WO2021087234A1 - Reprogrammation de lignée en tant qu'immunothérapie anticancéreuse - Google Patents

Reprogrammation de lignée en tant qu'immunothérapie anticancéreuse Download PDF

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WO2021087234A1
WO2021087234A1 PCT/US2020/058170 US2020058170W WO2021087234A1 WO 2021087234 A1 WO2021087234 A1 WO 2021087234A1 US 2020058170 W US2020058170 W US 2020058170W WO 2021087234 A1 WO2021087234 A1 WO 2021087234A1
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cells
cancer cells
cancer
reprogrammed
myeloid
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Ravindra Majeti
Miles Hamilton LINDE
James S. MCCLELLAN
Christopher George DOVE
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The Board Of Trustees Of The Leland Stanford Junior University
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    • A—HUMAN NECESSITIES
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Definitions

  • Cancer immunotherapy comprises a variety of treatment approaches, incorporating the specificity of the adaptive immune system, as well as features of the innate immune system.
  • Immunotherapy strategies include, for example, antitumor monoclonal antibodies, cancer vaccines, adoptive transfer of ex vivo activated T and natural killer cells, administration of antibodies or recombinant proteins that co-stimulate immune cells or block immune inhibitory pathways, and blockade of phagocytic pathway inhibitors.
  • the success of immuno-oncology approaches is based on scientific advances relating to the tolerogenic nature of cancer and the role of the tumor immune microenvironment in suppressing antitumor immunity.
  • New therapies are designed to break tolerance and reactivate antitumor immunity to induce potent, long-lasting responses.
  • Vaccination strategies utilizing tumor cells include, for example, GVAX, whereby whole tumor cells are genetically modified to secrete GM-CSF, irradiated, and then re-administered to elicit dendritic cell and T cell activation, which can be administered in combination with anti-PD-1 for pancreatic cancer.
  • Some cancer vaccines have relied on the isolation of monocytes of dendritic cell precursors from a tumor-bearing patient, maturation of these cells into dendritic cells, loading of the dendritic cells with tumor-specific or tumor-associated antigens, and re-administration of the antigen-loaded dendritic cells to the patient.
  • the dendritic cell vaccine Provenge (Sipileucel-T)
  • Provenge is approved for use in treating patients with advanced hormone-resistant prostate cancer.
  • This vaccine is patient-specific, produced by transiently incubating the patient's own peripheral blood mononuclear cells with a fusion protein consisting of prostatic acid phosphatase linked to granulocyte macrophage colony-stimulating factor (GM-CSF).
  • GM-CSF granulocyte macrophage colony-stimulating factor
  • Other cancer vaccines in clinical trials have used peptides derived from tumor antigens to stimulate the immune response.
  • compositions and methods are provided for vaccination with cancer cells.
  • the cancer cells are treated by direct reprogramming to generate antigen presenting cells, which can present tumor antigens and enhance immune responsiveness to the cancer.
  • the direct reprogramming utilizes genetic modification to force expression of transcription factors that drive cells into the myeloid lineage.
  • the transcription factors are selected from one or more of C/EBPoc, PU.1 , BATF3 and IRF8.
  • the transcription factors comprise or consist of one or both of C/EBPoc and PU.1.
  • cancer cells from both hematologic cancers such as leukemias and lymphomas
  • non-hematologic cancers such as carcinomas, fibrosarcomas, osteosarcomas, etc.
  • the methods can be applied to cancer cell lines, and to primary cancer cells, including patient-derived cancer cells.
  • Methods of direct reprogramming of cancer cells utilize introduction of genetic construct(s) encoding myeloid lineage transcription factors operably linked to a promoter that is active or can be activated in the cancer cell; or mRNA coding sequences.
  • Reprogramming can be performed ex vivo, e.g. with patient-derived cancer cells, or in vivo.
  • Direct in vivo reprogramming of cancer cells in situ can utilize delivery of reprogramming factors by viral vectors, mRNA, etc., for example by introducing the reprogramming factors at the site of the tumor.
  • the myeloid lineage transcription factors may comprise or consist of one or both of C/EBPoc and PU.1.
  • the cancer cells are transduced with genes encoding both C/EBPa and PU.1.
  • the genetic construct is integrated into the cancer cell genome.
  • the construct is a viral vector, e.g. a retroviral or lentiviral vector.
  • the promoter may be constitutive or inducible, e.g. a tet-inducible promoter.
  • the cancer cell genome is edited, e.g. by CRISPR technology, to operably link the transcription factors to a constitutive or inducible promoter.
  • the genetically modified cancer cells if cultured ex vivo, can be cultured in the presence of cytokines that enhance myeloid differentiation.
  • Cytokine culture conditions include, without limitation, one or more of Flt3-L, IL-7, IL-3, GM-CSF, m-CSF and IL-4. Combinations of cytokines may be provided.
  • the cancer cells are found to express cell-surface markers characteristic of myeloid, antigen-presenting cells.
  • markers include, without limitation, CD11b, CD14, SIRPa, Ly6c and CD115.
  • the reprogrammed cancer cells are CD11 b + and CD14 + .
  • the reprogrammed cells are selected for expression of one or both of CD11b and CD14 prior to use as a vaccine, e.g. by flow cytometry or magnetic selection methods.
  • T cells usually autologous T cells
  • T cells are stimulated ex vivo by contact with the reprogrammed cancer cells, and the T cells thus activated are re-introduced into the patient.
  • a therapeutic method comprising introducing into a recipient in need thereof, e.g. a cancer patient, an effective dose of an reprogrammed cancer cell population, wherein the cell population comprises cancer cells that have been genetically modified to force expression of transcription factors that drive cells into the myeloid lineage.
  • the transcription factors are one or both of C/EBPa and PU.1.
  • the initial cell population may be reprogrammed ex vivo, and is usually autologous or allogeneic with respect to the recipient.
  • the starting cell population may be derived from the patient to be treated.
  • the reprogrammed cancer cell population may be selected prior to administration for expression of one or both of CD11b and CD14.
  • the dose of reprogrammed cancer cells is effective in increasing immune responses against the cancer.
