP-binding Tg H chain was detected as described by
Grosschedl et al., suyra. Since the Tg VH region binds with high affinity to
the NIP
hapten in combination with ~.1 light chains, functional 12-26-IgG was detected
indirectly with a NIP-binding IgG, ELISA Although probably representing a
fraction
(only ~, light chain-associated) of expressed Tg serum protein, N1P-binding
IgG,
assays revealed that Line 17 and 5 expressed between 1000-25000 nglml and 50-
1000
ng/ml, respectively. 'The higher serum expression for Line 17 mice correlated
with
increased expression of surface IgG, in splenocytes as compared to Line 5 or
NTg
littermates.
Direct presentation of endogenously synthesized 12-26 by Tg B cells was
demonstrated with a ;peptide-specific hybridoma (9C 127). More importantly, 12-
26
peptide expression was demonstrated directly via immunoprecipitation of Tg
serum
with NIP-sepharose beads and immunoblot analysis with mAb B3.11. B-lymphoid
expression of the transgene mediated by a specific Ig promoter/enhancer was
demonstrated as significantly increased expression following activation by
bacterial
LPS, but not by Con A stimulation of splenocytes. These results collectively
show
that the Tg 12-26-Ig(s, H chain readily combines with endogenously-synthesized
light
chains to be expressed specifically as a self molecule by a large fraction of
B cells
without perturbing e:{pression of endogenous IgM rearrangements. Thus, the 12-
26
peptide is secreted as; well as processed and presented via the Ig endocytic
pathway as
an endogenous B cell self epitope.
Profound Peptide-Specific Cellular and Humoral Immune Tolerance in Mice
Expressing 12-26-IgG, During Development or in Adult Bone Marrow Chimeras
Tg mice expressing foreign "neo" self antigens have firmly established that
tolerance induction can readily occur for membrane-bound and soluble proteins
which
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are expressed ubiquitously, or in a tissue-specific manner during normal
development
(Goodnow, C.C. (1992) Ann. Rev. Immunol. 10:489-518; Miller, J.F.A.P. et al.
(1992)
Ann. Rev. Immunol. 10:51-69). In our initial experiments, we wished to
establish that
Tg animals expressing a model immunodominant epitope as part of serum IgG
protein
were similarly unresponsive to an immunogenic challenge with the epitope.
Since the
12-26 peptide contains both a T-cell and a B-cell epitope, we could measure
both
cellular and humoral immune responses to this relatively simple determinant
with
immunization assays. Draining LN cells from subjects who received SC injection
of
synthetic peptide in CFA and which were subsequently restimulated with antigen
displayed a profound proliferative unresponsiveness and IL-2 production.
Furthermore, NTg mice (H-2d) primed with peptide in adjuvant and followed by a
subsequent boost of peptide in saline 2 weeks later, developed an extremely
high titer
serum antibody response dominated by antibodies of the (Th2-mediated) IgG,
isotype
(Soloway. et al., supra). Profound humoral unresponsiveness was observed in Tg
animals immunized in this manner (Fig. 9A). This could not be due to immune-
complex binding with circulating serum flg (12-26-IgG) since these tolerant
animals
had diminished splenic memory T cell responses to 12-26 peptide (Figure 9B).
The
extent of cellular and humoral unresponsiveness was comparable for both Line 5
and
Line 17 suggesting that even lower levels of expression (Line S) efficiently
satisfied
antigenic thresholds for tolerance induction.
In the immunocompetent adult, solid induction of tolerance to foreign
transplantation antigens or viral CTL epitopes.has previously been shown to be
most
effective in subjects in whom hematopoietic or lymphoid ablation is followed
by
reconstitution with antigen-expressing BM-derived APC (IIdstad, S. T. et al.
(1984)
Nature 307:168-170; Cobbold, S.P. et al. (1984) Nature 312:548-551; Roberts,
J.L.
et al. (i990) J. Exp. Med. 171:935-940; Oehen, S.V. et al. (1994) Cellular
Immunol.
158:342-352; Nemazee, D. et al. (1989) Proc. Nat1 Acad. Sci. USA. 86:8039-
8043).
To ascertain whether tolerance to a class II-restricted T cell and B cell
epitope could
similarly be achieved, we constructed Tg BM chimeras.
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Sublethally irradiated adult BALB/c mice were injected with 1:1 mixtures of
Line 17 Tg and NTg iittermate BM, and the recipient's immune system was
allowed
to redevelop for 7-8 weeks in the presence of flg (12-26-IgG)-producing
lymphoid
tissue. Such treatment followed by immunogenic challenge with synthetic
peptide
revealed profoundly suppressed cellular and humoral (Figure 10) peptide-
specific
immunity in these normal adult recipients.
The magnitudle of tolerance as well as serum NIP-binding IgG, levels observed
for these BM chimeras was comparable to that observed in Tg animals expressing
12-
26 IgG continuously during ontogeny. More interestingly, even injection of
nonirradiated subjects with large numbers of crude syngeneic Line 17 BM
resulted in
high levels of serum NIP-binding IgG, which could be detected as long as one
year
post-infusion.
Tolerogenicity of flg-Expressing Lymphoid Tissue Transferred to Unmanipulated
Adult Subjects
Although tolerance in the neonate or hematopoietically-ablated adult recipient
is believed to involvf: thymic participation (central tolerance), we analyzed
the
potency of peptide-flg-expressing lymphoid tissue for the induction of
tolerance in
mature peripheral lymphocytes. To induce peripheral tolerance in an
unmanipulated
immune-competent adult, we injected iv various preparations of Line 17 Tg
hematopoietic tissue into normal adult BALB/c subjects. We first compared the
in
vivo tolerogenic efficacy of injecting large numbers of resting vs. activated
B cells.
Resting B cells are known to be competent in antigen processing and
presentation
functions, but have been described to possess defective costimulatory ability,
in
contrast to LPS- or surface Ig-activated B cell blasts which express abundant
B7-1,
B7-2, and CD40. Stuprisingly, injection of a variety of different 12-26-IgG-
expressing lymphoid preparations, including Percoll gradient-purified resting
B cells,
LPS-activated blasts, crude BM , and even crude splenocyte preparations, were
all
highly effective in diminishing humoral (Figure 11 ) and cellular immune
responses to
the 12-26 peptide in adult recipients.
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Analysis of B-Cell Tolerance Induction in Transgenic or Normal Tolerized
Recipients
Although processed 12-26 peptide induced efficient tolerance in T cells, the
bivalent epitope-containing IgG molecule could potentially induce B cell
tolerance
(Chiller, J.M. et al. (1970) Proc. Natl. Acad. Sci. USA. 65:551-556; Parks,
D.E. et al.
(1980) J. Immunol. 124:1230-1236; Tighe, H. et al. (1995) J. Exp. Med. 181:599-
606). To test for the potency of such an effect, line 5 and 17 Tg mice were
challenged
with peptide conjugated to a different carrier, hen egg lysozyme (HEL), as a
source of
T cell help for potentially tolerized B cells. Potentially self reactive anti-
peptide B
cells can receive foreign-reactive T cell help from HEL-specific T cells to
produce
autoantibodies. Immunization with I2-26-HEL in adjuvant, followed by a boost,
revealed that both Tg lines displayed B-cell tolerance, manifest as a
reduction in anti-
peptide IgG titers (Figure 12A). In contrast, all animals expressed similar
levels of
anti-HEL IgG titters (>105). Interestingly, although the lower expressing Line
5 was
solidly unresponsive to 12-26 when challenged with peptide alone (Figure 12B),
immunization with the conjugate revealed low but significant anti-peptide
responses
not evident in the higher expressing Line 17. Thus, although both lines are
solidly
tolerant at the T cell level, a more potent B cell tolerance appeared to have
been
associated with higher self peptide concentrations in Line 17 mice.
We also assessed the effect on B-cell tolerance of transferring 12-26-IgG-
expressing lymphoid tissue to normal, unconditioned recipients. Normal
immunocompetent subjects that were rendered tolerant by various preparations
of
Line 17 lymphoid cells, displayed similar levels of humoral immune tolerance
(Figure
11 ). Since BM cells, resting B cells, and activated LPS B cell blasts secrete
or
express on their surface varying amounts of the flg, it is conceivable that,
although
they are all effective in inducing T-cell tolerance (via similar tolerogen
presentation
pathways), they may have differing effects on B cell tolerance induction.
Thus, we
adoptively transferred splenocytes from previously-tolerized animals immunized
with
12-26 peptide and HEL (as a specificity control), and which had previously
displayed
unresponsiveness (Figure 11 ), into irradiated BALB/c recipients. These
recipients
were boosted with peptide-HEL (to stimulate T-cell help for potentially
tolerized B
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cells). Anti-peptide IgG titers were measured. Surprisingly, although all
animals
displayed relatively lower titers than non-tolerant controls, recipients of
splenocytes
from LPS-blast tolerized donors appeared to have a more profound B cell
tolerance
(Figure 12B).
5 Splenocytes from tolerized animals were also restimulated in vitro with
peptide and analyzed for IL-2/IL-4 cytokine responses. This experiment
confirmed
that the various lymphoid treatments resulted in similarly decreased cytokine
responses , and thus similar T-cell tolerance induction as was suggested by
results
described above.
10 Induction of Tolerance in Previously Primed Adult Subjects
Various experimental models of tolerance induction have established that it is
possible to diminish specific immunity in a naive, antigen-inexperienced
recipient
(Eynon, E.E. et al. (1991) Transplant. Proc. 23:729-730; Eynon, E.E. et al.
(1992) J.
Exp. Med. 175:131-138; Fucks, E.J. et al. (1992). Science. 258:1156-1159). In
15 contrast, inducing unresponsiveness in an antigen-primed (immunized) adult
has been
more difficult (Fucks et al., supra; Eynon, E.E. et al. (1993) J. Immunol.
151:2958-
2964).
We tested the ability of tolerogen-synthesizing B cells to modulate an ongoing
immune response by SC immunization of recipients with peptide in adjuvant and
20 waited 1-2 weeks before injecting of Line 17 Tg B cell preparations as
tolerogen.
Primed recipients received one of four preparations: (1) Percoll purified
resting B
cells, (2) crude BM cells, (3) LPS-activated B cell blasts, or (4) chemically-
fixed B
cells. One week lai:er, they were boosted with peptide in saline, and humoral
immune
responses were subsequently determined.
25 Although both resting B cells and crude BM cells produce specific
unresponsiveness in antigen-naive recipients, both were ineffective in
diminishing
peptide-specific humoral immunity in previously primed subjects (Figure 13A).
LPS-
activated Tg B cells completely reversed the ongoing immune response (Figure
16B).
A significant reduction in anti-peptide antibody titers was also produced by
treatment
30 with fixed Tg B cells (Figure 13C). Thus, the most potent tolerogenic
treatment for
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already immune subjects, which caused them to become as unresponsive as
antigen-
naive subjects, was the infusion of activated flg-synthesizing B cells.
Furthermore,
diminution of anti-peptide antibody responses by activated or fixed fig-
expressing
Line 17 B cells was observed not only for total IgG levels. Rather antibodies
of the
S IgG, isotype, a Thl-dependent response, and antibodies of the IgGZb isotype,
a Th2
dependent response, were also diminished, thus ruling out a possible "immune-
deviation" or class-switching effect (Asherson, G.L. et al. ( 1965)
Immunology. 9:206-
21 S).
III. DISCUSSION
A variety of protein engineering strategies have established the efficacy of
expressing heterologous epitopes in immunoglobulin frameworks for the
enhancement
of specific immunity (Billetta, R. et al. (1991) Proc. Natl. Acad. Sci. USA.
88:4713-
4717.; Zaghouani, H. et al. (1993) Science. 259:224-227). The present
inventors
have shown (see Example I) that similarly constructed peptide-Ig molecules can
induce specific tolerance to a foreign immunodominant epitope. The above
studies
extend this approach to a novel strategy in which antigen-presenting B cell
may be
engineered to express immunoglobulins which contain within their structure
tolerogens that can be employed to manipulate an undesired immune responses.
Expression of otherwise immunogenic determinants in an IgG fusion protein
which is
synthesized, secreted, and also directly presented by lymphoid tissue, was
shown to be
a highly efficient tool for the induction of immune self tolerance in mature,
immunocompetent subjects.
In the model described herein , iv injection of any of a variety of crude or
purified lymphoid cell preparations were effective for tolerance induction. We
were
surprised to find that even unfractionated splenocytes were effective, since
such a
population includes dendritic cells which have been reported to prevent
induction of
unresponsiveness (to the H-Y antigen) (Fuchs et al. supra). This is most
likely due to
the restricted expression of the self antigen in the present system to the B-
lymphoid
cell compartment whereas H-Y is expressed by B cells and other APC.
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Transgenic peptide-Ig chimeric molecules have the potential to be presented
directly or secreted and re-presented, making it likely that tolerance
induction by
injected of peptide-a:Kpressing lymphoid tissue occurs via multiple pathways.
This
may also explain the potency of the fIg tolerogens. Secretion of the fIg
tolerogen by
S activated transgenic B cells and re-presentation by non-transgenic APC may
provide
an additional tolerogenic pathway. This is supported by our observations that
high
doses of soluble peptide-IgG, or very low doses of flg from secreting
transfected cells,
upon injection d in vivo, are sufficient for inducing tolerance (Zambidis et
al., supra).
T-cell clonal deletion has been described in other transgenic models in which
soluble
self Ig antigenic determinants were presented in the periphery or in the
thymus (53,
64). Thus, although direct presentation of self antigens by B cells may be
sufficient
for peripheral tolerance induction, additional pathways using other APC such
as
macrophages (especially for soluble IgG antigens) (Phillipis, J.A. et al.
(1996) J. Exp.
Med. 183:1339-1344) may also tolerize independently.
BM-derived B cells or purified resting B cells are deficient in costimulatory
function. Hence, for these cells, direct presentation of endogenously
synthesized 12-
26 peptide (signal 1 ) is the most likely primary tolerogenic pathway. Such
cells
express very little membrane or secreted IgG,. Thus, a relatively low level of
production of soluble and/or membrane flg capable of interacting with surface
IgM
molecules specific fir the foreign epitope may explain why such cells were
relatively
less tolerogenic for the B cell compartment. In contrast, activated B cells
with
increased secretion of fIg were more efficient B cell tolerogens, and were the
only
preparation we tested which could shut down an ongoing immune response.
Recently, hil;h doses of antigen given to naive or even primed recipients was
shown to cause clonal deletion of both peripheral T and B lymphocytes via
mechanisms of programmed cell death, or apoptosis. Peripheral deletion of
mature
lymphocytes resulted from an exhaustive immune response ("propriocidal
regulation") which was IL-2-dependent and mediated by the apoptosis-regulating
surface molecules Fas and Fas ligand {Crispe, LN. (1994) Immunity. 1:347-349;
Critchfield, J.M. et .al. (1994) Science. 263:1139-1143; Singer, G.G. et al.
(1994)
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Immunity. 1:365-371; Pulendran, B. et al. (1995) Nature 375:331-334; Shokat,
K.M.
et al. (1995) Nature 375:334-338; Lenardo, M.J. (1991) Nature 353:858-861).
One interpretation of the present findings is that prolonged presentation of
peptide/MHC complexes provided by live transgenic B cells, combined with
sustained
production and re-presentation of secreted flg activates those mechanisms
responsible
for high dose tolerance. The existence of an activation-driven apoptosis
mechanism
may explain our observation of tolerance induction in subjects during an
ongoing
immune response when treated with transgenic LPS blasts expressing the flg.
Abundant evidence exists that transmittal of signal I alone (for example by
using fixed APC) may be important in tolerance induction in vitro or in vivo
of
preactivated T cell clones, or tolerance induction in a naive immune system.
We now
add the notion that resting or fixed (costimulation-deficient) transgenic B
cells, which
are not expected to secrete significant amounts of Ig, may evoke a different
tolerogenic pathway than does a large dose of activated B cell APC injected
iv. In the
present studies, resting B cells were less efficient at curtailing an ongoing
immune
response.
It is concluded that the expression of a flg construct comprising a selected
foreign epitope or epitopes in peripheral B cells using gene therapy
strategies has
great practical utility for modulating humoral and cellular immune responses.
In
comparison to currently used methods of high dose tolerance or oral tolerance,
genetic
transfer and expression of tolerogens in lymphoid APC requires only knowledge
of
the DNA sequence encoding the target epitope towards which tolerance is
desired.
The present method avoids the cumbersome antigen purification/synthesis steps
.
