EP3277291A1 - Compositions and methods of treating acute myeloid leukemia - Google Patents
Compositions and methods of treating acute myeloid leukemiaInfo
- Publication number
- EP3277291A1 EP3277291A1 EP16718545.3A EP16718545A EP3277291A1 EP 3277291 A1 EP3277291 A1 EP 3277291A1 EP 16718545 A EP16718545 A EP 16718545A EP 3277291 A1 EP3277291 A1 EP 3277291A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cells
- cell
- aml
- patients
- population
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4615—Dendritic cells
-
- 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
- A61K39/46—Cellular immunotherapy
- A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
- A61K39/4622—Antigen presenting cells
-
- 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
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
-
- 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 A61K39/46
- A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 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 A61K39/46
- A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
-
- 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 A61K39/46
- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
- A61K2239/48—Blood cells, e.g. leukemia or lymphoma
Definitions
- the present invention relates generally to cellular immunology and more particularly to and methods for treating acute myeloid leukemia (AML).
- AML acute myeloid leukemia
- Acute myelogenous leukemia is a hematological malignancy characterized by the proliferation and accumulation of immature hematopoietic cells in the bone marrow and peripheral blood. These leukemic blasts replace the bone marrow, and inhibit the growth and maturation of normal erythroid, myeloid, and megakaryocytic precursors. Presenting signs and symptoms are usually related to decreased production of normal hematopoietic cells. Untreated, AML is usually fatal within weeks to months from diagnosis. AML causes approximately 1.2% of all cancer deaths in the United States, and represents approximately 90% of adult acute leukemias.
- the invention features methods of treating acute myeloid leukemia (AML) cancer in a patient by administering to said patient in post chemotherapy induced remission or active disease a composition containing a population of autologous dendritic cell/ AML tumor cell fusions (DC/AML fusions).
- AML acute myeloid leukemia
- the composition is administered 4 to 12 weeks following the completion of chemotherapy.
- the composition contains about 1 xlO 6 to 1 x 10 7 DC/AML cell fusions.
- the composition contains about 5 x 10 6 DC/AML cell fusions.
- the composition is administered at 4 week intervals.
- the method further includes administering GM-CSF.
- the GM-CSF is administered daily for 4 days.
- the GM-CSF is administered at a dose of 100 ug.
- the GM-CSF is administered at each dose of said DC/MM cell fusions.
- the method further includes administering to the subject a checkpoint inhibitor.
- the checkpoint inhibitor is administered one week after the DC/MM fusions.
- the checkpoint inhibitor is a PD1, PDL1, PDL2, TIM3, LAG3 inhibitor.
- the checkpoint inhibitor is a PD1, PDL1, TIM3, LAG3 antibody.
- the method further includes administering to the subject an agent that target regulatory T cells [0009]
- the method further includes administering to the subject an immunomodulatory agent.
- the immunomodulatory agent is lenalidomide or pomalinomide or apremilast.
- he method further includes administering to the subject a TLR agonist, CPG ODN, polylC, or tetanus toxoid.
- the invention also features a method of producing a fused cell population by providing a population of AML cells and a population of dendritic cells (DC) obtained from the same subject; mixing the tumor cells and the DC at a ratio of 1 : 10 to 1 :3 to produce a mixed cell population; adding polyethylene glycol (PEG) to the mixed cell population in an amount sufficient to mediate fusion of the tumor cell and DC cell to produce a fused cell population.
- the invention further includes culturing the fused cell population in a culture media with 10% heat inactivated autologous plasma and GM-CSF and quantifying the number of cells in the fused cell population that co-express unique DC or leukemia markers.
- a cell is provided that is produced by providing a population of AML cells and a population of dendritic cells (DC) obtained from the same subject; mixing the tumor cells and the DC at a ratio of 1 : 10 to 1 : 3 to produce a mixed cell population; adding polyethylene glycol (PEG) to the mixed cell population in an amount sufficient to mediate fusion of the tumor cell and DC cell to produce a fused cell population.
- the cell population is substantially free of endotoxin microbial contamination and mycoplasma.
- Figure 1 is a series of histological micrographs of a biopsy taken from a vaccine site. The histological sections have been prepared counterstained with hematoxylin and eosin, or processed for immunological staining to detect CD4, CD8, or CDla expression.
- Figures 3A and 3B are a series of graphs that depict the percentage progression free survival (Figure 3A) or the percentage overall survival over a two-year period.
- Figures 4A-4D are a series of graphs that depict the expansion of WT-1, NY- ESO, MUCl, Survivin pentamer positive cells following vaccination.
- CD8+ T cells binding the MUCl (Figure 4C), WT-1 ( Figure 4A), Survivin (Figure 4D) and NY-ESO ( Figure 4B) pentamers were quantified at serial time points (prior to each vaccination and at 1, 3, 6 months post vaccination) in patients who are HLA-A2.1. Binding to a control tetramer was quantified in parallel and the control value was subtracted from that obtained for the indicated tetramer. Mean values of 4 patients are presented demonstrating a marked increase in MUCl, WT1, and NY-ESO tetramer+ cells following vaccination.
