Title of Invention


Abstract The present invention relates generally to the field of immunotherapy and immunodiagnosis of autoimmune conditions. More particularly, the present invention provides agents which are recognized by or are specific for proinsulin- or insulin-sensitized T-cells. The present invention further contemplates the use of the these agents in therapeutic and diagnostic applications for Type 1 diabetes.
The present invention relates generally to the 'field of immunotherapy and
imnuinodiagnosis of autoimmune conditions. More particularly, the present invention
provides agents which are recognized by or are specific for proinsulin- or insulinsensitized
T-cells. The present invention further contemplates the use of the these agents
in therapeutic and diagnostic applications for Type 1 'diabetes.
Bibliographic details of the publications referred to in this specification are also collected
at the end of the description.
Reference to any prior art in mis specification is not. and should not be taken as. an
acknowledgmentor any form of suggestion that that prior an forms part of the common
general knowledge in a n y country. Type 1 diabetes (T1D) is caused by autoimmune destruction of the insulin-producing
pancreatic p cell. The onset of clinical diabetes is preceded by the appearance of antibodies
to (pro)insulin, glutamic acid, decarboxylase (GAD) and tyrosine phosphatase-Hke
insulmoma antigen-2 [IA-2] (Verge, et al, Diabetes 47:1857-1866, 1998; Harrison.
Pediatr Diabetes, 2:71-82, 2001). Serum and T-cell transfer experiments in the non-obese
diabetic (NOD) mouse, a spontaneous model of T1D, have shown that T-cells, not
antibodies, mediate the destruction of the,p cells (Kitutani et al. Adv. Imimmol, 57:285-
322,1992). Furthermore, CD4+ T-cells are absolutely required for the development of TlD
in the NOD mice (Yagi et al., Eur. J. Immimol. 22:2387-2393, 1992). Many genetic loci
have been associated with risk of TlD, but the--highest risk is with the major
hislocoinpatibiliry locus llDDM I ) , in particular with class J) J-ILA genes in numaiis.
specifically the haplotypes H1.A-D1G-DQ2 and HLA-DR4-DQ8 (Pugliese el aL Type 1
diabcicx. Molecular, cellular, and cJinictil:immmolqgy:l3-\S2,\996; Tail et al, Him.
Jmnniniil. 42: \ 16-122. 1995). HLA class 11 molecules present processed protein antigens in
the form of short (1 lamino acids) peptides; to CD4H T-cells. underlining the importance
of Cl 34" T-cells in the development of T1D.
Proinsulin is the major protein product of the p. cell and. with the possible exception of rare
self-anligen expressing cells in lymphoid tissues, is the only known human islet
autoantigcn expressed solely in (3 cells. An increasing hody of evidence implicates
autoimmune reactivity to proinsulin as a major mechanism of P cell destruction
(Narendran et al., 2004. supra). Autoantibodies to insulin are associated with early onset
of disease (Ziegler et al., Diabetes 40:709-714, 1991), and interestingly with HLA DR4
(Eisenbarth et al., J. Ai/toimmiin.. SSuppl. A:241246, 1992). The second strongest genetic
susceptibility locus for T1D, IDDM 2; maps to a variable nucleotide tandem repeat
(VNTR) minisatellite upstream of theinsiuin,gene;(Lucassene/'3/.).Ate. Genet, 4:305-310.
1993). The short class 1 VNTR (26-63 repeats) 'alleles are associated with lower levels of
proinsulin gene transcription in the thymus'and, it is postulated, with less deletion of
proinsulin-specific T-cells and an increased risk of developing T1D; "in contrast, the "long
class HI (140-210 repeats) alleles are associated with higher proinsulin transcription in the
thymus, greater deletion of proinsulin-specific T-cells and a lower risk of T1D (Pugliese et
al, Nat Genet, 75:293-297. 1997). These findings infer that immune responses to
proinsulin are criticaLfor the development of T1D.. ,
The ammo acid sequence of human proinsulin is shown in Figure 1, Proinsulin-derived
peptides that stimulate human T-cells have been,reported (Narendran et al., Autoimmun.
Rev, 2:204-210, 2003, Lieberman -ei al., Tissue Antigens (52:359-377, 2003), but
knowledge of these T-cell epitopes is fragmentary..Proinsulin-derived T-cell epitopes have
been identified hi three different ways: (1) by cloning insulin- or proinsulin-specific CD4+
T-cells and analyzing the epitope specificity of these cells in vitro; (2) by using synthetic
peptides identical to the sequence of proinsuliivtp recall T-cell proliferation responses from
peripheral blr. \l mononuclcar cells (T'BMC.1 in vin; or (3) by immunizing mice that
cxpivss transyenie HLA genes, isolating T-cell hybridomas specific for proinsulin and
analyzing their specificity in vitro. There are few -published reports that describe the
cloning of human CD4H insulin- or proinsulin -specific T-cells (see Table 3). Schloot et al.
J. Autoimimm 1 /:169-175, 1998 identified an HLA-DR- restricted epitope in the B-chain
of insulin (B:ll-27). Semana el al, J. Avtoimnnw 72:259-267, 1999 detected T-ccIl
proliferation in response to C35-50 of proinsulin. A single T-cell clone was isolated that
recognized this C-peptide epitope although the responses were weak. The first proinsulin
T-cell epitope identified from synthetic peptides was B24-C36. in individuals with islet
autoantibodies at risk fo; T1D (Rudy, et al, Mtl MecL 7:625-633, 1995). Others have
tested human T-cells for their capacity to respond to the B9-23 peptide recognized by
CD4+ T-cells in NOD mice (Alleva, et al., J. CUn. Invest. 707:173-180, 2001). Responses
to this peptide were detected in PBMC from diabetic and pre-diabetic donors, but not
healthy controls. ELlSpot assays have been used to detect T-cell responses to GAD and
proinsulin peptides from healthy and diabetic donors (Ott el al., J. CUn. Immunul. 24:321-
339, 2004). T-cell responses Were detected to an epitope C18-A1 .in healthy, diabetic and
pre-diabetic donors. Donors who had antibodies to islet autoantigens had responses to
B11-C24 and C28-A21; donors who had .clinical-diabetes had responses to B20-C4
(Durinovic-Bello et al, J. Autoimmun 75:55-66,2002,). HLA DRB 1-*0401-transgenic mice
were used to identify an HLA DRB 1*0401-restricted proinsulin epitope [C56-64] (Congia
et al., Proc. Natl Acad. Sci USA P5:3833r3838, 1998). Raju et al., Hwn. Immimol 55:21-
29, 1997 found that pre-proinsulin 1-24, the entire leader sequence, and B21-C39 were
dominant epitopes after immunization of HLA DQ8 transgenic mice. In similar
experiments, HLA DQ6 transgenic mice responded to leader 14-B9 and C60-A5 (Raju el
al., ] 997. supra). In summary, several T-cell epitopes in proinsulin have been reported, but
only two have been identified using T-cell clones, and HLA restriction has only been
defined with HLA transgenic mice.
Modifications of amino acids that occur after translation of mRNA into polypeptide are
known as post-translationaJ modifications.; (PTMs). PTMs. such as phosphorylation,
glycosylation and disulphide bond,formation arj:; critical for correct protein folding and
function. \\'-hile T-cells thai recognize self-pcptidc antigens are deleted during
development or regulated post-natal))', PTMs; .particularly il' induced by inflammation
and/or cellular stress, could create 'nep-antigens' that might .trigger T-ccll responses to
modified self, leading to autoimmune disease (reviewed by Doyle and Mamula, Trends,
hnmunot. 22:443-449. 2001). The paradigm that PTMs create target autoantigens is
alUtctivc. hut there are few examples of PTMs that create human T-cell epitopes. Coeliac
disease, although strictly speaking a food intolerance rather than an autoimmune disease,
is the clearest example of a human disease in which a PTM of the target antigen leads to a
pathogenic T-cell response (Molbcrg et.al.,Nat.'Med. 4:713-717. 1998; Anderson et «/..
Nat. Med. 6:337-442, 2000). Glutamine .(Q) residues in the cereal protein gliadin are
dcamidated by tissue transglutaminase to glutamic acid (E) residues, which are then
recognized by pathogenic T-cells.
There is a iwed to identify major T-cell epitopes in protnsulin and insulin in order to
develop therapeutic and diagnostic agents for Til). i ,
The present invention provides iinmunotherapcutic and imtnunudiagnostic methods and
agei-is useful for T1D. The- present invention is predicated in part on the identification of a
class ol'pvoinstilin- or insulin-derived A-ohain .epitppe requiring a PTM in order to be fully
reactive to proinsulin or insulin-sensitive T-cells and... in particular CD4"1 T-cells. The PTM
is preferably an intra-chain disuliiile bond between two adjacent cysteine residues.
According! \ . the present invention provides an isolated peptide derivable from the A-chain
of proinsulin or insulin and comprising an amino acid sequence having two adjacent
cysteine residues which, when both participate in,an jntra-chain disulfide bond, enables the
peptide, to activate proinsulin- or insulin-sensitive CD4+ T-cells or a homolog of • said
The peptide or its homologs, analogs, orthologs, mutants or derivatives represent a T-cell
epitope from proinsulin or .insulin which requires a:PTM in order io.be fully reactive with
sensitized T-cells. For the sake of .brevity,, the peptide and its homologs, analogs,
orthologs, mutants and derivatives are all.encompassed by the term "T-cell reactive agent"
The prescnl invention contemplates, therefore, methods for diagnosing T1D or a
susceptibility for development of same in asubject. The present invention further provides
methods of treatment or prophylaxis of a subject with or having a pre-disposition to
develop T1D. The present invention also provides diagnostic and therapeutic, including
prophylactic, agents to treat or help prevent T.1D oriat least ameliorate the symptoms of
T1D. The present invention further contemplates a method for diagnosing and/or
preventing immune T-cell responses that could attack transplanted (pro)insulin-producing
cells o r tissue.
Vaccines and tolerance-inducing compositions are .also provided in the treatment or
prevention of T1D. Generally, the vaccines or .compositions comprise the TCRAs of the
present .invention or agents capable of interapting -with same.
Throughout this specification, unless the context requires otherwise, the word "comprise",
or variations such as 'comprises1' or 'comprising", will be understood to imply the
inclusion of a stated clement or integer or group of elements or integers but not the
exclusion of am other element or integer or group of elements or integers.
Nuelcolide and amino acid sequences are related to by a sequence identifier number (SEQ
ID "NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers 4001
(SEQ ID N0:l), 4002 (SEQ ID N0;2), etc. A summary of the sequence identifiers is
provided in Table 1. A sequence listing is provided after the claims.
(Figure Removed) is ;i representation of the ami no :icid sequence of human p:.iinsulin (SEQ ID
Figure 2 is a graphical representation showing that the epitope is in the first 12 amino
acids of the A-chair of insulin. Proinsulin specific CD4'T-cell clones were tested against
a panel of peptides corresponding to the sequence of human proinsulin. (A) Preliminary
epitopc mapping. ISiner peptides shifted by three amino acids, comprising the entire
sequence of proiusulin, were grouped into 8 pools of 3-4 peptides each. T-cell clones (5 x
lO4 cells/well) were cultured in the presence of irradiated autologous PBMC (5 x 104/weH)
and each peptide pool. Individual peptides were at a final concentration of 5u.g/ml and^
recombinant human proinsulin at 10|o.g/ml. (B) Fine epitope mapping. The three peptides
that comprised pool 8 were tested separately it'a final concentration of 5^ig/ml. Other
conditions were the same as for (A) above. (C) Cloned T-cells were cultured without
antigen or with lOug/ml proinsulin or 10(ig/ml recombinant human insulin. Anti-HLA DR
monoclonal antibody (L243) was added to a final concentration of 5ug/ml. Proliferation
(mean of triplicate +/- SD) was measured in all experiments by the addition of 0.5nCi/well
3H-thymidine for the final 18 hours of a 72-hour culture. One representative ol'five clones
is shown.
3 is a graphical representation showing that T-cell clones recognize native insulin.
