Classifications

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  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70517—CD8
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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/7051—T-cell receptor (TcR)-CD3 complex
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70514—CD4
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2815—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD8
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood 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
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/515—Animal cells
  • A61K2039/5158—Antigen-pulsed cells, e.g. T-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
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
  • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K2121/00—Preparations for use in therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
  • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

• Immunotherapy has become a cornerstone in cancer therapy that includes a broad array of strategies aiming to unleash, direct, and boost the patient’s own immune system through adoptive transfer of expanded naturally circulating or genetically engineered cytotoxic lymphocytes.
• current adoptive immunotherapies encounter several challenges. For example, many adoptive immunotherapies cannot generate a sufficient level of engineered cytotoxic lymphocytes of clinical or therapeutic value from peripheral blood, or have an inability to uniformly engineer effector cells, or cannot provide a sustained, long lasting therapeutic effect for the patient, resulting in tumor re-occurrence and other complications.
• the invention provides an isolated cell composition, which is suitable for adoptive immunotherapy and/or genetic engineering of T cells.
• the invention further provides methods of manufacturing the cell compositions and methods of treatment with the cell compositions.
• the composition comprises, in a pharmaceutically acceptable carrier, at least about 10 6 CD8+ T cells specific for target peptide antigen(s), and which comprises T memory stem (TSCM) cells.
• TSCM T memory stem
• the CD8+ T cells are at least about 1% T memory stem cells.
• T memory stem cells are isolated, thereby preparing a composition substantially comprising T memory stem cells (e.g., 70% to 100% TSCM).
• the compositions of the invention by virtue of the presence of significant levels of TSCM, can provide for a robust and durable adoptive therapy.
• the cell composition need not comprise T cells expressing a chimeric antigen receptor or a recombinant TCR, and therefore, in various embodiments, provides an alternative to these technologies that often produce more exhausted T cell phenotypes and less durable responses and greater toxicities.
• the TSCM are used to recombinately express a chimeric antigen receptor or heterologous TCR, thereby preparing engineered T cells with high proliferative capacity and less exhausted phenotype.
• the cell composition comprises at least 1%, or at least 15% T memory stem cells and about 10 6 CD8+ T cells specific for the target peptide antigens, or at least about 10 7 , or at least about 10 8 , or at least about 10 9 , or at least about 10 10 CD8+ T cells specific for the target peptide antigens, to provide robust destruction of target cells and a long persistence in vivo.
• the cell composition may comprise T cells specific for WT1, PRAME, Survivin, and Cyclin Al peptide antigens, among others.
• the cell composition comprises from about 1% to about 50% T memory stem cells, or from about 5% to about 25% T memory stem cells.
• the T cells are at least 5% T memory stem cells, or the T cells are at least about 10% T memory stem cells, or the T cells are at least 20% T memory stem cells, or at least 25% T memory stem cells, which provides an adoptive immunotherapy composition with significant proliferative potential, as well as immune-reconstitution capacity and longevity.
• the memory phenotype comprises, in addition to TSCM, one or more (or all) of central memory T cells (TCM), effector memory T cells (TEM), and effector memory RA+ T cells (TEMRA). In some embodiments, at least 80% of the memory phenotype is TSCM, TCM, and TEM.
• the CD8+ T cells in the composition comprise, in addition to TSCM, central memory and effector memory T cells.
• the T cells in the composition are at least about 30% central or effector memory T cells, or in some embodiments are at least about 50% central or effector memory T cells, or in some embodiments are at least about 70% central or effector memory cells, or in some embodiments are at least about 80% central or effector memory T cells, or in some embodiments are at least about 90% central or effector memory T cells.
• the CD8+ T cells specific for the one or more target antigens are at least 50% central and effector memory T cells, or in some embodiments are at least 80% central and effector memory T cells.
• the combination of TscM and TCM IS from about 40% to about 70% of the CD8+ T cells.
• the cell composition comprises less than about 20%, or less than about 10%, terminally differentiated memory T cells (e.g., TEMRA cells), and less than about 30% naive cells, or in some embodiments less than about 15%, or in some embodiments less than about 5%, or in some embodiments less than 1.5% naive cells.
• the cell phenotype disclosed herein can be created and/or controlled using an enrichment and expansion process with paramagnetic artificial Antigen Presenting Cells (aAPCs) and a recombinant T cell growth factor cocktail.
• aAPCs paramagnetic artificial Antigen Presenting Cells
• the cell composition is at least about 70%, or at least about 80%, or at least about 90% CD8+ or CD4- T cells (e.g., CD3+ CD8+ or CD3+ CD4- cells).
• the isolated cell composition may be characterized by having less than about 10%, or less than about 5% CD4+ T cells.
• the antigen-specific T cells display a polyfunctional phenotype upon activation.
• at least 10% of the CD8+ T cells, or in some embodiments at least 20% of the CD8+ T cells, or in some embodiments at least 40% of the CD8+ T cells display a polyfunctional phenotype upon activation.
• the T cells upon activation the T cells are positive for two or more of: intracellular staining for IL-2, IFN-g production, production of TNF-a, and CD107A.
• at least 20% of the antigen-specific T cells display at least two of these markers.
• at least 20% of the antigen-specific T cells display at least three of these markers, or in some embodiments all four of these markers.
• at least 5% of the CD8+ T cells are multi-antigenic, meaning the CD8+ T cells are capable of responding to multiple tumor or viral antigens in vitro or in vivo.
• the cell composition further comprises gd T cells gd T cells have a distinctive T-cell receptor (TCR) on their surface gd T cells may have a role in recognition of lipid antigens and phospho antigens, and can provide anti-pathogen and anti -tumor mechanisms that are not HLA-dependent. Further, gd T cells can provide help to the CD8+ cells. Clinical significance of gd T cells in the context of hematopoietic stem cell transplantation (HSCT) has been observed, and in particular, higher frequencies of gd T cells after transplantation were associated with favorable outcomes.
• HSCT hematopoietic stem cell transplantation
• CD8+ cells are enriched that are specific for the target antigen(s) (e.g., tumor associated antigens or viral- associated antigens).
• target antigen(s) e.g., tumor associated antigens or viral- associated antigens.
• This cell population even when predominately naive cells in the source lymphocytes, can be rapidly expanded in culture to arrive at the cell compositions described herein. Enrichment can take place using paramagnetic beads to positively select cell populations, and which can have the added advantage of activating naive cells and other T cell populations due to potent magnetic clustering of T cell surface receptors.
