U.S. patent application number 14/963308 was filed with the patent office on 2016-11-17 for novel antigen peptide and uses thereof.
This patent application is currently assigned to INSERM (Institut National de la Sante et de la Recherche Medicale). The applicant listed for this patent is Marc LOPEZ, Daniel OLIVE. Invention is credited to Marc LOPEZ, Daniel OLIVE.
Application Number | 20160331818 14/963308 |
Document ID | / |
Family ID | 46197273 |
Filed Date | 2016-11-17 |
United States Patent
Application |
20160331818 |
Kind Code |
A1 |
LOPEZ; Marc ; et
al. |
November 17, 2016 |
NOVEL ANTIGEN PEPTIDE AND USES THEREOF
Abstract
The invention relates to antigen peptide derived from the
Nectin4 and its use for preventing and treating cancer.
Inventors: |
LOPEZ; Marc; (Marseille,
FR) ; OLIVE; Daniel; (Marseille, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LOPEZ; Marc
OLIVE; Daniel |
Marseille
Marseille |
|
FR
FR |
|
|
Assignee: |
INSERM (Institut National de la
Sante et de la Recherche Medicale)
Paris
FR
UNIVERSITE D'AIX MARSEILLE
Marseille
FR
INSTITUT JEAN PAOLI & IRENE CALMETTES
Marseille
FR
|
Family ID: |
46197273 |
Appl. No.: |
14/963308 |
Filed: |
December 9, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14122937 |
Feb 18, 2014 |
9266922 |
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PCT/EP2012/060224 |
May 31, 2012 |
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14963308 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 7/06 20130101; C12N
2501/998 20130101; C12N 2510/00 20130101; C07K 14/4748 20130101;
A61K 35/17 20130101; C12N 5/0638 20130101; C07K 16/18 20130101;
A61P 35/00 20180101; C07K 14/78 20130101; A61K 2039/5158 20130101;
A61K 39/0011 20130101; A61K 2039/5156 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C12N 5/0783 20060101 C12N005/0783; A61K 35/17 20060101
A61K035/17 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 1, 2011 |
EP |
11305677.4 |
Claims
1-11. (canceled)
12. A method for producing genetically modified T lymphocytes, said
method comprising the steps of: (a) stimulating PBMCs or tumor
infiltrating lymphocytes obtained from a subject with at least one
antigen peptide, wherein said antigen peptide comprises an amino
acid motif having an amino acid sequence: TABLE-US-00007 (SEQ ID
NO: 2) VLVPPLPSL,
wherein the amino acid sequence of the amino acid motif may differ
from VLVPPLPSL by 1, 2 or 3 amino acids, (b) enriching the
population of T lymphocytes specific for the antigen peptide(s)
used in (a), and (c) optionally cloning said population of T
lymphocytes specific for the antigen peptide(s) used in (a).
13-14. (canceled)
15. A method of treating cancer in a subject in need thereof,
comprising the steps of (a) stimulating PBMCs or tumor infiltrating
lymphocytes obtained from the subject with at least one antigen
peptide, wherein said antigen peptide comprises an amino acid motif
having an amino acid sequence: TABLE-US-00008 (SEQ ID NO: 2)
VLVPPLPSL,
wherein the amino acid sequence of the amino acid motif may differ
from VLVPPLPSL by 1, 2 or 3 amino acids, (b) enriching the
population of genetically modified T lymphocytes specific for the
antigen peptide(s) used in (a), (c) optionally cloning said
population of genetically modified T lymphocytes specific for the
antigen peptide(s) used in (a), and (d) administering to the
subject a therapeutically effective amount of a composition for
adoptive therapy comprising the genetically modified T lymphocytes
produced in (b) or (c).
Description
FIELD OF THE INVENTION
[0001] The invention relates to antigen peptide derived from the
Nectin4 and its use for preventing and treating cancer.
BACKGROUND OF THE INVENTION
[0002] Over the recent years, the understanding of tumor biology
processes lead to the development of targeted therapies like
tyrosine kinase inhibitors or monoclonal antibodies. Recent results
obtained with monoclonal antibodies validated the immunotherapy
approach. Induction of immunity by cancer vaccines is thought to
induce immune memory and thereby prevent tumor development. Many
immunotherapeutic approaches are aimed to stimulate anti tumor CD8+
CTL responses. New cell surface expressed tumor associated antigens
(TAA) that are shared among different tumors and not expressed in
normal tissues are attractive candidates for used in vaccines.
[0003] The inventors recently described a new tumor associated
antigen named Nectin4 [S. Fabre-Lafay et al., 2007] which have been
described as a new TAA in 50%, 49% and 86% of breast, ovarian and
lung carcinomas respectively. Moreover, a soluble form of Nectin4
is found in sera of patients and Nectin4 expression is mainly found
in tumors with adverse prognosis. These characteristics assigned
Nectin4 as a potent candidate for used in vaccination.
[0004] Nectin4 is already known in cancer. For example, patent
application WO2004016799 discloses 9mer peptides obtainable from
the Nectin4 sequence and their possible use as vaccine against
cancer. However, this patent application doesn't disclose
experimental results and doesn't give any information about
relevant peptides.
SUMMARY OF THE INVENTION
[0005] In order to identify new Nectin4 antigens, the inventors
have done a multiplex approach aiming to select the most relevant
peptide antigen candidate using a combination of biochemical
(iTopia Epitope Discovery System), cellular (CEM-T2 cell line) and
algorithmic (SYFPEITHI)-based methods.
[0006] The critical steps of this approach are: 1) peptide
sequence; 2) demonstration of appropriate processing of the peptide
inside cell; 3) peptide binding to the relevant HLA class 1 or
Class 2 molecule; 4) recognition by T cell receptor.
[0007] Based on the Nectin4 sequence, the inventors have
synthesized all the relevant 9-mer peptides (502). Among all the
candidate peptides, they have identified peptides bound with high
avidity to HLA-A2, recognized by T cells and finally efficiently
processed and presented by tumor cells.
[0008] The study led the inventors to identify a new antigen
peptide (SEQ ID NO: 2) named N4-145 that are recognized by
HLA-A*0201-restricted CTL.
[0009] Thus, the invention relates to an antigen peptide comprising
the amino acids motif:
TABLE-US-00001 (SEQ ID No 2) VLVPPLPSL,
[0010] wherein the peptide may differ from 1, 2 or 3
aminoacids.
[0011] Another object of the invention relates to an expression
vector comprising a nucleic acid sequence encoding an antigen
peptide according to the invention.
[0012] Another object of the invention relates to a host cell
comprising an expression vector according to the invention.
[0013] Another object of the invention relates to an antibody or
fragment thereof that binds to the antigen peptide according to the
invention.
[0014] Another object of the invention relates to an MHC/peptide
multimer comprising an antigen peptide according to the
invention.
[0015] Another object of the invention relates to an immunising
composition comprising
[0016] (a) at least one antigen peptide according to the invention
or
[0017] (b) at least one expression vector according to the
invention, or
[0018] (c) at least one host cell according to the invention,
or
[0019] (d) at least one antibody according to the invention, or
[0020] (e) at least one nucleic acid sequence that encodes at least
one antigen peptide according to the invention.
[0021] Another object of the invention relates to a T lymphocyte
that recognizes specifically an antigen peptide according to the
invention.
[0022] Another object of the invention relates to a composition for
adoptive therapy comprising T lymphocytes according to the
invention.
[0023] Another object of the invention relates to a method for
producing said T lymphocytes according to the invention, said
method comprising the steps of
[0024] (a) stimulating PBMCs or tumor infiltrating lymphocytes
obtained from a subject with at least one antigen peptide according
to the invention,
[0025] (b) enriching the population of T lymphocytes specific for
the antigen peptide(s) used in (a),
[0026] (c) optionally cloning said population of T lymphocytes
specific for the antigen peptide(s) used in (a).
[0027] Another object of the invention relates to a method for
monitoring a cancer in a subject in need thereof, comprising
determining the frequency of T lymphocytes that recognize
specifically an antigen peptide according to the invention.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0028] Throughout the specification, several terms are employed and
are defined in the following paragraphs.
[0029] As used herein, the term "Nectin4" denotes a new tumor
associated antigen which is a cell surface expressed adhesion
molecule and belongs to the nectin family [N. Reymond, et al., 2001
and S. Fabre-Lafay, et al., 2005]. Nectins are widely expressed in
tissues and play a key role in different biological processes
during development and adult life [Y. Takai et al., 2003, Y. Takai
et al., 2008, N. Reymond et al., 2004 N. Takai et al., 2008, and
Fournier et al 2010 (med sciences)]. Nectins have been involved in
different pathological processes in humans [D. Pende et al., 2005,
M. Kuramochi et al., 2001 and F. Brancati et al., 2010]. An
exemplary sequence for human nectin4 gene is deposited in the
database under accession number AF426163 (SEQ ID NO. 1).
[0030] As used herein, the term "peptide" refers to an amino acid
sequence having less than 50 amino acids. As used herein, the term
"peptide" encompasses amino acid sequences having less than 50
amino acids, less than 40 amino acids, less than 30 amino acids,
less than 25 amino acids, less than 20 amino acids, less than 15
amino acids or less than 10 amino acids.
[0031] As used herein, the term "antibody" refers to a protein
capable of specifically binding an antigen, typically and
preferably by binding an epitope or antigenic determinant or said
antigen. The term "antibody" also includes recombinant proteins
comprising the binding domains, as well as variants and fragments
of antibodies. Examples of fragments of antibodies include Fv, Fab,
Fab', F(ab')2, dsFv, scFv, sc(Fv)2, diabodies and multispecific
antibodies formed from antibody fragments.
