U.S. patent application number 14/063164 was filed with the patent office on 2014-08-14 for recombinant adenylate cyclase toxin of bordetella induces t cell responses against tumoral antigens.
This patent application is currently assigned to Ludwig Institute for Cancer. The applicant listed for this patent is Cecil Bauche, Gilles DADAGLIO, Daniel Ladant, Claude Leclerc, Sandra Morel, Benoit Van Den Eynde. Invention is credited to Cecil Bauche, Gilles DADAGLIO, Daniel Ladant, Claude Leclerc, Sandra Morel, Benoit Van Den Eynde.
Application Number | 20140227323 14/063164 |
Document ID | / |
Family ID | 34652257 |
Filed Date | 2014-08-14 |
United States Patent
Application |
20140227323 |
Kind Code |
A1 |
DADAGLIO; Gilles ; et
al. |
August 14, 2014 |
RECOMBINANT ADENYLATE CYCLASE TOXIN OF BORDETELLA INDUCES T CELL
RESPONSES AGAINST TUMORAL ANTIGENS
Abstract
An immunogenic composition comprising a recombinant protein
comprising a Bordetella CyaA, or a fragment thereof, and a peptide
that corresponds to a tumor antigen is provided as a cancer
treatment. Methods of treatment with this immunogenic composition
are also provided. In an embodiment, the therapeutic composition is
a treatment for melanoma, and comprises epitopes from the HLA*0201
epitope. These epitopes include Tyr or GnT-V, and are present in
the recombinant proteins CyaA-E5-Tyr and CyaA-E5-GnT-V.
Inventors: |
DADAGLIO; Gilles;
(Chatillon, FR) ; Leclerc; Claude; (Paris, FR)
; Ladant; Daniel; (Cachan, FR) ; Van Den Eynde;
Benoit; (Genval, FR) ; Morel; Sandra;
(Bruxelles, BE) ; Bauche; Cecil; (Paris,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DADAGLIO; Gilles
Leclerc; Claude
Ladant; Daniel
Van Den Eynde; Benoit
Morel; Sandra
Bauche; Cecil |
Chatillon
Paris
Cachan
Genval
Bruxelles
Paris |
|
FR
FR
FR
FR
BE
FR |
|
|
Assignee: |
Ludwig Institute for Cancer
New York
NY
Institut Pasteur
Paris Cedex 15
|
Family ID: |
34652257 |
Appl. No.: |
14/063164 |
Filed: |
October 25, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12731198 |
Mar 25, 2010 |
8465208 |
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14063164 |
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10994204 |
Nov 22, 2004 |
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12731198 |
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60523632 |
Nov 21, 2003 |
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Current U.S.
Class: |
424/240.1 ;
424/253.1; 435/188 |
Current CPC
Class: |
F16C 27/066 20130101;
A61K 38/164 20130101; F16C 33/7886 20130101; A61K 2039/6037
20130101; A61P 37/04 20180101; A61K 39/0011 20130101; F16C 19/06
20130101; A61K 47/646 20170801; C12Y 406/01001 20130101; A61K 39/39
20130101; A61K 39/099 20130101; A61K 47/6415 20170801; A61P 31/12
20180101; A61P 37/00 20180101; A61P 35/00 20180101; F16C 25/083
20130101; A61K 2039/6068 20130101; C12N 9/88 20130101 |
Class at
Publication: |
424/240.1 ;
424/253.1; 435/188 |
International
Class: |
A61K 39/02 20060101
A61K039/02 |
Claims
1. An immunogenic composition comprising a recombinant protein,
wherein the recombinant protein comprises a Bordetella CyaA, or a
fragment thereof, and a peptide that corresponds to a tumor
antigen.
2. The immunogenic composition as claimed in claim 1, wherein the
Bordetella CyaA, or fragment thereof, and the peptide are
genetically fused or chemically bound together.
3. The immunogenic composition as claimed in claim 1, wherein the
Bordetella CyaA is detoxified.
4. The immunogenic composition as claimed in claim 1, wherein the
tumor is a melanoma.
5. The immunogenic composition as claimed in claim 1, wherein the
tumor antigen is an HLA*0201 epitope.
6. The immunogenic composition as claimed in claim 5, wherein the
HLA*0201 epitope is Tyr or GnT-V.
7. The immunogenic composition as claimed in claim 5, wherein the
HLA*0201 epitope is region 369-377 of tyrosinase.
8. The immunogenic composition as claimed in claim 5, wherein the
HLA*0201 epitope is from an intron of the
N-acetylglucosominyl-transferase V gene.
9. The immunogenic composition as claimed in claim 5, wherein the
HLA*0201 epitope is YMDGTMSQV.
10. The immunogenic composition as claimed in claim 5, wherein the
HLA*0201 epitope is VLPDVFIRC.
11-22. (canceled)
23. A method of treating a patient with cancer comprising (1)
administering a immunogenic composition to the patient, wherein the
immunogenic composition comprises a recombinant protein, wherein
the recombinant protein comprises a Bordetella CyaA, or a fragment
thereof, and a peptide that corresponds to a tumor antigen, and (2)
inducing a T cell response in the patient.
24. The method as claimed in claim 23, wherein the T cell response
is a CTL response.
25. The method as claimed in claim 23, wherein the cancer is a
melanoma.
26. The method as claimed in claim 23, wherein the tumor antigen is
an HLA*0201 epitope.
27. The method as claimed in claim 23, wherein the HLA*0201 epitope
is Tyr or GnT-V.
28-33. (canceled)
34. A method of treating a patient with cancer comprising (1)
administering an immunogenic composition to the patient, wherein
the immunogenic composition comprises a vector expressing a
recombinant protein, wherein the recombinant protein comprises a
Bordetella CyaA, or a fragment thereof, and a peptide that
corresponds to a tumor antigen, and (2) inducing a T cell response
in the patient.
35. The method as claimed in claim 34, wherein the T cell response
is a CTL response.
36. The method as claimed in claim 34, wherein the cancer is a
melanoma.
37. The method as claimed in claim 34, wherein the tumor antigen is
an HLA*0201 epitope.
38. The method as claimed in claim 34, wherein the HLA*0201 epitope
is Tyr or GnT-V.
39-44. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The application claims the benefit of priority of U.S.
Provisional Application No. 60/523,632 (attorney docket number
03495-6095), filed Nov. 21, 2003, which is incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The invention relates to a recombinant adenylate cyclase
toxin of Bordetella which induces T cell responses against tumoral
antigens.
BACKGROUND OF THE INVENTION
[0003] This invention relates to compositions and methods for
treating cancers.
[0004] In many animal tumor models, T cells play an important role
in tumor rejection. A variety of tumor antigens recognized by
CD4.sup.+ or CD8.sup.+ tumor reactive T cells have been identified
on both murine and human tumors (1). CD8.sup.+ cytotoxic
lymphocytes (CTL) are of particular interest because these cells
specifically recognize tumor cells and kill them. Therefore, an
important goal in cancer immunotherapy is to activate
tumor-specific CTL.
[0005] Study of antigens recognized by CD8.sup.+ T cells on human
melanoma has identified several MHC-restricted tumor epitopes that
correspond to nonmutated or mutated peptides derived from various
self proteins (2). Several of these peptides are derived from
nonmutated differentiation proteins such as tyrosinase,
Melan-A/Mart-1, and gp100. These proteins are specifically
expressed in most melanocytes/melanomas, and thus, the
HLA-restricted epitopes are presented by most melanoma cells from
patients expressing the relevant HLA molecules. Therefore, these
antigens could be the targets of immunotherapeutic strategies that
are based on immunization against tumor epitopes.
