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WO1998033888A1 - Cellules a peptides ou a antigenes charges de peptides - Google Patents

Cellules a peptides ou a antigenes charges de peptides Download PDF

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Publication number
WO1998033888A1
WO1998033888A1 PCT/US1998/001959 US9801959W WO9833888A1 WO 1998033888 A1 WO1998033888 A1 WO 1998033888A1 US 9801959 W US9801959 W US 9801959W WO 9833888 A1 WO9833888 A1 WO 9833888A1
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Prior art keywords
cells
peptide
ctl
peptides
antigen
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PCT/US1998/001959
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English (en)
Inventor
Van Tsai
Scott Southwood
John Sidney
Alessandro Sette
Esteban Celis
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Epimmune, Inc.
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Publication date
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Priority to AU61409/98A priority Critical patent/AU6140998A/en
Priority to JP53317398A priority patent/JP2001509677A/ja
Priority to CA002278189A priority patent/CA2278189A1/fr
Priority to EP98906086A priority patent/EP1012238A4/fr
Publication of WO1998033888A1 publication Critical patent/WO1998033888A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4205—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4267—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K40/4268—MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to compositions and methods for preventing or treating pathological states such as viral diseases and cancer through ex vivo therapy.
  • it provides methods for inducing cytotoxic T lymphocytes (CTL) using antigen presenting cells (APC) with a peptide of choice bound to selected major histocompatibility complex (MHC) molecules.
  • CTL cytotoxic T lymphocytes
  • APC antigen presenting cells
  • MHC major histocompatibility complex
  • CTLs or CD8 cells as they are also known specifically recognize cells that bear foreign antigens, and kill them. They constitute a main line of defense against diseased states such as those caused by viral infections and tumor cells.
  • antigen In contrast to antibodies, antigen must first be presented to the T cell receptors for activation to occur. CTLs recognize, not the intact foreign antigen itself, but rather 8-12 residue peptide fragments bound to MHC class I molecules and presented on the surface of antigen-presenting cells (APCs).
  • APCs antigen-presenting cells
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • MHC molecules are classified as either Class I, Class II or class HI molecules.
  • Class I MHC molecules are expressed on almost all nucleated cells and are recognized by CTLs.
  • Class ⁇ MHC molecules are expressed primarily on cells involved in initiating and sustaining immune responses, such as T lymphocytes, B lymphocytes, macrophages, etc.
  • Class ⁇ MHC molecules are recognized by helper T lymphocytes and induce proliferation of helper T lymphocytes and amplification of the immune response to the particular immunogenic peptide that is displayed.
  • CTL responses are not directed against all possible determinants but only against a selected few "immunodominant" epitopes. According to the nomenclature developed by Sercarz et al., Annu. Rev. Immunol.
  • an epitope is immunodominant when it is normally recognized in the course of a natural infection, or upon immunization with whole or naturally processed antigens.
  • Subdominant epitopes mediate immune responses if 1) synthetic peptides are used as immunogens, or 2) the dominant epitopes are removed (or altered) from the whole antigen.
  • the subdominant epitopes are also processed by the APC and presented as MHC complexes on the surface.
  • a theoretically attractive approach for the treatment of diseases as cancer, AIDS, hepatitis and other infectious disease is to activate only those CTLs that recognize diseased cells.
  • One possible approach is to immunize a healthy individual, isolate the CTLs from this individual, and inject these cells into the diseased person.
  • the MHC haplotype of the donor must be identical to that of the recipient. This is important because the CTLs of the recipient can only interact with peptides bound to one of the three to six Class I molecules present in the individual.
  • CTLs react violently with all Class I molecules which are different from those expressed in the individual from whom the CD8 cells are obtained, regardless of what peptides the Class I molecules contain. This reactivity is the underlying cause of the immune rejection of transplanted organs.
  • TAA tumor-associated antigens
  • CTLs from cancer patients were used to screen expression gene libraries or peptides eluted from tumor cell MHC molecules. Most of these CTL were from melanoma patients, and the majority of CTL epitopes defined to date are from melanomas. Few epitopes are available to address other more commonly found type of malignancies such as breast, lung, colon and other gastrointestinal carcinomas.
  • This invention is generally directed to methods of activating cytotoxic T cells (CD8 cells) in vitro or in vivo.
  • the method comprises associating desired immunogenic peptides from an antigen with class I MHC molecules on an antigen presenting cells pretreated with a growth factor; and incubating the antigen presenting cells with the cytotoxic T cells in the presence of two or more growth factors, thereby producing activated cytotoxic T cells.
  • a preferred APC is a dendritic cell, especially a dendritic cell that prepared by incubating dendritic cell precursors in the presence of GM-CSF and IL-4.
  • one growth factor is IL-7 and another is IL-10.
  • IL-7 is preferably added at the start of the incubation step and IL- 10 is added one day later.
  • the invention also provides a method of specifically killing target cells in a human patient, comprising obtaining a fluid sample containing cytotoxic T cells from a patient; contacting the cytotoxic T cells with antigen presenting cells pretreated with one or more pre-treatment growth factors, wherein the antigen presenting cells comprise class I MHC molecules incubating said pretreated antigen presenting cells with the cytotoxic T cells in the presence of desired immunogenic peptides and two or more incubation growth factors, thereby producing activated cytotoxic T cells; contacting the activated cytotoxic T cells with an acceptable carrier, thereby forming a pharmaceutical composition; and administering the pharmaceutical composition to a patient.
  • the antigen presenting cells having empty MHC class I molecules on their surface are capable of inducing cytotoxic T cells which are useful in the treatment of chronic infectious diseases and cancer.
  • this invention provides methods of producing empty MHC class I molecules on antigen presenting cells, loading those empty MHC class I molecules with selected immunogenic peptides, activating cytotoxic T cells which are specific for killing specific antigen targets.
  • This invention has broad therapeutic application in the treatment of cancers, certain immune diseases and viral diseases.
  • the method may further comprise: separating activated CTLs from the antigen presenting cells having the empty MHC class I molecule on its surface; suspending the activated CTLs in an acceptable carrier or excipient as a pharmaceutical composition; and administering the pharmaceutical composition to a patient having the disease.
  • IL-7, IL-10 and peptide-pulsed dendritic cells were used to induce primary cytotoxic T lymphocyte (CTL) responses in vitro.
  • DC were prepared from peripheral blood mononuclear cells isolated from normal volunteers and pulsed with various HLA-A1 and -A2.1 binding peptides corresponding to known viral and tumor CTL epitopes. These antigen-presenting cells were quite efficient in inducing CTL responses to a hepatitis B virus epitope restricted by the HLA-A2 molecule. Most of the CTLs were not only capable of killing peptide-coated target cells but also endogenously processed antigen, indicating that the CTL possessed both high specificity and affinity to the immunogen.
  • CTL induction by DC was significantly enhanced by the addition of recombinant IL-7 and IL-10, but not by IL-12.
  • the peptide-induced CTL were very effective in recognizing and killing tumor cell lines expressing the appropriate tumor antigens, and could be expanded 1000 fold in tissue culture without loss of activity and specificity.
  • the methods for inducing CTL with peptide-coated DC and the combination of cytokines are valuable for both the identification of novel CTL epitopes and for the development of CTL-based adoptive immunotherapies.
  • the invention also provides methods for defining the entire immunogenic potential of a given antigen in terms of CTL responses, irrespective of whether a given epitope is or is not recognized in the course of a natural disease or infection (that is, whether it is immunodominant or not).
  • the gp 100 melanoma-associated tumor antigen was selected as a model system to study the diversity of human anti-tumor cytotoxic T cell responses.
  • Peptides corresponding to dominant gplOO HLA-A2.1 -restricted CTL epitopes were tested using lymphocytes from normal volunteers and an in vitro priming protocol which utilizes peptide-pulsed dendritic cells as APC, and IL-7 and EL- 10 as immune enhancing cytokines.
  • High CTL activity towards both peptide-pulsed target cells and gpl00 + melanoma cells was also obtained with 4 out of 5 peptides tested.
  • the present invention comprises the peptides detected using the methods described herein.
  • HLA-A2.1 -binding peptides from gplOO which do not appear to represent CTL epitopes in melanoma patients, were also tested for their capacity to induce CTL using the in vitro priming protocol.
  • Three of six peptides tested induced CTL in lymphocytes from normal volunteers.
  • One of these peptides was also immunogenic for lymphocytes derived from a melanoma patient in remission. Because these three CTL epitopes were not recognized in the natural immune response in melanoma patients but do appear as immunogens when peptides are used to induce the T-cell response, they may be considered as typical "subdominant" epitopes.
  • the present invention provides an alternative approach to tumor epitope identification which does not require the use of patient's CTL, but relies instead on the identification of MHC-binding peptides from known TAA and on novel in vitro priming protocols. Specifically, peptides selected on the basis of MHC binding motifs and on their binding capacity to MHC molecules, are tested for their ability to elicit tumor-reactive CTL in vitro using lymphocyte cultures from normal individuals. Following this "reverse immunology" approach, several CTL epitopes from TAA expressed in melanoma tumors have been identified. In the examples, this methodology is applied to identify CTL epitopes derived from various TAA expressed on solid adenocarcinomas (Weynants, P. et al.
  • CD8 + lymphocytes from a normal volunteer were stimulated in vitro with G9 2S0 -pulsed autologous DC.
  • CD8+ lymphocytes from a normal volunteer were stimulated in vitro with gplOO peptides loaded onto autologous DC.
  • CTL lines were produced and tested with various "Cr labeled target cell lines in the presence and absence of unlabelled cold targets (Inhibitors) at an inhibitor: target ratio of 20: 1.
  • CTLs elicited with peptide G9 178 , (A) or G10 177 (B) were tested for their capacity to kill these targets: D , .221(A2.1) plus G10 177 ; O , .221 (A2. 1) plus G9 178 ; ⁇ , .221 (A2. 1) - no peptide; A , 624mel (A2 + , gpl00+); O , A375mel (A2 + , gplOO).
  • CTL were elicited using CD8+ lymphocytes from a normal volunteer using peptide-pulsed autologous DC.
  • FLPSDFFPSV at an inhibitor: target ratio of 20: 1.
  • Figure 6 Induction of melanoma-reactive CTL with patient's lymphocytes using a peptide corresponding to a subdominant epitope.
  • Blood lymphocytes from a melanoma patient were stimulated in vitro with autologous DC pulsed with peptide G10 177 , and after 3 cycles of antigen-restimulation, the T-cell line was assayed for its lytic activity against these targets: D ,.221(A2.1) plus G10 177 ; ⁇ ,.221(A2.1) - no peptide; O , 624mel (A2 + , gpl00+); A , A375mel (A2 + , gplOO).
  • Antigen specificity was further demonstrated by cold target inhibition of the lysis of 624mel cells by the addition of a 20:1 excess of unlabeled .221(A2.1) pulsed with G10 177 , ( 0 ); or unlabeled .221 (A2.1) pulsed with an HLA-A2.1 -binding control peptide ( ⁇ , FLPSDFFPSV).
  • FIG. 7 Recognition of various tumor types by a MAGE2 specific CTL clone.
  • the MAGE2[10 1S1 ] specific CTL clone was tested for its lytic activity against the following target cells: O, .221A2.1 pulsed with MAGE2[lQ l51 ] ⁇ • ,.221A2.1 without peptide; ⁇ , 624mel (melanoma, A2 + , MAGE2 + ); , KATO-m (gastric Ca, A2 + ,
  • MAGE2 + O , SW403 (colon Ca, A2 + , MAGE21); U , WiDr (colon Ca, A2, MAGE21); A , 888mel (melanoma, A2, MAGE2).
  • the tumor cell lines 624mel (A), SW403 (B) or KATO-m (C) were 31 Cr labeled and mixed at various Inhibitors / Target ratios with following cold targets: O, .221A2.1 pulsed with 4G£2[10 137 ]; A , .221A2.1 pulsed with irrelevant A2.1 binding peptide (HB i 8 - 27 ); • > .221A2.1 without peptide.
  • the effectors/target ratio used were 8: 1 (A), 4: 1 (B) or 5: 1 (C).
  • MAGE3[9 1 1 ] specific CTL clone was tested for cytotoxicity using following targets: O, .221A2.1 pulsed with MAGE3[9 112 ]; • , .221A2.1 without peptide; ⁇ , 624mel (melanoma, A2 + , MAGE3 + ); D, KATO-m (gastric Ca, A2 + , MAGE3 + ); M , SW403 (colon Ca, A2 + , MAGE3 + ); ⁇ . WiDr (colon Ca, A2 ⁇ MAGE3+); A , 888mel (melanoma, A2 ⁇ MAGE3-).
  • the tumor cell lines 624mel (A), SW403 (B) or KATO-m (Q were 31 Cr labeled and mixed at various Inhibitors / Target ratios with following cold targets: O, .221 A2.1 pulsed with MAGE3[9 U2 ]; A , .221A2.1 pulsed with irrelevant A2.1 binding peptide (HBc 18 . 27 ); • , .221A2.1 without peptide.