[0032] "Function-conservative variants" as used herein refer to
those in which a given amino acid residue in a protein or enzyme
has been changed (inserted, deleted or substituted) without
altering the overall conformation and function of the polypeptide.
Such variants include protein having amino acid alterations such as
deletions, insertions and/or substitutions. A "deletion" refers to
the absence of one or more amino acids in the protein. An
"insertion" refers to the addition of one or more of amino acids in
the protein. A "substitution" refers to the replacement of one or
more amino acids by another amino acid residue in the protein.
Typically, a given amino acid is replaced by an amino acid with one
having similar properties (such as, for example, polarity, hydrogen
bonding potential, acidic, basic, hydrophobic, aromatic, and the
like). Amino acids other than those indicated as conserved may
differ in a protein so that the percent protein or amino acid
sequence similarity between any two proteins of similar function
may vary and may be, for example, from 70% to 99% as determined
according to an alignment scheme such as by the Cluster Method,
wherein similarity is based on the MEGALIGN algorithm. A
"function-conservative variant" also includes a polypeptide which
has at least 60% amino acid identity as determined by BLAST or
FASTA algorithms, preferably at least 75%, more preferably at least
85%, still preferably at least 90%, and even more preferably at
least 95%, and which has the same or substantially similar
properties or functions as the native or parent protein to which it
is compared. Two amino acid sequences are "substantially
homologous" or "substantially similar" when greater than 80.degree.
A, preferably greater than 85%, preferably greater than 90% of the
amino acids are identical, or greater than about 90%, preferably
greater than 95%, are similar (functionally identical) over the
whole length of the shorter sequence. Preferably, the similar or
homologous sequences are identified by alignment using, for
example, the GCG (Genetics Computer Group, Program Manual for the
GCG Package, Version 7, Madison, Wis.) pileup program, or any of
sequence comparison algorithms such as BLAST, FASTA, etc.
[0033] The term "Major Histocompatibility Complex" (MHC) is a
generic designation meant to encompass the histo-compatibility
antigen systems described in different species including the human
leucocyte antigens (HLA).
[0034] The term "breast cancer" as used herein includes, but is not
limited to, all types of breast cancers at all stages of
progression like metastatic breast cancer or breast carcinomas.
[0035] The term "ovarian cancer" as used herein includes, but is
not limited to, all types of ovarian cancers at all stages of
progression like metastatic ovarian cancer or ovarian
carcinomas.
[0036] The term "lung cancer" as used herein includes, but is not
limited to all types of lung cancers at all stages of progression
like lung carcinomas metastatic lung cancer, non-small cell lung
carcinomas or Small cell lung carcinoma.
[0037] The term "treating" a disorder or a condition refers to
reversing, alleviating or inhibiting the process of one or more
symptoms of such disorder or condition. The term "preventing" a
disorder or condition refers to preventing one or more symptoms of
such disorder or condition.
[0038] As used herein, the term "subject" denotes a mammal, such as
a rodent, a feline, a canine, and a primate. Preferably a subject
according to the invention is a human.
[0039] A "therapeutically effective amount" as used herein is
intended for a minimal amount of active agent which is necessary to
impart therapeutic benefit to a subject. For example, a
"therapeutically effective amount of the active agent" to a subject
is an amount of the active agent that induces, ameliorates or
causes an improvement in the pathological symptoms, disease
progression, or physical conditions associated with the disease
affecting the subject.
[0040] The term "adjuvant" as used herein refers to a compound or a
mixture that may be non-immunogenic when administered in the host
alone, but that augments the host's immune response to an antigen
when administered conjointly with that antigen.
Peptide and Uses Thereof
[0041] A first object of the invention relates to an antigen
peptide comprising the amino acids motif:
TABLE-US-00002 (SEQ ID NO: 2) VLVPPLPSL
[0042] wherein the peptide may differ from 1, 2 or 3
aminoacids.
[0043] In one embodiment of the invention, by "antigen peptide" is
meant a peptide capable of binding to HLA molecule and causing a
cellular or humoral response in a subject.
[0044] In a preferred embodiment of the invention, said antigen
peptide may comprise a specific motif such that the polypeptide
binds an HLA molecule and induces a CTL response.
[0045] In another preferred embodiment of the invention, said
antigen peptide may comprise a specific motif such that the
polypeptide binds an HLA molecule and induces a helper T cell
response.
[0046] In another embodiment of the invention, said antigen
peptides as described here above are HLA-A*0201 restricted.
[0047] In one embodiment of the invention, said antigen peptide is
an amino acid sequence of less than 50 amino acids long that
comprises the amino acid motif SEQ ID NO: 2 as defined here
above.
[0048] In another embodiment of the invention, said antigen peptide
is an amino acid sequence of less than 45 amino acids long that
comprises the amino acid motif SEQ ID NO: 2 as defined here
above.
[0049] In another embodiment of the invention, said antigen peptide
is an amino acid sequence of less than 40 amino acids long that
comprises the amino acid motif SEQ ID NO: 2 as defined here
above.
[0050] In another embodiment of the invention, said antigen peptide
is an amino acid sequence of less than 30 amino acids long that
comprises the amino acid motif SEQ ID NO: 2 as defined here
above.
[0051] In another embodiment of the invention, said antigen peptide
is an amino acid sequence of less than 20 amino acids long that
comprises the amino acid motif SEQ ID NO: 2 as defined here
above.
[0052] In another embodiment of the invention, said antigen peptide
is an amino acid sequence of less than 15 amino acids long that
comprises the amino acid motif SEQ ID NO: 2 as defined here
above.
[0053] In another embodiment of the invention, said antigen peptide
is an amino acid sequence of 10 or 11 amino acids long that
comprises the amino acid motif SEQ ID NO: 2 as defined here
above.
[0054] The invention also encompasses peptides that are
function-conservative variants of antigen peptides comprising SEQ
ID NO: 2 as described here above.
[0055] Typically, the invention encompasses peptides substantially
identical to antigen peptides comprising SEQ ID NO: 2 in which one
or more residues have been conservatively substituted with a
functionally similar residue and which displays the functional
aspects of the antigen peptides comprising SEQ ID NO: 2 as
described here above, i.e. being still able to bind to an HLA
molecule in substantially the same way as a peptide consisting of
the given amino acid sequence.
[0056] Examples of conservative substitutions include the
substitution of one non-polar (hydrophobic) residue such as
isoleucine, valine, leucine or methionine for another, the
substitution of one polar (hydrophilic) residue for another such as
between arginine and lysine, between glutamine and asparagine,
between glycine and serine, the substitution of one basic residue
such as lysine, arginine or histidine for another, or the
substitution of one acidic residue, such as aspartic acid or
glutamic acid or another.
[0057] The term "conservative substitution" also includes the use
of a chemically derivatized residue in place of a non-derivatized
residue. "Chemical derivative" refers to a subject peptide having
one or more residues chemically derivatized by reaction of a
functional side group. Examples of such derivatized molecules
include for example, those molecules in which free amino groups
have been derivatized to form amine hydrochlorides, p-toluene
sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,
chloroacetyl groups or formyl groups. Free carboxyl groups may be
derivatized to form salts, methyl and ethyl esters or other types
of esters or hydrazides. Free hydroxyl groups may be derivatized to
form O-acyl or O-alkyl derivatives. The imidazole nitrogen of
histidine may be derivatized to form N-im-benzylhistidine. Chemical
derivatives also include peptides which contain one or more
naturally-occurring amino acid derivatives of the twenty standard
amino acids. For examples: 4-hydroxyproline may be substituted for
proline; 5-hydroxylysine may be substituted for lysine;
3-methylhistidine may be substituted for histidine; homoserine may
be substituted for serine; and ornithine may be substituted for
lysine.
[0058] According to the invention, the antigen peptides of the
invention can be obtained by synthesizing the peptides according to
the method for peptide synthesis known in the art.
[0059] In another embodiment, the antigen peptides of the invention
may be incorporated into polytopes. Two or more peptides of the
invention can be joined together directly, or via the use of
flanking sequences. Thompson et al., Proc. Natl. Acad. Sci. USA 92
(13): 5845-5849 (1995), teaches the direct linkage of relevant
epitopic sequences. The use of polytopes as vaccines is well known.
See. e.g. Gilbert et al., Nat. Biotechnol. 15 (12): 1280-1284
(1997); Thomson et al., supra; Thomson et al., J. Immunol. 157 (2):
822-826 (1996); Tam et al., J. Exp. Med. 171 (1): 299-306 (1990),
all of which are incorporated by reference. The Tam et al.
reference in particular shows that polytopes, when used in a mouse
model, are useful in generating both antibody and protective
immunity.
[0060] In another embodiment, the antigen peptide according to the
invention may be use in the prevention or treatment of cancer.
[0061] In a preferred embodiment, cancer may be selected from the
group consisting of breast, ovarian or lung cancer.
Vectors, Recombinant Host Cells and Uses Thereof
[0062] Another object of the invention is an expression vector
comprising a nucleic acid sequence encoding an amino sequence
comprising SEQ ID NO: 2 as described here above.
[0063] In one embodiment of the invention, said expression vector
comprises the nucleic acid sequence corresponding to the open
reading frame 433 to 459 of SEQ ID NO: 1.