(A) Insulin-specific T-cell clones were cultured in dilutions of a freeze-thaw lysate of
hand-picked human islets or human spleen. Insulin"(lOug/ml) and cells in the absence of
antigen served as positive and negative controls, respectively. Similar results were
obtained with two other insulin-specific clones; '(B) Insulin-specific T-cell clones were
cultured without antigen or with 1/400 dilution tf islet lysate or 10u.g/ml of insulin. Anti-
HLA DR (L243, IgG2a) or anu'-HLA DQ (SPV-L3, IgG2a) were included at a final
concentration of 5u.g/ml.
Figure 4 is u graphical representation showing thai ihe response of T-cell clones is HLADK4-
rcstricK'il (A) Insulin-specific T-cell clones were incubated with irradiated
autologou? PBIvlC (1 x 105/well) without antigen (no antigen) or with lOig/ml proinsulin.
Anlibodies specific for HI.A DR (L243); HLA DQ (SPV-L3) or HLA DP (B7/2V) were
added to a final concentration of 5u,g/ml. .Similar results were obtained with three other
insulin-specific clones. (TV) Insulin-specific T-cell clones were incubated with irradiated
(50 Gy). HLA-transfected BLS lines, that were pulsed with lOOuM A-chain pcptidc
(KRGWEQCCTSICSL) or an equal volume of solvent. Peptide-pulsed BLS cells (1 x
104/well) were cultured with 2.5 x 10" insulin-specific T-cell clone/well. Proliferation of
the BLS cells without T-cells (1,000-5,000 cpm) was subtracted. Siinilar results were
obtained with three other insulin-specific clones.
Figure 5 is a graphical representation showing that adjacent cysteines are required to
stimulate T'-call clones. The effect of substituting each cysteinc with serine was
investigated. (A) Insulin-specific T-cell clones (2.5: x 104 cells/well) were cultured hi the
presence of 10 to 0.001 (XM A-chain epitope or variants that have each cysteine. _(C)
substituted with serine (S). The substituted amino acid is shown in bold type. Similar
results were obtained with three other insulin-specific clones. (B) In separate experiments,
responses to the native sequence (KRGIVEQGCTSICSL; SEQ ED N0:23) and a more
potent variant (S-13, KRGIVEQCCTSISSL,; SEQ ID NO:26) were compared to the murine
homologue (KRGIVDQCCTSICSL; SEQ ID N0:27).
Figure 6 is a graphical representation showing that the A-chain epitope contains an intrachain
disulphide bond. (A) To identify the modified epitope ihe S-13 peptide was
incubated in serum-containing culture medium for Ih at 37°C. This mixture was separated
by RP-HPLC and 0.5ml fractions collected. The.profilc shows absorbance at214nm. Solid
bars show the proliferation of an insulin-specific.-T--cell clone in response to each fraction
(1/400 dilution). A paraformaldehyde-fixedB-cell.h'ne that expresses HLA DRB1*0404
was used as the APC (1 x 104/well). Proliferation was measured by 3H-thymidine
incorporation during the final 18 ,hours of a 72hr culture. (B) MALDI-QTOF mass
spectrometry analysis of the parental peptide .(fCRGIVEQCCTSlSSL; SEQ IDNO-.26) and
ibo aoti\e Iractiun (# 7'i From (he medium •modification'experiment in (A). The active
fraction contains a siuult peptide species two Daltons smaller than the S-13 peptidc,
L-onsistent with an infra-chain disulphide bond between the cysteinc.-. in A6 and A7.
Figure 7 is a graphical representation showing that reduction destroys the epitope. S-13
peptide (final l|.iM) was treated with freshly-prepared TCEP and diluted to the linul
concentrations shown. Each well included irradiated autologous PBMC (5 x 104) and Tcell
clone (2.5x10'Vwell). PHA fl.25j.ig/ml), IL-2 (2.5LVml) and solvent alone were
similarly prepared.
Figure 8 is a graphical representation showing that T-cell clones from a pre-clinical type 1
diabetes donor recognize the same epitope. Three of 11 proinsulin-specific clones
proliferated in response to insulin. (A) Proinsulin-specific T-cell clones were cultured with
proinsulin (10u.g/ml) or insulin (10|ag/ml) or without antigen. One of three insulin-specific
clones is shown, (B) Irradiated (50 Gy) B-cell lines transfected with ihc HLA genes shown
were pulsed with S-13 peptide (lOOuM) or:solvent,,alone, for 2 h at 37"C and washed.
Each well contained 2.5 x 104 T-cells and 2.5 x 104 BLS lines. (C) A paraformaldehydefixed
B cell line transfected with HLA DRB1*0404 was cultured with samples of RPHPLC
fractions used to identify the modification ,of the epitope recognized by tile first
series of clones. Each well contained 1 x 104'HLA;DRBr*0404 transfected B cells, III00
dilution of each fraction and 2.5 x 104 cloned insulin-specific T-cells derived from the preclinical
diabetic donor.
Figure 9 is a graphical representation, showing that clones from healthy subjects do not
recognize the Al-13 epitope.
Figure 10 is a graphical representation showing that Al-13 peptide prevents diabetes in
NOD mice.
Before describing the present invention in detail, i; :.s to be understood thai unless
otherwise indicated, the subject invention is not limited to specific formulations of
components, manufacturing methods, dosage or diagnostic regimes, or the like, as such
may vary. It is also to be understood that tin*-terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be limiting.
The singular '.urns "a", "an" and "the" include plural aspects unless the context clearly
dictates otherwise. Thus, for example, reference to 'a peptide" includes a single peptide, as
well as two or more peptides; reference to "a T-cell" includes a single '[-cell as well as two
or more T-cells; reference to "a TCRA" includes-a-single TGRA as well as two or more
TCRAs and so forth.
In describing and claiming the present invention, the following terminology is used in
accordance with the definitions set forth below.
The terms "peptide", "compound", TCRA,, "active agent", "chemical agent",
"pharmacologically active agent", "medicament", 'active" and "drug" are used
interchangeably herein to refer to a TCRA that induces a desired pharmacological and/or
physiological effect, The term "TCRA" also includes agonists and antagonists of TCRAs.
The terms also encompass phamaceuticaUy acceptable and pharmacologically active
ingredients of those active agents specifically mentioned herein including but not limited
to salts, esters, amides, prodrags, active metabolites, analogs and the like. Whenthe-terms
"peptide", "compound". TCRA, "active agent",, "chemical agent" "pharmacologically
active agent"', "medicament", "active" and "drug" are used, then it is to be understood that
this includes the active agent per se as well as pharmaceutically acceptable,
pharmacologically active salts, esters, amides, prodrugs. metabolites, analogs, etc.
Reference to a "peptide". "compound", TCRA, "active agent", "chemical agent"
"pharmacologically active agent", "medicament", "active" and "drug" includes
combinations of two or more actives such as two or more peptides. A "combination" also
includes multi-panuch as a twpart composition where the agents are provided
separately and »ivcn or dispensed separately or admixed together prior to dispensation.
.For example, a multi-pan pharmaceutical pack may have two or more TCRAs maintained
separate! y or aTCRA and an immunosuppressantagcut.
The terms "effective amount" and "therapeuticully effective amount" of an agent as used
herein mean a sufficient amount of the agent (eg. TCRA) to provide the desired therapeutic
or physiological effect or outcome including the desired immunological outcome (e.g.
immunological tolerance). Undesirable effects, e.g. side effects, are sometimes manifested
along with the desired therapeutic effect; hence, a practitioner balances the potential
benefits against the potential risks in determining what is an appropriate "effective
amount". The exact amount of agent required will vary from subject to subject, depending
on the species, age and general condition of the subject, mode of administration and the
like. Thus, it may not be possible 10 specify an exact "effective amount". However, an
appropriate "effective amount" in any individual case may be determined by one of
ordinary skill in the art using only routine experimentation.
By "pbarmaceutically acceptable" carrier, excipient or diluent is meant a pharmaceuticalvehicle
comprised of a material that is not biologically or otherwise undesirable, i.e. the
material may be administered to a subject along with the selected active agent without
causing any or a substantial adverse reaction. Carriers, may include excipients and other
additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH
buffering agents, preservatives, and the like. .
Similarly, a "pharmacologically acceptable"'salty/ester, emide. prodrug or derivative of a
compound as provided herein is a salt, ester, amide, prodrug or derivative that this not
biologically or otherwise undesirable.
The terms "treating" and "treatment" as used herein refer to reduction in severity and/or
frequency of symptoms of T1D, elimination of symptoms and/or underlying cause,
prevention ol' the occurrence of symptoms of T1D and/or their underlying cause and
.improvement nr remedial ion or amelioration of damajK following ;i T1D.
"Treating" a subject may involve prevention of T1D or other adverse physiological event
in a .susceptible subject as well as treatment of a clinically symptomatic subject by
amdioratiny the symptoms of T1D.
A "subject" as used herein refers to an animal, preferably a mammal and'more preferably a
human who can benefit from the pharmaceutical formulations and methods of the present
invention. There is no limitation on the type of animal that could benefit from the presently
described pharmaceutical formulations and methods, A subject regardless of whether a
human or non-human animal may be referred to as an individual, patient, animal, host or
recipient as well as subject. The compounds and methods -of the present invention have
applications in human medicine, veterinary medicine as well as in .general, domestic or
wild animal husbandryAs indicated above, the preferred animals are humans or other primates such as
orangutangs, gorillas, marmosets, livestock animals, laboratory test animals, companion
animals or captive-wild animals.
Examples of laboratory test animals include mice,,rats, rabbits, guinea pigs and hamsters.
Rabbits and rodent animals, such as rats and mice, provide a convenient test system or
animal model. Livestock animals include sheep, cows, pigs, goats, horses and donkeys.
The present invention identifies an epitope in the A-chain of proinsulin or insulin which
comprises a T-cell reactive portion to proinsulin- or insulin-sensitized CD4* T-cells. The
epitopes are conveniently on a peptide, or a .hpmolog, analog, ortholog, mutant or
derivative of the peptide, collectively referred to herein as a T-cell reactive agent or
TCRA, The peptides or functional equivalents are, therefore, T-cell epitopes or have the
capacity for T-cell epitopes. The present iriventipn.provides, therefore, T-cell pre-epitopes
which, through PTM form T-cell neoepitopes frun ;proinsulin and insulin.
j he "neoepitopc" in this context means that M is a newly formed epitope. The neocpitope
forms Jiom a pre-epilopi1. In a preferred embodiment, a I'TM transforms a T-cell precpitope
to an active T-cell neoepi lope which is capable of stimulating proinsulin or insulinscnsiti/
ed T-cells, and in particular CD4" T-cells.
In a preferred embodiment, the PTM is the .formulation of a disulfide bond between two
adjacent cystcine residues. The disulfide bond is, therefore an intra-chain disulfide bond.
The present invention extends however, to other PTMs whether naturally occurring or
ariificially induced.
Accordingly, the present invention provides an isolated peptide comprising at least 10
amino acid residues in length forming a sequence substantially homologous to at least 10
contiguous amino acids within amino acid residues 1 through 21 of the A-chain of human
proinsulin or insulin or their mammalian homolbgs wherein said amino acid sequence
comprises two adjacent cysteine residues whiph when participating in disulfide bond
formulation between each other, renders .the peptide is capable of*stimulating proinsulin- or
insulin-sensitized T-cells, or a homolog, analog, ortholog, mutant or derivative of said
By "substantially homologous" includes the situation where within the homologous amino
acid sequence on proinsulin or insulin, the sequence contains one or more amino acid
substitutions, additions or deletions.
The peptide of the present invention is regarded .as a T-cell epitope reactive with
proinsulin- or insulin-sensitized T-cells and in particular CD4+ T-cells when the two
adjacent cysteine residues participate in disulfide bond formulation. Prior to disulfide
bond formulation, the peptide (or its functional equivalents) is said to be a T-cell preepitope.