• paramagnetic beads or nanoparticles may contain monomeric or multimeric (e.g., dimeric) HLA ligands presenting peptide antigens, along with a co-stimulation signal on the same or different particles, such as an agonist for CD28 (e.g., an antibody agonist of CD28).
• CD28+ cells are also enriched, which can be simultaneous with antigen-specific enrichment.
• the target peptide antigens are tumor or cancer associated antigens, including tumor-derived, tumor-specific antigens, and neoantigens.
• T cells specific for tumor-associated antigens are often very rare, and in many cases undetectable, in the peripheral blood of healthy individuals. This is often a distinction observed between viral-specific and tumor antigen-specific T cells.
• the target peptide antigens include at least one that is associated with or derived from a pathogen, such as a viral, bacterial, fungal, or parasitic pathogen.
• a pathogen such as a viral, bacterial, fungal, or parasitic pathogen.
• at least one peptide antigen may be associated with HIV, hepatitis (e.g., B, C, or D) CMV, Epstein-Barr virus (EBV), influenza, herpes virus (e.g., HSV 1 or 2, or varicella zoster), and Adenovirus.
• CMV for example, is the most common viral pathogen found in organ transplant patients and is a major cause of morbidity and mortality in patients undergoing bone marrow or peripheral blood stem cell transplants. Viral activation is known to be implicated in cancer biology.
• the cell composition comprises T cells specific for tumor-associated antigens, with pathogen-associated antigen specific T cells provided as bystander cells.
• Other bystander cells include gd T cells.
• bystander cells will be enriched, and expanded, particularly when using a T cell growth factor cocktail that can drive some non-specific expansion of these cells without antigen-specific activation.
• T cells specific for the target peptides e.g., from 5% to 75%)
• remaining T cells from about 0.25% to about 25%
• T cell growth factors during expansion, which affect proliferation and/or differentiation of T cells.
• Particularly useful cytokines include MIP- 1b, IE-1b, IL-2, IL-4, IL-6, IL-7, IL-10, IL-21, and INF-g.
• the cells are expanded in culture in the presence of a cytokine cocktail comprising one, two, or three cytokines selected from MIR-Ib, IE-1b, and IL-6.
• the cytokines further comprise IL-10.
• the growth factors comprise or consist essentially of IL-2, IL-4, IL-6, INF-g, and IE-1b.
• Cells can be expanded in culture from 1 to 4 weeks, such as from about 10 to about 21 days.
• the invention provides methods for manufacturing the cell compositions, including by enrichment and expansion with aAPCs as described herein.
• aAPCs as described herein.
• source lymphocytes e.g., from a healthy donor or from a patient in need of adoptive immunotherapy
• antigen-specific CD8+ T cells are enriched for T cells specific for the target peptide antigens, as well as CD28+ cells in some embodiments.
• Target cells can be enriched using nanoparticle or microparticle aAPCs, such as superparamagnetic nanoparticles that activate T cells ex vivo by magnetic field induced clustering of cell surface receptors.
• Enriched T cells can then be rapidly expanded ex vivo, including with the use of reconstituted T cell growth factors (e.g., comprising factors selected from MIR-Ib, I ⁇ - ⁇ b, IL-2, IL-4, IL-6, IL-7, IL-10, IL- 12, IL-15, IL-21, IFN-g).
• T cell growth factors e.g., comprising factors selected from MIR-Ib, I ⁇ - ⁇ b, IL-2, IL-4, IL-6, IL-7, IL-10, IL- 12, IL-15, IL-21, IFN-g.
• the cells are expanded in culture in the presence of one, two, or three cytokines selected from MIR-Ib, IE-1b, IL-6, and IL-10.
• the growth factors comprise or consist essentially of IL-2, IL-4, IL-6, INF-g, and IL- I b.
• these cytokines are used in conjunction with artificial or natural antigen presenting cells to expand antigen specific T cells.
• the invention provides methods for adoptive cell therapy, including methods for treating a patient with cancer, and/or patients that have undergone allogeneic stem cell transplantation, with or without lympho-deleting therapy, cyto reductive therapy, immunomodulatory therapy (prior to administration of the cell therapy).
• the cell therapy may be further provided with or without cytokine support post treatment.
• the patient has a hematological cancer, which in some embodiments has relapsed after allogeneic stem cell transplantation.
• the patient has acute myelogenous leukemia (AML) or myelodysplastic syndrome.
• the cell composition comprises T cells specific for WT1, PRAME, Survivin, and Cyclin Al peptide antigens.
• the cancers include various types of solid tumors, including carcinomas, sarcomas, and lymphomas. Exemplary target peptide antigens are described herein.
• the patient has an infectious disease or is at risk for an infectious disease.
• patients that have undergone HSCT are at particular risk for infectious disease, given the immunocompromised state.
• Infectious diseases that can be treated or prevented include those caused by bacteria, viruses, prions, fungi, parasites, helminths, etc.
• Such diseases include AIDS, hepatitis B/C, CMV infection, Epstein-Barr virus (EBV) infection, influenza, herpes virus infection (including shingles), and adenovirus infection.
• EBV Epstein-Barr virus
• the invention provides a method for making a population of gd T cells.
• the method comprises expanding a population of cells comprising gd T cells in the presence of two or more of IL-2, IL-4, IL-6, INF-g, and IL- 1b. Before expansion, the population of cells comprises less than about 20% or less than about 10% or less than about 8% gd T cells.
• the population of cells is CD28 enriched. In some embodiments, the population of cells is CD4+ depleted.
• T cells can be separated from other cells using known methods, such as cell sorting, and can be provided as a cell composition for adoptive transfer or research use, and alternatively may be modified to express one or more heterologous or engineered genes, such as a heterologous or engineered T cell receptor (e.g., ab TCR), including a chimeric antigen receptor (CAR).
• a heterologous or engineered T cell receptor e.g., ab TCR
• CAR chimeric antigen receptor
• FIGURE 1 is an image showing the Artificial Immune Modulation (AIM) platform for the generation of CD8+ antigen specific T cells.
• AIM Artificial Immune Modulation
• FIGURE 2 is an image showing AIM ACT (Adoptive Cellular Therapy) and the Enrichment and Expansion (E+E) cellular expansion system that enables rapid in vitro enrichment and expansion of antigen-specific T cells.