[0064] According to the invention, expression vectors suitable for
use in the invention may comprise at least one expression control
element operationally linked to the nucleic acid sequence. The
expression control elements are inserted in the vector to control
and regulate the expression of the nucleic acid sequence. Examples
of expression control elements include, but are not limited to, lac
system, operator and promoter regions of phage lambda, yeast
promoters and promoters derived from polyoma, adenovirus,
retrovirus, lentivirus or SV40. Additional preferred or required
operational elements include, but are not limited to, leader
sequence, termination codons, polyadenylation signals and any other
sequences necessary or preferred for the appropriate transcription
and subsequent translation of the nucleic acid sequence in the host
system. It will be understood by one skilled in the art that the
correct combination of required or preferred expression control
elements will depend on the host system chosen. It will further be
understood that the expression vector should contain additional
elements necessary for the transfer and subsequent replication of
the expression vector containing the nucleic acid sequence in the
host system. Examples of such elements include, but are not limited
to, origins of replication and selectable markers. It will further
be understood by one skilled in the art that such vectors are
easily constructed using conventional methods or commercially
available.
[0065] Another object of the invention is a host cell comprising an
expression vector as described here above.
[0066] According to the invention, examples of host cells that may
be used are eukaryote cells, such as animal, plant, insect and
yeast cells and prokaryotes cells, such as E. coli. The means by
which the vector carrying the gene may be introduced into the cells
include, but are not limited to, microinjection, electroporation,
transduction, or transfection using DEAE-dextran, lipofection,
calcium phosphate or other procedures known to one skilled in the
art.
[0067] In a preferred embodiment, eukaryotic expression vectors
that function in eukaryotic cells are used. Examples of such
vectors include, but are not limited to, viral vectors such as
retrovirus, adenovirus, adeno-associated virus, herpes virus,
vaccinia virus, poxvirus, poliovirus; lentivirus, bacterial
expression vectors, plasmids, such as pcDNA3 or the baculovirus
transfer vectors. Preferred eukaryotic cell lines include, but are
not limited to, COS cells, CHO cells, HeLa cells, NIH/3T3 cells,
293 cells (ATCC# CRL1573), T2 cells, dendritic cells, or
monocytes.
Antibodies and Uses Thereof
[0068] Another object of the invention relates to an antibody or
fragment thereof that binds to the antigen peptide according to
invention.
[0069] In one embodiment of the invention, said antibody or
fragment thereof binds to the peptide of SEQ ID NO: 2.
[0070] In one embodiment of the invention, said antibody is
monoclonal. In another embodiment of the invention, said antibody
is polyclonal.
[0071] Such antibodies may be easily prepared, for example,
according to the method described in "Antibodies: A laboratory
manual", Lane H. D. et al. eds, Cold Spring Harbor Laboratory
Press, New York, 1989 or Antibody Engineering: Methods and
Protocols, 2003, Benny K. Lo.
MHC/Peptide Multimer
[0072] Another object of the invention is a MHC/peptide multimer
comprising an antigen peptide as described here above. According to
the invention, said MHC/peptide multimer include, but are not
limited to, a MHC/peptide dimer, trimer, tetramer or pentamer.
[0073] In one embodiment of the invention, said MHC/peptide
multimer is a HLA-A*0201/peptide multimer.
[0074] Methods for obtaining MHC/peptide tetramers are described in
WO96/26962 and WO01/18053, which are incorporated by reference.
[0075] In one embodiment of the invention, said MHC/peptide
multimer can be used to visualise T cell populations that are
specific for the complex HLA-A*0201/antigen peptide as described
here above.
[0076] In another embodiment of the invention, said MHC/peptide
multimer can be used for the detection and/or isolation by
screening (in flow cytometry or by immunomagnetic screening) of T
cell population that are specific for a complex HLA/antigen peptide
as described here above.
[0077] In another embodiment of the invention, said
HLA-A*0201/peptide multimer can be used for the detection and/or
isolation by screening (in flow cytometry or by immunomagnetic
screening) of T cell population that are specific for a complex
HLA-A*201/antigen peptide as described here above.
[0078] Another object of the invention is beads coated with
MHC/peptide multimers as described here above.
Immunising Composition
[0079] Another object of the invention is an immunising composition
comprising
[0080] (a) at least one antigen peptide as described here above
or
[0081] (b) at least one expression vector as described here above,
or
[0082] (c) at least one host cell as described here above, or
[0083] (d) at least one antibody as described here above, or
[0084] (e) at least one nucleic acid sequence that encodes at least
one antigen peptide as described here above.
[0085] In one embodiment, said immunising composition comprises the
antigen peptide N4-145 having the sequence SEQ ID NO: 2.
[0086] The prophylactic administration of the immunising
composition of the invention should serve to prevent or attenuate
cancer in a mammal. In a preferred embodiment mammals, preferably
human, at high risk for cancer are prophylactically treated with
the immunising composition of the invention. Examples of such
mammals include, but are not limited to, humans with a family
history of cancer.
[0087] When provided therapeutically, the immunising composition of
the invention is provided to enhance the patient's own immune
response to the cancer antigen present on the cancer or metastatic
cancer.
[0088] In one embodiment of the invention, the peptides of the
invention may be conjugated with lipoprotein or administered in
liposomal form or with adjuvant.
[0089] In one embodiment, said immunising composition is a
pharmaceutical composition.
[0090] In such embodiment, said immunising composition, for human
use, comprises at least one antigen peptide as described here above
or at least one antibody as described here above, together with one
or more pharmaceutically acceptable carriers and, optionally, other
therapeutic ingredients. The carrier(s) must be "acceptable" in the
sense of being compatible with the other ingredients of the
composition and not deleterious to the recipient thereof. The
immunising compositions may conveniently be presented in unit
dosage form and may be prepared by any method well-known in the
pharmaceutical art.
[0091] Immunising compositions suitable for intravenous,
intradermal, intramuscular, subcutaneous, or intraperitoneal
administration conveniently comprise sterile aqueous solutions of
the active agent with solutions which are preferably isotonic with
the blood of the recipient. Such compositions may be conveniently
prepared by dissolving solid active ingredient in water containing
physiologically compatible substances such as sodium chloride (e.g.
0.1-2.0M), glycine, and the like, and having a buffered pH
compatible with physiological conditions to produce an aqueous
solution, and rendering said solution sterile. These may be present
in unit or multi-dose containers, for example, sealed ampoules or
vials.
[0092] The immunising compositions of the invention may incorporate
a stabilizer. Illustrative stabilizers are polyethylene glycol,
proteins, saccharides, amino acids, inorganic acids, and organic
acids which may be used either on their own or as admixtures. These
stabilizers are preferably incorporated in an amount of 0.11-10,000
parts by weight per part by weight of active agent. If two or more
stabilizers are to be used, their total amount is preferably within
the range specified above. These stabilizers are used in aqueous
solutions at the appropriate concentration and pH. The specific
osmotic pressure of such aqueous solutions is generally in the
range of 0.1-3.0 osmoles, preferably in the range of 0.8-1.2. The
pH of the aqueous solution is adjusted to be within the range of
5.0-9.0, preferably within the range of 6-8.
[0093] Additional pharmaceutical methods may be employed to control
the duration of action. Controlled release preparations may be
achieved through the use of polymer to complex or absorb the
peptides of the invention. The controlled delivery may be exercised
by selecting appropriate macromolecules (for example polyester,
polyamirio acids, polyvinyl, pyrrolidone, ethylenevinylacetate,
methylcellulose, carboxymethylcellulose, or protamine sulfate) and
the concentration of macromolecules as well as the methods of
incorporation in order to control release. Another possible method
to control the duration of action by controlled-release
preparations is to incorporate the antigen peptides of the
invention into particles of a polymeric material such as
polyesters, polyamino acids, hydrogels, poly(lactic acid) or
ethylene vinylaceiate copolymers. Alternatively, instead of
incorporating these agents into polymeric particles, it is possible
to entrap these materials in microcapsules prepared, for example,
by coacervation techniques or by interfacial polymerization, for
example, hydroxy-methylcellulose or gelatin-microcapsules and
poly(methylmethacylate) microcapsules, respectively, or in
colloidal drug delivery systems, for example, liposomes, albumin
microspheres, microemulsions, nanoparticles, and nanocapsules or in
macroemulsions.
[0094] When oral preparations are desired, the compositions may be
combined with typical carriers, such as lactose, sucrose, starch,
talc magnesium stearate, crystalline cellulose, methyl cellulose,
carboxymethyl cellulose, glycerin, sodium alginate or gum arabic
among others.
[0095] Immunisation of a subject with the immunising composition of
the invention can be conducted by conventional methods, for
example, in the presence of conventional adjuvants. Examples of
conventional adjuvant include, but are not limited to, metal salts,
oil in water emulsions, Toll like receptors agonists, saponins,
lipid A, alkyl glucosaminide phosphate, Freund's adjuvant, keyhole
limpet haemocyanin (KLH), mannan, BCG, alum, cytokines such as
IL-1, IL-2, macrophage colony stimulating factor, and tumor
necrosis factor; and other substances that act as immunostimulating
agents such as muramyl peptides or bacterial cell wall components,
toxins, toxoids and TLR ligands.
[0096] The immunising composition can be administered by any route
appropriate for antibody production and/or T cell activation such
as intravenous, intraperitoneal, intramuscular, subcutaneous, and
the like. The immunising composition may be administered once or at
periodic intervals until a significant titre of anti-Nectin4 immune
cells or anti-Nectin4 antibody is produced. The presence of
anti-Nectin4 immune cells may be assessed by measuring the
frequency of precursor CTL (cytoxic T-lymphocytes) against the
antigen peptides of the invention prior to and after immunization
by specific tetramer labelling or by a CTL precursor analysis
assay. The antibody may be detected in the serum using an
immunoassay.