More importantly, since clinically useful tolerance would require that the
antigen
(tolerogen) persist, its genetic expression in long-lived APC or
pluripotential
hematopoietic stem cell precursors provides a means for achieving the
requisite
persistence. The present inventors have also induced peptide-specific
tolerance by
expression a flg construct in peripheral B cells or hematopoietic stem cells
using
retroviral-mediated gene transfer.
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EXAMPLE IV
Genetically-Transferred Central and Peripheral Immune Tolerance Via Retroviral-
Mediated Expression of Immunogenic Epitopes in Hematopoietic Progenitors or
Peripheral B Lymphocytes
One potential strategy for the induction of clinically relevant tolerance is
indirectly related to 'the original demonstration by Medawar's group of
tolerance
induction to foreign MHC antigens via injection of allogeneic hematopoietic
cells into
neonates (Billingharn et al., supra). In adults, attempts to induce tolerance
to foreign
grafts by injecting accessory-cell depleted splenocytes (Ryan, J.J., et al. (
1984) J.
Immunology 133:2343-2350; Hori, S., et al. (1989) J. Immunology 143:1447-1452)
or
syngeneic transfected cells (Madsen, J.C., et al. (1988) Nature 332:161-164)
has met
with, at best, limited. success. The advent of efficient methods for gene
transfer into
hematopoietic cells may, in theory, enable the expression of foreign antigens
for the
induction of tolerance via pathways similarly used for tolerance induction to
naturally-expressed epitopes (e.g. MHC, Mls antigens) in bone marrow chimeras
(Ramsdell, F., et al. (1989) Science 246:1038-1041; Roberts, J.L., et al.
(1990) J. Exp.
Med. 332:161-164; Gao, E-K, et al. ( 1990) J. Exp. Med. 171: I 1 O 1-1121;
Sachs, D.H.,
et al. (1993) Transplantation Proc. 25:348-349). Since such approaches have
usually
required some degree of myeloablation, a more desirable approach would be to
adoptively transfer l;enetically modified peripheral APC (Sutkowski, N., et
al. (1994)
Proc. Natl. Acad. Sci. USA 91:8875-8879), which engraft more efficiently in an
unconditioned host. An excellent candidate for such a strategy would be the
peripheral B lymphocyte which has been described to possess immune-modulating
characteristics. Previous studies have shown that antigen-presenting B cells
are
capable in some circumstances of inducing peripheral tolerance of (a) mature,
naive T
cells in vivo (Webb, S., et al. (1990) Cell 63:1249-1256; Eynon, E.E., et al.
(1992) J.
Exp. Med. 175:131-138; Fuchs, E.J., et al. (1992) Science 258:1156-1159;
Buhlmann,
J.E., et al. (1995) Immunity 2:645-653), or (b) previously activated T cell
clones in
vitro (Gilbert, K.M., et al. ( 1994) J. Exp. Med. 179:249-258).
To test the potential for gene-transfer of a target antigen into autologous
APC
for the induction of specific immune tolerance, we created a recombinant,
replication-
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defective retroviral vector for the expression of a foreign class II MHC-
restricted
immunodominant model epitope, I 2-26 (Soloway, P., et al. ( 1991 ) J. Exp.
Med.
17;1:847-858; Lai, M-Z, et al. (1987) J. Immunology 139:3973-3980), fused at
the N-
terminus of a murine IgG H chain, as described above. Engineered Ig expressing
5 heterologous epitopes has been described for the potentiation of peptide-
specific
immunity (Zaghouani, H., et al. (1993) Science 259:224-227), and the Examples
above expanded this approach by describing the tolerogenic properties of a
soluble
engineered 12-26-IgG fusion protein. The genetic transfer and expression of
immunogenic epitopes, or whole complex antigens by appropriate "non-
professional"
10 APC has great utility for the specific elimination of undesirable immunity
associated
with HIV infection, as describe herein, autoimmune states (Tisch, R. et al.
supra;
Higgins, P.J. et al. supra; Critchfield, J.M., e1 al.. supra), recombinant
clotting factor
administration (Allain, J.P. et al. (1976) Blood ~t7:973), and gene therapy
protocols
(Yang, Y., et al. (1995) J. virol. 69:2004-2015; Tripathy, S.K. et al. (1996)
Nature
15 Medicine 2:545-550).
A. Materials and Methods
1. Replication-defective retroviral vectors and gene-transfer protocols
12-26-IgGI H chain cDNA was derived by RT-PCR from J558L myeloma
cells, transfected with the rearranged genomic construct (Examples I-III;
Zambidis et
20 al., supra) and subcloned into retroviral vector MBAE (Kang, J., et al.
(1990) Proc.
Natl. Acad. Sci. USA 87:9803-9087) containing long terminal repeats (LTR), y~+
packaging signals, a neomycin resistance gene, and cloned human b-actin
promoter
sequences. PCR primers encoded 5' Ig H chain leader and 3' IgGI as well as Sal
I
restriction site sequences:
25 VH 5' primer: TGGACTAAGTCGACACCATGGGATGGAGC (SEQ ID N0:207)
G1 3' primer: TCGGAAGGGTCGACGGATCATTTACCAGGAGA (SEQ ID N0:208)
A high titer (105-106 neomycin-resistant NIH 3T3 CFU/ml) yr-2 packaging line
(F6P)
was prepared with recombinant plasmid MBAE.BAK, and assayed for helper virus
via horizontal spread of neomycin resistance with NIH 3T3 cells. Ecotropic F6P
was
30 prepared by "ping-pong" amplification using amphotropic line PA317.
Producer lines
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were stored in liquid nitrogen and freshly thawed cells were utilized for each
individual experiment.
B cell lines CH31, A20, J558L, and NS-1 (ATCC, Rockville, MD) were
transduced with recombinant retrovirus via co-culture with adherent F6P cells
for 24-
48 hours in the presence of 6 pg/ml polybrene (Sigma). Cells in suspension
were
washed and recultured in 1 mg/ml 6418 for selection of stable transductants
prior to
genomic Southern blot, RT-PCR, ELISA, or antigen-presentation studies.
Infection
of BM progenitors and quantitation of 6418-resistant colony-forming cells
(CFC) has
been described (Keller, G., et al. (1985) Nature 318:149-154; Bodine, D.M., et
al.
( 1989) Proc. Natl. Acad. Sci. USA 86:8897-8901 ). BM was harvested from
femurs
and tibiae of 6-8 week old BALB/c donors injected IV with 150 mg/kg 5-
fluorouracil
3-4 days previously. Erythrocyte-depleted BM was co-cultured (Sx106/ml) with
irradiated (2000 rads) F6P or y~-2 parental cells (mock transduction). Ten ml
cultures
in complete RPMI 1640 with 1 S% FCS were incubated at 37° C, 5% C02 for
48
hours, and included 200 U/ml each of IL-3, IL-6, and IL-7 (Genzyme). 4 pg/ml
polybrene was added to co-culture during the last 24 hours of infection.
Splenic B cells were similarly infected in vitro via co-culture with viral-
producing F6P or parental W-2 (mock transduction). Peripheral B cells were
purified
with anti-T cell antibody cocktail plus complement and Percoll density
gradients (60-
70% layers). Purified B cells were pre-stimulated with 50 pg/ml bacterial
lipopolysaccharide (LPS, E. coli OSS:BS, Sigma) overnight, and recultured
(3x106/ml, 5 ml cultures) with irradiated F6P in the presence of 4 ~g/ml
polybrene
and 50 ~g/ml LPS for an additional 24 hours.
2. Tolerance induction and measurement of peptide-specific cellular and
humoral immuni
Adult (6-8 week old) BALB/c recipients (Jackson Labs) were sublethally
irradiated (200-600 cads total body irradiation) and injected intravenously
(lateral tail
vein) with 1-2x106 gene-transferred or mock-transduced BM progenitor cells.
Unconditioned, noanal BALB/c were similarly injected with >Ix107 gene-
transferred
LPS blasts. All cells were washed extensively in serum-free medium and
injected IV
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in a volume of 500 pl. Recipients were analyzed for expression of recombinant
retrovirus as well as immune tolerance to 12-26 peptide 2-12 weeks later.
Tolerized
recipients were immunized for cellular or humoral immune responses as
described
above (see also: Soloway, P. et al. (1991) J. Exp. Med. 174:847-858; Zambidis
et al.,
supra). Animals were injected SC with 50 ~g synthetic I2-26 peptide emulsified
1:1
CFA. and in some experiments, also ip with 10 pg hen egg lysozyme (HEL) in CFA
as a specificity control. Two weeks later, mice received an additional ip
boost of 50
ug peptide and 10 ug HEL in saline. Antibody titers were determined from serum
bleeds 8 days after secondary boosts. Splenic memory T cell responses were
measured in vitro 6-8 weeks following these secondary challenges by
reculturing
purified T cells (3x106/ml) with irradiated (2500 rads) BALB/c splenocvtes
(1x106/ml) and dilutions of synthetic peptide. Serum peptide-specific or HEL-
specific IgG responses were determined by ELISA as described (supra). Cellular
responses from draining popliteal and inguinal LN cells were assayed 9 days
after SC
immunization with 20 pg peptide in CFA. Cultures were pulsed with
(3H]thymidine,
harvested and counted as described above) IL-2 and IL-4 cytokine production
was
quantitated as above. Dilutions of anti-IL-2 mAb S4B6 and anti-IL-4 mAb 11 B
11
(ATCC) were included to confirm specificity. IFN-y was measured using a
commercial ELISA kit (Intertest-y, Genzyme).
3. RT-PCR and immunologic methods
Detection of 12-26-IgG transcripts in transduced cell lines or hematopoietic
tissue
from BM-injected mice was performed with a~ 12-26 sequence RNA-PCR assay.
Primers were designed to amplify S' immunoglobulin leader sequence, "VH 5'
primer", as above, and 3' 12-26 sequence, ("3' pep primer"):
GGC AAC AGA AGC TTT CAC TTC TTC TTC TCG TAT (SEQ ID N0:209).
Briefly, 1-5 pg of total RNA from various tissue was reverse-transcribed (2
rounds)
with AMV reverse transcriptase, dNTP's, and oligo dT and random hexamer
primers
(Invitrogen cDNA cycle kit) at 42o C. The resultant cDNA was amplified with 5'
and
3' primers described above and Taq polymerase (Perkin-Elmer Cetus). PCR
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conditions were 45 seconds at 93o C, 2 min at 47o C, and 2 min at 72o C for 35-
40
cycles. Amplified DNA products were loaded (1/10-1/100 sample) onto 2%
agarose/TBE gels and subsequently transferred onto nylon membranes for
Southern
blot analysis. 12-26 sequences were confirmed in RT-PCR amplified DNA samples
with a g-32P-labeled oligonucleotide ("oligo Ig-one") encoding 12-26 but which
does
not overlap with the 3' PCR primer: TGATCTACTGCAGCTGGAGGACGCGCGGCGG
(SEQ ID N0:210). Tissue RNA samples were compared via b-actin RT-PCR using
commercially available primers (Stratagene).
12-26-IgG H chain protein was detected in culture supernatants of transduced
celi lines. or in sera of mice injected with gene-transferred cells, via its
ability to bind
to the NIP hapten using a modified NIP-binding ELISA as above. Briefly,
dilutions
of culture supernatants or sera were incubated on ELISA plates coated with NIP-
gelatin conjugate (and subsequently probed with goat anti-mouse IgGI-AP.
Standard
curves with affinity-purified 12-26-IgG from supernatants of transfected J558L
were
used for quantitation.
B. Results and Discussion
A recombinant retroviral vector (Kang, J., et al., supra ) was modified by
inserting a PCR-derived cDNA encoding the 12-26-IgG H chain sequence (Figure
14),
and a high titer ecotropic packaging line (F6P) was generated for the in vitro
infection
of cell lines and hematopoietic tissue via co-culture methods (Keller, G., et
al. (1985)
Nature 318:149-154;. Dick, J.E., et al. (1985) Cell 42:71-79; Bodine, D.M., et
al.
( 1989) Proc. Natl. A~:ad. Sci. USA 86:8897-8901 ). For initial studies, a
variety of B
cell lines at various stages of differentiation were transduced, including
CH31
(immature), A20 (mature, activated), NS-1, and J558L (plasmacytomas). Intact
proviral integration in transduced 6418-resistant A20 cells could be verified
by
genomic Southern blotting using a DNA probe specific for 12-26 sequence
(Figure
14). More importantly, 12-26-IgG H chain can assemble with endogenous light
chains in transduced B cell lines, to be expressed as a membrane surface
protein, or
secreted into cultured supernatants (50-80 ng/ml) in NS-1 and J558L myelomas.
Immunoprecipitation of secreted 12-26-IgG and immunoblot analysis with a
peptide-
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specific monoclonal antibody (B3.11) could directly demonstrate the expression
of
12-26 peptide.
Although retrovirally-synthesized gene products are expected to give rise
primarily to processed peptides presented by MHC class I molecules,
endogenously-
derived peptides can also be routed to endocytic class II MHC compartments in
some
cases (Weiss, S., et al. ( 1991 ) Cell 64:767-776). Such a pathway should be
enhanced
for retrovirally-encoded 12-26-IgG H chain due to the efficient nature of the
Ig
secretory pathway in targeting the endosomal compartment. To test for direct
presentation of this model immunodominant class II-restricted peptide, we
tested the
ability of 12-26-IgG-transduced A20 cells to directly activate peptide-
specific T-cell
hybrid 9C1?7, which recognizes 12-26 in the context of I-Ad (Lai, M-Z, et al.,
supra).
Efficient presentation of endogenously synthesized peptide was demonstrated,
and this
effect was blocked with antibodies to CD4 or class II MHC molecules. These
results
predict that, in vivo, the 12-26 peptide could be recognized directly by T
cells from a
variety of gene-transferred APC (both lymphoid and non-lymphoid).
Additionally,
synthesis, L chain assembly and secretion of 12-26-IgG H chain by B cells can
potentially result in re-presentation of the molecules by endogenous host APC.
The next experiments tested the potential of genetically modified BM cells to
specifically tolerize a regenerating immune repertoire ("central tolerance"
induction).
BM chimeras were produced in sublethally irradiated (200-650 tads) BALB/c mice
by
infusing 5-fluorouracil (FU)-pretreated donor BM which had been co-cultured
with
F6P. This protocol leads to newly developing lymphocytes and APC (lymphoid and
non-lymphoid) that are derived from both the host, as well as the transplanted
BM
progenitors expressing 12-26-IgG. Mice were immunized 4-12 weeks post-infusion
and specific immune responses were measured. Analysis of hematopoietic tissue
(collected at sacrifice) using a peptide sequence-specific RT-PCR assay
indicated that
transcripts were expressed consistently and reproducibly in the BM of all gene-
transferred recipients (Figure 15), although variably in the thymus or spleen.
A very
sensitive NIP-binding IgGI ELISA was used that detects secreted 12-26-IgG in
the
serum which is a result of pairing of the fusion protein H chain with
endogenous ~, L
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chains in B cells derived from the gene-transferred stem cells. Although this
assay
detects only a fraction of H chain secreted by B cells (~, light chain-
paired), expression
could be detected in approximately one-third of ali gene-transferred BM
recipients
(Table V). Despite These variable serum expression patterns, dramatic and
5 reproducible peptide-specific tolerance was observed in all 22 studied
recipients of 12-
26-expressing progenitor cells, which tolerance was demonstrated following
immunization with synthetic peptide in adjuvant. Specific T-cell proliferative
and
cytokine responses of draining LN cells were significantly diminished (Figure
16), as
were anti-peptide antibody levels following priming and boosting (Figure
17A,B).
10 Since the present BM transduction protocol was designed to effect viral
integration
into early hematopoietic progenitors, and both the 5' viral LTR and ~i-actin
promoters
are non-specific as to cell lineage, a variety of differentiated cells with
APC capacity
may directly tolerizc; developing lymphocytes. We predicted that non-lymphoid
APC
derived from transduced stem cells (e.g. monocytes, macrophages, dendritic
cells),
15 which cannot synthesize Ig L chains, and thus cannot secrete the flg, may
nevertheless
play a critical role in direct presentation of transgenic peptide to
developing
lymphocytes. To test this, syngeneic BM from SCID mice was gene-transferred
the
tolerogenic activity of myeloid APC was analyzed. Although hematopoietic
tissue
from SCID mice is deficient in developing mature lymphoid cells, the APC
function
20 of cells of the myeloid (non-lymphoid) lineage remains intact (Dorshkind,
K. et al.
(1984) J. Immunolo,~y 132:1804-1808). 5-FU-pretreated SCID or normal BALB/c
donor BM cells were co-cultured with F6P, and stem cells were injected into
sublethally irradiated normal BALB/c recipients. (With SCID BM donors, normal
lymphocytes can regenerate only from the recipients' stem cells). BM chimeras
were
25 rested over 2 months and subsequently immunized with 12-26 peptide for
measurement of humoral immune tolerance.
