U.S. patent application number 09/312338 was filed with the patent office on 2002-06-27 for verotoxin b subunit for immunization.
Invention is credited to GREEN, ALLAN M..
Application Number | 20020081307 09/312338 |
Document ID | / |
Family ID | 22193333 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020081307 |
Kind Code |
A1 |
GREEN, ALLAN M. |
June 27, 2002 |
VEROTOXIN B SUBUNIT FOR IMMUNIZATION
Abstract
Methods for stimulating an immune response in a mammal by
administering a toxin-antigen conjugate are provided.
Pharmaceutical compositions and methods for treating an
antigen-related state are also described.
Inventors: |
GREEN, ALLAN M.; (CAMBRIDGE,
MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Family ID: |
22193333 |
Appl. No.: |
09/312338 |
Filed: |
May 14, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60085693 |
May 15, 1998 |
|
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|
Current U.S.
Class: |
424/184.1 ;
424/236.1; 424/241.1 |
Current CPC
Class: |
A61K 39/0011 20130101;
C07K 14/25 20130101; A61K 2039/541 20130101; C07K 19/00 20130101;
A61K 2039/6037 20130101; A61P 35/00 20180101; A61P 37/04 20180101;
C07K 14/4748 20130101; C07K 14/245 20130101; C07K 2319/00
20130101 |
Class at
Publication: |
424/184.1 ;
424/236.1; 424/241.1 |
International
Class: |
A61K 039/00; A61K
039/38; A61K 039/02; A61K 039/108 |
Claims
What is claimed is:
1. A method for stimulating an immune response in a mammal,
comprising administering to said mammal a toxin-antigen conjugate
such that an immune response in said mammal is stimulated.
2. The method of claim 1, wherein said toxin-antigen conjugate is
administered to said mammal transcutaneously through the skin or a
mucous membrane.
3. The method of claim 2, wherein said toxin-antigen conjugate is
administered transcutaneously through the skin of said mammal.
4. The method of claim 2, wherein said mucous membrane is located
in the respiratory tract, gastrointestinal tract or reproductive
tract of said mammal.
5. The method of claim 4, wherein said mucous membrane of the
respiratory tract is selected from the mucous membranes of said
mammal's nose, throat or lungs.
6. The method of claim 4, wherein said mucous membrane of the
gastrointestinal tract is selected from the mucous membranes of
said mammal's mouth, throat, stomach, small intestine, large
intestine, colon, urethra, or rectum.
7. The method of claim 1, further comprising administering to said
mammal an adjuvant.
8. The method of claim 1, wherein said toxin-antigen conjugate
comprises a tumor antigen, a viral antigen, or a bacterial
antigen.
9. The method of claim 8, wherein said tumor antigen is derived
from a tumor lysate.
10. The method of claim 9, wherein said tumor antigen is derived
from lung tissue, skin tissue, breast tissue, stomach tissue, colon
tissue, rectal tissue or brain tissue.
11. The method of claim 8, wherein said tumor antigen is a melanoma
antigen.
12. The method of claim 1, wherein said toxin-antigen conjugate
comprises a toxin selected from the group consisting of a shiga
toxin, a verotoxin, and a cholera B toxin.
13. The method of claim 12, wherein said toxin is a verotoxin or a
fragment thereof.
14. The method of claim 13, wherein said verotoxin is verotoxin
B.
15. The method of claim 1, wherein said toxin-antigen conjugate is
produced recombinantly.
16. The method of claim 1, wherein said toxin-antigen conjugate is
associated through a non-covalent interaction or a covalent
linkage.
17. The method of claim 16, wherein said covalent linkage is a
cyanogen bromide linkage.
18. The method of claim 16, wherein said non-covalent interaction
is a protein-protein interaction, a hydrophobic interaction, a Van
der Waals interaction, or an ionic interaction.
19. The method of claim 1, wherein said toxin-antigen conjugate
further comprises an active or inactive endoplasmic reticulum
retrieval signal.
20. The method of claim 1, wherein said toxin-antigen conjugate
comprises verotoxin B and a tumor antigen.
21. The method of claim 1, wherein said immune response involves
stimulation of dendritic cells.
22. The method of claim 21, wherein said dendritic cells include
Langerhans cells.
23. The method of claim 1, wherein said mammal is a human.
24. The method of claim 1, wherein said toxin-antigen conjugate is
administered to said mammal non-invasively.
25. A method for treating an antigen-related state in a mammal
comprising administering to said mammal an effective amount of said
antigen-toxin conjugate, stimulating an immune response in said
mammal, thereby treating said antigen-related state in said
mammal.
26. The method of claim 25, wherein said toxin-antigen conjugate is
administered to said mammal transcutaneously through the skin or a
mucous membrane of said mammal.
27. The method of claim 26, wherein said toxin-antigen conjugate is
administered transcutaneously through the skin of said mammal.
28. The method of claim 26, wherein said mucous membrane is located
in said mammal's respiratory tract, gastrointestinal tract or
reproductive tract.
29. The method of claim 25, wherein said antigen-related state is
an infection or a tumor.
30. The method of claim 29, wherein said tumor is a skin tumor,
brain tumor, lung tumor, colon tumor, lung tumor, rectal tumor, or
breast tumor.
31. The method of claim 29, wherein the tumor is a melanoma
tumor.
32. The method of claim 25, wherein said toxin-antigen conjugate
comprises a melanoma tumor antigen.
33. The method of claim 25, wherein said mammal is a human.
34. The method of claim 25, further comprising administering an
adjuvant.
35. The method of claim 25, wherein said mammal is suffering from
said antigen-related state.
36. The method of claim 25, wherein said mammal is not suffering
from said antigen-related state.
37. The method of claim 25, wherein said toxin-antigen conjugate is
administered non-invasively.
38. A pharmaceutical composition comprising a toxin-antigen
conjugate and a pharmaceutically acceptable carrier.
39. The pharmaceutical composition of claim 38, wherein said
composition is suitable for administration to a mammal
transcutaneously through the skin or a mucous membrane of said
mammal.
40. The pharmaceutical composition of claim 39, wherein said
composition is suitable for administration transcutaneously through
the skin of said mammal.
41. The pharmaceutical composition of claim 39, wherein said mucous
membrane is located in the mammal's respiratory tract,
gastrointestinal tract or reproductive tract.
42. The pharmaceutical composition of claim 38, wherein said
toxin-antigen conjugate comprises a melanoma antigen.
43. The pharmaceutical composition of claim 38, wherein said
pharmaceutically acceptable carrier is suitable for administration
orally, transdermally, or intrabronchially.
44. The pharmaceutical composition of claim 38, wherein said
antigen conjugate comprises a verotoxin.
45. The pharmaceutical composition of claim 44, wherein said
verotoxin is verotoxin B.
46. The pharmaceutical composition of claim 38, wherein said
pharmaceutically acceptable carrier is suitable for non-invasive
administration.
Description
RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. 119(e) to
co-pending U.S. provisional application Serial No. 60/085,693,
entitled Verotoxin B Subunit for Immunization, filed May 15, 1998,
the entire contents of which are hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] Dendritic cells are sentinels of the immune system. They
originate from a bone marrow progenitor, travel through the blood
and are seeded into non-lymphoid tissue, e.g., skin. Dendritic
cells capture and process exogenous antigens for presentation as
peptide-MHC complexes at the cell surface and then migrate via the
blood and afferent lymph to secondary lymph nodes. In the lymph
nodes, they interact with T-lymphocytes to facilitate activation of
helper and killer T cells (Steinman, R. (1991) Annu. Rev. Immunol.
9:271; Cella et al. (1997) Curr. Opin. ImmunoL 9:10).
[0003] Dendritic have been named according to their appearance and
distribution in the body. For example, dendritic cells located in
the epidermis are known as Langerhans cells. Dendritic cells
located in the dermis and interstitium are known as interstitial
dendritic cells. Blood, veiled, and lymphoid dendritic cells are
found, respectively, in the circulatory system, afferent lymph and
the lymph nodes. Dendritic cells have further been characterized by
lineage, by maturation stage, by functional and phenotypic
characteristics of these stages, and by mechanisms involved in
their migration and function (Cella et al., supra; Austyn, J.
(1996) J. Exp. Med. 183:1287).
[0004] Recent work has demonstrated that cholera toxin can act as a
potent transcutaneous adjuvant in noninvasive, transcutaneous
immunization in mice (Glenn, G. M. et al. Nature (1998) 391:851).
Cholera toxin applied to the surface of the skin stimulates a
strong immune response to coadministered antigens such as
diphtheria or tetanus toxoids. This method is particularly
important given the large skin surface area and the existence of
potent immune cells within it (see, e.g., Bos, J. D., Clin. Exp.
Immunol., 107 (Suppl. 1), 3-5, 1997).
[0005] Studies with cholera toxin suggest that the holotoxin
enhances the presentation of soluble peptides on macrophages,
however it inhibited intracellular processing of soluble or
bacterial antigens. In contrast, the recombinant B subunit enhanced
surface presentation of the antigens, but did not inhibit
intracellular processing (Matousek, M. P., J. G. Nedrud and C. V.