  • the effectors/target ratio used were 8: 1 (A), 8:1 (B) or 5: 1 (C).
  • FIG. 11 Recognition of various tumor types by a CEA specific CTL clone.
  • the CEA[9 691 ] specific CTL clone was tested for its lytic activity against following target cell lines: O, .221A2.1 pulsed with CEA[9 691 ]; • , .221A2.1 without peptide; ⁇ , KATO-m (gastric Ca, A2 + , CEA + ); O , SW403 (colon Ca, A2 + , CEA + ); A , HT-29 (colon Ca, A2 ⁇ CEA + ).
  • CEA[9 691 ] specific CTL were tested at the effectors/target ratio of 15: 1 in both experiments.
  • the tumor cell lines KATO-m (A) or SW403 (B) were 51 Cr labeled and mixed at different Inhibitors / Target ratios with following cold targets: 0, .221A2.1 pulsed with CEA[9, 691 + ]; A , .221A2.1 pulsed with irrelevant A2.1 binding peptide (HBc 18 . 2 ).
  • HER2[9 43S ] specific CTL can kill tumor cells.
  • Lysis of Cr labeled SW403 cells at an effectors / target ratio of 10: 1 by the HER2[9 433 ] specific CTL was blocked at various Inhibitors / Target ratios by the following cold targets: (D, .221A2.1 pulsed with HER2[9 433 ]; A . 221A2.1 pulsed with irrelevant A2.1 binding peptide (HBC 18 - 27 ); • . .221A2.1 without peptide.
  • Lysis of 51 Cr-labeled SW403 at an effector/target ratio of 10: 1 by the HER2[9 3 ] -specific CTL line was blocked at various Inhibitors / Target ratios by the following cold targets: O, .221A2.1 pulsed with HER2[9 3 ]; A , .221A2.1 pulsed with irrelevant A2.1 binding peptide (HBc 18 . 27 ); • , .221A2.1 without peptide.
  • Figure 15 Peptide analogs can induce tumor-reactive CTL.
  • peptide is used interchangeably with “oligopeptide” in the present specification to designate a series of residues, typically L-amino acids, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids.
  • immunogenic peptide is a peptide which comprises an allele-specific motif such that the peptide will bind the MHC allele and be capable of inducing a CTL response.
  • immunogenic peptides are capable of binding to an appropriate class I MHC molecule and inducing a cytotoxic T cell response against the antigen from which the immunogenic peptide is derived.
  • residue refers to an amino acid or amino acid mimetic inco ⁇ orated in an oligopeptide by an amide bond or amide bond mimetic.
  • pretreatment growth factors refers to growth factors added to antigen presenting cells prior to incubating the antigen presenting cells with CTLS, peptides and growth factors that are used to expand the CTLs.
  • incubation growth factors refers to growth factors that are added as APCs and CTLs are incubated together to expand specific CTLS.
  • Our invention further comprises a general strategy to define the full immunological potential of a given antigen.
  • the dominance and subdominance of CTL epitopes associated to pathological states such as chronic viral infections and cancer can now be defined.
  • experimental strategies similar to the one detailed herein could help to shed some light on the mechanisms involved in the pathogenesis of infection, cancer, and autoimmune diseases.
  • the present invention provides a new in vitro primary immunization assay and its application to identify human CTL epitopes derived from tumor-associated antigens.
  • the invention entails stimulation of human PBMC with peptide-pulsed dendritic cells in the presence of IL-7 and IL-10. Utilizing this protocol we have defined 3 novel subdominant HLA-A2.1 -restricted CTL epitopes derived from the gplOO melanoma associated tumor antigen (G9 178 , G10 1T7 and G10 370 ).
  • the present invention also provides methods for ascertaining the role of i munodominance in the specific area of anti-tumor CTL responses. It has been pointed out that some of the CTL epitopes recognized by cancer patients appear to bind their MHC class I restriction element with relatively weaker affinities than epitopes recognized, for example, in acute viral infections (Kawakami, et al., J. Immunol. 754:3961 (1995); Celis, et al. Sem. Cancer Biol. 6:329 (1995)).
  • Epitopes binding in the 11 to 500 nM range were found in both dominant and subdominant groups (Tables ⁇ and IV), and the 2 highest affinity binding peptides G9 134) and G10 476 (Table I) correspond to immunodominant CTL epitopes recognized by melanoma patient's ⁇ L. Furthermore, the dominant gplOO epitopes elicited anti-melanoma CTL responses in 10-38% of all lymphocyte cultures, while the subdominant epitopes induced CTL that could kill melanoma cells less frequently (2-3 % of the cultures).
  • the present invention also provides that it is possible to induce tumor-reactive CTL from blood of cancer patients specific for both dominant and subdominant epitopes. These CTL could be expanded in culture to approximately 1 X 10 9 cells (data not shown), still retained antigen specificity and could thus be used for adoptive therapies. Immune therapy using peptide-primed CTL from blood may be a convenient (since no tumor biopsy is required), and reliable (because of the high degree of specificity) alternative to the already promising current TEL based immunotherapies (Rosenberg, et al. , J. Natl Cancer Inst. 86: 1159 (1994)).
  • the procedures of the present invention depend in part upon the determination of epitopes recognized by CTLs capable of eliminating target infected cells.
  • One approach to identification of these epitopes is the identification of allele-specific peptide motifs associated with a particular disease for human Class I MHC allele subtypes.
  • the MHC class I antigens are encoded by the HLA-A, B, and C loci.
  • HLA-A and B antigens are expressed at the cell surface at approximately equal densities, whereas the expression of HLA-C is significantly lower (perhaps as much as 10-fold lower).
  • Each of these loci have a number of alleles.
  • a large number of cells with defined MHC molecules, particularly MHC Class I molecules, are known and readily available. These cells can be used to identify particular allele specific motifs associated with target diseases.
  • the allele-specific motifs are then used to define T cell epitopes from any desired antigen, particularly those associated with human viral diseases or cancers, for which the amino acid sequence of the potential antigen targets is known. This general approach is described in detail in copending and commonly assigned applications U.S.S.N. 07/926,666 and U.S.S.N. 08/027,146, which are incorporated herein by reference.
  • PSA prostate specific antigen
  • HBVc hepatitis B core
  • HBVp hepatitis C antigens
  • Epstein-Barr virus antigens Epstein-Barr virus antigens
  • melanoma antigens e.g. , MAGE-1
  • HAV human immunodeficiency virus
  • HPV human papilloma virus
  • CMV cytomegalovirus
  • HSV he ⁇ es simplex virus
  • HSV oncogene products
  • motifs specific for different class I alleles allows the identification of potential peptide epitopes from an antigenic protein whose amino acid sequence is known. Typically, identification of potential peptide epitopes is initially carried out using a computer to scan the amino acid sequence of a desired antigen for the presence of motifs. The epitopic sequences are then synthesized. The capacity to bind MHC Class molecules is measured in a variety of different ways using, for example, purified class I molecules and radioiodinated peptides and/or cells expressing empty class I molecules by, for instance, immunofluorescent staining and flow microfluorimetry, peptide-dependent class I assembly assays, and inhibition of CTL recognition by peptide competition.
  • peptides that test positive in the MHC class I binding assay are assayed for the ability of the peptides to induce specific primary or secondary CTL responses in vitro.
  • antigen-presenting cells that have been incubated with a peptide can be assayed for the ability to induce CTL responses in responder cell populations.
  • antigen-presenting cells can be normal cells such as peripheral blood mononuclear cells or dendritic cells (Inaba, et al. , J. Exp. Med. , 166:182 (1987); Boog, Eur. J. Immunol, 18:219 [1988]).
  • mutant mammalian cell lines that are deficient in their ability to load class I molecules with internally processed peptides, such as the mouse cell lines RMA-S (Karre, et al. Nature, 319:675 (1986); Ljunggren, et al, Eur. J. Immunol , 21:2963-2970 (1991)), and the human somatic T cell hybridoma, T-2 (Cerundolo, et al , Nature, 345:449-452 (1990)) and which have been transfected with the appropriate human class I genes are conveniently used, when peptide is added to them, to test for the capacity of the peptide to induce in vitro primary CTL responses.
  • RMA-S mouse cell lines
  • T-2 human somatic T cell hybridoma
  • MHC-peptide complexes are preferable for inducing a primary response since the density of MHC-peptide complexes on the surface of the antigen presenting cell will be greater.
  • Other eukaryotic cell lines which could be used include various insect cell lines such as mosquito larvae (ATCC cell lines CCL 125, 126, 1660, 1591, 6585, 6586), silkworm (ATTC CRL 8851), armyworm (ATCC CRL 1711), moth (ATCC CCL 80) and Drosophila cell lines such as a Schneider cell line that have been transfected with the appropriate human class I MHC allele encoding genes and the human B- microglobulin genes.
  • immunogenic peptides comprising the motif required for MHC binding and the epitope recognized by the CTL are synthesized.
  • the immunogenic peptides can be prepared synthetically, or by recombinant DNA technology or isolated from natural sources such as whole viruses or tumors.
  • One of skill will recognize that the immunogenic peptides can be a variety of lengths, either in their neutral (uncharged) forms or in forms which are salts, and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications, subject to the condition that the modification not destroy the biological activity of the polypeptides as herein described.
  • the peptide will be as small as possible while still maintaining substantially all of the biological activity of the large peptide.
  • Peptides having the desired activity may be modified as necessary to provide certain desired attributes, e.g. , improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide to bind the desired MHC molecule and activate the appropriate T cell.
  • the peptides may be subject to various changes, such as substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use, such as improved MHC binding.
  • conservative substitutions is meant replacing an amino acid residue with another which is biologically and/or chemically similar, e.g. , one hydrophobic residue for another, or one polar residue for another.
  • substitutions include combinations such as Gly, Ala; Val, He, .Leu, Met; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • the effect of single amino acid substitutions may also be probed using D-amino acids. Such modifications may be made using well known peptide synthesis procedures.
  • the peptides of the invention can be prepared in a wide variety of ways. Because of their relatively short size, the peptides can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed. , Pierce Chemical Co. (1984), supra.
  • recombinant DNA technology may be employed wherein a nucleotide sequence which encodes an immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • a nucleotide sequence which encodes an immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • Fusion proteins which comprise one or more peptide sequences of the invention can also be used to present the appropriate T cell epitope.
  • the immunogenic peptides are then used to activate CTL ex vivo.
  • ex vivo therapy methods of the present invention and pharmaceutical compositions thereof are useful for treatment of mammals, particularly humans, to treat and/or prevent viral infection, immune disorders and cancer.
  • diseases which can be treated using the ex vivo therapy methods of the invention include prostate cancer, hepatitis B, hepatitis C, AIDS, renal carcinoma, cervical carcinoma, lymphoma, CMV, condyloma acuminatum, breast and ovarian cancer, colon, lung cancer and HSV.
  • therapy should begin at the first sign of viral infection or the detection or surgical removal of tumors or shortly after diagnosis in the case of acute infection. This is followed by boosting levels of CTL at least until symptoms are substantially abated and for a period thereafter. In chronic infection, loading doses followed by boosting doses may be required.
  • Treatment of an infected individual with the methods of the invention may hasten resolution of the infection in acutely infected individuals. For those individuals susceptible (or predisposed) to developing chronic infection the methods are useful for preventing the evolution from acute to chronic infection. Where the susceptible individuals are identified prior to or during infection, the compositions can be targeted to them, minimizing the need for administration to a larger population.
  • the methods of the present invention can also be used for the treatment of chronic infection and to stimulate the immune system to eliminate virus-infected cells in carriers.
  • CTL responses to a particular pathogen are induced by incubating in tissue culture the patient's CTL precursor cells (CTLp) together with a source of antigen-presenting cells (APC) loaded with the appropriate immunogenic peptide.
  • CTLp CTL precursor cells
  • APC antigen-presenting cells
  • the cells are infused back into the patient, where they will destroy their specific target cell (an infected cell or a tumor cell).
  • Infusion of the cells into the patient may include a T cell growth factor such as interieukin 2 (IL-2).
  • IL-2 interieukin 2
  • the culture of stimulator cells is maintained in an appropriate serum-free medium which may include one or more growth factors such as IL-2, IL-4, IL-7 and IL-12.
  • Peripheral blood lymphocytes are conveniently isolated following simple venipuncture or leukapheresis of normal donors or patients and used as the responder cell sources of CTLp.
  • the appropriate APC are incubated with 10-100 ⁇ M of peptide in serum-free media for 4 hours under appropriate culture conditions.
  • the peptide-loaded APC are then incubated with the responder cell populations in vitro for 7 to 10 days under optimized culture conditions.
  • APC expressing empty MHC would be used to stimulate naive CTLp. In this case the CTL would be stimulated more frequently (1-2 times).