Peptide-specific tolerance was comparable in recipients of either normal or
lymphoid-deficient BM. In contrast recipients of mock-transduced BM (not
expressing the 12-26 peptide from the flg) had high-titer antibody responses
(Figure
30 17A,B). Analysis of 6418-resistant hematopoietic colony forming cells (CFC)
from
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recipients of transduced normal BM or transduced SCID BM showed that the
proportion of BM-derived myeloid stem cells expressing the fIg construct was
similar
(1-5%, Table V). These results indicate that in addition to tolerogenesis by
lymphoid
APC, transduced myeloid BM-derived APC can also share this activity. This
result
may explain the consistently solid tolerance observed in all recipients of
transduced
normal BM. regardless of detectable levels of 12-26-IgG (the B-lymphocyte-
derived
transgene) in the subjects' serum.
Although efficient induction of tolerance in newly arising lymphocytes was
readily and reproducibly accomplished with genetically-modified BM, a more
clinically practical approach for gene-transfer tolerogenesis would be the
induction of
peptide-specific peripheral tolerance in a mature immune repertoire without
the need
for prior myeloablation. It is known that potent tolerance in normal,
immunocompetent subjects can be induced by injecting large numbers of mature B
lymphocytes expressing a "foreign" antigen (e.g., H-Y or Mls). Therefore, the
present
inventors tested the tolerogenicity of fIg -transduced peripheral B cells.
The approach comprised stimulating Percoll~ gradient-purified splenic B cells
to proliferate with bacterial LPS, brief co-culture with F6P, and subsequent
iv
injection into normal, immunocompetent (non-irradiated) BALB/c recipients.
This
treatment resulted in an efficient suppression of peptide-specific humoral
immunity
comparable to that observed in the BM chimera experiments described above.
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TABLE V
Expression of Serum 12-26-IgG and 6418-resistant BM Progenitors in
Genetically-Tolerized Mice
BM Ciene Serum NIP-Binding Tolerance
Mouse Transfer lgG1 % G418R Induction
# (ng/ml) CFC/ml
Expt.
1
1, 2, - <O.l 3, 7 wks) NT* -
3
4 + 20, 2 (3, 7 wks) NT +
+ <0.1, 20 (3, 7 wks)NT +/-
6 + 60, 2 (3, 7 wks) NT +
7 + 120, 20 (3, 7 wks) NT +
g + 10, <0.1 (3, 7 wks)NT +
Expt.
2
9, 1 - <0.1 (6 wks) NT -
U, I
I
12 + 135 (6 wks) NT +
13 + <0.1 (6 wks) NT +
14 + 15 (6 wks) NT +
+ <0.1 (6 wks) NT +
16 + <p.l (6 wks) NT +
Expt.
3
17, 18, - <0.1 (3 wks) NT -
19
+ <p.l (3 wks) NT +
21 + 30 (3 wks) NT +
22 + <0.1 (3 wks) NT +
Expt.
4
23, 24, - NT 0% ( 11 wks)-
26 + NT 2.2% ( 11 +
wks)
27 +~ NT 2.8% ( 11 +
wks)
28 +~ NT 2.8% ( 11 +
wks)
29 + (S(:ID)NT 5.4% ( 11 +
wks)
+ (S(:ID)NT 1.3% (11 +
wks)
31 + (S(:ID)NT ~ 4.7% ( 11 +
wks)
Legend to Table V: Recipients of F6P or mock-infected 5-FU-treated BM
progenitors were
assayed for transgene expression in serum or BM at indicated times. BM CFC
were assayed at
sacrifice time in 0.3~% semisolid agar cuitures in long-term recipients. BM
cells were cultured at
106 /well in complete 1MDM plus 15% FCS, 200 U/ml IL-3, and 10% ORIGEN
conditioned
medium (GIBCO, BRL) containing IL-I, G-CSF, GM-CSF, M-CSF, and IL-6. Erythro-
myeloid
colonies were grown with and without 1 mg/ml 6418, and the percentage which
were viable and
neomycin-resistant (G418R) were counted after 7-10 days. BM recipients were
conditioned with
either 200 rads (Expt. l ) or 600 rads (Expts. 2-4). Detailed experimental
results for Expt. l and
4 are presented in Figures 17A and 17B, respectively. * NT: not tested
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Splenic memory T cell responses measured approximately 3 months after
immunization in these subjects were markedly reduced (IL-2 and IL-4 cytokine
reduction), indicative of effective tolerization in both T helper cell
compartments (Thl
and Th2). See Figure 18). Furthermore, 6418-resistant hybridomas could be
generated from LPS-activated spleen cells of these tolerized mice by fusing
the spleen
cells with A20 lymphoma cells in the presence of PEG. These hybrids stimulated
peptide-specific 9C 127 T cells directly (Figure I 9), thus proving that gene-
transferred
peripheral B cells are capable of persisting and presenting 12-26-IgG self
antigen for
prolonged periods in a way which results in induction and maintenance of
peptide-
specific tolerance. Such long-term persistence (>3-6 months) is consistent
with results
obtained after injection of either normal peripheral B cells (Sprent, J. et
al. ( 1991 ) J.
Exp. Med. 17;1:717-728) or genetically-modified LPS-activated peripheral B
cells
(Sutkowski, N., et al., supra).
The foregoing results show that the present inventors have in hand a novel,
efficient strategy for delivery of a foreign peptide, which would otherwise be
an
immunogen, to an adult immune system in a tolerogenic manner in the form of a
soluble fIg protein expressed in hematopoietic tissue. Genetic transfer of a
selected
target single or mufti-epitope sequence into a multipotential stem cell or
into a
peripheral B cell permits the induction of, and more importantly, the long-
term
maintenance of, specific immune self tolerance in the autologous host. The
choice of
a model immunodominant peptide, 12-26, capable of inducing both high titer IgG
antibody responses (Th2-mediated), as well as. vigorous cellular (Thl-
mediated)
responses underscores the versatility of the present method. Although gene-
transfer of
BM expressing class I MHC-restricted CTL epitopes efficiently induced
tolerance
(Ally, B.A., et al. (1995) J. Immunology 155:5404-5408), the present approach
of
fusing an antigenic sequence to an Ig molecule allows for the eff cient
presentation of
a retrovirally-synthesized class II MHC-restricted epitope. Furthermore, in
addition to
effective Th tolerance induction, the bivalent nature of the secreted form of
the
tolerogenic epitope on the two H chains of the Ig-molecule can independently
mediate
effective peptide-specific B cell tolerance, probably via Fc-mediated antibody
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feedback mechanisms (Zambidis et al., supra). Thus, the potency of tolerance
induction using the present invention can exploit multiple pathways in the
immune
mechanism.
The use of to:lerogenic peptide-Ig constructs facilitates "tailor-designing"
the
immune response to a whole antigen by selectively inducing immunity
(Zaghouani,
H., et al., supra ) or tolerance to selected epitopes be they immunodominant
or
cryptic. In contrast to expressing a heterologous epitopes in the CDR3 region
of the
Ig H chains, fusing an foreign antigenic sequences at the N-terminus is not
limited by
size restrictions, and can thus be adapted for expressing large mufti-epitope
antigens,
for example, autoantigenic proteins such as factor VIII (Allain et al.,
supra), myelin
basic protein (I-Iiggins et al., supra; Critchfield et al., supra ), or
glutamic acid
decarboxylase (Tisch et al., supra). Delivery of the tolerogen as a gene
sequence has
many advantages over present tolerance induction protocols, since only the
cDNA
sequence of the target antigen, for example, one or more HIV gp120 epitopes,
needs
to be known. This avoids the need for a protein purification strategy. More
importantly, since experimentally acquired tolerance eventually wanes,
expression and
persistence of the tolerogen in long-lived or multipotential hematopoietic
tissue has
the potential to modulate permanently a specific immune response.
As described herein, an important application of the genetic tolerogenesis
method of the present invention is to help eliminate genetically-altered cells
encountered in gene therapy protocols. Autologous cells genetically modified
with
adenoviral and retroviral vectors are known to, induce immunity in a competent
recipient due to immune recognition of vector-encoded products leading to
subsequent
elimination of transduced cells via both cellular and humoral immunity (Yang
et al.,
supra). Although, for example, immunity to low-level expression of viral
proteins of
first-generation, El-deleted adenovirus can undoubtedly be reduced with
further
genetic manipulation of the vectors, rejection of the foreign transgenes
expressed by
such vectors remains; an even more significant obstacle (Tripathy et al.,
supra). The
present results suggest that tolerogenic pretreatment of immunocompetent
recipients
with vector-transduced autologous APC expressing viral or foreign transgenes
will
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allow the prolonged expression and multiple administration of therapeutic
transgenes
in immunocompetent recipients without need for generalized immunosuppressive
drugs
Although solid evidence exists for the tolerogenic role of
lymphohematopoietic APC in irradiated bone marrow chimeras,
The foregoing studies exploited the ability of mature peripheral B cells to
induce efficient peripheral tolerance in unconditioned adults. These findings
represent
the first example of using transduced LPS blasts as tolerogenic vehicles.
Others have
reported tolerance induction via presentation of antigen by resting B cells to
naive T
and have implicated the poor expression of costimulatory molecules such as B7-
1 and
B7-2 (Hathcock, K.S., et al. ( 1994) J. Exp. Med. 180:631-640). Antigen-
presentation
by resting B cells has thus far been successful in inducting tolerance in
naive
recipients, but has proven ineffective in primed (Fucks et al., supra) or alto-
MHC-
reactive recipients (Buhlmann et al., supra) unless an anti-gp39 (CD40-ligand)
1 S antibody was simultaneously injected to prevent upregulation of B cell
costimulatory
function. Paradoxically, costimulation-competent LPS blasts, as in the present
experiments, could serve as efficient tolerogenic APC in vivo in antigen-naive
recipients, or could induce tolerance in vitro in previously activated T cell
clones
(Gilbert et al., supra).
In studies with transgenic mice expressing the flg construct specifically in
the
B cell compartment, both purified resting flg-expressing B cells or their LPS-
activated
counterparts were highly tolerogenic in normal, antigen-naive adults. In
contrast, only
the activated transgenic B cells were effective in tolerizing an ongoing
response in a
previously-immunized recipient.
EXAMPLE V
HIV gp120 Crosslinking In Vivo Induces apoptosis of T Cells
Studies were performed in mice transgenic for the human CD4 gene. Normal
BALB/c mice or mice transgenic mice for human CD4 ("CD4hu") were immunized
with 20 pg of gp120 in complete Freund's adjuvant, boosted with gp120 in
incomplete
adjuvant and then injected intravenously with 1 p,g of gp120 in PBS.
Peripheral blood
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lymphocytes were harvested at various times after the last injection (of
soluble
gp120). Total number of T cells in the peripheral blood were evaluated using
flow
cytometry to enumerate CD3' cells. Table VI, below shows the results as
percent of
total blood lymphocytes which are CD3+
- 5 Table VI
HIV gp120 crosslinking induces apoptosis of CD3 cells in vivo
in human CD4 transgenic mice
Number
of
CD3+
Celts
in
PBL
Days
after
gp120
Donor mice 0 3 6 9 25 73
Immunized CD4hu 46 43 23 37 45 49
Non-immune CD4hu 44 49 53 45 49 45
Immunized BALB/c: 41 43 57 48 48 47
Non-immune BALH~/c61 62 66 71 59 47
The only significant reduction in numbers of T cells was observed in the
transgenic mice which had been immunized, that is, mice expressing the human
CD4
molecule which can bind gp120 or gp120-anti-gp120 complexes.
Experiments were performed to study the apoptosis resulting from the ligation
of T cell receptors. Mice were immunized as above. Spleens were harvested 9
days
1 S after this last injection and were cultured with medium or with anti-CD3
mAb
( 145.2C 1; 50 pg/ml coated wells) for 24 hours; cells were then harvested,
fixed and
assayed for apoptosiis by propidium iodide uptake and flow cytometry. The
percent of
hypodiploid, apoptotic cells at 24 or 48 hours with anti-CD3 and at 24 hours
with
anti-IgM are shown in Table VII.
_ 20
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Table VII
Induction of Apoptosis After T Cell Receptor Ligation
Percent
Apoptotic
Cells
at
24
or
48
Hours
Donor and treatment Bkgrnd Medium Anti-CD3
Oh 24h 48h 24h 48h
gp 120-immunized - CD4hu5.9 39.0 47.0 41.1 61.2
Non-immune - CD4hu 1.7 29.5 58.0 29.0 24.6
gp120-immunized - BALB/c0.8 38.8 42.6 18.1 16.1
Non-immune - BALB/c 2.1 51.6 47.0 24.9 8.8
"Bkgrnd" = Background values of apoptosis of freshly isolated cells (as
opposed to
cells cultured 24 or 48 hours).
The results show that apoptosis is increased significantly only in T cells of
immunized CD4 transgenic mice whose T cells receptors have been ligated
polyclonally with anti-CD3 mAb. This effect is most dramatic after 48 hours
.of
culture.
Collectively, the foregoing results prove that gp120 epitopes have the
capacity
to prime T cells for apoptosis shown as direct observation of apoptosis in
vitro and as
a Ioss of T cells in vivo which occurred selectively only in subjects who were
both
immunized with gp 120 and who had the appropriate target T cells (bearing
transgenic
human CD4) to which complexes of gp120 and anti-gp120 antibodies could bind,.
These results further support the findings of Finkel's group cited above and
provide an
even stronger basis for the utility of inducing epitope specific tolerance as
described
herein.
EXAMPLE VI
Ongoing Immune Responses to HIV gp120 in Human CD4 Transgenic Mice
Contributes to T Cell Decline upon LV. Administration of gp120
(most references are cited in this section as numbers and appear in a list at
the end of the section)
The HIV retrovirus interacts with the host immune system in a puzzling way.
Virtually everyone infected with the virus synthesizes antibodies directed
against a
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number of the viral envelope epitopes. However, much of this humoral response
has
little if any protective value over the course of HIV pathogenesis [5-16].
Titers of
neutralizing antibodies in AIDS patients are low [6], and the antibodies might
cross-
react with self components due to molecular mimicry and structural/genetic
similarities [7-9]. Fwrthermore, crosslinking CD4 by anti-CD4 antibodies or gp
120
and anti-gp 120 antibodies can upregulate Fas expression and prime Th cells
for
activation-induced apoptosis [l, 10-16].
Crosslinking of human CD4 coreceptor (huCD4), by HIV gp120 and anti-
gp120 antibody in vitro as well as in vivo, upregulate Fas (CD95) expression
and
prime T cells for activation-induced apoptosis (Banda et al., supra: Wang et
al.,
supra). Based on theae observations, we hypothesized that an ongoing humoral
immune response to gp 120 might not serve the host in a protective or virus-
neutralizing manner upon exposure to HIV gp120. Rather , the response might
sensitize even nonini:ected cells for apoptosis. Immunization mice transgenic
for
huCD4 ("huCD4 Tg"') and control mice with 20 ~g gp120 in CFA led to titers
exceeding 1:105 within three weeks. We injected i.v. 1 pg of rgp120s~ into
huCD4 Tg
and non-transgenic BALB/c and BALB/c x C57BI/6 F, mice (CB6 F 1 ) mice that
had
been immunized with rgp120s~. The same amount of rgp120 was also administered
i.v. to unprimed huCD4 Tg mice and nontransgenic controls. Boosting gp120-
primed
control mice with gp 120 gave rise to increased numbers of T and B cell as
well as in
the antibody titers. In sharp contrast, boosting the primed huCD4 Tg mice
(which
express huCD4 on both T and B cells) with soluble gp120 resulted in lower
secondary
antibody titers than in controls. The response to an irrelevant antigen, HEL,
was also
reduced in the gp 120 -primed and boosted huCD4 Tg mice. Furthermore, on day 6
after a single bolus of gp120, the number of peripheral T cells and B cells in
immunized huCD4 '.Cg decreased to 50% of the control levels. Moreover,
compared to
the control groups, t:he splenocytes from gp120-pretreated immunized huCD4 Tg
had
a lower level of CD3+ T cells and underwent extensive apoptosis after anti-CD3
treatment. These in vivo results were consistent with the in vitro findings:
Crosslinking of huCD4 on the spleen lymphocytes of huCD4 Tg mice using
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rgp120SFZ and anti-gp120 antibody not only sensitized T cells for apoptosis,
but also
induced apoptosis per se. Thus, precautions should be taken when employing HIV
envelope gp120 as one of the HIV vaccine components. Tolerogenic therapies
should
be considered when treating HIV infected subjects in this manner.