Harding, J. Immunol., 156, 4137-4145, 1996). Animal studies have
also demonstrated the potent capacity of dendritic cells to induce
anti-tumor immunity (Nestle, F. O. et al. Nature Medicine,
4:328-332, 1998).
SUMMARY OF THE INVENTION
[0006] In one embodiment, the present invention pertains to a
method for stimulating an immune response in a mammal, by
administering to the mammal a toxin-antigen conjugate such that an
immune response in the mammal is stimulated. Preferably, the
toxin-antigen conjugate is administered to the mammal
transcutaneously through the skin or a mucous membrane.
[0007] In an advantageous embodiment, the toxin-antigen conjugate
includes, for example, a tumor antigen, a viral antigen, or a
bacterial antigen. The tumor antigen may be derived from lung
tissue, skin tissue, breast tissue, stomach tissue, colon tissue,
rectal tissue or brain tissue. In one preferred embodiment, the
tumor antigen is from, for example, a melanoma tumor.
Advantageously, the toxin-antigen conjugate of the invention may
include a toxin such as a shiga toxin, a verotoxin, or a cholera B
toxin. Preferably, the toxin is the B fragment of verotoxin.
[0008] In another embodiment, the invention features a method for
treating an antigen-related state in a mammal, by administering to
the mammal an effective amount of an antigen-toxin conjugate and
stimulating an immune response in the mammal. Preferably, the
toxin-antigen conjugate is administered to the mammal
transcutaneously through the skin or a mucous membrane, e.g., a
mucous membrane located in the mammal's respiratory tract,
gastrointestinal tract or reproductive tract. In a particularly
preferred embodiment, the mammal is a human. Advantageously, an
adjuvant may also be administered with the conjugate of the
invention.
[0009] The invention also pertains to a pharmaceutical composition
comprising a toxin-antigen conjugate and a pharmaceutically
acceptable carrier. Preferably, the composition is suitable for
administration to the mammal transcutaneously through the skin or a
mucous membrane. Advantageously, the pharmaceutically acceptable
carrier is suitable for administration orally, transdermally, or
intrabronchially. Preferably, the pharmaceutical composition is
suitable for non-invasive administration.
[0010] The present invention relates to recombinant proteins such
as toxin-antigen conjugates which comprise a receptor-binding
non-toxic fragment of a toxin, e.g., a Shiga toxin or a verotoxin B
subunit, and an epitope of a tumor antigen, e.g., Mage 1. The
invention also features methods for using the claimed conjugates.
The toxin-antigen conjugates can be used, for example, to stimulate
an immune response in a mammal, or for treating an antigen-related
state in a mammal. The toxin-antigen conjugates can also be used
for presenting antigens on antigen-presenting cells and for the
formulation of pharmaceutical compositions.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1: Biochemical characterization of Shiga B-Mage 1 and
Ant-Mage 1 fusion proteins. The B-fragment of Shiga toxin (=B) or
recombinant B-Glyc-KDEL, B-Mage 1-Glyc-KDEL, B-Mage 1-Glyc-KDEL, or
B-Mage 1-Glyc-KDELGL proteins were analyzed by electrophoresis on
Tris-tricine gels under reducing conditions. The gels were then
stained with Coomassie blue (A) or revealed by Western blot
analysis with the monoclonal antibody 13C4 directed against the
B-fragment of Shiga toxin (B). Antennapedia-Mage 1 (Antp-Mage 1, C)
fusion protein was analyzed by Coomassie blue staining (a) or
Western blot analysis with anti-His Tag antibody (b).
[0012] FIG. 2: MHC class 1 presentation of soluble Shiga B-Mage 1
fusion proteins to PBMC: role of KDEL sequence. PBMC
(5.times.10.sup.4) were pulsed overnight with either peptide 191
age 1 (1 .mu.M) or Mart 1 (11 .mu.M) or the Shiga B-Mage 1 fusion
proteins (1 .mu.M) with active (B-Mage 1-Glyc-KDEL) or inactive
(B-Mage 1-Glyc-KDELGL) ER retrieval sequence. After washing,
2.times.10.sup.4 Mage 1 specific cytotoxic T cells (Clone B2/30)
were incubated with the pulsed PBMC for 24 hours. Supernatants were
then harvested and tested for IFN.lambda. production. The data are
means of triplicate .+-. standard deviation (bars) and are
representative of at least two similar experiments.
[0013] FIG. 3: MHC class 1 presentation ofsoluble Shiga B-Mage 1
fusion protein by different kinds of Antigen Presenting Cells. B
Lymphoblastoid cells, dendritic cells, and cloned T cells were
pulsed as described in FIG. 2 with soluble Shiga B-Mage 1 fusion
protein. Peptide Mage 1 presentation was tested with the 82/30
CTL.
[0014] FIG. 4: Analysis of the specificity of M1 IC class I
presentation ofsoluble Shiga B-Mage 1 fusion protein by B
lymphoblastoid cell lines. The B lymphoblastoid cell lines BM21
(HLA-A1) or B-V0.1 (HLA-42) were pulsed overnight with medium
alone, synthetic peptide Mage 1 (1 .mu.M), the synthetic peptide
Mart 1 (1 .mu.M), the Shiga B-Mage 1 fusion protein (1 .mu.M), the
recombinant Antp-Mage 1 fusion protein, and the wild type Shiga
toxin B-fragment. After washing, Mage 1 specific CTL 82/30 (A) or
Mart 1 specific LB 373 CTL (B) were incubated with the pulsed B-EBV
for 24 hours. Supernatants were then harvested and tested for
IFN.sub..lambda.production. The data are means of triplicate .+-.
standard deviation (bars) and are representative of at least two
similar experiments.
[0015] FIG. 5: MHC class I presentation of soluble Shiga B-Mage 1
fusion protein by B lymphoblastoid cell lines. Pole of
internalization (A) and intracellular processing (B-C): A:
Paraformaldehyde fixed B lymphoblastoid cell line (BM21) was pulsed
overnight with synthetic Mage 1 peptide (1 .mu.M), Shiga B-Mage 1
fusion protein (1 .mu.M) or medium alone. After washing, Mage 1
specific CTL were incubated with the pulsed B-EBV for 24 hours.
Supernatants were then harvested and tested for IFN.sub.80
production. B and C: Some unfixed B-EBV (BM21) were pretreated
chloroquine (250 .mu.M) or Brefeldin A (2 .mu.g/ml) for 30 min
before they were pulsed for 4 hours with synthetic Mage 1 peptide
(1 .mu.M) or Shiga B-Mage 1 fuision protein (1 .mu.M) or medium
alone. After washing, Mage 1 specific CTL were incubated with the
pulsed B-EBV for 24 hours. Supernatants were then harvested and
tested for IFN.sub..lambda.productio- n. The data are means of
triplicate .+-. standard deviation (bars) and are representative of
at least two similar experiments.
[0016] FIG. 6. Analysis of Shiga B-Mage 1 fusion protein
localization after internalization into B-EBV cells. B-EBV cells
were incubated with DTAF-labeled B-Mage 1-Glyc-KDEL (A) on ice for
45 min, washed and left for 1 hour at 37.degree. C. Cells were then
fixed with paraformaldehyde, permeabilized with saponin and stained
with a monoclonal anti-lamp-2 antibody (B). In C, B-fragment
specific labeling (A) and Lamp-2 labeling (B) are superimposed.
Representative images obtained by confocal microscopy are
shown.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention pertains, at least in part, to
pharmaceutical compositions and methods for stimulating an immune
response. The invention features a recombinant protein, e.g., a
toxin-antigen conjugate, comprising an antigen or an antigen
epitope, e.g., Mage 1, which is associated, e.g., by covalent
linkage, to a toxin, e.g., a Shiga toxin, a verotoxin B. In a
preferred embodiment, the pharmaceutical compositions are suitable
for transcutaneous administration either through the skin or a
mucous membrane, e.g., of the respiratory system, gastrointestinal
system or the reproductive system.
[0018] The term "antigen" includes agents which provoke an immune
response independently and those which are provoke an immune
response when incorporated in to a conjugate of the invention. The
term "antigen epitope" includes fragments of proteins capable of
determining antigenicity. An epitope may comprise, for example, a
peptide of six to eight residues in length (Berzofsky, J. and I.
Berkower, (1993) in Paul, W., Ed., Fundamental Immunology, Raven
Press, N.Y., p.246). Some epitope may be significantly larger. The
affinity of an antibody molecule for its cognate epitope ranges
from low, e.g. 10.sup.-6 M, to high, e.g., 10.sup.-11 M.