[0006] Other antigens expressed on tumor cells have also been
described, for example, a peptide derived from an intron sequence
of the gene that codes for N-acetylglucosaminyl-transferase V
(GnT-V) (3). This intron is specifically expressed in melanoma
cells and is present in about 50% of melanoma cells.
[0007] Various vaccination protocols designed to induce specific
anti-tumor CTL responses against these epitopes have been
developed, including protocols that use free peptide in IFA (4),
recombinant viral vectors (5-7), or dendritic cells (8-11). The
application of these approaches to human vaccination remains
limited due to potential toxicity of adjuvants, bias towards the
response against vector-derived epitopic peptide, or because they
are "labor-demanding" (in vitro manipulated DC).
[0008] Previously, recombinant plasmids have been used for the
expression of Bordetella sp. adenylate cyclase (cyaA) and a
heterologous DNA inserted in a permissive site of CyaA. These
plasmids and resulting recombinant proteins have been useful for
inducing immune responses. The immune responses elicited have been
in CD8+ T lymphocytes with class I major histocompatibility
complexes, as well as in CD4+ T lymphocytes with class II major
histocompatibility complexes. (See U.S. Pat. Nos. 5,503,829,
5,679,784, and 5,935,580.) More specifically, the recombinant
proteins can be delivered to CD11b expressing cells, such as
dendritic cells. (See European Patent Application EP1 188 446 A1,
"Proteinaceous vectors for molecule delivery to CD11b expressing
cells", and WO/2122169 A2 "Vectors for Molecule Delivery to CD11b
Expressing Cells", corresponding to U.S. Pat. No. 387,486, and
European Patent Application No. 03291486.3, "Modified Bordetella
Adenylate Cyclase Comprising or Lacking CD11b/CD18 Interaction
Domain and Uses thereof".) See also, El-Azami-El-Idrissi, et al.,
2003, Interaction of Bordetella pertussis Adenylate Cyclase with
CD11b/CD18, J. Biol. Chem., vol. 278, pp. 38514-21.
[0009] There exists a need in the art for new antitumor treatments
that allow for specific targeting to immune cells and T cell
responses. These new strategies should result in specific
amplification of immune responses against tumoral antigens.
BRIEF SUMMARY OF THE INVENTION
[0010] This invention aids in fulfilling the needs in the art by
providing recombinant CyaA proteins that induce immune responses.
These responses can be directed towards tumoral antigens.
[0011] The invention provides novel methods of treating and
immunomonitoring cancers.
[0012] The invention provides an immunogenic composition comprising
a recombinant protein, wherein the recombinant protein comprises a
Bordetella adenylate cyclase (CyaA) and a peptide that corresponds
to a tumor antigen.
[0013] An embodiment of the invention is a method of treating a
patient with cancer comprising (1) administering an immunogenic
composition to the patient, wherein the immunogenic composition
comprises a recombinant protein, wherein the recombinant protein
comprises a Bordetella CyaA or a specific fragment thereof, and a
peptide that corresponds to a tumor antigen, and (2) inducing an
immune response, such as a T cell response, in the patient.
[0014] An embodiment of the invention is a method of treating a
patient with cancer comprising (1) administering an immunogenic
composition to the patient, wherein the immunogenic composition
comprises a vector expressing a recombinant protein, wherein the
recombinant protein comprises Bordetella CyaA or a specific
fragment thereof, and a peptide that corresponds to a tumor
antigen, and (2) inducing a T cell response in the patient.
[0015] The T cell response is a CTL response or a T helper response
or a CTL and a T helper response.
[0016] In an embodiment of the invention, the tumor is a
melanoma.
[0017] In another embodiment of the invention, the tumor antigen is
an HLA*0201 epitope.
[0018] Encompassed in the invention is the recombinant protein is
CyaA-E5-Tyr or CyaA-E5-GnT-V.
[0019] In a further embodiment of the invention, the recombinant
protein comprises more than one tumor antigen. In a particular
embodiment, at least one tumor antigen is different from the
other(s).
[0020] The tumor antigen is localized to any permissive site of
CyaA.
[0021] In an embodiment of the invention CyaA is from Bordetella
pertussis, Bordetella parapertussis, or Bordetella bronchiseptica.
In a preferred embodiment CyaA is from Bordetella pertussis.
[0022] The invention also provides for an immunogenic composition
comprising a recombinant protein, wherein the recombinant protein
comprises at least one specific fragment of the adenylate cyclase
protein that is recognized as a ligand on human and animal cells,
and at least one epitope specific for a tumoral antigen. In the
recombinant protein of the immunogenic composition CyaA and the
tumoral antigen can either be genetically fused or chemically bound
(PCT/EPO1/11315).
[0023] Furthermore, the invention provides a recombinant protein
wherein the recombinant protein comprises Bordetella CyaA, or a
specific fragment thereof, and a peptide that corresponds to an
antigen comprising the GnTV epitope. The antigen is either fused or
chemically bound to the CyaA protein or a specific fragment
thereof.
[0024] The invention also provides a nucleic acid sequence coding
for a fusion protein comprising Bordetella CyaA, or a specific
fragment thereof, and a peptide that corresponds to an antigen
comprising the GnTV epitope. In a particular embodiment, said
sequence is included in the plasmid deposited at C.N.C.M., Paris,
France, on Oct. 16, 2003 under accession number I-3111.
[0025] Also included in the invention is a vector expressing a
recombinant protein which comprises Bordetella CyaA, or a specific
fragment thereof, and a peptide that corresponds to an antigen
comprising the GnTV epitope. In a particular embodiment, said
vector has been deposited at C.N.C.M., Paris, France, on Oct. 16,
2003 under accession number I-3111.
[0026] The invention further encompasses a host cell that expresses
a recombinant protein comprising Bordetella CyaA, or a specific
fragment thereof, and a peptide that corresponds to an antigen
comprising the GnTV epitope. In a particular embodiment, the host
cell expresses the vector that has been deposited at C.N.C.M.,
Paris, France, on Oct. 16, 2003 under accession number I-3111.
[0027] The invention also provides a nucleic acid sequence coding
for a fusion protein comprising Bordetella CyaA, or a specific
fragment thereof, and a peptide that corresponds to an antigen
comprising the Tyr epitope. In a particular embodiment, said
sequence is included in the plasmid deposited at C.N.C.M., Paris,
France, on May 31, 2001 under accession number I-2679.
[0028] Another embodiment of the invention is a vector expressing a
recombinant protein that comprises Bordetella CyaA, or a specific
fragment thereof, and a peptide that corresponds to an antigen
comprising the Tyr epitope. In a particular embodiment, said vector
has been deposited at C.N.C.M., Paris, France, on May 31, 2001,
under accession number I-2679.
[0029] The invention further encompasses a host cell that expresses
a recombinant protein comprising Bordetella CyaA, or a specific
fragment thereof, and a peptide that corresponds to an antigen
comprising the pTyr epitope. In a particular embodiment, the host
cell expresses the vector that has been deposited at C.N.C.M.,
Paris, France, on May 31, 2001, under accession number I-2679.
DESCRIPTION OF THE DRAWINGS
[0030] This invention will be described with reference to the
drawings in which:
[0031] FIG. 1 depicts in vivo induction of CTL responses by
recombinant CyaA carrying HLA*0201 restricted melanoma epitopes.