The peptide and its homologs, analogs, prthologs, mutants and derivatives are
referred to as a "TCRA".
in' concepi of :i "'1-cell cpitope" signifies an antigenic/immunogenic sequence of a
protein which brings about an activati n of T-cells and comprises in accordance with the
present invciuiun the primary sequence of the T-cell epitopc or the primary sequence of an
(untigenic) polypcptidc or protein or antigen which contains at least one primary sequence
.if a T-cell epitope. T-cdls recognize this stimulus normally in the form of a peptide
hound to MHC molecules and in particular MHC class II molecules. T-ccll epitopes can
also bring about only a partial activation in certain instances, in which a decoupling of
various processes associated with the T-cell activation can take place. Full or partial
activation of T-cells may result in release .of mediator substances (e.g. cylokines).
A homolog, analog, ortholog, mutant or derivative of the subject peptide also includes an
altered peptide ligand (APL). An APL of the present invention includes a single or
multiple amino acid substitution, deletion or addition of an amino acid residue in the
peptide or a change in PTM. Changes.in PTM include the introduction or removal of
particular glycosylation patterns, covalent bonds, ionic bonds, disulfide bonds or the
introduction of other groups such as unsaturated or saturated fatty acid moieties or chains.
The present invention provides, therefore, an isolated T-cell neoepiiope created by a PTM
characterized-by (i) comprising a peptide backbone of. at least 10 amino acids having an amino acid
sequence substantially homologous to an arninp acid sequence of the A-chain of proinsulin
or insulin;
(ii) wherein the amino acid sequence of the peptide comprises at least two adjacent
cysteine residues of which at least onexorresponds to Cys6 or Cys7 in human proinsulin or
insulin A-chain or a non-human .mammalian equivalent thereof;
(iii) when functioning as a T-cell epitope for proinsulin- or insulin-sensitized CD4+ Tcells,
the two adjacent cysteine residues participate in disulfide bond formulation between
each other;
or a homolog. analog, ortholog. mutant or denvalivc ol said peptidc.
The preierred T-cclJ epitope of the present invention comprises the amino acid sequence
set forth in SHQ ID N0:26. However, the present invention extends to a range of
fragments of the insulin A-diain which comprise at least two adjacent cysteine residues
provided that at least one corresponds to Cys6 or Cys7 although most preferably both Cys6
and Cys7.
For example, the peptide may comprise amino acids 1 through 20, 2 through 20, 3 through
20, 4 through 20, 5 through 20 or 2 through 19. 2 through 18, 2 through 17,2 through 16,2
through IS, 2 through 14.. 2 through 13, 2 through 12, 2 through 11, 2 through 10, 2
through 9, 2 through 8 or 3 through 19, 3 through 18, 3 through 17, 3 through 16, 3
through 15, 3 through 14, 3 through 13, 3 through 12, 3 through 11, 3 through 10, 3
through 9 or 4 through 19. 4 through 18, 4 through 17, 4 through 16, 4 through 15, 4
through 14,4 through 13,4 through 12. 4 through 11,4 through 10, 4 through 9,4 through
8 or 5 through 19, 5 through 18, 5 through 17, 5 through 16, 5 through 15, 5 through 14, 5
through 13, 5 through 12, 5 through 11, 5 through 10, 5 through 9, 5 through 8 or 6
through 19, -6 through tS, 6 "through 17, ~6"through 1'6, ~6 "through 1'5, ~6 -through H, '6
through 13, 6 through 12.. 6 through 11, 6 through 10, 6 through 9 or 6 through 8 of the Achain
of human proinsulin or insulin or non-human mammalian equivalents or homologs
As stated above, the peptide defined by ,SEQ ID NQ:26 is most preferred as well as
homologs, analogs, orthologs, mutants and derivatives thereof. Reference to the above
"TCRAs" include peptides having at least 80% similarity to SEQ ID NO:26 after optimal
alignment provided that there are at lest two adjacent .cysteine residues of which at least
one corresponds to Cys6 or Cys7 of the human A-chain of proinsulin or insulin of its nonhuman
mammalian equivalent At least 80% includes 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90,91, 92,93, 94, 95, 96, 97, 98,99,100%. :
Aindicated above, in acconJnncc with the present .invention. PTivl creates a neocphope
for proinsulin- or insulin-sensitized CD4'1 T-cells wherein the ?TM is the formulation of a
disulfide bond between Cys6 and Cys7. Tho present invention extends lo trie introduction
of non-naturallv occurring amino acid residues to facilitate the same conformational
constraints imposed by ihe disulOde bond between Cys6 and Cys7. Alternatively, the use
of non-naturally occurring amino acids can be used to stabilize the peptide for use in in
vitro or in vivo diagnostic or therapeutic testing.
An "analog" is generally a chemical analog and include, but are not limited to,
modification lo side chains, incorporation of unnatural .amino acids and/or their derivatives
during peptick. polypeptide or protein synthesis and the use of crosslinkcrs and other
methods which impose conformational constraints on the peptides.
Examples of side chain modifications contemplated by the present invention include
modifications of amino groups such as by reductive alkylation by reaction with an
aldehyde followed by reduction with NaBHU; amidination with methylacetimidate;
acylation with acetic anhydride; carbamoylation of amino groups with cyanate;
trinitrobenzylation of amino groups with ,2, 4, 6-trinitrobenzene sulphonk.acid (TNBS);
acylation of amino groups with.succinic anhydride and-tetrahydrophthalic anhydride;-and
pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH^,
The guanidine group of arginine residues, may be modified by the formation of
heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal
a n d glyoxal
The carboxyl group may be modified by carbodiimide activation via O-acylisourea
formation followed by subsequent derivitization, lor example, to a corresponding amide.
Suiphydryl groups may be modified by methods such as cafboxymethylation with
iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid: formation of a
mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride
or other substituted malcimide; formation of mercurial derivatives using 4-
chlnromercuntienzoatc. chloronierciiripbcnylsulphpnic acid, phenylmercury chloride, 2-
chloronjereuri-l-nilropheno) anil other mercurials; carbauioylation with cyanalc at alkaline
Tryptophan residues may be modified by, for example, oxidation with Nbromosr.
ecinhiride or ulkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide
or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with
iciranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accornplisbed by
alkylation -with iodoacetic acid derivatives or N-carbethoxj'lation with
diethy Ipyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during peptide synthesis
include, but are not limited to, use of norleucine, 4-arnino butyric acid, 4-amino-3-
hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine. non'ailine,
phenylglycine, ornithine, sarcosine, 4-amino-3-hydrbxy-6-methylheptanoic acid, 2-thienyl
alanine and/or D-isomers o"f amino acids. A list of "onnabiral 'amino "acid, "contemplated
herein is shown in
(Table Removed)osslinkers can be used, for example, to stabilize 3D conformations, using homobifunctional
crosslinkers such as the bifunctional:irriido'esters having (CHz)n spacer groups
with n=l to n=6, glutaraldehyde, N-hydroxysuccinimidc esters and hetero-bifunctional
reagents which usually contain an amino-reactive moiety such as? N-hydroxysuccinimide
and another group specific-reactive moiety sueh as maleirhido or dithio moiety (SI-I) or
carbodiimide (COOH). In addition, peptides can bc conforniationally constrained by, for
example, incorporation of Ca and "N 0-raethylamrnp acids, introduction of double bonds
between Ca and Cp atoms of amino acids and the formation of cyclic peptides or analogues
by introducing covalent bonds such as forming an amide bond between the N and C
termini, between two side chains or between a side chain and the N or C terminus.
The present invention further contemplates chemical analogs of the subject polypeptide
capable of acting as antagonists or agonists of the T-cell epitope peptide or other TCRA.
Chemical analogs may not necessarEy be derived from the instant peptide molecules but
may share certain conformational similarities. Alternatively, chemical analogs may be
specifically designed 10 mimic certain physiochcinical properties of the subject peptide Tce-
11 cpitopi1. Chemical analogs may be chemically synthesized or may be delected
following, for example, natural product screening or screening of chemical libraries. The
latter refers to molecules identified from various environmental sources such a river beds.
coral, plants, microorganisms and insects.
These types of modifications raaj be important to stabilize the subject TCRAs if
administered to an individual or for use as a diagnostic reagent.
Other derivatives contemplated by the present invention include a range of PTMs such as
glycosylation and sulfide bond changes.
The designing of mimetics to a TCRA is a known approach to Ihe development of
Pharmaceuticals based on a "lead" compound such as the:peptide defined by SEQ ID
NO.26. This might be desirable where the .active peptide .compound is difficult or
expensive to synthesize or where it is unsuitable for. a particular, method of administration,
e.g. peptides are generally unsuitable active agents'for oral compositions as they tendlohe
quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and
testing is 'generally -used -to avoid -randomly-screening large -numbers -of-molecules_for_atarget
There are several steps commonly taken in the design of a mimetic from a compound
having a given target property. First, the particular, .parts of the compound that are critical
and/or important in determining the target property are determined. In the case of a
peptide, this can be done by systematically varying the amino acid residues in the peptide,
e.g. by substituting each residue in turn. Alanine scans of peptides are commonly used to
refine such peptide motifs. These parts or residues constituting the active region of the
compound are known as its "pharmacophore".
Once the pharmacophore has been found, .its structure is modeled according lo its physical
properties, e.g. stereochemistry bonding, size and/or charge, using data from a range of
'scurces. c.ii. spectroscopic techniques, x-ray diffraction data and !\VIR. Computational
analysis, similarity mapping i which models the charge and/or volume of a phurmacophore,
ralher than Ihf bonding hchvcen atoms) and oilier techniques can be used in this modeling
In :i variani of this approach, the three-dimensional structure of the MHC class II molecule
or the pcptidc is determined. This can be especially useful where the MIC molecule
and/or peptidee change conformation on binding, allowing the model to take account of
this hi the design of the mimetic. Modeling can be used to generate inhibitors which
interact with the linear sequence or a three-dimensional configuration.
A template molecule is then selected onto which chemical groups which mimic the
pharmacophore can be grafted. The template molecule and the chemical groups .grafted
onto it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be
pharmacologically acceptable, and does not degrade in vivo, while retaining the biological
activity of the lead compound. Alternatively, the stability can be achieved by cyclizing the
peptidee, increasing its rigidity. The mimetic or mimctics found by this approach can then
be screened to see whether they have the target property, or to what extent they exhibit it.
Further optimization or modification can then-be carried-out-to arrive at -one-or more final
mimetics for in vivo or clinical testing
The goal of rational drug design is to produce structural analogs of biologically active
polypeptides of interest or of small molecules with which they interact {e.g. agonists,
antagonists, inhibitors or enhancers) in order to fashion drugs which are, for example,
more active or stable forms of the polypeptide, or-which, e.g. enhance or interfere with the
function of a polypeptide in vivo. Sec, e.g. Hodgson (BioTechnolog)' 9: 19-21..1991). In
one approach, one first determines the three-dimensional structure of a protein of interest
by x-ray crystallography, by computer modeling or most typically, by a combination of
approaches. Useful information .regarding the structure of a polypeptide may also be
gained by modeling based on the structure of homologous proteins. An example of rational
drug design is the development of HIV protease inhibitors (Erickson et a!., Science 249:
527-5?". 1990). In addition, target molecule.- may be analyzed by an alanine scan (Wells,
Metliods Enzyniol. 202: 2699-270.. 1991). In this technique, an ;imino acid residue is
replaced by Ala and itF effect on-ihe pcptide's activity is determined. Bach of the amino
acid residues ol' the pcplide is analyzed in this manner to determine the important regions
oftlic peptide.
It is also possible to isolate a peplide-speeific antibody, selected by a functional assay and
then to solve its crysul strueture. In principle, this approach yields a pharmacore upon
which subsequent drug design can be based. It'is possible to bypass protein crystallography
altogether by generating anti-idiotypic antibodies (anti-ids) to a functional,
pharmacologically active antibody. As a mirror image of a mirror image, the binding site
of the anti-ids would he expected to be an analog of the original receptor. The anti-id could
then, be used to identify and isolate peptides from banks of chemically or biologically
produced bunks of peptides. Selected peptides would then act as the pharmacore.