- the invention features immune system-stimulating compositions that contain cells formed by fusion between autologous dendritic cells (DCs) and tumor cells.
- DCs dendritic cells
- the invention provides cell fusion of autologous DCs and acute myelogenous leukemia (AML) cells obtained from a subject that has AML.
- AML acute myelogenous leukemia
- AML offers a unique therapeutic challenge.
- a majority of patients achieve a remission following chemotherapy; however, only a small minority experience durable responses particularly in patients over the age of 60 in which only 15-20% of patients remain free of leukemia at two years.
- the high rate of recurrence following induction chemotherapy is thought to arise from the persistence of clonal populations intrinsically resistant to cytotoxic injury, including the malignant stem cell population resulting in the repopulation of disease within 6-12 months.
- the unique efficacy of cellular immunotherapy for AML is highlighted by the observation that allogeneic transplantation is potentially curative for a subset of patients due to the immunologic capacity of alloreactive lymphocytes to broadly eradicate the malignant clonal population.
- the application of this strategy is limited by often prohibitive treatment associated morbidity and mortality due to targeting of normal tissues in the context of graft versus host disease.
- a personalized vaccine in which patient derived AML cells are fused with autologous dendritic cells (DCs), incorporating antigens that capture the heterogeneity of the leukemia cell population and presenting them in the context of the potent antigen presenting machinery of the DC. Additionally, DC/ AML fusions induce a polyclonal helper and cytotoxic T cell immune response that includes targeting of the leukemia stem cell population.
- DC/ AML fusions induce a polyclonal helper and cytotoxic T cell immune response that includes targeting of the leukemia stem cell population.
- DCs can be obtained from bone marrow cultures, peripheral blood, spleen, or any other appropriate tissue of a mammal using protocols known in the art.
- Bone marrow contains DC progenitors, which, upon treatment with cytokines, such as granulocyte- macrophage colony-stimulating factor (“GM-CSF”) and interleukin 4 ("IL-4"), proliferate and differentiate into DCs.
- cytokines such as granulocyte- macrophage colony-stimulating factor (“GM-CSF) and interleukin 4 (“IL-4"
- TNF Tumor necrosis cell factor
- DCs obtained from bone marrow are relatively immature (as compared to, for instance, spleen DCs).
- GM-CSF/IL-4 stimulated DC express MHC class I and class II molecules, B7-1, B7-2, ICAM, CD40 and variable levels of CD83. These immature DCs are more amenable to fusion (or antigen uptake) than the more mature DCs found in spleen, whereas more mature DCs are relatively more effective antigen presenting cells. Peripheral blood also contains relatively immature DCs or DC progenitors, which can propagate and differentiate in the presence of appropriate cytokines such as GM-CSF and-which can also be used in fusion.
- the DCs are obtained from peripheral blood.
- the DCs are obtained from the patients peripheral blood after it has been documented that the patient is in complete remission.
- the DCs must have sufficient viability prior to fusion.
- the viability of the DCs is at least 70%, at least 75%, at least 80% or greater.
- the population of the DCs Prior to fusion the population of the DCs are free of components used during the production , e.g., cell culture components and substantially free of mycoplasm, endotoxin, and microbial contamination .
- the population of DCs has less than 10, 5, 3, 2, or 1 CFU/swab. Most preferably the population of DCs has 0 CFU/swab.
- the tumor cells used in the invention are acute myelogenous leukemia cells.
- the acute myelogenous leukemia cells are obtained from a patient having acute
- the patient has are newly diagnosed with AML or in their first relapse.
- the tumor cells must have sufficient viability prior to fusion.
- the viability of the tumor cells is at least 50%, at least 60%, at least 70%, at least 80% or greater.
- the population of tumor cells Prior to fusion the population of tumor cells are free of components used during the production , e.g., cell culture components and substantially free of mycoplasm, endotoxin, and microbial contamination .
- the population of tumor cell population has less than 10, 5, 3, 2, or 1 CFU/swab.
- Most preferably the population of tumor cells has 0 CFU/swab.
- the endotoxin level in the population of tumor cells is less than 20 EU/mL, less than 10 EU/mL or less than 5 EU/mL.
- the post-fusion cell mixtures containing the fused as well as the parental cells may optionally be incubated in a medium containing this reagent for a period of time sufficient to eliminate most of the unfused cells.
- the fusion product is used directly after the fusion process (e.g., in antigen discovery screening methods or in therapeutic methods) or after a short culture period.
- Fused cells are irradiated prior to clinical use.
- primary fused cells can be refused with dendritic cells to restore the DC phenotype.
- the refused cells i.e. , secondary fused cells
- the fused cells can be refused with the dendritic or non-dendritic parental cells as many times as desired.