HHD-mice received i.p. injections on days 0, 21 and 42 of either 50
.mu.g control CyaA toxin ( , .largecircle.) or recombinant CyaA
toxins carrying melanoma epitopes (.box-solid., .quadrature.)(A,
CyaA-Tyr; B, CyaA-GnT-V) in the presence of 1 mg alum. Seven days
after the last injection, spleen cells from immune mice were
stimulated in vitro with the priming peptide pTyr (A), or pGnT-V
(B) in the presence of irradiated syngenic spleen cells. The
cytotoxic activity of these effector cells was measured on
.sup.51Cr-labeled RMA-S-HHD target cells pulsed with the respective
peptide ( , .box-solid.) or incubated with medium alone
(.largecircle., .quadrature.). The data represent mean values of
duplicates (SD<10%). Quadrants represent the number of positive
mice versus the number of tested mice, and curves represent mean
values.+-.SD of responder mice per group from three
experiments.
[0032] FIG. 2 depicts induction of melanoma-specific CTL responses
by recombinant CyaA carrying melanoma epitopes using different
routes of immunization. Panels A and B: HHD mice were immunized
i.p. twice on days 0 and 21 with 50 .mu.g wild-type CyaA ( ,
.largecircle.) or recombinant CyaA-Tyr (.box-solid., .quadrature.)
in the presence (A) or in the absence of 1 mg alum (B). Panels C
and E: HHD mice were immunized by one i.v. injection with 50 .mu.g
control wild type CyaA ( , .largecircle.) or recombinant CyaA-Tyr
(.box-solid., .quadrature.) (C) or recombinant CyaA-GnT-V
(.box-solid., .quadrature.) (E) in the absence of adjuvant. Panels
D and F: HHD mice were immunized by one i.v. injection with 50
.mu.g control detoxified CyaA-E5 ( , .largecircle.) or detoxified
recombinant CyaA-E5-Tyr (.box-solid., .quadrature.) (D) or
recombinant CyaA-E5-GnTV (.box-solid., .quadrature.) (F) in the
absence of adjuvant. Seven days after the last injection, spleen
cells from immune mice were stimulated in vitro with priming
peptides in the presence of irradiated syngeneic spleen cells. The
cytotoxic activity was measured on .sup.51Cr-labeled RMA-S-HHD
target cells pulsed with the priming peptide ( , .box-solid.) or
incubated with medium alone (.largecircle., .quadrature.). The
results show cumulative data from 2-4 experiments. Quadrants
represent the number of positive mice versus the number of tested
mice, and curves represent mean values.+-.SD of responder mice per
group. The results obtained after immunization with toxic and
detoxified CyaA are not statistically different using a t test.
[0033] FIG. 3 demonstrates that immunization of mice with CyaA-Tyr
induces a long-lasting specific memory CTL activity. HHD mice were
immunized i.p. twice on days 0 and 21 with 50 .mu.g wild-type CyaA
( , .largecircle.) or recombinant CyaA-Tyr (.box-solid.,
.quadrature.) in the presence of 1 mg alum. Three months (A) or
five months (B) after the last injection, spleens were removed and
specific CTL activity was measured after in vitro stimulation as
described in FIG. 1 on .sup.51Cr-labeled RMA-S-HHD target cells
pulsed with the peptide pTyr ( , .box-solid.) or incubated with
medium alone (.largecircle., .quadrature.). Quadrants represent the
number of positive mice versus the number of tested mice. Curves
represent mean values.+-.SD of responder mice per group from one
experiment.
[0034] FIG. 4 depicts stimulation of human specific CTL clones by
human dendritic (DC) cells incubated with recombinant CyaA-E5-Tyr
or CyaA-E5-GnT-V. Due to the cytotoxicity of CyaA, only detoxified
recombinant CyaA were tested in vitro. Immature HLA*0201.sup.+ DC
derived from human monocytes were incubated with CyaA-E5
(.largecircle.), recombinant CyaA-E5-Tyr ( ) (A), CyaA-E5-GnT-V ( )
(B) or with the relevant antigenic peptide (.tangle-solidup.), and
were used as APC (Antigen Presenting Cells) to stimulate
anti-tyrosinase CTL clone IVS-B (A) or anti-GnT-V CTL clone CMU 579
6/3 (B). The secretion of IFN-.gamma. by the CTL clones was
assessed by ELISA. The results are expressed as the mean
concentration of IFN-.gamma. released in the supernatants from
duplicate wells and are representative of three independent
experiments. Standard errors of the mean are indicated.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0035] A new approach for CTL activation has recently been
developed based on bacterial toxins capable of delivering antigenic
epitopes across the plasma cell membrane into the cytosol, where
appropriate processing and interaction with MHC-class I molecules
can occur. The adenylate cyclase toxin (CyaA) of Bordetella
pertussis (Glasser, P., et al. 1988 Bordetella pertussis adenylate
cyclase: the gene and the protein, Tokai J. Exp. Clin. Med., 13
Supp.: 239) has the capacity to deliver its catalytic domain into
the cytosol of eukaryotic cells (12). Delivery of a CM.sup.+ T cell
epitope inserted into the catalytic domain of CyaA results in
intracellular processing and presentation of the epitope by
MHC-class I molecules at the surface of antigen presenting cells
(13). Furthermore, CyaA specifically binds to
.alpha..sub.M.beta..sub.2 integrin (CD11b/CD18) (14), and thus,
targets the CD11b+ DC subpopulation, which very efficiently induces
primary immune responses (15). Therefore, immunization of mice with
a recombinant CyaA toxin bearing a viral epitope leads to the
induction of strong CTL responses and to a full protection against
a lethal viral challenge (16).
[0036] Moreover, CyaA toxins carrying a single CTL epitope can also
stimulate efficient protective and therapeutic antitumor immunity
in mice (17). Importantly, genetically detoxified CyaA toxoids
retain the property to induce protective antiviral or antitumoral
immunity (17, 18). Thus, CyaA seems to be a safe and efficient
non-replicating vector to induce specific immune responses in mice.
However, in the view of elaborating cancer immunotherapy using
CyaA, it is of particular importance to demonstrate that human
tumoral epitopes inserted into CyaA are efficiently processed and
presented in association with human MHC molecules.
[0037] In one embodiment of the invention, two recombinant CyaA
carrying HLA*0201 restricted melanoma epitopes derived either from
tyrosinase or from GnT-V were constructed. The potency of these
recombinant CyaA to induce in vivo HLA*0201 restricted CTL
responses against the inserted epitopes and the ability to deliver
these epitopes to human antigen presenting cells is demonstrated in
the Examples below.
[0038] It was discovered that CyaA of Bordetella pertussis is able
to deliver CD8.sup.+ T cell epitopes into the cytosol of
CD11b.sup.+ dendritic cells following its specific interaction with
the .alpha..sub.M.beta..sub.2 integrin (CD11b/CD18). This delivery
results in intracellular processing and presentation by MHC-class I
molecules of the CD8.sup.+ T cell epitopes inserted into CyaA.
Indeed, CyaA toxins carrying a single CTL epitope can induce
efficient protective, and therapeutic antitumor immunity in
mice.
[0039] It was further discovered that these recombinant CyaA
proteins induce strong anti-melanoma CTL responses in
HLA*0201-transgenic mice, even after a single intravenous
immunization without adjuvant. The responses are long lasting,
being detected as long as five months after the last injection.
[0040] Finally, it was discovered that human dendritic cells,
treated with the recombinant CyaA, process and efficiently present
melanoma epitopes to human CTL clones. The recombinant CyaA
proteins of the invention demonstrate that tumoral epitopes
inserted into CyaA are efficiently processed and presented in
association with human MHC molecules. Therefore, CyaA is capable of
activating antitumoral CTL in humans, and is a novel factor for
cancer immunotherapy.
[0041] As used herein, the term "immunogenic composition" relates
to a composition that leads to an immunological response and that
is associated with therapeutic treatments, such as treatments
against cancers.