Two-hybrid screening is also useful in .identifying.other members of a biochemical or
genetic pathway associated with a target. Two-hybrid screening conveniently uses
Saccliaromyces cerevisiae and Saccharomyces pombe. Target interactions and screens for
iiflubitors can~be carried out using the yeasfrwo-hyfarid system, -which-takes advantage-oftranscriptional
factors that are composed of two physically, separable, functional domains.
The most commonly used is the yeast GAL4 transcriptional activator consisting of a DNA
binding domain and a transcriptional activation domain. Two different cloning vectors are
used to generate separate fusions iof the GAL4 domains to genes encoding potential
binding proteins. The fusion proteins are co-expressed, targeted to the nucleus and if
interactions occur, activation of a reporter gene (e.g. lacZ) produces a detectable
phenotype. In the present case, for example, S. .cerevisiae is co-transformed with a library
or vector expressing a cDNA GAL4 activation domain fusion and a vector expressing a
holocyclot5dn-GAL4 binding domain fusion. If lacZ is used as the reporter gene, coexpression
of the fusion proteins will produce a blue color. Small molecules or other
candidate compounds which interact with a target will result in loss of colour of the cells.
Reference may be made to the yeast two-hybrid systems as disclosed by Munder el al.
.W/c.-iifr/n/. Biotechw'l. ?2: HI 1-320. 1999) and Young et a! (Nat. tiiotechnol. 16:
y4d-950. 1998). Molecules Ihus identified by this system are then rc-tesied in animal cells.
Another technique for drug screening provides high throughput screening for compounds
having suitable binding affinity to a target and is described in detail in Geysen
(International Patent Publication No. "WO 84/03564). Briefly stated, large numbers of
different small peptide test compounds are synthesized on a solid substrate, such as plastic
pins or some other surface. The peptide test compounds arc reacted with a target and
washed. Bound target molecule is then detected by methods well known in the art. This
method may be adapted for screening for non-peptidc. chemical entities. This aspect,
therefore, extends to combinatorial approaches to screening for target antagonists or
Purified target can be coated directly onto plates!for use in the aforementioned drag
screening techniques. However, non-neutralizing antibodies to the target may also be used
to immobilize the target on the solid phase.
The present invention also contemplates the use .of competitive drug screening assays in
whiclrneutralizmg-antibodies capable-of-specifically^binding the^arget-eompete-with-a-testi
compound for binding to the target or fragments thereof. In this manner, the antibodies can
be used to detect the presence of any peptide which shares one or more antigenic
determinants of the target.
As stated above TCRAs have a range of diagnostic and therapeutic utilities.
Any number of methods may be employed to detect T-cells sensitized to the subject T-cell
epitopc. Immunological testing is one paitficularnnethod screening for T-cells 'restricted'
by particular MHC class n molecules. Acqprdingly,, the present invention extends to
antibodies and other immunological agents directed to or preferably specific for the T-cell
epitope or the disulfide bond or its binding MHC class H molecule. The antibodies may be
monoclonal or polyclonal or may comprise Fab fragments or synthetic forms.
Specific antibodies can be used to screen for the subject T-ocll epitope and its fragments.
Techniques for the assays contemplated herein arc known in the art and include, for
example, sandwich assays a n d EL1SA. It is within the scope of this invention to include any second antibodies (monoclonal.
polyclonal or fragments of antibodies or synthetic antibodies) directed to the first
mentioned antibodies referred to above. Both the first and second antibodies may be used
in detection assays or a first antibody may be used with a commercially available antiimmunoglobulin
antibody. An antibody as contemplated herein includes any antibody
specific ip any region of the peptide at or near the T-cell epitope.
Both polyclonal and monoclonal antibodies are obtainable by immunization with the
subject peptide or larger forms such as an A-chain or a complex between an MHC class II
molecule and the peptide and either type is utjlizable for imrnunoassays. The methods of
obtaining both types of antibodies.are well known in the art. Polyclonal sern are less
preferred but are relatively easily prepared by injection of a suitable laboratory animal with
an effective amount of subject polypeptide, or antigenic parts thereof, collecting serum
from the 'animal and isolating specific sera .by-any of the -known immunoadsorbent
techniques. Although antibodies produced by this method are utilizable in virtually any
type of immunoassay, they arc generally less favoured because of the potential
heterogeneity of the product.
The presence of the instant T-cell epitope may be detected in a number of ways such as by
Western blotting and ELISA procedures. A wide range of immunoassay techniques are
available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and
4,018,653The T-cell epitope of the present invention iand its neoepitope has a range of uses in
For example, the 1'CKA may he used in therapy to induce immunological tolerance. This
may be -achieved in various ways including administering the pcptide by a lulerogenic
route (eg vin rnucosHej.or in .a tolerogenic form (eg via immature dendritic colls), exposing
the thyimiK to the peptide, using Ihe pepvidc to induce deletional tolerance such as by
stimulating apoptosis in T-cells or by linking a eytotbxic moiety to- the peptide or to induce
T-cell anergy. The TGRA may also be used to generate antibodies or other
immunoteractivc molecules specific lo the TCRA.
Accordingly, another aspect of ihc present invention contemplates a method for preventing
or reducing the risk of onset of T1D in a subject said method comprising introducing into
said subject a T-cell tolerance-inducing effective amount of a peptide .having the
characteristics of:
(i) comprising a pepTlDe backbone of at least, 10 amino acids having an amino acid
sequence substantially homologous to an amino acidiseauence of the A-chain of proinsulin
o r insulin
(ii) wherein the amino acid sequence, of the, peppClDe comprises at least two adjacent
cysteiiic residues of\vhich~atleast •one-corresponds'to"Gys6-or-eys7-in-human-proinsulin-erinsulin
A-chain or a non-human mammalian; equivalent thereof;
(iii) when functioning as a T-cell epitope for proinsulin- or insulinrsensitized CD Tcells,
the two adjacent cysteine residues .participate in disulfide bond formulation between
each cnher; .
or a homolog, analog, orthobg, mutant or derivative of said peptide.
hi an alternative method, the peptide is used to induce. selective depletion of proinsulin- or
insulin-sensitized CD4+ T-cells. In this regard, the T-cell epitope peptide or other TCRA
of the present invention and its neoepitopic form is fused or otherwise associated with a
cytotoxic molecule including an apoptptic molecule or radioactive isotope.
In siill further alternative, catalytic antibodies"£ire generated which specifically target the
disulfiUc- bond. In this regard, although not-wishing to limit the invention to any one
hypothesis, it is conceivable that the disulfide bond formed between two adjacent existing
rcsidiics results in .1 formation of substantial bend in the protein backbone. The omega
torsion angle for such a bend may range from 180 degrees to negative 130 degrees. The
result of this bending of the peptide backbone,is the possibility of generating an antibody
such as a catalytic antibody which is specific for that conformation.
Accordingly, another aspect of the present invention provides a peptide:
(i) comprising a peptide backbone of at least. -10 amino acids having an amino acid
sequence substantially homologous to an amino.acid sequence of the A-chain of proinsulin
or insulin;
(ii) wherein the amino acid sequence qf the peptide comprises at least two adjacent
cysteine residues of which at least one corresponds; to Cys6 or Cys7 in human proinsulin or
insulin A-chain or a non-human mammalian equivalent thereof:
(iii) when functioning as a T-cell epitope .for prpinsulin- or insulin-sensitized 004* Ttvlls,
the two adjacent cysteine residues participate in disulfide bond formulation between
each other:
or a homolog, analog, ortholog, mutant or derivative of said peptide;
wherein said peptide is fused or otherwise bound:,onassociated with a cytotoxic.moiety.
Such a peptide is referred to herein as a cytptoxic TVcell targeting agent but is also
encompassed by the term TCRA. Accordingly, another aspect of the present invention provides a method for treating a
subject will) 'i'lD or a predisposition for the development of same said method comprising
administration to said subject of apepiide'vrtiiclii-'js-an amount sufficient lo be cytoloxic to
CD4'1 T-cells wlu-rem in said peptidcs is characterized by:
(i) comprising a peptidc backbone bl'at least I'O amino acids having an amino acid
sequence substantially homologous to an amino acid sequence of the A-ehain of proinsulin
or insulin; , .,
(ii) wherein the amin" acid sequence of the peptide comprises at least two adjacent
cystcine residues of which at least one corresponds to Cys6 or Cvs7 in human proinsulin or
insulin A-chain or a non-human mammalian equivalent thereof;
(iii) when functioning us a T-cell epitope.for,proinsulin- or insuluvsensitizcd GD4f Tcells,
the two adjacent cysteine residues participate in disulfide bond formulation between
each other
or a homolog. analog, oftliolog, mutant or derivative of said peptide.
The present invention further contemplates •& inethod \for diagnosing and/or preventing
immune T-cell -responses -that could attack "transplanted "(pro")insu1in-pf6ducii;(g cells "or"
In another aspect, the present invention, contemplates MHC class II molecules are loaded
with peptides comprising the subject T-cell epitope. The.peptides,ate typically .loaded into
the binding groove formed by the Talpha und tbeta.l .domains and bind to the MHC class n
molecules through non-covalent interactions. The peptides can be from about 9 to 10 lo
about 20 amino acids, or more, in length. The peptides of the present invention are conveniently synthetically produced but the
sequence is derived from proinsulin or insulin.
The peptides can be prepared in a variety of, ways. For example, peptides can be
synthcsi/cd using an automated pcptide synthesiser. The peptides can aiso DC
synthesized (HauuUapiller c; ul. Nature 370:105-11. 1984, Siewarl and Young. Solid Phase
f'epiiilt Synthesis, T" Ed., fierce Chemical Co.; Rockford III, 1984, Houben-Weyl,
Methoclun ckr organisi-'hen Chemie. Vol. IS/! and i'5'2, Bodanszky, Principles vf Pepticle
Synthesis. Springer Ve.rlag 1984), alternatively, peptides can be synthesized by proteolytic
cleavage (e.g. by trypsin, chymotrypsin. papain, VS protease, and the like) or. specific
chemical cleavage (e.g. cyanogen bromide). The peptides can also be synthesized by
expression of overlapping nucleic acid sequences in vivo or in vitro, each nucleic acid
sequence encoding a particular peptide.
The peptides optionally can be isolated and purified prior to contacting with the MI1C
class II molecules. Suitable methods include, for example, chromatography (e.g. ion
exchange chromatography, affinity chromatography, sizing column chromatography. high
pressure liquid chromatography. and the like), centrifugation, differential solubility, or by
any other suitable technique for the purification of peptides or proteins. In certain
embodiments, thejpeptides can be labeled (e.g.-with-a radioactive label, a luminescent label,
an affinity tag, and the like) to facilitate purification of the peptides (infra).
The peptides are "typically 'not CToss'linked rtp the-MHC class II molecules. ~In-.jQthet--
embodiments, the peptides optionally ,can be cross-linked to the binding groove of the
MHC class II molecules. For example, bi-functional crosslinking reagents (e.g. heterobifunctional,
homo-bifunctional. etc.) can be used to covalentiy link the peptides to the
MHC class II moelcules (Kunkel et al., Mol. Cell. Biochem. 34:3, 1981). Suitable
crosslinking reagents include, for example, dimethylsuberimidate, glutaraldehyde,
succinimidyloxycarbonyl-.alpha.-methyl-.alpha,(2-pyridyldithio)-toluene (SMTP). Nsuccinimidyl
3-(2-pyridyldithio_-propionate (SPDP), sulfosuccinimidyl 4-(Nmaleimidomethyl)
cyclohexane-l-carboxylatesulfprSMCC), m-maleimidobenzoyl-Nhydroxysuccinimide
ester (MBS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester
(sulfo-MBS), N-succiiiimidyl(4-iodoacetyl);. ; aminobenzoate (SIAB), Nsulfosuccinimidyl(
4-I-odoacetyl)aminobenzoate (sulfo-SIAB), succinimid)-l-4-(pfSMPBsulfosuccinimidyl-4-(p-maleunidophyenyl) butyrate
(sullb-SivlPB). 1 -cihyl -.'i-(3-diiTicihylaminopropylcarbodiimidc hydrochloride (EDC),
dithbbissuccinimidylpropionate (DSP), 3,3'dithiobis(su]lbsuccinim-ich Ipropionute)
'DTSSP,) and the; liui- (Pierce Chemical Co. Pierce Immvno Technology Catalog ; ul
Handbonk. 1990). In one embodiment, one or more anchor residues of a peptide can be
cross-linked to the MHC class II molecule. In other embodiments, any suitable residue(s)
of a pcpu'de can be cross-linked to the MHC class ,11 molecule.