- Fused cells that express MHC class II molecules, B7, or other desired T-cell stimulating molecules can also be selected by panning or fluorescence-activated cell sorting with antibodies against these molecules.
- DCs are autologous or allogeneic.
- the ratio of DCs to tumor cells in fusion can vary from 1 : 100 to 1000: 1, with a ratio higher than 1 : 1 being preferred.
- the ratio is 1 : 1, 5: 1, or 10: 1.
- the ratio of DCs to tumor cells is 10: 1 or 3: 1.
- unfused DCs After fusion, unfused DCs usually die off in a few days in culture, and the fused cells can be separated from the unfused parental non-dendritic cells by the following two methods, both of which yield fused cells of approximately 50% or higher purity, i.e. , the fused cell preparations contain less than 50%, and often less than 30%, unfused cells.
- one method of separating unfused cells from fused cells is based on the different adherence properties between the fused cells and the non-dendritic parental cells. It has been found that the fused cells are generally lightly adherent to tissue culture containers. Thus, if the non-dendritic parental cells are much more adherent, e.g. , in the case of carcinoma cells, the post-fusion cell mixtures can be cultured in an appropriate medium for a short period of time (e.g., 5-10 days). Subsequently, the fused cells can be gently dislodged and aspirated off, while the unfused cells grow firmly attached to the tissue culture containers.
- a short period of time e.g. 5-10 days
- Fused cells obtained by the above-described methods typically retain the phenotypic characteristics of DCs.
- these fused cells express T-cell stimulating molecules such as MHC class II protein, B7-1, B7-2, and adhesion molecules characteristic of APCs such as ICAM-1.
- T-cell stimulating molecules such as MHC class II protein, B7-1, B7-2, and adhesion molecules characteristic of APCs such as ICAM-1.
- the fused cells also continue to express cell-surface antigens of the tumor cells such as MUC-1, CD34, CD 117, and CD38 are therefore useful for inducing immunity against the cell-surface antigens.
- the fused cells lose certain DC characteristics such as expression of the APC-specific T-cell stimulating molecules, they (i.e., primary fused cells) can be re-fused with dendritic cells to restore the DC phenotype.
- the re-fused cells i.e., secondary fused cells
- the fused cells can be re-fused with the dendritic or non-dendritic parental cells as many times as desired.
- DC/AML fusions co-express: a) CD86 and CD117 or CD 34 or MUC-1 or b) CD83 and CD117 or CD34 or MUC-1; c) if the leukemia cells do not express DR, then DR and CD 117 or CD34 or MUC- 1.
- the fused cells may be frozen before administration.
- the fused cells are frozen in a solution containing 10% DMSO in 90% autologous heat inactivated autologous plasma.
- the fused cells of the invention can be used to stimulate the immune system of a mammal for treatment or prophylaxis of acute myeloid leukemia.
- a composition containing fused cells formed by his own DCs and tumor cells can be administered to him, e.g., at a site near the lymphoid tissue.
- the subject is in post chemotherapy induced remission.
- Another criteria for determining the timing of the administration of the fused cell is at a time post-transplant in which there is expansion of AML specific T cells post-chemotherapy as measured by the percentage of CD4 and/or CD8 T cells that express IFNy in response to ex vivo exposure to autologous tumor lysate or the percentage of T cells that bind to tetramers or pentamers expressing AML specific antigens such as WT1, Survivin, NY-ESO, MUC1, and PRAME.
- the vaccine is administered to four different sites near lymphoid tissue.
- the composition may be given multiple times (e.g., two to five, preferably three) at an appropriate intervals, preferably, four weeks and dosage (e.g. , approximately 10 5 -10 8 , e.g. , about 0.5 X 10 6 to 1 X 10 6 , fused cells per administration).
- each dosage contains approximately 1 xlO 6 to 1 x 10 7 fused cells. More preferably each dosage contains approximately 5 x 10 6 fused cells.
- the patient further receives GM-CSF.
- the GM-CSF is administered on the day the fused cells are administered and for daily for three subsequent days.
- the GM-CSF is administered subcutaneously at a dose of 100 ug.
- the GM-CSF is administered at the site where the fused cells are administered.
- the patient further receives a checkpoint inhibitor.
- the check point inhibitor is administered contemporaneously with the fused cell, prior to administration of the fused cells or after administration of the fused cells.
- the checkpoint inhibitor is administered 1 week prior to the fused cells.
- the checkpoint inhibitor is administered 1 week after the fused cells.
- the checkpoint inhibitor is administered at 1, 2, 3, 4, 5, 6 week intervals.
- checkpoint inhibitor it is meant that at the compound inhibits a protein in the checkpoint signally pathway.
- Proteins in the checkpoint signally pathway include for example, PD-1, PD-L1, PD-L2, TIM3, LAG3, and CTLA-4.