  • Figure 1 Ectopic expression of CEBPa and PU.1 reprograms hematologic and solid tumor cell lines into myeloid lineage cells
  • Figure 2 Ectopic expression of CEBPa and PU.1 reprograms hematologic cell lines into myeloid lineage cells.
  • Figure 3 Ectopic expression of CEBPa and PU.1 reprograms hematologic cell lines into myeloid lineage cells.
  • Figure 4 Ectopic expression of CEBPa and PU.1 reprograms hematologic cell lines into myeloid lineage cells.
  • FIG. 5 Myeloid-reprogrammed tumor cells become efficient antigen presenting cells capable of stimulating T cell mediated immunity.
  • FIG. 6 Myeloid-reprogrammed tumor cells become efficient antigen presenting cells capable of stimulating T cell mediated immunity.
  • Figure 7 Myeloid-reprogramming of solid tumor significantly extends media life expectancy and attenuates tumor growth.
  • Figure 8 Myeloid-reprogramming of solid tumor significantly extends media life expectancy and attenuates tumor growth.
  • antigen presenting cells have been directly generated from cancer cells.
  • myeloid-differentiation promoting cytokines or the ectopic expression of myeloid lineage-defining transcription factors (C/EBPa and PU.1) cancer cells, including without limitation precursor B cell acute lymphoblastic leukemia, osteosarcoma, fibrosarcoma, and mammary carcinoma cells have been reprogrammed into myeloid lineage antigen presenting cells (APCs) that are no longer tumorigenic.
  • APCs myeloid lineage antigen presenting cells
  • cancer-derived APCs exhibit myeloid characteristics, including the expression of antigen presentation and co stimulatory molecules, phagocytic activity, and most importantly, lead to the in vivo development of tumor-eradicating immune responses. Since these reprogrammed cells are derived from cancer cells, they retain all genetic abnormalities present in the cell of origin, eliminating the need for identification or loading of exogenous tumor antigens for immune stimulation. These findings demonstrate that tumor cell-derived APCs generated via myeloid transdifferentiation provide a cancer vaccination approach with broad utility across many cancer types.
  • cancer vaccination has relied on the isolation of monocytes or dendritic cell precursors from a tumor-bearing patient, maturation of these cells into dendritic cells, loading of the dendritic cells with tumor-specific or tumor-associated antigens, and re-administration of the antigen-loaded dendritic cells to the patient.
  • Direct reprogramming of tumor cells into antigen presenting cells for the purpose of cancer vaccination as described herein offers benefits relative to the previous methodologies. In particular, identification of tumor-specific or tumor-associated antigens is not required for the methods described herein.
  • the reprogrammed cells are derived from the initial tumor, they share genetic and cytogenetic abnormalities present in the bulk tumor burden and are capable of eliciting immune responses directed against multiple tumor antigens without prior identification of the antigens. Further, because the transdifferentiation process is mediated by defined factors, the culture and activation conditions are straightforward. Finally, dendritic cell vaccinations have been thought to be limited in efficacy to tumor types with a high mutational burden, primarily due to the limitations of antigen identification, processing, and presentation during the development of the vaccine. Flowever, myeloid reprogramming has shown remarkable efficacy in a preclinical model of B cell acute lymphoblastic leukemia, a cancer type with low mutational frequency. Thus, vaccination immunotherapy can be efficacious for even low mutation cancers.
  • Vaccine refers to a composition including at least one antigen, and preferably more than one antigen, that is administered to a patient to create an immune response and preferably T cell response to the antigen(s) delivered.
  • the vaccine can include various other components such as a carrier or other stimulatory molecules.
  • the vaccines of the present invention are cell-based, where a reprogrammed cancer cell provide stimulus for an immune response targeted to antigens present on the cancer cells.
  • Tumor cells taken from a patient or from allogeneic cell lines are used as the immunogen. Unmodified tumor cells are poorly immunogenic, but the immunogenicity is greatly enhanced by the reprogramming methods described herein. With tumor cell vaccines, many normal cellular antigens and tumor antigens are presented to the immune system. A strength of the approach is that any tumor-specific mutated antigens are presented to the immune system, and both CD4 and CD8 epitopes are presented to the immune system.
  • Cell-based cancer vaccines can be combined with additional anti-cancer therapies, including without limitation chemotherapy, radiation therapy, surgery, targeted therapy and immuno-oncology therapy.
  • additional anti-cancer therapies including without limitation chemotherapy, radiation therapy, surgery, targeted therapy and immuno-oncology therapy.
  • the administration of the vaccine is combined with other biological anti-cancer drugs such as checkpoint inhibitors (e.g. PD1 or PDL1 inhibitors, CD47/SIRPoc blockade, anti-CTLA4, etc) or therapeutic monoclonal antibodies (e.g Avastin, Herceptin).
  • checkpoint inhibitors e.g. PD1 or PDL1 inhibitors, CD47/SIRPoc blockade, anti-CTLA4, etc
  • therapeutic monoclonal antibodies e.g Avastin, Herceptin.
  • the terms “recipient”, “individual”, “subject”, “host”, and “patient”, are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • "Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In some embodiments the mammal is human.
  • a "therapeutically effective amount” refers to that amount of the vaccine, e.g. reprogrammed cells, sufficient to prevent, treat or manage a disease or disorder.
  • a therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the progression of disease, e.g., delay or minimize the spread of cancer, or to reduce tumor size, tumor cell burden, etc.
  • a therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.
  • a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.
  • the term "survival” refers to a length of time following the diagnosis of a disease and/or beginning or completing a particular course of therapy for a disease (e.g., cancer).
  • the term “overall survival” includes the clinical endpoint describing patients who are alive for a defined period of time after being diagnosed with or treated for a disease such as cancer.
  • the term “disease-free survival” includes the length of time after treatment for a specific disease (e.g., cancer) during which a patient survives with no sign of the disease (e.g., without known recurrence).
  • disease-free survival is a clinical parameter used to evaluate the efficacy of a particular therapy, which is usually measured in units of 1 or 5 years.
  • progression-free survival includes the length of time during and after treatment for a specific disease (e.g., cancer) in which a patient is living with the disease without additional symptoms of the disease. In some embodiments, survival is expressed as a median or mean value.
  • the term "in combination” refers to the use of more than one therapeutic agent.
  • the use of the term “in combination” does not restrict the order in which therapeutic agents are administered to a subject with a disorder.
  • a first therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent to a subject.
  • biopsy refers to the process of removing a tissue sample for diagnostic or prognostic evaluation, and to the tissue specimen itself. Any biopsy technique known in the art can be applied to the methods and compositions of the present invention. The biopsy technique applied will generally depend on the tissue type to be evaluated and the size and type of the tumor (i.e., solid or suspended (i.e., blood, thoracentesis aspirate, or ascites)), among other factors. Representative biopsy techniques include excisional biopsy, incisional biopsy, needle biopsy (e.g., core needle biopsy, fine-needle aspiration biopsy, etc.), surgical biopsy, and bone marrow biopsy.
  • Biopsy techniques are discussed, for example, in Harrison's Principles of internal Medicine, Kasper, et al., eds., 16th ed., 2005, Chapter 70, and throughout Part V.
  • biopsy techniques can be performed to identify cancerous and/or precancerous cells in a given tissue sample.
  • a cancer biopsy is of particular interest, where the cancer cells are maintained in culture for reprogramming.
  • An antigen-presenting cell (APC) or accessory cell is a cell that displays antigen complexed with major histocompatibility complexes (MHCs) on the cell surfaces; this process is known as antigen presentation.
  • MHCs major histocompatibility complexes
  • T cells can recognize these complexes using their T cell receptors. While almost all cell types can present antigens in some way, the co-stimulatory molecules and MHC antigens present on professional antigen-presenting cells, including macrophages, B cells and dendritic cells, present foreign antigens to both helper T cells, including naive T cells, as well as cytotoxic T cells; and provide for an enhanced response.
  • Characteristics of professional APCs include phagocytosis or receptor-mediated endocytosis of antigen, processing the antigen into peptide fragments and then displaying those peptides, bound to a class II MHC molecule, on their membrane.
  • the T cell recognizes and interacts with the antigen-class II MHC molecule complex on the membrane of the antigen- presenting cell.
  • An additional co-stimulatory signal can be produced by the antigen-presenting cell, leading to activation of the T cell.
  • the expression of co-stimulatory molecules and MHC class II are defining features of professional APCs.
  • PU.1 Transcription factor PU.1 is encoded by the SPI1 gene.
  • This gene encodes an ETS- domain transcription factor that activates gene expression during myeloid and B-lymphoid cell development.
  • the nuclear protein binds to a purine-rich sequence known as the PU-box found on enhancers of target genes, and regulates their expression in coordination with other transcription factors and cofactors. The protein can also regulate alternative splicing of target genes. Multiple transcript variants encoding different isoforms have been found for this gene.
  • the ETS domain is the DNA-binding module of PU.1 and other ETS-family transcription factors.
  • SPI1 has been shown to interact with FUS, GATA2, IRF4 and NONO.
  • the reference sequence for the human PU.1 protein and gene may be found at Genbank, NP 003111 ; and NM 003120, respectively.
  • C/EBPoc CCAAT-enhancer-binding proteins (orC/EBPs) is a family of transcription factors composed of six members, named from C/EBRa to C/EBRz.
  • CCAAT/enhancer-binding protein alpha is involved in the differentiation of myeloid cells.
  • the protein encoded by this intronless gene is a bZIP transcription factor which can bind as a homodimer to certain promoters and gene enhancers. It can also form heterodimers with the related proteins CEBP-beta and CEBP-gamma, as well as distinct transcription factors such as c-Jun.
  • CEBPA is essential for myeloid lineage commitment and therefore required both for normal mature granulocyte formation and for the development of myeloid leukemia.
  • the reference sequence for the human C/EBPoc protein and gene may be found at Genbank, NP 004355.
  • polypeptide refers to any chain of amino acid residues, regardless of its length or post-translational modification (e.g., glycosylation or phosphorylation).
  • identity refers to the sequence identity between two molecules.
  • the similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. In general, the sequences are aligned so that the highest order match is obtained. If necessary, identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof.
  • myeloid reprogramming factors are ectopically expressed in cancer cells by introducing an expression vector into the cells, or by genome editing the cells to operably link the transcription factors to a promoter of interest.
  • the transcription factor coding sequences may be introduced on an expression vector into the cell to be reprogrammed.
  • DNA encoding the transcription factors may be obtained from various sources as desired during the engineering process.
  • the nucleic acid encoding the transcription factors is inserted into a replicable vector for expression.
  • the vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • Vectors include viral vectors, plasmid vectors, integrating vectors, and the like. Lentiviral vectors are of particular interest. Other viral vectors of interest include adenovirus vectors, AAV vectors, and the like.
  • Expression vectors usually contain a selection gene, also termed a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media.
  • Expression vectors will contain a promoter that is recognized by the host organism and is operably linked to the transcription factor coding sequences. Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of particular nucleic acid sequence to which they are operably linked. Such promoters typically fall into two classes, inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature. A large number of promoters recognized by a variety of potential host cells are well known.
  • Transcription from vectors in mammalian host cells may be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus (such as murine stem cell virus), hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter, PGK (phosphoglycerate kinase), or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus,
  • each of the genes encoding myeloid lineage transcription factors are operably linked to a conditional promoter.
  • the promoter is a tet promoter, e.g. comprising a tet responsive element (TRE).
  • TRE tet responsive element
  • the conditional expression is driven by the presence of tetracycline or an analog thereof, e.g. doxycycline.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, which act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent, having been found 5' and 3' to the transcription unit, within an intron, as well as within the coding sequence itself. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, oc-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus.
  • Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the expression vector at a position 5' or 3' to the coding sequence, but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. Construction of suitable vectors containing one or more of the above-listed components employs standard techniques.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
  • useful mammalian host cell lines are mouse L cells (L-M[TK-], ATCC#CRL-2648), monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO); mouse sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver
  • Cancer cells can be transfected or transduced with the above-described expression vectors.
  • Cells may be cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Mammalian cells may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the reprogrammed cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • Nucleic acids are "operably linked" when placed into a functional relationship with another nucleic acid sequence.
  • DNA for a signal sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous. However, enhancers do not have to be contiguous.
  • the coding sequences of interest are editing in the cancer cell genome, using a sequence specific endonuclease for genome editing.
  • the sequence specific endonuclease is used to create a double-stranded break at a specific site in the genome, wherein the NPCEC-specific promoter construct is used to localize production of the sequence-specific endonuclease to NPCECs.
  • the double stranded breaks can then be repaired by non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or homology-directed repair (HDR) pathways.
  • Desired genome edits can be introduced into the genome using donor DNA to repair double-strand breaks by homologous recombination.
  • sequence-specific endonucleases can be used in genome editing for creation of double-strand breaks in DNA, including, without limitation, engineered zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) Cas9.
  • ZFNs zinc-finger nucleases
  • TALENs transcription activator-like effector nucleases
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Precise control over the timing of production of the genome editing enzyme can be achieved by including a regulatory sequence in the promoter construct that makes the minimal BEST2 promoter inducible and operably linking a recombinant polynucleotide encoding the genome editing enzyme to the inducible promoter to allow turning on and off of expression as desired.
  • an reprogrammed cell in which the cell has been modified by genetic engineering to force expression of myeloid lineage transcription factors.
  • Any cancer cell can be used for this purpose, particularly patient-derived cancer cells, e.g. obtained from a tumor biopsy, bone marrow sample, blood sample, and the like.
  • the cell is genetically modified in an ex vivo procedure, prior to transfer into a subject, to introduce the genetic construct encoding the transcription factors.
  • Cells for reprogramming as described above can be collected from a subject or a donor, and may be separated from a mixture of cells by techniques that enrich for desired cells or may be reprogrammed and cultured without separation.
  • An appropriate solution may be used for dispersion or suspension.
  • Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hank’s balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM.
  • Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc.
  • Techniques for affinity separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, e.g., complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., a plate, or other convenient technique.
  • Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • the cells may be selected against dead cells by employing dyes associated with dead cells ⁇ e.g., propidium iodide).
  • the affinity reagents may be specific receptors or ligands for the cell surface molecules indicated above.
  • peptide-MHC antigen and T cell receptor pairs may be used; peptide ligands and receptor; effector and receptor molecules, and the like.
  • the separated cells may be collected in any appropriate medium that maintains the viability of the cells, usually having a cushion of serum at the bottom of the collection tube.
  • Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove’s medium, etc., frequently supplemented with fetal calf serum (FCS).
  • FCS fetal calf serum
  • FCS fetal calf serum
  • the collected and optionally enriched cell population may be used immediately for genetic modification, or may be frozen at liquid nitrogen temperatures and stored, being thawed and capable of being reused.
  • the cells will usually be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.
  • the preparation of cancer cells useful in the practice of the present invention is achieved by transforming or transducing isolated cells with an expression vector comprising a nucleic acid sequence encoding the desired transcription factors.
  • the cancer cells take on characteristics of myeloid cells.
  • the reprogrammed cells are maintained in culture for a period of time sufficient to induce myeloid lineage programming.
  • Culture may be from about 1 day, 2 days, 3 days, 4 days, 5 days, up to about 3 weeks, about 2 weeks, about 12 days, about 10 days, about 1 week.
  • Myeloid characteristics may include expression of CD11b, CD14, SIRPoc, Ly6c, CD115, Class II MHC, and the like.
  • the cells may be initially selected for the presence of the expression vector, with a suitable selective agent appropriate for the vector. Examples of cytokines include Flt3-L, IL-7, IL- 3, GM-CSF, M-CSF, IL-4.
  • the concentrations may be conventional.
  • Non-limiting examples of combinations include, for example, 100ng/ml_ Flt3-L, 10ng/ml_ IL-7, 20ng/mL IL-3, 25 ng/mL GM- CSF, 25 ng/mL M-CSF; or 100ng/mL Flt3-L, 10ng/mL IL-7, 100 ng/mL GM-CSF, 50 ng/mL IL-4.
  • Immunoselection may then be used to purify reprogrammed cells from non-reprogrammed cancer cells by selecting for expression of one or more myeloid markers, including without limitation CD11b and CD14.
  • Positive immunoselection utilizes a reagent that selectively binds to these markers on the cells surface.
  • Negative immunoselection is optionally performed to deplete cells of lineages other than myeloid cells.
  • binding member refers to a member of a specific binding pair, i.e. two molecules, usually two different molecules, where one of the molecules (i.e., first specific binding member) through chemical or physical means specifically binds to the other molecule (i.e., second specific binding member).
  • the complementary members of a specific binding pair are sometimes referred to as a ligand and receptor; or receptor and counter-receptor. Such specific binding members are useful in positive and negative selection methods.
  • Specific binding pairs of interest include carbohydrates and lectins; complementary nucleotide sequences; peptide ligands and receptor; effector and receptor molecules; hormones and hormone binding protein; enzyme cofactors and enzymes; enzyme inhibitors and enzymes; etc.
  • the specific binding pairs may include analogs, derivatives and fragments of the original specific binding member.
  • a receptor and ligand pair may include peptide fragments, chemically synthesized peptidomimetics, labeled protein, derivatized protein, etc.
  • Especially useful reagents are antibodies specific for markers present on the desired cells (for positive selection) and undesired cells (for negative selection).
  • lectins can be used for selection.
  • Whole antibodies may be used, or fragments, e.g. Fab, F(ab).sub.2, light or heavy chain fragments, etc.
  • selection antibodies may be polyclonal or monoclonal and are generally commercially available or alternatively, readily produced by techniques known to those skilled in the art.
  • Antibodies selected for use will have a low level of non-specific staining and will usually have an affinity of at least about 100 mM for the antigen.
  • antibodies for selection are coupled to a plate, bead, magnetic reagent, and the like.
  • the exact method for coupling is not critical to the practice of the invention, and a number of alternatives are known in the art.
  • Direct coupling attaches the antibodies to the plate, particles, magnetic reagent, etc.
  • Indirect coupling can be accomplished by several methods.
  • the antibodies may be coupled to one member of a high affinity binding system, e.g. biotin, and the particles attached to the other member, e.g. avidin.
  • One may also use second stage antibodies that recognize species-specific epitopes of the antibodies, e.g. anti-mouse Ig, anti-rat Ig, etc.
  • Indirect coupling methods allow the use of a single magnetically coupled entity, e.g. antibody, avidin, etc., with a variety of separation antibodies. Selection may use flow cytometry, MACs, columns, etc.
  • the cell compositions thus obtained can be highly purified, where the desired cells may be at least about 50% of the desired cell type, at least about, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least 99%, or more.
  • the reprogrammed cells may be infused to the subject in any physiologically acceptable medium by any convenient route of administration, normally intravascularly, although they may also be introduced by other routes, where the cells may find an appropriate site for growth, e.g. to the lymph nodes, intratumor, etc.
  • a convenient route of administration normally intravascularly, although they may also be introduced by other routes, where the cells may find an appropriate site for growth, e.g. to the lymph nodes, intratumor, etc.
  • at least 1x10 6 cells/kg will be administered, at least 1x10 7 cells/kg, at least 1x10 8 cells/kg, at least 1x10 9 cells/kg, at least 1 x10 10 cells/kg, or more.
  • T cells refers to mammalian immune effector cells that may be characterized by expression of CD3 and/or T cell antigen receptor, which cells may respond to the reprogrammed APC by priming for an effector cell response appropriate for the subset of T cells.
  • the T cells are cytotoxic T cells, (CTL), which may be characterized as CD8+ T cells.
  • CTL cytotoxic T cells
  • the T cells are contacted with reprogrammed APC in vivo, i.e. where an effective dose of the reprogrammed APC are injected into the recipient and allowed to interact with T cells in their native environment, e.g. in lymph nodes, etc.
  • the contacting is performed in vitro.
  • the contacting may be performed in any suitable culture medium.
  • the reprogrammed APC processes proteins from the cancer cell and presents them on the cell surface.
  • the reprogrammed APC is contacted with a population of T cells.
  • an isolated T cell population is added to the reprogrammed APC in a dose and for a period of time sufficient to prime the T cells.
  • the ratio of T cell to APC is anywhere from 1 :10 to 10:1 , and is not critical so long as the number of reprogrammed APC is not limiting. Any suitable culture medium may be used. A period of from up to 8 days, up to 10 days, up to 12 days, up to 14 days may be sufficient (see, for example, Dudley et al, JCO 2005; 23(10):2346-2357).
  • the T cells thus primed may be used for any desired purpose, including experimental purposes relating to determination of antigen specificity, cytokine profiling, and the like, and for delivery in vivo.
  • CD3 + T cells can be isolated by CD3 + selection and magnetic isolation.
  • T cells are plated at 10 6 cells/mL and anti-CD3/CD28 Dynabeads are added at a bead-to-cell ratio of 1 :1.
  • T cell activation media also contains 100 U/ml_ IL-2, 20 ng/ml_ IL-7, and 10 ng/ml_ IL-12.
  • Dynabeads are removed and T cells are rested for 48 hrs in media containing 30 U/mL IL-2. After resting of T cells, they are cocultured with reprogrammed tumor cells at a ratio of 1 :2 tumor: T cells.
  • CD69 and CD25+ T cells are sorted for re-infusion.
  • the enhanced immune response may be manifest as an increase in the cytolytic response of T cells towards the target cells present in the recipient, e.g. towards elimination of tumor cells, infected cells; combining the primed T cells with HLA-matched targets to test for cytotoxic activity; determination of intracellular cytokine analysis; and the like.
  • Methods and compositions are provided for enhancing cellular responses, by reprogramming cancer cells, which may be obtained from the cancer cells of the vaccine recipient.
  • the subject methods include a step of obtaining the targeted cancer cells, which may be isolated from a biological sample.
  • the cells are transduced or transfected with an expression vector comprising a sequence encoding myeloid lineage transcription factors, which step may be performed in any suitable culture medium.
  • a therapeutic method is provided, the method comprising introducing into a recipient in need thereof of a reprogrammed cell population, wherein the cell population has been modified by introduction of a sequence encoding myeloid lineage transcription factors.
  • the cell population may be reprogrammed ex vivo, and is usually autologous or allogeneic with respect to the recipient.
  • An effective dose of reprogrammed cells is infused to the recipient and allowed to contact cells in their native environment, e.g. in lymph nodes, etc. Dosage and frequency may vary depending on the agent; mode of administration; nature of the cancer; and the like. It will be understood by one of skill in the art that such guidelines will be adjusted for the individual circumstances.
  • the dosage may also be varied for localized administration, e.g. intranasal, inhalation, etc., or for systemic administration.
  • Parenteral infusions include intramuscular, intravenous (bolus or slow infusion), intranodal, intraarterial, intraperitoneal, intrathecal, intratumoral, subcutaneous administration; etc.
  • Reprogrammed cells can be provided in pharmaceutical compositions suitable for therapeutic use, e.g. for human treatment.
  • Therapeutic formulations comprising such cells can be frozen, or prepared for administration with physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of aqueous solutions.
  • the cells will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • At least about 10 4 reprogrammed cells/kg are administered, at least about 10 5 reprogrammed cells /kg; at least about 10 6 reprogrammed cells /kg, at least about 10 7 reprogrammed cells/kg, at least about 10 8 reprogrammed cells/kg, at least 1x10 9 cells/kg, at least 1x10 1 ° cells/kg, or more.
  • typical ranges for the administration of cells for use in the practice of the present invention range from about 1x10 5 to 5x10 8 viable cells per kg of subject body weight per course of therapy.
  • typical ranges for the administration of viable cells in human subjects ranges from approximately 1 x10 6 to approximately 1x10 13 viable cells, alternatively from approximately 5x10 6 to approximately 5x10 12 viable cells, alternatively from approximately 1x10 7 to approximately 1 x10 12 viable cells, alternatively from approximately 5x10 7 to approximately 1x10 12 viable cells, alternatively from approximately 1 x10 8 to approximately 1x10 12 viable cells, alternatively from approximately 5x10 8 to approximately 1x10 12 viable cells, alternatively from approximately 1 x10 9 to approximately 1x10 12 viable cells per course of therapy.
  • the dose of the cells is in the range of 2.5-5x10 9 viable cells per course of therapy.
  • a course of therapy may be a single dose or in multiple doses over a period of time.
  • the cells are administered in a single dose.
  • the cells are administered in two or more split doses administered over a period of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 21 , 28, 30, 60, 90, 120 or 180 days.
  • the quantity of reprogrammed cells administered in such split dosing protocols may be the same in each administration or may be provided at different levels. Multi-day dosing protocols over time periods may be provided by the skilled artisan (e.g. physician) monitoring the administration of the cells taking into account the response of the subject to the treatment including adverse effects of the treatment and their modulation as discussed above.
  • compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • diluents are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • the diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
  • macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
  • Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, his
  • kits for use in the methods are provided in a dosage form (e.g., a therapeutically effective dosage form), in liquid or solid form in any convenient packaging (e.g., stick pack, dose pack, etc.).
  • a dosage form e.g., a therapeutically effective dosage form
  • liquid or solid form in any convenient packaging (e.g., stick pack, dose pack, etc.).
  • Reagents for the selection or in vitro derivation of cells may also be provided, e.g. growth factors, differentiation agents, tissue culture reagents; and the like.
  • the subject kits may further include (in certain embodiments) instructions for practicing the subject methods.
  • These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
  • One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like.
  • Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), flash drive, and the like, on which the information has been recorded.
  • Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.
  • the condition is cancer.
  • cancer or “cancerous”
  • hyperproliferative or “hyperproliferative”
  • neoplastic to refer to cells having the capacity for autonomous growth (e.g., an abnormal state or condition characterized by rapidly proliferating cell growth).
  • Hyperproliferative and neoplastic disease states may be categorized as pathologic (e.g., characterizing or constituting a disease state), or they may be categorized as non- pathologic (e.g., as a deviation from normal but not associated with a disease state).
  • Pathologic hyperproliferative cells occur in disease states characterized by malignant tumor growth.
  • non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.
  • cancer or "neoplasm” are used to refer to malignancies of the various organ systems, including those affecting the lung, breast, thyroid, lymph glands and lymphoid tissue, gastrointestinal organs, and the genitourinary tract, as well as to adenocarcinomas which are generally considered to include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • carcinoma is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas.
  • An "adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • tumor cells include but are not limited to AML, ALL, CML, adrenal cortical cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain cancers, central nervous system (CNS) cancers, peripheral nervous system (PNS) cancers, breast cancer, cervical cancer, childhood Non-Hodgkin's lymphoma, colon and rectum cancer, endometrial cancer, esophagus cancer, Ewing's family of tumors (e.g.
  • Ewing's sarcoma eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, lung carcinoid tumors, Non-Hodgkin's lymphoma, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, nasal cavity and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, melanoma
  • uterine sarcoma transitional cell carcinoma, vaginal cancer, vulvar cancer, mesothelioma, squamous cell or epidermoid carcinoma, bronchial adenoma, choriocarinoma, head and neck cancers, teratocarcinoma, or Waldenstrom's macroglobulinemia.
  • Any cancer, where the cancer cells exhibit increased expression of CD47 compared to non-cancer cells, is a suitable cancer to be treated by the subject methods and compositions.
  • compositions and method of the present invention may be combined with additional therapeutic agents.
  • the methods may be combined with conventional chemotherapeutic agents or other biological anti-cancer drugs such as checkpoint inhibitors (e.g. PD1 or PDL1 inhibitors) or therapeutic monoclonal antibodies (e.g Avastin, Herceptin).
  • checkpoint inhibitors e.g. PD1 or PDL1 inhibitors
  • therapeutic monoclonal antibodies e.g Avastin, Herceptin
  • Examples of chemical agents identified in the art as useful in the treatment of neoplastic disease include without limitation, abitrexate, adriamycin, adrucil, amsacrine, asparaginase, anthracyclines, azacitidine, azathioprine, bicnu, blenoxane, busulfan, bleomycin, camptosar, camptothecins, carboplatin, carmustine, cerubidine, chlorambucil, cisplatin, cladribine, cosmegen, cytarabine, cytosar, cyclophosphamide, cytoxan, dactinomycin, docetaxel, doxorubicin, daunorubicin, ellence, elspar, epirubicin, etoposide, fludarabine, fluorouracil, fludara, gemcitabine, gemzar, hycamtin, hydroxyurea,
  • Targeted therapeutics that can be administered in combination may include, without limitation, tyrosine-kinase inhibitors, such as Imatinib mesylate (Gleevec, also known as STI- 571), Gefitinib (Iressa, also known as ZD1839), Erlotinib (marketed as Tarceva), Sorafenib (Nexavar), Sunitinib (Sutent), Dasatinib (Sprycel), Lapatinib (Tykerb), Nilotinib (Tasigna), and Bortezomib (Velcade), Jakafi (ruxolitinib); Janus kinase inhibitors, such as tofacitinib; ALK inhibitors, such as crizotinib; Bcl-2 inhibitors, such as obatoclax, venclexta, and gossypol; FLT3 inhibitors, such as midostaurin (Rydapt
  • Examples of biological agents identified in the art as useful in the treatment of neoplastic disease include without limitation, cytokines or cytokine antagonists such as IL-12, INFa, or anti- epidermal growth factor receptor, radiotherapy, irinotecan; tetrahydrofolate antimetabolites such as pemetrexed; antibodies against tumor antigens, a complex of a monoclonal antibody and toxin, a T-cell adjuvant, bone marrow transplant, or antigen presenting cells (e.g., dendritic cell therapy), anti-tumor vaccines, replication competent viruses, signal transduction inhibitors (e.g., Gleevec® or Herceptin®) or an immunomodulator to achieve additive or synergistic suppression of tumor growth, cyclooxygenase-2 (COX-2) inhibitors, steroids, TNF antagonists (e.g., Remicade® and Enbrel®), interferon ⁇ la (Avonex®), and interferon ⁇ l
  • Tumor specific monoclonal antibodies that can be administered in combination with an reprogrammed cell may include, without limitation, Rituximab (marketed as MabThera or Rituxan), Alemtuzumab, Panitumumab, Ipilimumab (Yervoy), etc.
  • compositions and methods of the present invention may be combined with immune checkpoint therapy.
  • immune checkpoint therapies include inhibitors of the binding of PD1 to PDL1 and/or PDL2.
  • PD1 to PDL1 and/or PDL2 inhibitors are well known in the art.
  • Examples of commercially available monoclonal antibodies that interfere with the binding of PD1 to PDL1 and/or PDL2 include nivolumab (Opdivo®, BMS-936558, MDX1106, commercially available from BristolMyers Squibb, Princeton NJ), pembrolizumab (Keytruda®MK-3475, lambrolizumab, commercially available from Merck and Company, Kenilworth NJ), and atezolizumab (Tecentriq®, Genentech/Roche, South San Francisco CA).
  • PD1 inhibitory antibodies include but are not limited to durvalumab (MEDI4736, Medimmune/AstraZeneca), pidilizumab (CT-011 , CureTech), PDR001 (Novartis), BMS-936559 (MDX1105, Bristol Myers Squibb), and avelumab (MSB0010718C, Merck Serono/Pfizer) and SHR-1210 (Incyte).
  • Additional antibody PD1 pathway inhibitors are described in United States Patent No. 8,217,149 (Genentech, Inc) issued July 10, 2012; United States Patent No. 8,168,757 (Merck Sharp and Dohme Corp.) issued May 1 , 2012, United States Patent No.
  • Immunoregulatory modulating agents also include an agent that blockades CD47 activity.
  • the term “anti-CD47 agent” or “agent that provides for CD47 blockade” refers to any agent that reduces the binding of CD47 (e.g., on a target cell) to SIRPa (e.g., on a phagocytic cell).
  • suitable anti-CD47 reagents include SIRPa reagents, including without limitation high affinity SIRPa polypeptides, anti-SIRPa antibodies, soluble CD47 polypeptides, and anti-CD47 antibodies or antibody fragments.
  • a suitable anti-CD47 agent e.g.
  • Immunoregulatory modulating agents also include an agent that agonizes an immune costimulatory molecule, e.g. CD40, 0X40, etc.; and/or an agent that antagonizes an immune inhibitory molecule, e.g. CTLA-4, PD1 , PDL1 , etc.
  • the active agents are administered within a period of time to produce an additive or synergistic effect on depletion of cancer cells in the host. Methods of administration include, without limitation, systemic administration, intra-tumoral administration, etc.
  • Checkpoint inhibitors include, for example, e.g.
  • such an immunoregulatory agent is a CTLA-4, PD1 or PDL1 antagonist, e.g. avelumab, nivolumab, pembrolizumab, ipilimumab, and the like.
  • limmune response agonists include, e.g. a CD28 agonist, an 0X40 agonist; a GITR agonist, a CD137 agonist, a CD27 agonist, an HVEM agonist, etc.
  • such an immunoregulatory agent is an 0X40, CD137, or GITR agonist e.g. tremelimumab, and the like.
  • B-ALL Precursor B-cell acute lymphoblastic leukemia
  • B-ALL is an aggressive hematopoietic neoplasm characterized by recurrent genetic lesions resulting in B-cell maturation arrest and malignant transformation.
  • targeted therapies to conventional treatment regimens, prognosis for adults with high risk disease remains poor, particularly for those patients with relapsed or refractory disease.
  • human B-ALL blasts retain the capacity for reprogramming to the myeloid lineage.
  • No differentiation therapies have been effective in the treatment of B-ALL, thus, we sought to investigate the therapeutic implications of myeloid lineage reprogramming of B-ALL cells.
  • B-ALL cells into antigen presenting cells could induce tumor-specific T cell responses through effective presentation of aberrant tumor-associated self peptides.
  • APCs antigen presenting cells
  • MHC-II 8.6-fold, p ⁇ 0.0001
  • CD80 (62.1 -fold, p ⁇ 0.0001
  • CD86 107.6-fold, p ⁇ 0.0001
  • CD40 92-fold, p ⁇ 0.0001 .
  • B-ALL cells reprogrammed to the myeloid lineage can operate as potent APCs capable of presenting both endogenous and exogenous tumor- associated antigens
  • in vivo B-ALL reprogramming elicits robust immune activation, dependent on both CD4+ and CD8+ T cells
  • B-ALL reprogramming-induced immune activation is potent, durable, tumor-eradicating, and systemic.
  • reprogramming of B-ALL cells into APCs provides a novel immunotherapeutic strategy with clinical benefit for the management of B-ALL disease progression.
  • B-ALL Primary B cell acute lymphoblastic leukemia
  • B-ALL blasts were sorted (CD19+CD34+CD11b- CD14-) from three patient samples and cultured in the presence of GM-CSF, M-CSF, IL-3, FLT3L, and IL-6) for twelve days.
  • Cells were co-stained with APC conjugated anti-CD11b and APC-Cy7-conjugated anti-CD19.
  • a mammalian lentiviral expression vector encoding the transcription factor CEBPoc was introduced into sorted primary B-ALL blasts via nucleofection and cultured in the absence of cytokines for four days.
  • CEBPccand PU.1 initiate myeloid reprogramming in diverse tumor types.
  • Murine tumor cell lines RAW-112 B cell lymphoma, 2F3 BCR-ABL+ B cell leukemia, K-BALB fibrosarcoma, and 4T1 mammary carcinoma were dually transduced with the lentiviral vectors.
  • Cell lines were cultured in the absence or presence of 1 mg/mL doxycycline for four days. Resulting cultures were co-stained with APC-conjugated anti-CD11b (Mac-1) and APC-Cy7-conjugated CD14.
  • Myeloid reprogrammed tumor cells acquire myeloid phenotypic and functional markers and lose cell-of-origin markers.
  • RAW-112 B cell lymphoma cells were reprogrammed via doxycycline-induced expression of CEBPa and PU.1 in the presence of myeloid cytokines GM- CSF, M-CSF, and IL-3 over the course of seven days.
  • CD11b+CD14+ cells were sorted via FACS and cytospun and compared to CD11b-CD14- RAW- 112 cells treated with vehicle alone.
  • Wright-Giemsa staining showed a morphology consistent with mature phagocytic cells in the CD11b+CD14+ reprogrammed cells.
  • Myeloid reprogrammed tumor cells acquire myeloid phagocytic function AW-112.
  • B cell lymphoma cells were reprogrammed via doxycycline-induced expression of CEBPoc and PU.1 in the presence of myeloid cytokines GM-CSF, M-CSF, and IL-3 for seven days.
  • Doxycycline or vehicle treated cells were incubated with fluorescently labelled pHrodo Red E. coli particles.
  • Phagocytic cells were quantified as a percentage of total population by negative gating on RAW- 112 cells prior to incubation with E. coli particles.
  • Treatment with cytochalasin D to block phagocytosis demonstrated the specificity of the assay.
  • Cells reprogrammed as above were either stimulated with 1 mg/mL LPS or left unstimulated. Stimulated, unstimulated, and non-reprogrammed cells were co-stained with FITC- conjugated anti-l-A/l-E, APC-conjugated anti-H-2Dd, PerCP-Cy5.5-conjugated anti-CD80, APC- Cy7-conjugated anti-CD86, BV605-conjugated anti-CD40, and PE-conjugated anti-CD16/32.
  • Myeloid reprogrammed tumor cells express antigen presentation and costimulatory molecules.
  • Ectopic expression of CEBPa and PU.1 reprograms hematologic and solid tumor cell lines into myeloid lineage cells.
  • Stable tumor cell lines representing diverse cancer cell types, including osteosarcoma, carcinoma (mammary), fibrosarcoma, B-cell leukemia and B-cell lymphoma (RAW-112, 2F3, K-BALB, 4T1 , or K7M2 cells) were transduced with doxycycline-inducible CEBPa and PU.1 constructs.
  • Cells were incubated in vitro for 4 days with 1 ug/mL doxycycline to drive the expression of CEBPa and PU.1 , or with a vehicle control.
  • Flow cytometry was used to assess the expression of myeloid-specific lineage markers CD11b (Mac-1) and CD14.
  • Ectopic expression of CEBPa and PU.1 reprograms hematologic cell lines into myeloid lineage cells.
  • B cell lymphoma RAW-112 cells were incubated in vitro for 4 days with 1 ug/mL doxycycline to drive the expression of CEBPa and PU.1 , or with a vehicle control.
  • FACS was used to sort CD11b+CD14+ cells from doxycycline treated cultures. Cells were cytospun onto a microscope slide and stained with Wright-Giemsa stain.
  • Ectopic expression of CEBPa and PU.1 reprograms hematologic and solid tumor cell lines into myeloid lineage cells.
  • RAW-112 and K-BALB (fibrosarcoma) cells were incubated in vitro for 4 days with 1 ug/mL doxycycline to drive the expression of CEBPa and PU.1 , or vehicle. Cultures were then incubated in the presence of pHrodo Red E. coli particles with or without the phagocytosis inhibitor Cytochalasin D. Flow cytometry was used to measure the fluorescent signal from phagocytosed E. coli particles. Shown in Figure 4, the reprogrammed cells not only adopt myeloid phenotypic features, but also become functional myeloid cells.
  • phagocytose Compared to vehicle treated cells, doxycycline-treated cultures phagocytose significantly higher amounts of pHrodo E. coli particles. This signal is diminished in the presence of Cytochalasin D, implicating phagocytosis as the biological process responsible for the observed result. Thus, reprogrammed cells acquire the functional capacity for phagocytosis.
  • Myeloid-reprogrammed tumor cells become efficient antigen presenting cells capable of stimulating T cell mediated immunity.
  • Reprogrammable RAW-112 were stably transduced with chicken ovalbumin and were incubated in vitro for 4 days with 1 ug/mL doxycycline to drive the expression of CEBPa and PU.1 , or vehicle.
  • Co-cultures were then generated with vehicle or doxycycline-treated RAW-112-OVA cells and Proliferation Dye eFluor450 loaded transgenic D011 .10 T cells. T cell proliferation was measured after 72 hours of co-culture by flow cytometry. Data shown in Figure 5.
  • An assay was designed to probe whether or not myeloid-reprogrammed cells can function as antigen presenting cells.
  • a model antigen chicken ovalbumin, as a surrogate tumor antigen.
  • DO11.10 T cells are derived from a transgenic mouse that expresses a T cell receptor designed to recognize ovalbumin peptide presented in BALB/c MHCII molecules.
  • BMDCs bone marrow dendritic cells
  • Myeloid-reprogrammed tumor cells become efficient antigen presenting cells capable of stimulating T cell mediated immunity.
  • Reprogrammable RAW-112, K-BALB (fibrosarcoma), or 4T1 (mammary carcinoma) cells were incubated in vitro for 4 days with 1 mg/mL doxycycline to drive the expression of CEBPa and PU.1 , or vehicle. Both vehicle and doxycycline treated cultures were then incubated with DQ-OVA for 4 hours. DQ-OVA fluorescent signal was assessed after 4 hours by flow cytometry, shown in Figure 6.
  • This assay is designed to determine if exogenous antigens can be taken up from the extracellular space, processed, and loaded into MHC molecules.
  • DQ-OVA reagent which emits not fluorescent signal in the absence of the proteolytic cleavage associated with peptide processing for MHC presentation.
  • DQ-OVA become fluorescent.
  • RAW-112 and K-BALB and to a lesser extent, 4T 1 cells, are much more efficient at antigen uptake, processing, and presentation upon reprogramming.

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Abstract

Cellules cancéreuses traitées par reprogrammation directe pour générer des cellules présentatrices d'antigène, qui peuvent présenter des antigènes tumoraux et améliorer la réactivité immunitaire au cancer. La reprogrammation directe utilise une modification génétique pour forcer l'expression de facteurs de transcription qui entraînent des cellules dans la lignée myéloïde. L'invention porte sur des compositions et méthodes de vaccination avec des cellules cancéreuses.
PCT/US2020/058170 2019-11-01 2020-10-30 Reprogrammation de lignée en tant qu'immunothérapie anticancéreuse WO2021087234A1 (fr)

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