Alternatively, the pepiideb can be prepared as fusion proteins with a soluble MHC class
Il.beui. subunii. For example, nucleic acids encoding a peptide, or a mixture of peptides,
can be expressed as a fusion protein comprising a peptide. spacer or linker region (e.g 10
to 20 amino acid linker), a soluble MHC class II subunit, and a ligand binding domain.
The peptide can be linked for example, to the amino terminal end of the MHC class Il.beta.
subunit. In one embodiment the fusion protein is expressed from an expression cassette.
The expression cassette can included, for example, a promoter operably associated with,
and in a 5' to 3' direction relative to the direction of transcription,, a nucleic acid encoding a
polylinker cloning region, a nucleic acid encoding a spacer region, and a nucleic acid
encoding an MHC class Il.beta. subunit. The expression cassette can be expressed in any
suitable host organism and can be part of aa expression vector. In another embodiment,
pools of MHC-classII/peptide fusion protein pairs. semi-degenerate or non-degenerate nucleic acids into the polylinker region of an
expression cassette, such as those described above. Alternatively, nucleic acids encoding a
single peptide can be inserted into the polylinker region.
The MHC class II molecules and peptides can be formed into multimeric MHC class II
complexes. As used herein, forming multimeric MHC class Il/peptide complexes can
include forming multimeric MHC class II/peptide complexes from MHC class II
molecule/peptide pairs and/or forming multimeric 'MHC class II molecules from MHC
class II molecules, which can be loaded with peptides.
The multimeric complexes can comprise two, three, four, or more MHC class II/peptide
complexes. Such complexes can be formed by interaction between a ligand on the MHC
class 11 molecules anJ a polyvalent binding partner. As used herein, ihe phrase "ligandlitiand
binding partner pair" refers to a ligand and its liganc1 binding partner that arc
capnble of re. ognizing and binding in each other. The term "polyvalent" refers to a ligand
binding partner that has at Icasi two binding sites, typically three or four, ligand binding
sites. The lig;md(s) and bindiiu' partner can be any moieties that are capable of
rccongin/ing and binding to each other to form a mullinicric complex. Additionally, the
ligand and binding partner can interact via the binding of a third intermediary substance.
Typically, the ligand and ligand binding partner constituting the ligand-binding partner
pair are binding molecules that undergo a specific noncovalent interaction wife each other.
The ligand ant! ligand binding partner can be naturally occurring or artificially produced,
and optionally can be aggregated with other species of molecules.
The binding partner can be free in solution or can be attached to a solid support. Examples
of suitable solid supports include beads (e.g. .magnetic b.eads), membranes, mcirotitcr
plates, and the like. The support can be glass, plastic (e.e. polystrene), polysaccharide,
nylon, nitrocellulose. PVDF. and the like. The use of a binding partner linked to a solid
support can be useful for immobilization, and/or isolation of T-cells (e.g. such as from a
population of PBMC) that recognize the multimeric MHC class II molecule and the bound
peptideIn an exemplary embodiment, one of the MHC. class II submits includes a modification
sile (e.g. a BirA recognition sequence); BirA catalyzes biotinylation of the protein
substrate. The biotinylated MHC class II moleculeis then bound to a polyvalent binding
partner (e.g. streptavidin or avidin), to which biotin binds .with extremely high affinity.
The multimers can then be stored until needed.
The MHC class II molecules typically are loaded with peptide by incubation at
37.degree.C. in a phosphate buffer at slightly acidic pH (.e.g lOOmM sodium phosphate,
pH 6.0) in the presence of 0.2% n-octyl-D-glucopyranoisde (OG). A protease inhibitor is
optionally added to the mixture. Suitable peptide loading times range from about 48 to
about 72 hours, although greater and lesser .times are within the scope of the present
invention. Suitable peptide: NJHC. class molecule molar ratios are in excess of !0:1,
although L-roatcr and lesser ratios are within the scope of the present invention. Other
liiiflVrs ;i:,.l pITs can be useJ. us will be appreciaiccl by the skilled artisan.
In certain embodiments, the multimeric MHC class H/peptide complexes :••..,: labeled. As
used herein, the terms "label" or "labeled" refer to n molecule or groups of molecules
which can provide a detectable signal when the label is incorporated into, or attached to, a
polypeptide, such as a MHC class II molecule or a polyvalent binding partner. For
example, a polypeptide or a polyvalent binding partner can be labeled with a radioactive
molecule, a luminescent molecule,;: fluorescent molecule, a chemi-luminesccnl molecule,
an enzyme, or by biotinyl moieties. Methods of labeling polypeptides and binding partners
are well known in the art. Examples of detectable labels include, but are not limited to, the
following: radioisotopes (e.g. 3H, 14C., 32P, 35s, I25I, !3II, and the like), fluorescent
molecules (e.g. fluorescein isothiocyanate (FITC), rhodamine, phycoerythrin (PK),
phycocyanin, allophycocyanin, ortho-phthaldehyde, fluorescamine, peridinin-chlorophyll a
fPerCP), Cy3 (indocarbocyanine), Cy5 (mdDdiacarbocyanine), lanthanide phosphors, and
the like), enzymes (e.g. horseradish peroxidase, .beta.-galactosidase, lucifcrase, alkaline
phosphatase). biotinyl groups, and the like. In. some embodiments, detectable labels are
attached-by spacerarms-of various -lengths to reduce; potential steric hindrance.
In specific embodiments, the binding partner can be labeled. For example, a biotinylatcd
MHC class II molecule can be detected with .labeled avidin or streptavidin (e.g streptavidin
containing a fluorescent molecule or a colored molecule produced by enzymatic activity
that can be detected by optical or colorimetric methods). Alternatively, the MHC ckss JI
molecule can be detected, for example, with a labeled antibody or other binding agent that
will bind specifically bind to the multimeric MHC class II complex. Suitable labels
include any of those described above or known toithe skilled artisan.
In another aspect, the multimeric MHC class D/peptide complexes are contacted with Tcells
to determine whether the complexes bind the T-cells in an epitope-specific manner.
In certain embodiments, the multimeric 'MHC class Il/peptide complexes can be used to
stain or deloaabh Libel the "J -cells. Abused'herein, "stain" refers to the ability of the
multimeric Ml 1C class ll/'pcptide complexes to detecUihly label T-cells that can hind the
complexes ii; .in fpitope-specifk manner.
Human 1 cells can be isoiatul from fresh samples from a human subject, from an in vitro
culture of cells from a human subject, from a frozen sample of cells, and the like. Suitable
samples can include, for example, blood, lymph, lymph nodes, spleen, liver, kidney,
pancreas, lonsil. thyrnus. joints, synovia, and other tissues from which T-cells can be
isolated. Typically, the T-cells are isolated ,as..peripheral blood mononuclear cells
(PBMO. PBMC can be partially purified,.for example, by ccntrifugation (e.g from a buffy
coat), by density gradient centrifugation (e.g. through-a Ficoll-Hypaque), by panning,
affinity separation, cell sorting (e.g. using antibodies specific for one or more cell surface
markers) mid other techniques that provide enrichment of PBMC and/or Ttcells.
In one exemplary embodiment, PBMC are isolated from a blood sample by standard
Ficoll-Hypaque method. The blood .sample is treated with heparin and underlain with :a__
Ficoll solution. Following centrifugation, the recovered cells can be washed, for example,
in PBS or T-cell culture medium (e.g. RPM1 1640 supplemented with 2mL L-glutamine,
100.-mu:g/ml -penicillin/streptomycin, ImM sodium pyruvate and 15% pooled human"
serum; A1M-V; and the like). The washed cells can be resuspendcd in T-cell culture
medium, and the like. .
The multimeric class Il/peptide complexes can be contacted with the T-cells to identify one
or more MHC class II epitopes of a predetennmed antigen. The epitopes can be
determined according to the specificity of the .alpha, and .beta, subunits comprising the
MHC class II molecules. ,,
Generally, the multimeric class Il/peptide complexes are contacted with a sample of Tcells
of interest. In some embodiments, the T-cells are cultured for between about 1 to 10
days, or more, in T-cell culture media in the presence of the predetermined antigen to
stimulate proliferation of T-cells that are specific for that antigen. The media optionally
can be supplemented other components-Tor the culture and.'or viability of T-cells (e,g.
serum, antibiotics, cyiokines. co-stimulatory receptor agonists, and the like). In other
embodiments, the T-cells are contacled with the multimerie class H/peptide complexes
\vithoul antigen stimulation amU'r culturing (e.g. for patient monitoring).
The T-cells are contacled with the multimeric MHC class II/peptide pools under suitable
binding conditions. In one embodiment, the binding conditions are 37.degree.C. in any
suitable T-cell culture media (c,g. ,RPMI 1640 or .A1M-V). phosphate buffered saline,
Dulbecco's phosphate buffered saline, Dulbecco's Modified Eagle Medium, Iscove's
medium, and the like. The media can be supplemented with other components for the
culture and/or viability of T-cells (e.g. serum, antibiotics, cytokines. and the like). The
multimeric complexes are typically contacted with the T-cells for at least about 5 minutes
and typically within the range of about. 1 to 2 hours. The appropriate concentration of.
multimeric complexes can be determined by titration.
The amount ofmurtimeric complex bound to the T-cells is determined. For example, if the
T-cells are substantially homogenous, then the amount of labeled multimeric.
Still another-aspect -of -the present invention provides a -method -for-inducing tolerance -oranergy.
This may be achieved in a number of ways including providing MHC class II/Tcell
epitope complexes, introduction of the T-cell epitopes into the thymus, providing suboptimal
levels of the T-cell epitope and providing antagonists of T-cell eptiope-TGR
interaction. Examples of suitable antagonists .are antibodies or recombinant forms or
chimcric forms or derivatives thereof. ;.
The T-cell epitope peptides or their homologs, analogs, ortholpgs, mutants and derivatives
(i.e. the TCRAs) pr antibodies, MHC class II complexes or T-cell receptor-TCR antagonist
(also referred to herein as "active compounds") used in the methods of the invention can be
incorporated into pharmaceutical compositions suitable for administration to a subject, e.g.
a human. Such compositions typically also comprise a phannaceutically acceptable
carrier. As used herein the language "phannaceutically acceptable carrier" is intended to
include am and all solvents, dispersion medin. coating, antibacterial and antifungal
agents, isotonic ;ind absorption delaying ayents. and the like, compatible \vitii
pharmaceutical administration. The use of such media and agents for pharmnciiutically
active substances is well known in the an. Except insofar as any conventional media or
agent is incompatible with (he active compound, such media can be useci in the
compositions of the invention. Supplementary active compounds can also b incorporated
into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its
intended route of administration. Examples of routes of administration include parenternl,
e.g. intravenous, intradermal, subcutaneous, oral (e,g. inhalation), transdermal (topical),
transmucosal, and rectal administration. Solution or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following components: a sterile
diluent such as water for injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl
alcohol or methyl paprabens; antioxidants.such as ascorbic acid or sodium bisulfite;
chelating agents such as ethylenedianuneletraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as sodium chloride 01
dextrose. ipH can be adjusted with acids or -bases, such as-hydrochloric acid .or sodium..
hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or
multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions
(where water soluble) or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. For intravenous administration,
suitable carriers include physiological. saline, bacteriostatis water, Cremophor EL.TM.