[0019] For example, antigens include proteins and other molecules
which are specifically associated with surfaces of particular types
of cancer cells, e.g. tumor cells. Many forms of cancer can be
characterized by production of proteins associated with that form
of the disease, and are not found in normal tissue. Often these
proteins are used at a specific stage of embryonic development, and
are not observed during normal adult lifetime. These antigens are
particularly useful as a source of epitopes for anti-cancer
vaccines. Examples of tumor antigens that are envisioned as
antigens for the conjugates of the present invention include those
corresponding to cancers affecting the breast, ovarian, lung, skin,
and brain. For example, breast tumors may be characterized by
abnormally expressed receptors, e.g. those of the human-EGF-like
receptor family (HER). Additionally, the nestin protein, which is
expressed by neuroepithelial stem cells during normal mammalian
fetal development, is also expressed on tumors of the central
nervous system, including most forms of brain cancer (McKay, D. G.
Ronald, U.S. Pat. No. 5,338,839, Aug. 16, 1994). It is also
expressed on melanomas found on the skin and on those which have
metastasized to other tissues (V. A. Florenes, R. Holm, 0.
Myklebost, U. Lendahl, 0. Fodstad, 1994, Cancer Res. 54:354-6). The
present invention contemplates incorporating these antigens or
epitopes of these antigens into compounds of the invention.
Preferably, the antigens of the toxin-antigen conjugates of the
invention are peptides associated with melanoma which may be
derived, for example, recombinantly or from tumor cell lysate.
[0020] Other examples of tumors expressing antigens contemplated by
the present invention include Wilm's tumor (A. J. Buckler, K. M.
Call, T. M. Glaser, D. A. Haber, D. E. Housman, C. Y. Ito, J.
Pelletier, Rose, E. A. Rose, U.S. Pat. No. 5,350,840),
gastrointestinal cancer (R. Fishel et al., International
Application WO 95/14085, May 26, 1995), cancers characterized by
development of multiple drug resistance during chemotherapy (J. M.
Croop et al., U.S. Pat. No. 5,198,344), and cancers characterized
by the presence of at least one of a large number of oncogenes well
known to the skilled artisan, such as Rb, ras, and c-myc, the
sequences of which are available for analysis to those with skill
in the art.
[0021] Alternatively, antigens of the invention may be associated
with the surfaces or secretion products of micro-organisms or
pathogens. The term "pathogen" is meant to include organisms that
cause disorders, such disorders produced by one or more particular
species of bacteria, viruses, fungi, and protozoans which are
disease-producing organisms. Examples of pathogens include
gram-negative bacterial species such as Escherichia coli serotype
0157:H7, Helicobacter pylori, H. mustelae, Haemophilus influenzae
and H. ducreyi, Pseudomonas aeruginosa, Shigella dysenteria,
Salmonella typhi and S. paratyphi; Gram-positive bacterial species
such as Mycobacterium tuberculosis, M. leprae, Clostridium tetani,
Staphylococcus aureus, and Streptococcus hemolyticus; obligate
intracellular bacterial organisms such as Rickettsia and Chlamydia
species; retroviruses, which are RNA containing viruses that use
reverse transcriptase to synthesize complementary DNA, including
but not limited to HIV-1, and -2; other pathogenic viruses such
HSV-I and -II, non-A non-B non-C hepatitis virus, pox viruses, and
rabies viruses; fungi such as Candida and Aspergillus species;
protozoa such as Cryptosporidiumparvum, Entamoeba histolytica and
Giardia lamblia; and animal pathogens such as Newcastle disease
virus. Obtaining unique epitopes from these organisms by screening
proteins and by assaying peptides in vitro are commonly known to
those skilled in the art; many examples have been described and the
appropriate amino acid residue sequence may be accessed from
Genbank.
[0022] The term "infection" is meant to include persistence and
growth of a pathogen in a subject host. While symptoms used to
diagnose the presence of infection include fever, inflammation,
pain, joint and muscular sensations at or near sites of infection,
the absence of one or more of these symptoms do not preclude
infection in a subject host organism. The term "inflammation"
indicates a set of host reactions that accompany infection, and may
also be present in the absence of infection, for example, as a
symptom of autoimmune reactions, degenerative diseases, tissue
remodeling disorders, exposure to allergens, and/or other
conditions. Inflammatory responses include cellular processes such
as neutrophil, mast cell and basophil degranulation with associated
release of proteases, histamines, and superoxide generation, and
production of and responses to cytokines such as interferons and
tumor necrosis factor.
[0023] One type of antigen can be an allergen. An "allergen" refers
to a substance that can induce an allergic or asthmatic response in
a susceptible subject. The number of allergens that elicit a
sensitive response in a proportion of a population is enormous, and
includes pollens, insect venoms, animal dander, dust mite proteins,
fungal spores and drugs (e.g. penicillin). Examples of natural
animal and plant allergens include proteins specific to the
following genera: Felis (Felis domesticus); Canis (Canis
familiaris); Dermatophagoides (e.g. Dermatophagoides farinae);
Periplaneta (e.g. Periplaneta americana); Ambrosia (Ambrosia
artemiisfolia; Lolium (e.g. Lolium perenne or Lolium multiflorum);
Cryptomeria (Cryptomeria japonica) ; Alternaria (Alternaria
alternata); Alnus (Alnus gultinosa); Betula (Betula verrucosa);
Quercus (Quercus alba); Olea (Olea europa); Artemisia (Artemisia
vulgaris); Plantago (e.g. Plantago lanceolata); Parietaria (e.g.
Parietaria officinalis or Parietaria judaica); Blattella (e.g.
Blattella germanica); Apis (e.g. Apis multiflorum); Cupressus (e.g.
Cupressus sempervirens, Cupressus arizonica and Cupressus
macrocarpa); Juniperus (e.g. Juniperus sabinoides, Juniperus
virginiana, Juniperus communis and Juniperus ashei); Thuya (e.g.
Thuya orientalis); Chamaecyparis (e.g. Chamaecyparis obtusa);
Agropyron (e.g. Agropyron repens); Secale (e.g. Secale cereale);
Triticum (e.g. Triticum aestivum); Dactylis (e.g. Dactylis
glomerata); Festuca (e.g. Festuca elatior); Poa (e.g. Poa pratensis
or Poa compressa); Avena (e.g. Avena sativa); Holcus (e.g. Holcus
lanatus); Anthoxanthum (e.g Anthoxanthum odoratum); Arrhenatherum
(e.g. Arrhenatherum elatius); Agrostis (e.g. Agrostis alba); Phleum
(e.g. Phleum pratense); Phalaris (e.g. Phalaris arundinacea);
Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghum
halepensis); and Bromus (e.g. Bromus inermis). An "allergen
associated state" includes states which are the resulting from an
allergic or asthmatic response to an allergen.
[0024] The term "toxin" includes compounds which are capable of
facilitating an immune response of the antigen. Exemplary toxins
include cholera b chain, Shiga toxins, and preferably, shiga-like
toxins, e.g., verotoxin. Shiga-toxin is a bacterial protein toxin
of the AB.sub.5 subunit family that is secreted by Shigella
dysenteriae. The A-subunit inhibits protein biosynthesis in higher
eukaryotic cells after transfer into the cytoplasm by modifying a
conserved residue of 28S rRNA. The B-subunit, a homopentamer (%-B
fragments) is responsible for toxin binding to and internalization
into target cells by interacting with the glycolipid Gb.sub.3 found
in the plasma membrane of these cells. The B-fragment is not toxic
but conserves the intracellular transport characteristics of the
holotoxin which in many Gb.sub.3 expressing cells is transported in
a retrograde fashion from the plasma membrane via endosomes into
the biosynthetic/secretory pathway.
[0025] In a related embodiment, the toxin is a Shiga-like toxin,
e.g., verotoxin. Currently known verotoxins include verotoxin 1,
verotoxin 2, verotoxin 2c and verotoxin 2e of subunit toxins
elaborated by some strains of E. coli. These toxins are involved in
the etiology of the hemolytic uremic syndrome and haemorrhagic
colitis. Cell cytotoxicity is mediated via the binding of the B
subunit of the holotoxin to the receptor glycolipid,
globotriaosylceramide, in sensitive cells. Advantageously, the
toxin of the invention is non-toxic, e.g., preferably, shiga-toxin
B or verotoxin B.
[0026] The term "associated" includes covalent linkages between the
toxin and the antigen. Preferred covalent linkages include, for
example, peptide linkages and cyanogen bromide activation. The term
also includes protein-protein interactions, hydrophobic
interactions, Van der Waals interactions and ionic interactions.
Examples include conjugates which comprise biotin.
[0027] In one embodiment, the toxin-antigen conjugate is produced
recombinantly. Methods for producing compounds of the invention
recombinantly are well known to the skilled artisan and are
elaborated in the Example.
[0028] In a further embodiment, the toxin-antigen conjugate may
further comprise an active or inactive endoplasmic reticulum
retrieval signal. The term "endoplasmic reticulum retrieval signal"
includes peptide sequences which enhance the ability of a conjugate
of the invention to interact with cells involved in immune
response. An example of an active endoplasmic reticulum retrieval
signal is KDEL, which may, for example, advantageously be attached
to the C-terminal of the toxin. An example of a suitable inactive
endoplasmic reticulum retrieval signal is KDELGL. Advantageously,
KDELGL is attached to the C-terminal of the toxin.