[0097] Antibodies directed to the antigens of the invention can
also be used directly as anti-cancer agents. To prepare antibodies,
a host animal may be immunized using the Nectin4 protein or others
antigen peptides as described here above. The host serum or plasma
is collected following an appropriate time to provide a composition
comprising antibodies reactive to said antigen peptides. The gamma
globulin fraction or the IgG antibodies can be obtained, for
example, by use of saturated ammonium sulfate or DEAE Sephadex, or
other techniques known to those skilled in the art. The antibodies
are substantially free of many of the adverse side effects which
may be associated with other anti-cancer agents such as
chemotherapy.
[0098] The immunising composition of the invention comprising
antibodies as described here above can be made even more compatible
with the host system by minimizing potential adverse immune system
responses. This is accomplished by removing all or a portion of the
Fc portion of a foreign species antibody or using an antibody of
the same species as the host subject, for example, the use of
antibodies from human/human hybridomas. Humanized antibodies (i.e.,
nonimmunogenic in a human) may be produced, for example, by
replacing an immunogenic portion of an antibody with a
corresponding, but nonimmunogenic portion (i.e., chimeric
antibodies). Such chimeric antibodies may contain the reactive or
antigen binding portion of an antibody from one species and the Fc
portion of an antibody (nonimmunogenic) from a different species.
Examples of chimeric antibodies, include but are not limited to,
nonhuman mammal-human chimeras, rodent-human chimeras, murine-human
and rat-human chimeras.
[0099] Methods for obtaining said antibodies, chimeric antibodies
and humanized chimeric antibodies are well-known in the art.
[0100] The immunising composition comprising the antibodies of the
invention can also be used as a means of enhancing the immune
response. The antibodies can be administered in amounts similar to
those used for other therapeutic administrations of antibody. For
example, pooled gamma globulin is administered at a range of about
1 mg to about 100 mg per subject. Thus, antibodies reactive with
the antigen peptides of the invention can be passively administered
alone or in conjunction with other anti-cancer therapies to a
mammal afflicted with cancer. Examples of anti-cancer therapies
include, but are not limited to, chemotherapy, radiation therapy,
adoptive immunotherapy therapy with TIL.
[0101] The antibodies or chimeric antibodies described herein may
also be coupled to toxin molecules, radioisotopes and drugs by
conventional methods. Examples of toxins to which the antibodies
may be coupled to included, but are not limited to, ricin or
diphtheria toxin. Examples of drugs or chemotherapeutic agents
include, but are not limited to, cyclophosphamide or doxorubicin.
Examples of radioisotopes, include, but are not limited to, 1311.
Antibodies covalently conjugated to the aforementioned agents can
be used in cancer immunotherapy for treating cancer.
[0102] If the subject to be immunized is already afflicted with
cancer or metastatic cancer, the immunising composition of the
invention can be administered in conjunction with other therapeutic
treatments. Examples of other therapeutic treatments includes, but
are not limited to, adoptive T cell immunotherapy, coadministration
of cytokines or other therapeutic drugs for cancer.
[0103] The dose of antigen peptide of the invention to be
administered to a subject may be adjusted as appropriate depending
on, for example, the disease to be treated, the age and the body
weight of said subject. Ranges of antigen peptides of the invention
that may be administered are about 0.001 to about 100 mg per
subject, preferred doses are about 0.01 to about 10 mg per
subject.
[0104] The immunising composition of the invention may be evaluated
first in animal models, initially rodents, and in nonhuman primates
and finally in humans. The safety of the immunization procedures is
determined by looking for the effect of immunization on the general
health of the immunized animal (weight change, fever, appetite
behavior etc.) and looking for pathological changes on autopsies.
After initial testing in animals, cancer patients can be tested.
Conventional methods would be used to evaluate the immune response
of the patient to determine the efficiency of the immunising
composition.
[0105] Another object of the invention is an immunising composition
comprising
[0106] (a) at least one antigen peptide as described here above
or
[0107] (b) an expression vector comprising a nucleic acid sequence
encoding a antigen peptide defined in (a) as described here above,
or
[0108] (c) a host cell comprising an expression vector defined in
(b) as described here above, or
[0109] (d) an antibody that recognizes specifically a antigen
peptide defined in (a) as described here above, or
[0110] (e) at least one nucleic acid encoding at least one antigen
peptide of the invention,
[0111] for preventing or treating cancer in a subject in need
thereof.
[0112] In one embodiment, the cancer may be breast, ovarian or lung
cancer.
Antigen Presenting Cell
[0113] Another object of the invention is an antigen presenting
cell comprising a complex HLA antigen and an antigen peptide of the
invention.
[0114] In one embodiment of the invention, said complex HLA antigen
is a HLA-A*0201 antigen.
[0115] In one embodiment of the invention, said antigen presenting
cell is derived from the subject to be treated.
[0116] The term "antigen presenting cell" (APCs) refers to any cell
that expresses an HLA antigen capable of presenting the antigen
peptide of the invention on its surface. Dendritic cells, which are
reported to have an especially high antigen-presenting ability, are
preferred. In another embodiment, artificial APCs may also be used
such mammalian cells (fibroblast, endothelial cells,
keratinocytes), insect cells, or cell lines.
[0117] In order to prepare such APCs of the invention, cells having
an antigen-presenting ability are isolated from the subject to be
treated, and pulsed ex vivo with at least one antigen peptide of
the invention to form a complex with the HLA-A*0201 antigen.
[0118] In case dendritic cells are used, the APC of the invention
can be prepared as follows. Lymphocytes are isolated from
peripheral blood of the subject to be treated by Ficoll method;
adherent cells are separated from non-adherent cells; the adherent
cells are then cultured in the presence of GM-CSF and IL-4 to
induce dendritic cells; and the dendritic cells are pulsed by
culturing with at least one antigen peptide of the invention to
obtain the APCs of the invention. The dendritic cells should be
exposed to the antigen peptide for sufficient time to allow the
antigens to be internalized and presented on the dendritic cells
surface. The resulting dendritic cells can then be re-administrated
to the subject to be treated. Such methods are described in
WO93/208185 and EP0563485, which are incorporated by reference.
[0119] Another object of the invention is a composition for active
immunotherapy comprising antigen presenting cells comprising a
complex HLA antigen and an antigen peptide of the invention.
[0120] In one embodiment of the invention, said antigen presenting
cells comprise a complex HLA-A*0201 antigen and a antigen peptide
of the invention.
[0121] Said APCs may be preferably contained in physiological
saline, phosphate buffered saline (PBS), culture medium, or the
like. Administration may be achieved, for example, intravenously,
hypodermically, or intradermally.
Lymphocytes T and Uses Thereof
[0122] Another object of the invention is a T lymphocyte that
recognizes specifically the antigen peptide of the invention.
[0123] In one embodiment of the invention, said T lymphocyte is a
cytotoxic T lymphocyte.
[0124] In another embodiment of the invention, said T lymphocyte is
HLA-A*0201 restricted.
[0125] In another embodiment of the invention, said T lymphocyte is
a T cell clone.
[0126] In another embodiment, said T lymphocyte is a genetically
modified T lymphocyte that expresses a TCR that recognizes
specifically the antigen peptide of the invention.
[0127] Another object of the invention is a composition for
adoptive therapy comprising said T lymphocytes as described here
above that recognizes specifically the antigen peptide of the
invention.
[0128] In the case of cancer, and preferable in breast, ovarian or
lung cancer, it has been observed that an adoptive immunotherapy
wherein intratumoral T cell infiltrate taken from the subject to be
treated are cultured ex vivo in large quantities, and then returned
into the patient achieves a therapeutic gain.
[0129] It is preferred that the T cells are contained in
physiological saline, phosphate buffered saline (PBS), culture
medium, or the like in order to their stable maintain.
Administration may be achieved, for example, intravenously or
intra-tumoraly. By returning the T cells that recognizes
specifically the antigen peptide of the invention into the
subject's body, the toxicity of said T cells on tumor cells is
enhanced in the patient who is positive for Nectin4. The tumor
cells are destroyed and thereby the treatment of tumor is
achieved.
[0130] Examples of where T-lymphocytes can be isolated, include but
are not limited to, peripheral blood cells lymphocytes (PBL), lymph
nodes, or tumor infiltrating lymphocytes (TIL).
[0131] Such lymphocytes can be isolated from tumor or peripheral
blood of the individual to be treated by methods known in the art
and cultured in vitro. Lymphocytes are cultured in media such as
RPMI or RPMI 1640 for 2-5 weeks, preferably for 2-3 weeks.
Viability is assessed by trypan blue dye exclusion assay. The
lymphocytes are exposed to the antigen peptide of the invention for
all of the culture duration.
[0132] In a preferred embodiment the lymphocytes are exposed to the
antigen peptide of the invention at a concentration of about 1 to
about 10 micrograms(n)/ml per 107 cells for all the duration of
lymphocyte culture. Cytokines may be added to the lymphocyte
culture such as IL-2.
[0133] The antigen peptide of the invention may be added to the
culture in presence of antigen presenting cells such as dendritic
cells or allogeneic irradiated cancer cell line cells.
[0134] After being sensitized to the peptide, the T-lymphocytes are
administered to the subject in need of such treatment.
[0135] Examples of how these sensitized T-cells can be administered
to the mammal include but are not limited to, intravenously,
intraperitoneally or intralesionally. Parameters that may be
assessed to determine the efficacy of these sensitized
T-lymphocytes include, but are not limited to, production of immune
cells in the subject being treated or tumor regression.
Conventional methods are used to assess these parameters. Such
treatment can be given in conjunction with cytokines or gene
modified cells (Rosenberg, S. A. et al. (1992) Human Gene Therapy,
3: 75-90; Rosenberg, S. A. et al. (1992) Human Gene Therapy, 3:
57-73).