(BASF, Parsippany, N.J.) or phosphate .buffered saline (PBS). In all cases, the
composition must be sterile and should be fhiid to the extent that easy syringeability exists.
It must be stable under the conditions of manufacture and storage and must be preserved
against the contaminating action of microorganisms such as bacteria and fungi. The carrier
can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for
cxampk. glyr..-ivl, propylene glycoK and liquid po'yetheylene giycol. and the like), and
suitable mixtures thcrcul'. The proper fluidity can be maintained, for example, by the use
of :i coating M.ic:h as lecithin, by (he maintenance of the required panicle size in the case of
dispersion and by the use of surfactants. Prevention of the action of mciroorganisrns can
be achieved by various antibacterial and antifungal agents, for example, parabcns,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars, poly-alcohols such as manitol.
sorbitol, sodium chloride in the composition. Prolonged absorption of the injectahle
compositions can be brought about by including in the composition an apent which delays
absorption, for example, aluminium monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g. a
small molecule, nucleic acid molecule, or peptide) in the required amount in an appropriate
solveni with one or a combination of ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are .prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion medium and the required
other ingredients from those enumerated .above. In the case of sterile powders for the
preparation of sterile injectable. solutions, the preferred methods of preparation are vacuum
drying and freeze-drying which-yields a powder, of the active-ingredient plus any .additional
desired ingredient from a previously sterilerfiltered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be
enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with excipients and used in the
form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid
carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orallj
and swished and expectorated or swallowed. Pharmaceutically compatible binding agents
and. or adjuvant materials can be included as part,of the .composition. The tablets, pills
capsules, troches and the like can contain any of ihe following ingredients, or compounds
of a similar nature: a binder such as mierpcystalline cellulose, gum tragacanth or gelat4in;
an excipient such as starch or lactose, a distingegrating agent as alginic acid, Primogel, or
.•urn stare;, a lubricant such as mayneMuni stearate or Sterotes; a ^Want such as colloidal
silicon .'inside; a sweetening agenl such a* sucrose or saccharin; or a flav .ring agent such
as peppermint, methyl salicylaic. or orange flavoring.
For administrating by inhalation., the compounds arc delivered in the form of an aerosol
spray from pressured container or dispenser which contains a suitable propellant. e.g. a gas
such us cai ivw dioxide, or a neubulizer.
Systemic administration can also be by transmucosal or transdermal means. For
transmucosal or Iransdennal administration, penetrants appropriate, to the barrier to be
permeated are used in the formulation. Such penetrants are general! y known in thwart, and
include, for exampk. for Iransmucosul administration, detergents, bile salts, and fusidic
acid derivatives. Transmucosal administration can be accomplished through the use of
nasal sprays or suppositories. For transdermal administration, the active compounds are
formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositiories (;e.g with conventional
suppository bases such as cocoa buttCTvand other, glycerides) or retention enemas-for rectal
delivery: - ••-•-• ,.,;.'-'-....:;.r,,,-.....„...i.:.:_._ _ _..
In one embodiment, the active compounds are prepared with carriers that will protect the
compound against rapid elimination from .the body, such as a .controlled release
formulation, including implants and microencapsulated delivery .systems. Biodegradable,
biocompatable polymers can be used, such, as ethylene vinyl acetate, pblyanhdrides,
polyglycolic acid, collagen, polyorthoesters, .and polylactic acid. Methods for preparation
of such formulations will be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza iL orporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with monoclonal antibodies to
viral antigens) can Also be used as pharmaceutically acceptable carriers. These can be
prepared according to methods known to those skilled in the art, for example, as described
in US Patent No. 4,522,81.1.
H is-especially advantauuiii'.* to formulate/oral or parenteral compositions in dosage unit
Conn for ease ol'adrninisinui.<: and. uniformity of dosage. dosage unit form as used herein> iclcrs to physically discrete units-suited'as nnitury;dosages for the subject lo be treated;
each unit containing a predetenninevl..quantitv ufiactive corapouuil calculated to-produee
ihe desired therapeutic effect in association-withithc'tequircd^phamiuceuticBl carrier. The
specification for the dosage unit forms "-of the 'invention .are dictated by and directly
dependent on the unique characteristics of -the ; active compound and the particular
therapeutic effect -to be achieved, and the liniitationsfiinherent in the -art oI' compounding
such an active compound for the treatment of individuals.
The present invention is further described'by the toiiowmgmon-aiminng example
Blood donors and PBMC isolation
Blood was obtained by veucpuncture. The HLA type of the donors is shown in Table 6.
PBMC were isolated over Ficoll/Hypaque (Amersham Pharmacia Biotech.AB. Uppsala
Sweden) and washed twice in PBS, Cells were cultured in Iscove's modified Dulbecco's
medium (1MDM) (Gibco. Rockville, MA,'USA) supplemented with 5% v/v pooled male
human serum, 2raM glutamine (Glutamax, Gibco, Rockville, MA. USA), 5 x 10"5M 2-
mercaptoethanol (Sigma. St.-Louis, MO), penicillin (100U/nU), streptomycin (1 OOng/ml)
(Gibco, Rockville, MA, USA) and 100|aM non-essential amino acids (Gibco, Rockville,
MA5USA). Mets and spleeirsamples were obtained^
Transplantation Program. St Vincent's Institute, Melbourne, Australia. Islets were isolated
by ductal perfusion with collagenase, digestion and gradient centrifugation as described
(Shapiro et al, NEngl. J. Med. 3^:230-238,2000).
Pi-prides used in this study are shown in Table 7. Recoml/mnnV human proinsulin was
produced by inoclillcalion of a published protocol .(CV-wle\ nnd Mackin. FEES Lett
402:124-130, 1997). Briefly, after anion exchange chroinatography, refolding and
reversed phase HPLC purification, the protein resolved as a single species of correct
molecular weight in matrix assisted laser .desorpiion/ionisation - time of flight mass
spectrometry. The endotoxin concentration of proinsulin stock measured by Limulus
lysate assay (BioWhiltaker. Walkerville, MD), -was 0.51 EU.'mg/ml. Synthetic peptides
were purchased from Mimotopes (Clayton, Victoria, Australia) or Auspep (Parkville,
Victoria, Australia) and reconstituted to 5mM in 0.5% v/v acetic acid, 40% v/v acetoniuile
- water, aliqiioted and stored at -70"C. Clinical grade recombinant human insulin was
HUMULIN (Novo. Nordisk, Copenhagen, Denmark). Human islet-cell lysate was prepared
from 100 hand-picked islets that were resuspended in 0.5ml of serum free IMDM then
frozen, thawed-and sonicated three times. Spleen lysate from the same donor was prepared
(Figure Removed)
For .staining with ihe dye 5,6-carbox'ylfluorcscein dincetatc succinimidyl ester (CFSE)
(Molecular Probes. Eugene, Or) PBMC at,l * Hi7/ml in PBS were incubated at 37°C for 5
minutes with the concentrations of CFSE indicated in the Brief Description of the Figures.
Staining was terminated by adding culture medium containing 5% pooled human serum,
the cells washed oace in PBS/1% pooled human serum and resuspended in culture medium
at 2 x 106 /ml. Stained cells (2 x 105/well, lOOul) were cultured in 96-well round bottom
plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA) with medium alone or with
tetanus toxoid or proinsulin. Unstained cells were included in all experiments and were
used to set the compensations on the flow cytometer.
After 7-10 days of culture, cells for each antigen concentration were pooled, washed in
PBS and stained on ice with anti-human GP4-PE (}gG2a, clone RPA-T4), (BD
Pharmingen, San Diego, Ca). Optimal compensation and gain settings were determined for
each experiment based on unstained and single stained samples. Propidium iodide was
used to exclude "dead cells. A single "CESE'lim. CB^1", propidiirar iodide-=negative cell -was
sorted into all wells, except the outer wells, ,af a 96-well tray. Each well contained 1 x 10s
freshly prepared, irradiated (20 Qy), allpgeneic PBMC from two unrelated donors and 5 x
10", irradiated (50 Gy) JY Epstein Ban Virus transformed B cell line (EBV), IL-2
(lOU/ml), IL-4 (5ng/ml) and phytohaemagluttinin (PHA) (2.5|ig/ml), to a final volume of
100ul Fresh IL-2 and IL-4 was added after 7 and 14 days of culture. Growing clones were
visible after 2-3 weeks and transferred to a 48-well plate. fed with cytokines and screened
for antigen specificity. For screening, cells were .cultured with autologous irradiated PBMC
with or without proinsulin and proliferation was measured by 3H-thymidine incorporation
as described in Example 4.
Proliferation assays
All assay;- were performed in 96-wcl) round bottom plates'in :5% PHb/lMDM. Antigen
presenting cells (A PC) were either (i) irradiated (20Gy) autologous or iLLA-matched
PBMC (fresh or thawed), (ii) HLA-typed EBV transformed B lines from the 9th
International H1..A Typing Workshop or (iii) EBV-,transformed B cell lines (from a donor
with Bare Lymphocyte Syndrome), tiunsfectcd with different HLA genes. EBV lines were
irradiated at 50 Gy. In sorm: experiments APC were fixed with 1% v/v paraformaldehyde
for 20 minutes at room temp, washed twice, in PB,S and once in culture medium before use
in proliferation assays.
Cloning and sequencing TCR genes
Total RNA was isolated from 1-5 x 106 cloned insulin-specific T-cells with RNAeasy
columns (Qiagen, Maryland, USA). First strand synthesis was carried out with oligo (dT)
primers and MMLY reverse transcriptatse (Promega, Wisconsin, USA). cDNA was
purified using silica and a 5' poly-G anchor was added with terminal transferasc (Promega.
Wisconsin, USA). TCR genes were amplified with a forward anchor ^primer (cac-teg ^gc
ggc ccc ccc ccc ccc cc; SEQ ID WO: 28) and either alphA-chain (cag caa cgt etc tgt etc tg;
SEQ ID NO.29) or betA-chain (get eta gcg teg acg get gel cag gca gta tct gga; SEQ ID
NO:30) reverse primers. PCR products were purified, cloned into pGEM and sequenced by
Big Dye. Several clones were sequenced in each direction for each clone.
ffPLC fractionation and mass spedrometry
An aliquot of l.Smg of peptide was added to L8ml of serum for llir and incubated at 37°C.
The mixture was then fractionationated by RP-HPLC using an AKTA Basic HPLC
(Amersham Biosciences) equipped with a multi wavelength tuneable UV detector and a
Frac 950 fraction collector. Proteins were separated on a 300A, 4.6 x 250 mm Yydac
protein and peptide C18 column, using a linear gradient of buffer A (0.1% v/v TFA) to
60% v/v B (acetonitri)L'.'0.(i(.>% v. •> 1TA; O.SiV'o/mirij, at 'a flow rate of Iml/min. Fractions
(500 ul) were collected and I(.U aliquots were mixed with lul of 2,5-dihydroxyben/.oic
acid (Agilent) and dried onto a sample stage .for analysis by MA1.DI-QTOF mass
spcctromciry (Applied Biosystems QSTAR pulsar i). Selected ions were subject to further
M.SMS analysis. Fragment ions generated in (his way were manually assigned based on the
known sequence of the parental peptide and modified amino acid residues identified within
the sequence.
identification ofproinsutin A-chain epitope
Fifteen proinsulin-specific CD4" T-cell clones were isolated from the peripheral blood of a
donor with established T1D3 as described in Example 1. The epitope was mapped against
an overlapping panel of 15-mer peptides, each shifted .by three amino acids. First, the
clones were eachjncubated with 8 pools of peptides, each comprising 3-4 peptides. Five of
15 clones recognized a peptide witliin pool 8 (Figure'2A). The remaining clones failed to
respond to any peptide. Second^ when the three peptides in pool 8 were tested separately, a
peptide comprising the last two amino acids of the C-peptide (underlined) and the first 13
amino acids of the A-chain oTinsulin (^GflVEQeCTSieSL-; -SEQiD-N0:23-)-strrrralated
the clones as well as pool 8 or recombinant proinsulin (Figure 2B). The epitope is within
the A-chain of insulin as clinical grade insulin was able to stimulate the clones. The
epitope was confirmed to be witliin the first 12 amino acids of the A-chain of insulin using
a panel of peptides. Finally, the response to insulin was due to antigen-specific recognition
since it was blocked by anti-HLA DR specific mAb (Figure 2C).