[0042] As used herein the terms "Bordetella CyaA" or "Bordetella
adenylate cyclase" encompass the CyaA or a fragment thereof, either
modified or not. The modifications can include deletion of some
internal amino acids. For example, CyaA may have no catalytic
activity, but the specific binding to CD11b/CD18 receptor and the
process of translocation of the catalytic domain are not affected.
The term "Bordetella" refers to the adenylate cyclase protein of a
pathogen of Bordetella species. Said pathogen can be Bordetella
pertussis, Bordetella parapertussis, or Bordetella
bronchiseptica.
[0043] As used herein, the term "antigen" or "epitope" refers to a
peptide including a protein that can induce an immune response. The
term "heterologous" refers to the nature of the antigen bound to
the CyaA protein, which induces an immune response different from
that of the CyaA protein. A heterologous antigen or epitope can be
fused to CyaA or chemically bound to CyaA, for instance.
[0044] As used herein, the term "immunogenic" refers to a
characteristic of a protein as being able to elicit an immune
response in a mammal, particularly in a human. The term "immune
response" refers to many effects that are caused by cells of the
immune system, such as, for instance, a CTL response and/or a T
helper response, and in the context of the invention includes, but
are not limited to, activation of tumor-specific cytotoxic
lymphocytes. As used herein, the term "immunotherapy" refers to a
therapy for a disease that relies on an immune response.
[0045] In addition to the recombinant protein or vector of the
invention, the immunogenic composition of the invention can include
adjuvants and excipients to allow an increase and modulation in the
immune response. These adjuvants are diverse in nature. They can,
for example, comprise liposomes, oily phases, for example, the
Freund type of adjuvants, which are generally used in the form of
an emulsion with an aqueous phase, or, more commonly, can comprise
water-insoluble inorganic salts. These inorganic salts can
comprise, for example, aluminum hydroxide, zinc sulfate, colloidal
iron hydroxide, calcium phosphate, or calcium chloride. Aluminum
hydroxide (Al(OH).sub.3) is the most commonly used adjuvant.
[0046] The invention also encompasses recombinant proteins
comprising Bordetella CyaA, or a specific fragment thereof, and the
peptide pTyr (YMDGTMSQV). Said peptide may comprise extended
flanking sequences. The pTyr peptide corresponds to the melanoma
HLA*0201 restricted epitope from the 369-377 region of tyrosinase.
Note that the amino acids 369-377 of human tyrosinase are
YMNGTMSQV. However, the Asn residue at position 371 (N) of
tyrosinase is naturally deamidated to Asp (D) of this epitope in
living cell so that the true epitope recognized by the CTL clones
in vivo are recognizing sequence YMDGTMSQV.
[0047] Furthermore, an epitope with an extended flanking sequence
of amino acid 360-385 of human Tyrosinase is
SSMHNALHIYMNGTMSQVQGSANDPI (SEQ ID NO: 1) (with N371 converted to
D).
[0048] An epitope derived from the human
N-acetylglucosaminyltransferase V gene is VLPDVFIRC (Y Guilloux, et
al. A peptide recognized by human cytolytic T lymphocytes on HLA-A2
melanomas is encoded by an intron sequence of the
N-acctylglucosaminyltransferase V gene, J. Exp. Med. 1996 183:
1173-1183.) The Gnt-V epitope is encoded by an intron sequence that
may code for a 74 amino acid long polypeptide (H. sapiens DNA for
exon encoding for N-acetylglucosaminyltransferase V; Accession
#X91652). Furthermore, the epitope with extended flanking sequences
from human N-acetylglucosaminytransferase V is MVLPDVFIRCVVFCL (SEQ
ID NO: 2).
[0049] The invention also encompasses the recombinant fusion
protein comprising Bordetella CyaA, or a specific fragment thereof,
and the peptide pGnT-V (VLPDVFIRC). Said peptide may comprise
extended flanking sequences. The peptide pGnT-V corresponds to the
HLA*0201 restricted epitope NA17-A derived from an intron of the
N-acetylglucosaminyl-transferase V gene.
[0050] In one preferred embodiment of the invention the recombinant
protein is CyaA-Tyr. The term "CyaA-Tyr" means a fusion protein
comprising the tyrosinase melanoma epitope of HLA*0201, which can
be prepared as described in Example 1, and Bordetella pertussis
CyaA. The term "CyaA-E5-Tyr" refers to the CyaA-Tyr protein in
which the catalytic activity of CyaA has been genetically
inactivated. See, for instance, Example 1.
[0051] In another preferred embodiment of the invention, the
recombinant protein is CyaA-E5-GnT-V. The term "CyaA-E5-GnT-V"
means a fusion protein comprising the NA17-A melanoma epitope of
HLA*0201 derived from an intron of the
N-acetylglucosaminyl-transferase V gene, which can be prepared as
described in Example 1, and Bordetella pertussis CyaA. The term
"CyaA-E5-GnT-V" refers to the Cya-GnT-V protein in which the
catalytic activity of CyaA has been genetically inactivated. Once
again, see, for instance, Example 1.
[0052] In yet another embodiment of the invention, the recombinant
protein between CyaA and pTyr or pGnT-V is modified from the
structure of CyaA-Tyr, CyaA-GnT-V, CyaA-E5-Tyr, or CyaA-E5-GnT-V.
Modification of these embodiments can include the addition of
flanking regions, which are sequences of amino acids that surround
the peptides comprising the recombinant protein, and were described
above. These flanking sequences can enhance processing. Flanking
sequences can also be sequences which is not naturally surround the
antigen but which specifically enhance the antigen processing by
antigen preventing cells.
[0053] In yet another embodiment, the recombinant proteins can be
modified by including multiple identical heterologous epitopes. For
instance, Tyr or. GnT-V epitope, as described above, or other
melanoma epitopes.
[0054] In further embodiments of the invention, the recombinant
protein can include at least one specific fragment of the adenylate
cyclase protein, such as, but not limited to, CyaA the 373-1706
region or the 1166-1281 region which are recognized as a ligand on
human and animal cells, such as, dendritic cells, and at least one
epitope specific for a cancer antigen, such as, but not limited to,
pTyr or GnT-V.
[0055] In another embodiment of the invention, the recombinant
protein can include multiple epitopes from one or more tumoral
antigens.
[0056] Another embodiment of the invention includes permissive
sites of CyaA that differ from those provided in the Examples. The
antigen portion of the recombinant protein used in the tests of the
invention can be localized to any permissive site of the CyaA
adenylate cyclase protein WO 93/21324. In addition, the invention
encompasses tests and immunogenic compositions that utilize only
fragments of the CyaA adenylate cyclase in the recombinant protein
(see EPO 03/291486.3).
[0057] As used herein, the term "permissive site" relates to a site
where the heterologous peptide can be inserted without
substantially affecting the desired functional properties of the
adenylate cyclase toxin, i.e. without affecting the domains
necessary for the specific binding to CD11b/CD18 receptor and
advantageously without affecting the process of translocation of
the catalytic domain.
[0058] Permissive sites of the Bordetella pertussis adenylate
cyclase include, but are not limited to, residues 137-138
(Val-Ala), residues 224-225 (Arg-Ala), residues 228-229 (Glu-Ala),
residues 235-236 (Arg-Glu), and residues 317-318 (Ser-Ala) (see
Sebo et al., 1985). The following additional permissive sites are
also included in embodiments of the invention: residues 107-108
(Gly-His), residues 132-133 (Met-Ala), residues 232-233 (Gly-Leu),
and 335-336 (Gly-Gln) and 336-337. (See generally, Glaser et al.,
1988 Bordetella pertussis adenylate cyclase: the gene and the
protein, Tokai J. Exp. Clin. Med., 13 Suppl.: 239-52.)