[0029] In a particularly preferred embodiment, the toxin-antigen
conjugate of the invention, comprises verotoxin B and a tumor
antigen, e.g., for example, a melanoma-associated peptide.
Advantageously, the conjugate may be produced recombinantly.
[0030] The invention also features a method for stimulating an
immune response in a mammal by administering to a mammal a
toxin-antigen conjugate. Preferably, the stimulation of the immune
response includes involvement of dendritic, e.g., Langerhans,
cells. The term "immune response" includes any immunological
response of the mammal to the conjugate of the invention.
Advantageously, the immune response may include, for example, the
promotion of T cells, generation of antibodies against the antigen,
and/or the presentation of the antigen by dendritic cells, e.g.
preferably, Langerhans cells. The term includes any response of the
immunological system of the mammal to the conjugate of the
invention. The term "stimulating" includes, for example, initiation
or enhancement of an immune response.
[0031] Processing and presentation of antigens to T lymphocytes are
critical events in the development of an immune response. As a
general rule, Major histocompatibility Complex (MHC) class I
molecules present endogenous peptide epitopes, mainly derived from
cytosolic and nuclear proteins to CD8+ T lymphocytes (CTL) (Monaco,
J. J. Immunol. Today (1992) 13:173-179; Heemels, M. T. and Ploagh,
H., Annu. Rev. Biochem. (1995) 64:463491), whereas peptides derived
from exogenous proteins are generated in the endocytic pathway and
presented on MHC class II molecules to CD4. T lymphocytes (Janeway,
C. A. Jr., et al., Inf. Rev. Imunol. (1993) 10:301-311; Pieters,
J., Curr. Opin, Immunol. (1997) 9:89-96). However, different
reports showed that self endogenous antigens expressed by normal or
tumor tissues can be efficiently processed via an exogenous class I
restricted pathway for presentation to CD8+ T cells.
[0032] In transgenic mice expressing a membrane bound form of
ovalbumin (OVA) only in the pancreatic islet cells and in renal
tubular cells, OVA derived peptides are presented in a class I
restricted manner by cells from bone marrow origin in the draining
lymph node of kidney or pancreas (Kurts, C., et al. J. Exp. Med.
(1996) 184:923-930). Huang et al. demonstrated that priming of T
cells to MHC class I restricted tumor antigens required the
transfer and processing of these antigens from tumor cells to bone
marrow derived antigen presenting cells (Huang, A. Y., et al.
Science (1994) 264:961-965). In spite of these arguments
demonstrating presentation in the context of MHC class I molecules
and generation of CTL via an exogenous processing pathway, most
studies failed to demonstrate efficient in vitro class I restricted
presentation or induction of CTL in vivo with foreign exogenous
soluble antigens (Moore, M. W., et al., Cell (1988) 54:777-785;
Rock, K, L., Immunol. Today (1996) 17:131-137; Watts, C., Annu.
Rev. Immunol. (1997)15:821-850).
[0033] Since CTL are an important component of the protective and
therapeutic immune responses to viral infections and tumors
(Sabzovari, H. et al. Cancer. Res. (1993) 53:4933-4937; Rosenburg,
S. A., et al. J. Natl. Cancer. Inst. (1994) 86:1159-1166;
Stevenson, P. G., et al. Virology (1997) 232:158-166; Feltkarnp, M.
C. Eur. J. Immunol. (1995) 25:2638-2642), different strategies have
been developed to allow MHC class I restricted presentation and
stimulation of CTL by exogenous soluble antigens.
[0034] Recombinant live vectors such as Vaccinia, Listeria or
Salmonella expressing viral or tumor antigens efficiently delivered
these antigens to the cytosol, thereby allowing their introduction
to the class I restricted presentation pathway and sensitization of
CTL in vivo (Gao, X. M. et al. Infect. Immunity (1992)
60:3780-3789; Pan, Z. K. et al. Cancer. Res. (1995) 55:4776-4779;
Tsang, K. Y. et al., J. Natl. Cancer. Inst. (1995) 87:952-990).
However, the use of like vectors poses risks for the recipient due
to the potential pathogenicity of the vectors used, especially In
immunosuppressed patients such as cancer and HIV infected subjects
(Kavanaugh, D. Y. et al. Hematol. Oncol. Clin. North. Arnenca
(1996) 10:927-951; Redfield, R. R. et al. N. Eng. J. Med (1987)
316:673-676).
[0035] Other approaches using particulate antigens linked to latex
beads (Kovacsovics-Bankowski, M. et al., Pro. Nat. Acad. Sci. USA
(1993) 90:4942-4946; Harding, C. V. et al. J. Immnunol. (1994)
153:4925-4933), fused with liposomes (Nair, S., et al., J. Exp. Med
(1992) 175:609-612) or associated with adjuvants (Ke, Y. et al.
Eur. J. Immunol. (1995) 25:549-553; Lipford, G. B. et al. Eur. J.
Immunol. (1997) 27:2340-2344) succeeded in introducing foreign
antigen in the MHC class I pathway in vitro and in vivo. In most
cases, phagocytosis was involved in this process and it appeared
that this class I presentation pathway required high antigen
concentrations and its efficiency was low (Reis e Sousa, C. et al.
J. Exp. Med (1995) 1B2:841-851).
[0036] The term "mammal" includes warm blooded animals such as, for
example, rodents (e.g. rats, mice, hamsters, squirrels), horses,
cows, pigs, sheep, cats, dogs, bears, goats, and primates (e.g.,
monkeys, chimpanzees, gorillas, and, preferably, humans).
[0037] The term "dendritic cells" include Langerhans cells,
interstitial dendritic cells, interdigitating dendritic cells,
follicular dendritic cells and circulating dendritic cells.
Langerhans cells are found in the epidermis and mucous membranes.
Interstitial dendritic cells populate most organs such as the
heart, lungs, liver, kidney, and gastrointestinal tract.
Interdigiting dendritic cells are present in T-cell areas of the
secondary lymphoid tissue and the thymic medulla. Circulating
dendritic cells include "veiled cells" which constitute about 0.1%
of the blood leukocytes.
[0038] In general, dendritic cells are covered with a maze of long
membrane processes resembling dendrites of nerve cells. Due to
their long dendritic processes, dendritic cells have been
challenging to study using conventional procedures for isolating
lymphocytes and accessory immune-system cells. Dendritic cells tend
to express high levels of both class II MHC molecules and the
co-stimulatory B7 molecule. For this reason, they are more potent
antigen-presenting cells than macrophages and B cells, both of
which need to be activated before they can function as APCs. After
capturing an antigen in the tissues by phagocytosis or by
endocytosis, dendritic cells migrate into the blood of lymph and
circulate to various lymphoid organs where they present the antigen
to T lymphocytes.
[0039] The invention also pertains to a method for treating an
antigen-related state in a mammal by administering to the mammal a
therapeutically effective amount of an antigen-toxin conjugate and,
thereby, stimulating an immune response in the mammal. For example,
this method includes coadministering an antigen with verotoxin B
subunit or administering a antigen coupled to a verotoxin B subunit
to a subject, e.g., a mammal, to stimulate the antigen presenting
capabilities of dendritic cells.
[0040] The term "antigen-related state" includes micro-organism or
pathogenic infections, allergen associated states, and, preferably,
tumors such as, for example, breast, ovarian, brain, skin, lung,
etc. Preferably, the antigen-related state is melanoma.
[0041] The term "treating" includes preventing and curing as well
as ameliorating at least one symptom of the antigen-related state.
It also includes the initiation of an immune response against an
antigen-related state that the mammal may be susceptible to, but
not necessarily suffering from. For example, in a mammal at risk
for melanoma, a conjugate of the invention may be administered to
said mammal, thus generating an immune response so as to prevent or
delay the initiation of the potential melanoma.
[0042] The term "administering" includes routes of administration
which allow the conjugate of the invention to perform its intended
function, e.g. stimulate an immune response. Preferred routes of
administration include, but are not limited to, orally,
intrabronchially, and transdermally. Depending on the route of
administration, the conjugate of the invention can be coated with
or disposed in a selected material to protect it from natural
conditions which may detrimentally effect its ability to perform
its intended function. The conjugate of the invention can be
administered alone or with a pharmaceutically acceptable carrier.
Further, the conjugate of the invention can be administered as a
mixture of conjugates of the invention, which also can be
coadministered with a pharmaceutically acceptable carrier. The
conjugate of the invention can be administered prior to the onset
of an antigen-related state, or after the onset of an
antigen-related state.