Materials and Methods
Mice -- BALB/cByJ and CB6 F 1 mice were purchased from the Jackson
Laboratories (Bar Harbor, ME) at 6-10 weeks of age, and housed in pathogen-
free,
microisolater cages. Line 313 huCD4 Tg mice were obtained from Dr. Terri
Finkel,
Denver, CO). These transgenics were originally produced by Dr. Richard Flavell
by
injecting a huCD4 transgene into fertilized eggs and were maintained by
repeated
backcrosses on the C57B1/6 background [17]. The F, offspring between huCD4 Tg
mice and BALB/cByJ are produced in our animal facility by crossbreeding female
BALB/cByJ with male huCD4 Tg mice to yield huCD4 expressing mice
histocompatible with CB6 F 1 mice. The huCD4 molecule was shown to be
functional
in calcium signal transduction and in overcoming the block in positive
selection
induced by in vivo injection of mAbs to the endogenous mouse CD4 [17]. Since
in
these transgenic mice, expression of huCD4 is driven by CD2 regulatory
elements,
both B and T cells express huCD4. Approximately, 85% of splenic cells from
Line
313 huCD4 transgenic mice and more than 50% of spleen cells from the F,
offspring
between huCD4 Tg mice and BALB/cByJ expressed the huCD4 receptor on their
surface. Recombinant wild type gp120 (rgp120SFZ), from Dr. K. Steimer (Chiron
Corporation, Emeryville, CA), binds to the huCD4 molecules in a dose-dependent
manner and competes with huCD4 mAb (Leu-3a; Becton Dickinson, Mountain View,
CA) for huCD4 binding.
Cell Culture
RPMI 1640 medium (GIBCO-BRL, Gaithersburg, MD) supplemented with
heat-inactivated S% fetal calf serum (Hyclone, Logan, UT), 501.~M
2-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml
streptomycin,
MEM nonessential amino acids, and 1 mM sodium pyruvate was used.
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Antibodies
The following antibodies were purchased from PharMingen ( San Diego, CA): FITC-
labeled anti-hamster IgG, PE-labeled anti-mouse CD3, biotin-labeled anti-mouse
CD19 and anti-Fas antibody Jo2) FITC- labeled mouse anti-human CD4 mAb (FITC-
Leu-3a) was obtainc;d from Becton Dickinson (View Mountain, CA). The following
biotin- or FITC- labeled antibodies were purified in our lab by standard
protocols:
anti-mouse CD3 ( 145.2C 11 ), anti-mouse CD4 (GK1.5), anti-mouse CD8 (53-6.72)
and anti-mouse CD45R (RA36B2, B220). Anti-gp120 antibodies used for huCD4
crosslinking were obtained as follows: human monoclonal anti-gp120 antibodies
directed against gp 120 C-terminal peptide, (450-30D 100,100,1,1; abbreviated
"450-
30")) and against the V3 loop (694/98-D 100,100,10,1; abbreviated "694/98")
were
from Dr. Susan Zoll.a-Pazner (VA Medical Center, NY); sheep anti-gp120
antibody
(6205) directed against the 15 C-terminal residues was obtained from
lnternational
Enzymes, Inc. (Falllbrook, CA); human HIV-1 gpi20 monoclonal antibodies (F105)
[Dr. M. Posner, refs.. 18-21] and (AD3) [Drs. K. Ugen and D. Weiner, ref. 22],
as well
as goat polyclonal HIV-1SF2 gp120 antibodies raised against glycosylated and
non-
glycosylated gp120 [Dr. K. Steimer, refs. 23-27] were obtained from the AIDS
Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. Mouse
polyclonal HIV-1SF;; gp120 antisera were produced in our lab by immunization
of
BALB/cJ mice with HIV-ISFZ gP120 in CFA intradermally and boosting twice with
HIV-I sFZ gP120 in IFA intramuscularly after 2 weeks and 1 month of primary
immunization, respectively. The antibody titer was >1/105 as determined by
ELISA
using rgp120 coated plates (1 pg/ml in Tris coating buffer, pH 9.0).
In vitro crosslinking lhuCD4 by gp120 and anti-gp120 followed by anti-CD3
activation
Viable splenocytes from Line 313 huCD4 Tg were incubated (S x 106/ml) with
rgp120srz (20 (g/ml) on ice for 30 min., washed twice, and reincubated with
various
anti-gp120 antibodies (2 (g/rnl or 1:1000 dilution) at 37°C for 45 min.
While an
aliquot of the cells was checked for surface levels of Fas expression by flow
cytometry, 1 x 106 cells were transferred onto anti-CD3 antibody ( 145.2C 11 )-
precoated 96 well plates in a 200 (1 volume and incubated at 37°C, 5%
COz for further
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24-72 hr. The cells were then harvested and assayed for apoptosis by DNA
content
analysis, as described below [28].
Immunization protocol and intravenous administration of gp120
BALB/cJ mice and Line 313 huCD4 transgenic mice were immunized
intradermally with 20 pg HIV-ISFZ rgP120 emulsified in CFA and boosted
intramuscularly with 20 pg rgp120 in IFA 9-12 days later. Ten days after
boosting,
when high titers of anti gp120 sera were detectable, a single dose of 1 pg of
rgp120
was administered intravenously into the immunized animals, as well as animals
that
had not received rgp120 immunization. The percentages of human CD4+, mouse
CD3+, CD8+ and B220+ cells in the peripheral blood were followed using dual
color
flow cytometry described below. In some experiments. the spleens were
harvested
within 9 days after gp120 iv injection and the percentages of splenic CD3+ T
cells
were determined by flow cytometry. The apoptotic cells in freshly harvested
spleens
were assessed by in situ terminal deoxynucleotidyl transferase-mediated dUTP-
biotin
nick end labeling staining (TUNEL). Spontaneous apoptosis and anti-CD3
activation-
induced apoptosis was measured after anti-CD3 in vitro treatment for 24-72 hr.
To
search for depletion of antigen-specific T cells by repeated gp120 iv
injection, the F,
offspring between huCD4 Tg and BALB/cByJ mice, as well as CB6 F1 control mice,
were immunized intradermally with 20 ltg HIV-1SF2 rgp120 and 20 ~tg hen egg-
white
lysozyme (HEL, Sigma Chemical Co., St. Louis, MO) emulsified in CFA. Three
weeks after primary immunization, the animals were boosted intravenously with
1 p,g
rgp120SFZ and 1 pg HEL in PBS, and the iv injection was repeated three times
at 10
day intervals. One week after each iv injection, the CD3+ T cells were
determined by
flow cytometric analysis and the gp120 and HEL specific IgG responses were
measured by ELISA by coating plates with 1 pg/ml gp120 or 1 ~g/ml HEL,
respectively. Antibody titers were determined using CA-Cricket Graph software
and
were expressed as the serum dilution that would bring the OD to pre-
immunization
levels (OD4os = 0.04), assuming parallelism of curves. In other experiments,
gp120-
primed mice were sacrificed on day 1, 4, 7, 11, and 20 after a single bolus of
gp120
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iv, and lymph nodes and spleens were harvested,and determinations made of cell
phenotypes, spontan~:ous apoptosis and anti-CD3 stimulation index.
Flow cytometric anal
The surface level of Fas expression on the splenocytes from Line 313 huCD4
transgenic mice after huCD4 crossiinking was measured by staining cells with
hamster anti-Fas antibody (Jo2), followed by FITC-labeled anti-hamster IgG. To
determine the percentage of peripheral CD3+ T cells, blood was removed from
the
retroorbital plexus. White blood cells were prepared by lysing red blood cells
with
Tris-buffered ammonium chloride buffer (pH 7.2). After washing with PBS, the
cells
were stained with FI'TC-labeled Leu-3a. or anti-B220, or anti-CD8 antibody
plus
biotin-labeled anti-mouse CD3 antibody, followed by Streptavidin-PE staining.
The
same procedure was applied to spleens for measurement of CD3+ T cells. To
measure
Annexin V positive 't cells, lymph node and spleen cells were washed twice
with PBS
and resuspended in binding buffer ( 10 mM Hepes/NaOH, pH 7.4,140 mM NaCI, 2.5
mM CaCI,) [41 ]. To the cell resuspension was added fluorescein labeled
Annexin V
(R&D SYSTEMS, Inc., Minneapolis, MN) and biotin-labeled anti-CD3 antibody,
followed by Streptavidin-PE staining. The FITC and PE fluorescence analysis
were
performed by CELLQuest software in FACScan flow cytometry (Becton Dickinson).
Apoptosis analysis
The percentage of cells undergoing apoptosis was quantitated by a flow
cytometric method described earlier [28]. Briefly, cells ( 1 x 106) were fixed
in 70%
ethanol for 1 hour at 4 C. The cells were then washed and resuspended in 1 ml
PBS, to
which 1 ~i RNase solution (10 mg/mi in PBS) was added and incubated at
37°C for 1
hour. Following the addition of S l,tl of propidium iodide (PI, 10 mg/ml in
PBS), the
PI fluorescence of individual cells was measured using flow cytometry. Cell
debris
and clumps were excluded by gating for single cells by forward and side light
scatter
and by FL-2 area vs. FL-2 width. A distinct cell cycle region of apoptosis
(A.o) could
be identified below the G°/G, diploid peak and the percentage of cells
in the A°
region was quantitated.
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Statistical Analysis
The difference among different treatment groups was analyzed by one-way
ANOVA (StatView, BrainPower, Inc., Calabasas, CA).
RESULTS
In vitro induction orapoptosis by gp120 crossli»king of huCD4
We hypothesized that gp120 crosslinking of huCD4 on splenic cells from
huCD4 transgenic mice would induce apoptosis or prime for apoptosis despite
the fact
that these cells cannot be infected by HIV [34] due to the lack of a necessary
cofactor
(such as Eosin) in mice [35]. We cultured splenocytes from Line 313 huCD4
transgenic mice with rgp120sFZ, and then crosslinked huCD4 by the addition of
marine
hyperimmunc anti-gp 120 antibody, and finally stimulated aliquots of these
cells with
anti-CD3 antibody. Data from three sets of 24 hr in vitro apoptosis induction
experiments showed that crosslinking huCD4 by gpI20 and anti-gp120 antibody
prior
to TCR ligation primed huCD4 Tg splenocytes for anti-CD3 activation-induced
apoptosis. In contrast, anti-CD3 antibody alone reduced spontaneous apoptotic
cell
death. This phenomenon of reduction of apoptosis in unprimed T cells
presumably
reflects cell cycle entry induced by anti-CD3 activation. Furthermore,
crosslinking
huCD4 by gp120 and anti-gp120 antibody per se induced apoptosis in the huCD4
Tg
splenocytes, though to a lesser extent than that with additional anti-CD3
treatment.
Neither gp120 alone nor anti-gp120 antibody alone had any effect on the
priming and
apoptosis induction, suggesting that anti-gp120 antibody was required for
huCD4
crosslinking-mediated apoptosis induction in.vitro.
Crosslinking huCD4 on spleencells of these transgenic mice via gp120 and
anti-gp120 antibody modestly upregulated surface levels of Fas expression
(Table
VIII), as shown by others [10, 13, 14]. Furthermore, the induction of
apoptosis and
upregulation of Fas expression by huCD4 crosslinking is not dependent on the
specificity of anti-gp120 antibody. Thus, we tested a variety of anti-gp120
antibodies
for huCD4 crosslinking, either mAbs raised against a number of gp 120
antigenic
domains or polyclonal antibodies. The results on induction of apoptosis and
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upregulation of Fas expression using different kinds of anti-gp 120 antibodies
for CD4
crosslinking are summarized in Table VIII.
gp120 Induces Acute Loss of CD3+ T cells in Immunized huCD4 Transgenic Mice
A paradox in HIV pathogenesis is that the virus appears to cause AIDS after
the onset of antiviral immunity [11]. Wang et al [15] reported that injection
into
huCD4 transgenic mice of HIV-gp120 and subsequent gp120-specific antibodies
from
AIDS patients (passive transfer of immunity) induced massive long-lasting T
lymphocyte deletion. To investigate if an ongoing immune response to gp120
(active
immunity) could crosslink huCD4 and lead to T cell depletion upon exposure to
HIV-
gp120, we administered gp120SFZ intravenously into gp120SF, immunized huCD4
transgenic mice.
The time course of CD3+ T cell loss in an in vivo experiment was analyzed
Though the number of peripheral CD3+ T cells varied in individual mice, there
was a
drop of peripheral CD3+ T cells only in immunized huCD4 Tg mice after a single
i.v.
injection of gp120. TJone of the other three control groups showed a drop of
CD3+ T
cells. This CD3+ T cell loss occurred acutely, reached its peak (SO% of the
pre-
injection level) on day 6 after i.v. gp120 injection and then gradually
recovered.
Although we did observe CD3+ T cell depletion for more than two weeks in
individual animals , the CD3 + T cell drop in the majority returned to its
preinjection
level within two weeks.
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Table VIII. Apoptosis
induction and
Fas upregulation
on splenocytes
of huCD4
transgenic mice
by CD4 crosslinking
using various
anti-gp120 antibodies
directed
against a number
of g120 antigenic
domains
Apoptosis Fas
Anti-gp120 Induction Upregulation
with
antibodies Specificity & Speciesanti-CD3 (%) % of Control
450-30 CS domain (PTKAKRR) 56% 13%
human mAb
594/98 V3 loop Human mAb 64% 16%
F 105 Conformational epitope71 % 12%
human mAb
AD3 First 204 amino acids70% 15%
marine mAb
6205 CS domain (aa 497-511)74% 13%
sheep Ab
Anti-gp 120 ab Glycosylated gp 120 67% 10%
#6
goat polyclonal Ab
Anti-gp120 ab Non-glycosylated 64% 13%
#7 gp120
goat polyclonal Ab
Anti-gp120 ab Rgp120SFZ 75% 2%
#8
marine polyclonal
Ab
Medium Control 29% 0%
no CD4 crosslink.
no anti-CD3
Anti-CD3-Control 20%
no CD4 crosslink.
Splenocytes from huCD4 transgenic mice were treated and analyzed as described.
Apoptosis induction with anti-CD3 (%) refers to the percentages of apoptotic
cells
after crosslinking of huCD4 crosslinking with anti gp120 antibodies and after
24 hr of
treatment with anti-CD3. Fas upregulation by huCD4 crosslinking for 45 min.
was
calculated as the % increase in median fluorescent channel over medium
control.
The mean percentages of peripheral blood CD3+ T cells on day 0 and day 6
after i.v. gp120 injection from all in vivo experiments were compared. On day
6,
injection of gp120 induced significant peripheral CD3+ T cell loss in
immunized
huCD4 Tg, compared to the other three groups.
The first bolus of gp120 induced a significant, though transient, loss of
peripheral CD3+ T cells in huCD4 transgenic mice, but not in CB6 F 1 control
mice.
Surprisingly, repeated gp120 iv injections afterwards were not able to produce
a state
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of long-lasting T cell loss, neither to induce the T cells to decline again
after recovery
from the first drop, though a slight lower level of CD3+ T cells were
constantly
observed after gp120 injections in the huCD4 transgenic mice than in the
controls.
Interestingly, gp120 i..v. injection also resulted in a loss of peripheral
blood CD19+ B
cells which express huCD4 driven by CD2 promoter but not the CD3-/CD19-cells
in
the gp120-immunized huCD4 transgenic mice, in the same pattern as the loss of
CD3+ T cells.
T cell depletion via apoptosis is not restricted to peripheral blood but also
occurs in
spleen and Iymph nodes
To investigate if T cells in spleen and lymph node are also deleted in
immunized huCD4 transgenic mice receiving gp120 iv injection. we harvested
spleens
and lymph nodes 1 - 20 days after the first gp120 i.v. injection and measured
CD3+ T
cells by flow cytometry. The numbers of splenic CD3+ T cells in all gp 120-
immunized and -pretreated huCD4 Tg were decreased to 50-75% of those in
control
groups. Moreover, lower levels of CD3+ T cells were also observed in the lymph
nodes from gp 120 - immunized and -pretreated huCD4 Tg. The decrease in CD3+ T
cells in spleens and lymph nodes suggested that the T cell drop was not
restricted to
peripheral blood. Furthermore, as measured in a T(JNEL assay, while no
difference
from control groups was noted in some gp120-pretreated immunized huCD4 Tg, a
slightl increase (by 3-5%) in apoptotic cell death was observed in freshly
harvested
spleens from other gp120-pretreated immunized huCD4 Tg, as shown by others
[16].