[0136] Another object of the invention is a method for producing T
lymphocytes that recognize specifically an antigen peptide of the
invention, said method comprising the steps of:
[0137] (a) stimulating peripheral blood mononuclear cells (PBMCs)
or tumor infiltrating lymphocytes (TIL) obtained from a subject
with at least one antigen peptide of the invention,
[0138] (b) enriching the population of T lymphocytes specific for
the antigen peptide(s) used in (a),
[0139] (c) optionally cloning said population of T lymphocytes
specific for the antigen peptide(s) used in (a).
[0140] Enrichment and/or cloning may be carried out by using an
MHC/peptide multimer as described here above. Cloning may also be
carried out by conventional methods.
[0141] In one embodiment of the invention, the T lymphocytes that
recognize specifically an antigen peptide of the invention are
HLA-A*0201 restricted. In such embodiment, enrichment and/or
cloning may be carried out by using an HLA-A*0201/peptide multimer
as described here above.
[0142] Stimulation of PBMCs may be carried out with at least one
antigen peptide of the invention alone, or presented by an antigen
presenting cell such as dendritic cells or allogeneic irradiated
cancer cell line cells. Typically, cytokines such as IL-2 may also
be added to the culture.
[0143] Another object of the invention is a composition for
adoptive therapy that comprises lymphocytes that recognizes
specifically the antigen peptide of the invention for preventing or
treating cancer in a subject in need thereof, wherein said T
lymphocytes are to be re-administrated to the subject.
[0144] In one embodiment, the cancer may be breast, ovarian or lung
cancer.
[0145] In one embodiment, said lymphocytes that recognizes
specifically the antigen peptide of the invention are HLA-A*0201
restricted.
[0146] The invention also relates to a method for treating cancer
in a subject in need thereof, comprising administering a
therapeutically effective amount of
[0147] (a) at least one antigen peptide as described here above
or
[0148] (b) an expression vector as described here above, or
[0149] (c) a host cell as described here above, or
[0150] (d) an antibody as described here above, or
[0151] (e) at least one nucleic acid encoding at least one antigen
peptide of the invention.
[0152] The invention also relates to a method for treating cancer
in a subject in need thereof, comprising administering a
therapeutically effective amount of T lymphocytes that recognizes
specifically the antigen peptide of the invention. In one
embodiment, said T lymphocytes are HLA-A*0201 restricted.
[0153] The invention also relates to a method for treating cancer
in a subject in need thereof, comprising administering a
therapeutically effective amount of antigen presenting cells
comprising a complex HLA antigen and a antigen peptide of the
invention. In one embodiment, said complex HLA/peptide is a complex
HLA-A*0201/antigen peptide of the invention.
Monitoring Assay
[0154] Another object of the invention is a method for monitoring a
cancer, especially an ovarian, breast or lung cancer in a subject
in need thereof, comprising determining the frequency of T
lymphocytes that recognize specifically a antigen peptide of the
invention.
[0155] In one embodiment of the invention, said T lymphocytes are
HLA-A*0201 restricted.
[0156] In one embodiment of the invention, the frequency of T
lymphocytes that recognize specifically an antigen peptide of the
invention may be determined by using an MHC/peptide multimer as
described here above.
[0157] According to the invention, an increase in the frequency of
T lymphocytes that recognize specifically an antigen peptide of the
invention correlates with relapse prevention.
Kit
[0158] Another object of the invention is a kit comprising: [0159]
an antibody that recognizes specifically the antigen peptide and/or
[0160] primers or probes for Nectin4 mRNA detection, and/or [0161]
an MHC/peptide multimer comprising a antigen peptide of the
invention.
[0162] In one embodiment, said kit further comprises a solid
support, wherein said solid support is selected from the group
consisting of wells of reaction trays, test tubes, polystyrene
beads, strips, membranes and microparticles.
[0163] In another embodiment, said kit further comprises a label,
wherein said label is selected in the group consisting of enzymes,
radioisotopes, fluorescent compounds and chemiluminescent
compounds.
[0164] The invention will be further illustrated by the following
figures and examples. However, these examples and figures should
not be interpreted in any way as limiting the scope of the present
invention.
FIGURES
[0165] FIG. 1: Frequency of peptide binding to 8 HLA alleles. The
502 nonamers overlapping by 8 aa, were tested in the iTopia EDS
system. Cut-off values were as defined by the manufacturer.
[0166] FIGS. 2A-2D: Off-rates and binding curves for selected
peptides binding HLA-A*0201.
[0167] The number associated with peptides represent where the
peptide starts in the protein sequence. Curves are fitted using the
Graphpad's Prism software. A: Peptide titration curves for selected
nectin4 peptides, B: Peptide titration curves for reference
peptides, C: Off-rate curves for selected nectin4 peptide, D:
Off-rate curves for reference peptides.
[0168] FIGS. 3A-3D: HLA stabilization assay using the CEMT2 cell
line was assessed by flow cytometry. Examples of binding and
dissociation histograms of Nectin4 and HBV positive control.
[0169] Binding of peptide 145 (white histogram), peptide 122 (grey
histogram) or no peptide (dotted histogram) (A) and HBV positive
control peptide (white histogram) (B). Kinetic of dissociation for
peptide 145 (C) and HBV positive control peptide (D). T0h (solid
line), T1h (dotted line), T4h (dashed line), peptide 122 (grey
histogram).
[0170] FIGS. 4A-4F: Characterization of anti-nectin4 CTL derived
from healthy PBMC donors.
[0171] Labeling of CTL 21 and CTL 145 with HLA-A2/Nectin4 tetramers
loaded with peptide 21 (A) and peptide 145 (B). CTL 21 and CTL 145
mediated lysis of HLA-A2 nectin4 negative MDA-MB-231 target cells
loaded or not with Nectin4 peptide 21 (4C) or peptide 145 (4E)
respectively. CTL 21 and CTL 145 mediated lysis of HLA-A2 nectin4
expressing MDA-MB-231 target cells loaded or not with Nectin4
peptide 21 (4D) or peptide 145 (4F) respectively.
[0172] FIGS. 5A-5B: Characteristics of N4-145 peptide.
[0173] A: Nectin4 is a type-I transmembrane glycoprotein that
belongs to the immunoglobulin superfamily. Nectin4 exhibits 3
Ig-like domains of V, C, C type in the extracellular region.
Peptide N4-145 localized at the edge of the second Ig-like type C
domain. Transmembrane region is in grey. B: Characteristics of
N4-145 peptide for the 8 different HLA alleles.
TABLE-US-00003 TABLE 1 List of control peptides used in the study.
Peptide No Sequence Origin SEQ ID NO 503 NLSASVATV Proteinase3 3
513 SLLMWITQV NY-ESO-1 4 514 LYVDSLFFL PRAME 5 515 KVAELVHFL
MAGE-A3 6 516 ASSTLYLVF MAGE-C2 7 517 GLYDGMEHL MAGE-A10 8 518
ALKDVEERV MGAE-C2 9 519 YMDGTMSQV Tyrosinase 10 520 VLPDVFIRC GnTV
11
[0174] All these peptides have been referenced and validated as
naturally processed and recognized by specific CTL. Some of these
control peptides are used in pre-clinical or clinical settings.
These peptides were selected to compare with selected nectin4
peptides in the different assays.
TABLE-US-00004 TABLE 2 Selection and binding characteristics of the
22 high rank HLA-A2 nectin4 peptides based on iTopia analysis.
Peptide SEQ % T1/2 Value Affinity Rank No ID NO Sequence Binding
(Hours) (ED50) iScore 1 238 12 HILHVSFLA 87 6.7 6.00E-08 1.426 2
355 13 VIAALLFCL 59 5.2 3.00E-08 0.888 3 354 14 GVIAALLFC 61 6.2
9.00E-08 0.845 4 80 15 ALLHSKYGL 87 7.8 1.00E-06 0.815 5 351 16
VVVGVIAAL 58 3.4 3.00E-08 0.784 6 145 2 VLVPPLPSL 67 7.4 5.00E-07
0.783 7 21 17 LLASFTGRC 91 6 6.00E-07 0.727 8 358 18 ALLFCLLVV 53
6.1 2.00E-07 0.71 9 347 19 SASVVVVGV 64 3.7 2.00E-07 0.708 10 215
20 SMNGQPLTC 81 6.8 7.00E-07 0.689 11 359 21 LLFCLLVVV 67 7.4
5.00E-07 0.674 12 266 22 AMLKCLSEG 74 6.8 7.00E-07 0.667 13 137 23
FQARLRLRV 69 7.3 8.00E-07 0.66 14 19 24 LLLLASFTG 75 2.1 6.00E-08
0.659 15 345 25 LVSASVVVV 51 2.3 6.00E-08 0.626 16 357 26 AALLFCLLV
56 3.8 1.00E-07 0.619 17 35 27 GTSDVVTVV 63 2.9 3.00E-07 0.539 18
255 28 DQNLWHIGR 62 5.4 5.00E-07 0.539 19 244 29 FLAEASVRG 78 2.8
7.00E-07 0.533 20 203 30 AVTSEFHLV 64 3.5 4.00E-07 0.533 21 443 31
RSYSTLTTV 68 2.4 3.00E-07 0.521 22 239 32 ILHVSFLAE 61 2.3 2.00E-07
0.51
TABLE-US-00005 TABLE 3 Summary of iTopia, CEM T2, SYFPEITHI and
BIMAS data obtained for the 22 Nectin4 peptides with iScore
.gtoreq. 0.50. CEM.T2 Peptide SEQ iTopia % % Residual Algorithms No
Sequence ID NO iScore Binding Binding SYFPEITHI BIMAS 19 LLLLASFTG
24 0.659 17 0 16 2 21 LLASFTGRC 17 0.727 44 86 17 4 35 GTSDVVTVV 27
0.539 60 0 20 3 80 ALLHSKYGL 15 0.815 53 86 26 79 137 FQARLRLRV 23
0.66 30 72 13 32 145 VLVPPLPSL 2 0.783 76 54 31 83 203 AVTSEFHLV 30
0.533 48 0 16 11 215 SMNGQPLTC 20 0.689 21 0 16 3 238 HILHVSFLA 12
1.426 37 66 14 0 239 ILHVSFLAE 32 0.51 10 0 14 0 244 FLAEASVRG 29
0.533 12 0 17 1 255 DQNLWHIGR 28 0.539 6 0 1 0 266 AMLKCLSEG 22
0.667 31 39 18 0 345 LVSASVVVV 25 0.626 11 0 22 9 347 SASVVVVGV 19
0.708 40 0 23 2 351 VVVGVIAAL 16 0.784 82 50 24 7 354 GVIAALLFC 14
0.845 10 0 12 5 355 VIAALLFCL 13 0.888 18 24 26 66 357 AALLFCLLV 26
0.619 15 18 20 13 358 ALLFCLLVV 18 0.71 26 26 28 242 359 LLFCLLVVV
21 0.674 7 23 30 412 443 RSYSTLTTV 31 0.521 37 0 17 3
TABLE-US-00006 TABLE 4 Ranking of the best five nectin4 peptides
selected on the basis of the F-score (iTopia and CEM T2).