• AIM ACT Aloptive Cellular Therapy
• E+E Enrichment and Expansion
• FIGURE 3 has two graphs showing the enriched and expanded antigen-specific cell product for the AIM ACT platform.
• the graph on the left shows the total number of CD8+ T cells generated from fresh PBMCs of four healthy donors after the T cells were enriched and expanded ex vivo for AML-specific antigens WT137-45, 126-134, PRAME425, and Cyclin AI227-235, 341-351.
• the graph on the right shows the percentage total of the same acute myeloid leukemia (AML) specific antigens after the CD8+ T cells were enriched and expanded ex vivo.
• AML acute myeloid leukemia
• FIGURE 4A and FIGURE 4B show that the CD8+ T cells generated by the AIM ACT platform comprise memory T cells.
• Figure 4A shows the AML antigen-specific CD8+ T cell population after enrichment but prior to expansion at Day 0 (top), and after expansion at Day 14 (bottom).
• TCM central memory T cells (CD62L+, CD45RA-);
• TN naive T cells (CD62L+, CD45RA+);
• TEM effector memory T cells (CD62L-, CD45RA- );
• TEMRA effector memory RA+ T cells (CD62L-, CD45RA+);
• TSCM T memory stem cells (CD62L+, CD45RA+, CD95+).
• FIGURE 4B shows the memory T cell phenotypes for TSCM, TCM, TEM, and TEMRA on day 14 after AML-specific enrichment and expansion for AML specific antigens WT137-45, 126-134, PRAME425, Cyclin AI227-235, 341-351.
• FIGURE 5A and FIGURE 5B show that AML specific T cells enriched and expanded ex vivo have a high degree of polyfunctional phenotype, including intracellular staining for IL-2 (proliferation and memory), IFN-g (activating other T cells, memory, upregulation of MHC), TNF-a (pro-inflammatory), and CD107A (granzyme release, cytotoxic activity).
• the majority of AML-specific T cells i.e., about 62%) demonstrated 3-4 effector functions upon non-specific stimulation (FIGURE 5A, top).
• FIGURE 5A (bottom) the graph shows the percentage of T cells expressing IL-2, TNF-a, IFN-g, and CD107A.
• FIGURE 5 A the T cells were stimulated by non-specific stimulation of peptide-pulsed T2 cells.
• FIGURE 5B a graph is shown of T cell mediated tumor specific killing of AML cell line U266 at two effector to target (E:T) ratios, 10: 1 (left bar) and 20: 1 (right bar), using CTLs generated from fresh PBMCs of healthy donors with AML specific antigens WT137-45, 126-134, PRAME425, and Cyclin Al 227-235, 341-351.
• E:T effector to target
• FIGURE 6 consists of four graphs comparing the specificity of Mart- 1 specific T cells generated by the enrichment and expansion process between melanoma patient derived PBMCs (top) and healthy donor derived PBMCs (bottom).
• the enrichment and expansion process produces a consistent cellular composition regardless of the donor source.
• the data in this experiment was generated from frozen PBMCs.
• FIGURE 7 is a graph showing that the AIM ACT based E+E process generated a TCR repertoire that mimics the natural immune response, thereby providing a robust adoptive therapy from a natural T cell repertoire that has undergone natural selection.
• the breadth of the polyclonal TCR repertoire enables a natural and durable immune response.
• FIGURE 8 has graphs showing that the E+E process generated significant amounts of multiple myeloma antigen-specific T memory stem (TSCM) cells ((CD62L+, CD45RA+, CD95+).
• the graphs show the phenotype of multiple myeloma-specific antigenic T cells pre and post expansion from a healthy donor leucopak.
• FIGURE 9A shows the phenotype of T cells enriched and expanded ex vivo in batch for multiple myeloma antigen-specific T cells from a healthy donor leucopak.
• the graphs show that the E+E process generated significant amounts of antigen-specific CD8+ T cells ( ⁇ l.6xl0 9 CD8+ T cells based on hinge dimer staining) that comprise T memory stem (TSCM) cells, central memory T cells TCM, and effector memory T (TEM) cells.
• FIGURE 9B shows the phenotype of T cells enriched and expanded ex vivo in batch for multiple myeloma antigen-specific T cells from four different clinical multiple myeloma patients.
• the graphs show that the enrichment and expansion process generated significant amounts of antigen-specific CD8+ T cells that comprise T memory stem (TSCM) cells, central memory T cells TCM, and effector memory T (TEM) cells.
• TSCM T memory stem
• TEM effector memory T
• FIGURE 10 shows production of gd T cells, including V51 and V52 TCR subtypes.
• FIGURE 11 shows the percent gd T cells at Day 14, after expanding for various antigen-specific T cells (AML, MM, EBV, MART-l).
• the percent gd T cells at Day 14 broadly correlates with the number of gd T cells at Day 0.
• T cell memory is heterogeneous in composition, comprised of stable, resting, phenotypically-distinct subsets of surface markers capable of unique functional responses upon stimulation.
• Subsets related by differentiation include central memory T cells (TCM), effector memory T cells (TEM), effector memory RA+ T cells (TEMRA), and T memory stem cells (TSCM).
• TCM central memory T cells
• TEM effector memory T cells
• TEMRA effector memory RA+ T cells
• TSCM T memory stem cells
• Memory T cells develop when antigen-specific naive CD4+ or CD8+ T cells become activated upon antigen exposure and subsequently undergo proliferative expansion and differentiation. Accordingly, persistent memory is essential for long-term protection against infections and malignancies. Only the TSCM subset cells of T memory cells have been shown to differentiate into central memory T cells (TCM), effector memory (TEM), and terminal effector T cells (TTE).
• T memory stem cells are scarce and represent a small proportion of circulating lymphocytes. Generating clinically relevant amounts of T memory stem cells, e.g., for adoptive immunotherapy, is currently not feasible. Therefore, technologies are needed that can generate, expand, and enable the redirection of TSCM cells against cancer and infectious disease antigens.
• isolated cell compositions having at least about 10 6 CD 8+ T cells specific for target peptide antigen(s), and which comprise TSCM cells.
• the TSCM cells of the present disclosure express surface markers that are similar to naive T cells, but express elevated levels of the CD95 surface marker.
• Such TSCM cells are the least differentiated and expanded memory subset.