[0043] Preferably, the conjugates of the invention are administered
to the mammal transcutaneously through the skin or through a mucous
membrane. Mucous membranes include lubricated membranes of the
inner linings of many internal systems of mammals. Examples of
systems with mucous membranes include, but are not limited to, the
respiratory system, the gastrointestinal tract and the reproductive
tract. Preferred mucous membranes include, for example, the
membranes of a mammals'nose, nasal passages, throat, lungs, mouth,
stomach, intestine, colon, rectum, urethra, and vagina. Not to be
bound by theory, it is thought that by administering a compound of
the invention to a mammal by application or other means to a mucous
membrane or the skin of a mammal, is advantageous, for example, due
to the presence of specialized and powerful immune cells such as,
for example, dendritic cells, e.g., Langerhans cells.
[0044] In a particularly preferred embodiment, the present
invention contemplates methods, preferably non-invasive, for
immunizing a mammal from a specific antigen by transcutaneously
administering a conjugate of the invention. One advantage of
non-invasive forms of immunization, is that the need for injection
of the pharmaceutical composition into the mammal is, at least in
part, obviated thus potentially reducing the need for injections
and reducing, for example, the mammals' risk of exposure to foreign
pathogens, e.g., HIV. The term non-invasive includes methods of
administration such as transcutaneous administration through skin
and mucous membranes. Examples include, inhalation of the
pharmaceutical composition for transcutaneous administration
through the mucous membranes of the respiratory tract, swallowing
the pharmaceutical composition for administration through, for
example, transcutaneous administration, through, for example, the
mucous membranes, e.g., of the gastrointestinal tract, e.g., the
membranes of the mouth, throat, stomach, intestines, colon and
rectum. The invention also pertains to pharmaceutical compositions
for the immunization of mammals comprising a toxin-antigen
conjugate of the invention and a pharmaceutically acceptable
carrier suitable for transcutaneous administration of the
conjugate.
[0045] In a further embodiment, the invention pertains to
pharmaceutical compositions and methods which further comprise
adjuvants. An example of an adjuvant is KLH. A critical role in
enhancing the ability of compounds to be successfully administered
transcutaneously is played by adjuvants (Edelman Rev. Infec. Dis.
(1980) 2:370-383). Adjuvants have been important in the development
of the transcutaneous, e.g., transdermal or mucousal, routes as
useful and easily accessible non-invasive methods for
administration of compounds to mammals (Snider, Crit. Rev. Immunol.
(1995) 14:317-348). Suitable adjuvants are well known to those
skilled in the relevant arts.
[0046] In a further embodiment, the present invention contemplates
a method of inducing immunity in a mammal by administering to a
mammal a therapeutically effective amount of a toxin-antigen
conjugate of the invention and pharmaceutical carrier suitable for
administration to the mammal. Preferably, the carrier is suitable
for administration nasally, orally, or topically. Advantageously,
the method may further comprise the administration of an adjuvant.
The invention also pertains to pharmaceutical compositions capable
of inducing immunity in a mammal comprising an effective amount of
a toxin-antigen conjugate and a pharmaceutical carrier. Preferred
carriers include those suitable for administering the conjugate of
the invention to the mammal nasally, orally, or topically.
[0047] The language "therapeutically effective amount" of the
compound is that amount necessary or sufficient to treat or prevent
an antigen-related state, e.g. prevent the various morphological
and somatic symptoms of an antigen-related state. The effective
amount can vary depending on such factors as the size and weight of
the subject, the type of illness, or the particular conjugate of
the invention. For example, the choice of the conjugate of the
invention can affect what constitutes an "effective amount". One of
ordinary skill in the art would be able to study the aforementioned
factors and make the determination regarding the effective amount
of the conjugate of the invention without undue
experimentation.
[0048] The invention also pertains to a method for presenting
antigens on antigen-presenting cells, comprising contacting the
antigen-presenting cells with a toxin-antigen conjugate such that
the antigen-presenting cells present said antigens. Preferably, the
antigens are tumor antigens, e.g., melanoma antigens. It is
believed, for example, that the B subunit of verotoxin can
stimulate both surface presentation of the antigen by the dendritic
cells as well as intracellular antigen processing.
[0049] The term "antigen-presenting cells" include those cells
which display antigens or antigenic fragments. Examples of
antigen-presenting cells include some peripheral blood mononuclear
cells and, preferably, dendritic cells, e.g., Langerhans cells
[0050] In another embodiment, the invention features a
pharmaceutical composition which includes a toxin-antigen conjugate
and a pharmaceutically acceptable carrier. The phrase
"pharmaceutically acceptable carrier" as used herein means a
pharmaceutically acceptable material, composition or vehicle, such
as a liquid or solid filler, diluent, excipient, solvent or
encapsulating material, involved in carrying or transporting a
compound(s) of the present invention within or to the subject such
that it can performs its intended finction. Typically, such
compounds are carried or transported from one organ, or portion of
the body, to another organ, or portion of the body. Each carrier
must be "acceptable" in the sense of being compatible with the
other ingredients of the formulation and not injurious to the
patient. Some examples of materials which can serve as
pharmaceutically acceptable carriers include: sugars, such as
lactose, glucose and sucrose; starches, such as corn starch and
potato starch; cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer
solutions; and other non-toxic compatible substances employed in
pharmaceutical formulations.
[0051] As set out above, certain embodiments of the present
conjugates can contain a basic functional group, such as amino or
alkylamino, and are, thus, capable of forming pharmaceutically
acceptable salts with pharmaceutically acceptable acids. The term
"pharmaceutically acceptable salts" in this respect, refers to the
relatively non-toxic, inorganic and organic acid addition salts of
compounds of the present invention. These salts can be prepared in
situ during the final isolation and purification of the compounds
of the invention, or by separately reacting a purified compound of
the invention in its free base form with a suitable organic or
inorganic acid, and isolating the salt thus formed. Representative
salts include the hydrobromide, hydrochloride, sulfate, bisulfate,
phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate,
laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate,
fumarate, succinate, tartrate, napthylate, mesylate,
glucoheptonate, lactobionate, and laurylsulphonate salts and the
like. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J.
Pharm. Sci. 66:1-19).
[0052] In other cases, the compounds of the present invention may
contain one or more acidic functional groups and, thus, are capable
of forming pharmaceutically acceptable salts with pharmaceutically
acceptable bases. The term "pharmaceutically acceptable salts" in
these instances refers to the relatively non-toxic, inorganic and
organic base addition salts of compounds of the present invention.
These salts can likewise be prepared in situ during the final
isolation and purification of the compounds, or by separately
reacting the purified compound in its free acid form with a
suitable base, such as the hydroxide, carbonate or bicarbonate of a
pharmaceutically acceptable metal cation, with ammonia, or with a
pharmaceutically acceptable organic primary, secondary or tertiary
amine. Representative alkali or alkaline earth salts include the
lithium, sodium, potassium, calcium, magnesium, and aluminum salts
and the like. Representative organic amines useful for the
formation of base addition salts include ethylamine, diethylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine and the
like.
[0053] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0054] Examples of pharmaceutically acceptable antioxidants
include: water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0055] Formulations of the present invention include those suitable
for oral, nasal, topical, transdermal, buccal, sublingual, rectal,
vaginal and/or parenteral administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any methods well known in the art of pharmacy. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will generally be that amount of the
compound which produces a therapeutic effect. Generally, out of one
hundred percent, this amount will range from about 1 percent to
about ninety-nine percent of active ingredient, preferably from
about 5 percent to about 70 percent, most preferably from about 10
percent to about 30 per cent.
[0056] Methods of preparing these formulations or compositions
include the step of bringing into association a compound of the
present invention with the carrier and, optionally, one or more
accessory ingredients. In general, the formulations are prepared by
uniformly and intimately bringing into association a compound of
the present invention with liquid carriers, or finely divided solid
carriers, or both, and then, if necessary, shaping the product.
[0057] Formulations of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, lozenges (using a flavored basis, usually sucrose and
acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of a compound of the
present invention as an active ingredient. A compound of the
present invention may also be administered as a bolus, electuary or
paste.
[0058] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the active ingredient is mixed with one or
more pharmaceutically acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; humectants, such as glycerol; disintegrating
agents, such as agar-agar, calcium carbonate, potato or tapioca
starch, alginic acid, certain silicates, and sodium carbonate;
solution retarding agents, such as paraffin; absorption
accelerators, such as quaternary ammonium compounds; wetting
agents, such as, for example, cetyl alcohol and glycerol
monostearate; absorbents, such as kaolin and bentonite clay;
lubricants, such a talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and coloring agents. In the case of capsules, tablets and
pills, the pharmaceutical compositions may also comprise buffering
agents. Solid compositions of a similar type may also be employed
as fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugars, as well as high molecular
weight polyethylene glycols and the like.
[0059] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent.
[0060] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions which
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain portion of the gastrointestinal tract, optionally, in
a delayed manner. Examples of embedding compositions which can be
used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or
more of the above-described excipients.
[0061] Liquid dosage forms for oral administration of the compounds
of the invention include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluents commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof. Besides inert
dilutents, the oral compositions can also include adjuvants such as
wetting agents, emulsifying and suspending agents, sweetening,
flavoring, coloring, perfuming and preservative agents.