Comparison with reference peptides listed in table 1. RANK F-Score
No Peptide SEQ ID NO 1 520 (GnTV) 11 2 513 (NY-ESO-1) 4 3 519
(Tyrosinase) 10 4 80 15 5 351 16 6 145 2 7 238 12 8 517 (MAGE-A10)
8 9 503 (PR3) 3 10 515 (MAGE-A3) 6 11 21 17
Example
Material & Methods
[0175] Cell Lines
[0176] MDA-MB-231 breast carcinoma cell line (ATCC, Manassas) was
cultivated in DMEM supplemented with 10% fetal calf serum, 50 IU/ml
penicillin, 50 .mu.g/ml streptomycin, and 2 mM glutamine. Cells
were cultivated in a 5% CO2 atmosphere at constant humidity. The
MDA-MB-231 nectin4 cell line was obtained after transfection with
the p3XFLR4.C1 [S. Fabre et al, 2002].
[0177] The Epstein Barr Virus-transformed B (EBV-B) cell lines were
cultured in IMDM supplemented with 10% fetal calf serum, with 0.24
mM L-asparagine, 0.55 mM L-arginine, 1.5 mM L-glutamine (AAG), 100
U/ml penicillin and 100 .mu.g/ml streptomycin. Human recombinant
IL-2 was purchased from Novartis. Human recombinant IL-4, IL-7 and
GM-CSF were from R&D Systems, MN, USA.
[0178] Peptides
[0179] A library of 502 nonapeptides which span the entire 510
amino acid sequence of human nectin4, was synthesized (JPT Peptide
Technologies GmbH, Berlin Germany). A series of 9 peptides
described in the literature was selected and used as reference
(Table 1). Peptides (purity >80%) were dissolved in DMSO at a
concentration of 10 mM and stored at -20.degree. C. Specific
control peptides for each allele were supplied by the manufacturer
of the iTopia Epitope Discovery System (EDS) (Beckman Coulter Inc.,
San Diego, Calif., USA). The 502 peptides were tested for binding
to eight alleles (HLA-A*0101; HLA-A*0201; HLA-A*0301; HLA-A*1101;
HLA-A*2402; HLA-A*0702; HLA-B*0801; HLA-B*1501) with the iTopia
EDS.
[0180] MHC Peptide Binding Assay Using iTopia EDS
[0181] Microtiter 96 well plates containing HLA class I MHC monomer
loaded with the human .beta.2-microglobulin and a "placeholder"
peptide were used to evaluate peptide binding, affinity, and
off-rate.
[0182] The 502 nectin4 nonapeptides were first evaluated for their
ability to bind to each MHC molecule in the peptide binding assay.
In this assay, the placeholder peptides were stripped from the MHC
class I monomer complexes coated on plastic, which leaves the MHC
class I heavy chain available as a binding partner for candidate
peptides in the presence of human .beta.2-microglobulin.
[0183] Peptide binding was performed at 21.degree. C. for 18 h.
Revelation was done using an anti HLA-ABC-FITC mAb that binds to
refolded MHC complex conformation. The relative fluorescent
intensity was read using a fluorimeter (Molecular Devices,
SpectraMax Gemini, Sunnyvale, Calif., USA).
[0184] Peptides with a binding >30% of the positive control
peptide were selected as "binder" and subsequently analyzed for
affinity (ED50) and off-rate (T1/2).
[0185] Off-Rate Assay
[0186] This assay evaluates the dissociation rate of "binder"
peptides as a function of time. As for binding assay, peptides were
incubated with MHC class I monomer at 21.degree. C. for 18 h.
Plates were then incubated at 37.degree. C. and the MHC
classI/peptide/.beta.2-microglobulin complex was measured at 0.5,
1, 1.5, 2, 4, 6, 8 hours under agitation. The t1/2 value
corresponds to the time in hours required to reach a 50% reduction
of fluorescence intensity, i.e., a 50% of complex dissociation.
[0187] Affinity Assay
[0188] "Binder" peptides were incubated with MHC class I monomer at
21.degree. C. for 18 h at concentrations ranging from 10-4 to 10-9
M. Affinity (ED50 value) is expressed as the peptide concentration
that gives 50% of the optimal binding.
[0189] iScore Calculation
[0190] The iTopia system software was used to include, binding,
off-rate (T1/2 value) and affinity (ED50 value) data collected for
each peptide, in a multiparametric score called iScore. The
corresponding graphs were generated using the GraphPad Prism.RTM..
Peptides with iScore >0.50 were considered as good candidates
for HLA A*0201 binding.
[0191] Cellular Binding Assay
[0192] The CEM.T2 cell line (T2) is defective in the processing of
proteins for presentation by MHC class I molecules. This cell line
expresses HLA A*0201 but is deficient for TAP transporter. T2 is
classically used to assess the Peptide/HLA-A*0201 Complex
Stability[H. W. Nijman et al., 1993]
[0193] For HLA-A2*0201 stabilization, T2 cells (2.times.105/well)
were incubated in serum-free RPMI (Gibco.RTM.-France) with 100
.mu.M of selected peptide, at 37.degree. C. for 4 h. Cells were
washed two times with PBS supplemented with 1% BSA and stained with
fluorescein isothiocyanate-conjugated anti HLA-A2 (Clone BB7.2) (BD
Bioscience Pharmingen-France-) at 4.degree. C. for 45 min. After 3
washes, cells were fixed in PBS supplemented with 2%
paraformaldehyde and analyzed by FACS (FACScan, BD Bioscience). The
percentage of peptide binding was calculated as follows:
% Binding={(MFI peptide sample-MFI Neg control)/(MFI Pos
control-MFI Neg control)}.times.100
[0194] The peptide FLPSDFFPSV from Hepatitis B Virus [I. A.
Doytchinova et al., 2004] was used as positive control peptide.
Negative control was done without peptide.
[0195] Cellular Dissociation Assay
[0196] After HLA-A2*0201 stabilization with peptides at 37.degree.
C. for 4 h, T2 cells were incubated for 1 h, in serum-free RPMI
containing Brefeldin A (10 .mu.g/ml), an inhibitor of the transport
of secretory and lysosomal proteins [G. J. Strous et al., 1993].
After 3 washes, T2 cells were incubated for 3 h in serum-free RPMI
at 37.degree. C. Binding was then measured by FACS and after 4 h
incubation. Percentage of Residual Binding (% RB) was calculated as
follows:
% RB = 100 - ( MFI peptide sample - MFI Neg control ) T 0 - ( MFI
peptide sample - MFI Neg control ) T 4 ( MFI Pos control - MFI Neg
control ) T 0 - ( MFI Pos control - MFI Neg control ) T 4 .times.
100 ##EQU00001##
[0197] Multimer Production
[0198] Recombinant HLA-A*0201 molecules were folded in vitro with
.beta.2-microglobulin and nectin4 peptides and the EBV lytic cycle
antigen BMLF1 peptide; VSDGGPNLY [M. DiBrino, et el., 1993].
Purification was achieved by gel fitration. Biotinylated tetramers
were then labeled with either Extravidin-PE (Sigma, St Louis,
Mont., USA) for the HLA-A2/Nectin4 multimers, or streptavidin-APC
(Molecular Probes, Eugen, Oreg., USA) for the HLA-A2/EBV multimer
control.
[0199] Generation and Stimulation of Dendritic Cells
[0200] PBMC from HLA-A2+ healthy volunteers were isolated by
Ficoll-Plaque density gradient centrifugation. To generate
autologous dendritic cells. PBMC were left to adhere for 1 h at
37.degree. C. in culture flasks in RPMI 1640 supplemented with
L-Arginine, L-Asparagine L-Glutamine (AAG), 1%
streptomycin/penicillin and 10% FBS (complete RPMI medium).
Non-adherent cells were removed and cultivated in IMDM supplemented
with AAG, 1% streptomycin/penicillin, 10% human serum and IL-2 (5
U/ml) for 6 days. Adherent-cells were cultured in presence of
GM-CSF (70 ng/ml) and IL-4 (200 U/ml) in complete RPMI medium.
Cultures were fed on days 2 and 4 by removing 1/3 of the volume and
adding fresh medium with cytokines.