- Checkpoint inhibitor are known in the art.
- the checkpoint inhibitor can be a small molecule.
- a "small molecule” as used herein, is meant to refer to a composition that has a molecular weight in the range of less than about 5 kD to 50 daltons, for example less than about 4 kD, less than about 3.5 kD, less than about 3 kD, less than about 2.5 kD, less than about 2 kD, less than about 1.5 kD, less than about 1 kD, less than 750 daltons, less than 500 daltons, less than about 450 daltons, less than about 400 daltons, less than about 350 daltons, less than 300 daltons, less than 250 daltons, less than about 200 daltons, less than about 150 daltons, less than about 100 daltons.
- Small molecules can be, e.g.
- the checkpoint inhibitor is an antibody is an antibody or fragment thereof.
- the antibody or fragment thereof is specific to a protein in the checkpoint signaling pathway, such as PD-1, PD-Ll, PD-L2, TIM3, LAG3, or CTLA-4.
- the checkpoint inhibitor is an antibody specific for PD-1, PD-Ll, PD-L2, TIM3, LAG3, or CTLA-4.
- the patient may receive concurrent treatment with an
- immunomodulatory agent include lenalidomide, pomalinomide or apremilast.
- Lenalidomide has been shown to boost response to vaccination targeting infectious diseases and in pre-clinical studies enhances T cell response to the fusion vaccine.
- the patient may undergo vaccination in combination with strategies to reduce levels of regulatory T cells.
- These strategies may include combining vaccination with chemotherapy, during the period of lymphopoietic reconstitution following autologous or allogeneic transplantation, and with antibodies or drugs that target regulatory T cells.
- cytotoxic T lymphocytes obtained from the treated individual can be tested for their potency against cancer cells in cytotoxic assays. Multiple boosts may be needed to enhance the potency of the cytotoxic T lymphocytes.
- compositions containing the appropriate fused cells are administered to an individual (e.g. , a human) in a regimen determined as appropriate by a person skilled in the art.
- the composition may be given multiple times (e.g. , three to five times, preferably three) at an appropriate interval (e.g. , every four weeks) and dosage (e.g., approximately 10 5 -10 8 , preferably about 1 xlO 6 to 1 x 10 7 , more preferably 5 x 10 6 fused cells per administration).
- composition of fused cells prior to administration to the patient must have sufficient viability.
- the viability of the fused cells at the time of administration is at least 50%, at least 60%, at least 70%, at least 80% or greater.
- 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 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.
- DCs Dendritic cells
- APCs are potent APCs.
- DCs are minor constituents of various immune organs such as spleen, thymus, lymph node, epidermis, and peripheral blood.
- DCs represent merely about 1% of crude spleen (see Steinman et al. (1979) J. Exp. Med 149: 1) or epidermal cell suspensions (see Schuler et al. (1985) J. Exp. Med 161 :526; Romani et al. J. Invest. Dermatol (1989) 93: 600) and 0.1-1% of mononuclear cells in peripheral blood (see Freudenthal et al. Proc. Natl Acad Sci USA (1990) 87: 7698).
- DCs Dendritic cells
- a complex network of antigen presenting cells that are primarily responsible for initiation of primary immunity and the modulation of immune response.
- Partially mature DCs are located at sites of antigen capture, excel at the internalization and processing of exogenous antigens but are poor stimulators of T cell responses. Presentation of antigen by immature DCs may induce T cell tolerance. (See Dhodapkar et al, J Exp Med. 193:233-38 (2001)).
- DCs can be generated by cytokine mediated differentiation of DC progenitors ex vivo. DC maturation and function can be further enhanced by exposure to the toll like receptor 9 agonist, CPG ODN. Moreover, DCs can be manipulated to present tumor antigens potently stimulate anti-tumor immunity. (See Asavaroenhchai et al, Proc Natl Acad Sci USA 99:931-36 (2002); Ashley et al, J Exp Med 186: 1177-82 (1997)).
- “Foster antigen presenting cells” refers to any modified or naturally occurring cells (wild-type or mutant) with antigen presenting capability that are utilized in lieu of antigen presenting cells (“APC”) that normally contact the immune effector cells they are to react with. In other words, they are any functional APCs that T cells would not normally encounter in vivo.
- APC antigen presenting cells
- 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 costimulatory signals, are 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.
- cytokine refers to any of the numerous factors that exert a variety of effects on cells, for example, inducing growth or proliferation.
- Non-limiting examples of cytokines include, IL-2, stem cell factor (SCF), IL-3, IL-6, IL-7, IL-12, IL-15, G-CSF, GM-CSF, IL-1 a, IL-1 ⁇ , MIP-1 a, LIF, c-kit ligand, TPO, and flt3 ligand.