In addition, when fluorescein labeled Annexin V was employed to detect
phosphatidylserine e:rcpression on early apoptotic cells [41 ], a higher
percentage of
Annexin V positive T cells was observed in the spleens and the lymph nodes
from
gp120-immunized huCD4 transgenic mice than in the organs of control mice,
indicating that gp 12(1-induced apoptosis occurs in peripheral lymphoid organs
as well
as blood. Taken together, these results suggest that a higher number of T
cells in the
gp120-immunized huCD4 transgenic mice undergo apoptosis after receiving gp120
iv
injection.
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gp120 injections lead to a lower secondary antibody titers in primarily
immunized
huCD=l transgenic mice presumably by depletion of antigen-specific T and B
cells
To further confirm that crosslinking in vivo of huCD4 by gp 120/anti-gp 120
binding causes depletion and/or sensitizes huCD4-expressing T and/or B cells
for
activation-induced apoptosis, we injected gp120 and an irrelevant antigen
(HEL) i.v.
into huCD4 Tg mice and CB6 F 1 mice that had received primarily immunization
with
gp 120 and HEL in CFA. We then measured the secondary antibody titers against
gp120 and HEL. Boosting with gp120 not only boosts specific T and B cells for
secondary responses, but the gp120 can also bind to huCD4 receptors to prime
for
apoptosis on all huCD4+ cells. We used the response to HEL as an irrelevant
control
response although HEL-specific T and B cells would be expected to bind gp120
to
their huCD4 receptors like gp120 specific cells. The results showed that huCD4
Tg
mice primed with soluble gp120 had lower secondary titers than did controls,
and the
response to an irrelevant antigen, HEL, was also reduced in the gp 120 -
primed/boosted huCD4 Tg mice. These results indirectly support our hypothesis
that
gp120 injection depletes a population of antigen-specific T cells and/or B
cells.
TCR ligation induces further apoptosis in the spleens of immunized huCD4
transgenic
mice
As mentioned above, in immunized huCD4 Tg mice, the total CD3+ T cell
population was depleted by SO%. Numbers of CD3+ cells then recovered to pre-
injection levels within two weeks after a bolus of gp120 injection. To test if
the
undeleted / recovered CD3+ T cells were primed for apoptosis by in vivo huCD4
crosslinking, we harvested spleens after a single bolus of gp120 and assayed
in vitro
the apoptosis stimulated by anti-CD3 antibody. Anti-CD3 treatment resulted in
lower
stimulation in spleen cells from gp120 - immunized and -pretreated huCD4 Tg
compared to CB6 F 1 control mice. Anti-CD3 treatment also increased the
percentage
of apoptotic cells compared to the medium control treatment only in immunized
huCD4 Tg mice receiving a bolus injection of gpI20 . These ex vivo results
indicated
that the undeleted / recovered CD3+ T cells in the gp 120-pretreated immunized
huCD4 Tg mice were also primed for apoptosis by crosslinking of huCD4 in vivo
.
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DISCUSSION
Efforts are currently underway to elucidate the mechanisms responsible for
AIDS pathogenesis and to establish a protective vaccine for HIV. The precursor
envelope glycoprotein of HIV, gp160, and mature proteins, gp120 and gp4l, have
been considered to be important if not essential as vaccine components,
because
epitopes of these proteins induce both antibody and cytotoxic T cell responses
in man.
Although usually viewed as potentially protective, the role of humoral immune
responses to viral envelope gp120 in HIV pathogenesis was investigated in the
current
study. Our findings , as well as those of others [11, 14-16, 29, 30], suggest
that
endogenous anti-gp1.20 antibody in AIDS patients' serum may actually promote
rather
than neutralizing and inhibitng HIV pathogenesis. Therefore, to avoid
provoking
even greater T cell depletion in AIDS, tolerogenic therapies should be
considered in
HIV vaccine design . This concept is supported by the results of Finkel et al.
[16],
who used huCD4 and HIV gp120 double transgenic mice to address the role of
anti-
gp120 antibody in T cell depletion. Their findings, that antibodies
crosslinking
huCD4/gp120 complexes are a determinant for the outcome of the T cell
responses to
stimuli in vitro and ~'n vivo, are consistent with the present results.
Here, we followed the numbers of CD3+ T cells in human CD4 transgenic
mice in vivo after immunization and a single intravenous exposure to soluble
gp 120.
In such mice, a bolus of gp120 led to rapid depletion of CD3+ T cells in the
periphery
and in the spleen of gp 120 immunized huCD4 transgenic mice; non-immune
transgenic or immunized non-transgenic mice, were unaffected by this
treatment. We
measured CD3 T cells instead of human CD4 expressing cells since, in
preliminary
studies, we found that membrane expression of huCD4 was either downregulated
or
blocked by gp 120:anti-gp 120 binding. Expression of huCD4 is driven in these
mice
by the CD2 promoter, which results in expression in both T and B cells (over
80% of
splenocytes). Our results indicate that huCD4-expressing B cells may also be
depleted in vivo. In humans, a very small ntunber of B cells (0.1-1 %) express
CD4
molecules annd their function in human is still unclear, as is their fate in
HIV
infection. More importantly, the present results suggest that huCD4
crosslinking-
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transmitted death signal might not necessarily require association with
TCR/CD3
signaling pathway and that, under appropriate circumstances, huCD4
crosslinking is
enough to send the death signal and induce the cells to die.
Because we cannot exclude Fas involvement in huCD4 crosslinking-mediated
apoptosis, it is important to know the threshold of Fas expression in
apoptosis
induction. While there is a higher percentage of Annexin V positive T cells in
the
lymph nodes and spleens from gp120-pretreated immunized huCD4 transgenic mice
than those in the control mice, we have not detected a significant increase in
apoptotic
cells in vivo in these tissues using in situ TLJNEL staining. Annexin V binds
to
phosphatidylserine, which is translocated from the inner side of the plasma
membrane
to the outer layer and becomes exposed at the external surface on early
apoptotic cells
[41 ]. This event occurs well before the DNA fragmentation as measured by in
situ
TUNEL staining, therefore, it is possible that rapid phagocytosis of early
stage
apoptotic T cells may be occurring so that an increased level of apoptotic
cells may be
evanescent.
Experiments in which repeated gp120 injections are given to the immunized
huCD4 transgenic mice suggest that at least some antigen specific T cells and
B cells
have been depleted by gp120/anti-gp120 antibody crosslinking huCD4 via
apoptosis.
However, to our surprise, multiple gp120 injections did not induce a long-
lasting T
cell loss from the periphery, though the splenocytes from these mice were
still primed
for TCR activation-induced apoptosis in vitro. Given the fact that huCD4
transgenic
mice produced a large amount of anti-gp120 antibodies after the first bolus of
gp120,
circulating in the bloodstream, gp120 injected thereafter may not be able to
compete
for binding to huCD4 molecules. tthe relatively rapid recovery of CD3 T cells
in vivo
may reflect the small amount of available gp120 when delivered to the
bloodstream of
immunized mice asa bolus, in contrast to the small but steady production of
viral
gp120 by HIV in infected individuals. Recent studies [16] with non-tolerant
gp120
transgenic mice are encouraging for the validity of this model. Transgenic
mice in
which production of gp120 can be controlled by an inducible promoter (to avoid
partial tolerance seen in other work [16]) would be helpful in this direction.
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In summary, our studies demonstrate that an ongoing humoral immune
response to gp120 in huCD4 transgenic mice can participate in priming T cells
to die
upon exposure to gp120. While transient, these primed T cells remain
hypersensitive
to activation-induced death in vitro by antiCD3 crosslinking, and undergo
apoptosis in
S vivo when exposed to a specific antigen that can bind to their TCR. We
believe this
system will be useful for evaluating one of the fundamental processes
underlying HIV
pathogenesis. Using the present invention, it will be possible to test
potential immune
modulation therapies to reverse this sensitivity to apoptosis by inducing
tolerance to
gp120 epitopes [40J,.
Re erences
( 1 ) Ameisen, J. C. et al., Immunol. Today 1991. 12:102; (2) Starcich, B. R.
et al., Cell
1986. 45:637; (3) Ab~acioglu et al., supra; (4) Modrow, S.et al., J. Virol.
1987. 61:570:
(5) Willey, R. L.et al., Proc. Natl. Acad. Sci. USA 1986. 83:5038; (6) Weiss,
R. A.et al.,
Nature 1985. 316:69; (7) Katz, D. H., AIDS Res. Hum. Retro. 1993. 9:489; (8)
Golding, H.
1 S et al., J. Exp. Med. 1988. 167:914; (9) Kion, T. A. et al., Science 1991.
253:1138. ( 10)
Desbarats et al. supra; ( 11 ) Banda et al., supra (J. Exp. Med. 1992.
176:1099; ( 12) Newell
et al., supra.[Nature 1990. 347:286; (13) Oyaizu et al., 1994, supra [Blood
1994. 84:2622;
(14/15) Wang et al., supra; (16) Finco, O. et al., Eur. J. Immunol. 1997.
27:1319; (17)
Paterson, R. K. et al., J. Immunol. 1994. 153:3491; ( 18) Posner, M. R. et
al., Acquired
Immune Defic. Syndr. 1993. 6:7; (19) Cavacini, L. A. et al., J. Acquired
Immune Defic.
Syndr. 1993. 6:353; (.'0) Posner, M. R. et al., Hybridoma 1987. 6:611; (21 )
Posner, M. R. et
al., J. Immunol. 1991. 146:4325; (22) Ugen, K. E. et al., In: Vacines 93, Cold
Spring Harbor,
NY: Cold Spring Harbor Laboratory Press. 1993. p. 215; (23) Haigwood, N. L. et
al., AIDS
Res. Hum. Retro. 1990. 6:855; (24) Steimer, K. S. et al., In: Vacines 88, Cold
Spring Harbor,
NY: Cold Spring Harbor Laboratory Press. 1988. p. 347; (25) Levy, J. A. et
al., Science
1984. 225:840; (26) ;ianchez-Pescador et al., supra; (27) Haigwood, N. L. et
al." In:
Vacines 90, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. 1990.
p. 313;
(28) Ezhevsky, S. A. et al., Mol. Biol. Cell 1996. 7:553; (29) Goronzy, J.et
al., J. Exp. Med.
1986. 164:911; (30) Jamali, Let al., J. Immunol. 1992. 148:1613; (31 ) Muro-
Cacho, C. A. et
al., J. Immunol. 1995.. 154:5555; (32) Chun, T.-W. et al., Nature 1997.
387:183; (33)
Harper, M. E. et al., P'roc. Natl. Acad. Sci. USA 1986. 83:772; (34) Lores, P.
et al., AIDS
Res. Hum. Retro. 1992. 8:2063; (35) Feng, Y. et al., Science 1996. 272:872;
(36) Scott, D.
W. et al., J. Immunol. 1996. 156:2352; (37) Lanzavecchia, A., J. Exp. Med.
1995. 181:1945;
(38) Saiemi, S. et al., J. Exp. Med. 1995. 181:2253; (39) Siliciano, R. F. et
al., Cell 1988.
54:561; (40) Zambid:is, E. T. et al., Mol. Med. 1997. 3:212; (41 ) Vermes, I.
et al., J.
Immulnol. Meth. 199:5. 184:39.
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EXAMPLE VII
Induction of Immune Tolerance to Foreign Immunogenic Epitopes via Retroviral
_Mediated E~cpression of Foreign Protein- the IgG Scaffold is Important for
Induction
and Maintenance of Humoral Immunological Tolerance
$ Results described above showed that recipients of bone marrow or of LPS-
stimulated B-cell blasts, both of which were retrovirally gene-transferred
with an
immunodominant peptide in-frame to the V region of a murine IgG H chain, were
rendered profoundly unresponsive to that epitope. To further investigate
whether
tolerance to larger molecules can be achieved via this approach and whether
the IgG
scaffold is important for induction and maintenance of immunoiogical
tolerance, we
engineered two retroviral constructs (MBAE-1-102 and MBAE-1-102-IgG). The
first
of these included the DNA encoding the pl-102 peptide of bacteriophage ~.. The
second had DNA encoding that peptide fused to the murine Ig y chain DNA such
that
the peptide was expressed at the N-terminus of the H chain. These vectors were
used
for gene transfer.
Specif city of pl-102 humoral tolerance in genetically tolerized bone marrow
recipients was examined. CB6 F, mice were sublethally irradiated with 400 rads
and
injected with mock-transduced or 1-102 -IgG gene-transduced bone marrow cells.
Mice were primed and boosted with pl-102 and HEL in CFA. Antibody responses
were measured in ELISA by coating plates with 50 p.g/ml synthetic peptides
(peptides
12-26, 73-88 or 55-69). The titers were determined by using CA-Cricket graph
software and expressed as the dilution which brings the OD4os to the pre-
immune
level. Each experiment had 3-4 mice per group. The efficacy in induction and
maintenance of tolerance by pl-102 and pl-102-IgG gene transfer in bone marrow
recipients was also examined. CB6 F, mice were sublethally irradiated as above
and
injected with mock-transduced or pl-102 transduced or pl-102 -IgG gene-
transduced
bone marrow cells. Mice were later primed and boosted with pl-102 and HEL in
CFA. Antibody responses were assayed and analyzed as above.
The results showed that recipients of bone marrow cells or peripheral B cells
that had been gene-transferred with MBAE-1-102-IgG were specifically
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hyporesponsive to the pl-102 protein (and this occurred in a strain-specific
manner).
That is, Balb/c and C57B1/6 mice recognize epitopes contained in residues 12-
26 and
73-88, respectively, whereas F, hybrid mice between these strains recognize
epitopes
at both sites. Gene-transfer produced tolerance in F, mice to the whole pl-102
protein, as well as to the major determinants. No "epitope spreading" to minor
epitopes was observed. The results suggest that the self IgG scaffold is
necessary for
long-lasting unresponsiveness because recipients of pl-102-IgG fusion protein
construct remained tolerant to secondary challenge whereas controls given
cells
transfected with pl-102 construct (not fused to the IgG) regained
responsiveness.
These results demonstrated that the host can then present the relevant
epitopes in a
MHC-hapiotype-specific manner to the immune system and induce profound
tolerance. This results is directly applicable to treatment of autoimmune
diseases, as
well as for creating a receptive environment for foreign or otherwise
immunologically
"unacceptable" proteins to be administered in the context of gene therapy.
1 S The references cited above are all incorporated by reference herein,
whether
specifically incorporated or not.
Having now fully described this invention, it will be appreciated by those
skilled in the art that the same can be performed within a wide range of
equivalent
parameters, concentrations, and conditions without departing from the spirit
and scope
of the invention andl without undue experimentation.
While this invention has been described in connection with specific
embodiments
thereof, it will be understood that it is capable of further modifications.
This
application is intended to cover any variations, uses, or adaptations of the
invention
following, in gener<rl, the principles of the invention and including such
departures
from the present disclosure as come within known or customary practice within
the art
to which the invention pertains and as may be applied to the essential
features
hereinbefore set forth as follows in the scope of the appended claims
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SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: American National Red Cross
(ii) TITLE OF THE INVENTION: IMMUNOLOGICAL TOLERANCE TO
HIV EPITOPES
(iii) NUMBER OF SEQUENCES: 210
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Fetherstonhaugh & Co.
(B) STREET: Box 11560, Vancouver Centre, 2200-650 W. Georgia
Street
(C) CITY: Vancouver
(D) STATE: British Columbia
(E) COUNTRY: Canada
(F) ZIP: V6B 4N8
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US98/02766
(B) FILING DATE: 13-FEB-1998
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/040,581
(B) FILING DATE: 13-FEB-1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Fetherstonhaugh & Co.