• the TSCM cells of the present disclosure demonstrate an enormous proliferative capability, are capable of reconstituting the full repertoire of memory and effector T cells, and are a long-term, stable population of cells endowed with superior homeostatic and differentiation capabilities.
• compositions disclosed herein having at least about 10 6 CD8+ T cells specific for target peptide antigen(s), and which comprise TSCM cells provide a highly effective anti-tumor composition that can generate, expand, and enable the redirection of TSCM cells against cancer cells at clinically relevant amounts for therapeutic applications.
• the invention provides an isolated cell composition, as well as methods of manufacturing the cell compositions and methods of treatment with the cell compositions.
• the cell compositions are used for adoptive cell therapy.
• the composition comprises, in a pharmaceutically acceptable carrier, at least about 10 6 CD8+ T cells specific for target peptide antigen(s), and which comprise TSCM cells.
• the CD8+ T cells are at least about 1% TSCM cells.
• TSCM cells are isolated, thereby preparing a composition comprising nearly 100% T memory stem cells (e.g., at least 90% TSCM cells).
• a composition of from about 70% to about 100% T memory stem cells is created.
• compositions of the invention provide for a robust and durable adoptive therapy.
• the cell composition need not comprise T cells expressing a chimeric antigen receptor (CAR) or a recombinant TCR, and therefore, in various embodiments, provides an alternative to these technologies that often produce more exhausted T cell phenotypes and less durable responses.
• the TSCM are used to recombinately express a chimeric antigen receptor or heterologous TCR (e.g., ab TCR or gd TCR), thereby preparing engineered T cells with high proliferative capacity and less exhausted phenotype than previously described.
• the TSCM cells can be used for engineering T cells with CARs or heterologous TCRs.
• target peptide antigen(s)” or“target antigens” refers to peptide antigens employed ex vivo to enrich and/or expand the desired CD8+ cell population, for example in connection with artificial Antigen Presenting Cell (aAPC) or professional Antigen Presenting Cell (pAPC) platforms (e.g., dendritic cells).
• aAPC Antigen Presenting Cell
• pAPC professional Antigen Presenting Cell
• the aAPCs or pAPCs are employed to activate and expand CTLs from donor or patient lymphocytes.
• the target peptide antigens are peptide epitopes loaded onto aAPCs for ex vivo enrichment and expansion of specific CD8+ T cells.
• the term “specific for the target peptide antigen” means that the T cell is antigen experienced with the target antigen.
• the cell composition comprises at least about 10 6 CD8+ T cells specific for the target peptide antigens, or at least about 10 7 CD8+ T cells specific for the target peptide antigens, or at least about 10 8 , at least about 10 9 , or at least about 10 10 CD8+ T cells specific for the target peptide antigens, to provide robust destruction of target cells.
• the cell composition contains from 1 x 10 7 to 1 x 10 9 CD8+ T cells specific for the target antigens, or in some embodiments from 5 x 10 7 to 5 x 10 8 CD8+ T cells specific for the target antigens.
• the composition can comprise from about 5 x 10 5 to about 5 x 10 6 cells per ml, in a volume of from 50 to 200 ml. In certain embodiments, the volume of the composition is ⁇ 100 ml (e.g., from 50 to 100 ml).
• the cells of the composition in various embodiments are at least 70% viable or at least about 80% or about 90% viable, and provided in a sterile medium, which may be a cryoprotectant medium (e.g., 10% DMSO).
• the medium can be an aqueous medium suitable for intravenous infusion, e.g., including water and electrolytes.
• An exemplary medium is PLASMALYTE.
• a cell composition comprising CD8+ cytotoxic lymphocytes (CTLs) and memory T cells.
• CTLs include the following T cell populations: naive, T memory stem cell, central memory, effector memory, and terminally differentiated memory cells.
• T cells specific for the target antigens include a significant amount of TSCM cells.
• the T cells specific for the target antigens further include central memory T cells and effector memory T cells.
• the cell composition provides a durable response, including in vivo persistence of antigen-specific T cells for at least about 6 months, or at least about 12 months, or at least about 18 months, or at least about two years in some embodiments.
• naive T cell has differentiated in bone marrow, and successfully undergone the positive and negative processes of central selection in the thymus.
• a naive T cell is considered mature and, unlike activated or memory T cells, has not encountered its cognate antigen.
• Naive T cells can be characterized by the surface expression of L- selectin (CD62L) and the absence of activation surface markers. In the naive state, T cells are generally quiescent and non-dividing. In accordance with this disclosure, naive T cells are defined as CD62L+ and CD45RA+.
• Memory T cells include T memory stem cells (TSCM), central memory and effector memory T cells. Memory T cells have previously responded to their cognate antigen. At a second encounter with the cognate antigen, memory T cells can reproduce to mount a faster and stronger immune response. Memory T cells include at least T memory stem cells, effector memory T cells, and central memory T cells. Memory T cell subtypes are long-lived and can quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen.
• T memory stem cells are defined herein as CD45RA+ and as having at least the following surface markers: CD62L+, CD45RA+, and CD95+.
• the T memory stem cells disclosed herein are CD62L+, CD45RA+, CD95+ and may have one or more of the following surface markers: CD28+, CD27+, CXCR3+ CD1 la+, IL-2R +, CD58+, and CD57-.
• the T memory stem cells comprise cells that are CD62L+, CD45RA+, CD28+, CD27+, and CD95+.
• the T memory stem cells comprise cells that are CD62L+, CD45RA+, CD95+ and CXCR3+.
• the T memory stem cells comprise cells that are CD62L+, CD45RA+, CD95+ and CDl la+. In some embodiments, the T memory stem cells comprise cells that are CD62L+, CD45RA+, CD95+ and IL-2R +. In some embodiments, the T memory stem cells comprise cells that are CD62L+, CD45RA+, CD95+ and CD58+. In some embodiments, the T memory stem cells comprise cells that are CD62L+, CD45RA+, CD95+ and CD57-. This memory subpopulation has the stem cell-like capacity for self-renewal, as well as the multipotent capacity to reconstitute the memory and effector T cell subpopulations.
• TSCM cells typically represent a small fraction of circulating T lymphocytes (e.g., >5%), and have the ability to proliferate rapidly and release inflammatory cytokines in response to antigen re-exposure. Accordingly, TSCM cells are a subset of the memory T cell subpopulation.
• the TSCM cells can be created and/or controlled using, as disclosed herein, an enrichment and expansion process with paramagnetic artificial Antigen Presenting Cells (aAPCs) and a recombinant T cell growth factor cocktail.
• aAPCs paramagnetic artificial Antigen Presenting Cells
• central memory T cells are defined herein as CD62L+ and CD45RA-. This memory subpopulation is commonly found in the lymph nodes and in the peripheral circulation.
• Effector memory T cells are defined herein as CD62L- and CD45RA-. These memory T cells lack lymph node homing receptors and are thus found in the peripheral circulation and tissues.
• TEMRA stands for terminally differentiated effector memory cells re-expressing CD45RA (Temra). These cells do not have the capacity to divide, and are CD62L- and CD45RA+.
• T central memory (TCM) cells display a capacity for self-renewal, and in accordance with embodiments of the invention, are also important for obtaining a long- lived effect.
• TEM cells also have some capacity for self-renewal, and strongly express genes essential to the cytotoxic function.
• TEMRA cells also provide robust cytotoxic function, but do not display a capacity for self-renewal.
• compositions in various embodiments comprise CTLs that are substantially composed of TSCM, TCM and TEM cells to balance duration of the effect versus potent destruction of the malignancy or other target cells.
• these cells make up at least about 75% or at least about 80% or at least about 90% of the memory phenotype.
• the T cells in the composition are at least about 30% central and effector memory cells, or at least about 40% central or effector memory cells, or at least about 50% central or effector memory T cells, or in some embodiments are at least about 70% central or effector memory cells, or at least about 80% central or effector memory T cells, or at least about 90% central or effector memory T cells.
• the cell composition comprises less than about 20% terminally differentiated memory T cells (e.g., TEMRA cells), or less than about 10% or less than about 5% or less than about 4% terminally differentiated memory T cells in some embodiments.
• the CD8+ T cells contain no more than about 30% naive cells, or in some embodiments, no more than about 15% naive cells, or no more than about 10% naive cells, or no more than about 5% naive cells, or no more than about 4% naive cells, or no more than about 3% naive cells, or no more than about 2% naive cells, or no more than about 1.5%, or no more than about 1% naive cells.
• the CD8+ T cells contain from about 1% to about 100% T memory stem cells, or in some embodiments, from about 1% to about 50% T memory stem cells, or in some embodiments, from about 1% to about 25% T memory stem cells, or from about 5% to about 25% T memory stem cells, or from about 5% to about 15% T memory stem cells (i.e., with the total of naive, TSCM, TCM, TEM Temra cells as 100%)
• the TSCM and TCM cells make up from about 30% to about 80% of the memory phenotype, or in some embodiments about 40% to about 80% of the memory phenotype, or in some embodiments about 40% to about 70% of the memory phenotype.
• the T cells specific for the target antigens are at least about 30% central and effector memory cells, or at least about 40% central or effector memory cells, or at least about 50% central or effector memory T cells, or in some embodiments are at least about 70% central or effector memory cells, or at least about 80% central or effector memory T cells, or at least about 90% central or effector memory T cells.
• these memory cells are about 10:90 to about 90: 10 central to effector memory cells.
• these T cells are from about 25:75 to about 75:25 central to effector memory cells.
• the memory T cells are from about 40:60 to about 60:40 central to effector memory T cells.
• the T cells specific for the target antigen(s) are less than about 20% terminally differentiated memory T cells (e.g., TEMRA cells), or less than about 10% or less than about 5% or less than about 4% terminally differentiated memory T cells.
• the T cells specific for target antigens contain no more than about 30% naive cells, or in some embodiments, no more than about 20% naive cells, or in some embodiments, no more than about l5% naive cells, orno more than about 10% naive cells, or no more than about 5% naive cells, or no more than about 2%, or 1.5%, or 1% naive cells.
• the antigen-specific T cells contain from about 1% to about 100% T memory stem cells, or in some embodiments, from about 1% to about 50% T memory stem cells, or in some embodiments, from about 5% to about 25% T memory stem cells, or from about 5% to about 15% T memory stem cells.
• These memory T cells can be created by the enrichment and expansion process with paramagnetic artificial Antigen Presenting Cells (aAPCs). Populations containing predominately TSCM cells can be created be further isolating or enriching for TSCM cells using known techniques, including magnetic enrichment or cell sorting.
• the cell composition is at least 90% T cells, or at least 95% T cells, or at least 98%, or at least 99% T cells.
• T cells are characterized by CD3+ cells.
• the T cells are generally CD8+ or CD4-.
• the terms“CD8+” and“CD4-” are interchangeable unless stated otherwise.
• the isolated cell composition may be characterized by having less than about 10%, or less than about 5% CD4+ T cells, or in some embodiments, less than about 2%, less than about 1.5%, or less than about 1% CD4+ T cells.
• CD4+ cells When expanding CD8+ T cells ex vivo, CD4+ cells have a tendency to overgrow the CD 8+ cells and compete for growth signals, and exogenous CD4+ T cells are not necessary for a robust and durable response upon adoptive transfer.
• CD4+ and CD8+ T cells correlates with response to cancer vaccine therapy with peptide neoantigens.
• Ott PA et al, An immunogenic personal neoantigen vaccine for patients with melanoma. Nature 547(7662):2l7-22l (2017).
• CD4+ and CD8+ T cells are further described as being important for mediating tumor cell destruction. See, Tran E, Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science 344, 641-645 (2014); Sahin U, et al., Personalized RNA mutanome vaccines mobilize nolv- specific therapeutic immunity against cancer. Nature 547(7662):222-226 (2017).
• adoptive cell compositions need only provide substantial numbers of antigen-specific CD8+ T cells, particularly where the phenotype can support a robust and durable response, and particularly where the antigen-specific CD8+ T cells are provided in sufficient numbers.
• the cell composition is substantially CD28+.
• the cell composition is at least about 25%, or at least about 50%, or at least about 75%, or at least about 90% CD28+.
• the antigen-specific T cells display a polyfunctional phenotype upon activation.
• the T cells upon activation the T cells are positive for two or more of: intracellular staining for IL-2, which is a marker for proliferation and memory; IFN-g production, which activates other T cells, and induces memory and upregulation of MHC); production of TNF-a, a pro-inflammatory marker; and CD107A, which is a marker for granzyme release and cytotoxic activity.
• at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the antigen-specific T cells display at least three of these markers.
• At least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the antigen-specific T cells display all four of these markers.
• polyfunctionality is assessed or quantified using target killing assays, which assess the ability of CD8+ cytotoxic T cells to lyse target cells presenting the peptide antigen in complex with MHC.
• the cell composition further comprises gd T cells gd T cells have a distinctive T-cell receptor (TCR) on their surface.
• TCR T-cell receptor
• gd T cells have a TCR that is made up of one g chain and one d chain gd T cells are believed to not require antigen processing and major-histocompatibility-complex (MHC) presentation of peptide epitopes for activation gd T cells may have a role in recognition of lipid antigens and phospho antigens, and can play a role in anti-viral and anti-tumor protection. See, Kalyan and Arthuritz, Defining the nature of human gd T cells: a biographical sketch of the highly emnathetic.
• gd T cells can provide help to the CD8+ cells through release of cytokines, e.g., contributing to the activation, proliferation, and differentiation of CD8+ cells. Further, clinical significance of gd T cells in the context of hematopoietic stem cell transplantation (HSCT) has been observed, and in particular, higher frequencies of gd T cells after transplantation were associated with favorable outcomes.
• HSCT hematopoietic stem cell transplantation
• the cell composition comprises at least about 2% gd T cells, or at least about 5% gd T cells. In some embodiments, the cell composition comprises at least about 10% gd T cells, or at least about 20% gd T cells. In some embodiments, the cell composition comprises at least about 25% gd T cells, or at least about 30%, or at least about 35%, or at least about 40% gd T cells, or at least about 45% gd T cells. In these embodiments, the gd T cells may comprise one or both of Ud ⁇ and V52 cells. In some embodiments, a portion of the gd T cells are CD8+. In various embodiments, the gd T cells are predominately CD28+.
• Cell compositions in accordance with various embodiments can be prepared by enrichment of CD8+ cells that are specific for the target antigen(s) (e.g., tumor associated antigens or viral-associated antigens).
• target antigen(s) e.g., tumor associated antigens or viral-associated antigens.
• This cell population even when predominately naive cells in the source lymphocytes, can be rapidly expanded in culture to arrive at the cell compositions described herein.
• CD4+ cells can be depleted (pre- or post- antigen- specific enrichment) from the lymphocytes using CD4+ cell depletion microbeads.
• paramagnetic beads or nanoparticles may contain monomeric or multimeric (e.g., dimeric) HLA ligands presenting peptide antigens, along with a co-stimulation signal in some embodiments, such as an agonist for CD28 (e.g., an antibody agonist of CD28).
• agonist for CD28 e.g., an antibody agonist of CD28.
• CD28+ cells are also enriched, which can be simultaneous with antigen-specific enrichment.
• CD28 is expressed on T cells, and is a co-stimulatory signal required for T cell activation and survival.
• CD28 is the only B7 receptor constitutively expressed on naive T cells. Association of the TCR of a naive T cell with MHC-antigen complex without CD28 co-stimulation can result in a T cell that is anergic.
• CD28+ cells are not enriched, but a CD28 agonist is added in soluble form during the enrichment process, or added as conjugated to non-paramagnetic beads.
• CD28 in conjugated or non-conjugated form
• the T cells specific for target antigens are specific for from 1 to about 100 target antigens, or from 1 to about 75 target antigens, or from 1 to about 50 target antigens, or from 1 to about 25 target antigens, or from 1 to about 20 target antigens, or from 1 to about 15 target antigens, or from 1 to 10 target antigens, or from 1 to 5 target antigens.
• the distinct target antigens can include overlapping peptide epitopes in some embodiments.
• T cells specific for these peptide antigens can be enriched and expanded in batch, allowing for rapid, parallel production of cell compositions.
• the composition contains T cells specific for from 5 to 15 or from 5 to 10 peptide antigens.
• T cell specificity toward a target peptide antigen in the composition is defined by MHC multimer staining (e.g., dimer or tetramer staining) as is well known in the art.
• a cocktail of nano-aAPCs each aAPC presenting a different, distinct target antigen, can be used to enrich T cells against multiple antigens simultaneously.
• T cells specific for from 2 to 10 antigens can be enriched simultaneously from the lymphocyte source.
• a number of different nano-aAPC batches, each bearing a different MHC-peptide would be combined and used to simultaneously enrich T cells against each of the antigens of interest.
• the resulting T cell pool would be activated against each of these antigens, and expanded together in culture.
• These antigens could be related to a single therapeutic intervention; for example, multiple antigens present on a single tumor or malignant cell.
• the target peptide antigens are generally suitable for presentation by an HLA-A, B, or C molecular complex, and in some embodiments an HLA-A2 molecular complex.
• the target peptide antigens are tumor or cancer associated antigens, including tumor-derived or tumor-specific antigens.
• T cells specific for tumor associated antigens are often very rare, and in many cases undetectable, in the peripheral blood of healthy individuals. Further, the cells are often of a naive phenotype, particularly when using donor T lymphocytes. See, Quintarelli et al, Cytotoxic T lymphocytes directed to the preferentially expressed antigens of melanoma (PRAME) target chronic myeloid leukemia. Blood 2008; 112: 1876-1885. This is often a distinction observed between viral-specific and tumor antigen specific T cells.
• Tumor-associated antigens or“cancer specific antigens” include unique tumor or cancer antigens expressed exclusively by the tumor or malignant cells from which they are derived, shared tumor antigens expressed in many tumors but not in normal adult tissues (oncofetal antigens), and tissue-specific antigens expressed also by the normal tissue from which the tumor arose.
• Tumor associated antigens can be, for example, embryonic antigens, antigens with abnormal post-translational modifications, differentiation antigens, products of mutated oncogenes or tumor suppressors, fusion proteins, or oncoviral proteins.
• the target peptide antigens include one or more associated with or derived from hematological cancer, such as leukemia, lymphoma, or myeloma.
• the hematological malignancy may be acute myeloid leukemia, chronic myelogenous leukemia, childhood acute leukemia, non-Hodgkin’s lymphomas, acute lymphocytic leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, malignant cutaneous T-cells, mycosis fungoids, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, and T-cell rich cutaneous lymphoid hyperplasia.