- SCF stem cell factor
- IL-6 IL-6
- IL-7 IL-12
- IL-15 G-CSF
- GM-CSF GM-CSF
- IL-1 a IL-1 ⁇
- MIP-1 a LIF
- c-kit ligand TPO
- flt3 ligand flt3 ligand
- cytokines e.g., recombinantly produced cytokines
- Costimulatory molecules are involved in the interaction between receptor- ligand pairs expressed on the surface of antigen presenting cells and T cells.
- One exemplary receptor-ligand pair is the B7 co-stimulatory molecules on the surface of DCs and its counter-receptor CD28 or CTLA-4 on T cells.
- Other important costimulatory molecules include, for example, CD40, CD54, CD80, and CD86. These are commercially available from vendors identified above.
- hybrid cell refers to a cell having both antigen presenting capability and also expresses one or more specific antigens.
- these hybrid cells are formed by fusing, in vitro, APCs with cells that are known to express the one or more antigens of interest.
- hybrid cell and fusion cell are used interchangeably.
- the term "culturing” refers to the in vitro propagation of cells or organisms on or in media of various kinds, it is understood that the descendants 30 of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or
- 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.
- an effective amount of hybrid cells is that amount which promotes expansion of the antigenic-specific immune effector cells, e.g. , T cells.
- An “isolated” population of cells is “substantially free” of cells and materials with which it is associated in nature. By “substantially free” or “substantially pure” is meant at least 50% of the population is the desired cell type, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90%.
- An “enriched” population of cells is at least 5% fused cells. Preferably, the enriched population contains at least 10%, more preferably at least 20%, and most preferably at least 25% fused cells.
- autogeneic indicates the origin of a cell.
- a cell being administered to an individual is autogeneic if the cell was derived from that individual (the "donor") or a genetically identical individual (i.e., an identical twin of the individual).
- An autogeneic cell can also be a progeny of an autogeneic cell.
- the term also indicates that cells of different cell types are derived from the same donor or genetically identical donors.
- an effector cell and an antigen presenting cell are said to be autogeneic if they were derived from the same donor or from an individual genetically identical to the donor, or if they are progeny of cells derived from the same donor or from an individual genetically identical to the donor.
- allogeneic indicates the origin of a cell.
- a cell being administered to an individual is allogeneic if the cell was derived from an individual not genetically identical to the recipient.
- the term relates to non-identity in expressed MHC molecules.
- An allogeneic cell can also be a progeny of an allogeneic cell.
- the term also indicates that cells of different cell types are derived from genetically nonidentical donors, or if they are progeny of cells derived from genetically non-identical donors.
- an APC is said to be allogeneic to an effector cell if they are derived from genetically non-identical donors.
- 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.
- genetic modification refers to any addition, deletion or disruption to a cell's endogenous nucleotides.
- a "viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
- viral vectors include retroviral vectors, adenovirus vectors, adeno- associated virus vectors and the like.
- a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene.
- Retroviruses carry their genetic information in the form of RNA. However, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form that integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.
- a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a therapeutic gene.
- Ads are a relatively well characterized, homogenous group of viruses, including over 50 serotypes.
- Ads are easy to grow and do not integrate into the host cell genome.
- Recombinant Ad-derived vectors particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed.
- Wild-type AAV has high infectivity and specificity integrating into the host cells genome.
- polynucleotide can be operatively linked are well known in the art.
- Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI).
- Stratagene La Jolla, CA
- Promega Biotech Promega Biotech
- consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression.
- Suitable vectors are viruses, such as baculovirus 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 eucaryotie and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
- viruses such as baculovirus 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 eucaryotie and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
- Non-viral vectors including DNA/liposome complexes, and targeted viral protein DNA complexes.
- the nucleic acid or proteins of this invention can be conjugated to antibodies or binding fragments thereof which bind cell surface antigens, e.g. , TCR, CD3 or CD4.
- Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention.
- This invention also provides the targeting complexes for use in the methods disclosed herein.
- 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 transfectants 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 ColEI for proper episomal replication; versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA.
- a selectable marker gene such as the neomycin gene for selection of stable or transient transfectants 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 transcription termination and RNA
- expression refers to the process by which polynucleotides are transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA, if an appropriate eukaryotic host is selected. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding.
- a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG (Sambrook et al. (1989), supra).
- a eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome.
- RNA polymerase II a heterologous or homologous promoter for RNA polymerase II
- downstream polyadenylation signal a downstream polyadenylation signal
- start codon AUG the start codon AUG
- termination codon for detachment of the ribosome.
- 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 a chain encoded in the MHC associated noncovalently with p2-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 II MHC molecules also include membrane heterodimeric proteins consisting of noncovalently associated and J3 chains.
- Class II MHCs are known to function in CD4+ T cells and, in humans, include HLA-DP, -DQ, and DR.
- 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 in association with either a class I or class II MHC molecule. Methods of identifying and comparing MHC are well known in the art and are described in Allen M. et al. (1994) Human Imm. 40:25-32; Santamaria P. et al. (1993) Human Imm. 37:39-50; and Hurley C.K. et al. (1997) Tissue Antigens 50:401-415.