(C) REFERENCE/DOCKET NUMBER: 40478-132
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
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Asn Ala Asn Pro
1
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu Phe Cys Ala Ser Asp
1 5 10 15
Ala Lys Ala Tyr
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
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Glu Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp
1 5 10 15
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(A) LENGTH: 10 amino acids
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Tyr Asp Thr Glu Val His Asn Val Trp Ala
1 5 10
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Pro Gln Glu Val Val Leu Val Asn Val Thr
1 5 10
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(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Pro Gln Glu Val Val Leu Val Asn Val Thr Glu Asn Phe Asp Met Trp
1 5 10 15
Lys Asn Asp Met
(2) INFORMATION FOR SEQ ID N0:7:
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Pro Asn Asn Asn Thr Arg Lys Ser Ile Arg
1 5 10
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Asn Asn Asn Thr Arg Lys Arg Ile Arg Ile Gln Arg Gly Pro Gly Arg
1 5 10 15
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Arg Lys Ser Ile Arg
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Ile Gln Arg Gly Pro Gly Arg Ala Phe Val
1 5 10
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Gly Arg Ala Phe Val Thr Ile Gly Lys Ile
1 5 10
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Pro Gly Arg Ala Phe Tyr
1 5
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(A) LENGTH: 20 amino acids
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Asn Thr Arg Lys Ser Ile Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe
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Val Thr Ile Gly
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Pro Asn Asn Asn Thr Arg Lys Ser Ile Arg Ile Gln Arg Gly Pro Gly
1 5 10 15
Arg Ala Phe Val Thr Ile Gly Lys Ile Gly Asn Met Arg Gln Ala His
20 25 30
Cys
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Asn Asn Thr Arg Lys Ser Ile Arg Ile Gln Arg Gly
1 5 10
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Asn Lys Arg Lys Arg Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr
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Thr Lys Asn Ile Ile Gly Thr Ile Cys
20 25
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Arg Lys Ser Ile Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val
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Ile Arg Ile Gln Arg Gly Pro Gly Arg
1 5
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Lys Arg Ile Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile
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Gly
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Gln Arg Gly Pro Gly Arg Ala Phe
1 5
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(B) TYPE: amino acid
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Arg Gly Pro Gly Arg Ala Phe Val
1 5
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Arg Lys Arg Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr
1 5 10 15
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Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Gly
1 5 10
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Ser Ile Ser Gly Pro Gly Arg Ala Phe Tyr Thr Gly
1 5 10
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Lys Arg Ile His Ile
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
Lys Arg Ile His Ile Gly Pro
1 5
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
Ile His Ile Gly Pro Gly Arg
1 5
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
His Ile Gly Pro Gly Arg
1 5
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
His Ile Gly Pro Gly Arg Ala
1 5
(2) INFORMATION FOR SEQ ID N0:30:
CA 02279492 1999-09-02
1171.
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
His Ile Gly Pro
1
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
Arg Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Gly
1 5 10
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe
1 5 10
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
Ile Gln Arg Gly Pro Gly Arg Ala Phe
1 5
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
CA 02279492 1999-09-02
117j
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
Ile Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile
1 5 10
(2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Gly Lys Ile Gly
1 5 10
(2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
Gln Arg Gly Pro Gly Arg Ala
1 5
(2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:
Ile Xaa Xaa Gly Pro Gly Arg Ala
1 5
(2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
CA 02279492 1999-09-02
117k
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:
Ile Gly Pro Gly Arg
1 5
(2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
Gly Pro Gly Arg
1
(2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
Gly Pro Xaa Arg
1
(2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
Gly Pro Gly Arg Ala Phe
1 5
(2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
CA 02279492 1999-09-02
1171
Arg Ile His Ile Gly
1 5
(2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
His Ile Gly Pro Gly Arg Ala Phe
1 5
(2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
Gly Arg Ala Phe
1
(2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr
1 5 10 15
Lys Val Val Lys
(2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:
Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Lys
1 5 10 15
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117m
Ala Lys Arg Arg
(2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
Leu Gly Val Ala Pro Thr Lys Ala Lys Arg
1 5 10
(2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:
Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys
1 5 10 15
Val Val Lys Ile
(2) INFORMATION FOR SEQ ID N0:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
Ile Glu Pro Leu Gly Val Ala Pro Thr Lys
1 5 10
(2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:
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117n
Arg Arg Val Val Gln Arg Glu
1 5
(2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
Pro Thr Lys Ala Lys Arg Arg
1 5
(2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:
Val Val Gln Arg Glu Lys Arg
1 5
(2) INFORMATION FOR SEQ ID N0:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
Glu Gln Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val
1 5 10
(2) INFORMATION FOR SEQ ID N0:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:54:
Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala
1 5 10
CA 02279492 1999-09-02
1170
(2) INFORMATION FOR SEQ ID N0:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
Gly Val Pro Val Trp Lys Glu Ala Thr Thr Leu Phe Cys
1 5 10
(2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:56:
Ala His Lys Val Trp Ala Thr His Ala Cys Val
1 5 10
(2) INFORMATION FOR SEQ ID N0:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:57:
Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp
1 5 10
(2) INFORMATION FOR SEQ ID N0:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:58:
Cys Val Pro Thr Asn Pro Val Pro Gln Glu Val Val
1 5 10
(2) INFORMATION FOR SEQ ID N0:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
CA 02279492 1999-09-02
117p
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:59:
Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp
1 5 10
(2) INFORMATION FOR SEQ ID N0:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:60:
Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Gln
1 5 10
(2) INFORMATION FOR SEQ ID N0:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:61:
Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:62:
His Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys
1 5 10
(2) INFORMATION FOR SEQ ID N0:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
CA 02279492 1999-09-02
117q
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:63:
Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr Thr Thr
1 5 10 15
Leu Phe Cys
(2) INFORMATION FOR SEQ ID N0:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:64:
Val Val Leu Val Asn Val Thr Glu Asn Phe Asn Met
1 5 10
(2) INFORMATION FOR SEQ ID N0:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:65:
Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Tyr
1 5 10
(2) INFORMATION FOR SEQ ID N0:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:66:
Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Arg Ile Gln Arg
1 5 10 15
Gly Pro Gly Tyr
(2) INFORMATION FOR SEQ ID N0:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
CA 02279492 1999-09-02
117r
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:67:
Asn Thr Arg Lys Ser Ile Arg Ile Gln Arg Gly Pro Gly Arg
1 5 10
(2) INFORMATION FOR SEQ ID N0:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:68:
Glu Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Gly Lys Ile
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:69:
Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Gly Lys
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:70:
Arg Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Lys Asn
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:71:
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1175
Gly Arg Ala Phe Val Thr Ile Gly Lys Ile Gly Asn Met Arg Gln
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:72:
Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Gly Lys Ile Gly Asn
1 5 10 15
Met Arg Gln Ala His
(2) INFORMATION FOR SEQ ID N0:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:73:
Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln Ile Arg
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:74:
Gly Asn Ser Asn Asn Glu Ser Glu Ile Phe Arg Pro Gly Gly Gly
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:75:
CA 02279492 1999-09-02
117t
Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:76:
Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:77:
Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:78:
Cys Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:79:
Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro
1 5 10 15
CA 02279492 1999-09-02
117u
(2) INFORMATION FOR SEQ ID N0:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:80:
Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys
1 5 10 15
Arg Arg Val Val Gln Arg Glu Lys Arg Cys
20 25
(2) INFORMATION FOR SEQ ID N0:81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:81:
Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Glu
1 5 10 15
Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln Ile Arg Cys
20 25 30
(2) INFORMATION FOR SEQ ID N0:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:82:
Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala
1 5 10 15
Pro Thr Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg
20 25 30
(2) INFORMATION FOR SEQ ID N0:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 481 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:83:
CA 02279492 1999-09-02
117v
Xaa Glu Xaa Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys
1 5 10 15
Glu Ala Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp
20 25 30
Thr Glu Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp
35 40 45
Pro Asn Pro Gln Glu Val Val Leu Xaa Asn Val Thr Glu Asn Phe Asn
50 55 60
Met Trp Lys Asn Asn Met Val Glu Gln Met His Glu Asp Ile Ile Ser
65 70 75 80
Leu Trp Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys
85 90 95
Val Thr Leu Asn Cys Thr Asp Xaa Xaa Xaa Xaa Xaa Asn Thr Asn Xaa
100 105 110
Xaa Xaa Xaa Xaa Xaa Xaa Met Glu Xaa Gly Glu Ile Lys Asn Cys Ser
115 120 125
Phe Asn Ile Thr Thr Ser Ile Arg Asp Lys Val Gln Lys Glu Tyr Ala
130 135 140
Leu Phe Tyr Lys Leu Asp Val Val Pro Ile Asp Xaa Xaa Xaa Xaa Xaa
145 150 155 160
Tyr Arg Leu Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro
165 170 175
Lys Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly
180 185 190
Phe Ala Ile Leu Lys Cys Asn Asp Lys Lys Phe Asn Gly Thr Gly Pro
195 200 205
Cys Thr Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val
210 215 220
Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val
225 230 235 240
Val Ile Arg Ser Glu Asn Phe Thr Asp Asn Ala Lys Thr Ile Ile Val
245 250 255
Gln Leu Asn Glu Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn
260 265 270
Thr Arg Lys Ser Ile His Ile Xaa Xaa Gly Pro Gly Arg Ala Phe Tyr
275 280 285
Thr Thr Gly Xaa Ile Gly Asp Ile Arg Gln Ala His Cys Asn Ile Ser
290 295 300
Arg Ala Lys Trp Asn Asn Thr Leu Lys Gln Ile Val Xaa Lys Leu Arg
305 310 315 320
Glu Gln Phe Xaa Xaa Asn Lys Thr Ile Val Phe Asn Gln Ser Ser Gly
325 330 335
Gly Asp Pro Glu Ile Val Met His Ser Phe Asn Cys Gly Gly Glu Phe
340 345 350
Phe Tyr Cys Asn Thr Thr Gln Leu Phe Asn Ser Thr Trp Xaa Asn Xaa
355 360 365
Thr Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Thr Xaa Xaa Xaa Xaa Xaa Ile
370 375 380
Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val
385 390 395 400
Gly Lys Ala Met Tyr Ala Pro Pro Ile Xaa Gly Gln Ile Arg Cys Ser
405 410 415
Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Asn Xaa Xaa
420 425 430
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117w
Xaa Xaa Xaa Glu Ile Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn
435 440 445
Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu
450 455 460
Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys
465 470 475 480
Arg
(2) INFORMATION FOR SEQ ID N0:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 615 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:84:
Met Arg Val Met Gly Ile Gln Xaa Asn Tyr Gln Xaa Leu Trp Arg Xaa
1 5 10 15
Xaa Xaa Xaa Trp Gly Thr Met Ile Leu Gly Xaa Xaa Ile Ile Cys Asn
20 25 30
Ala Xaa Xaa Glu Xaa Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val
35 40 45
Trp Lys Asp Ala Glu Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala
50 55 60
Tyr Asp Thr Glu Xaa His Asn Val Trp Xaa Ala Thr His Ala Cys Val
65 70 75 80
Pro Thr Asp Pro Asn Pro Gln Glu Ile Xaa Leu Glu Asn Val Thr Glu
85 90 95
Xaa Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln Met His Glu Asp
100 105 110
Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr
115 120 125
Pro Leu Cys Val Thr Leu Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
130 135 140
Xaa Xaa Xaa Asn Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
145 150 155 160
Asn Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa
165 170 175
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Met Xaa Xaa Glu Ile Lys Asn Cys
180 185 190
Ser Phe Asn Met Thr Thr Glu Leu Arg Asp Lys Lys Gln Lys Val Tyr
195 200 205
Ser Leu Phe Tyr Arg Leu Asp Val Val Gln Ile Xaa Xaa Xaa Xaa Xaa
210 215 220
Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa
225 230 235 240
Xaa Xaa Xaa Xaa Tyr Arg Leu Ile Asn Cys Asn Thr Ser Ala Ile Thr
245 250 255
Gln Ala Cys Pro Lys Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys
260 265 270
Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Xaa Asp Lys Xaa Phe Asn
275 280 285
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117x
Gly Thr Gly Pro Cys Lys Asn Val Ser Thr Val Gln Cys Thr His Gly
290 295 300
Xaa Ile Lys Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu
305 310 315 320
Ala Glu Xaa Xaa Xaa Val Xaa Ile Arg Ser Glu Asn Ile Thr Asn Asn
325 330 335
Ala Lys Thr Ile Ile Val Gln Leu Xaa Xaa Pro Val Xaa Ile Asn Cys
340 345 350
Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Xaa Xaa Xaa Val Arg Ile
355 360 365
Xaa Xaa Xaa Gly Pro Gly Gln Xaa Xaa Ala Phe Tyr Ala Thr Gly Asp
370 375 380
Ile Ile Gly Asp Ile Arg Gln Ala His Cys Asn Val Ser Arg Xaa Glu
385 390 395 400
Trp Asn Xaa Thr Leu Gln Xaa Val Ala Xaa Gln Leu Arg Xaa Xaa Phe
405 410 415
Xaa Xaa Xaa Asn Lys Thr Xaa Xaa Ile Ile Phe Xaa Asn Ser Ser Gly
420 425 430
Gly Asp Leu Glu Ile Thr Thr His Ser Phe Asn Cys Gly Gly Glu Phe
435 440 445
Xaa Phe Tyr Cys Asn Thr Ser Xaa Leu Phe Asn Ser Thr Trp Xaa Xaa
450 455 460
Xaa Xaa Xaa Xaa Xaa Asn Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
465 470 475 480
Asn Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Asn Asp Thr Ile Thr
485 490 495
Leu Gln Cys Arg Ile Lys Gln Ile Val Asn Met Trp Gln Arg Val Gly
500 505 510
Gln Ala Met Tyr Ala Pro Pro Ile Gln Gly Xaa Ile Arg Cys Xaa Ser
515 520 525
Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Xaa Xaa Asn Asn
530 535 540
Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Glu Thr Phe Arg Pro Gly Gly
545 550 555 560
Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val
565 570 575
Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Arg Ala Lys Arg Arg
580 585 590
Val Val Glu Arg Glu Lys Arg Ala Xaa Xaa Val Gly Leu Gly Ala Val
595 600 605
Phe Leu Gly Phe Leu Gly Ala
610 615
(2) INFORMATION FOR SEQ ID N0:85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:85:
Ile Leu Gly Phe Trp Met Leu Met
1 5
CA 02279492 1999-09-02
117y
(2) INFORMATION FOR SEQ ID N0:86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:86:
Thr Met Lys Ala Met Xaa Lys Arg Asn Arg Lys Leu
1 5 10
(2) INFORMATION FOR SEQ ID N0:87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:87:
Xaa Leu Tyr Leu Ala Met Ala Leu Ile
1 5
(2) INFORMATION FOR SEQ ID N0:88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:88:
Asn Leu Thr Ser
1
(2) INFORMATION FOR SEQ ID N0:89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:89:
Asn Val Ser Asn Ile Ile Gly
1 5
(2) INFORMATION FOR SEQ ID N0:90:
CA 02279492 1999-09-02
1172
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:90:
Xaa Thr Leu Lys Glu
1 5
(2) INFORMATION FOR SEQ ID N0:91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:91:
Asn Asn Ser Thr Val