[0059] As used herein, the terms "specific region of the adenylate
cyclase protein" or "fragment of the CyaA adenylate cyclase"
relates to a fragment of said protein including the protein wherein
some amino acids which are not on the tumoral parts of the protein
have been deleted, and the desired functional properties of
adenylate cyclase toxin are not substantially affected, i.e. the
domains necessary for the specific binding to CD11b/CD18 receptor
and the process of translocation of the catalytic domain are not
affected.
[0060] The terms "tumor antigen" or "cancer antigen" refer to any
substance from a tumor that elicits an immune response and reacts
specifically with antibodies or T cells. Said substance can be from
any origin, either spontaneous or from a virus, which transforms
cells to form a tumor. Examples of such viruses are HHV8, HCV, and
HBV. The antigen or epitope must be present on the surface of the
tumor cell.
[0061] As used herein, the term "a peptide that corresponds to an
antigen" encompass an antigen, an epitope, or an antigen or an
epitope flanked by naturally or non-naturally occurring flanking
regions, which specifically enhance antigen processing by antigen
presenting cells.
[0062] The term "epitope" refers to the minimal peptide sequence of
an antigen that can induce an immune response.
[0063] The term "peptide" refers to a series of amino acids linked
by amide bonds, comprising at least 3 amino acids, and preferably
at least 6 amino acids.
[0064] The immunogenic composition of the invention can be used in
solution, for example, but not limited to, in PBS, or with
adjuvants, for example, but not limited to alum. The immunogenic
composition can be administered intramuscularly, subcutaneously,
intravenously, or intradermally. The immunogenic composition can be
administered in amounts from 0.5-10 mg, preferably 1-5 mg, 1.5-3
mg, or more preferably 1.50 mg. The effects of these treatments can
be monitored by assaying the levels of IFN-.gamma. with ELISPOT,
ELISA, or CTL activation assays, or other appropriate
immunoassays.
[0065] Publications illustrate the use of recombinant adenylate
cyclase of Bordetella sp. for diagnosis and immunomonitoring, i.e.,
Vordermeier H. Martin et al (Infection and Immunity, November 2004,
p. 6255-2261) and Schlecht G. et al (The Journal of Immunology
2004, p. 6089-6097). A separate US patent application is filed by
Leclerc et al. on the same day as the present application is filed
concerning "Recombinant Adenylate Cyclase of Bordetella sp. For
Diagnostic and Immunomonitoring Uses, Method of Diagnostic or
Immunomonitoring Using Said Recombinant Adenylate Cyclase, Kit for
Diagnosing or Immunomonitoring Comprising said Recombinant
Adenylate Cyclase" (Attorney Docket No. 03495-0326) and claiming
priority to U.S. Provisional Application 60/523,704, filed Nov. 21,
2003. These references and application are hereby incorporated by
reference as regards the use of recombinant adenylate cyclase of
Bordetella sp. for diagnosis and immunomonitoring.
[0066] The adenylate cyclase of Bordetella Sp. represents a new
delivery system able to specifically stimulate CD8.sup.+ T
lymphocytes leading to protective antiviral and antitumoral
immunity in mice (16, 17). CyaA is a powerful non-replicating
vector for induction of adaptive immunity and is useful in
vaccines. Demonstration, according to this invention, that the
inserted epitopes can be processed and presented in association
with human MHC molecules is an indispensable prerequisite for the
use of this vector in humans.
[0067] By using recombinant CyaA in which human melanoma epitopes
expressing the human HLA*0201 class I molecule were present, strong
and lasting melanoma specific CTL responses could be induced in HLA
transgenic mice. Similar results were obtained with recombinant
detoxified CyaA devoid of adenylate cyclase activity. CyaA
represents an efficient vector to induce specific CTL responses in
vivo because more than 80% of immunized HHD mice responded to the
tyrosinase epitope inserted into CyaA following one i.v. injection
without adjuvant, while only 26% of HHD mice respond to this
epitope following one injection of 100 .mu.g of peptide in the
presence of IFA (partially in 26). In addition, it was surprisingly
observed according to this invention that human DC efficiently
processed these recombinant molecules for antigenic peptide
presentation to human CTL. Strikingly, the recombinant CyaA-Tyr was
much more efficient than the synthetic peptide in delivering the
tyrosinase epitope to DC.
[0068] Alternative antigen delivery systems based on recombinant
viruses usually result in an in vitro presentation efficiency that
is lower than the synthetic peptide. The surprising results from in
vivo and in vitro experiments according to the invention underline
the power of CyaA as delivery system, and show that CTL responses
can be obtained in humans after immunization with recombinant CyaA
and thus, that efficient immunotherapy can be achieved with this
vector. However, the immunogenicities of the two recombinant CyaA
tested in this study were quite different. Indeed, strong CTL
responses in HHD mice were induced with only one i.p. injection of
CyaA-Tyr in the absence of adjuvant, while three i.p. injections of
CyaA-GnT-V, in the presence of alum, were required to generate
specific CTL responses. The weak efficiency of CyaA-GnT-V to
deliver GnT-V melanoma epitope was also evidenced in vitro, since
human DC incubated with this vector poorly stimulated an anti-GnT-V
CTL clone as compared to CyaA-Tyr, which efficiently stimulated a
specific anti-tyrosinase CTL clone.
[0069] This difference can be explained by the fact that the GnT-V
peptide grafted into CyaA-GnT-V was poorly processed, as compared
to the tyrosinase peptide inserted into CyaA-Tyr. Indeed, flanking
regions of a given epitope are known to influence the proteolytic
generation of the mature peptide (27-29) and particularly for
subdominant and/or cryptic epitopes (30). Therefore, it is expected
that modification of the molecular context of GnT-V epitope into
CyaA can enhance the efficiency of processing of this epitope by
APC. On the contrary, the sequence flanking the tyrosinase epitope
in CyaA-Tyr appears to allow its efficient processing.
[0070] Furthermore, CyaA-Tyr is very efficient in activating
HLA*0201-restricted CD8.sup.+ T cell in vivo, because a single
intravenous immunization or two i.p. injections without adjuvant
were sufficient to generate strong specific CTL responses. This is
explained by the fact that CyaA targets specifically CD11b.sup.+
DC, the most potent APC to induce primary response, as a result of
its interaction with the .alpha..sub.M.beta..sub.2 integrin
expressed by these cells (14). Thus, CyaA has the exceptional
property of specifically delivering antigens to the cytosolic Ag
class I presentation pathway of professional APC.
[0071] Further improvements of the CyaA recombinant strategy are
also possible. First, multiple insertions of CD8.sup.+ T cell
epitopes into the same recombinant molecule has already been
successfully achieved. Indeed, immunization of mice with
recombinant CyaA carrying three different epitopes, including a
LCMV epitope, leads to the induction of specific CTL responses for
each of the three epitopes, as well as protection against a lethal
LCMV challenge (31). Detoxified CyaA carrying multiple melanoma
epitopes constitute a good alternative to induce multispecific CTL
responses. Furthermore, additional insertion of CD4.sup.+ T cell
epitopic peptides is also possible. Although the implication of
CD8.sup.+ T cells in eradication of established tumors has been
clearly demonstrated (32), T helper cells can also be required to
induce efficient anti-tumoral responses (33-35). Recombinant CyaA
can also deliver epitopes into the MHC class II processing pathway
(36) and is able to induce, in vivo, both specific Th1 and CTL
responses (37). This characteristic is of great interest for
vaccination strategies where both kinds of T cell responses have to
be induced, noticeably in the context of cancer immunotherapy.