T-cell clones to the A-chain epilope recognize native insulin
To confirm thai the T-cell clones recognize an epitope derived from native human insulin,
their capacity to proliferate in response ID a human islet lysalc was tested. The results from
two clones are shown (Figure '\A). Both proliferated in response to islet lysate, but not to
spleen lysate from the same donor. Further experiments (Figure 3B) showed that this
response was blocked hy antibodies specific for HLA DR. but not for HLA DQ.
TheA-cliain epitope is presented by HLA DRBJ*0401, 0404, 0405
The HLA type of the donor is shown in Table 5. The HLA molecule(s) that present(s)4the
epitope to the clones was determined in two steps. First, blocking with HLA-isotypespecific
antibodies showed that proliferation of all clones was prevented by anti HLA-DR
specific mAb (L243), but not by HLA DQ ;(SP¥-L3)- or HLA DP (B7/21)-specific
antibodies (Figure 4A). Second, HLA restriction was confirmed using the panel of B*cell
lines transfected with HLA pdiain genes (Figure 4B). Cells transfected with DRB 1*0401
(DR4), DRB 1*0404 (DR4) or DRB1*0405 (DR4) were all able to present peptide,
whereas cells transfected with DRB4*0101 (DR52), DQB1*0201 (DQ2), DQB1*0302
(DQ8) were unable to present the agoniw peptide. Similar results were obtained with a
panel of HLA-typed EBV-transformed B cell lines. Hence, the HLA restriction is HLA
All clones that require the. A-chain epitope use the same Va Ja and Vf5 Jf3 sequences
The TCR usage of A-chain epitope-specific T-cell clones was determined (Table 8). One
clone uses VgtiSll while the other '.hree use Vo:i3.-2i, The same Ja 401 is expressed by
all clones. All clones use V0 7-801 and Jp.lp:l The CDR3 sequence was identical for
all but clone 4.19, which had different Va,and Vp GDR3 sequences.
(Table Removed) TheA-chain epitope requires adjacent cysteines
Peptides were tested in which cysteine was substituted by serine to determine the role of
cysteine in creating the epitope recognized by the A-chain-specific clones. Substitution oi
either of the adjacent cysteines (A-6 and A-7) with serine completely abolished the
capacity of the peptide to stimulate any of. the T-cell clones (Figure 5A), at peptide
concentrations of up to SOfoM. Surprisingly, substitution of the cysteine at position Al 1
with serine increased the activity -of the .'peptide 100-fold. A synthetic peptide
corresponding to iho sequence in mouse '.insulin 11. u'fhich has aspartic acid instead of
ghiumiic ucid at position VI. was 10-fold 'loss poien1 than the human peptide (Figure 5B).
Adjacent cysleines in the A-chain epitope form an infra-chain disulphide bond
It is proposed that the epitope recognized by the T-cell clones requires PTM of the
cysieines at positions A6 and A7. To investigate this, the capacity of paraibrmaldehydefixed
and -unfixed APC to present the epitope (as a synthetic peptide) to the T-ccll clones
was compared. Fixation did not affect the response to the peptide. Therefore, it v^as
concluded that the modification occurred spontaneously in the culture medium. To define
the modification more exactly, peptide with a serine for cysteine substitution at residue 13
(A-11), referred to as S-13 was incubated, in culture medium and then used RP-HPLC to
separate the components. The resultant fractions were tested for their capacity lo stimulate
proliferation of the clones. A single fraction (#7, Figure 6A) was able to stimulate
proliferation of the T-cell clones. No fractions stimulated proliferation when peptide with a
serine for cysteine substitution at position A-6 was used. Analysis of the active fraction by
mass spectroscopy (Figure 6B) showed that it contained a single species that was 2 Daltons
i ..,
smaller than the parental S-13 peptide. Which is consistent with a loss of 2 .hydrogen
atoms. MS/MS analysis showed that this loss of 2 Daltons arose at the adjacent cysteine
residues (Table 9). From this, it is concluded that the .cognate epitope recognized by the Tcell
clones contained an intra-chain disulphide bond between the two adjacent cysteines at
positions A-6 and A-7.
(Table Removed)Recognition of fin1 -l-chniu upitope is blocked.-by reduction :of disulphide bonds
To confirm thai an intrachain disulphidc bond is required for .stimulation of the T-ccll
clones, the affect of the disulphide reducing ageiv, Tris (2-earboxyethyl) phosphine
hydrochliride (TCEP) on the re spouse-of T-ccll clones to peptide S-13 was tested. With
increasing concentrations of TCEP a dose-dependent inhibition of the response of the Tcell
clones to peptide S-13 was observed (Figure 7). The reduction in proliferation was not
due to toxicity as the responses to PFIA or IL-2 were not reduced, but rather slightly
increased, by TCEPi • -
T-cell clones to the A-chain epitope isolated from a healthy donor with pre-clinical T1D
Eleven proinsulin-specific CD4T T-cell'clones were isolated from an islet autoantibody-
• positive HLA DR4+ donor at risk of developing TiD. Three of the clones proliferated in
response to both proinsulin and insulin (see for example Figure 8 A). These clones
recognized the S-13 peptide in association with HLA DRB 1*0404-0405 (Figure 8B). The
response was targeted to the A-chain epitope with an intra-chain disulphide bond because
this fraction from the RP-HLC column was the only one that stmnilatcd this clone (Figure
BC). Hence, T-cell clones specific for the post-translationally modified A-chain epitope
were isolated from a donor at high risk for T1D never exposed to exogenous insulin.
r I-Ag7 binding experiment
ofl-Ag7. l-Ag7 protein was affinity-purified from detergent lysatcs of 4G4.7
II ceil hybridcmia cells by clesoiption IVorn OX-6 mouse monoclonal antibody. The 4G4.7
B cell hybridorna was derived by polyethylene glycol (PBG)-induced fusion of NOD
mouse T-ccll-clepleted splenocytes with the HAT-sensitivc A20.2J lymphoma line. OX-6
is a mouse monoclonal IgGl antibody against an invariant determinant of rat la, which also
recognizes I-Ag7 but not I-Ad. Approximately 1 5 mg of OX-6 antibody was first "bound to
4ml oi' protein A-Sepharose 4Fastflow (Pharmacia, Uppsala, Sweden) and then
chemically cross-linked to the protein A with dimethyl piuielimidate dihydrochloride
(Sigma Chemical Co., St. Louis. MO) in sodium borate buffer, pH 9.0. After 60 min at
room temperature (RT), the reaction was quenched by incubating the Sepharose in 0.2 M
ethanolarnine. pH 8.0, for 60 min at RT. The ..suspension was washed thoroughly in PBS
and stored in PBS, 0.02% sodium azide (NaNS). 4G4.7 cells were harvested by ccntrifugation, washed in -PBS, resuspended at 108 cells/ml
of lysis buffer, and then allowed to stand.at 4°C for 120 min. The lysis buffer was 0.05 M
sodium phosphate. -pH 7.5, containing 0.15 M-Nad, l%-(-vo]/vol).HP4.0,detergenLand ihe
following protease inhibitors: 1 mM phenylmethylsulphonyl fluoride, 5mM -araino-ncaproic
acid and 10 ug/ml each of soybean trypsin inhibitor, untipain, pepstatin, leupeptin
and chymotrypsin. Lysates were cleared of nuclei and debris by centrifugation at 27,000 g
for 30 min and stored as such if not immediately processed further. To the postnuclear
supernatant was added 0.2 vol of 5% sodium depxyc.holate (DQC). After mixing at 4°C for
10 min, the supernatant was centrifuged at lOOjOOO^ at 4°C for 120 min, carefully
decanted, and filtered through a 0.45-um nylon. membrane. The ly sate of 5 * 1010 4G4.7
cells was gently mixed overnight at 4°C with 4, ml of OX6rprotein A-Sepharose, and the
suspension then poured into a column and washed \Yiuh at least 50 vol each of buffers A, B,
and C. Buffer A was 0.05 M Tris3 pH 8.0, .0.15 M NaCl, 0.5% MHO, 0.5% DOC, 10%
glyoerol, and 0.03% NaN3; buffer B was 0.05 M Tris, pH 9.0, 0.5 M NaCl, 0.5% NP-40,
0.5% DOC, 10% glycerol, and Q.03% NaN3; buffer C was 2 mM Tris, pH 8.0, 1% octyl
b-glucopvrimosidc K)GP). 1 0%lyccrol, and 0.03% NaNl Bound [.-Ag7 was clutcd with
50 mM dicthylamint 1ICL p] I r. .5 in 0.!'^ M NaCl. 1. mM EDTA, 1% OOP, 10% glycevol,
and 0.03% NaN3. and inuuediii1cl neutralized with 1 MTris.
Implicit: Synthesis. Peptides were synthesized with a multiple pcptide synthesizer (model
396; Advanced ChmiTech. I.ouis\ille. K.Y) using Fmnc chemistry and solid phase
synthesis on Rink Amide icsin. All acvlaliun reactions were effected with a threefold
excess of activated Fmoc amino acids, and a standard coupling lime of 20min was used.
Each Fmoc amino acid was coupled at least twice. Cleavage and side chain deprotection
was achieved by treating the resin with 90% trifluoroacetio acid, 5% thioanisole, 2.5%
phenol, 2.5% water. The indicator peptide for the 'binding assay was biotinylated before
being cleaved from resin by coupling two 6aminocaproic acid spacers on the NH2
terminus and one biotin molecule sequentially, using the above-described procedure.
Individual peptides were analyzed by reverse-phase HP1.C and those used in this studywere
routinely 85% pure. . .
I-Ag7 Peptide-binding Assay. Peptides were dissolved at 10 mM in DMSO and diluted
into 20% DMSO/PBS for assay. Indicator l-Ag7 binding peptide, HEL 10-23, was
synthesized with a biotin molecule and two spacer residues at the NH2 terminus.
Approximately 200 nM of this biotinylated HEL^peptide and each test peptide in seven
concentrations ranging from 50 ^Mjo'50..pM/wer.e'.'.cpincubated with -200 ng of I-Ag7
protein in U-bottomed polypropylene 96-well plates (Costar Serocluster, Costar Corp.,
Cambridge, MA) in binding buffer at RT, Tlie binding buffer was 6.7 mM citric phosphate,
pH 7.0, with 0.15 M NaCl, 2% NP-40,2 mM EDTA, and the protease inhibitors as used in
the lysis buffer. After a minimum of 24 h, each incubate was transferred to the
corresponding well of an ELISA plate (Nunc Maxisorp, Nunc, Roskilde, Denmark)
containing prebound OX-6 antibody (5 fig/ml overnight at 4°C, followed by washing),
After incubation at RT for at least 2 h, and washing, bound biotinylated peptide-I-Ag7
complexes were detected colorimetrically at 405 nm after reaction with streptavidinalkaline
phosphatase and paranitrophenolphosphate. Competition binding curves were
plotted and the affinity of peptide for I-Ag7 was expressed as an inhibitory concentration
5i; 11C'50). the concentration t.i'peptiuc iY..iuired 10 inhibii the binding ofbio-HEI. 10-23 by
50': i).
l~Ag7 Purification and Binding Assay
Approximately 2 mg of protein, estimated by Coomassie blue binding (Bio Rad Protein
assay), was purified from 5 x 1010 4G4.7 cells. In'SDS-PAGE. the majority (>95%i of the
protein was resolved as two bands of molecular weight --33,000 and ~28,000 that
correspond to the and subunits, respectively, of mouse class II MHC molecules. The
competition binding assay with purified l-Ag7 was sensitive and specific, and highly
reproducible; in 15 separate assays the mean ± SB of the 1C50 for competition between
biotinylated and unlabeled HEL 10-23 was 295 ± 72 nM.