[0062] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0063] Formulations of the pharmaceutical compositions of the
invention for rectal or vaginal administration may be presented as
a suppository, which may be prepared by mixing one or more
compounds of the invention with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or a salicylate, and which
is solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release
the active compound.
[0064] Formulations of the present invention which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate.
[0065] Dosage forms for the topical or transdermal administration
of a compound of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches and inhalants.
The active compound may be mixed under sterile conditions with a
pharmaceutically acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0066] The ointments, pastes, creams and gels may contain, in
addition to an active compound of this invention, excipients, such
as animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0067] Powders and sprays can contain, in addition to a compound of
this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0068] Transdermal patches have the added advantage of providing
controlled delivery of a compound of the present invention to the
body. Such dosage forms can be made by dissolving or dispersing the
compound in the proper medium. Absorption enhancers can also be
used to increase the flux of the compound across the skin. The rate
of such flux can be controlled by either providing a rate
controlling membrane or dispersing the active compound in a polymer
matrix or gel.
[0069] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0070] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise one or more compounds of the
invention in combination with one or more pharmaceutically
acceptable sterile isotonic aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions, or sterile powders which may
be reconstituted into sterile injectable solutions or dispersions
just prior to use, which may contain antioxidants, buffers,
bacteriostats, solutes which render the formulation isotonic with
the blood of the intended recipient or suspending or thickening
agents.
[0071] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0072] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents which delay
absorption such as aluminum monostearate and gelatin.
[0073] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0074] Injectable depot forms are made by forming microencapsule
matrices of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions which are
compatible with body tissue.
[0075] The preparations of the present invention may be given
orally, parenterally, topically, or rectally. They are of course
given by forms suitable for each administration route. For example,
they are administered in tablets or capsule form, by injection,
inhalation, eye lotion, ointment, suppository, etc. administration
by injection, infusion or inhalation; topical by lotion or
ointment; and rectal by suppositories. Oral administration is
preferred.
[0076] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrastemal injection and
infusion.
[0077] The phrases "systemic administration," "administered
systematically," "peripheral administration" and "administered
peripherally" as used herein mean the administration of a compound,
drug or other material other than directly into the central nervous
system, such that it enters the patient's system and, thus, is
subject to metabolism and other like processes, for example,
subcutaneous administration.
[0078] These compounds may be administered to humans and other
animals for therapy by any suitable route of administration,
including orally, nasally, as by, for example, a spray, rectally,
intravaginally, parenterally, intracistemally and topically, as by
powders, ointments or drops, including buccally and
sublingually.
[0079] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms by conventional methods known to those of skill in the
art.
[0080] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this invention may be varied so as
to obtain an amount of the active ingredient which is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient.
[0081] The selected dosage level will depend upon a variety of
factors including the activity of the particular compound of the
present invention employed, or the ester, salt or amide thereof,
the route of administration, the time of administration, the rate
of excretion of the particular compound being employed, the
duration of the treatment, other drugs, compounds and/or materials
used in combination with the particular compound employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0082] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds of the invention
employed in the pharmaceutical composition at levels lower than
that required in order to achieve the desired therapeutic effect
and gradually increase the dosage until the desired effect is
achieved.
[0083] While it is possible for a compound of the present invention
to be administered alone, it is preferable to administer the
compound as a pharmaceutical composition. Preferred pharmaceutical
compositions include those suitable for administration orally,
transdermally, or intrabronchially.
[0084] According to the invention, antigens, e.g., Mage 1, either
coadministered with a toxin, e.g., the verotoxin B subunit, or
coupled, e.g., by covalent linkage, to the toxin are administered
to a subject to stimulate the antigen-presenting capabilities of
dendritic cells, e.g., Langerhans cells, and act as a vaccine. Such
vaccines can be administered orally, transdermally or
intrabronchially, and are capable of stimulating dendritic cells in
the tissue to which they are exposed.
[0085] The invention further pertains to a pharmaceutical
composition for treating an antigen-related state in a mammal. The
pharmaceutical composition includes an effective amount of a
toxin-antigen conjugate and a pharmaceutically acceptable carrier.
Preferably, the antigen-related state is a tumor, e.g., melanoma,
and the toxin-antigen conjugate comprises, for example, a B subunit
of a verotoxin.
[0086] The invention also pertains to a vaccine for vaccinating a
mammal, e.g., a human, for a antigen-related state, e.g. a tumor,
comprising a toxin-antigen conjugate and a pharmaceutically
acceptable carrier.
[0087] The present invention relates to the use of verotoxin B
subunits, and hybrid compositions which include all or part of a
verotoxin B subunit, to stimulate immune cells, e.g., to stimulate
the antigen-presenting function of immune cells, e.g., to provide
an effective vaccination strategy.
[0088] In certain embodiments, the B toxin of verotoxin (VT) is
administered by administering a holotoxin (e.g., VT1, VT2, VT2c,
VT2e) to the subject. However, in a preferred embodiment, the B
subunit of verotoxin is administered without the toxic portions of
VT holotoxin, thereby avoiding holotoxin toxicity. It is believed
that the B subunit of verotoxin can stimulate both surface
presentation of antigen by the dendritic cells as well as
intracellular antigen processing.
[0089] In another embodiment, melanoma-associated peptides linked
to the verotoxin B chain or to cholera B chain are administered to
the subject as anti-tumor vaccines by stimulating dendritic cells
and providing active presentation of such antigens to lymphocytes.
In a preferred embodiment, tumor lysates are linked to the
verotoxin B chain using techniques such as covalent linkages, e.g.,
cyanogen bromide activation. In yet another preferred embodiment,
adjuvants, e.g., KLH, are co-administered.
[0090] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patent applications, patents, and published patent
applications cited throughout this application are hereby
incorporated by reference.
EXAMPLE
[0091] Major histocompatibility complex class I presentation of
exogenous soluble tumor antigen fused to the B fragment of Shiga
toxin: Shiga toxin B-fragment targets exogenous antigen into the
MHC class I presentation pathway.
[0092] Recombinant proteins composed of the Shiga toxin B. fragment
fused to a CD8 epitope derived from the Mage 1 tumor antigen (van
der Bruggen, P. et al., Science (1991) 254:1643-1647) were
constructed. This antigen, initially cloned from human melanoma is
expressed in tumors of different origin. No expression of this gene
was found on a large panel of normal tissue except for testis which
did not express MHC class I molecules (van der Bruggen, P. et al.,
Science (1991) 254:1643-1647). The in vitro ability of this
engineered tumor antigen to be processed and presented in a class I
restricted pathway was investigated.
Materials and Methods
[0093] Cells
[0094] Peripheral blood mononuclear cells (PBMC) were separated
from peripheral blood of HLA-A1+healthy donors by centrifuigation
on Ficoll-Hypaque gradients. The dendritic cells used in the
present study were generated from PBMC according to the protocols
described previously (Sallusto, F. and Lanzavecchia, A., J. Exp.
Med. (1994) 179:1109-1118). Briefly, adherent cells were obtained
after 2 hours incubation of the Ficoll/Hypaque gradient-separated
mononuclear population on plastic dishes. These cells were then
cultured in RPMI 1640 medium supplemented with 10% heat inactivated
Fetal Calf Serum, 2 mM L-Glutamine, 50 IU/ml penicillin, 50
.mu.g/ml streptomycin, 5% sodium pyruvate ("complete medium") and
500 U/ml recombinant GM-CSF (Leucomax, Molgramostin, Sandoz Pharma,
Basel, Switzerland) and 100 IU/ml IL4 (Diaclone, Besangon, France).
On day 3, GM-CSF and IL4 were added once again and dendritic cells
obtained at day 6-7 were used for antigen presentation assays.
[0095] The B lymphoblastoid cell line LB 705 (HLA-A1, A2, B8, B27)
was previously described (Van der Bruggen, P. et al. Eur. J.
Immunol. (1994) 24:3038-3043). The EBV-transformed B cell line BM
21 was derived from an homozygous HLA-A1 individual (Yang, S. Y. et
al., Immunobiology of HLA. (Springer-Verlag, N.Y.:1989)). The
HLA-A2 EBV-transformed B cell line V0.1 was a gift of Dr. U. Blank.
All the B lymphoblastoid cell lines were maintained in complete
medium supplemented with 1 .mu.M 2-mercaptoethanol.
[0096] MZ2 CTL82/30 and LB373 CTL 246/15 clones recognized
respectively the 160-168 peptide of the Mage 1 protein in an HLA-A1
restricted manner (Traversari, C. et al., J. Exp. Med. (1992)
176:1453-1457) and the 27-35 peptide of the Mart 1 protein in an
HLA-A2 restricted manner (Coulle, P, G. et al., J. Exp. Med. (1994)
180:35-42).