[0072] Plasmid pTRACE5-GnTV is a derivative of the expression
vector pTRACG that expresses the cyaC and cyaA genes from
Bordetella pertussis under the control of the .lamda. phage Pr
promoter (pTRCAG also harbors an ampicillin resistance selectable
marker and the thermosensitive .lamda. repressor CI.sup.857). In
pTRACE5-GnTV, the cyaA gene is modified by insertion of a dipeptide
Leu-Gln between codons 188 and 189 of wild-type CyaA (resulting in
the inactivation of the adenylate cyclase activity) and by
insertion of a DNA sequence encoding the following peptide sequence
PASVLPDVFIRCGT (SEQ ID NO: 3) inserted between codons 224 and 240
of CyaA. The underlined peptide (VLPDVFIRC) (SEQ ID NO: 4)
corresponds to the HLA-A2 restricted melanoma epitope NA17-A
derived from the N-acetylgiucosaminyl-transferase V gene. (G.
Dadaglio, et al. (2003) Recombinant adenylate cyclase of Bordetella
pertussis induces CTL responses against HLA-A2-restricted melanoma
epitope. Int. Immuno.) Plasmid XL1/pTRACE5-GnTV was deposited at
C.N.C.M. on Oct. 16, 2003, with accession number I-3111.
[0073] Plasmid pTRACE-5-Tyros369 is a derivative of the expression
vector pTRACG that expresses the cyaC and cyaA genes from
Bordetella pertussis under the control of the .lamda. phage Pr
promoter (pTRCAG also harbors an ampicillin resistance selectable
marker and the thermosensitive .lamda. repressor CI.sup.857). In
pTRACE5-Tyros369, the cyaA gene is modified by insertion of a
dipeptide Leu-Gln between codons 188 and 189 of wild-type CyaA
(resulting in the inactivation of the adenylate cyclase activity)
and by insertion of a DNA sequence encoding the following peptide
sequence PASYMDGTMSQVGTRARLK (SEQ ID NO: 5) inserted between codons
224 and 240 of CyaA. The underlined peptide (YMDGTMSQV) (SEQ ID NO:
6) corresponds to the amino acids sequence 369-377 of tyrosinase.
Plasmid XL1/pTRACE5-Tyros369 was deposited at C.N.C.M. on May 31,
2001, with accession number I-2679.
[0074] The abbreviations used are as follows: CTL: cytotoxic T
lymphocytes; DC: dendritic cells; PBMC: peripheral blood
mononuclear cells; CyaA: adenylate cyclase of Bordetella sp; Tyr:
tyrosinase; GnT-V: N-acetylglucosaminyl-transferase V; GM-CSF:
granulocyte-macrophage colony-stimulating factor; IFN: interferon;
i.p.: intraperitoneal; i.v.: intravenous.
[0075] This invention will be described in greater detail in the
following Examples.
Example 1
Materials and Methods
[0076] Mice. HHD mice are H-2D.sup.-/- .beta.2.sup.-/- double knock
out mice expressing the HHD transgene comprising the .alpha.1 (H)
and .alpha.2 (H) domains of HLA*0201 linked to .alpha.3
transmembrane and cytoplasmic domains of H-2D.sup.b (D), with the
.alpha. 1 domain linked to human .beta.2-microglobulin. Thus, the
only MHC class I molecule expressed by the HHD mice is the modified
HLA*0201 molecule (19). HHD mice were bred and housed in animal
facilities of Institut Pasteur.
[0077] Peptides. The synthetic peptides pTyr (YMDGTMSQV) (SEQ ID
NO: 6) corresponding to the melanoma HLA*0201 restricted epitope
from the 369-377 region of tyrosinase (20, 21) and pGnT-V
(VLPDVFIRC) (SEQ ID NO: 4) corresponding to the HLA*0201 restricted
epitope NA17-A derived from an intron of the
N-acetylglucosaminyl-transferase V gene (3) were purchased from
Neosystem (Strasbourg, France).
[0078] Construction of recombinant Bordetella pertussis adenylate
cyclase toxins and toxoids carrying melanoma epitopes. The
recombinant CyaA toxin, CyaA-Tyr, harbors a 14 amino acid long
polypeptide sequence (PASYMDGTMSQVGT (SEQ ID NO: 7), one-letter
code for amino acid) genetically inserted between residues 224 and
225 of CyaA. This sequence contains a single copy of the HLA*0201
restricted melanoma epitope derived from tyrosinase (amino acid
369-377, underlined sequence above). Recombinant CyaA toxin
CyaA-GnT-V harbors a 14 amino acid long (PASVLPDVFIRCGT) (SEQ ID
NO: 3) insert at the same position and contains a single copy of
the HLA*0201 restricted melanoma epitope NA17-A derived from the
N-acetylglucosaminy-transferase V gene (underlined sequence
above).
[0079] These recombinant toxins were produced in the E. coli strain
BLR (Novagen) by using expression vectors that are derivatives of
the pTRACG plasmid (22), modified by the insertion between the NheI
and KpnI restriction sites of appropriate synthetic double stranded
oligonucleotides encoding the indicated polypeptide sequences. The
E. coli strain XL1-Blue (Stratagene) was used for all DNA
manipulations that were done according to standard protocols. The
recombinant proteins were purified to homogeneity from inclusion
bodies by a two-step procedure that includes DEAE-Sepharose and
Phenyl-Sepharose chromatographies, as described previously
(23).
[0080] The recombinant toxins CyaA-Tyr and CyaA-GnT-V are
enzymatically active and therefore cytotoxic. The recombinant
toxoids CyaA-E5-Tyr and CyaA-E5-GnT-V are enzymatically inactive,
detoxified variants of CyaA-Tyr and CyaA-GnT-V respectively. They
are unable to synthesize cAMP as a result of a dipeptide insertion
within a critical region of the catalytic site (23). CyaA-E5-Tyr
and CyaA-E5-GnT-V toxoids were produced in E. coli by using
expression vectors that are derivatives of the pTRACE5 plasmid:
this plasmid was obtained by insertion of the hexanucleotide CTGCAG
(SEQ ID NO: 8) in an EcoRV site located in the 5' part of the cyaA
DNA sequence. This results in an in-frame insertion of the
dipeptide Leu-Gln between Asp188 and Ile189 of CyaA (23). The same
synthetic double stranded oligonucleotides described above were
inserted between the NheI and KpnI sites of pTRACE5 to create
plasmids pTRAC-E5-Tyr and pTRAC-E5-GnT-V. The recombinant
CyaA-E5-Tyr and CyaA-E5-GnT-V toxoids were purified to homogeneity
as described (23).
[0081] All purified recombinant toxins and toxoids were more than
90% pure as judged by SDS-gel analysis. The toxin concentrations
were determined spectrophotometrically from the absorption at 280
nm using a molecular extinction coefficient of 142,000 M.sup.-1
cm.sup.-1.
[0082] Oligonucleotide synthesis and DNA sequencing were performed
by Genaxis (France). Cultures in fermentors were performed by the
Service des Fermentations facility from Institut Pasteur.
[0083] Mouse immunization. Six to ten week old female HHD mice were
immunized with two or three i.p. injections at 21 day intervals of
either 50 .mu.g CyaA or recombinant CyaA carrying melanoma epitopes
in the presence or in the absence of 1 mg alum. In some
experiments, mice were immunized with one i.v. injection of 50
.mu.g of the recombinant CyaA in PBS. Detoxified recombinant
CyaA-E5 were used in the same conditions. Spleens were surgically
removed seven days after the last injection, except for the
analysis of long lasting responses, where spleens were removed
three or five months after the last injection.