  • Positive CTL activation can be determined by assaying the cultures for the presence of CTLs that kill radiolabeled target cells, both specific peptide-pulsed targets as well as target cells expressing endogenously processed form of the relevant virus or tumor antigen from which the peptide sequence was derived.
  • Specificity and MHC restriction of the CTL of a patient can be determined by a number of methods known in the art. For instance, CTL restriction can be determined by testing against different peptide loaded target cells expressing human MHC class I alleles shared with the HLA phenotype of the donor CTL. The peptides that test positive in the MHC binding assays and give rise to specific CTL responses are identified as immunogenic peptides.
  • the induction of CTL in vitro requires the specific recognition of peptides that are bound to allele specific MHC class I molecules on APC.
  • the number of specific MHC/peptide complexes per APC determines the level of stimulation of CTL, particularly during the primary immune response. While small amounts of peptide/MHC complexes per cell are sufficient to render a cell susceptible to lysis by CTL, or to stimulate a secondary CTL response, the successful activation of a CTLp during primary response requires a significantly higher number of MHC/peptide complexes.
  • mutant cell lines capable of expressing empty MHC do not exist for every human MHC allele, it is advantageous to use a technique to remove endogenous MHC-associated peptides from the surface of APC, followed by loading the resulting empty MHC molecules with the immunogenic peptides of interest.
  • the use of non-transformed (non-tumorigenic), non-infected cells, and preferably, autologous cells of patients as APC is desirable for the design of CTL induction protocols directed towards development of ex vivo CTL therapies.
  • This present invention provides novel methods generating empty class I MHC which can then be loaded with an appropriate immunogenic peptide by stripping the endogenous MHC-associated peptides from the surface of APC or through cold temperature incubation (37 * C ⁇ 26 "* C)followed by the loading of desired peptides.
  • a stable MHC class I molecule is a trimeric complex formed of the following elements: 1) a peptide usually of 8 - 10 residues, 2) a transmembrane heavy polymo ⁇ hic protein chain which bears the peptide-binding site in its l and ci2 domains, and 3) a non-covalently associated non-polymo ⁇ hic light chain, ⁇ 2 microglobulin. Removing the bound peptides and/or dissociating the ⁇ 2 microglobulin from the complex renders the MHC class I molecules nonfunctional and unstable, resulting in rapid degradation at 37 "C. Almost all MHC class I molecules isolated from PBMCs have endogenous peptides bound to them. Therefore, the first step to prepare APC for primary CTL induction is to remove all endogenous peptides bound to MHC class I molecules on the APC without causing degradation or cell death before exogenous peptides can be added.
  • Two possible ways to generate free MHC class I molecules include lowering the culture temperature from 37°C to 26°C overnight to allow MHC class I without peptides to be expressed and stripping the endogenous peptides from the cell using a mild acid treatment.
  • the mild acid treatment releases previously bound peptides into the extracellular environment allowing new exogenous peptides to bind to the empty class I molecules.
  • the overnight cold-temperature incubation at 26°C which may slow the cell's metabolic rate enables expression of stable empty class I molecules which then bind exogenous peptides efficiently. It is also likely that cells not actively synthesizing MHC molecules (e.g. , resting PBMC) would not produce high amounts of empty surface MHC molecules by the cold temperature procedure.
  • Extraction of the peptides is accomplished by harsh acid stripping using trifluoroacetic acid, pH 2, or acid denaturation of the immunoaffmity purified class I-peptide complexes. These methods are not feasible for CTL induction, since it is important to remove the endogenous peptides while preserving APC viability and an optimal metabolic state which is critical for antigen presentation.
  • Mild acid solutions of pH 3 such as glycine or citrate-phosphate buffers have been used to identify endogenous peptides and to identify tumor associated T cell epitopes.
  • the treatment is especially effective, in that only the MHC class I molecules are destabilized (and associated peptides released), while other surface antigens remain intact, including MHC class ⁇ molecules.
  • an amount of antigenic peptide is added to the APCs or stimulator cell culture, of sufficient quantity to become loaded onto the human Class I molecules to be expressed on the surface of the APCs.
  • a sufficient amount of peptide is an amount that will allow about 200 or more human Class I MHC molecules loaded with peptide to be expressed on the surface of each stimulator cell.
  • the stimulator cells are incubated with 5-100 ⁇ g/ml peptide.
  • the CTLs are then incubated in culture with the appropriate APCs for a time period sufficient to activate the CTLs.
  • the CTLs are activated in an antigen-specific manner.
  • the ratio of precursor CTLs to APCs may vary from individual to individual and may further depend upon variables such as the amenability of an individual's lymphocytes to culturing conditions and the nature and severity of the disease condition or other condition for which the within-described treatment modality is used.
  • the CTL:APC (i.e. responder to stimulator) ratio is in the range of about 10: 1 to 100:1.
  • the CTL/ APC culture may be maintained for as long a time as is necessary to stimulate a therapeutically useable or effective number of CTL.
  • Activated CTL may be effectively separated from the APC using one of a variety of known methods.
  • monoclonal antibodies specific for the APCs, for the peptides loaded onto the stimulator cells, or for the CTL (or a segment thereof) may be utilized to bind their appropriate complementary ligand.
  • Antibody-tagged cells may then be extracted from the admixture via appropriate means, e.g. , via well-known immunoprecipitation or immunoassay methods.
  • Effective, cytotoxic amounts of the activated CTLs can vary between in vitro and in vivo uses, as well as with the amount and type of cells that are the ultimate target of these killer cells. The amount will also vary depending on the condition of the patient and should be determined via consideration of all appropriate factors by the practitioner. Preferably, however, about 1 X 10 6 to about 1 X 10 12 , more preferably about 1 X 10 s to about 1 X 10 u , and even more preferably, about 1 X 10 9 to about 1 X 10 10 activated CTLS are utilized for adult humans, compared to about 5 X 10 6 - 5 X 10 7 cells used in mice.
  • the activated CTLS may be harvested from the cell culture prior to administration of the cells to the individual being treated. It is important to note, however, that unlike other present treatment modalities, the present method uses a cell culture system that does not contain transformed or tumor cells. Therefore, if complete separation of antigen-presenting cells and activated CTLS is not achieved, there is no inherent danger known to be associated with the administration of a small number of stimulator cells, whereas administration of mammalian tumor-promoting cells may be extremely hazardous.
  • the APC uses the APC generated by the in vitro techniques of this application for therapy against CTL in vivo.
  • the APC are a patient's cells (e.g. , the peripheral blood cells) which are stripped of their natural antigenic peptides and loaded with a peptide of choice which is conjugated to a toxin (e.g. ricin A chain or pseudomonas toxin).
  • the APCs are then re-introduced into the patient, where they will be bound by the endogenous CTLs that are specific for the antigenic peptide.
  • the coupled toxin will kill the activated CTL that are harmful i.e. those which stimulate transplant rejection after it binds the APC.
  • Such directed CTL killing is broadly useful for treating tissue-transplantation rejection and auto-immune disorders, which are mediated through CTL.
  • the treatment regime will vary depending upon the specific disorder to be treated and the judgement of the treating physician.
  • CTLp CTL precursors
  • pan DR-binding peptides of the present invention and pharmaceutical and vaccine compositions thereof can be administered to mammals, particularly humans, for prophylactic and/or therapeutic pu ⁇ oses.
  • the pan DR peptides can be used to enhance immune responses against other immunogens administered with the peptides.
  • CTL pan DR mixtures may be used to treat and/or prevent viral infection and cancer.
  • immunogens which induce antibody responses can be used. Examples of diseases which can be treated using the immunogenic mixtures of pan DR peptides and other immunogens include prostate cancer, hepatitis B, hepatitis C, AEDS, renal carcinoma, cervical carcinoma, lymphoma, CMV and condyloma acuminatum.
  • pan DR-binding peptides may also be used to treat a variety of conditions involving unwanted T cell reactivity.
  • diseases which can be treated using pan DR-binding peptides include autoimmune diseases (e.g. , rheumatoid arthritis, multiple sclerosis, and myasthenia gravis), allograft rejection, allergies (e.g. , pollen allergies), lyme disease, hepatitis, LCMV, post-streptococcal endocarditis, or glomerulonephritis, and food hypersensitivities.
  • the immunogenic compositions or the pan DR- binding peptides of the invention are administered to an individual already suffering from cancer, autoimmune disease, or infected with the virus of interest. Those in the incubation phase or the acute phase of the disease may be treated with the pan DR- binding peptides or immunogenic conjugates separately or in conjunction with other treatments, as appropriate.
  • compositions comprising immunogenic compositions are administered to a patient in an amount sufficient to elicit an effective CTL response to the virus or tumor antigen and to cure or at least partially arrest symptoms and/or complications.
  • compositions comprising pan DR-binding peptides are administered in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications.
  • An amount adequate to accomplish this is defined as "therapeutically effective dose.” Amounts effective for this use will depend on, e.g. , the peptide composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.
  • Therapeutically effective amounts of the immunogenic compositions of the present invention generally range for the initial immunization (that is for therapeutic or prophylactic administration) from about 1.0 ⁇ g to about 10,000 ⁇ g of peptide for a 70 kg patient, usually from about 100 to about 8000 ⁇ g, and preferably between about 200 and about 6000 ⁇ g. These doses are followed by boosting dosages of from about 1.0 ⁇ g to about 1000 ⁇ g of peptide pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition by measuring specific CTL activity in the patient's blood.
  • compositions of the present invention may generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and the relative nontoxic nature of the conjugates, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these compositions.
  • administration should be given to risk groups.
  • protection against malaria, hepatitis, or AIDS may be accomplished by prophylactically administering compositions of the invention, thereby increasing immune capacity.
  • Therapeutic administration may begin at the first sign of disease or the detection or surgical removal of tumors or shortly after diagnosis in the case of acute infection. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. In chronic infection, loading doses followed by boosting doses may be required.
  • Treatment of an infected individual with the compositions of the invention may hasten resolution of the infection in acutely infected individuals.
  • the compositions are particularly useful in methods for preventing the evolution from acute to chronic infection.
  • the susceptible individuals are identified prior to or during infection, for instance, as described herein, the composition can be targeted to them, minimizing need for administration to a larger population.
  • the peptide mixtures or conjugates can also be used for the treatment of chronic infection and to stimulate the immune system to eliminate virus-infected cells in carriers. It is important to provide an amount of immuno-potentiating peptide in a formulation and mode of administration sufficient to effectively stimulate a cytotoxic T cell response.
  • a representative dose is in the range of about 1.0 ⁇ g to about 5000 ⁇ g, preferably about 5 ⁇ g to 1000 ⁇ g for a 70 kg patient per dose. Immunizing doses followed by boosting doses at established intervals, e.g. , from one to four weeks, may be required, possibly for a prolonged period of time to effectively immunize an individual.
  • administration should continue until at least clinical symptoms or laboratory tests indicate that the viral infection has been eliminated or substantially abated and for a period thereafter.
  • compositions for therapeutic or prophylactic treatment are intended for parenteral, topical, oral or local administration.
  • the pharmaceutical compositions are administered parenterally, e.g. , intravenously, subcutaneously, intradermally, or intramuscularly.
  • the vaccine compositions of the invention are particularly suitable for oral administration.
  • the invention provides compositions for parenteral administration which comprise a solution of the peptides or conjugates dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
  • an aqueous carrier may be used, e.g. , water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like.
  • compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • concentration of CTL stimulatory peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1 % , usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • the peptides and conjugates of the invention may also be administered via liposomes, which serve to target the conjugates to a particular tissue, such as lymphoid tissue, or targeted selectively to infected cells, as well as increase the half-life of the peptide composition.
  • liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the peptide to be delivered is inco ⁇ orated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g.
  • liposomes filled with a desired peptide or conjugate of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the selected therapeutic/immunogenic peptide compositions.
  • Liposomes for use in the invention are formed from standard vesicle- forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g. , liposome size, acid lability and stability of the liposomes in the blood stream.
  • a ligand to be inco ⁇ orated into the liposome can include, e.g. , antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells.
  • a liposome suspension containing a peptide or conjugate may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the conjugate being delivered, and the stage of the disease being treated.
  • DNA or RNA encoding one or more PADRE peptides and a polypeptide containing one or more CTL epitopes or antibody inducing epitopes may be introduced into patients to obtain an immune response to the polypeptides which the nucleic acid encodes.
  • Wolff et. al Science 247: 1465-1468 (1990) describes the use of nucleic acids to produce expression of the genes which the nucleic acids encode.
  • conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95 % of active ingredient, that is, one or more conjugates of the invention, and more preferably at a concentration of 25% -75% .
  • the peptides are preferably supplied in finely divided form along with a surfactant and propellant.
  • Typical percentages of conjugates are 0.01 %-20% by weight, preferably 1 %-10% .
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
  • Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • the surfactant may constitute 0.1 % -20% by weight of the composition, preferably 0.25-5 %.
  • the balance of the composition is ordinarily propellant.
  • a carrier can also be included, as desired, as with, e.g. , lecithin for intranasal delivery.
  • the present invention is directed to vaccines which contain as an active ingredient an immunogenically effective amount of an immunogenic pan DR peptide or a CTl ⁇ pan DR peptide conjugate as described herein.
  • the conjugate(s) may be introduced into a host, including humans, linked to its own carrier or as a homopolymer or heteropolymer of active peptide units.
  • a polymer has the advantage of increased immunological reaction and, where different peptides are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the virus or tumor cells.
  • Useful carriers are well known in the art, and include, e.g.
  • the vaccines can also contain a physiologically tolerable (acceptable) diluent such as water, phosphate buffered saline, or saline, and further typically include an adjuvant.
  • Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are materials well known in the art.
  • CTL responses can be primed by conjugating peptides of the invention to lipids, such as P 3 CSS.
  • lipids such as P 3 CSS.
  • the immune system of the host responds to the vaccine by producing large amounts of CTLs specific for the desired antigen, and the host becomes at least partially immune to later infection, or resistant to developing chronic infection.
  • Vaccine compositions containing the pan DR peptides of the invention are administered to a patient susceptible to or otherwise at risk of disease, such as viral infection or cancer to elicit an immune response against the antigen and thus enhance the patient's own immune response capabilities.
  • a patient susceptible to or otherwise at risk of disease such as viral infection or cancer to elicit an immune response against the antigen and thus enhance the patient's own immune response capabilities.
  • Such an amount is defined to be an "immunogenically effective dose.”
  • the precise amounts again depend on the patient's state of health and weight, the mode of administration, the nature of the formulation, etc. , but generally range from about 1.0 ⁇ g to about 5000 ⁇ g per 70 kilogram patient, more commonly from about 10 ⁇ g to about 500 ⁇ g per 70 kg of body weight.
  • PADRE peptides of the invention can be combined with hepatitis vaccines to increase potency or broaden population coverage.
  • Suitable hepatitis vaccines that can be used in this manner include, Recombivax HB ® (Merck) and Engerix-B (Smith-Kline).
  • the peptides of the invention can also be expressed by attenuated viral hosts, such as vaccinia or fowlpox.
  • attenuated viral hosts such as vaccinia or fowlpox.
  • This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the peptides of the invention.
  • the recombinant vaccinia virus Upon introduction into an acutely or chronically infected host or into a non-infected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL response.
  • Vaccinia vectors and methods useful in immunization protocols are described in, e.g. , U.S. Patent No. 4,722,848, inco ⁇ orated herein by reference.
  • BCG Bacillus Calmette Guerin
  • BCG vectors are described in Stover et al, Nature 351, 456-460 (1991)) which is inco ⁇ orated herein by reference.
  • Other vectors useful for therapeutic administration or immunization of the peptides of the invention e.g. , Salmonella typhi vectors and the like, will be apparent to those skilled in the art from the description herein.
  • Antigenic conjugates may be used to elicit CTL ex vivo, as well.
  • the resulting CTL can be used to treat chronic infections (viral or bacterial) or tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a peptide vaccine approach of therapy.
  • Ex vivo CTL responses to a particular pathogen are induced by incubating in tissue culture the patient's CTL precursor cells (CTLp) together with a source of antigen-presenting cells (APC) and the appropriate immunogenic peptide. After an appropriate incubation time (typically 1-4 weeks), in which the CTLp are activated and mature and expand into effector CTL, the cells are infused back into the patient, where they will destroy their specific target cell (an infected cell or a tumor cell).
  • PBMC peripheral blood mononuclear cells
  • APC peptide-pulsed antigen presenting cells
  • HLA-A2.1 -binding peptide from hepatitis B virus HLA-A2.1 -binding peptide from hepatitis B virus (HBV) we observed that although procedure was quite effective in inducing primary CTL responses to peptide-pulsed target cells, only a few of the resulting CTL ( ⁇ 10%) were capable of killing targets that naturally produced and processed the antigen. In some instances the inability of the peptide-reactive CTL to recognize the endogenously processed antigen could be attributed to a low avidity of the CTL-target cell interaction (Wentworth, et al., Molec. Immunol. 32:603 (1995)).
  • Peptides were prepared on an Applied Biosystems machine (Foster City, CA). Briefly, after removal of the ⁇ -amino-rer-butylocarbonyl protecting group, the phenylacetamidomethyl resin peptide was coupled with a 4 fold excess of preformed symmetrical anhydride (hydroxybenzyltriazole esters for arginine, histidine, asparagine, and glutamine) for 1 hr in dimethylformamide. For arginine, histidine, asparagine, glutamine and histidine residues, the coupling step was repeated in order to obtain a high efficiency coupling. Peptides were cleaved by treatment with hydrogen fluoride in the presence of the appropriate scavengers. Synthetic peptides were purified by reverse phase high pressure hquid chromatography (RV-HPLC). The purity and identity of the peptides was determined by amino acid sequence and mass spectrometric analysis respectively, and was routinely > 95 %.
  • Peptide binding assay to purified HLA-A2.1 molecules was measured as previously described. Rupert et al , Cell: 74:929 (1993); Sette et al , Molec. Immunol. 31 : 813 (1994). Briefly, the assay is based on the inhibition of binding of radiolabeled standard peptides to detergent solubilized MHC molecules.
  • a standard peptide used for the HLA-A2.1 binding studies was FLPSDYFPSV (HBC 18 . 27 ), a CTL epitope from hepatitis B virus core (nucleoprotein) antigen, positions 18-27 (Bertoletti, et al., J. Virol. 67:2316 (1996)).
  • Peptide MAGE3 16l corresponds to CTL epitope recognized in the context of the HLA-A 1 allele (Celis, et al , Proc. Natl. Acad. Sci. (USA) 97:2105 (1994); Gaugler, er al., J. Exp. Med. 779:921 (1994)).
  • Peptide G9 280 corresponds to CTL epitope from the melanosomal gplOO molecule which is recognized by HLA-A2.1 melanoma patients (Cox et al , Science 264:116 (1994); Kawakami, er al, J. Immunol. 754:3961 (1995)).
  • a standard peptide for A68.2 binding assays was FTQAGYPAL.
  • the standard peptide was radioiodinated using 12i I (ICN, Irvine, CA) by the chloramine T method. HLA-A2.1 concentration yielding approximately 15 % of bound peptide (approximately in the 10 nM range) was used in the inhibition assays.
  • Various doses of the test peptides (10 ⁇ M to 1 nM) were incubated together with 5 nM radiolabeled standard peptide and HLA-A2.1 molecules for 2 days at room temperature in the presence of a cocktail of protease inhibitors and I ⁇ M 02-microglobulin (Scripps Labs., San Diego, CA). At the end of the incubation period, the % MHC bound radioactivity was determined by gel filtration, and the concentration of the test peptides that inhibited 50% of the binding of the label peptide (IC 30 ) was calculated.
  • the .221(A2.1) cell line was produced by transferring the HLA-A2.1 gene into the HLA-A, -B, -C-null mutant human B lymphoblastoid cell line .221 (Celis, E. et al , Proceedings of the National Academy of Sciences of the United States of America 91:2105-2109 (1994)). Flow cytometry of anti-class I MHC antibody binding showed that .221 (A2.1) cells express 50-100 % as much as cell surface HLA-A molecules as do normal lymphoblastoid cells expressing single copies of the same HLA alleles in their native chromosomal locations.
  • the .221(A2.1) cells express 50-100% as much as cell surface HLA-A molecules as do normal lymphoblastoid cells as determined by flow cytometry using anti-class I MHC specific antibodies.
  • the .221(A2.1) cell line was transfected with the gene coding for the hepatitis B virus core (nucleoprotein) antigen as described (Wentworth, et al, Molec. Immunol. 32:603 (1995)).
  • the Steinlin EBV-LCL (HLA-A1/1, B8/8) was used to test CTL recognition of peptide in the context of HLA-A1 molecules.
  • HLA-typed melanoma cell lines used in these studies (624mel (A2.1, A3, B7, B14, gpl00 + ); A375mel (Al, A2.1 , B17, gplOO * ); 938mel (Al, A24, B7, B8, MAGE-3 + ); 888mel (Al , A24, B52, B55, MAGES ' ) have been previously described. Kawakami et al, Proc. Natl. Acad. Sci. 91: 6458 (1994); Kawakami er al , J. Immunol. 154: 3961 (1995).
  • the colon adenocarcinoma cell lines SW403 and HT-29 were obtained from the American Type Culture Collection (Rockville, MD) and were maintained in culture as recommended by the supplier.
  • the gastric cancer cell line KATO-m was obtained from the Japanese Cancer Research Resources Bank. All tumor lines were maintained in culture using RPMI-1640 medium supplemented with antibiotics and 10% (V/V) FBS (and 400 ⁇ g/ml G418 for the .221(A2.1) cell lines).
  • Melanoma colon and gastric cancer cells were treated with 100 U/ml ⁇ -IFN (Genzyme) for 48 hr at 37°C before testing their susceptibility to lysis by the CTL.
  • ⁇ -IFN-treated cells increased the level of MHC class I expression by 2-3 fold (unpublished). Although ⁇ -EFN treatment increased the levels of CTL mediated cytotoxicity, untreated melanoma cells were still recognized by the CTL (data not shown).
  • MAGE2 and MAGE3 were determined by RT-PCR as described (De Smet, C. et al , Immunogenetics 39: 121-129 (1994); Gaugler, B. et al, J Exp Med. 179:921-930 (1994); Zakut, R. et al., Cancer Research 53:5-8 (1993)).
  • the expression of MAGE3 protein was determined by immunohistochemistry using specific monoclonal antibodies (Kocher, T. et al, Cancer Research 55:2236-2239 (1995)).
  • the expression of CEA and ⁇ ES2/neu was measured by cytofluorometric analysis using the monoclonal antibodies CEMOIO (Takara Shuzo, Japan) and c-neu Ab-5 (Oncogene Science, Uniondale, NY) respectively.
  • PBMC Peripheral blood mononuclear cells
  • peripheral blood mononuclear cells from HLA-A2.1 + normal volunteers and a melanoma patient were purified by centrifugation in Ficoll-Paque (Pharmacia, Piscataway, NJ) from leukopheresis products.
  • PBMC peripheral blood mononuclear cells
  • Ficoll-Paque Pulcoa, Piscataway, NJ
  • the isolated and purified PBMC are co-cultured with an appropriate number of APC expressing empty MHC molecules, previously incubated ("pulsed") with an appropriate amount of synthetic peptide (containing the HLA binding motif and the sequence of the antigen in question).
  • PBMC are usually incubated at 1-3 X 10 6 cells/ml in culture medium such as RPMI-1640 (with autologous serum or plasma) or the serum-free medium AIM-V (Gibco).
  • APC are usually used at concentrations ranging from l X lO' to l X lO 6 cells/ml, depending on the type of cell used.
  • Possible sources of APC include: autologous PBMCs, SAC-I activated PBMCs, PHA blasts; autologous dendritic cells (DC) which are isolated from PBMC and purified as described (Inaba, et al. , J. Exp. Med. , 166: 182 (1987)); and mutant and genetically engineered mammalian cells such as the mouse RMA-S cell line or the human T2 cell line transfected with the appropriate MHC genes that express "empty" HLA molecules which are syngeneic to the patient's allelic HLA form).
  • DC dendritic cells
  • APC containing empty HLA molecules are known to be potent inducers of CTL responses, possibly because the peptide can associate more readily with empty MHC molecules than with MHC molecules which are occupied by other peptides (DeBruijn, er al., Eur. J. Immunol, 21:2963-2970 (1991)).
  • DC Tissue culture-derived dendritic cells
  • DC dendritic cells
  • monocyte-enriched cell fractions as described (Tsai er al, Cell. Immunol. 144:203 (1992)) or by adherence to plastic (2 hr at 37°C in serum-containing medium). Briefly, PBMC were separated on multi-step Percoll gradients (30, 40, 50.5 and 70%) and the monocyte-enriched population was harvested in the light density fraction at the 40-50.5 % interface.
  • the washed monocytes containing the DC precursors were cultured at 5 X 10 5 cells/ml in the presence of 50 ng/ml of GM-CSF and 1 ,000 U/ml of IL-4 (both from R&D Systems, Minneapolis, MN) as described in Romani, er al, Exp. Med. 180:83 (1994); Sallusto, et al, J. Exp. Med. 779: 1109 (1994), in complete medium (see below).
  • the adherent monocyte population was treated in the same manner. After 7 days the cells, which display typical cell surface markers of DC (CD3 " , CD14 ' , CD88 + , HLA-A,-B,-C ra , HLA-DQ + ; data not shown) were harvested.
  • “Fresh” DC were isolated directly from a pheresis product as described (Tsai, et al, J. Clin. Invest 82: 1131 (1988)). Briefly, monocytes and DC were first purified with a Percoll gradient as described above and the majority of monocytes were removed by adherence to plastic for 2 hr at 37 C C. The DC fraction, which is released from the substrate is further purified by eliminating contaminating monocytes and lymphocytes using a single step Metrizamide gradient. Between 50 and 70% of the resulting cells have typical DC markers and can be distinguished from other cell types in phase contrast microscopy.
  • CTL lines were expanded in tissue culture following a method similar to the one described by Riddell, er al , Nature Medicine 2:216 (1996)) for CTL clones.
  • a total of 5 X 10 CTL were resuspended in 25 ml of complete medium with 25 X 10 6 autologous irradiated (4200 rads) PBMC (The APC are gamma irradiated to prevent their proliferation and to facilitate the expansion of the CTLp.) and 5 X 10 6 allogeneic (non HLA-A2) and irradiated (8000 rads) Epstein-Barr virus-transformed lymphoblastoid B-cells in the presence of 30 ng/ml of anti-CD3 monoclonal antibodies (OKT3).
  • CTL lines were also expanded using peptide-pulsed PBMC (instead of anti-CD3 antibodies), in order to maintain antigen specificity.
  • PBMC, APC and peptide were kept in an appropriate culture vessel such as plastic T-flasks, gas-permeable plastic bags, or roller bottles, at 37 * centigrade in a humid air/C0 2 incubator.
  • an appropriate culture vessel such as plastic T-flasks, gas-permeable plastic bags, or roller bottles.
  • 200 IU/ml of recombinant IL-2 were added to the cultures.
  • the cultures were fed with fresh medium containing 50 IU/ml IL-2 on days 5, 8 and 11 and were split if the T-cell concentration reached numbers > 1.5 X lOVml.
  • the resulting effector CTL can be further expanded, by the addition of recombinant growth factors such as interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin- 7 (IL-7), or interieukin- 10 (IL-12) to the cultures.
  • IL-2 interleukin-2
  • IL-4 interleukin-4
  • IL-7 interleukin- 7
  • IL-12 interieukin- 10
  • An expansion culture can be kept for an additional 5 to 12 days, depending on the numbers of effector CTL required for a particular patient.
  • expansion cultures may be performed using hollow fiber artificial capillary systems (Cellco), where larger numbers of cells (up to 1X10") can be maintained.
  • CTL priming cultures were set up in 48-well tissue culture plates (Costar) by mixing 0.25 ml cytokine-generated DC (at 1 X lO 3 cells/ml) with 0.25 ml of CD8 + T-cells (at 20 X 10 *5 cell/ml) in each well in the presence 10 ng/ml of IL-7 (Genzyme). 24 hrs. after the cultures were initiated, 10 ng/ml of recombinant IL-10 (Endogen, Cambridge, MA) were added to each well. CTL cultures (each individual well) were re-stimulated every 7 days (up to 6 cycles) with irradiated (3,300 rad) autologous monocytes pulsed with peptide as described.
  • Recombinant IL-10 (10 ng/ml) and recombinant IL-2 (10 IU/ml) were added 1 day and 2 days respectively after each re-stimulation cycle.
  • medium supernatants were sampled (100 ⁇ l) just prior the second antigen restimulation cycle and 24 hours after antigen restimulation, to determine the production of GM-CSF, which was measured using an ELISA kit (R&D Systems, Minneapolis, MN).
  • Complete culture medium consisted of RPMI- 1640 (Gibco, Grand Island, NY) supplemented with 5 % AB male human serum, L-Glutamine, sodium pyruvate non-essential amino acids (all from Gibco) at the recommended concentrations.
  • expanded cells are used (e.g. , infused into a patient), they are tested for activity, viability, toxicity and sterility.
  • the cytotoxic activity of the resulting CTL can be determined by a standard 51 Cr-release assay (Biddison, W.E. (1991), Current Protocols in Immunology, p7,17.1-7.17.5, Ed. J. Coligan et al, J. Wiley and Sons, New York), using target cells that express the appropriate HLA molecule, in the presence and absence of the immunogenic peptide.
  • peptide-pulsed targets were prepared incubating the cells at about 5 X 10 s cells/ml with 10 ⁇ g/ml synthetic peptide overnight at 37°C. All cells were labeled with 100-300 ⁇ Ci 51 Cr(ICN) per 3 X 10 6 cells for l. hr at 37°C.
  • Adherent target cells e.g. , melanomas
  • Target cells were detached from tissue culture flasks with Trypsin-EDTA.
  • Target cells were washed by centrifugation and mixed with effectors in a final volume of 0.2 ml of RPMI-1640 containing 10% fetal bovine serum (FBS, GEBCO) in round-bottom microtiter plates.
  • the plates were centrifuged (5 min at 400 x g) to increase cell to cell contact and placed in a 37°C C0 2 incubator. After 4-6 hr incubation time, and 0.1 ml of the supernatant was removed from each well, and the radioactivity was determined in a ⁇ -counter.
  • effector cells were routinely not counted. However, sample counting of these cells indicated that effector: target ratios in these assays were in the range of 10: 1 to 1: 1.
  • Antigen specificity was determined by cold target inhibition analyses by determining the capacity of peptide pulsed unlabelled .221 (A2.1) cells to block the lysis of melanoma cells.
  • % Specific Cytotoxicity was determined by calculating the % Specific 51 Cr release by the formula: [(cpm of the test sample - cpm of the spontaneous 31 Cr release)/(cpm of the maximal 3l Cr release - cpm of the spontaneous Cr release)] x 100.
  • the spontaneous 51 Cr release was determined by incubating the targets alone, in the absence of effectors, and the maximal -"Cr release was obtained by incubating the targets with 0.1 % Triton X-100 (Sigma Chem. Co. , St. Louis, MO). Determinations were performed in duplicate. The standard error of the means were typically below 10% of the value of the mean.
  • Viability was determined by the exclusion of trypan blue dye by live cells. Cells are tested for the presence of endotoxin by conventional techniques. Finally, the presence of bacterial or fungal contamination was determined by appropriate microbiological methods (chocolate agar, etc.). Once the cells pass all quality control and safety tests, they are washed and placed in the appropriate infusion solution (Ringer/glucose lactate/human serum albumin) which may include a T-cell growth factor such as IL-2 and infused intravenously into the patient.
  • infusion solution Rostagar, etc.
  • This example demonstrates the use of cold temperature incubation and acid stripping for generation of empty MHC class I molecules to enable peptide loading method to prepare effective HLA-allele-specific antigen presenting cells (APC) for use in diagnostic or ex vivo therapy applications.
  • APC antigen presenting cells
  • PHA blasts and CTL inductions were done in RPMI 1640 + Hepes + glutamine (Gibco) supplemented with 2 mM L-glutamine (Irvine Scientific), 50 ⁇ g/ml gentamicin (Gibco), and 5 % heat inactivated pooled human Type AB serum (Gemini Bioproducts) [RPMI/5 % HS].
  • EBV transformed lymphoblastoid cell lines (LCL) were maintained in RPMI 1640 + Hepes + glutamine (BioWhittaker) supplemented with L-glutamine and gentamicin as above and 10% heat inactivated fetal calf serum (Irvine Scientific) [RPMI/10% FCS]. Chromium release assays were performed in RPMI/10% FCS.
  • JY a HLA A2.1 expressing human EBV-transformed B-cell line
  • K562 a NK cell sensitive erythroblastoma line was grown in RPMI/10% FCS.
  • K562 was used to reduce background killing by NK and LAK cells in the chromium release assays. Peptides.
  • the immunogenic peptides used in these studies were synthesized as described above using motifs for HLA alleles for specific target antigens as described in copending commonly assigned applications U.S.S.N. 07/926,666 and U.S.S.N. 08/027,146. Their sequences are shown in Table I. Peptides were routinely dissolved in 100% DMSO at 20 mg/ml, aliquoted, and stored at -20°C.
  • PBMC Peripheral Blood Mononuclear Cells
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • CD4+ lymphocyte depletion was performed using antibody-coated flasks: MicroCELLector T-150 flasks for the selection of CD4+ cells (Applied Immune Sciences) were washed according to the manufacturer's instructions with 25 ml PBS CMF (calcium magnesium free) + 1 mM EDTA (Sigma) by swirling flasks for 30 sec followed by incubation for 1 hour at room temperature on a flat surface. Buffer was aspirated and flasks were washed 2 additional times by shaking the flasks for 30 seconds and maintaining coverage of the binding surface. To each washed flask, 25 ml culture medium were added and incubated for 20 minutes at room temperature on a flat surface.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • Culture medium was aspirated from the flask and then the cell suspension was gently added to the MicroCELLector.
  • Flasks containing the cells were incubated for 1 hour at room temperature on a flat surface. At the end of the incubation, the flask was gently rocked from side to side for 10 seconds to resuspend the nonadherent cells.
  • Nonadherent CD4+ T cell depleted cells were harvested and then flasks were washed twice with PBS CMF to collect the nonadherent cells.
  • Harvested CD4+ T cell depleted cells were pelleted by centrifugation and resuspended in culture medium.
  • PBMC peripheral blood mononuclear cells
  • Frozen cells were washed twice before use.
  • Cells were cultured at 2 x 10 6 /ml in RPMI/5 % HS containing 1 ⁇ g/ml PHA (Wellcome) and 10 U/ml rIL-2.
  • PHA blasts were maintained in culture medium containing 10 U/ml rIL-2 with feeding and splitting as needed. PHA blasts were used as APCs on day 6 of culture. Generation of empty class I molecules and peptide loading was only performed by the acid strip method when using PBMCs as APCs.
  • PBMC peripheral blood mononuclear cells
  • Cells were resuspended in 1 volume cold sterile neutralizing buffer #2 [PBS CMF, 1 % BSA, 30 ⁇ g/ml DNAse, 3 ⁇ g/ml 0, microglobulin, 40 ⁇ g/ml peptide] and incubated for 4 hours at 20°C. Cells were diluted with culture medium to approximately 5 x lCrVml and irradiated with 6000 rads. Cells were then centrifuged at 1500 ⁇ m for 5 minutes at room temperature and resuspended in culture medium. The acid stripped/peptide loaded cells were used immediately in the CTL induction cultures (below).
  • JY cells were either acid stripped (i.e. treated with citrate-phosphate buffer and neutralizing buffer #1 as described above) or incubated at a reduced temperature. JY control cells were left untreated in tissue culture media. After treatment both cell populations were washed twice with serum free RPMI and loaded with 125 I-radiolabelled 941.01 (HBc 18-27) peptide (standard chloramine T iodination). To determine binding specificity, 2 x 10 6 cells were resuspended in 200 ⁇ l neutralizing buffer #2 (described above) containing 1 5 1-941.01 (10 5 cpms) +/- 100 ⁇ g unlabelled 941.01.
  • Cells were incubated for 4 hours at 20 °C and washed twice with serum free RPMI to remove free peptide. Cells were resuspended in 200 ⁇ l of serum free RPMI. In a microfuge tube the cell suspension was layered over an 800 ⁇ l FCS and pelleted by centrifugation for 5 seconds. Supematants were aspirated and the radioactivity remaining in the pellet was measured (Micromedic automatic gamma counter, 1 minutes per tube).
  • JY cells an HLA-A2.1 EBV -transformed B cell line
  • JY cells were preincubated at 26 * C overnight or acid-stripped to remove the endogenous MHC-associated* peptides and the loading of exogenous peptide was determined using a 125 I-radiolabelled HLA-A2.1 binding peptide.
  • the specificity of this reaction was determined by measuring the inhibition of labelled peptide binding using a cold peptide of the same sequence. Results presented in Table 2 demonstrate that acid-treatment of the cells increased significantly (approximately 10-fold) the amount of labelled peptide binding to the JY cells. Furthermore, the binding of labelled peptide was completely blocked by the addition of the cold peptide, demonstrating specific binding (data not shown).
  • PHA- induced T-cell blasts were acid stripped/peptide loaded according to the methods described above.
  • the resulting cells were stained for FACS analysis using anti-HLA-A2 (BB7.2) and anti-HLA alpha chain-specific (9.12.1) monoclonal antibodies.
  • Controls for this experiment included the same cell population which was not treated at pH 3 (but treated with PBS buffer at pH 7.2), and cells treated with citrate-phosphate buffer (to strip the MHC) but neutralized in the absence of ⁇ 2 microglobulin and peptide.
  • Acid stripping/peptide loading of PBMC and PHA blasts are described above.
  • the responder cell population was prepared: Responders were PBMC that were depleted of CD4+ T cells (described above). Responder cells were resuspended in culture medium at 3 x 10 6 /ml and 1 ml of the responder cell suspension was dispensed into each well of a 24-well tissue culture plate (Falcon, Becton Dickinson). The plates were placed in the incubator at 37°C, 5 % C0 2 until the stimulator population was ready.
  • stimulator APCs were resuspended in culture medium containing 20 ng/ml rIL-7 at 10 6 /ml for the PBMC, or at 3 x 10 5 /ml for the PHA blasts, 1 ml of stimulator cell suspension was added per well to the plates containing the responders.
  • 100 ⁇ l culture medium containing 200 ng/ml rIL-7 was added to each well (10 ng/ml rIL-7 final).
  • 100 ⁇ l of culture medium containing 200 U/ml rIL-2 was added to each well (10 U/ml rIL-2 final).
  • Additional critical parameters for the induction of primary CTL are: 1) enrichment of CD8+ T-cells in the responder cell population (by depletion of CD4+ T-cells), 2) addition of rIL-7 to the CTL induction cultures from day 0, and 3) restimulation of the cultures with antigen on day 12-14 using autologous adherent cells pulsed with peptide.
  • the primary CTL were restimulated with peptide using autologous, adherent APCs.
  • Autologous PBMC were thawed and washed as described above.
  • Cells were irradiated at 6000 rads.
  • Cells were pelleted and resuspended in culture medium at 4 x lOVml and 1 ml of cell suspension was added to each well of a 24-well tissue culture plate, and incubated for 2 hours at 37°C, 5% C0 2 .
  • Nonadherent cells were removed by washing each well three times with serum free RPMI.
  • a 0.5 ml culture medium containing 3 ⁇ g/ml )3 2 microglobuIin and 20 ⁇ g/ml total peptide was added to each well.
  • APC were incubated for 2 hrs at 37°C, under 5 % C0 2 with the peptide and /3 2 microglobulin.
  • Wells were aspirated and 1 ml of responder cells at 1.5 x 10 6 /ml in culture medium was added to each well.
  • 1 ml of culture medium containing 20 U/ml rEL-2 was added to each well. Cultures were supplemented with 10 U/ml rEL-2 (final) every three days thereafter.
  • Effector Cell Preparation The responders were centrifuged and resuspended at 10 7 /ml in RPMI/10% FCS. Three-fold serial dilutions of effectors were performed to yield effector to target ratios of 100:1 , 33:1, 11 :1, and 3:1. Effector cells were aliquoted at 100 ⁇ l well on 96 well U-bottomed cluster plates (Costar), in duplicate. b.
  • Target Cell Preparation Approximately 16-20 hours prior to the assay, target cells were resuspended at 3 x lOVml in RPMI/10% FCS in the presence or absence of 3 ⁇ g/ml /3 2 microglobulin and 10 ⁇ g/ml total peptide. After preincubation, target cells were centrifuged and pellets were resuspended in 200 ⁇ l (300 ⁇ Ci) sodium ( 51 Cr) chromate (NEN). Cells were incubated at 37°C for 1 hour with agitation. Labelled target cells were washed 3 times with RPMI/10% FCS. c.
  • Target cell concentration was adjusted to 10 5 /ml in RPMI/10% FCS and 100 ⁇ l aliquots were added to each well containing responders.
  • K562 cells cold targets, to block NK, and LAK activity
  • RPMI/10% FCS were washed and resuspended in RPMI/10% FCS at 10 7 /ml.
  • Aliquots of 20 ⁇ l were added per well, yielding a 20: 1 cold K562 target to labelled target ratio.
  • 100 ⁇ l/well of RPMI/10% FCS were added to 100 ⁇ l/well of labelled target cells, and 20 ⁇ l/well of K562.
  • CTL were stimulated by SAC-I activated PBMCs as APC.
  • Cold temperature enhanced expression of empty MHC enabling loading of antigenic peptide to generate SACT activated PBMC APC.
  • This method presents an alternative protocol to the methods described above for the generation of the APC which are used to stimulate CTL.
  • This example also presents an alternative protocol for the stimulation of CTL by the APC.
  • the tissue culture medium used in this study consisted of RPMI 1640 with Hepes and L-glutamine (Gibco) (Biowhittaker) supplemented with 2 mM L-glutamine (Irvine Scientific), 0.5 mM sodium pyruvate (Gibco), 100 U/100 ug/ml penicillin/streptomycin (Irvine), and 5 % * heat- inactivated Human Serum Type AB (RPMI/5 % HS; Gemini Bioproducts).
  • Culture media used in the growth of EBV-transformed lines contained 10% heat-inactivated fetal calf serum (RPMI/ 10% FCS, Irvine) instead of human serum.
  • rIL-2 Recombinant human Interleukin-2 (rIL-2) and Interleukin-4 (rIL-4) were obtained from Sandoz and used at a final concentration of 10 U/ml and 10 ng/ml, respectively.
  • Peptides Peptides. Peptides were synthesized as described above and are described in Table I. Peptides were routinely dissolved in 100% DMSO at 20 mg/ml, aliquoted, and stored at -70°C until used.
  • JY, Steinlein, EHM, BVR, and KT3 are homozygous human EBV-transformed B cell lines expressing HLA A 2 1 , A ⁇ A 3 , A u , and A 24 , respectively. They are grown in RPMI/10% FCS and used as targets in the CTL assays. K562, an NK cell sensitive, erythroblastoma line grown in RPMI/10% FCS, was used for reduction of background killing in the CTL assays.
  • PBMCs Peripheral Blood Mononuclear Cells
  • the interface between the Ficoll and the plasma containing the PBMCs was recovered with a transfer pipet (two interfaces per 50 ml tube) and washed three times with 50 ml of RPMI (1700, 1500, and 1300 RPM for 10 minutes). Cells were resuspended in 10-20 ml of culture medium, counted, and adjusted to the appropriate concentration.
  • PBMCs peripheral blood mononuclear cells
  • SAC-I cells expressing Protein A; Calbiochem
  • 20 ⁇ g/ml Immunobeads Rabbit anti-Human IgM; Biorad
  • 20 ng/ml of human rIL-4 Two ml of cells per well were plated in a 24-well plate (Falcon, Becton Dickinson) and cultured at 37°C. After 3 days, the medium was removed and the cells were washed three times followed by addition of RPMI/ 10% HS. The cells were used after culturing for an additional 2 days in RPMI/ 1096 HS.
  • the cells were irradiated at 6100 rads (5 x 10 6 / ml; 25 million cells/tube), washed and adjusted to the appropriate concentration for addition to the induction culture (see below).
  • Acid stripping This was used as an alternative method for generating empty MHC on the surface of the APCs.
  • the SAC-I activated PBMCs were washed once in cold 0.9% sodium chloride (J.T. Baker) containing 1 % BSA.
  • the cells were resuspended at 10 7 /ml in cold citrate-phosphate buffer (0.13M citric acid [J.T. Baker], 0.06M sodium phosphate monobasic [Sigma], pH 3) containing 1 % BSA and 3 ⁇ g/ml ⁇ 2 m and incubated on ice.
  • the cells were irradiated at 6100 rads (5 x 10 s / ml; 25 million cells/tube), washed, then adjusted to the appropriate concentration for addition to the induction culture (see below).
  • AIS MicroCellector T-150 flasks (specific for the depletion of CD4+ T cells; Menlo Park, CA) were primed by adding 25 ml of PBS/1 mM EDTA, swirling for 30 seconds so that all surfaces were moistened, and then incubating with the binding surface down at room temperature for 1 hour. Following this incubation, flasks were shaken vigorously for 30 seconds, washed 1 time with PBS/EDTA, 2 additional times with PBS and then incubated with 25 ml of culture medium for 15 minutes.
  • PBMCs were thawed in serum-free RPMI (+ L-glutamine + Hepes) containing 30 ⁇ g/ml DNAse, washed once, and incubated for 15 minutes in culture medium. Following aspiration of culture medium from the flasks, up to 180 million PBMCs were added in 25 ml of culture medium containing 30 ⁇ g/ml DNAse. After 1 hour at room temperature, the flasks were rocked gently for 10 seconds to resuspend the nonadherent cells. The nonadherent cell suspension containing the CD8+ T cells was collected and the flasks were washed 2 times with PBS.
  • the CD4+ T cell depleted PBMCs were centrifuged and counted for addition to the induction culture.
  • the CD4+ and CD8+ phenotype of the CD4+ depleted cell population was determined by FACS analysis (see below). In general, this technique resulted in a two-fold enrichment for CD8+ T cells with an average of approximately 40-50% CD8+ T cells and 15-20% remaining CD4+ T cells following depletion of CD4+ T cells.
  • Depletion of CD4+ T cells can also be accomplished by using antibody and complement methods or antibody coated magnetic beads (Dynabeads). Depletion of CD4+ T cells enriched the CTLp and removed cells which competed for cell nutrients.
  • CD4+ depleted PBMC to be used as the responder population were prepared utilizing AIS flasks for selection of CD8+ T cells through the depletion of CD4+ T cells (above).
  • the responder cells were plated at 3 x lOVml in a 1 ml volume (24 well plate) and placed at 37 °C until the peptide loaded stimulator APCs were prepared.
  • the irradiated, peptide loaded APCs were washed 1 time in serum-free RPMI (+ L-glutamine and Hepes), adjusted to the appropriate concentration in complete medium, and plated into a 24 well plate at 1 ml plate:
  • For PBMC and SAC-I activated PBMCs as APCs 1 x 10 6 stimulator cells (1 ml volume) were plated into the wells containing the responder cells;
  • For PHA blasts as APCs 1 ml of 3 x lOVml stimulator cells were plated in each well.
  • a final concentration of 10 ng/ml of rIL-7 (2 ml total volume) was added.
  • PBMCs Autologous PBMCs were thawed into serum-free RPMI (+ L-glutamine and Hepes) containing 30 ⁇ g/ml DNAse, washed 2 times, and adjusted to 5 x 10 6 /ml in culture medium containing DNAse.
  • PBMCs (25 million cells/tube in 5 ml) were irradiated at 6100R. After 1 wash, the PBMCs were resuspended in culture medium and adjusted to 4 x 10 6 /ml and 1 ml of irradiated PBMCs was added per well of a 24-well plate.
  • the PBMC were incubated for 2 hours at 37°C, washed 3 times to remove nonadherent cells, and cultured in medium containing 20 ⁇ g/ml total peptide and 3 ⁇ g/ml ⁇ 2 microglobulin added in a 0.5 ml volume and again incubated for 2 hours at 37°C.
  • the peptide was aspirated and 1.5 x 10 6 responder cells resuspended in culture medium were added in a 1 ml volume. After 2 days, 1 ml of culture medium containing 20 U/ml rIL-2 was added.
  • Target cell preparation Approximately 16-20 hours prior to the CTL assay, target cells (Class I matched EBV-transformed lines) were washed once and resuspended in a 10 ml volume at 3 x lOVml in RPMI/5 % FCS in the presence or absence of 10 ⁇ g/ml total peptide.
  • Target cells were centrifuged and resuspended in 200 ⁇ l tube sodium 51 Cr chromate (NEN), then incubated at 37°C for 1 hour on a shaker.
  • Targets were washed 3 times (10 ml wash) with RPMI/10% FCS and resuspended in 10 ml (to determine the efficiency of labelling, 50 ⁇ l target was counted on the Micromedic automatic gamma counter).
  • CTL assay Target cells were adjusted to 2 x lOVml and 50 ⁇ l of the cell culture was added to each well of a U-bottomed 96-well plate (Costar Co ⁇ .) for a final concentration of 1 X lOVwell.
  • K562 cells were washed once, resuspended at 4 x 10 6 /ml, and 50 ⁇ l well was added for a final concentration of 2 x lOVwell (ratio of cold K562 to target was 20:1).
  • Responder cells were washed once, resuspended at 9 x lOVml, and three fold serial dilutions were performed for effector to target ratios of 90: 1 , 30: 1, 10: 1 , and 3: 1.
  • Responder cells were added in a volume of 100 ⁇ l in duplicate wells. For spontaneous release, 50 ⁇ l/well of labelled target cells, 50 ⁇ l/well K562, and 100 ⁇ l/well of medium was added.
  • % specific lysis cpm experimental release - cpm spontaneous release/cpm maximum release - cpm spontaneous release X 100.
  • a cytotoxicity assay was considered positive if the lysis by CTL of targets sensitized with a specific peptide at the two highest effector to target (E:T) ratios was 15 % greater than lysis of control targets (i.e. target cells without peptide).
  • a cytotoxicity assay was considered borderline if the lysis by CTL of targets sensitized with a specific peptide at the two highest effector to target (E:T) ratios was 6% greater than lysis of control targets (i.e. target cells without peptide).
  • PBMC from 12 HLA-A2.1 normal volunteers all negative for HBV serological markers were used to generate DC and CTL responses to a known HBV CTL epitope.
  • the in vitro generated DC were pulsed with the HBV peptide HBC 18 (which binds with high affinity to HLA-A2.1), and subsequently used to induce CTL from autologous CD8 + purified lymphocytes.
  • the lymphocyte cultures were re-stimulated with antigen (peptide-pulsed autologous adherent monocytes) on days 7 and 14, and cytolytic activity was measured on day 21.
  • the CTL induction cultures in each experiment were maintained in separate microcultures (in 48 well-plates) throughout the entire procedure.
  • Cytotoxic activity was measured in a 4-6 hr 51 Cr release assay using .221 (A2.1) cells that were either pulsed with peptide or not, and with a .221 (A2.1) cell line transfected with the HBV core gene.
  • the results presented in Table I show that in all experiments performed with the 12 individuals, at least one culture had CTL that recognized peptide-pulsed target cells.
  • Table I Summary of results obtained in the induction of CTL using peptide-pulsed DC and EL-7 to a known HBV epitope in normal individuals
  • GM-CSF concentrations in culture supematants were determined p ⁇ or Ag restimulation and 24 hr after Ag restimulation using an ELISA specific for human GM-CSF. Results are expressed as pg/ml
  • GM-CSF Ratio GM-CSF produced after Ag restimuIation GM-CSF produced before Ag restimulation
  • Cytotoxic activity was determined in a 5-hr 3l Cr release assay using .221(A2.1) cells with and w/o peptide and .221(A2.1) cells transfected with the HBV core gene [.221(A2.1).HBc]. Effector:
  • Target ratios were between 10.1 and 1.1.
  • IL-7 enhances the in vitro generation of CTLs and promotes their growth (Celis, et al , Proc. Natl. Acad. Sci. (USA) 91:2105 (1994)); Wentworth, et al , Molec. Immunol. 32:603 (1995); Alderson, et al. , J. Exp. Med. 773:923)).
  • Other lymphokines such as IL-12 and IL-10 have also been reported to enhance the production of CTL responses and to function as growth factor for CTLs (Chen, et al, J. Immunol. 747:528 (1991); Yang, et al , J. Immunol. 755:3897 (1995)).
  • IL-7, IL-10 and IL-12 could further enhance the induction of CTL by peptide-pulsed DC, we selected an experimental model system where, in our experience, suboptimal CTL responses were generally obtained.
  • CTL induction to melanoma-associated peptides using SAC- 1 -generated APC or DC is in general more difficult to achieve than CTL responses to the HBC 18 , peptide (V. Tsai, unpublished). This could be due to a lower precursor frequency for CTL specific for melanoma-associated antigens, which in most cases represent tissue-specific "self' antigens that may induce some level of immunological tolerance.
  • EL-10 was studied using DC pulsed with an HLA-A 1 -binding peptide from the melanoma-associated antigen MAGE-3.
  • Lysis' Lysis' 1 No. Lysis (•* Tumor Cells)
  • Lysis Lysis 4 No. Lysis" (A Tumor Cells) ( ⁇ Peptide) ( ⁇ Tumor Cells) ( ⁇ Peptide)
  • DC were prepared from PBMC as described in Materials and Methods and pulsed with the MAGE m peptide.
  • IL-10 (10 ng/ml) was added on day 1 of the CTL induction cultures, and during the antigen re-stimulation procedure.
  • Cultures marked with an "*" are considered to have specific CTL activity against both peptide-sensitized targets and melanoma cells.
  • Target Cells IL-10(ng/ml) 0 10 0 10 30 100 0 10 0 10 30 100
  • Numbers represent the % of wells, from a total of 48 that were set up for each condition.
  • Cultures were scored as positive for peptide-reactive CTL if the difference in the % specific 3l Cr release obtained with targets loaded with peptide minus the value obtained with targets w/o peptide was 20% or more.
  • Lysis 4 . Tumor Cells
  • Lysis ⁇ . Tumor Cells
  • Lysis ⁇ . Tumor Cells
  • ⁇ Peptide ⁇ Peptide
  • One of the applications of the methods described here is in the area of adoptive therapy using antigen-specific CTL. If the peptide-induced CTL can be expanded in tissue culture in a similar way as tumor-infiltrating lymphocytes ( ⁇ Ls) and T cell clones, and maintain their specificity for antigen, these cells could be used for treating patients with on-going disease.
  • ⁇ Ls tumor-infiltrating lymphocytes
  • T cell clones T cell clones
  • HLA-A2-restricted CTL lines derived from melanoma patients recognize various peptides derived from the gplOO melanocyte-derived protein.
  • gplOO-reactive CTL lines derived from tumor infiltrating lymphocytes (TIL) of 4 patients were screened for reactivity with a large panel of peptides containing HLA-A2.1 binding motifs (Kawakami, et al, J. Immunol. 154:3961 (1995)). Only 7 of the 169 peptides tested were recognized by at least one of the CTL lines.
  • the G9 280 was also reported by others as recognized by CTL from five melanoma patients (Cox, et al , Science 264:716 (1994)).
  • the cytolytic activity of individual cultures was measured in a 5 hr 3 'Cr release against four targets: .221(A2. 1) plus peptide, .221 (A2.1) w/o peptide, 624mel (A2+ , grpl00 + ) and A375 mel (A2+ , gplOO-). Effectors: target ratios were between 1: 1 and 10: 1. Numbers represent % specific cytotoxicity as defined in "Materials and Methods. " Results in each row represent examples of individual wells from the same experiment performed with a specific peptide.
  • This CTL line was restimulated with antigen and expanded for further characterization (Fig. 2).
  • Each culture represents an individual well of a 48-well tissue culture plate.
  • the cytoiytic activity of individual cultures was measured in a 5 hr 31 Cr release against four targets: .221(A2.1) plus peptide, .221(A2.1) w/o peptide, 624mel (A2 + , grpl00 + ) and A375 mel (A2-I- , gplOO-). Effectors: target ratios were between 1 : 1 and 10: 1. Numbers represent % specific cytotoxicity as defined in "Materials and Methods. " Results in each row represent examples of individual wells from the same experiment performed with a specific peptide.
  • Table XI indicates that CTL responses to these peptides were somewhat more difficult to obtain than responses to the immunodominant epitopes recognized by melanoma patients (Table El), possibly indicating a less abundant TCR repertoire directed towards these peptide specificities. 888
  • Percentages represent the number of positive wells/total X 100.
  • Each culture represents an individual well of a 48-well tissue culture plate.
  • the peptide G10 570 was also effective in eliciting anti-melanoma specific CTL.
  • the data presented in Figure 5 illustrates that the G10 570 CFL line was capable of killing melanoma cell lines that express gplOO and that this lytic activity was inhibitable by cold targets loaded with G10 570 , but not by cold targets sensitized with an irrelevant peptide.
  • the G10 177 epitope was tested for its ability to induce melanoma-reactive CTL using lymphocytes obtained from an HLA-A2.1 melanoma patient. Forty eight individual CTL cultures were established and after three rounds of antigen stimulation one culture gave forth to melanoma-reactive CTL. The results presented in Figure 6 show that the melanoma patient CTL induced with peptide G10 I77 effectively and specifically recognized both peptide-sensitized targets and gpl00 + melanoma cells and that the lytic activity against melanoma cells was blocked by cold target cells pulsed with the relevant peptide.
  • the present invention led to the identification of at least 3 new gplOO epitopes capable of eliciting tumor- reactive CTE ⁇ . Since only 6 of 12 potential subdominant epitopes have been tested to date following our dendritic cell priming in vitro immunization protocol, it is likely that additional gplOO derived epitopes will also be identified.
  • the experimental protocol described herein allows to define the CTL epitopes contained within a given tumor antigen (regardless of whether they are immunodominant or not) and fully exploit the immunological potential of the tumor antigen itself. In vivo and in vitro CTL inductions using both dominant and subdominant epitopes will yield a more vigorous and diverse immune response.
  • Polyvalent vaccines containing mixtures of epitopes from several TAA will overcome the need for complex mixtures of whole proteins or genes. Immunization with subdominant epitopes will focus CTL responses toward specific conserved sequences and thus prevent the emergence of tumor variants during the course of disease.
  • results presented here demonstrate that it is possible to induce tumor-reactive CTL from blood of cancer patients specific for both dominant and subdominant epitopes.
  • These CTL could be expanded in culture to approximately 1 X 10 9 cells (data not shown), still retained antigen specificity and could thus be used for adoptive therapies.
  • Immune therapy using peptide-primed CTL from blood may be a convenient (since no tumor biopsy is required), and reliable (because of the high degree of specificity) alternative to the already promising current ⁇ L based immunotherapies (Rosenberg, et al, J. Natl. Cancer Inst. 86:1159 (1994)).
  • MAGE2 and MAGE3 antigens are not only frequently expressed in melanoma tumors, but also in approximately 40-60% of epithelial tumors (colon, breast, lung) (Weynants, P. et al, International Journal of Cancer 56:826-829 (1994); De Plaen, E. et al , Immuno genetics 40: 360-369 (1994); Russo, V. et al , International Journal of Cancer 64:216-221 (1995)).
  • HLA-A1 (Celis, E. et al. , Proceedings of the National Academy of Sciences of the United States of America 91:2105-2109 (1994); Gaugler, B. er al , J Exp Med.
  • Selected CTL cultures were expanded by repeated antigen restimulations (peptide and APC) to obtain sufficient effector cells for more extensive cytotoxicity assays.
  • peptide-reactive CTL lines were studied for their ability to recognize tumor cells expressing the corresponding MAGE antigens. In the majority of cases, the reactivity towards peptide-sensitized target cells remained evident.
  • MAGE2[10 157 ]-reactive CTL lines exhibited the capacity to kill melanoma tumor cells, which naturally produce and process the MAGE2 protein.
  • CEA carcinomaembryonic antigen
  • CEA is a TAA frequently expressed in epithelial tumors such as lung, colon, gastric and breast carcinomas
  • CEAUO-aJ VLYGPDAPT1 454.5 NT NT
  • Numbers in parentheses represent peptide size and the relative position in the protein sequence
  • Cultures were considered containing CTL when specific cytotoxicity towards antigen-containing target (Plus Peptide or CEA+ tumor, SW403) was > 15 % above cytotoxicity with negative control target. Values represent the number of positive cultures out of 48 that were tested.
  • This peptide was described as an HLA-A2 restricted epitope (13). Although cytotoxicity 10 tumor cells was observed, specificity could not be demonstrated by cold target inhibition (not shown).
  • HLA-2.1 -binding peptides derived from CEA were studied for their capacity to elicit CTL using DC as antigen-presenting cells. Three of 5 peptides tested, stimulated CTL responses which recognized peptide-sensitized target cells. Moreover, after restimulation with antigen and APC, killing of CEA-expressing tumor cells was demonstrated for CEA ⁇ ] and CEA[9 691 ] specific CTL (Table XHI).
  • the CTL line reactive with peptide CEA[9 691 ] was cloned and studied further in terms of specificity and recognition of various CEA-expressing tumor cells.
  • Cloned CTL specific for CEA[9 691 ] had high specific lytic activity towards tumor target cells (colon and gastric) that express HLA-A2 and CEA ( Figure 11).
  • the killing activity directed against gastric and colon tumor cells was specific since it was blocked with "cold targets" pulsed with peptide CEA[9 691 ] but not with targets pulsed with a control HLA-A2.1 binding peptide (Fig. 12).
  • TAA tumor necrosal growth factor-2/neu gene
  • cerbB-2 a tumor necrosis factor-2/neu gene
  • This TAA is a cell surface protein often overexpressed by breast and ovarian carcinomas, and also expressed by colon and lung carcinomas (Slamon, D. J. et al , Science 244:707-712 (1989); Yokota, J. et al , Lancet 1 :765-767 (1986)).
  • Table 14 List cfHLA-A2.1 binding peptides from HER-2/neu fFootnotes for Table 14. a Numbers in parentheses represent peptide size and the relative position in the protein sequence
  • HLA-A2.1 binding peptides HER2[9 435 ], HER2[9 789 ], HER2[9 48 ] and HER2[9 5 ]
  • HER2[9 369 ] was included in this analysis to confirm that this epitope was capable of inducing tumor-reactive CTL in our system. All 5 HLA-A2.1 binding peptides tested elicited CTL that killed peptide-sensitized target cells.
  • HER2[9 369 ], HER2[9 435 ] and HER2[9 5 ] elicited CTL which recognized and specifically killed tumor cells expressing the HLA-A2.1 and ⁇ ER.-2/neu antigens (Table XIV). Since HER2[9 369 ] has been described in the past as an epitope for tumor-reactive CTL (Fisk, B. et al., J Exp Med, 181:2109-2117 (1995)), we did not further characterize these CTL. By contrast, CTL lines specific for HER2[9 435 ] and HER2[9 5 ] were expanded for further analysis.
  • HLA-A2.2 and -A68.2 were selected because of their high frequency in the black population and -A2.3 and -A2.6 were chosen because of their high frequency in Oriental ethnicity (del Guercio, M. F. et al , Journal of Immunology 154: 685-693 (1995); Sidney, J. , Grey er al , Immunology Today 17:261-266 (1996)).

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Abstract

L'invention porte sur un procédé d'obtention in vitro de lymphocytes T cytotoxiques (CTL) spécifiques d'antigènes présenté en association avec les méthodes d'utilisation in vivo desdits lymphocytes à des fins thérapeutiques. Les cellules porteuses d'antigènes (APC) utilisées dans ces travaux comportent notamment des cellules dendritiques prétraitées par du GM-CSF et de l'IL-4. Les cellules CTL spécifiques d'un antigène ont été obtenues par incubation de préparations de CD8 en présence de cellules APC qui fixent les peptides désirés. L'IL-7 était généralement ajouté au jour 1 de l'incubation, et l'IL-10 était généralement ajouté un jour plus tard, et pendant la restimulation. L'invention porte également sur des procédés d'utilisation de cellules dendritiques pulsées par peptides pour identifier des antigènes tumoraux restreints au CMH de classe I, et des épitopes subdominants.
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WO2001000225A1 (fr) * 1999-06-29 2001-01-04 Epimmune Inc. Peptides de liaison a hla et leurs utilisations
WO2001042270A1 (fr) * 1999-12-10 2001-06-14 Epimmune Inc. Induction de reponses immunes cellulaires a l'antigene carcinoembryonnaire a l'aide des compositions renfermant des peptides et des acides nucleiques
WO2002072627A2 (fr) * 2001-03-09 2002-09-19 Callistogen Ag Induction de lymphocytes t cytotoxiques antitumoraux chez les humains au moyen de determinants antigeniques peptidiques decouverts au moyen d'algorithmes informatiques afin d'effectuer des vaccinations
EP1303287A1 (fr) * 2000-06-16 2003-04-23 Argonex Pharmaceuticals Peptides mhc surexprimes sur des cellules cancereuses de la prostate et methodes d'utilisation
JP2003516344A (ja) * 1999-12-13 2003-05-13 エピミューン， インコーポレイテッド Ｈｌａクラスｉａ２腫瘍関連抗原ペプチドおよびワクチン組成物
EP1312670A1 (fr) * 2000-08-16 2003-05-21 Takara Bio Inc. Procede de culture extensive de lymphocytes t cytotoxiques specifiques de l'antigene
US6602510B1 (en) 2000-04-05 2003-08-05 Epimmune Inc. HLA class I A2 tumor associated antigen peptides and vaccine compositions
EP1343819A1 (fr) * 2000-09-01 2003-09-17 Epimmune Inc. Peptides de fixation de hla-a2.1 et leurs utilisations
EP1715343A1 (fr) * 2005-04-20 2006-10-25 F. Hoffmann-La Roche Ag Méthode d'identification des épitopes en rapport avec l'immunogénicité des produits bio-pharmaceutiques
US7252829B1 (en) 1998-06-17 2007-08-07 Idm Pharma, Inc. HLA binding peptides and their uses
EP2177534A3 (fr) * 1999-11-18 2010-07-14 Pharmexa Inc. Analogues hétéroclites de classe 1 épitopes
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US7795381B2 (en) 2001-04-09 2010-09-14 Mayo Foundation For Medical Education And Research Methods and materials for cancer treatment
US7910368B2 (en) 2001-08-15 2011-03-22 Takara Bio Inc. Method of extended culture for antigen-specific cytotoxic lymphocytes
US8728811B2 (en) 2002-03-25 2014-05-20 Takara Bio Inc. Process for producing cytotoxic lymphocyte
US8765469B2 (en) 2005-08-17 2014-07-01 Takara Bio Inc. Method of producing lymphocytes
US8927273B2 (en) 2003-08-22 2015-01-06 Takara Bio Inc. Process for producing cytotoxic lymphocytes
CN105175527A (zh) * 2015-10-09 2015-12-23 深圳市康尔诺生物技术有限公司 一种乳腺癌特异性的热休克蛋白复合物及其应用
US9394350B2 (en) 2003-04-18 2016-07-19 Ose Pharma International Sa HLA-A2 tumor associated antigen peptides and compositions
WO2017117418A1 (fr) * 2015-12-30 2017-07-06 Anthrogenesis Corporation Procédés de production de lymphocytes t et lymphocytes t ainsi produits
US10351824B2 (en) 2011-12-12 2019-07-16 Cell Medica Limited Process of expanding T cells
WO2020086927A1 (fr) * 2018-10-26 2020-04-30 Saint Louis University Peptides pour induire des réponses hétérosubtypiques de lymphocytes t de la grippe
CN111529697A (zh) * 2011-05-26 2020-08-14 金纽斯生物科技投资有限责任公司 调节的免疫优势疗法
US11931408B2 (en) 2015-09-18 2024-03-19 Baylor College Of Medicine Immunogenic antigen identification from a pathogen and correlation to clinical efficacy
US11963979B2 (en) 2011-12-12 2024-04-23 Allovir, Inc. Process for T cell expansion
US11981923B2 (en) 2012-02-09 2024-05-14 Baylor College Of Medicine Pepmixes to generate multiviral CTLS with broad specificity

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2005007694A1 (ja) * 2003-07-16 2007-09-20 株式会社グリーンペプタイド ＨＥＲ２／ｎｅｕペプチドおよびその治療上の用途

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994003205A1 (fr) * 1992-08-07 1994-02-17 Cytel Corporation Peptides de liaison de hla, et leurs utilisations

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994003205A1 (fr) * 1992-08-07 1994-02-17 Cytel Corporation Peptides de liaison de hla, et leurs utilisations

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BENDER A., ET AL.: "IMPROVED METHODS FOR THE GENERATION OF DENDRITIC CELLS FROM NONPROLIFERATING PROGENITORS IN HUMAN BLOOD.", JOURNAL OF IMMUNOLOGICAL METHODS., ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM., NL, vol. 196., 1 January 1996 (1996-01-01), NL, pages 121 - 135., XP002910822, ISSN: 0022-1759, DOI: 10.1016/0022-1759(96)00079-8 *
BUELENS C., ET AL.: "INTERLEUKIN-10 DIFFERENTIALLY REGULATES B7-1 (CD80) AND B7-2 (CD86) EXPRESSION ON HUMAN PERIPHERAL BLOOD DENDRITIC CELLS.", EUROPEAN JOURNAL OF IMMUNOLOGY, WILEY - V C H VERLAG GMBH & CO. KGAA, DE, vol. 25., 1 January 1995 (1995-01-01), DE, pages 2668 - 2672., XP002910824, ISSN: 0014-2980, DOI: 10.1002/eji.1830250940 *
MACATONIA S. E., ET AL.: "PRIMARY PROLIFERATIVE AND CYTOTOXIC T-CELL RESPONSES TO HIV INDUCED IN VITRO BY HUMAN DENDRITIC CELLS.", IMMUNOLOGY., WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 74., 1 January 1991 (1991-01-01), GB, pages 399 - 406., XP002910826, ISSN: 0019-2805 *
ROMANI N., ET AL.: "GENERATION OF MATURE DENDRITIC CELLS FROM HUMAN BLOOD AN IMPROVED METHOD WITH SPECIAL REGARD TO CLINICAL APPLICABILITY.", JOURNAL OF IMMUNOLOGICAL METHODS., ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM., NL, vol. 196., 1 January 1996 (1996-01-01), NL, pages 137 - 151., XP002910827, ISSN: 0022-1759, DOI: 10.1016/0022-1759(96)00078-6 *
See also references of EP1012238A4 *
STEINMAN R. M.: "DENDRITIC CELLS AND IMMUNE-BASED THERAPIES.", EXPERIMENTAL HEMATOLOGY, ELSEVIER INC., US, vol. 24., 1 January 1996 (1996-01-01), US, pages 859 - 862., XP002910825, ISSN: 0301-472X *
WENTWORTH P. A., ET AL.: "IN VITRO INDUCTION OF PRIMARY, ANTIGEN-SPECIFIC CTL FROM HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS STIMULATED WITH SYNTHETIC PEPTIDES.", MOLECULAR IMMUNOLOGY., PERGAMON, GB, vol. 32., no. 09., 1 January 1995 (1995-01-01), GB, pages 603 - 612., XP002910823, ISSN: 0161-5890, DOI: 10.1016/0161-5890(95)00037-F *

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US7252829B1 (en) 1998-06-17 2007-08-07 Idm Pharma, Inc. HLA binding peptides and their uses
WO2000052045A2 (fr) * 1999-02-26 2000-09-08 Fondazione Centro San Raffaele Del Monte Tabor Peptides immunogenes derives de mage-3 presentes par le complexe majeur d'histocompatibilte (mch) de categorie ii et utilisations associees
WO2000052045A3 (fr) * 1999-02-26 2001-01-04 San Raffaele Centro Fond Peptides immunogenes derives de mage-3 presentes par le complexe majeur d'histocompatibilte (mch) de categorie ii et utilisations associees
WO2001000225A1 (fr) * 1999-06-29 2001-01-04 Epimmune Inc. Peptides de liaison a hla et leurs utilisations
JP2003535024A (ja) * 1999-06-29 2003-11-25 エピミューン インコーポレイテッド Ｈｌａ結合ペプチドおよびそれらの使用
US8741576B2 (en) 1999-11-18 2014-06-03 Epimunne Inc. Heteroclitic analogs and related methods
EP2177534A3 (fr) * 1999-11-18 2010-07-14 Pharmexa Inc. Analogues hétéroclites de classe 1 épitopes
WO2001042270A1 (fr) * 1999-12-10 2001-06-14 Epimmune Inc. Induction de reponses immunes cellulaires a l'antigene carcinoembryonnaire a l'aide des compositions renfermant des peptides et des acides nucleiques
JP2003516344A (ja) * 1999-12-13 2003-05-13 エピミューン， インコーポレイテッド Ｈｌａクラスｉａ２腫瘍関連抗原ペプチドおよびワクチン組成物
US6602510B1 (en) 2000-04-05 2003-08-05 Epimmune Inc. HLA class I A2 tumor associated antigen peptides and vaccine compositions
EP1303287A1 (fr) * 2000-06-16 2003-04-23 Argonex Pharmaceuticals Peptides mhc surexprimes sur des cellules cancereuses de la prostate et methodes d'utilisation
EP1303287A4 (fr) * 2000-06-16 2006-02-08 Argonex Pharmaceuticals Peptides mhc surexprimes sur des cellules cancereuses de la prostate et methodes d'utilisation
US7816134B2 (en) 2000-08-16 2010-10-19 Takara Bio Inc. Method of extensive culture of antigen-specific cytotoxic T cells
EP1312670A1 (fr) * 2000-08-16 2003-05-21 Takara Bio Inc. Procede de culture extensive de lymphocytes t cytotoxiques specifiques de l'antigene
EP1312670A4 (fr) * 2000-08-16 2005-11-30 Takara Bio Inc Procede de culture extensive de lymphocytes t cytotoxiques specifiques de l'antigene
EP1343819A1 (fr) * 2000-09-01 2003-09-17 Epimmune Inc. Peptides de fixation de hla-a2.1 et leurs utilisations
EP1343819A4 (fr) * 2000-09-01 2005-03-23 Epimmune Inc Peptides de fixation de hla-a2.1 et leurs utilisations
WO2002072627A2 (fr) * 2001-03-09 2002-09-19 Callistogen Ag Induction de lymphocytes t cytotoxiques antitumoraux chez les humains au moyen de determinants antigeniques peptidiques decouverts au moyen d'algorithmes informatiques afin d'effectuer des vaccinations
WO2002072627A3 (fr) * 2001-03-09 2003-08-07 Callistogen Ag Induction de lymphocytes t cytotoxiques antitumoraux chez les humains au moyen de determinants antigeniques peptidiques decouverts au moyen d'algorithmes informatiques afin d'effectuer des vaccinations
US7795381B2 (en) 2001-04-09 2010-09-14 Mayo Foundation For Medical Education And Research Methods and materials for cancer treatment
US7910368B2 (en) 2001-08-15 2011-03-22 Takara Bio Inc. Method of extended culture for antigen-specific cytotoxic lymphocytes
US8975070B2 (en) 2002-03-25 2015-03-10 Takara Bio Inc. Process for producing cytotoxic lymphocyte
US8728811B2 (en) 2002-03-25 2014-05-20 Takara Bio Inc. Process for producing cytotoxic lymphocyte
US9394350B2 (en) 2003-04-18 2016-07-19 Ose Pharma International Sa HLA-A2 tumor associated antigen peptides and compositions
US9913884B2 (en) 2003-04-18 2018-03-13 Ose Pharma International Sa HLA-A2 tumor associated antigen peptides and compositions
US8927273B2 (en) 2003-08-22 2015-01-06 Takara Bio Inc. Process for producing cytotoxic lymphocytes
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US8765469B2 (en) 2005-08-17 2014-07-01 Takara Bio Inc. Method of producing lymphocytes
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US11723921B2 (en) 2011-05-26 2023-08-15 Geneius Biotechnology, Inc. Modulated immunodominance therapy
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US11834675B2 (en) 2015-12-30 2023-12-05 Celgene Corporation T lymphocyte production methods and t lymphocytes produced thereby
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WO2020086927A1 (fr) * 2018-10-26 2020-04-30 Saint Louis University Peptides pour induire des réponses hétérosubtypiques de lymphocytes t de la grippe
US12247053B2 (en) 2018-10-26 2025-03-11 Saint Louis University Peptides for inducing heterosubtypic influenza T cell responses

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