[0097] Culture of CTL followed the protocols described by
Traversari et al. with slight modifications (Traversari, C. et al.,
Immunogenetics (1992) 35:145-152). Briefly, 3.times.10.sup.5 CTL
were cultured in 2 ml complete Iscove medium containing L-arginine
(116 .mu.g/ml), L-asparagine (36 .mu.g/ml) and L-glutamine (216
.mu.g/ml) supplemented with 10% human pool A, B and O serum (SAB)
from healthy donors and in the presence of 10.sup.6 irradiated (100
Gy) LG-2 EBV feeder cells, 10.sup.6 irradiated (30 Gy) allegoric
PBMC, PHA-L (5 .mu.g/ml) and IL-2 (150 IU/ml). Three or four days
after stimulation, CTL were diluted in culture medium supplemented
with IL-2 (50 IU/ml). Stimulation of CTL with feeder cells was
repeated once a week. For IFN.sub..lambda.assays, CTL cultures were
used six days after the last restimulation.
[0098] Plasmid constructions and fusion peptide production
[0099] The construction of the pSU108 based plasmids expressing the
B-fragment of Shiga toxin or fusion proteins in which a
N-glycosylation site and an active (KDEL) or inactive (KDELGL)
endoplasmic reticulum (ER) retrieval signal were introduced at the
C-terminus of the B-fragment, were described previously (Johannes,
L. et al., J. Biol. Chem. (1997) 272:19554-19561). A PCR based
strategy was used to introduce the Mage 1 epitope and its 5' and 3'
flanking sequences (DVKEADPTGHSYVLG) into the Not 1 site of
previously constructed B-Glyc-KDEL and B-Glyc-KDELGL expression
vectors. The resulting fusion proteins were termed B-Mage
1-Glyc-KDEL and B-Mage 1-Glyc-KDELGL. Sequences were checked by
double-stranded DNA sequencing. The proteins were expressed in E.
coli strain DH5.alpha. and purification was essentially done as
described (Johannes, L. et al., J Biol. Chem. (1997)
272:19554-19561).
[0100] Briefly, after preparation of periplasmic extracts, they
were loaded on a QFF column (Pharmacia) and eluted by a linear NaCl
gradient (120 to 400 mM) in 20 mM Tris/HCl, pH 7.5. Shiga B-Mage 1
fusion protein containing fractions were dialyzed against 20 mM
Tris/HCl, pH 7.5, reloaded on a Mono Q column (Pharmacia), and
eluted as before.
[0101] The Antennapedia-Mage 1 (Antp-Mage 1) fusion gene was
obtained by insertion of synthetic oligonucleotides encoding the
Mage 1 epitope and its 5' and 3' flanking sequences (see above)
between the Xhol and BamH1 restriction sites of the PAH61S plasmid
in replacement of the Rab3 coding sequence (Perez, F. et al., J.
Cell. Sci. (1992) 102:717-722). An Ndel-BamHI restriction fragment
containing the Antp-Mage 1 sequence was then subcloned into the
Novagen plasmid pET 2gb (+) (R&D System, London, UK) in which
the fusion protein expression was under the control of T7 promoter
and His-tagged at its C-terminus. The fusion peptides were
expressed in E. coli strain BL21 (DE3)Lys after IPTG induction as
described (Studier, F. W. et al., Methods in EnzymoL (1990)
185:60-89). The Antp-Mage 1 protein was then purified on Ni-NTA
agarose gel (QIAGEN GmbH, Hilden, Germany) under denaturing
conditions according to the supplier's protocols. The resulting
fusion proteins were estimated to be 95% pure by SDS-polyacrylamide
gel electrophoresis and Coomassie blue staining (FIG. 1A, B, and
C).
[0102] Western Blot
[0103] After high resolution SDS-polyacrylamide gel
electrophoresis, proteins were electro-transferred onto a
nitrocellulose membrane (BA85, 0.45 mm, Schleicher & Schuell,
Darsell Germany) in transfer buffer (192 mM glycine, 25 mM Tris
base pH 8.3, 20% ethanol) using the transblot cell (Bio-Rad). The
membrane was then saturated for 1 hour at 37.degree. C. in 20 mM
Tris (pH 7.4), 150 mM NaCl (Western buffer (WB)), 5% bovine serum
albumin (BSA), 0.1% Tween 20 and incubated for 18 hours at
4.degree. C. with either the mouse monoclonal antibody 13C4 (4
.mu.g/ml) (ATCC, Rockville, USA) directed against the B-fragment of
Shiga toxin for Shiga B-Mage 1 Fusion proteins or the mouse
anti-(His).sub.6-tag antibody (1 .mu.g/ml) (Dianova, Takara
Biomedical Europe S. A. Genevilliers, France) for the Antp-Mage 1
fusion protein. The membranes were washed with WB and 0.1% Tween 20
and incubated for 1 hour with anti-mouse immunoglobulin coupled to
horseradish peroxidase (Amersham, Les Ulis, France). After washing
with WB and 0.1% Tween 20, the filters were incubated with the
Western-blotting reagent ECL (Amersham), and chemiluminescence was
detected by exposure of the membranes to Biomax MR films
(Kodak).
[0104] Antigenic Peptides
[0105] Synthetic peptides 27-35 Mart 1 (AAGIGILTV) and 160-168 Mage
1 (EADPTGMSY) were obtained from Neosystem (Strasbourg, France) and
derived from previously published sequences (Traversari, C. et al.
J. Exp. Med. (1992) 176:1453-1457; Kawakami, Y. et al. J. Exp. Med.
(1994) 180:347-352).
[0106] Antigen presentation assays
[0107] Antigen presenting cells (PBMC, B-EBV, T cells or dendritic
cells) were plated in 96-well flat bottom microplates at 10.sup.5
cells/well and pulsed at 37.degree. C. for 4 hours or 15 hours with
antigen in 100 .mu.l Iscove medium without SAB. At the end of the
incubation, the medium was removed and 20,000 CTL clones were added
to each well in 100 .mu.l CTL culture medium containing 25 units/ml
of IL2. After 24 hours, 50 .mu.l of supernatant were harvested and
IFN.sub..lambda.was measured by ELISA (Diaclone, Besangon,
France).
[0108] In some experiments, the cells were fixed in 1%
paraformaldehyde for 10 min at room temperature and extensively
washed before transfer to microplates. Where appropriate, Brefeldin
A (Sigma) or Chloroquine (Sigma) were added at 2 .mu.g/ml and 250
.mu.M respectively for 30 min prior to addition of antigen and were
present during the antigen processing incubation at the same final
concentrations.
[0109] DTAF coupling
[0110] B-Mage1-Glyc-KDEL was coupled to DTAF
(5-(4,6-dichlorotriazin-2-yl)- amino)fluorescein) essentially as
described previously. Briefly, 60 .mu.g of recombinant
B-Mage1-Glyc-KDEL in 20 mM HEPES, pH 7.4, 150 mM NaCl were added to
250 mM NaHCO.sub.3 and a 10 fold molar excess of DTAF (Sigma) and
incubated by end-over-end rotation for 30 min at room temperature.
0.2 mM NH.sub.4Cl was then added and the coupled protein was
purified on PD10 columns (Pharmacia).
[0111] Internalization and Immunofluorescence staining
[0112] 10.sup.5 B-EBV BM 21 cells, grown on polylysine pretreated
12-mm round glass coverslips, were incubated on ice for 45 min with
1 .mu.g/ml of DTAF-labeled recombinant B-Mage1-Glyc-KDEL. After
washing, the cells were incubated for 1 hour at 37.degree. C.,
fixed with 3% paraformaldehyde for 10 min, permeabilized with
saponin (0.01%), stained with the monoclonal anti-Lamp-2 antibody
H4B4 (Pharmingen, San Diego) and revealed with a Texas Red coupled
anti-mouse IgG antibody (Jackson, West Grove, USA).
[0113] Confocal laser scanning microscopy and immunofluorescence
analysis were performed using a TCS4D confocal microscope based on
a DM microscope interfaced with an argon/krypton laser (Johannes,
L. et al., J. Biol. Chem. (1997) 272:19554-19561).
Results
[0114] Biochemical characterization of Shiga B-Mage 1 and Antp-Mage
1 fusion proteins
[0115] As shown in FIG. 1A, Mage 1 containing B-fragment fusion
proteins carrying an active ER retrieval signal (the tetrapeptide
KDEL) or an inactive version of this signal (the hexapeptide
KDELGL) migrate under reducing conditions with molecular weights of
about 11.3 kDa corresponding to their expected sizes. Western blot
analysis with a monoclonal anti-B-fragment antibody (13C4)
confirmed the identity of all purified Shiga B fusion proteins
(FIG. 1B).
[0116] The two Shiga B-Mage 1 fusion proteins were already, after
purification, partially cleaved, yielding 9.5 kDa fragments (FIG.
1A-B). It should be noted, however, that the Mage 1 sequence is
internal within B-Mage 1-Glyc-KDEL and B-Mage1-Glyc-KDELGL. Thus,
even if C-terminal cleavage was responsible for the production of
the 9.5 kDa fragments, as judged from their molecular weights, they
still contain the Mage 1 epitope.