[0084] In vitro cytotoxic assay. Spleen cells from immunized mice
were stimulated in vitro with 10 .mu.g/ml of pTyr or pGnT-V
peptides corresponding to the priming epitope in the presence of
syngeneic irradiated naive spleen cells in complete medium (RPMI
1640 medium containing L-Alanyl-L-Glutamine dipeptide supplemented
with 10% fetal calf serum, 5.times.10.sup.-5 M 2-mercaptoethanol,
100 U/ml penicillin, 100 .mu.g/ml streptomycin and 20 mM Hepes) for
five days. The cytotoxic activity of these effector cells was
tested in a 4-h .sup.51Cr-release assay on HHD transfected
TAP.sup.-/- RMA-S cells (RMA-S-HHD) loaded with the relevant
peptides as target cells. .sup.51Cr-labeling was performed as
follows: one day before the cytotoxic test, RMA-S-HHD cells were
incubated overnight at room temperature in 7% CO.sub.2 equilibrated
RPMI 1640 medium supplemented with 20 mM Hepes. Then, cells were
incubated 3 h at room temperature with or without 20 .mu.g/ml of
the relevant peptide, washed once and radiolabeled with 100 .mu.Ci
of .sup.51Cr for 1 h at 37.degree. C.
[0085] Various effectors to target ratios were used and all assays
were done in duplicate. The radioactivity released in the
supernatant of each well was measured. The percentage of specific
lysis was calculated as 100.times.(experimental release-spontaneous
release)/(maximum release-spontaneous release). Maximum release was
generated by adding 10% triton X-100 to target cells and
spontaneous release was obtained with target cells incubated in
medium alone. Mice are considered to be responders when at least
20% specific lysis was observed at the highest E/T ratio. The
results are expressed as mean values.+-.SD of responder mice per
group. No specific CTL activity was obtained with splenocytes from
immunized mice stimulated in vitro with an irrelevant peptides.
[0086] Human dendritic cells. Human dendritic cells were derived
from adherent monocytes as follows: Peripheral blood mononuclear
cells were isolated from buffy coats obtained from the HLA*0201
hemochromatosis patient LB2050 by centrifugation on Leucosep tubes
(Greiner, Frickenhausen, Germany) previously centrifuged 30 sec at
2200 rpm with 15 ml of Lymphoprep (Nycomed Pharma, Oslo, Norway).
These tubes were centrifuged at 2200 rpm for 20 min at room
temperature and the top part containing plasma was discarded. The
interphase containing the PBMC was harvested and washed at least
three times in cold phosphate buffer with 1 mM EDTA in order to
eliminate the remaining platelets. The PBMC were then left to
adhere for 1 h in culture flasks at a density of 2.times.10.sup.6
cells/cm.sup.2 in RPMI 1640 supplemented with amino-acids
(L-arginine 116 .mu.g/ml, L-asparagine 36 .mu.g/ml, L-glutamine 216
.mu.g/ml), antibiotics, and 10% fetal calf serum (hereafter
referred as culture medium). Non-adherent cells were discarded,
adherent cells were washed twice carefully with 20 ml of medium and
incubated in culture medium with 200 U/ml human IL-4 and 70 ng/ml
human GM-CSF. On day two and four, 5 ml of medium were removed and
10 ml containing 700 ng of GM-CSF were added. IL-4 was also
supplemented at 200 U/ml for the total volume of the flask. The
cells were used between day five and seven.
[0087] Human CTL clones. CTL clone IVS-B directed against the
HLA*0201 restricted Tyr epitope (positions 369-377) of tyrosinase
was previously described (24). The clone was stimulated weekly with
50 U/ml human IL-2, irradiated HLA*0201 transfected MZ2-MEL
melanoma cells pulsed with 2 .mu.g/ml pTyr peptide, and irradiated
LG2-EBV cells as feeder cells. The CTL clone CMU 579 6/3 specific
for the HLA*0201 restricted epitope derived from GnT-V was obtained
from the blood of a healthy donor following a recently described
method (25). Briefly, the PBMC were stimulated for two weeks with
the antigenic peptide pGnT-V, human IL-2, IL-4, and IL-7. On day
13, PBMC were stained with an HLA*0201 tetramer folded with the
pGnT-V peptide. Tetramer positive lymphocytes were cloned using
flow cytometry. They were stimulated for two weeks with irradiated
allogeneic HLA*0201-positive EBV-transformed B cells pulsed with
the peptide, irradiated allogeneic PBL, IL-2, IL-4, and IL-7, and
then maintained by weekly stimulation with irradiated
HLA*0201-positive peptide-pulsed allogeneic tumor cells and
irradiated allogeneic EBV-B cells. Both CTL clones were maintained
in Iscove's medium supplemented with 10% of human serum, amino
acids, and antibiotics.
[0088] In vitro stimulation assay of human CTL clones. For the
stimulation assay, 10,000 immature dendritic cells were seeded in
U-bottom microplates in 25 .mu.l of X-Vivo 10 medium (Whittaker
Bioprodutcs, Walkersville, USA). 25 .mu.l of CyaA preparations
diluted in X-Vivo 10 medium at different concentrations were added
to the wells. After 30 min of incubation, the corresponding CTL
clones were incubated with these cells (75 .mu.l of X-vivo medium
containing 10.sup.4 anti-tyrosinase CTL clone IVS-B or 10.sup.4
anti-GnT-V CTL clone CMU 579 6/3) and IL-2 (at a final
concentration of 25 U/ml). The supernatants were collected after 20
h and their IFN-.gamma. content was determined by ELISA (Biosource
International, Camarillo, Calif.). To control the ability of DC
incubated with the various detoxified recombinant toxoids to
stimulate the CTL clones, they were exogenously loaded with the
relevant antigenic peptides, incubated with the relevant CTL clones
and the production of IFN-.gamma. was similarly assessed (data not
shown).
Example 2
Induction of Melanoma-Specific CTL Responses by Immunization of HHD
Transgenic Mice with Recombinant CyaA Carrying HLA*0201-Restricted
Melanoma Epitopes
[0089] To determine whether the CyaA toxin is capable of inducing
specific CTL responses against human tumoral antigens, two
recombinant CyaA carrying HLA*0201-restricted human melanoma
epitopes were constructed. The first recombinant CyaA expresses the
epitope 369-377 from the tyrosinase antigen (CyaA-Tyr) and the
second one expresses the epitope NA17-A derived from an intron of
the N-acetylglucosaminyl-transferase V (CyaA-GnT-V). The ability of
recombinant CyaA to induce CTL responses against these two epitopes
in vivo was assessed in HHD mice, which are transgenic for the
human MHC class I molecule HLA*0201 and have been shown to develop
HLA*0201-restricted CTL responses against tumoral peptides (26).
HHD mice were immunized by 3 i.p. injections of 50 .mu.g of
recombinant CyaA with alum. After in vitro stimulation of
splenocytes with the corresponding peptide, CTL responses were
tested in a chromium release assay, using as targets peptide-pulsed
RMA-S-HHD cells, which express the same transgene as HHD mice. As
shown in FIG. 1, both recombinant toxins carrying either Tyr or
GnT-V epitopes induce strong CTL responses against target cells
loaded with the relevant peptide. These CTL responses were
antigen-specific since only peptide sensitized target cells were
killed and CTL activity was not detected on target cells loaded
with irrelevant peptides (data not shown). As expected, no
significant CTL responses were observed in mice immunized with the
wild type CyaA showing that the induction of specific CTL responses
required in vivo priming by the epitope inserted into recombinant
CyaA.