Peptides overlapping by four residues and spanning the entire sequence of human
proinsulin were tested for binding to I-Ag7, and were inspected for presence of binding
motif (see Table 10), The proinsulin peptide aia. 65-79, from the A-chain of insulin, in
which the cj'Steines were replaced for the "binding studies -by alanines., -bound with
moderately high affinity (400nM) to I-Ag7.
overlapping by Tour residue- and spanning ihc entire- sequence of human
proinsulin \sorc tesiccl for binding to T-Ag7, and inspected them for presence of the binding
motif. The proinsulin peptidc nn 65-79, from the A-chain of insulin, in which the cysteines
were replaced for the binding studies b alanines, bouiul with moderately high affinity
NOD/Jax (t')} 10-12 mice per group
Mouse proinsulin II ammo acids .C53-A7 iLQTLALBVAQQKRGIVDQCC (SEQ ID
Mouse proinsulin II amino acids C64-A13 (KRGJVDQCCTSICSL (SEQ ID NO-.32)).
Mouse proinsulin II ainino acids C64-A13 with.cysteine at A6, A7 and A 1 1 substituted
for serine (KUG1VDQSSTSISSL (SEQ ID NO:33)).
Mice were treatsi-wfthlOjig-od
series of treatments were administered, each for 10 consecutive days, starting -when the
mice were 21, 50 and 100 days old;
Once the mice reached 100 days of age their urine glucose was measured. Mice that had
elevated urine-glucose concentrations (1 ImM) 'Were re-tested, those with two consecutive
urine glucose concentrations above 1 ImM were considered diabetic.
(Table Removed)5 Residues well tolerated at the p6 or p9 anchor positions are bolded; weakly tolerate
underlined; non-tolerated are bolded in lower case. Cysteines have been substituteu
alanine (A in italics). •
Prevention of tiiabi-it& in »N O.D mice
The aim of this Example i. to investigate human 'i'-cell response to Al-13 epitope and the
use of the NOD mouse model to determine the efficacy of pepiide encompassing this
cpi tope for prevention of type 1 diabetes (T1D),
T-cell responses to the islet autoaniigcns proinsulin and GAD can be detected in healthy
subjects (Mannering at al, Ann A",)'. Acad Set J03":l6-2l, 2004). While the epitope
specificity of these responses has not been determined it is possible that certain epitopes
are associated with diabetes while other epitopes are recognized by T-cells from healthy
The non obese diabetic (NOD) mouse spontaneously develops autoimmune diabetes and is
a widely used model of human type 1 .diabetes. Intranasal delivery of insulin protein
(Harrison et al, J Exp Med 755:1013-1021 ,,1996) or a proinsulin peptide spanning the B-C
chain junction (Martinez el al, J Clin Invesi 111:}365-1371, 2003) has been shown to
prevent diabetes in the -N0D mouse. The -intranasaJ proinsulin -B-G chain -peptide-induees
regulator)', anti-diabetogenic CD4+ T-cells. To elicit a 004-0611 response a peptide must
first bind to an MHC class II molecule. This mouse expresses a single MHC class II
moleculeThe peptide KRGIVEQCCTSICSL (SEQ ID. N0:32), or the murine homologue
KRGIVDQCCTSICSL (SEQ ID NO:33) are referred to as the Al-13.epitope, because
these peptides encompass the minimum epitppe described herein (GTVEQCCTSICSL
(SEQ ID NO:34), or in the mouse QJVDQCCTSICSL (SEQ ID NO:35)).
Analysis of clones from healthy HLA DR4 subjects: ,CD4+ T-cell clones 1hat proliferate in
response to proinsulin were isolated as described (Mannering ci al, J Imnnmol Methods
?0iS:83-lJ2. 2005). TIacli clone was cultured with irradiated antigen presenting cells (A?C)
and either no antigen, proinsulin (lOng/ral) 6'r AM pcptide (KRGIVEQCGTR1CSL
(SFQ TD NO:32), lOjaM). Responses to a pepiide encompassing the Al-13 epitopc were
detected by incorporation oi'3H-thymidinc durinu the fimil 16 hours of culture.
I-Ag7 bindingii-Atil binding was determined by competition for a biotinylated reporter
peptide (bio- 1TEL10-23), known to bind l-Ag7. Proinsulin pcptides were incubated in
serial dilution with a fixed concentration of bio-HEL 10-23 (For full details sec
Attachment 1). High-affinity peptides inhibit binding of the reporter peptide at low
concentrations (~100-500nM), whereas low or non-binding peptides inhibited at high
concentrations (2.0 |iM). Cysteine residues in the proinsulin peptides used for the I-Ag7
binding were substituted for alanine to avoid oxidative modifications.
Peptide therapy: NOD mice were used to test the capacity of peptide encompassing the
Al-13 epitope to prevent diabetes (see protocol in Attachment 2). Mice were treated by
intranasal delivery of a peptide of the Joltowing,peptides (KRGIVDQCC'l SICSL (SEQ ID
NO:32 or a similar peptide whe.re the cysteines were substituted by serine
(KRGIVDQSSTSISSL (SEQ ID NO:33)), or a control peptide
(LQTLALEVAQQKRGrVDQCC (SEQ"BD N0:315).The cyste'ihe residues at positions A6-
and A7 are required for formation of a vicinal disulfide bond and recognition by human
CD44" T-cell clones (as shown previously). Binding of the peptulo encompassing the Al-
epitope to HLA DR4 is not affected by substituting cysteine for serine. The murine form
homologue of the Al-13 peptide binds to I-Ag7, as.shown below, when cysteine has been
replaced with alanine. Diabetes incidence was monitored for 240 days by testing for urine
glucose each week from 100 days of age. Diabetes was confirmed by two consecutive
daily blood glucose concentrations 1 ImM.
Results i ,
Analysis of clones from healthy HLA DRJ subjects: Fifteen clones that recognize
proinsulin from two HLA DR4+ healthy subjects were tested for their capacity to
proliferate in response to proinsulin and a peptide encompassing the Al-13 epitope
('KKGIVKQCC 1'SJCSI. (SEQ ID NQ;32)l,None o! the clones thai proliferated in response
• pwinsulin proliferated in response 10,a pcptik;., ericoinpassing the Al-13 epitope. sec
Figure 9.
Peptide binding 10 I-Ag? .
A peptide encompassing the murine homologue oflhe A .hain epitope with the cysteincs
replaced by alaninc (RGIVEQAATS1ASL (SEQ ID N0:55)) bound with high to-moderate
affinity, IC50of 400nM (See Table 11), to I-Ag7.
Prevention ofTID in the NOD mice
NOD mice were treated with peptide encompassing the Al-13 epilope, variants of the
epitope, or an irrelevant peptide from proinsulin. The incidence of diabetes was monitored
until the mice were 240 days old. Of the mice treated with the a peptide encompassing the
A]-13 epitope (KRGIVDQCCTSICSL (SEQ ID NO:32)), one of 12 developed diabetes
(8.3%), whereas four of 10 treated with a similar peptide with the cysteines replaced with
serine (KRGIVDQSSTSISSL (SEQ ID NO:33)) developed diabetes (40°/i,). When mice
were treated with another peptide from proinsulin (LQTLALEVAQQKRGrVBQGC (SEQ
ID NO:31)) four out of 11 (36.4%) developed diabetes (Figure 10).
(a) Analysis ofpruinsulin-specific T-cell clones from healthy donors
This shows that clones isolated from healthy subjects, who express HLA DR4, do not
respond to the Al-13 epitope. This suggests that responses to this epitope may only be
found in people at risk of T1D or those who already have T1D,
(b) Binding 1o I-Ag7
These data show that the peptide encompassing the Al-13 epitope binds to the MHC class
1J molecule, I-Ag7. Intranasal treatment with a peptide encompassing the Al-13 epitope
reduced diabetes development in NOD .mice, but only if cysteines were present. This
supports the role of adjacent cysteine residues at A6 and A7 forming a vicinal-disulfide
bond in the formation of the epitope recognized by T-cells that mediate protection against
(O !\'plnli' //HTii/M fur preventing T1D in A'^D /m'tv
The NOD mow.1 is a useful model Cur ana) \sing ihe mechanism of pepiide mediated
protection ugainsi type 1 diabetes.Thesc data show that treatment with this peptide can
prevent the development of diabetes in .'.susceptible"animals. This supports the use of
peptide(s) encompassing the Al-13 cpitope for the prevention of T1D in susceptible
Those skilled in the art will appreciate that the invention described herein is susceptible to
variations and modifications other than those specifically described. It i.s to be understood
that the invention includes all such variations and modifications. The invention also
includes all of the steps, features, compositions and compounds referred to or indicated in
this specification, individually or collectively, and any and all combinations of any two or
more of said steps or features.

1. An isolated peptidf derivable from the A-chain of proinsulin or insulin and
comprising an amino acid sequence having two adjacent cysteine residues which, when
hath participate in an intra-chain disulfide bond, enables the peptide to activate proinsulinor
insulin-sensitive CD4"" T-eells or a homolog of said peptide.
2. The isolated peptide of Claim 1 wherein the proinsulin or insulin is human derived.
3. The isolated peptide of Claim 1 wherein the proinsulin or insulin is irmrine derived.
4. The isolated peptide of Claim 1 wherein the two adjacent cysteine residues
participating in the intra-chain disulfide bond forms part of a T-eell epitope.
5. The isolated peptide of Claim 1 or .2 or 3 wherein the T-cell epitqpe is on the Achain
of the proinsulin or insulin.
6. The isolated peptide of Claim 5 comprising at least 10 amino acid residues in
lehph forming a sequence-suljistawithin ammo acid residues 1 through 21 of the A-chain of human proinsulin or insulin or a
mammalian homolog thereof.
7. The isolated peptide of Claim 5 wherein the two adjacent cysteine residues
correspond to at least one of Cys6 or Cys7 of human proinsulin or insulin A-chain or a
mammalian homolog thereof.
8. The isolated peptide of Claim 7 comprising the amino acid sequence set forth in
SEQ ID N0:26 or an amino acid sequence .having at least 80% similarity to SEQ ID
N0:26 after optimal alignment.
9. An isolated antibody specific for the T-cell epitope on the peptide of Claim 8.
I (.). The solaied antibody of Claim 9 wherein the antibody is a monoclonal antibody.
! 1. The isolated antibody of Claim 9 in humanized form.
12. The isolated antibody of Claim 9 wherein the antibody is a catalytic antibody.
I j. An isolated derivative of the antibody of Claim 9 or 10 or i 1 or 12.
14. A method for preventing or reducing the risk of onset of Type 1 diabetes in a
subject, said-method comprising -administering to said subject a T^ccl] tolerance-inducing
effective amount of a peptidc of any one of Claims 1 to 8 for a tune and under conditions
to induce T-cell tolerance to proinsulin or insulin.
15. The method of Claim 14 wherein the subject is a human.
16. The method of Claim 14 or 15 wherein.the ^peptide induces selective depletion of
proinsulin- or insulin-sensitized CD4+ T-cells.
I1. A cytotoxic T-cell targeting agent comprising the peptide of an)- one of Claims 1 to
8 bound, fused or otherwise associated with a cytotoxic moiety.
.IB., A method for preventing or reducing Type. 1 diabetes in a subject, said method
comprising administering to said subject a T-cell depleting effective amount of the
cytotoxic T-cell targeting agent of Claim 17.
19. The method of Claim 18 wherein the subject is a human.
20. A pharmaceutical composition comprising the peptide of any one of Claims 1 to 8
and one or more pharmaceutically acceptable carriers and/or diluents.







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Patent Number 258215
Indian Patent Application Number 1116/DELNP/2007
PG Journal Number 51/2013
Publication Date 20-Dec-2013
Grant Date 18-Dec-2013
Date of Filing 09-Feb-2007
# Inventor's Name Inventor's Address
PCT International Classification Number C07K 4/12
PCT International Application Number PCT/AU2005/001086
PCT International Filing date 2005-07-22
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 2004904133 2004-07-23 Australia