1 5
(2) INFORMATION FOR SEQ ID N0:92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:92:
Leu Xaa Lys Xaa Asn
1 5
(2) INFORMATION FOR SEQ ID N0:93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:93:
Glu Glu Ile Ile
1
(2) INFORMATION FOR SEQ ID N0:94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
CA 02279492 1999-09-02
117aa
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:94:
Glu Asp Ile Ile
1
(2) INFORMATION FOR SEQ ID N0:95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:95:
Asn Lys Ser Ile Glu
1 5
(2) INFORMATION FOR SEQ ID N0:96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:96:
Ser Lys Gly Lys Ile Arg
1 5
(2) INFORMATION FOR SEQ ID N0:97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:97:
Xaa Asp Ser Gly
1
(2) INFORMATION FOR SEQ ID N0:98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
CA 02279492 1999-09-02
117bb
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:98:
Glu Ile Asn Gly Thr Lys
1 5
(2) INFORMATION FOR SEQ ID N0:99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:99:
Gln Pro Xaa Pro
1
(2) INFORMATION FOR SEQ ID N0:100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:
Leu Xaa Xaa Xaa Asn Xaa Lys Xaa Xaa Xaa Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO:101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:101:
Xaa Tyr Asn Ala Thr Asp
1 5
(2) INFORMATION FOR SEQ ID N0:102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:102:
CA 02279492 1999-09-02
117cC
Glu Arg Tyr Leu Glu
1 5
(2) INFORMATION FOR SEQ ID N0:103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:103:
Xaa Xaa Xaa Xaa Val Thr Met Xaa
1 5
(2) INFORMATION FOR SEQ ID N0:104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:104:
Xaa Xaa Asn Xaa Thr Xaa
1 5
(2) INFORMATION FOR SEQ ID N0:105:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 512 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:105:
Met Arg Val Lys Gly Lys Tyr Gln His Leu Xaa Trp Arg Trp Xaa Xaa
1 5 10 15
Xaa Xaa Gly Thr Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Xaa
20 25 30
Glu Xaa Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu
35 40 45
Ala Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr
50 55 60
Glu Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro
65 70 75 80
Asn Pro Gln Glu Val Val Leu Asn Val Thr Glu Asn Phe Asn Met Trp
85 90 95
Lys Asn Asn Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp
100 105 110
Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr
115 120 125
CA 02279492 1999-09-02
117dd
Leu Asn Cys Thr Asp Asn Xaa Thr Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa
130 135 140
Xaa Xaa Xaa Xaa Xaa Met Glu Gly Glu Ile Lys Asn Cys Ser Phe Asn
145 150 155 160
Ile Thr Thr Ser Ile Arg Asp Lys Val Gln Lys Glu Tyr Ala Leu Phe
165 170 175
Tyr Lys Leu Asp Val Val Pro Ile Asp Asn Xaa Xaa Xaa Tyr Arg Leu
180 185 190
Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Val Ser
195 200 205
Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile
210 215 220
Leu Lys Cys Asn Asp Lys Lys Phe Asn Gly Thr Gly Pro Cys Thr Asn
225 230 235 240
Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr
245 250 255
Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Val Ile Arg
260 265 270
Ser Glu Asn Phe Thr Asp Asn Ala Lys Thr Ile Ile Val Gln Leu Asn
275 280 285
Glu Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys
290 295 300
Ser Ile His Ile Xaa Xaa Gly Pro Gly Arg Ala Phe Tyr Thr Thr Gly
305 310 315 320
Xaa Ile Xaa Xaa Ile Arg Gln Ala His Cys Asn Ile Ser Arg Ala Lys
325 330 335
Trp Asn Asn Thr Leu Lys Gln Ile Val Xaa Lys Leu Arg Glu Gln Phe
340 345 350
Xaa Xaa Asn Lys Thr Xaa Ile Phe Asn Gln Ser Ser Gly Gly Asp Pro
355 360 365
Glu Ile Val Met His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys
370 375 380
Asn Thr Thr Gln Leu Phe Asn Ser Thr Trp Xaa Asn Xaa Thr Xaa Xaa
385 390 395 400
Xaa Xaa Xaa Xaa Asn Xaa Thr Xaa Xaa Xaa Xaa Ile Thr Leu Pro Cys
405 410 415
Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met
420 425 430
Tyr Ala Pro Pro Ile Xaa Gly Gln Ile Arg Cys Ser Ser Asn Ile Thr
435 440 445
Gly Leu Leu Leu Thr Arg Asp Gly Gly Xaa Xaa Xaa Asn Xaa Xaa Xaa
450 455 460
Xaa Thr Glu Ile Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp
465 470 475 480
Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly
485 490 495
Val Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg
500 505 510
(2) INFORMATION FOR SEQ ID N0:106:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 511 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
CA 02279492 1999-09-02
117ee
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:106:
Met Arg Val Lys Glu Lys Tyr Gln His Leu Trp Arg Trp Gly Trp Arg
1 5 10 15
Trp Gly Thr Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Thr Glu
20 25 30
Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala
35 40 45
Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu
50 55 60
Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn
65 70 75 80
Pro Gln Glu Val Val Leu Val Asn Val Thr Glu Asn Phe Asn Met Trp
85 90 95
Lys Asn Asp Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp
100 105 110
Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Ser
115 120 125
Leu Lys Cys Thr Asp Leu Lys Asn Asp Thr Asn Thr Asn Ser Ser Ser
130 135 140
Gly Arg Met Ile Met Glu Lys Gly Glu Ile Lys Asn Cys Ser Phe Asn
145 150 155 160
Ile Ser Thr Ser Ile Arg Gly Lys Val Gln Lys Glu Tyr Ala Phe Phe
165 170 175
Tyr Lys Leu Asp Ile Ile Pro Ile Asp Asn Asp Thr Thr Ser Tyr Thr
180 185 190
Leu Thr Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Val
195 200 205
Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala
210 215 220
Ile Leu Lys Cys Asn Asn Lys Thr Phe Asn Gly Thr Gly Pro Cys Thr
225 230 235 240
Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser
245 250 255
Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Val Ile
260 265 270
Arg Ser Ala Asn Phe Thr Asp Asn Ala Lys Thr Ile Ile Val Gln Leu
275 280 285
Asn Gln Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg
290 295 300
Lys Ser Ile Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile
305 310 315 320
Gly Lys Ile Gly Asn Met Arg Gln Ala His Cys Asn Ile Ser Arg Ala
325 330 335
Lys Trp Asn Asn Thr Leu Lys Gln Ile Asp Ser Lys Leu Arg Glu Gln
340 345 350
Phe Gly Asn Asn Lys Thr Ile Ile Phe Lys Gln Ser Ser Gly Gly Asp
355 360 365
Pro Glu Ile Val Thr His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr
370 375 380
Cys Asn Ser Thr Gln Leu Phe Asn Ser Thr Trp Phe Asn Ser Thr Trp
385 390 395 400
CA 02279492 1999-09-02
117ff
Ser Thr Lys Gly Ser Asn Asn Thr Glu Gly Ser Asp Thr Ile Thr Leu
405 410 415
Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys
420 425 430
Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln Ile Arg Cys Ser Ser Asn
435 440 445
Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Asn Ser Asn Asn Glu
450 455 460
Ser Glu Ile Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg
465 470 475 480
Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val
485 490 495
Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg
500 505 510
(2) INFORMATION FOR SEQ ID N0:107:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 516 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:107:
Met Arg Val Lys Glu Lys Tyr Gln His Leu Trp Arg Trp Gly Trp Lys
1 5 10 15
Trp Gly Thr Met Leu Leu Gly Ile Leu Met Ile Cys Ser Ala Thr Glu
20 25 30
Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala
35 40 45
Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu
50 55 60
Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn
65 70 75 80
Pro Gln Glu Val Val Leu Val Asn Val Thr Glu Asn Phe Asn Met Trp
85 90 95
Lys Asn Asp Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp
100 105 110
Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Ser
115 120 125
Leu Lys Cys Thr Asp Leu Gly Asn Ala Thr Asn Thr Asn Ser Ser Asn
130 135 140
Thr Asn Ser Ser Ser Gly Glu Met Met Met Glu Lys Gly Glu Ile Lys
145 150 155 160
Asn Cys Ser Phe Asn Ile.Ser Thr Ser Ile Arg Gly Lys Val Gln Lys
165 170 175
Glu Tyr Ala Phe Phe Tyr Lys Leu Asp Ile Ile Pro Ile Asp Asn Asp
180 185 190
Thr Thr Ser Tyr Thr Leu Thr Ser Cys Asn Thr Ser Val Ile Thr Gln
195 200 205
Ala Cys Pro Lys Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala
210 215 220
Pro Ala Gly Phe Ala Ile Leu Lys Cys Asn Asn Lys Thr Phe Asn Gly
225 230 235 240
CA 02279492 1999-09-02
117gg
Thr Gly Pro Cys Thr Asn Val Ser Thr Val Gln Cys Thr His Gly Ile
245 250 255
Arg Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu
260 265 270
Glu Glu Val Val Ile Arg Ser Ala Asn Phe Thr Asp Asn Ala Lys Thr
275 280 285
Ile Ile Val Gln Leu Asn Gln Ser Val Glu Ile Asn Cys Thr Arg Pro
290 295 300
Asn Asn Asn Thr Arg Lys Ser Ile Arg Ile Gln Arg Gly Pro Gly Arg
305 310 315 320
Ala Phe Val Thr Ile Gly Lys Ile Gly Asn Met Arg Gln Ala His Cys
325 330 335
Asn Ile Ser Arg Ala Lys Trp Asn Ala Thr Leu Lys Gln Ile Ala Ser
340 345 350
Lys Leu Arg Glu Gln Phe Gly Asn Asn Lys Thr Ile Ile Phe Lys Gln
355 360 365
Ser Ser Gly Gly Asp Pro Glu Ile Val Thr His Ser Phe Asn Cys Gly
370 375 380
Gly Glu Phe Phe Tyr Cys Asn Ser Thr Gln Leu Phe Asn Ser Thr Trp
385 390 395 400
Phe Asn Ser Thr Trp Ser Thr Glu Gly Ser Asn Asn Thr Glu Gly Ser
405 410 415
Asp Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Phe Ile Asn Met Trp
420 425 430
Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln Ile
435 440 445
Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly
450 455 460
Asn Asn Asn Asn Gly Ser Glu Ile Phe Arg Pro Gly Gly Gly Asp Met
465 470 475 480
Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile
485 490 495
Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln
500 505 510
Arg Glu Lys Arg
515
(2) INFORMATION FOR SEQ ID N0:108:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 509 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:108:
Met Lys Val Lys Gly Thr Arg Arg Asn Tyr Gln His Leu Trp Arg Trp
1 5 10 15
Gly Thr Leu Leu Leu Gly Met Leu Met Ile Cys Ser Ala Thr Glu Lys
20 25 30
Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr
35 40 45
Thr Thr Leu Phe Cys Ala Ser Asp Ala Arg Ala Tyr Asp Thr Glu Val
50 55 60
CA 02279492 1999-09-02
117hh
His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro
65 70 75 80
Gln Glu Val Val Leu Gly Asn Val Thr Glu Asn Phe Asn Met Trp Lys
85 90 95
Asn Asn Met Val Glu Gln Met Gln Glu Asp Ile Ile Ser Leu Trp Asp
100 105 110
Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu
115 120 125
Asn Cys Thr Asp Leu Gly Lys Ala Thr Asn Thr Asn Ser Ser Asn Trp
130 135 140
Lys Glu Glu Ile Lys Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr
145 150 155 160
Thr Ser Ile Arg Asp Lys Ile Gln Lys Glu Asn Ala Leu Phe Arg Asn
165 170 175
Leu Asp Val Val Pro Ile Asp Asn Ala Ser Thr Thr Thr Asn Tyr Thr
180 185 190
Asn Tyr Arg Leu Ile His Cys Asn Arg Ser Val Ile Thr Gln Ala Cys
195 200 205
Pro Lys Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Thr Pro Ala
210 215 220
Gly Phe Ala Ile Leu Lys Cys Asn Asn Lys Thr Phe Asn Gly Lys Gly
225 230 235 240
Pro Cys Thr Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro
245 250 255
Ile Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu
260 265 270
Val Val Ile Arg Ser Asp Asn Phe Thr Asn Asn Ala Lys Thr Ile Ile
275 280 285
Val Gln Leu Asn Glu Ser Val Ala Ile Asn Cys Thr Arg Pro Asn Asn
290 295 300
Asn Thr Arg Lys Ser Ile Tyr Ile Gly Pro Gly Arg Ala Phe His Thr
305 310 315 320
Thr Gly Arg Ile Ile Gly Asp Ile Arg Lys Ala His Cys Asn Ile Ser
325 330 335
Arg Ala Gln Trp Asn Asn Thr Leu Glu Gln Ile Val Lys Lys Leu Arg
340 345 350
Glu Gln Phe Gly Asn Asn Lys Thr Ile Val Phe Asn Gln Ser Ser Gly
355 360 365
Gly Asp Pro Glu Ile Val Met His Ser Phe Asn Cys Arg Gly Glu Phe
370 375 380
Phe Tyr Cys Asn Thr Thr Gln Leu Phe Asn Asn Thr Trp Arg Leu Asn
385 390 395 400
His Thr Glu Gly Thr Lys Gly Asn Asp Thr Ile Ile Leu Pro Cys Arg
405 410 415
Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met Tyr
420 425 430
Ala Pro Pro Ile Gly Gly Gln Ile Ser Cys Ser Ser Asn Ile Thr Gly
435 440 445
Leu Leu Leu Thr Arg Asp Gly Gly Thr Asn Val Thr Asn Asp Thr Glu
450 455 460
Val Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu
465 470 475 480
Leu Tyr Lys Tyr Lys Val Ile Lys Ile Glu Pro Leu Gly Ile Ala Pro
485 490 495
CA 02279492 1999-09-02
117ii
Thr Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg
500 505
(2) INFORMATION FOR SEQ ID N0:109:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 512 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:109:
Met Arg Val Lys Gly Ile Arg Arg Asn Tyr Gln His Trp Trp Gly Trp
1 5 10 15
Gly Thr Met Leu Leu Gly Leu Leu Met Ile Cys Ser Ala Thr Glu Lys
20 25 30
Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr
35 40 45
Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val
50 55 60
His Asn Val Trp Ala Thr Gln Ala Cys Val Pro Thr Asp Pro Asn Pro
65 70 75 80
Gln Glu Val Glu Leu Val Asn Val Thr Glu Asn Phe Asn Met Trp Lys
85 90 95
Asn Asn Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp
100 105 110
Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu
115 120 125
Asn Cys Thr Asp Leu Arg Asn Thr Thr Asn Thr Asn Asn Ser Thr Ala
130 135 140
Asn Asn Asn Ser Asn Ser Glu Gly Thr Ile Lys Gly Gly Glu Met Lys
145 150 155 160
Asn Cys Ser Phe Asn Ile Thr Thr Ser Ile Arg Asp Lys Met Gln Lys
165 170 175
Glu Tyr Ala Leu Leu Tyr Lys Leu Asp Ile Val Ser Ile Asp Asn Asp
180 185 190
Ser Thr Ser Tyr Arg Leu Ile Ser Cys Asn Thr Ser Val Ile Thr Gln
195 200 205
Ala Cys Pro Lys Ile Ser Glu Pro Ile Pro Ile His Tyr Cys Ala Pro
210 215 220
Ala Gly Phe Ala Ile Leu Lys Cys Asn Asp Lys Lys Phe Ser Gly Lys
225 230 235 240
Gly Ser Cys Lys Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg
245 250 255
Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu
260 265 270
Glu Val Val Ile Arg Ser Glu Asn Phe Thr Asp Asn Ala Lys Thr Ile
275 280 285
Ile Val His Leu Asn Glu Ser Val Gln Ile Asn Cys Thr Arg Pro Asn
290 295 300
Tyr Asn Lys Arg Lys Arg Ile His Ile Gly Pro Gly Arg Ala Phe Tyr
305 310 315 320
Thr Thr Lys Asn Ile Ile Gly Thr Ile Arg Gln Ala His Cys Asn Ile
325 330 335
CA 02279492 1999-09-02
117j j
Ser Arg Ala Lys Trp Asn Asp Thr Leu Arg Gln Ile Val Ser Lys Leu
340 345 350
Lys Glu Gln Phe Lys Asn Lys Thr Ile Val Phe Asn Gln Ser Ser Gly
355 360 365
Gly Asp Pro Glu Ile Val Met His Ser Phe Asn Cys Gly Gly Glu Phe
370 375 380
Phe Tyr Cys Asn Thr Ser Pro Leu Phe Asn Ser Thr Trp Asn Gly Asn
385 390 395 400
Asn Thr Trp Asn Asn Thr Thr Gly Ser Asn Asn Asn Ile Thr Leu Gln
405 410 415
Cys Lys Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala
420 425 430
Met Tyr Ala Pro Pro Ile Glu Gly Gln Ile Arg Cys Ser Ser Asn Ile
435 440 445
Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Lys Asp Thr Asp Thr Asn
450 455 460
Asp Thr Glu Ile Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp
465 470 475 480
Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Thr Ile Glu Pro Leu Gly
485 490 495
Val Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg
500 505 510
(2) INFORMATION FOR SEQ ID NO:110:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 505 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:
Val Thr Glu Ile Arg Lys Asn Cys Gln His Trp Trp Arg Trp Gly Ile
1 5 10 15
Met Leu Leu Gly Met Leu Met Thr Cys Asn Asn Ala Glu Glu Ser Trp
20 25 30
Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr Thr Thr
35 40 45
Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val His Asn
50 55 60
Ile Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Glu
65 70 75 80
Val Val Leu Glu Asn Val Thr Glu Asn Phe Asn Met Trp Lys Asn Asn
85 90 95
Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser
100 105 110
Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cys
115 120 125
Thr Asp Leu Ser Asn Ala Thr Asn Thr Asn Ala Thr Thr Thr Thr Asn
130 135 140
Ser Ser Ala Gly Met Met Met Asp Arg Gly Glu Ile Lys Asn Cys Ser
145 150 155 160
Phe Asn Val Thr Ala Ser Ile Arg Asp Lys Met Gln Arg Glu Tyr Ala
165 170 175
CA 02279492 1999-09-02
117kk
Leu Phe Tyr Lys Leu Asp Val Ile Gln I1e Asp Asn Thr Ser Tyr Arg
180 185 190
Leu Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Val
195 200 205
Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala
210 215 220
Ile Leu Lys Cys Asn Asp Lys Lys Phe Asn Gly Thr Gly Pro Cys Lys
225 230 235 240
Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser
245 250 255
Ser Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Gly Val Val Ile
260 265 270
Arg Ser Ala Asn Leu Ser Asp Asn Ala Lys Ile Ile Ile Val Gln Leu
275 280 285
Asn Glu Ser Val Glu Met Asn Cys Ile Arg Pro Asn Asn Asn Thr Arg
290 295 300
Lys Ser Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Gly Asp
305 310 315 320
Ile Ile Gly Asp Ile Arg Lys Ala His Cys Asn Ile Ser Arg Ala Lys
325 330 335
Trp Asn Asn Thr Leu Lys Gln Ile Ala Ile Lys Leu Lys Glu Gln Phe
340 345 350
Glu Asn Lys Thr Ile Val Phe Asn Gln Ser Ser Gly Gly Asp Pro Glu
355 360 365
Ile Met Thr Leu Met Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn
370 375 380
Ser Thr Gln Leu Phe Asn Ser Thr Trp Asn Ser Thr Gln Leu Val Asn
385 390 395 400
Asp Thr Gly Gly Asn Ile Thr Leu Gln Cys Arg Ile Lys Gln Ile Ile
405 410 415
Asn Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Arg
420 425 430
Gly Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg
435 440 445
Asp Gly Gly Ile Asn Lys Ser Glu Asn Gly Thr Glu Ile Phe Arg Pro
450 455 460
Gly Gly Gly Asp Met Arg Glu Asn Trp Arg Ser Glu Leu Tyr Lys Tyr
465 470 475 480
Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys
485 490 495
Arg Arg Val Val Gln Arg Glu Lys Arg
500 505
(2) INFORMATION FOR SEQ ID NO:111:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:
CTGGAGGACG CGCGGCGGCT GAAGGCGATA TACGAGAAGA AGAAGCAGGT CCAACTGCAG 60
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11711
(2) INFORMATION FOR SEQ ID N0:112:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:112:
Leu Glu Asp Ala Arg Arg Leu Lys Ala Ile Tyr Glu Lys Lys Lys
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:113:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:113:
Ala Ala Ala Phe Asn Met Trp Lys Asn Asp Gly Gly Gly Cys
1 5 10
(2) INFORMATION FOR SEQ ID N0:114:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:114:
Ala Thr Glu Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp
1 5 . 10 15
Lys Glu Ala Thr Thr Thr
(2) INFORMATION FOR SEQ ID N0:115:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:115:
Thr Glu Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys
1 5 10 15
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117mm
Glu Ala Thr Thr
(2) INFORMATION FOR SEQ ID N0:116:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:116:
Gly Val Pro Val Trp Lys Glu Ala Thr Thr
1 5 10
(2) INFORMATION FOR SEQ ID N0:117:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:117:
Pro Asn Pro Gln Glu Val Val Leu Val Asn Val Thr Glu Asn Phe
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:118:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:118:
Glu Asn Phe Asp Met Trp Lys Asn Asp Met
1 5 10
(2) INFORMATION FOR SEQ ID N0:119:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:119:
Phe Asn Met Trp
1
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117nn
(2) INFORMATION FOR SEQ ID N0:120:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:120:
Phe Asn Met Trp Lys Asn
1 5
(2) INFORMATION FOR SEQ ID N0:121:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:121:
Val Glu Gln Met His Glu Asp Ile Ile Ser
1 5 10
(2) INFORMATION FOR SEQ ID N0:122:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:122:
Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu
1 5 10 15
Lys Pro Cys Val
(2) INFORMATION FOR SEQ ID N0:123:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:123:
Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys
1 5 10 15
Pro Cys Val Lys
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11700
(2) INFORMATION FOR SEQ ID N0:124:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:124:
Leu Trp Asp Gln Ser Leu Lys Pro Cys Val
1 5 10
(2) INFORMATION FOR SEQ ID N0:125:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:125:
Met His Glu Asp Ile Ile Ser Leu Trp Asp
1 5 10
(2) INFORMATION FOR SEQ ID N0:126:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:126:
Leu Thr Pro Leu Cys Val Ser Leu Lys Cys Thr Asp Leu Lys Asn Asp
1 5 10 15
Thr Asn Thr Asn
(2) INFORMATION FOR SEQ ID N0:127:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:127:
Ser Thr Ser Ile Arg Gly Lys Val
1 5
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117pp
(2) INFORMATION FOR SEQ ID N0:128:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:128:
Ser Thr Ser Ile Arg Gly Lys Val Gln
1 5
(2) INFORMATION FOR SEQ ID N0:129:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:129:
Gln Lys Glu Tyr Ala Phe Phe Tyr Lys Leu Asp
1 5 10
(2) INFORMATION FOR SEQ ID N0:130:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:130:
Glu Tyr Ala Phe Phe Tyr Lys Leu Asp Ile Ile Pro Ile Asp Asn Asp
1 5 10 15
Thr Thr Ser Tyr
(2) INFORMATION FOR SEQ ID N0:131:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:131:
Ser Thr Ser Ile Arg Gly Lys Val Gln Lys Glu Tyr Ala Phe Phe Tyr
1 5 10 15
Lys Leu Asp Ile
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117qq
(2) INFORMATION FOR SEQ ID N0:132:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:132:
Glu Tyr Ala Phe Phe Tyr Lys Leu Asp Ile
1 5 10
(2) INFORMATION FOR SEQ ID N0:133:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:133:
Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala
1 5 10
(2) INFORMATION FOR SEQ ID N0:134:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:134:
Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu
1 5 10 15
Lys Cys Asn Asn
(2) INFORMATION FOR SEQ ID N0:135:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:135:
Gly Phe Ala Ile Leu Lys Cys Asn Asn Lys
1 5 10
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117rr
(2) INFORMATION FOR SEQ ID N0:136:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:136:
Arg Pro Val Val Ser Thr Gln Leu Leu Leu
1 5 10
(2) INFORMATION FOR SEQ ID N0:137:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:137:
Arg Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu
1 5 10 15
Glu Glu Val Val
(2) INFORMATION FOR SEQ ID N0:138:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:138:
Thr Gln Leu Leu Leu Asn
1 5
(2) INFORMATION FOR SEQ ID N0:139:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:139:
Thr Gln Leu Leu Leu Asn Gly
1 5
(2) INFORMATION FOR SEQ ID N0:140:
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117ss
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:140:
Asn Gly Ser Leu Ala Glu Glu Glu Val Val Ile Arg Ser Val Asn Phe
1 5 10 15
Thr Asp Asn Ala
(2) INFORMATION FOR SEQ ID N0:141:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:141:
Val Ile Arg Ser Val Asn Phe Thr Asp Asn
1 5 10
(2) INFORMATION FOR SEQ ID N0:142:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:142:
Ile Asn Cys Thr Arg Pro
1 5
(2) INFORMATION FOR SEQ ID N0:143:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:143:
Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser
1 5 10 15
Ile
(2) INFORMATION FOR SEQ ID N0:144:
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117tt
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:144:
Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Ala His Cys Asn Ile Ser
1 5 10 15
Arg Ala Lys Trp
(2) INFORMATION FOR SEQ ID N0:145:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:145:
Gly Arg Ala Phe Val Thr Ile Gly Lys Ile Leu Gly Val Ala Pro Thr
1 5 10 15
Lys Ala Lys Arg
(2) INFORMATION FOR SEQ ID N0:146:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:146:
Ile Gly Phe Tyr Thr
1 5
(2) INFORMATION FOR SEQ ID N0:147:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:147:
Ile Xaa Ile Gly Pro Gly Arg
1 5
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117uu
(2) INFORMATION FOR SEQ ID N0:148:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:148:
Arg Ala Phe
1
(2) INFORMATION FOR SEQ ID N0:149:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:149:
Ile Phe Lys Gln Ser Ser Gly Gly Asp Pro Glu Ile Val Thr His Ser
1 5 10 15
Phe Asn Cys Gly Gly
(2) INFORMATION FOR SEQ ID N0:150:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:150:
Phe Lys Gln Ser Ser Gly Gly Asp Pro Glu Ile Val Thr His Ser Phe
1 5 10 15
Asn Cys Gly Gly Glu
(2) INFORMATION FOR SEQ ID N0:151:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:151:
Gly Glu Phe Phe Tyr Cys Asn Ser Thr Gln Leu Phe Asn Ser
1 5 10
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117W
(2) INFORMATION FOR SEQ ID N0:152:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:152:
His Tyr Gln
1
(2) INFORMATION FOR SEQ ID N0:153:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:153:
Arg Asn Ile Ser Phe Lys Ala
1 5
(2) INFORMATION FOR SEQ ID N0:154:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:154:
Ala Pro Gly Lys
1
(2) INFORMATION FOR SEQ ID N0:155:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:155:
Trp Phe Asn Ser Thr Trp
1 5
(2) INFORMATION FOR SEQ ID N0:156:
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117ww
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:156:
Ile Ile Asn Met Trp Gln Lys Val Gly Lys Ala Met Tyr Ala Pro
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:157:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:157:
Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln Ile
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:158:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:158:
Glu Val Gly Lys Ala Met Tyr Ala Pro Pro
1 5 10
(2) INFORMATION FOR SEQ ID N0:159:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:159:
Gly Lys Ala Met Tyr Ala Pro Pro Ile Ser
1 5 10
(2) INFORMATION FOR SEQ ID N0:160:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
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117xx
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:160:
Ala Met Tyr Ala Pro Pro Ile
1 5
(2) INFORMATION FOR SEQ ID N0:161:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:161:
Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln
1 5 10
(2) INFORMATION FOR SEQ ID N0:162:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:162:
Asn Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Ser
1 5 10 15
Gly
(2) INFORMATION FOR SEQ ID N0:163:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:163:
Ser Asn Asn Glu Ser Glu Ile Phe Arg Leu
1 5 10
(2) INFORMATION FOR SEQ ID N0:164:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
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117yy
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:164:
Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro Cys
1 5 10
(2) INFORMATION FOR SEQ ID N0:165:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:165:
Ser Val Ile Thr Gln Ala Cys Ser Lys Val Ser Phe Glu
1 5 10
(2) INFORMATION FOR SEQ ID N0:166:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:166:
Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Phe Pro Gly Phe
1 5 10
(2) INFORMATION FOR SEQ ID N0:167:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:167:
Ala Gly Phe Ala Ile Leu Lys Cys Asn Asn Lys Thr
1 5 10
(2) INFORMATION FOR SEQ ID N0:168:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:168:
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117zz
Glu Val Val Ile Arg Ser Ala Asn Phe Thr Asp Asn Ala Lys Thr
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:169:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:169:
Ser Ala Asn Phe Thr Asp Asn Ala Lys Thr Ile Ile Val Gln Leu
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:170:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:170:
Ile Ile Val Gln Leu Asn Gln Ser Val Glu
1 5 10
(2) INFORMATION FOR SEQ ID N0:171:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:171:
Asn Glu Ser Val Ala Ile Asn Cys Thr
1 5
(2) INFORMATION FOR SEQ ID N0:172:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:172:
Glu Ser Val Gln Ile Asn
1 5
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117aaa
(2) INFORMATION FOR SEQ ID N0:173:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:173:
Phe Val Thr Ile Gly Lys Ile Gly Asn Met Arg Gln Ala His Cys
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:174:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:174:
Arg Gln Ala His Cys Asn Ile Ser Arg Ala Lys Trp Asn Asn Thr
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:175:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:175:
Arg Ala Lys Trp Asn Asn Thr Leu Lys Gln Ile Cys Ser Lys Leu
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:176:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:176:
Gln Ile Val Lys Lys Leu Arg Glu Gln Phe Gly Asn Asn Lys
1 5 10
(2) INFORMATION FOR SEQ ID N0:177:
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117bbb
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:177:
Ser Ser Gly Gly Lys Pro Glu Ile Val Thr His Ser Phe Asn Cys
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:178:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:178:
Asn Lys Thr Ile Ile Phe Lys Gln Ser Ser
1 5 10
(2) INFORMATION FOR SEQ ID N0:179:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:179:
Pro Glu Ile Val Thr His Ser Phe Asn Cys Gly Gly Glu Phe Phe
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:180:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:180:
Thr Trp Phe Asn Ser Thr Trp Ser Thr Lys Gly Ser Asn Asn Thr
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:181:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
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117ccc
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:181:
Thr Trp Ser Thr Lys Gly Ser Asn Asn Thr Glu Gly Ser Asp Thr
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:182:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:182:
Gly Ser Asp Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Phe Ile Asn
1 5 10 15
Met Trp Gln Glu
(2) INFORMATION FOR SEQ ID N0:183:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:183:
Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:184:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:184:
Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:185:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
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117ddd
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:185:
Asn Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:186:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:186:
Thr Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Thr Glu Lys Leu
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:187:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:187:
Lys Val Gln Lys Glu Tyr Ala Phe Phe Tyr Lys Leu Asp Ile Ile Pro
1 5 10 15
Ile Asp
(2) INFORMATION FOR SEQ ID N0:188:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:188:
Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Val Ser Phe Glu Pro Ile
1 5 10 15
Pro
(2) INFORMATION FOR SEQ ID N0:189:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
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117eee
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:189:
His Gly Ile Arg Pro Val Val Ser Thr Gln Leu Leu Leu
1 5 10
(2) INFORMATION FOR SEQ ID N0:190:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:190:
Ile Ile Val Gln Leu Asn Gln Ser Val Glu Ile Asn Cys
1 5 10
(2) INFORMATION FOR SEQ ID N0:191:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:191:
Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Gly Lys Ile Gly Asn
1 5 10 15
Met Arg Gln Ala His
(2) INFORMATION FOR SEQ ID N0:192:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:192:
Glu Gln Phe Gly Asn Asn Lys Thr Ile Ile Phe Lys Gln
1 5 10
(2) INFORMATION FOR SEQ ID N0:193:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
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117fff
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:193:
Glu Phe Phe Tyr Cys Asn Ser Thr Gln Leu Phe Asn
1 5 10
(2) INFORMATION FOR SEQ ID N0:194:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:194:
Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala
1 5 10 15
Pro Thr Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg Ala Val
20 25 30
Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly
35 40 45
(2) INFORMATION FOR SEQ ID N0:195:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:195:
CGATCTGGAG GACGCGCGGC GGCTGAAGGC GATATACGAG AAGAAGAAGG 50
(2) INFORMATION FOR SEQ ID N0:196:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 52 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:196:
GATCCCTTCT TCTTCTCGTA TATCGCCTTC AGCCGCCGCG CGTCCTCCAG AT 52
(2) INFORMATION FOR SEQ ID N0:197:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
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117ggg
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:197:
TGATCTACTG CAGCTGGAGG ACGCGCGGCG G 31
(2) INFORMATION FOR SEQ ID N0:198:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:198:
CGACCTCCTG CAGTTGGACC TGCTTCTTCT TCTCGTATAT 40
(2) INFORMATION FOR SEQ ID N0:199:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:199:
Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Lys
1 5 10 15
Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg
20 25
(2) INFORMATION FOR SEQ ID N0:200:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:200:
Lys Ala Lys Arg Arg
1 5
(2) INFORMATION FOR SEQ ID N0:201:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
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117hhh
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:201:
Lys Tyr Lys Arg Gln Ala Gln Ala Asp Arg Val Asn Leu Arg Lys Leu
1 5 10 15
Arg
(2) INFORMATION FOR SEQ ID N0:202:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:202:
Ile Glu Pro Leu Gly Val Ala Pro Thr
1 5
(2) INFORMATION FOR SEQ ID N0:203:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:203:
Ala Ala Lys Tyr Lys Gly Gly Gly Gly Gly Lys Ala Lys Arg Arg Gly
1 5 10 15
Gly Cys
(2) INFORMATION FOR SEQ ID N0:204:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:204:
Ala Ala Lys Tyr Lys Gly Gly Gly Pro Thr Lys Ala Lys Arg Arg Gly
1 5 10 15
Gly Cys
(2) INFORMATION FOR SEQ ID N0:205:
(i) SEQUENCE CHARACTERISTICS:
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117iii
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:205:
Ala Ala Lys Tyr Lys Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Gly
1 5 10 15
Gly Cys
(2) INFORMATION FOR SEQ ID N0:206:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:206:
Ile Glu Pro Thr Gly Val Ala Pro Thr Lys Ala Lys Arg Arg
1 5 10
(2) INFORMATION FOR SEQ ID N0:207:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:207:
TGGACTAAGT CGACACCATG GGATGGAGC 2g
(2) INFORMATION FOR SEQ ID N0:208:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:208:
TCGGAAGGGT CGACGGATCA TTTACCAGGA GA 32
(2) INFORMATION FOR SEQ ID N0:209:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
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117jjj
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:209:
GGCAACAGAA GCTTTCACTT CTTCTTCTCG TAT 33
(2) INFORMATION FOR SEQ ID N0:210:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:210:
TGATCTACTG CAGCTGGAGG ACGCGCGGCG G 31