[0090] Induction of CTL responses by CyaA-Tyr was then analyzed
using different immunization protocols with or without alum as
adjuvant. As illustrated in FIGS. 2A and B, two i.p. injections of
CyaA-Tyr were enough to induce specific CTL responses against the
tyrosinase epitope, even in the absence of alum. Induction of
strong specific CTL responses was also observed following a single
injection of 50 .mu.g of CyaA-Tyr without adjuvant using the i.v.
route (FIG. 2C). As expected, these CTL responses were observed
only when using peptide pulsed target cells and splenocytes from
mice immunized with the recombinant CyaA-Tyr, showing the
specificity of the responses. These results demonstrate the high
efficiency of CyaA-Tyr to induce specific CTL responses against the
tyrosinase melanoma epitope. However, using similar conditions of
immunization (2 or 1 i.p. injections with or without alum), no
specific CTL response was observed with the recombinant toxin
CyaA-GnT-V, indicating that this toxin is less efficient to
generate specific CTL response than the CyaA-Tyr (data not shown).
However, by the intravenous route, one injection of CyaA-GnT-V was
sufficient to induce a strong CTL response (FIG. 2E).
[0091] Finally, CTL responses induced by genetically detoxified
mutants of CyaA carrying Tyrosinase or GnTV epitopes that are
devoid of adenylate cyclase activity following insertion of a
dipeptide into the catalytic site were analyzed. HHD mice immunized
with these detoxified molecules developed specific CTL responses
against both tyrosinase and GnTV epitopes (FIGS. 2D, 2F), which
were comparable to the responses of mice immunized with the toxic
forms of CyaA carrying the corresponding epitope. These results
indicate that HLA*0201-restricted CTL induction is independent of
the catalytic activity as it was clearly demonstrated for a viral
epitope from LCMV in BALB/C mice (18).
Example 3
Recombinant CyaA-Tyr Induces Long Lasting Memory CTL Responses
[0092] To analyze the persistence of the CTL responses induced by
the recombinant CyaA bearing melanoma epitope, HHD mice received
two i.p. injections of 50 .mu.g of CyaA-Tyr in the presence of
alum. Three and five months after the last injection, splenocytes
from immunized mice were stimulated in vitro over five days with
the peptide pTyr and then, their cytolytic activity was tested
against peptide pulsed RMA-S-HHD target cells. As illustrated in
FIG. 3, CyaA-Tyr induced a long-lasting specific CTL response
because specific cytotoxic activity could be detected in all mice
three months after the last injection, and even after five months
in one animal.
Example 4
HLA*0201-Restricted Peptides Inserted into CyaA are Processed and
Presented by HLA*0201.sup.+ Human DC
[0093] In vivo induction of specific CTL responses by recombinant
CyaA indicates that inserted epitopes are efficiently processed and
presented by murine APC. However, it is important to demonstrate
that human APC are also able to process and present these
HLA*0201-restricted epitopes inserted into CyaA. Because DC are the
most important APC to induce primary T cell responses, the ability
of HLA*0201.sup.+ DC incubated with the recombinant CyaA to
stimulate human CTL clones specific for the epitopes inserted into
the recombinant CyaA was determined. For these experiments, human
DC were generated in vitro from HLA*0201.sup.+ adherent PBMC in the
presence of GM-CSF and IL-4. Increasing doses of CyaA-E5-Tyr,
CyaA-E5-GnT-V or control CyaA-E5 were then added and presentation
of the antigenic peptides was assessed by measuring the ability of
the treated DC to stimulate the relevant CTL in an IFN-.gamma.
production assay.
[0094] As shown in FIG. 4A, human DC incubated with CyaA-E5-Tyr
induced a high production of IFN-.gamma. by the tyrosinase-specific
CTL clone, indicating that the tyrosinase epitope is efficiently
processed and presented in association with HLA*0201 molecules. The
specificity of this recognition was confirmed by the lack of
stimulation of two irrelevant CTL clones (data not shown) and by
the lack of stimulation of the tyrosinase-specific clone by DC
treated with the control toxoid CyaA-E5 (FIG. 4A). Presentation of
the tyrosinase epitope was proportional to the dose of CyaA-Tyr up
to 30 nM. Under these conditions, higher doses appeared to be toxic
for the DC, as indicated by the low recognition of the treated DC
and by their decreased ability to present the synthetic peptide
loaded exogenously (data not shown).
[0095] In order to assess the relative efficiency of antigen
presentation using CyaA as delivery system a titration curve of the
tyrosinase synthetic peptide, which was pulsed on similar DC, was
also performed. As shown on FIG. 4A, the CyaA-Tyr was up to 100
times more efficient than the synthetic peptide to induce the
presentation of the epitope by DC.
[0096] Human DC incubated with CyaA-E5-GnT-V induced a weak but
reproducible production of IFN-.gamma. by the GnT-V specific CTL
clone, as compared with DC incubated with the peptide pGnT-V (FIG.
4B). This result indicates that human DC are able to present the
GnT-V epitope inserted into CyaA, although with a moderate
efficiency.
[0097] In summary, these results clearly demonstrate the capacity
of human DC to process and present human epitopes inserted into
CyaA.
Example 5
CyaA Toxin Constructions which do not Induce a Response
[0098] CyaA-Mel 21, which comprises the epitope gp100-280, and
includes the inserted sequence YLEPGTVTA formed the GP 100
melanoma-associated tumor antigen, does not include a CTL response.
Similarly, CyaA-CEA 13, which comprises the epitope CEA 571-579,
and has the inserted sequence YLSGANLNL from the Carcinoma
Embryonic Antigen, does not induce a CTL response. Neither of these
toxins induce a CTL response specific for the inserted epitopes in
the HHD mouse. Furthermore, some human dendritic cells cannot
present the inserted epitope CyaA-Mel 21 (epitope gp100-280) to a
human CTL clone. Therefore, this toxin is probably also ineffective
in humans. These two toxins, CyaA-Mel21 and CyaA-CEA13, are
identical to CyaA-Tyr and CyaA-GnTV, but differ only in the
inserted sequences. Therefore, only the epitopes are different, and
so the response to CyaA-Tyr and CyaA-GNTV are epitope specific.
[0099] Finally, the Cya-Mage toxin, which comprises the Mage A10/A2
epitope with the inserted sequence GLYDGMEHL from the melanoma
protein Mage 10, induces very good CTL response in HHD mouse, but
is inefficient in humans under the protocol described above.
Therefore, a positive response in the mouse is not always
indicative of a positive response in humans. In general, human
responses are epitope and species specific.
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Sequence CWU 1
1
12126PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Ser Ser Met His Asn Ala Leu His Ile Tyr Met Asn
Gly Thr Met Ser1 5 10 15Gln Val Gln Gly Ser Ala Asn Asp Pro Ile 20
25215PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 2Met Val Leu Pro Asp Val Phe Ile Arg Cys Val Val
Phe Cys Leu1 5 10 15314PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 3Pro Ala Ser Val Leu Pro Asp
Val Phe Ile Arg Cys Gly Thr1 5 1049PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Val
Leu Pro Asp Val Phe Ile Arg Cys1 5519PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Pro
Ala Ser Tyr Met Asp Gly Thr Met Ser Gln Val Gly Thr Arg Ala1 5 10
15Arg Leu Lys69PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 6Tyr Met Asp Gly Thr Met Ser Gln Val1
5714PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Pro Ala Ser Tyr Met Asp Gly Thr Met Ser Gln Val
Gly Thr1 5 1086DNAArtificial SequenceDescription of Artificial
Sequence Synthetic hexanucleotide 8ctgcag 699PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 9Tyr
Leu Glu Pro Gly Thr Val Thr Ala1 5109PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 10Tyr
Leu Ser Gly Ala Asn Leu Asn Leu1 5119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Gly
Leu Tyr Asp Gly Met Glu His Leu1 5129PRTHomo sapiens 12Tyr Met Asn
Gly Thr Met Ser Gln Val1 5
* * * * *