[0117] Another fusion protein was constructed in which the Mage 1
peptide was fused to a polypeptide derived from the third segment
of the Antennapedia homeodomain (Perez, F. et al., J. Cell. Sci
(1992) 102:717-722). Coomassie blue polyacrylamide gel staining and
western blot analysis identified the recombinant Antp-Mage 1
protein as a 15 kDa polypeptide (FIG. 1C).
[0118] Presentation of exogenous soluble Shiga B-Mage 1 fusion
proteins by class I molecules
[0119] Role of the KDEL sequence
[0120] To assess the potential of exogenous Shiga B-Mage 1 fusion
protein to drive MHC class I presentation of internal Mage 1
epitope, PBMC were pulsed with B-Mage 1-Glyc-KDEL. As shown in FIG.
2, these PBMCs efficiently presented the Mage 1 peptide derived
from Shiga B-Mage 1 fusion protein in an HLA-A1 restricted manner
to specific CTLs. The influence of the KDEL signal on the
efficiency of presentation was then tested. Both the KDEL- and the
KDELGL-bearing Shiga B-Mage 1 fusion proteins were able to activate
a Mage 1 specific HLA class I restricted CTL response (FIG. 2).
Similar activity of the two molecules were also observed when
different concentrations of both protein were tested (data not
shown). Presence of the PBMC during the experiment was necessary
because direct CTL activation by Shiga B-Mage 1 fusion proteins did
not occur (data not shown).
[0121] For the following examples, only B Mage 1-Glyc-KDEL was used
since the antigen presentation abilities of KDEL- and
KDELGL-bearing Shiga B-Mage 1 fusion proteins were equivalent.
[0122] Analysis of MHC class I presentation of soluble BMage
1-Glyc-KDEL protein by different antigen presenting cells.
[0123] Homogeneous antigen presenting cells were sensitized with
Shiga B-Mage 1 fusion protein. After pulsing B lymphoblastoid cells
and dendritic cells, activation of the class I restricted Mage 1
specific CTL was demonstrated (FIG. 3). These results were observed
with two different B-EBV cell lines (EIM21 and LB 705) and
dendritic cells derived from two HLA-A1 donors. As low as 0.2 .mu.M
of Shiga B-Mage 1 protein was sufficient to sensitize B-EBV cells
and dendritic cells for Mage 1 peptide presentation (FIG. 3).
Conversely, T cell clones sensitized in vitro with Shiga B-Mage1
fusion protein could not activate autologous T cell clones (FIG.
3). The 82/30 cloned T cells used for this study as antigen
presenting cells expressed HLA-A1 molecules and presented synthetic
Mage 1 peptide (data not shown).
[0124] Analysis of the specificity of MHC class I presentation of
Mage 1 peptide derived from exogenous soluble Shiga B-Mage 1 fusion
protein by B lymphoblastoid cell lines.
[0125] An exogenous class I restricted antigen presentation pathway
is rarely observed in B-EBV cells (Rock, K, L., Immunol. Today
(1996) 17:131-137; Watts, C., Annu. Rev. ImmunoL (1997)
15:821-850). To demonstrate the role of Shiga toxin B-fragment in
this process, a recombinant protein was produced in which the B
fragment was replaced by the third segment of the antennapedia
homeodomain, a transcription factor from Drosophila. This domain
has been shown to translocate some peptides into the cytoplasm and
the nucleus of eukaryotic cells (Perez, F. et al., J. Cell. Sci.
(1992) 102:717-722). It also targeted exogenous antigen in the MHC
class I processing pathway in some cell types (Schutze-Redelmeier,
M. P. et al., J. Immunol. (1996) 157:650-655). As shown in FIG. 4A,
no presentation of Mage 1 peptide could be detected when the
antp-Mage 1 fusion protein was used to pulse HLA-A1 B-EBV cells.
When B-EBV cells with HLA-A2 haplotype were sensitized with Shiga
B-Mage 1 protein, no activation of Mage 1 specific CTL could be
observed (FIG. 4A). The specificity of the activation was also
supported by the absence of stimulation of Mart 1 specific CTL
clones when HLA-A1 or -A2 B-EBV cells were pulsed with Shiga B-Mage
1 fusion protein (FIG. 4B). B-Glyc-KDEL without Mage 1 epitope was
not recognized by the Mage 1 specific CTL clone (FIG. 4A).
Altogether, these data demonstrate a specific HLA-A1 restricted
presentation of Mage 1 peptide derived from the Shiga B-Mage 1
fusion protein.
[0126] Role of internalization and intracellular processing in the
MHC class I presentation of soluble Shiga B-Mage 1 fusion protein
by B lymphoblastoid cell lines.
[0127] The ability of recombinant Shiga B-Mage 1 fusion protein to
direct internal peptide epitopes into the class I restricted
pathway and its dependence on the internalization of this protein
was tested.
[0128] Paraformaldehyde fixed B lymphoblastoid cells did not allow
presentation of Shiga B-Mage 1 derived Mage 1 peptide to class 1
restricted Mage 1 specific CTL clone, whereas exogenous synthetic
Mage 1 peptide incubated with fixed B-ESBV cells activated this CTL
(FIG. 5A). This precluded extracellular Shiga B-Mage 1 processing
as an explanation for the observed exogenous HLA class I restricted
antigen presentation. To further demonstrate that intracellular
transport or processing were involved in this model of antigen
presentation, Brefeldin A (BFA) which blocks protein transport in
the biosynthetic/secretory pathway, or Chloroquine which raises the
pH of endosomes and thus inhibits endosomal proteolysis were used.
Neither chloroquine nor BFA interfered with presentation of
exogenous synthetic Mage 1 peptide (FIG. 5B). The antigen
presenting ability of these cells was maintained. In contrast, the
presence of BFA during the incubation of soluble Shiga B-Mage 1
fusion protein prevented the presentation of the Mage 1 T cell
epitope (FIG. 5C). This effect was not observed with chloroquine.
In control experiments, the same concentration of chloroquine was
efficient to inhibit HLA class II restricted presentation of
tetanus toxin derived peptides (data not shown).
[0129] Internalization of Shiga B-Mage 1 fusion protein in B-EBV
cells: Analysis of its colocalization with a lysosomal marker.
[0130] To follow the intracellular transport of B-Mage 1-Glyc-KDEL
in B-EBV cells, the protein was covalently linked to the
fluorophore DTAF. After its binding to cells on ice and subsequent
incubation for 1 hour at 37.degree. C., the protein was
internalized into cytoplasmic structures with a marked juxtanuclear
staining (FIG. 6A). The staining patterns corresponded to the Golgi
and ER compartments, consistent with the previous studies on HeLa
cells (Johannes, L. et al., J. Biol. Chem. (1997) 272:19554-19561).
Using an antibody against the lysosomal associated membrane
protein-2 (Lamp-2) (FIG. 6B) in double labeling immunofluorescence
experiment, the Shiga B-Mage 1 fusion protein was largely excluded
from Lamp-2 positive lysosomal compartments (FIG. 6C).
[0131] Approximately 10-20% of the B-EBV cells showed intermediate
to strong B-fragment specific staining after B-Mage-Glyc-KDEL
internalization. Such heterogeneity between cells of a given
population has already been described (Sandvig, K. et al. J. Cell
Biol. (1994) 126:53-64) and is due to variations in the toxin
receptor expression in function of the cell cycle (Pudymaitis, A.
and Lingwood, C. A., J. Cell. Physiol. (1992) 150:632-639).
Discussion
[0132] It has been demonstrated that a soluble CD8 tumor antigen
fused to the B fragment of Shiga toxin is efficiently presented in
an HLA class I restricted manner to specific CTL. Although some
partial proteolytic cleavage of the purified recombinant protein
was observed, extracellular processing is unlikely for the
following reasons: i) the CTL Mage 1 epitope is internal within the
Shiga B-Mage 1 fusion protein and therefore it requires two or more
separate and specific cleavage events to generate HLA-A1 binding
Mage 1 peptide; ii) the absence of presentation of Mage 1 peptides
derived from Shiga B-Mage 1 fusion protein by T cells which in the
same experiment kept the ability to bind and present synthetic Mage
1 peptides discounts extracellular cleavage (FIG. 3); iii)
paraformaldehyde fixed antigen presenting cells were able to
present synthetic exogenous Mage 1 peptides, yet they did not
process Shiga B-Mage 1 fusion proteins; and iv) Brefeldin A
prevented the presentation of Mage 1 epitope derived from soluble
Shiga B-Mage 1 protein. Since Brefeldin A inhibits both the
transport of Shiga B-Mage 1 to the endoplasmic reticulum (Johannes,
L. et al., J. Biol. Chem. (1997) 272:19554-19561) and the
association and transport of processed peptides with nascent class
I molecules to the plasma membrane (Monaco, J. J., Immunol. Today
(1992) 13:173-179), its exact mechanism of inhibition remains to be
established. However, the experiments with BFA indicate that
internalization of Shiga B-Mage 1 fusion protein is required for
efficient HLA-class I restricted presentation.
Equivalents
[0133] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *