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WO1999047687A1 - Immunite antitumorale amelioree - Google Patents

Immunite antitumorale amelioree Download PDF

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Publication number
WO1999047687A1
WO1999047687A1 PCT/US1999/006037 US9906037W WO9947687A1 WO 1999047687 A1 WO1999047687 A1 WO 1999047687A1 US 9906037 W US9906037 W US 9906037W WO 9947687 A1 WO9947687 A1 WO 9947687A1
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WO
WIPO (PCT)
Prior art keywords
cells
antigen
cell
dcs
apcs
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PCT/US1999/006037
Other languages
English (en)
Inventor
Johanne Kaplan
Abraham Scaria
Original Assignee
Genzyme Corporation
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Filing date
Publication date
Application filed by Genzyme Corporation filed Critical Genzyme Corporation
Priority to AU31027/99A priority Critical patent/AU3102799A/en
Priority to JP2000536870A priority patent/JP2002506886A/ja
Priority to EP99912714A priority patent/EP1064390A4/fr
Priority to CA002322660A priority patent/CA2322660A1/fr
Publication of WO1999047687A1 publication Critical patent/WO1999047687A1/fr
Priority to US09/663,072 priority patent/US7014848B1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K40/428—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule

Definitions

  • TECHNICAL FIELD This invention is in the field of molecular immunology and medicine. In particular, methods of inducing an antigen-specific immune response are provided.
  • the present invention provides a method of inducing an antigen-specific immune response in a subject by administering to that subject an effective amount of an allogeneic APC.
  • method further comprises administering an effective amount of an autologous APC.
  • the APCs are administered under conditions that provoke an antigen specific immune response in the subject.
  • the autologous APC such as dendritic cells, will present antigen to cytotoxic T lymphocytes and elicit .an antigen- specific response.
  • the allogeneic APC most suitably universal APCs (defined below) which also can be dendritic cells, will traffic to the same areas as the autologous cells, but will elicit a strong reaction from alloreactive T lymphocytes resulting in the local release of stimulatory cytokines that amplify the anti-antigen response and promote destruction of the antigen-expressing cells, such as tumor cells.
  • FIGURES Figure 1 shows induction of a mixed lymphocyte reaction (MLR) by dendritic cells. Varying numbers of bone-marrowed derived C57BL/6 dendritic cells (DCs) were used to stimulate allogeneic allogeneic BALB/c T lymphocytes.
  • MLR mixed lymphocyte reaction
  • DCs dendritic cells
  • the DCs were either unifected or infected with adenovirus vectors.
  • the level of proliferation induced was measured by tritiated thymidine incorporation after 5 days of culture.
  • An overall dose-dependent stimulation was observed with incre.asing numbers DCs and the stimulatory activity of Ad-transfected DCs was equal or greater to that of uninfected DCs.
  • FIG. 2 shows the results of B16 melamona immunization model.
  • Groups of 5 C57BL/6 mice were immunized against the gplOO or TRP-2 melanoma antigen by intraveneous injection of 5 X 10 5 bone marrow-derived dendritic cells (DCs) transfected with adenovirus vector encoding hum.an gplOO (Ad2/hugpl00 DCs) or mouse TRP-2 (Ad2/mTRP-2 DCs).
  • Uninfected DCs serve as a negative control.
  • Two weeks after immunization the mice were challenged with a subcutaneous injection of 2 X 10 4 B16 melanoma cells and tumor growth was monitored over time. The results indicate that pre-immunization against tumor antigen is effective in inducing anti-tumor immunity.
  • Figure 3 shows the results of an experiment with B16 melanoma active treatment model.
  • Groups of 5 C57BL/6 mice received a subcutaneous injection of
  • MOLECULAR BIOLOGY F. M. Ausubel et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.); ANTIBODIES: A LABORATORY MANUAL (E. Harlow and D. Lane eds (1988)); PCR2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)) and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)). Definitions
  • cancer refers to cells that have undergone a malignant transformation that makes them pathological to the host organism.
  • Primary cancer cells that is, cells obtained from near the site of malignant transformation
  • the definition of a cancer cell includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.
  • a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation. Biochemical or immunologic findings alone may be insufficient to meet this definition.
  • genetically modified means containing and/or expressing a foreign gene or nucleic acid sequence which in turn, modifies the genotype or phenotype of the cell or its progeny. In other words, it refers to .any addition, deletion or disruption to a cell's endogenous nucleotides.
  • cytokine refers to any one of the numerous factors that exert a variety of effects on cells, for example, inducing growth or proliferation.
  • Non-limiting examples of cytokines which may be used alone or in combination in the practice of the present invention include, interleukin-2 (IL-2), stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 12 (IL-12), G-CSF, granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-1 alpha (IL-l ⁇ ), interleukin-11 (IL-11), MlP-l ⁇ , leukemia inhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO) and flt3 ligand.
  • IL-2 interleukin-2
  • SCF stem cell factor
  • IL-3 interleukin 6
  • IL-12 interleukin 12
  • G-CSF granulocyte macrophage-colony stimulating factor
  • the present invention also includes culture conditions in which one or more cytokine is specifically excluded from the medium.
  • Cytokines are commercially available from several vendors such as, for example, Genzyme (Framingham, MA), Genentech (South San Francisco, CA), Amgen (Thousand Oaks, CA), R&D Systems and Immunex (Seattle, WA). It is intended, although not always explicitly stated, that molecules having similar biological activity as wild-type or purified cytokines (e.g., recombinantly produced or muteins thereof) .are intended to be used within the spirit and scope of the invention.
  • .antigen-presenting cells includes both intact, whole cells as well as other molecules which are capable of inducing the presentation of one or more antigens, preferably in association with class I MHC molecules.
  • suitable APCs include, but are not limited to, whole cells such as macrophages, dendritic cells, B cells; purified MHC class I molecules complexed to ⁇ 2-microglobulin; and foster antigen presenting cells.
  • Dendritic cells are potent antigen-presenting cells (APCs). It has been shown that DCs provide all the signals required for T cell activation and proliferation. These signals can be categorized into two types.
  • the first type which gives specificity to the immune response, is mediated through interaction between the T-cell receptor/CD3 ("TCR/CD3”) complex .and an antigenic peptide presented by a major histocompatibility complex (“MHC") class I or II protein on the surface of APCs. This interaction is necessary, but not sufficient, for T cell activation to occur.
  • MHC major histocompatibility complex
  • the first type of signals can result in T cell anergy.
  • the second type of signals called co- stimulatory signals, is neither antigen-specific nor MHC-restricted, and can lead to a full proliferation response of T cells and induction of T cell effector functions in the presence of the first type of signals.
  • dendritic cell is to include, but not be limited to a pulsed dendritic cell, a foster antigen presenting cell or a dendritic cell hybrid.
  • cytokine refers to any one of the numerous factors that exert a variety of effects on cells, for example, inducing growth or proliferation.
  • Non-limiting examples of cytokines which may be used alone or in combination in the practice of the present invention include, interleukin-2 (IL-2), stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 12 (IL-12), G-CSF, granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-1 alpha (IL-l ⁇ ), interleukin ll(IL-ll), MlP-l ⁇ , leukemia inhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO) and flt3 ligand.
  • IL-2 interleukin-2
  • SCF stem cell factor
  • IL-3 interleukin 6
  • IL-12 interleukin 12
  • G-CSF granulocyte macrophage-colony
  • the present invention also includes culture conditions in which one or more cytokines is specifically excluded from the medium.
  • Cytokines are commercially available from several vendors such as, for example, Genzyme (Framingham, MA), Genentech (South San Francisco, CA), Amgen (Thousand Oaks, CA), R&D Systems and Immunex (Seattle, WA). It is intended, although not always explicitly stated, that molecules having similar biological activity as wild-type or purified cytokines (e.g., recombinantly produced or biologically equivalent variants thereof) are intended to be used within the spirit and scope of the invention.
  • MHC major histocompatibility complex
  • HLA complex The proteins encoded by the MHC complex are known as "MHC molecules” and are classified into class I and class II MHC molecules.
  • Class I MHC molecules include membrane heterodimeric proteins made up of an chain encoded in the MHC associated noncovalently with ⁇ 2- microglobulin.
  • Class I MHC molecules are expressed by nearly all nucleated cells and have been shown to function in antigen presentation to CD8+ T cells.
  • Class I molecules include HLA-A, -B, and -C in humans.
  • Class I molecules generally bind peptides 8-10 amino acids in length.
  • Class II MHC molecules also include membrane heterodimeric proteins consisting of noncovalently associated ⁇ and ⁇ chains.
  • Class II MHC are known to participate in antigen presentation to CD4 + T cells and, in humans, include HLA-DP, -DQ, and DR.
  • Class II molecules generally bind peptides 12-20 amino acid residues in length.
  • MHC restriction refers to a characteristic of T cells that permits them to recognize antigen only after it is processed and the resulting antigenic peptides are displayed
  • Host cell is intended to include any individual cell or cell culture which can be or have been recipients for vectors or the incorporation of exogenous nucleic acid molecules, polynucleotides and/or proteins.
  • progeny of a single cell it also is intended to include progeny of a single cell, and the progeny may not necessarily be completely identical (in morphology or in genomic or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • the cells may be procaryotic or eucaryotic, and include but are not limited to bacterial cells, yeast cells, animal cells, and mammalian cells, e.g., murine, rat, simian or hum-an.
  • An "antibody” is an immunoglobulin molecule capable of binding an antigen.
  • the term encompasses not only intact immunoglobulin molecules, but also anti-idiotypic .antibodies, mutants, fragments, fusion proteins, humanized proteins and modifications of the immunoglobulin molecule that comprise an antigen recognition site of the required specificity.
  • antibody complex is the combination of antibody (as defined above) and its binding partner or ligand.
  • a native antigen is a polypeptide, protein or a fragment containing an epitope, which induces an immune response in the subject.
  • an "effective amount” is an amount sufficient to effect beneficial or desired results.
  • An effective .amount can be administered in one or more administrations, applications or dosages.
  • the polynucleotides of the present invention may be administered or applied transdermally, orally, subcutaneously, intramuscularly, intravenously or parenterally.
  • an effective amount of the polynucleotides is that amount which provokes an antigen-specific immune response in the subject.
  • polynucleotide and “nucleic acid molecule” are used interchangeably to refer to polymeric forms of nucleotides of any length.
  • the polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotide includes single-, double-stranded and triple helical molecules.
  • Oligonucleotide refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the .art. A "primer” refers to .an oligonucleotide, usually single-stranded, that provides a 3'-hydroxyl end for the initiation of enzyme-mediated nucleic acid synthesis.
  • polynucleotides a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs.
  • Analogs of purines and pyrimidines are known in the art, and include, but are not limited to, aziridinycytosine, 4-acetylcytosine, 5- fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5- carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine, 1- methyladenine, 1-methylpseudouracil, 1-methylguanine, 1 -methylinosine, 2,2- dimethylgu.anine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5- methylcytosine, pseudouracil, 5-pentylnyluracil and 2,6-diaminopurine.
  • uracil as a substitute for thymine in a deoxyribonucleic acid is also considered an analogous form of pyrimidine.
  • the polynucleotides encode a peptide, a ribozyme or an antisense sequence.
  • PCR primers refer to primers used in "polymerase chain reaction” or "PCR,” a method for amplifying a DNA base sequence using a heat-stable polymerase such as Taq polymerase, and two oligonucleotide primers, one complementary to the (+)-str.and at one end of the sequence to be amplified and the other complementary to the (- )-strand at the other end. Because the newly
  • PCR also can be used to detect the existence of the defined sequence in a DNA sample.
  • protein protein
  • oligopeptide polypeptide
  • peptide polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • culturing refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (either morphologically, genetically, or phenotypically) to the parent cell. By “expanded” is meant any proliferation or division of cells.
  • a "subject” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
  • composition is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.
  • a composition of this invention comprises the transduced APC and a pharmaceutically acceptable carrier suitable for administration to the subject.
  • pharmaceutical composition is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants see Martin, REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).
  • Co-stimulatory molecules are involved in the interaction between receptor-ligand pairs expressed on the surface of antigen presenting cells and T cells. Research accumulated over the past several years has demonstrated convincingly that resting T cells require at least two signals for induction of cytokine gene expression and proliferation (Schwartz R.H. (1990) Science 248:1349-1356 and Jenkins M.K. (1992) Immunol. Today 13:69-73).
  • One signal the one that confers specificity, can be produced by interaction of the TCR/CD3 complex with an appropriate MHC/peptide complex. The second signal is not antigen specific and is termed the "co-stimulatory" signal.
  • This signal was originally defined as an activity provided by bone-marrow-derived accessory cells such as macrophages and dendritic cells, the so called “professional” APCs.
  • bone-marrow-derived accessory cells such as macrophages and dendritic cells
  • APCs the so called "professional” APCs.
  • HSA heat stable antigen
  • Ii-CS chondroitin sulfate-modified MHC invariant chain
  • ICM-1 intracellular adhesion molecule 1
  • B7-l and B7-2/B70 (Schwartz R.H. (1992) Cell 71:1065-1068).
  • One exemplary receptor-ligand pair is the B7 co-stimulatory molecule on the surface of APCs and its counter-receptor CD28 or CTLA-4 on T cells (Freeman et al. (1993) Science 262:909-911 ; Young et al. (1992) J. Clin. Invest. 90:229; and Nabavi et al. (1992) Nature 360:266- 268).
  • co-stimulatory molecule encompasses any single molecule or combination of molecules which, when acting together with a peptide/MHC complex bound by a TCR on the surface of a T cell, provides a co- stimulatory effect which achieves activation of the T cell that binds the peptide.
  • the term thus encompasses B7, or other co-stimulatory molecule(s) on an antigen-presenting matrix such as an APC, fragments thereof (alone, complexed with another molecule(s), or as part of a fusion protein) which, together with peptide/MHC complex, binds to a cognate ligand and results in activation of the T cell when the TCR on the surface of the T cell specifically binds the peptide.
  • Co- stimulatory molecules are commercially available from a variety of sources, including, for example, Beckman Coulter. It is intended, although not always explicitly stated, that molecules having similar biological activity as wild-type or purified co-stimulatory molecules (e.g., recombinantly produced or muteins thereof) are intended to be used within the spirit and scope of the invention.
  • modulate an immune response includes inducing (increasing, eliciting) an immune response; and reducing (suppressing) an immune response.
  • An immunomodulatory method is one that modulates an immune response in a subject.
  • the term "inducing an immune response in a subject” is a term well understood in the art and intends that an increase of at least about 2- fold, more preferably at least about 5-fold, more preferably at least about 10-fold, more preferably at least about 100-fold, even more preferably at least about 500- fold, even more preferably at least about 1000-fold or more in an immune response to an antigen (or epitope) can be detected (measured), after introducing the antigen (or epitope) into the subject, relative to the immune response (if any) before introduction of the antigen (or epitope) into the subject.
  • An immune response to an antigen includes, but is not limited to, production of an antigen-specific (or epitope-specific) antibody, and production of an immune cell expressing on its surface a molecule which specifically binds to an antigen (or epitope).
  • Methods of determining whether an immune response to a given antigen (or epitope) has been induced are well known in the art.
  • antigen-specific antibody can be detected using any of a variety of immunoassays known in the art, including, but not limited to, ELISA, wherein, for example, binding of .an antibody in a sample to an immobilized antigen (or epitope) is detected with a detectably-labeled second antibody (e.g., enzyme-labeled mouse anti-human Ig
  • Immune effector cells specific for the antigen can be detected any of a variety of assays known to those skilled in the art, including, but not limited to, FACS, or, in the case of CTLs, 51 Cr-release assays, or 3 H-thymidine uptake assays.
  • assays known to those skilled in the art, including, but not limited to, FACS, or, in the case of CTLs, 51 Cr-release assays, or 3 H-thymidine uptake assays.
  • the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination.
  • compositions consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
  • the present invention provides a method for provoking antigen-specific immune responses, and in particular, immune responses against tumor antigens.
  • this invention exploits the fact that alloreactivity (reactivity against foreign MHC antigens) is a universally strong immune reaction leading to the activation of CD4 + and CD8 + T lymphocytes.
  • Dendritic cells (DC) which display high densities of MHC class I and class II antigens, are especially potent stimulators of alloreactive T cells which has been confirmed in a mixed lymphocyte reaction (See Figure 1).
  • the ability of DCs to elicit potent alloreactivity is harnessed to promote the activation of tumor-specific T lymphocytes which are stimulated concurrently with autologous, MHC- compatible DCs expressing a relevent tumor-associated antigen(s) (T.AA).
  • T.AA tumor-associated antigen
  • the APC are dendritic cells, which are purified from the same and unrelated subject. Alternatively, more primitive cells of the hematopoietic lineage are isolated and cultured in appropriate growth factors which enrich for a population of dendritic cells. The release of stimulatory
  • allogeneic APCs are loaded with with the antigen of interest, i. e. , against which the immune response is to be raised.
  • the method of this invention comprises co-administering to a subject allogeneic and autologous antigen presenting cells (APCs), wherein the autologous APC express the antigen against which the immune response is raised.
  • APCs autologous antigen presenting cells
  • the invention provides a means to amplify the host immune response against an antigen, and in particular, a TAA.
  • the APC are purified, autologous APC are purified from the subject and the allogeneic APC are purified from an unrelated subject and expressing the antigen of interest which are then administered intravenously or intradermally.
  • these APC are transduced with a polynucleotide that codes for the antigen of interest, e.g., gplOO, MARTI, MUC-1.
  • the polynucleotide coding for the antigen may be from the same or similar species, for example the gene coding for the human gplOO can be transduced into the autologous APC.
  • the polynucleotides encoding TAAs of this invention can be, in one embodiment, previously characterized tumor-associated antigens such as gplOO
  • PSMA prostate membrane specific antigen
  • the cells can be transduced with a polynucleotide coding for a yet unidentified antigen which is identified and sequenced using methods described herein.
  • the cells are transduced using a "gene delivery vehicle” which is any agent that can carry inserted polynucleotides into a host APC.
  • gene delivery vehicles are liposomes, viruses, such as baculovirus, adenovirus, and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
  • Polynucleotides are inserted into vector genomes using methods well known in the art.
  • insert and vector DNA can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase.
  • synthetic nucleic acid linkers can be ligated to the termini of restricted polynucleotide. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector DNA.
  • an oligonucleotide containing a termination codon and an appropriate restriction site can be ligated for insertion into a vector containing, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient tr.ansfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper
  • presentation of the antigens such as tumor associated antigens by the APCs elicits a strong immune response resulting in destruction of tumor cells by antigen-specific immune effector cells such as cytotoxic T lymphocytes (CTLs).
  • CTLs cytotoxic T lymphocytes
  • the induction of the CTL response is one method to assay for a positive response to the therapy and a means to confirm the biological activity of new factors useful in the methods of this invention.
  • the presence of a large number of T-cells in tumor has been correlated with a prognostically favorable outcome in some cases
  • the antigen can be a previously characterized antigen, e.g., gplOO, MART or MUC-1 , or it can be a yet unidentified antigen.
  • Tumor cells that can be used for isolation of TAA can be isolated by any method known in the art. In one embodiment, a biopsy sample is minced and a cell suspension created. Preferably, the tumor cells are separated from other cells (such as immune effector cells, e.g., T cells) using methods well known in the art.
  • the use of physical separation techniques include, but are not limited to, those based on differences in physical (density gradient centrifugation .and counter-flow centrifugal elutriation), cell surface (lectin and antibody affinity), and vital staining properties (mitochondria-binding dye rhol23 and DNA-binding dye Hoechst 33342).
  • Monoclonal antibodies are another useful reagent for identifying markers associated with particular cell lineages and/or stages of differentiation can be used.
  • the antibodies can be attached to a solid support to allow for crude separation.
  • the separation techniques employed should maximize the retention of viability of the fraction to be collected.
  • Various techniques of different efficacy can be employed to obtain "relatively crude” separations. Such separations are up to 10%, usually not more than about 5%, preferably not more than about 1%, of the total cells present not having the marker can remain with the cell population to be retained.
  • the particular technique employed will depend upon efficiency of separation, associated cytotoxicity, ease and speed of performance, and necessity for sophisticated equipment and/or technical skill.
  • Any conventional method e.g., subtractive library, comparative Northern and/or Western blot analysis of normal and tumor cells, Serial Analysis Gene
  • T-cells Another common strategy in the search for tumor antigens is to isolate tumor-specific T-cells and attempt to identify the antigens recognized by the T- cells.
  • specific CTLs have been derived from lymphocytic infiltrates present at the tumor site. Weidmann , et al., supra. These TILs .are unique cell population that can be traced back to sites of disease when they are labeled with indium and adoptively transferred. Alternatively, large libraries of putative antigens can be produced and tested. Using the "phage method" (Scott and Smith (1990) Science 249:386-390; Cwirla, et al. (1990) Proc. Natl. Acad.
  • SAGE analysis can be employed to identify the antigens recognized by expanded immune effector cells such as CTLs. Briefly, SAGE analysis begins with providing complementary deoxyribonucleic acid (cDNA) from (1) the antigen-expressing population and (2) cells not expressing that antigen. Both cDNAs can be linked to primer sites. Sequence tags are then created, for example, using the appropriate primers to amplify the DNA. By measuring the differences in these tags between the two cell types, sequences which are aberrantly expressed in the antigen-expressing cell population can be identified.
  • cDNA complementary deoxyribonucleic acid
  • SolidPHase Epitope REcovery (“SPHERE”, described in PCT WO 97/35035) can be used to identify tumor antigens.
  • SPHERE can be used to identify antigens by creating a library
  • this invention also provides a screen to identify novel wild-type antigens that can be further modified and used to induce a cellular and a humoral immune response in the subject.
  • the antigens gplOO, MART or MUC-1, their biological activity in vitro and in vivo are positive controls.
  • the second approach for isolating APCs is to collect the relatively large numbers of precommitted APCs already circulating in the blood.
  • Previous techniques for isolating committed APCs from human peripheral blood have involved combinations of physical procedures such as metrizamide gradients and adherence/nonadherence steps (Freudenthal, PS et al. (1990) PNAS 87:7698- 7702); Percoll gradient separations (Mehta-Damani, et al. (1994) J. Immunol. 153:996-1003); and fluorescence activated cell sorting techniques (Thomas, R. et al. (1993) J. Immunol. 151:6840-52).
  • CCE countercurrent centrifugal elutriation
  • cells are subject to simultaneous centrifugation and a washout stre.am of buffer which is constantly increasing in flow rate.
  • the constantly increasing countercurrent flow of buffer leads to fractional cell separations that are largely based on cell size.
  • the APC are precommitted or mature dendritic cells which can be isolated from the white blood cell fraction of a
  • the white blood cell fraction can be from the peripheral blood of the mammal.
  • This method includes the following steps: (a) providing a white blood cell fraction obtained from a mammalian source by methods known in the .art such as leukophoresis; (b) separating the white blood cell fraction of step (a) into four or more subfractions by countercurrent centrifugal elutriation, (c) stimulating conversion of monocytes in one or more fractions from step (b) to dendritic cells by contacting the cells with calcium ionophore or IL-4 and GM-CSF, (d) identifying the dendritic cell-enriched fraction from step (c), and (e) collecting the enriched fraction of step (d), preferably at about 4°C.
  • the white blood cell fraction can be treated with calcium ionophore in the presence of other cytokines, such as IL-12, GM-CSF or IL-4.
  • the cells of the white blood cell fraction can be washed in buffer and suspended in Ca ++ /Mg ++ free media prior to the separating step.
  • the white blood cell fraction can be obtained by leukapheresis.
  • the dendritic cells can be identified by the presence of at least one of the following markers: HLA-DR, HLA-DQ, or B7. 2, and the simultaneous absence of the following markers: CD3, CD14, CD16,56,57, and CD 19, 20. Monoclonal antibodies specific to these cell surface markers are commercially available.
  • the method requires collecting an enriched collection of white cells and platelets from leukapheresis that is then further fractionated by countercurrent centrifugal elutriation (CCE) (Abrahamsen TG et al. (1991) J. Clin. Apheresis. 6:48-53).
  • CCE countercurrent centrifugal elutriation
  • Cell samples are placed in a special elutriation rotor.
  • the rotor is then spun at a constant speed of, for example, 3000 rpm. Once the rotor has reached the desired speed, pressurized air is used to control the flow rate of cells.
  • Cells in the elutriator are subjected to simultaneous centrifugation .and a washout stream of buffer which is constantly increasing in flow rate. This results in fractional cell separations based largely but not exclusively on differences in cell size.
  • PBMCs 19 Large numbers of PBMCs (up to lxlO 10 ) can be obtained by leukapheresis and circulating immature DCs or hematopoietic progenitors can be subsequently isolated using several methods including but not limited to: metrizamide gradients (Freudenthal P.S. and Steinman, R.M. (1990) Proc. Natl. Acad. Sci.87:7698-7702); PercoU gradients (Mehta-Damani, et al. (1994) J. Immunol.
  • peripheral blood monocytes can also serve as a pool of DC precursors.
  • Monocytes can be recovered from PBMC by elutriation, Ficoll and PercoU gradients or through their ability to adhere to plastic. Adherent layers of monocytes are typically cultured in
  • GM-CSF + IL-4 or GM-CSF + IL-13 to promote differentiation into immature DCs. Further purification of DCs obtained in this manner can be achieved by depletion of contaminating lymphocytes and monocytes/macrophages with specific .antibodies plus complement or with antibody-coated magnetic beads (Sozzani, et al. (1997) J. Immunol. 9:271-96; Henderson, et al. (1997) J. Immunol.
  • CD34 + hematopoietic progenitors using magnetic beads coated with CD34-specific antibodies
  • the frequency of circulating progenitor cells can be increased by treating the host with various combinations of cytokines such as Flt-3 ligand (Sudo, et al. (1997) Blood 89:3186-91), IL-3, G-CSF (Siena, et al. (1995)) and GM-CSF (Siena, et al. (1989) Blood 74:1905- ).
  • cytokines such as Flt-3 ligand (Sudo, et al. (1997) Blood 89:3186-91), IL-3, G-CSF (Siena, et al. (1995)) and GM-CSF (Siena, et al. (1989) Blood 74:1905- ).
  • Treatment of cancer patients with cyclophosphamide also leads to mobilization of CD34 + stem cells into peripheral blood (Siena, et
  • a basic approach to deriving DCs from bone marrow consists of bone marrow collection followed by depletion of all non-DC cell types (lymphocytes, granulocytes, monocytes/macrophages) for example with a cocktail of specific antibodies and complement (Inaba, et al. (1992) J. Exp. Med. 176:1693-1702).
  • the remaining cells are then cultured in GM-CSF ⁇ IL-4 to promote the growth and differentiation of DCs.
  • CD34 + precursor stem cells can be positively selected from bone marrow.
  • CD34 + precursor stem cells isolated from blood or bone marrow can be differentiated into DCs by culture in various combinations of cytokines such as GM-CSF, SCF (stem cell factor), TNF- ⁇ and Flt-3 ligand (Siena, et al. (1995);
  • DCs are typically identified by their veiled morphology and by the presence of a set of characteristic surface markers (MHC Class I, MHC Class II, B7.1, B7.2, CD13, CD33, CD40, etc.) and the absence of surface markers typical of macrophages (CD14) or lymphocytes (CD3, CD4, CD8).
  • Immature DCs isolated directly from blood or derived from blood monocytes or bone marrow can effectively take up and process antigen for presentation to T lymphocytes. However, further maturation is required for the DCs to acquire the ability to effectively prime antigen-specific T cells (Steinman,
  • transduced or peptide-pulsed immature DCs could be used directly for immunization with the maturation process occurring in vivo or, alternatively, antigen-presenting DCs could be further matured in vitro prior to administration as desired.
  • antigen-presenting DCs could be further matured in vitro prior to administration as desired.
  • muteins of the antigen as well as allogeneic and antigens from a different species, of previously characterized antigens are useful in the
  • MARTI and gplOO are melanocyte differentiation antigens specifically recognized by HLA-A2 restricted tumor-infiltrating lymphocytes (TILs) derived from patients with melanoma, and appear to be involved in tumor regression (Kawakami, Y., et al. (1994) Proc. Natl. Acad. Sci. USA 91:6458-62; Kawakami, Y., et al. (1994) Proc. Natl. Acad. Sci. USA 91:91:3515-9). Recently, the mouse homologue of human MART-1 has been isolated.
  • TILs HLA-A2 restricted tumor-infiltrating lymphocytes
  • the full-length open reading frame of the mouse MARTI consists of 342 bp, encoding a protein of 113 amino acid residues with a predicted molecular weight of -13 kDa. Alignment of human and murine MARTI amino acid sequences showed 68.6% identity. The murine homologue of gp 100 has also been identified. The open reading frame consists of 1,878 bp, predicting a protein of 626 amino acid residues which exhibits 75.5% identity to human gplOO.
  • genetic modifications of cells employed in the present invention are accomplished by introducing a vector containing a polypeptide or cDNA encoding an antigen.
  • a vector containing a polypeptide or cDNA encoding an antigen A variety of different gene tr.ansfer vectors, including viral as well as non-viral systems can be used.
  • Viral vectors useful in the genetic modifications of this invention include, but are not limited to adenovirus, adeno- associated virus vectors, retroviral vectors and adeno-retroviral chimeric vectors.
  • Adenovirus and adeno-associated virus vectors useful in the genetic modifications of this invention may be produced according to methods already taught in the art. (see, e.g., Karlsson, et al. (1986) EMBO 5:2377; Carter (1992) Current Opinion in Biotechnology 3:533-539; Muzcyzka (1992) Current Top.
  • adenoviral vectors based on the human adenovirus 5 (Virology 163:614-617, 1988) .are missing essential early genes from the adenoviral genome (usually E1.A/E1B), and are therefore unable to replicate unless grown in permissive cell lines that provide the missing gene products in trans.
  • a transgene of interest can be cloned and expressed in cells infected with the replication deficient adenovirus.
  • adeno virus-based gene transfer does not result in integration of the transgene into the host genome (less than 0.1%> adenovirus- mediated transfections result in transgene incorporation into host DNA), and therefore is not stable, adenoviral vectors can be propagated in high titer and transfect non-replicating cells.
  • Human 293 cells which are human embryonic kidney cells transformed with adenovirus E 1 A/E 1 B genes, typify useful permissive cell lines .and are commercially available from the ATCC. However, other cell lines which allow replication-deficient adenoviral vectors to propagate therein can be used, including HeLa cells.
  • Additional references describing adenovirus vectors and other viral vectors which could be used in the methods of the present invention include the following: Horwitz, M.S., Adenoviridae and Their Replication, in Fields, B., et al. (eds.) VIROLOGY, Vol. 2, Raven Press New York, pp. 1679-1721, 1990); Graham, F., et al., pp. 109-128 in METHODS IN MOLECULAR BIOLOGY, Vol. 7: GENE TRANSFER AND EXPRESSION PROTOCOLS, Murray, E. (ed.), Humana Press, Clifton, N.J. (1991); Miller, N., et al. (1995) FASEB Journal 9:190-199 Schreier, H (1994)
  • adenovirus plasmids are also available from commercial sources, including, e.g., Microbix Biosystems of Toronto, Ontario (see, e.g.,
  • Microbix Product Information Sheet Plasmids for Adenovirus Vector Construction
  • retroviral vectors which are produced recombinantly by procedures already taught in the .art.
  • WO 94/29438 describes the construction of retroviral packaging plasmids and packaging cell lines.
  • retroviral vectors useful in the methods of this invention are capable of infecting the cells described herein. The techniques used to construct vectors, and transfix and infect cells are widely practiced in the art. Examples of retroviral vectors are those derived from murine, avian or primate retroviruses. Retroviral vectors based on the Moloney murine leukemia virus (MoMLV) are the most commonly used because of the availability of retroviral variants that efficiently
  • Suitable vectors include those based on the Gibbon Ape Leukemia Virus (GALV) or HIV.
  • GALV Gibbon Ape Leukemia Virus
  • the viral gag, pol and env sequences are removed from the virus, creating room for insertion of foreign DNA sequences.
  • Genes encoded by the foreign DNA are usually expressed under the control of the strong viral promoter in the LTR.
  • Such a construct can be packed into viral particles efficiently if the gag, pol and env functions are provided in trans by a packaging cell line.
  • the gag-pol and env proteins produced by the cell assemble with the vector RNA to produce infectious virions that are secreted into the culture medium.
  • the virus thus produced can infect .and integrate into the DNA of the target cell, but does not produce infectious viral particles since it is lacking essential packaging sequences.
  • the packaging cell line Most of the packaging cell lines currently in use have been transfected with separate plasmids, each containing one of the necessary coding sequences, so that multiple recombination events are necessary before a replication competent virus can be produced.
  • the packaging cell line harbors an integrated provirus.
  • the provirus has been crippled so that, although it produces all the proteins required to assemble infectious viruses, its own RNA cannot be packaged into virus. Instead, RNA produced from the recombinant virus is packaged. The virus stock released from the packaging cells thus contains only recombinant virus.
  • the range of host cells that may be infected by a retro virus or retroviral vector is determined by the viral envelope protein.
  • the recombinant virus can be used to infect virtually any other cell type recognized by the env protein provided by the packaging cell, resulting in the integration of the viral genome in the transduced cell and the stable production of the foreign gene product.
  • murine ecotropic env of MoMLV allows infection of rodent cells
  • amphotropic env allows infection of rodent, avian and some primate cells, including human cells.
  • Amphotropic packaging cell lines for use with MoMLV systems are known in the art and commercially available and include, but are not
  • VSV-G vesicular stomatitis virus
  • a marker gene can be included in the vector for the purpose of monitoring successful transduction and for selection of cells into which the DNA has been integrated, as against cells which have not integrated the DNA construct.
  • Various marker genes include, but are not limited to, antibiotic resistance markers, such as resistance to G418 or hygromycin. Less conveniently, negative selection may be used, including, but not limited to, where the marker is the HSV-tk gene, which will make the cells sensitive to agents such as acyclovir and gancyclovir.
  • selections could be accomplished by employment of a stable cell surface marker to select for transgene expressing cells by FACS sorting.
  • NeoR neomycin /G418 resistance
  • the viral vector can be modified to incorporate chimeric envelope proteins or nonviral membrane proteins into retroviral particles to improve particle stability and expand the host range or to permit cell type-specific targeting during infection.
  • the production of retroviral vectors that have altered host range is taught, for example, in WO 92/14829 and WO 93/14188.
  • Retroviral vectors that can target specific cell types in vivo are also taught, for example, in Kasahara, et al. (1994) Science 266:1373-1376. Kasahara, et al. describe the construction of a
  • MoMLV Moloney leukemia virus having a chimeric envelope protein consisting of human erythropoietin (EPO) fused with the viral envelope protein.
  • EPO erythropoietin
  • This hybrid virus shows tissue tropism for human red blood progenitor cells that bear the receptor for EPO, and is therefore useful in gene therapy of sickle cell anemia and thalassemia.
  • Retroviral vectors capable of specifically targeting infection of cells are preferred for in vivo gene therapy.
  • the viral constructs can be prepared in a variety of conventional ways. Numerous vectors are now available which provide the desired features, such as long terminal repeats, marker genes, and restriction sites, which may be further modified by techniques known in the art.
  • the constructs may encode a signal peptide sequence to ensure that cell surface or secreted proteins encoded by genes are properly processed post-translationally and expressed on the cell surface if appropriate.
  • the foreign gene(s) is under the control of a cell specific promoter. Expression of the transferred gene can be controlled in a variety of ways depending on the purpose of gene transfer and the desired effect. Thus, the introduced gene may be put under the control of a promoter that will cause the gene to be expressed constitutively, only under specific physiologic conditions, or in particular cell types.
  • the retroviral LTR (long terminal repeat) is active in most hematopoietic cells in vivo .and will generally be relied upon for transcription of the inserted sequences and their constitutive expression (Ohashi, et al. (1992) Proc. Natl. Acad. Sci. 89:11332; Correll, et al. (1992) Blood 80:331).
  • Other suitable promoters include the human cytomegalovirus (CMV) immediate early promoter and the U3 region promoter of the Moloney Murine Sarcoma Virus (MMSV),
  • Rous Sarcoma Virus (RSV) or Spleen Focus Forming Virus (SFFV).
  • RSV Rous Sarcoma Virus
  • SFFV Spleen Focus Forming Virus
  • promoters examples include Granzyme A for expression in T-cells and NK cells, the CD34 promoter for expression in stem and progenitor cells, the CD8 promoter for expression in cytotoxic T-cells, and the CD1 lb promoter for expression in myeloid cells.
  • Inducible promoters may be used for gene expression under certain physiologic conditions.
  • an electrophile response element may be used to induce expression of a chemoresistance gene in response to electrophilic molecules.
  • the therapeutic benefit may be further increased by targeting the gene product to the appropriate cellular location, for example the nucleus, by attaching the appropriate localizing sequences.
  • the vector construct is introduced into a packaging cell line which will generate infectious virions.
  • Packaging cell lines capable of generating high titers of replication-defective recombinant viruses are known in the art, see for example, WO 94/29438.
  • Viral particles are harvested from the cell supernatant and purified for in vivo infection using methods known in the art such as by filtration of supernatants 48 hours post transfection.
  • the viral titer is determined by infection of a constant number of appropriate cells (depending on the retrovirus) with titrations of viral supernatants.
  • the transduction efficiency can be assayed 48 hours later by a variety of methods, including Southern blotting.
  • Non- viral vectors such as plasmid vectors useful in the genetic modifications of this invention, can be produced according to methods taught in the art. References describing the construction of non- viral vectors include the following: Ledley, FD, Human Gene Therapy 6: 1129-1144, 1995; Miller, N., et al, FASEB Journal 9: 190-199, 1995; Chonn, A, et al, Curr. Opin. in Biotech. 6: 698-
  • the efficacy of gene transfer into the cells of the subject can be monitored by any method known in the art.
  • a reporter or marker gene can be included in the gene delivery vehicle to facilitate identification of those cells into which the vehicle is successfully incorporated.
  • marker genes may prove especially helpful.
  • Screening markers or reporter genes are genes that encode a product that can readily be assayed.
  • Non-limiting examples of screening markers include genes encoding for green fluorescent protein (GFP) or genes encoding for a modified fluorescent protein.
  • the marker gene included in the delivery vehicle is a selectable marker.
  • a "positive" selectable marker gene encodes a product that enables only the cells that carry the gene to survive and/or grow under certain conditions. For example, plant and animal cells that express the introduced neomycin resistance (Neo r ) gene are resistant to the compound G418. Cells that do not carry the Neo r gene marker are killed by G418.
  • Negative selectable marker genes encode a product that allows cells expressing that product to be selectively killed.
  • the conditionally activated cytotoxic agent may also be a selectable marker such as HSV-tk. Cells expressing this gene can be selectively killed using gancyclovir or acyclovir.
  • Peptide fragments from antigens must first be bound to peptide binding receptors (major histocompatibility complex class I and class II molecules) that display the antigenic peptides on the surface of the APCs. Palmer E. and
  • T lymphocytes produce an antigen receptor that they use to monitor the surface of APCs for the presence of foreign peptides.
  • the antigen receptors on CD4 + T cells recognize antigenic peptides bound to MHC class II molecules whereas the receptors on CD8 + T cells react with antigens displayed on class I molecules.
  • antigens can be delivered to antigen- presenting cells as protein/peptide or in the form of polynucleotides encoding the protein/peptide ex vivo or in vivo.
  • the methods described below focus primarily on DCs which are the most potent, preferred APCs.
  • APCs genetically modified APCs. These include: (1) the introduction into the APCs of polynucleotides that express antigen or fragments thereof; (2) infection of APCs with recombinant vectors to induce endogenous expression of antigen; and (3) introduction of tumor antigen into the DC cytosol using liposomes. (See, Boczkowski D. et al. (1996) J. Exp. Med. 184:465; Rouse et al. (1994) J. Virol 68:5685; and Nair et al. (1992) J. Exp. Med. 175:609).
  • any method which allows for the introduction and expression of the heterologous or non-self antigen and presentation by the MHC on the surface of the APC is within the scope of this invention.
  • Pulsing is accomplished in vitro/ex vivo by exposing APCs to antigenic protein or peptide(s).
  • the protein or peptide(s) are added to APCs at a concentration of 1-10 ⁇ m for approximately 3 hours.
  • Paglia et al. (1996) J. Exp. Med. 183:317, has shown that APC incubated with whole protein in vitro were recognized by MHC class I-restricted CTLs, and that immunization of animals with these APCs led to the development of antigen-specific CTLs in vivo.
  • Protein/peptide antigen can also be delivered to APC in vivo .and presented by the APC.
  • Antigen is preferably delivered with adjuvant via the intravenous, subcutaneous, intranasal, intramuscular or intraperitoneal route of delivery. Grant E.P. and Rock K.L. (1992) J. Immunol. 148:13; Norbury, C. C. et al. (1995)
  • Antigen Painting Another method which can be used is termed "painting". It has been demonstrated that glycosyl-phosphotidylinositol (GPI)-modified proteins possess the ability to reincorporate themselves back into cell membranes after purification. Hirose et al. (1995) Methods Enzymol 250:582; Medof et al. (1984) J. Exp. Med. 160:1558; Medof (1996) FASEBJ. 10:574; and Huang et al. (1994) Immunity 1:607, have exploited this property in order to create APCs of specific composition for the presentation of antigen to CTLs. Expression vectors for ⁇ 2- microglobulin and the HLA-A2.1 allele were first devised.
  • the proteins were expressed in Schneider S2 Drosophila melanogaster cells, known to support GPI- modification. After purification, the proteins could be incubated together with a purified antigenic peptide which resulted in a trimolecular complex capable of efficiently inserting itself into the membranes of autologous cells. In essence, these protein mixtures were used to "paint" the APC surface, conferring the ability to stimulate a CTL clone that was specific for the antigenic peptide. Cell coating was shown to occur rapidly and to be protein concentration dependent. This method of generating APCs bypasses the need for gene transfer into the APC and permits control of antigenic peptide densities at the cell surfaces.
  • Foster APCs are derived from the human cell line 174xCEM.T2, referred to as T2, which contains a mutation in its antigen processing pathway that restricts the association of endogenous peptides with cell surface MHC class I molecules (Zweerink et al. (1993) J. Immunol. 150:1763). This is due to a large homozygous deletion in the MHC class II region encompassing the genes TAP1, TAP2, LMP1, and LMP2, which are required for antigen presentation to MHC class 1 -restricted CD8 + CTLs. In effect, only "empty" MHC class I molecules are presented on the surface of these cells. Exogenous peptide added to the culture
  • T2 cells bind to these MHC molecules provided that the peptide contains the allele-specific binding motif.
  • T2 cells are referred to herein as "foster" APCs. They can be used in conjunction with this invention to present the heterologous, altered or control antigen. Transduction of T2 cells with specific recombinant MHC alleles allows for redirection of the MHC restriction profile. Libraries tailored to the recombinant allele will be preferentially presented by them because the anchor residues will prevent efficient binding to the endogenous allele.
  • MHC molecules make the APC more visible to the CTLs.
  • a powerful transcriptional promoter e.g., the CMV promoter
  • results in a more reactive APC most likely due to a higher concentration of reactive MHC-peptide complexes on the cell surface.
  • DCs can be transduced with viral vectors encoding a relevant TAA.
  • Viral vectors that can be used include recombinant poxviruses such as vaccinia and fowlpox virus (Bronte, et al. (1997) Proc. Natl. Acad. Sci. 94:3183-88; Kim, et al. (1997) J. Immunother. 20:276-86) and, preferentially, adenovirus (Arthur, et al. (1997) Cancer Gene Ther. 4:17-25; Wan, et al. (1997) Hum. Gene Ther. 8:1355-
  • virus vectors e.g. retrovirus
  • a retrovirus may also prove suitable for transduction of human DCs (Marin, et al. (1996) J Virol. 70:2957-62).
  • In vivo transduction of DCs can potentially be accomplished by administration of Ad vector via different routes including intravenous, intramuscular, intranasal, intraperitoneal or cutaneous delivery.
  • the preferred route of administration is cutaneous delivery at multiple sites using a total dose of approximately Ixl0 10 -lxl0 12 i.u.
  • Levels of in vivo transduction can be roughly assessed by co-staining with antibodies directed against DC marker(s) and the TAA being expressed.
  • the staining procedure can be carried out on biopsy samples from the site of administration or on cells from draining lymph nodes or other organs where DCs may have migrated (Condon, et al. (1996) Nature Med. 2:1122-28; Wan, et al. (1997) Human Gene Therapy 8:1355-1363).
  • the amount of TAA being expressed at the site of injection or in other organs where transduced DCs may have migrated can be evaluated by ELISA on tissue homogenates.
  • dendritic cells can also be transduced in vitro/ex vivo by non- viral gene delivery methods such as electroporation, calcium phosphate precipitation or cationic lipid/plasmid DNA complexes (Arthur, et al. (1997) Cancer Gene Therapy 4:17-25).
  • In vivo transduction of dendritic cells can potentially be accomplished by administration of cationic lipid/plasmid DNA complexes delivered via the intravenous, intramuscular, intranasal, intraperitoneal or cutaneous route of administration.
  • Gene gun delivery or injection of naked plasmid DNA into the skin also leads to transduction of DCs (Condon, et al. (1996), supra; Raz, et al. (1994) Proc. Natl. Acad. Sci. 91:9519-23).
  • transduction efficiency and levels of transgene expression can be assessed as described above for viral vectors.
  • compositions and Methods for Therapeutic Application In the context of cancer gene therapy, the invention consists of amplifying the host immune response against tumor by co-administering allogeneic and
  • Autologous DCs provide cognate presentation of a tumor antigen to MHC-compatible T lymphocytes while allogeneic DCs are used to stimulate high frequency alloreactive T lymphocytes and thereby induce local production of stimulatory cytokines that will support and amplify the specific host immune response against tumor.
  • the preferred method is isolation of monocytes from peripheral blood followed by in vitro differentiation into immature DCs.
  • Immature autologous DCs are transduced with a vector, preferably adenovirus, expressing a relevant TAA(s).
  • Allogeneic DCs are derived from a different individual in the same manner or, alternatively, a dendritic cell line as opposed to primary cells could be used as a source of allogeneic DCs. As mentioned above, the allogeneic DCs may or may not have to be transduced as well.
  • Allogeneic DCs should be pre-tested and selected for their ability to induce a strong allogeneic reaction on the part of the intended recipient. This can be accomplished by HLA-typing (select allogeneic DCs with the most mismatched HLA alleles) or in a functional mixed lymphocyte reaction (MLR).
  • HLA-typing selective allogeneic DCs with the most mismatched HLA alleles
  • MLR functional mixed lymphocyte reaction
  • lymphocytes or purified T lymphocytes from the patient to be treated are stimulated with inactivated (irradiated or mitomycin C-treated) allogeneic DCs and the proliferative response induced is measured by tritiated thymidine incorporation after 5-6 days of culture. Allogeneic DCs that elicit the highest levels of proliferation are selected for co-administration with transduced autologous DCs.
  • Autologous DCs are transduced ex vivo, preferably with an Ad vector encoding a TAA (500 MOI for 24h). At this point, the DCs can be administered or can be further matured by exposure to TNF- ⁇ prior to administration. The former option is preferred. At the time of administration, transduced autologous
  • DCs are mixed with allogeneic primary DCs or an allogeneic DC line and the mixture is delivered i.v. or s.c. It is estimated that doses of autologous and allogeneic DCs can range from approximately 5xl0 7 -5xl0 9 each with up to 6 doses being delivered at intervals of approximately 3-6 weeks.
  • the protocol is the same as described in example 1 , except that instead of being transduced, autologous DCs are pulsed with protein or peptide epitopes from a given TAA(s) (1-10 ⁇ m protein/peptide for approximately 3 h).
  • Autologous DCs are transduced in vivo, preferably by cutaneous administration of a TAA-encoding Ad vector at multiple sites (total dose of Ixl0 10 -lxl0 12 i.u.).
  • the allogeneic primary DCS or DC line are either mixed with the virus and administered concurrently or are administered separately, immediately after virus, at the same sites. The former option is preferred. It is estimated that the dose of allogeneic DCs can range from approximately 5x10 7 - 5x10 9 and that treatment can be repeated up to six times at intervals of approximately 3-6 weeks.
  • the relative potency of different preparations of allogeneic DCs to stimulate lymphocytes from the patient to be treated can be evaluated in vitro using the well-described mixed lymphocyte reaction (MLR).
  • MLR mixed lymphocyte reaction
  • the relative ability of transduced autologous DCs ⁇ allogeneic DCs to elicit cytolytic effector cells capable of lysing tumor cells can also be tested in vitro.
  • transduced autologous DCs expressing a relevant TAA ⁇ allogeneic DCs are used to stimulate autologous lymphocytes (or purified CD8 + T cells) and, after several rounds of stimulation, the effector cells generated are
  • TAA-specific effector cells can be measured by several methods including cytokine production (e.g. TNF- ⁇ , interferon- ⁇ ) upon recognition of TAA-expressing targets or lysis of TAA-expressing target cells as assessed by release of various intracellular labels/markers such as 51 Chromium or lactose dehydrogenase (LDH).
  • cytokine production e.g. TNF- ⁇ , interferon- ⁇
  • lysis of TAA-expressing target cells as assessed by release of various intracellular labels/markers such as 51 Chromium or lactose dehydrogenase (LDH).
  • LDH lactose dehydrogenase
  • the efficacy of the planned strategy is best assessed in an animal model.
  • the murine B16 melanoma model is one model.
  • B16 melanoma cells are injected into syngeneic C57BL/6 mice either s.c. or i.v. to give rise to a local subcutaneous tumor or produce lung metastases, respectively.
  • the relative immunizing potential of transduced autologous DCs ⁇ allogeneic DCs can be tested in a pre-immunization or active treatment setting.
  • Figure 3 shows the results of one experiment using this model.
  • DCs are given approximately 2 weeks before challenge with B16 cells to establish immunity and protect against the development of a s.c. tumor or lung metastases.
  • DCs are given 3-4 days after administration of B16 cells to induce host immune responses that will inhibit/prevent ongoing growth of the tumor cells.
  • the efficacy of treatment is assessed by measuring tumor size over time (B 16 cells s.c.) or the number of lung metastases (B 16 cells i.v.).
  • levels of TAA-specific immunity cytotoxic T lymphocytes, antibodies, NK cell lysis
  • the DCs are given at a dose of 5x10 5 each, either s.c. or i.v.

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Abstract

La présente invention concerne des méthodes permettant d'induire une réponse immunitaire spécifique de l'antigène, grâce à l'administration de cellules allogéniques présentatrices de l'antigène à un sujet. Dans un autre aspect de cette invention, une quantité efficace de cellules autologues présentatrices de l'antigène est co-administrée audit sujet.
PCT/US1999/006037 1998-03-20 1999-03-19 Immunite antitumorale amelioree WO1999047687A1 (fr)

Priority Applications (5)

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AU31027/99A AU3102799A (en) 1998-03-20 1999-03-19 Enhanced anti-tumor immunity
JP2000536870A JP2002506886A (ja) 1998-03-20 1999-03-19 抗腫瘍免疫亢進
EP99912714A EP1064390A4 (fr) 1998-03-20 1999-03-19 Immunite antitumorale amelioree
CA002322660A CA2322660A1 (fr) 1998-03-20 1999-03-19 Immunite antitumorale amelioree
US09/663,072 US7014848B1 (en) 1998-03-20 2000-09-15 Enhanced anti-tumor immunity

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US9044447B2 (en) 2009-04-03 2015-06-02 University Of Washington Antigenic peptide of HSV-2 and methods for using same
US9328144B2 (en) 2009-02-07 2016-05-03 University Of Washington HSV-1 epitopes and methods for using same

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JP2003512305A (ja) * 1999-09-30 2003-04-02 ユニバーシティ オブ ワシントン 免疫学的に重要な単純疱疹ウイルス抗原
WO2002009745A1 (fr) * 2000-07-28 2002-02-07 Liponova Gmbh Medicament destine a l"immunotherapie de tumeurs malignes
EP1509244B1 (fr) * 2002-06-06 2011-07-27 Immunicum AB Nouveau procede et composition servant a produire un vaccin allogenique cellulaire
US8673296B2 (en) 2002-06-06 2014-03-18 Immunicum Ab Method and composition for producing a cellular allogeneic vaccine
EP1712634A1 (fr) * 2005-04-13 2006-10-18 Wittycell SAS Procédé pour la sélection des cellules présentatrices d'antigène efficaces et leur utilisation pour la régulation de l'immunité
WO2006109193A2 (fr) * 2005-04-13 2006-10-19 Wittycell Sas Selection de cellules extremement efficaces presentant l'antigene pour la regulation de l'immunite et leurs utilisations
WO2006109193A3 (fr) * 2005-04-13 2006-11-30 Wittycell Sas Selection de cellules extremement efficaces presentant l'antigene pour la regulation de l'immunite et leurs utilisations
US9328144B2 (en) 2009-02-07 2016-05-03 University Of Washington HSV-1 epitopes and methods for using same
US9044447B2 (en) 2009-04-03 2015-06-02 University Of Washington Antigenic peptide of HSV-2 and methods for using same
US9579376B2 (en) 2009-04-03 2017-02-28 University Of Washington Antigenic peptide of HSV-2 and methods for using same

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EP1064390A1 (fr) 2001-01-03
AU3102799A (en) 1999-10-11
JP2002506886A (ja) 2002-03-05
EP1064390A4 (fr) 2002-06-12
CA2322660A1 (fr) 1999-09-23

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