- sequence motif refers to a pattern present in a group of 15 molecules (e.g. , amino acids or nucleotides).
- the present invention provides for identification of a sequence motif among peptides present in an antigen.
- a typical partem may be identified by characteristic amino acid residues, such as hydrophobic, hydrophilic, basic, acidic, and the like.
- peptide is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics.
- the subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g. ester, ether, etc.
- amino acid refers to either natural and/or 25 unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
- a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.
- solid phase support is used as an example of a “carrier” and is not limited to a specific type of support. Rather a large number of supports are available and are known to one of ordinary skill in the art.
- Solid phase supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels.
- a suitable solid phase support may be selected on the basis of desired end use and suitability for various synthetic protocols. For example, for peptide synthesis, solid phase support may refer to resins such as polystyrene (e.g.
- solid phase support refers to polydimethylacrylamide resin.
- abnormally expressed refers to polynucleotide sequences in a cell or tissue which are differentially expressed (either over-expressed or under-expressed) when compared to a different cell or tissue whether or not of the same tissue type, i.e. , lung tissue versus lung cancer tissue.
- “Host cell” or “recipient 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. 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 prokaryotic or eukaryotic, and include but are not limited to bacterial cells, yeast cells, animal cells, and mammalian cells, e.g. , murine, rat, simian or human.
- 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.
- an "antibody complex” is the combination of antibody 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.
- isolated means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature. As is apparent to those of skill in the art, a non-natural occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart.
- a "concentrated”, “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than “concentrated” or less than “separated” than that of its naturally occurring counterpart.
- composition is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent, carrier, solid support or label) or active, such as an adjuvant.
- a "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)).
- 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.
- 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 H-thymidine uptake assays.
- FACS fluorescence-activated cell sorting
- substantially free for mycoplasma and microbial contamination is meant as negative readings for the generally accepted tests know to those skilled in the art.
- mycoplasm contamination is determined by subculturing a cell sample in broth medium and distributed over agar plates on day 1, 3, 7, and 14 at 37°C with appropriate positive and negative controls. The product sample appearance is compared
- the sterility test to establish that the product is free of microbial contamination is based on the U.S. Pharmacopedia Direct Transfer Method. This procedure requires that a pre-harvest medium effluent and a pre-concentrated sample be inoculated into a tube containing tryptic soy broth media and fluid thiogly collate media. These tubes are observed periodically for a cloudy appearance (turbidity) for a 14 day incubation. A cloudy appearance on any day in either medium indicate contamination, with a clear appearance (no growth) testing substantially free of contamination.
- EXAMPLE 1 CLINICAL STUDY DESIGN TO ACCESS VACCINATION OF PATIENTS WITH ACUTE MYELOID LEUKEMIA WITH DENDRITIC CELL TUMOR FUSIONS
- First stage Patients will receive DC/AML fusion vaccination in conjunction with GM-CSF following a chemotherapy induced remission.
- Secondary objective To explore immunological response to DC/AML fusion vaccination in patients who have achieved a chemotherapy induced remission.
- Second stage To determine if cellular immunity is induced by treatment with monoclonal antibody PD-1 BLOCKADE and DC/AML fusion cells given in conjunction with GM-CSF following a chemotherapy induced remission.
- Participants must meet the following criteria on screening examination to be eligible to participate in the study: 1. Patients with AML at initial diagnosis or at first relapse; 2. Patients must be > 18 years old; 3. Patients must have ECOG performance status of 0-2; 4. Life expectancy of greater than 9 weeks; 5. Laboratories: Bilirubin ⁇ 2.0 mg/dL; AST/ALT ⁇ 3 x ULN; Creatinine ⁇ 2.0 mg/dl; 6. The effects of DC/AML fusion cells and PD-1 BLOCKADE on the developing human fetus are unknown. For this reason, women of child-bearing potential and men must agree to use adequate contraception (hormonal or barrier method of birth control; abstinence) prior to study entry and for the duration of study participation. Should a woman become pregnant or suspect she is pregnant while participating in this study, she should inform her treating physician immediately; 7. Ability to understand and the willingness to sign a written informed consent document
- Participants who exhibit any of the following conditions at screening will not be eligible for admission into the study.1. Patients must not have active or history of clinically significant autoimmune disorders/conditions, defined as requiring systemic therapy, including Type I diabetes. Type II diabetes, vitiligo, stable hypothyroidism, and thyroid disease well controlled with thyroid replacement will not be considered exclusion criteria; 2. Because of compromised cellular immunity, patients who have a known history of HIV will be excluded; 3. Patients must not have significant cardiac disease characterized by symptomatic congestive heart failure, unstable angina pectoris, clinically significant cardiac arrhythmia; 4. Patients must not be pregnant. All premenopausal patients will undergo pregnancy testing. Men will agree to not father a child while on protocol treatment.
- leukemia therapy will be administered according to current standards of practice at the discretion of the treating physician. Patients are permitted to receive hydroxyurea prior to initiation of tumor cell collection if clinically indicated. Patients who achieve remission status as defined by absence of circulating blasts, and less than 5% blasts on bone marrow exam following hematopoietic recovery will proceed with immunotherapy. When patients are in remission, leukemic blasts will be thawed, cultured and viability as well as gram stain will be assessed. An aliquot from this sample will undergo microbiological assessment.
- PBMC will be isolated from the leukapheresis product and cultured in the presence of autologous plasma for 1-2 hours.
- the adherent fraction will be cultured in the presence of 1% autologous plasma/RPMI medium with 12.5 ng/ml rhIL-4 and 1000 U/ml GM-CSF for five to seven days to generate dendritic cells.
- 25 ng/ml of TNFa will be then be added for 48-72 hours to enhance DC maturation. Viability and gram stain will be assessed prior to fusion.
- DC will be assessed for morphologic characteristics and expression of characteristic DC markers that include CDl lc, HLA DR, CD80, CD86, and CD83.
- characteristic DC markers that include CDl lc, HLA DR, CD80, CD86, and CD83.
- CDl 17 and MUC-1 will be determined.
- Functional properties will be assessed using MLR assays in which DC will be co-cultured with allogeneic T cells. T cell proliferation will be measured via tritiated thymidine incorporation
- Vaccine preparation may occur upon completion of induction/re-induction therapy, or upon completion of up to 4 cycles of post-remission chemotherapy.
- Samples will be frozen as outlined below and thawed at the time of vaccine administration.
- Tumor cells and DC at ratio of 1 : 10-1 : 3 (dependent on cell yields) will be mixed and extensively washed in serum-free medium (RPMI1640). After low speed centrifugation, the cell pellets will be re-suspended in 500 ⁇ of 50% solution of polyethylene glycol (PEG) in Dulbecco's phosphate buffered saline without Ca ++ , Mg ++ .
- PEG polyethylene glycol
- the PEG will be progressively diluted by the slow addition of serum-free medium.
- the cells will be washed free of PEG and cultured in RPMI 1640 with 10% autologous plasma and GM-CSF in a 5% CO2 atmosphere at 37° C.
- the percentage of the cell population that represent DC/tumor fusions will be determined by quantifying the cells as defined by dual expression of unique DC and leukemia markers such as: a) CD86 and CDl 17 or CD 34 or MUC-1 or b) CD83 and CDl 17 or CD34 or MUC-1; c) if the leukemia cells do not express DR, then DR and CDl 17 or CD34 or MUC-1 as measured by
- FACS analysis Dosing will be determined by the absolute number of fusion cells identified in this manner. Fusion cells must demonstrate >10% fusion efficiency to proceed with vaccination. The fusion cells will then be separated into appropriate aliquots of fusion cells. 2-3 doses of 5 xlO 6 fusion cells, depending on cell yields, will be prepared and will be frozen in 10% DMSO/90% autologous plasma in liquid nitrogen. At that time, appropriate aliquots will be sent for microbiological analysis.
- EXAMPLE 6 PATIENT MONITORING
- Bone marrow aspirate and biopsy may be done within 30 days of initiation of [000135] Immunotherapy
- LFTs including; ALT, AST, total bilirubin, direct bilirubin**, LDH, Alkaline
- EXAMPLE 7 VACCINATION SCHEDULE
- Cohort 1 Patients will be vaccinated with 5x10 6 fusion cells in the upper thigh region. The site will be alternated for each vaccine administration (right and left extremity). Vaccination will be administered subcutaneously using a 25-gauge 5/8-inch needle. On the day of vaccination, the clinical research nurse/physician assistant will administer 100 ug of GM-CSF subcutaneously at the site of the vaccine. The patient will be trained to inject the remaining three GM-CSF injections (lOOug dose once a day) for self-administration subcutaneously at home. Tumor vaccine will be administered first, followed by GM-CSF injection.
- GM-CSF may be held until the vaccine site reaction improves to grade 1 or less.
- patients will receive pre-medication with diphenhydramine (Benadryl) 25-50 mg and/or acetaminophen (Tylenol) 650 mg to minimize potential allergic related symptoms.
- a total of 3 vaccinations will be given at monthly intervals as outlined in the study schema. When cell yields allow for only two doses of vaccine to be generated, patients will receive two doses of vaccine at monthly intervals.
- Cohort 2 Patients will receive 3 doses of PD-1 BLOCKADE given at 6 week intervals.
- PD-1 BLOCKADE Patients will receive acetaminophen 500-1000 mg orally and anti-histamine (for eg. Diphenhydramine 25-50mg) intravenously 20-60 minutes prior to PD-1 BLOCKADE infusion.
- anti-histamine for eg. Diphenhydramine 25-50mg
- Blood pressure, heart rate, and temperature will be measured after the administration of anti-histamine, and before the initiation of PD-1 BLOCKADE infusion. Vitals signs will be reviewed prior to administration of the study drug.
- PD-1 BLOCKADE will be infused over approximately 2 hours, in cases where infusion rate is slowed due to an infusion related reaction, the overall infusion time should not exceed 10 hours.
- EXAMPLE 8 FOLLOW-UP PERIOD
- CBC with differential liver function tests (including; ALT, AST, total bilirubin, direct bilirubin*, LDH, Alkaline Phosphatase),electrolytes(Na, K, CI, CO2, Ca, Mg, P04) ,BU , creatinine; Direct bilirubin only required if Total bilirubin is not within normal limits
- cytogenetics/FISH will be assessed
- Mononuclear cells were isolated by Ficoll density gradient centrifugation and cryopreserved in 10% DMSO/90% autologous plasma. Patients eligible for vaccine generation underwent a single leukapheresis collection for DC generation and vaccine production. Adherent cells were cultured with 1000 U/mL GM-CSF (Sanofi) and 500 IU/ml IL-4 (Cellgenix USA, Antioch, IL) for 5-7 days and matured in the presence of 25 ng/ml TNFa (Cellgenix) for 2-3 days. The DC and thawed autologous AML preparations were analyzed by
- DC/AML fusions were (i) quantified by determining the percentage of cells that co-express unique DC (CD80, CD83 or CD86) and tumor-associated antigens (CD34, CD38, CD64, CD117, or MUC1) by
- T cells obtained from leukopak collections were co- cultured with either DC/AML fusion cells, DCs, or AML blasts at a ratio of 10: 1 for 5 days.
- T cell proliferation was determined by measuring incorporation of [3H] thymidine following overnight pulsing ( ⁇ ) of triplicate samples.
- PBMC samples were thawed and 1 x 106 cells were cultured with lysate generated by repeated freeze thaw cycles of 1 x 105 autologous leukemia cells for 5 days. Cells were re-stimulated with autologous tumor lysate for 6 hours and cultured overnight with 1 ⁇ g/ml GolgiStop.
- Intracellular expression of IFNy by CD4+ or CD8+ T cells was determined by FACS analysis of permeabilized cells.
- Leukemia reactive T cells were also quantified in the bone marrow prior to and 1 month following completion of vaccination in a subset of patients.
- the number of circulating CD8+ T cells binding the MUC1, WT1, PR1 pentamers were determined by bidimensional FACS analysis using CD8-FITC and the corresponding pentamer-PE antibody.
- Levels of regulatory T cells were quantified by determining the expression of FOXP3 by CD4/CD25 cells using intracellular FACS analysis. PD-1 expression on circulating CD4 and CD8 T cell populations was assessed by flow cytometry. In a subset of patients, vaccine site reactions underwent biopsy and immunocytochemical staining to assess infiltration of CD4, CD8 T cells. Recruitment of native DCs was assessed by CDla expression in the vaccine site.
- the mean yield of the DC and AML preparations was 171 and 87 x 10 6 cells, respectively.
- the mean fusion efficiency as determined by the percentage of cells that co- expressed unique DC (CD80, CD86, and CD83) and AML (CD38, CD34, CD117, CD64, or MUC1) antigens was 43%.
- the mean viability of the DC, AML, and fusion preparations was 91%, 91%, and 86%, respectively.
- the DC and fusion preparations potently stimulated allogeneic T cell proliferation (mean stimulation indices 3.6, 15.7, and 10.9, respectively). 14/16 patients received 3 doses while 2 patients received 2 doses, 1 due to limitations of cell yields and 1 due to disease relapse prior to the third vaccine.
- Vaccination was well tolerated and not associated with clinically significant autoimmunity. Possibly related adverse events were transient and of grade 1-2 intensity (Table 3). The most common adverse event was erythema, pruritis and/or induration at the vaccine site. Biopsy of vaccine site reactions demonstrated a dense infiltrate of CD4 and CD8 T cells consistent with recruitment of reactive T cell populations to the vaccine bed ( Figure 1). [000190] Cellular Immunologic Response to Vaccination
- Examples presented herein demonstrate the striking efficacy of this vaccine in AML patients who achieve a remission following chemotherapy. Twelve of 16 patients (75%) remain in remission with a median of nearly 4 years of follow up and no patients have relapsed after 1 year following chemotherapy. Patients remaining in remission include several over age 70. Remarkably, one patient who relapsed within 1 year of initial chemotherapy underwent vaccination after achieving a second chemotherapy -induced remission and remains in remission over 4 years after completing chemotherapy. The long term remission observed following chemotherapy and vaccination of a patient who had experienced early relapse following initial induction therapy is distinctly unusual in the absence of allogeneic transplantation.
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