[0201] Autologous monocyte-derived immature dendritic cells were
incubated for 6 h with 10 .mu.g/ml of a nectin4 peptide pool
comprising LLASFTGRC (Pep No: 21), ALLHSKYGL (Pep No: 80),
VLVPPLPSL (Pep No: 145), VVVGVIAAL (Pep No: 351), IL-4 (200 U/ml),
GM-CSF (70 ng/ml), in presence of 1 .mu.g/ml of Immucytal.RTM.
granulare (Pierre Fabre Medicament Production, Gien, France) and
500 U/ml of INF-.gamma. (R&D Systems). Dendritic cells were
used to sustain activation of sorted lymphocytes.
[0202] Isolation of CTL Populations
[0203] Non-adherent cells (10.times.108 cells/ml) were incubated
for 15 min at room temperature with HLA-A2 multimers loaded with
Nectin4 peptides (20 nM).
[0204] Multimer-labeled cells were incubated at 4.degree. C. with
anti-PE microbeads as recommended by the manufacturer (Miltenyi
Biotec, Bergisch-Gladbach, Germany). After three washes, cells were
magnetically sorted by AUTOMACS.TM. (Miltenyi Biotec). Efficacy of
cell sorting was measured on a FACS CANTO.TM. II (Becton-Dickinson,
Calif., USA). Sorted cells were incubated in 96 U-bottomed plates
(2.times.104/200 .mu.l) in IMDM medium containing IL-2 (20 U/ml),
IL-7 (10 ng/ml) and 1.104 peptide pulsed irradiated autologous
dendritic cells [S. Ottaviani, et al., 2005].
[0205] Cytotoxicity Assay
[0206] CTL cells were tested for cytolytic activity against the
indicated target cells in a 4 h 51 Cr-release assay as previously
described [C. Bottino et al., 2001]. The E/T ratios are indicated
in the text.
[0207] Results
[0208] Identification of Nectin4 Peptides that Bind MHC Class I
Alleles.
[0209] Nectin4 protein is a 510 aminoacids long protein. Screening
of 502 nectin4 nonapeptides, spanning the entire sequence, was
carried out on 8 different alleles (HLA-A*0101; HLA-A*0201;
HLA-A*0301; HLA-A*1101; HLA-A*2402; HLA-A*0702; HLA-B*0801;
HLA-B*1501) using the iTopia EDS. Peptide binding was analyzed
according to manufacturer recommendations, i.e., with an arbitrary
cut-off of 30% relative to the binding of allele specific control
peptides. The number of positive peptides over 502 tested was
represented for each allele in FIG. 1. We identified 5 HLA-A*0101,
130 HLA-A*0201, 24 HLA-A*0301, 38HLA-A*1101, 77 HLA-A*2402, 28
HLA-B*0702, 14 HLA-B*0801, 40 HLA-B*1501 binding peptides.
Percentage of binding peptides ranged from 1% for HLA-A*0101 to 25%
for HLA-A*0201. Interestingly, we found that the greatest rates of
peptides binding are in HLA-A*0201, HLA-A*2402 and HLA-B*1501 with
respectively 130, 77, 40 positive peptides. These alleles have the
similar binding motif. We decided to focus our study on HLA-A*0201
which represents the most frequent allele in US and Caucasian
populations. As shown, in FIG. 1, we found 130 nectin4 peptides
with relative binding affinity higher than 30% of HLA-A*0201
positive control peptides. In order to select the best "binders",
the cut-off value was increased to 50% and 63 peptides were
selected for the study.
[0210] Determination of Off-Rate Stability and Binding Affinity of
the Selected Nectin4 Peptides.
[0211] Analysis of binding affinity revealed that the peptides 21,
80, 145 present affinity close to the positive control peptide
(POS) (FIG. 2A) and better than the selected reference TAA peptides
(FIG. 2B). Indeed, over a peptide concentration ranging from 10-4
to 10-9 M, the estimated dose to reach 50% maximal binding (ED50)
was around 10-7 M for these three peptides.
[0212] The relative stability of each complex was evaluated for a
period of 8 h as described in material and methods. Off-rate
stability was determined by measuring the dissociation time of the
complex (t1/2). Three groups were found according to the t1/2
value. Among the 63 peptides, 16 have a t1/2>4 h, 19 a t1/2
between 2 h and 4 h, and 28 a t1/2<2 h. Decay curves are
represented in FIGS. 2C and 2D. t1/2 of five representative nectin4
peptides is showed and compared with the positive control peptide
(POS) (FIG. 2C) and the reference peptides listed in table 1 (FIG.
2D). Among the 16 peptides with a t1/2 greater than 4 h, we found 3
nectin4 peptides (21, 80, 145) with a t1/2 over 6 hours. These
values are close to the t1/2 of the positive control peptide and
the reference peptides 513, 517, 519 (FIGS. 2C and 2D). Peptide 351
(t1/2=3.4 h) and peptide 229 (t1/2=1.7 h) represent peptides with
intermediate and low off-rate stability, respectively.
[0213] The iScore is dedicated to define the overall peptide
binding properties to HLA alleles. This multiparametric analysis
includes binding, off-rate and affinity of the peptide/HLA complex.
The 63 peptides studied for HLA-A*0201 were classified in three
groups according to manufacturer recommendations: the first group
including 23 peptides with a low iScore .ltoreq.0.25; the second
group including 18 peptides with a iScore between 0.25 and 0.50;
the third group includes 22 peptides with an iScore .gtoreq.0.50.
These 22 Nectin4 peptides can be considered as "good binders"
(Table 2).
[0214] T2 Cell Binding and Dissociation Assay.
[0215] Because T2 cells lack TAP molecules, the HLA-A*0201 class I
level on the cell surface of T2 cells is low. In the presence of
binding peptides, HLA-A*0201 class I is stabilized, leading to
up-regulation on the cell surface. This assay is highly relevant to
peptide association with MHC class I molecule. However, this assay
cannot be used for large screening. For this reason, we first
restricted analysis on the 22 HLA-A*0201 class 1 binding peptides
selected from iTopia assay (Table 2). As shown in FIG. 3A, the
peptide 145 (white histogram) leads to increase the HLA-A*0201
class I level on T2 cells. The peptide 122, with a low iScore, does
not increase HLA level on T2 cells (FIG. 3A, grey histogram).
Positive control HBV peptide stabilized as well as peptide 145
(FIG. 3B, white histogram). In addition, dissociation assay was
performed in the presence of Brefeldin A as described in material
and methods. This assay is exemplified in FIGS. 3C and 3D for
peptide 145 and control HBV peptide, respectively. Among the 22
peptide tested, peptides 80, 145, 351 have a binding score higher
than 50% of positive control and a high residual binding. Overall
T2 assay results are presented in table 3.
[0216] Integration of Overall Results: Ranking of Nectin4
Epitopes.
[0217] Table 3 presents the different score related to the 22
selected peptides i.e.the iScore obtained with iTopia EDS, the T2
assay results that integrate T2 binding and T2 dissociation results
and the SYFPEITHI and BIMAS score.
[0218] Overall ranking of each peptide according to iTopia and
cellular assays (F-Score) is shown in table 4. Ranking also
includes control reference peptides presented in table 1. Peptides
80, 351, 145, 238 are ranked to the 4th, 5th, 6th, 7th position
respectively. These peptides present binding characteristics close
to the well characterized GnTV, NY-ESO-1 and tyrosinase peptides
used in clinics.
[0219] Generation and Detection of CTL Clones with HLA-A*0201
Tetramers Folded with Nectin4 Peptides
[0220] According to the above results, we then developed an
approach to characterize functional anti-nectin4 CTL from PBMC of
normal donors. Phycoerythrin (PE) labeled HLA-A*0201/nectin4
tetramers were used to select specific CTL by immuno-magnetic cell
sortings. After 21 days of culture, cells were screened with PE
labeled tetramers (for example see FIGS. 4A and 4B).
Tetramer-positive cells were tested for their capacity to kill
nectin4 expressing tumor cells. Four out of five peptides selected
and presented in table 4 were tested (80, 351, 145, 21). We used
the MDA-MB-231 breast carcinoma cell line, which is HLA-A*0201 and
does not express nectin4 as well as the MDA-MB-231 cell line
transfected with Nectin4 plasmid. All the CTL isolated from the
different tetramer/peptide were able to lyse the cell line when
loaded with the respective peptides (see example FIG. 4C). Among
the five different CTL, only the CTL isolated from
HLA-A2/nectin4-145 peptide were able to kill the MDA-MB-231 cell
line expressing nectin4 but not the wild-type MDA-MB-231 cells
(FIG. 4D). These results lead to the identification of a naturally
processed and HLA-A2 presented nectin4 peptide recognized by CD8+ T
lymphocytes. Peptide 145 sequence (VLVPPLPSL) is located at the
beginning of the second Ig-like domain (FIG. 5A). Interestingly,
iTopia EDS and SYFPEITHI analyzes highlight that this peptide
behave as a "strong binder" candidate for HLA-A*0201, HLA-A*2402,
HLA-B*1501 (FIG. 5B). This is of first importance regarding its
potential use in clinical trials.
REFERENCES
[0221] Throughout this application, various references describe the
state of the art to which this invention pertains. The disclosures
of these references are hereby incorporated by reference into the
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that trans-interacts with nectin1/PRR1 through V domain
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a new histological and serological tumor associated marker for
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Fabre-Lafay, S. Garrido-Urbani, N. Reymond, A. Goncalves, P.
Dubreuil, et M. Lopez, "Nectin-4, a new serological breast cancer
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"Prominent role of the Ig-like V domain in trans-interactions of
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2008.
Sequence CWU 1
1
3213520DNAHomo sapiens 1acctgttctg acctgctgag caggttccca ggtttctgcc
gtcgttgttg gccacagcgt 60gggaagcagc tctgggggag ctcggagctc ccgatcacgg
cttcttgggg gtagctacgg 120ctgggtgtgt agaacggggc cggggctggg
gctgggtccc ctagtggaga cccaagtgcg 180agaggcaaga actctgcagc
ttcctgcctt ctgggtcagt tccttattca agtctgcagc 240cggctcccag
ggagatctcg gtggaacttc agaaacgctg ggcagtctgc ctttcaacca
300tgcccctgtc cctgggagcc gagatgtggg ggcctgaggc ctggctgctg
ctgctgctac 360tgctggcatc atttacaggc cggtgccccg cgggtgagct
ggagacctca gacgtggtaa 420ctgtggtgct gggccaggac gcaaaactgc
cctgcttcta ccgaggggac tccggcgagc 480aagtggggca agtggcatgg
gctcgggtgg acgcgggcga aggcgcccag gaactagcgc 540tactgcactc
caaatacggg cttcatgtga gcccggctta cgagggccgc gtggagcagc
600cgccgccccc acgcaacccc ctggacggct cagtgctcct gcgcaacgca
gtgcaggcgg 660atgagggcga gtacgagtgc cgggtcagca ccttccccgc
cggcagcttc caggcgcggc 720tgcggctccg agtgctggtg cctcccctgc
cctcactgaa tcctggtcca gcactagaag 780agggccaggg cctgaccctg
gcagcctcct gcacagctga gggcagccca gcccccagcg 840tgacctggga
cacggaggtc aaaggcacaa cgtccagccg ttccttcaag cactcccgct
900ctgctgccgt cacctcagag ttccacttgg tgcctagccg cagcatgaat
gggcagccac 960tgacttgtgt ggtgtcccat cctggcctgc tccaggacca
aaggatcacc cacatcctcc 1020acgtgtcctt ccttgctgag gcctctgtga
ggggccttga agaccaaaat ctgtggcaca 1080ttggcagaga aggagctatg
ctcaagtgcc tgagtgaagg gcagccccct ccctcataca 1140actggacacg
gctggatggg cctctgccca gtggggtacg agtggatggg gacactttgg
1200gctttccccc actgaccact gagcacagcg gcatctacgt ctgccatgtc
agcaatgagt 1260tctcctcaag ggattctcag gtcactgtgg atgttcttga
cccccaggaa gactctggga 1320agcaggtgga cctagtgtca gcctcggtgg
tggtggtggg tgtgatcgcc gcactcttgt 1380tctgccttct ggtggtggtg
gtggtgctca tgtcccgata ccatcggcgc aaggcccagc 1440agatgaccca
gaaatatgag gaggagctga ccctgaccag ggagaactcc atccggaggc
1500tgcattccca tcacacggac cccaggagcc agccggagga gagtgtaggg
ctgagagccg 1560agggccaccc tgatagtctc aaggacaaca gtagctgctc
tgtgatgagt gaagagcccg 1620agggccgcag ttactccacg ctgaccacgg
tgagggagat agaaacacag actgaactgc 1680tgtctccagg ctctgggcgg
gccgaggagg aggaagatca ggatgaaggc atcaaacagg 1740ccatgaacca
ttttgttcag gagaatggga ccctacgggc caagcccacg ggcaatggca
1800tctacatcaa tgggcgggga cacctggtct gacccaggcc tgcctccctt
ccctaggcct 1860ggctccttct gttgacatgg gagattttag ctcatcttgg
gggcctcctt aaacaccccc 1920atttcttgcg gaagatgctc cccatcccac
tgactgcttg acctttacct ccaacccttc 1980tgttcatcgg gagggctcca
ccaattgagt ctctcccacc atgcatgcag gtcactgtgt 2040gtgtgcatgt
gtgcctgtgt gagtgttgac tgactgtgtg tgtgtggagg ggtgactgtc
2100cgtggagggg tgactgtgtc cgtggtgtgt attatgctgt catatcagag
tcaagtgaac 2160tgtggtgtat gtgccacggg atttgagtgg ttgcgtgggc
aacactgtca gggtttggcg 2220tgtgtgtcat gtggctgtgt gtgacctctg
cctgaaaaag caggtatttt ctcagacccc 2280agagcagtat taatgatgca
gaggttggag gagagaggtg gagactgtgg ctcagaccca 2340ggtgtgcggg
catagctgga gctggaatct gcctccggtg tgagggaacc tgtctcctac
2400cacttcggag ccatgggggc aagtgtgaag cagccagtcc ctgggtcagc
cagaggcttg 2460aactgttaca gaagccctct gccctctggt ggcctctggg
cctgctgcat gtacatattt 2520tctgtaaata tacatgcgcc gggagcttct
tgcaggaata ctgctccgaa tcacttttaa 2580tttttttctt ttttttttct
tgccctttcc attagttgta ttttttattt atttttattt 2640ttattttttt
ttagagatgg agtctcacta tgttgctcag gctggccttg aactcctggg
2700ctcaagcaat cctcctgcct cagcctccct agtagctggg actttaagtg
tacaccactg 2760tgcctgcttt gaatccttta cgaagagaaa aaaaaaatta
aagaaagcct ttagatttat 2820ccaatgttta ctactgggat tgcttaaagt
gaggcccctc caacaccagg gggttaattc 2880ctgtgattgt gaaaggggct
acttccaagg catcttcatg caggcagccc cttgggaggg 2940cacctgagag
ctggtagagt ctgaaattag ggatgtgagc ctcgtggtta ctgagtaagg
3000taaaattgca tccaccattg tttgtgatac cttagggaat tgcttggacc
tggtgacaag 3060ggctcctgtt caatagtggt gttggggaga gagagagcag
tgattataga ccgagagagt 3120aggagttgag gtgaggtgaa ggaggtgctg
ggggtgagaa tgtcgccttt ccccctgggt 3180tttggatcac taattcaagg
ctcttctgga tgtttctctg ggttggggct ggagttcaat 3240gaggtttatt
tttagctggc ccacccagat acactcagcc agaataccta gatttagtac
3300ccaaactctt cttagtctga aatctgctgg atttctggcc taagggagag
gctcccatcc 3360ttcgttcccc agccagccta ggacttcgaa tgtggagcct
gaagatctaa gatcctaaca 3420tgtacatttt atgtaaatat gtgcatattt
gtacataaaa tgatattctg tttttaaata 3480aacagacaaa acttgttctg
tcaaaaaaaa aaaaaaaaaa 352029PRTHomo sapiens 2Val Leu Val Pro Pro
Leu Pro Ser Leu 1 5 39PRTHomo sapiens 3Asn Leu Ser Ala Ser Val Ala
Thr Val 1 5 49PRTHomo sapiens 4Ser Leu Leu Met Trp Ile Thr Gln Val
1 5 59PRTHomo sapiens 5Leu Tyr Val Asp Ser Leu Phe Phe Leu 1 5
69PRTHomo sapiens 6Lys Val Ala Glu Leu Val His Phe Leu 1 5
79PRTHomo sapiens 7Ala Ser Ser Thr Leu Tyr Leu Val Phe 1 5
89PRTHomo sapiens 8Gly Leu Tyr Asp Gly Met Glu His Leu 1 5
99PRTHomo sapiens 9Ala Leu Lys Asp Val Glu Glu Arg Val 1 5
109PRTHomo sapiens 10Tyr Met Asp Gly Thr Met Ser Gln Val 1 5
119PRTHomo sapiens 11Val Leu Pro Asp Val Phe Ile Arg Cys 1 5
129PRTHomo sapiens 12His Ile Leu His Val Ser Phe Leu Ala 1 5
139PRTHomo sapiens 13Val Ile Ala Ala Leu Leu Phe Cys Leu 1 5
149PRTHomo sapiens 14Gly Val Ile Ala Ala Leu Leu Phe Cys 1 5
159PRTHomo sapiens 15Ala Leu Leu His Ser Lys Tyr Gly Leu 1 5
169PRTHomo sapiens 16Val Val Val Gly Val Ile Ala Ala Leu 1 5
179PRTHomo sapiens 17Leu Leu Ala Ser Phe Thr Gly Arg Cys 1 5
189PRTHomo sapiens 18Ala Leu Leu Phe Cys Leu Leu Val Val 1 5
199PRTHomo sapiens 19Ser Ala Ser Val Val Val Val Gly Val 1 5
209PRTHomo sapiens 20Ser Met Asn Gly Gln Pro Leu Thr Cys 1 5
219PRTHomo sapiens 21Leu Leu Phe Cys Leu Leu Val Val Val 1 5
229PRTHomo sapiens 22Ala Met Leu Lys Cys Leu Ser Glu Gly 1 5
239PRTHomo sapiens 23Phe Gln Ala Arg Leu Arg Leu Arg Val 1 5
249PRTHomo sapiens 24Leu Leu Leu Leu Ala Ser Phe Thr Gly 1 5
259PRTHomo sapiens 25Leu Val Ser Ala Ser Val Val Val Val 1 5
269PRTHomo sapiens 26Ala Ala Leu Leu Phe Cys Leu Leu Val 1 5
279PRTHomo sapiens 27Gly Thr Ser Asp Val Val Thr Val Val 1 5
289PRTHomo sapiens 28Asp Gln Asn Leu Trp His Ile Gly Arg 1 5
299PRTHomo sapiens 29Phe Leu Ala Glu Ala Ser Val Arg Gly 1 5
309PRTHomo sapiens 30Ala Val Thr Ser Glu Phe His Leu Val 1 5
319PRTHomo sapiens 31Arg Ser Tyr Ser Thr Leu Thr Thr Val 1 5
329PRTHomo sapiens 32Ile Leu His Val Ser Phe Leu Ala Glu 1 5
* * * * *