• the target peptide antigens include one or more associated with or derived from a solid tumor, including melanoma, colon cancer, duodenal cancer, prostate cancer, breast cancer, ovarian cancer, ductal cancer, hepatic cancer, pancreatic cancer, renal cancer, endometrial cancer, testicular cancer, stomach cancer, dysplastic oral mucosa, polyposis, head and neck cancer, invasive oral cancer, non-small cell lung carcinoma, small-cell lung cancer, mesothelioma, transitional and squamous cell urinary carcinoma, brain cancer, neuroblastoma, and glioma.
• a solid tumor including melanoma, colon cancer, duodenal cancer, prostate cancer, breast cancer, ovarian cancer, ductal cancer, hepatic cancer, pancreatic cancer, renal cancer, endometrial cancer, testicular cancer, stomach cancer, dysplastic oral mucosa, polyposis, head and neck cancer, invasive oral cancer, non-small cell lung carcinoma, small-cell
• Oncofetal and embryonic antigens include carcinoembryonic antigen and alpha-fetoprotein (usually only highly expressed in developing embryos but frequently highly expressed by tumors of the liver and colon, respectively), MAGE-l and MAGE-3 (expressed in melanoma, breast cancer, and glioma), placental alkaline phosphatase sialyl-Lewis X (expressed in adenocarcinoma), CA-125 and CA-19 (expressed in gastrointestinal, hepatic, and gynecological tumors), TAG-72 (expressed in colorectal tumors), epithelial glycoprotein 2 (expressed in many carcinomas), pancreatic oncofetal antigen, 5T4 (expressed in gastriccarcinoma), alphafetoprotein receptor (expressed in multiple tumor types, particularly mammary tumors), and M2A (expressed in germ cell neoplasia).
• carcinoembryonic antigen and alpha-fetoprotein
• Tumor-associated differentiation antigens include tyrosinase (expressed in melanoma) and particular surface immunoglobulins (expressed in lymphomas).
• Mutated oncogene or tumor-suppressor gene products include Ras and p53, both of which are expressed in many tumor types, Her-2/neu (expressed in breast and gynecological cancers), EGF-R, estrogen receptor, progesterone receptor, retinoblastoma gene product, myc (associated with lung cancer), ras, p53, nonmutant associated with breast tumors, MAGE-l, and MAGE-3 (associated with melanoma, lung, and other cancers). Fusion proteins include BCR-ABL, which is expressed in chromic myeloid leukemia. Oncoviral proteins include HPV type 16, E6, and E7, which are found in cervical carcinoma.
• Tissue-specific antigens include melanotransferrin and MUC1 (expressed in pancreatic and breast cancers); CD 10 (previously known as common acute lymphoblastic leukemia antigen, or CALLA) or surface immunoglobulin (expressed in B cell leukemias and lymphomas); the a chain of the IL-2 receptor, T cell receptor, CD45R, CD4+/CD8+ (expressed in T cell leukemias and lymphomas); prostate specific antigen and prostatic acid-phosphatase (expressed in prostate carcinoma); GP 100, MelanA/Mart-l, tyrosinase, gp75/brown, BAGE, and S-100 (expressed in melanoma); cytokeratins (expressed in various carcinomas); and CD 19, CD20, and CD37 (expressed in lymphoma).
• CALLA common acute lymphoblastic leukemia antigen
• CALLA surface immunoglobulin
• Tumor-associated antigens also include altered glycolipid and glycoprotein antigens, such as neuraminic acid-containing glycosphingolipids (e.g., GM2 and GD2, expressed in melanomas and some brain tumors); blood group antigens, particularly T and sialylated Tn antigens, which can be aberrantly expressed in carcinomas; and mucins, such as CA-125 and CA-19-9 (expressed on ovarian carcinomas) or the underglycosylated MUC-l (expressed on breast and pancreatic carcinomas).
• neuraminic acid-containing glycosphingolipids e.g., GM2 and GD2, expressed in melanomas and some brain tumors
• blood group antigens particularly T and sialylated Tn antigens, which can be aberrantly expressed in carcinomas
• mucins such as CA-125 and CA-19-9 (expressed on ovarian carcinomas) or the underglycosylated MUC-l (expressed on breast and pancre
• one or more target antigens are associated with bladder cancer, such as one or more of NY-ESO-l, MAGE-A10, and MUC-l antigens.
• one or more target antigens are associated with brain cancer, and may include one or more of NY-ESO-l, Survivin, and CMV antigens.
• one or more target antigens are associated with breast cancer, and may include one or more of MUC-l, Surivin, WT-l, HER-2, and CEA antigens.
• one or more target antigens are associated with cervical cancer, and may include HPV antigen.
• one or more target antigens are associated with colorectal cancer, and may include one or more of NY-ESO-l, Survivin, WT-l, MUC-l, and CEA antigens.
• one or more target antigens are associated with esophageal cancer, and may include NY-ESO-l antigen.
• one or more target antigens may be associated with head and neck cancer, and may include HPV antigen.
• the target antigen is associated with kidney or liver cancer, and may include NY-ESO-l antigen.
• the target antigen is associated with lung cancer, and may include one or more of NY- ESO-l, Survivin, WT-l, MAGE-A10, and MUC-l antigens.
• one or more target antigens is associated with melanoma, and may include one or more of NY-ESO-l, Survivin, MAGE-A10, MART-l, and GP-100.
• one or more peptide antigens are associated with ovarian cancer, and may include one or more of NY-ESO-l, WT-l, and Mesothelin antigen.
• one or more target antigens are associated with prostate cancer, and may include one or more of Survivin, hTERT, PSA, PAP, and PSMA antigens.
• the target antigen is associated with a sarcoma, and may include NY-ESO-l antigen.
• one or more target antigens are associated with lymphoma, and may include EBV antigen.
• one or more target antigens are associated with multiple myeloma, and may include one or more of NY-ESO-l, WT-l, XBP1-US, XBP1-SP, CD138, CS1 (SLAMF7), and SOX2 antigens.
• the target antigens associated with multiple myeloma are two or more of (or three, four, five, or six of) peptide antigens disclosed in US 9,096,681, which is hereby incorporated by reference in its entirety.
• Exemplary peptides comprising antigenic epitopes include XBP1 unspliced (UN)i85-i93, XBPl-US -i92, XBP1 spliced (SP)223-23i, XBPI-SP367-375, CD 138 265 -273, CD138260-268, CSU40-248, CS l 239-247 , NY-ESOl 157-165A, and SOX2ll8 - 127.
• the target antigens comprise NY-ESO-l, WT-l, SOX-2, CD138, and CS1. In some embodiments, the target antigens comprise NY-ESO-l, WT-l, SOX- 2, CD138, CS1, and XBP1-US and/or XBP1-SP. In some embodiments, the peptide antigens comprise NY-ESO-l, WT-l, and SOX-2. See Table 2.
• one or more target antigens are associated with acute myelogenous leukemia or myelodysplastic syndrome, and may include one or more of (including 1, 2, 3, 4, or 5 of) Survivin, WT-l, PRAME, RHAMM, PR3, and Cyclin Al antigens.
• the target antigens include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all target antigens from Table 1 below. Table 1 : Exemplary AML target peptide antigens
• one or more target antigens may include one or more of XBP1-US, XBP1-SP, CD138, CS1, NY-ESOl, SOX2, EBV, Influenza, CMV, RHAMM, PR3, Mart-l/Melan A, gplOO, CMVpp65, and Influenza Matrix Protein Ml antigens.
• the target antigens include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 target antigens from Table 2 below, which are useful for targeting multiple myeloma, melanoma, or various viral or infectious diseases.
• one or more target peptide antigens are neoantigens.
• neoantigens specific to the patient are identified, and synthesized for loading aAPCs.
• between three and ten neoantigens are identified through genetic analysis of the patient’s malignancy (e.g., by nucleic acid sequencing of malignant cells), followed by predictive bioinformatics.
• the antigens are natural, non-mutated, cancer antigens, of which many are known.
• At least one of the target peptide antigens is recognized by a low frequency precursor T cell.
• the invention enables rapid activation and expansion of these cells for adoptive therapy.
• the target peptide antigens include at least one that is associated with or derived from a pathogen, such as a viral, bacterial, fungal, or parasitic pathogen.
• a pathogen such as a viral, bacterial, fungal, or parasitic pathogen.
• at least one peptide antigen may be associated with HIV, hepatitis (e.g., A, B, C, or D) CMV, Epstein-Barr virus (EBV), influenza, herpes virus (e.g., HSV 1 or 2, or varicella zoster), and Adenovirus.
• CMV for example, is the most common viral pathogen found in organ transplant patients and is a major cause of morbidity and mortality in patients undergoing bone marrow or peripheral blood stem cell transplants.
• the patient may receive adoptive immunotherapy comprising T cells specific for pathogen antigens.
• the method can entail generation of virus-specific CTL derived from the patient or from an appropriate donor before initiation of the transplant procedure.
• At least one target antigen is a pathogen-associated antigen, including antigens associated with protozoa, bacteria, fungi (both unicellular and multicellular), viruses, prions, intracellular parasites, helminths, and other infectious agents.
• Bacterial antigens include antigens of gram-positive cocci, gram positive bacilli, gram-negative bacteria, anaerobic bacteria, such as organisms of the families Actinomycetaceae, Bacillaceae, Bartonellaceae, Bordetellae, Captophagaceae, Corynebacteriaceae, Enterobacteriaceae, Legionellaceae, Micrococcaceae, My cobacteriaceae, Nocardiaceae, Pasteurellaceae, Pseudomonadaceae, Spirochaetaceae,
• Antigens of protozoan infectious agents include antigens of malarial plasmodia, Leishmania species, Trypanosoma species and Schistosoma species.
• Fungal antigens include antigens of Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus, Histoplasma, Paracoccicioides, Sporothrix, organisms of the order Mucorales, organisms inducing choromycosis and mycetoma and organisms of the genera Trichophyton, Microsporum, Epidermophyton, and Malassezia.
• Viral peptide antigens include, but are not limited to, those of adenovirus, herpes simplex virus, papilloma virus, respiratory syncytial virus, poxviruses, HIV, influenza viruses, EBV, hepatitis, and CMV.
• HIV proteins such as HIV gag proteins (including, but not limited to, membrane anchoring (MA) protein, core capsid (CA) protein and nucleocapsid (NC) protein), HIV polymerase, influenza virus matrix (Ml) protein and influenza virus nucleocapsid (NP) protein, hepatitis B surface antigen (HBsAg), hepatitis B core protein (HBcAg), hepatitis e protein (HBeAg), hepatitis B DNA polymerase, hepatitis C antigens, and the like.
• HIV gag proteins including, but not limited to, membrane anchoring (MA) protein, core capsid (CA) protein and nucleocapsid (NC) protein
• HIV polymerase HIV polymerase
• HBsAg hepatitis B surface antigen
• HBcAg hepatitis B core protein
• HBeAg hepati
• the target peptide antigens include one or more tumor associated antigens, and one or more virus-associated antigens (such as CMV, EBV, influenza, or Adenovirus), to provide an antitumor response while protecting against common pathogens that complicate recovery after HSCT.
• virus-associated antigens such as CMV, EBV, influenza, or Adenovirus
• EBV infection is believed to be present in approximately 90% of the adult population in the United States. Active viral replication and infection is kept in check by the immune system, but, as in cases of CMV, individuals immunocompromised by transplantation therapies lose the controlling T cell populations, which permits viral reactivation. This represents a serious impediment to transplant protocols. EBV may also be involved in tumor promotion in a variety of hematological and non-hematological cancers.
• the cell composition comprises T cells specific for tumor associated antigens, with pathogen-associated T cells provided as bystander cells. Specifically, by enriching for CD8+ T cells based on selection with both HLA-peptide complexes and anti-CD28, bystander cells will be enriched, and expanded, particularly when using a T cell growth factor cocktail that can drive some non-specific expansion of these cells without antigen-specific activation.
• T cells specific for the target peptides e.g., from 5% to 75%, or from 10 to 50%
• the remaining T cells provide some reconstitution of the immune system against common pathogens, which is particularly beneficial after transplant.
• the composition may comprise T cells specific for CMV, EBV, influenza, and Adenovirus.
• pathogen-specific T cells may be present at from 0.1% to about 4% of the composition.
• the invention involves compositions prepared by enrichment and expansion of antigen-specific CD8+ T cells.
• Precursor T cells can be obtained from the patient or from a suitable HLA-matched donor.
• Source T cells can be either fresh or frozen samples.
• Precursor T cells can be obtained from a number of sources that comprise WBCs, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, spleen tissue, buffy coat fraction, and tumors.
• PBMC peripheral blood mononuclear cells
• precursor T cells are obtained from a unit of blood collected from a subj ect using any number of techniques known to one or skill in the art.
• precursor T cells from the circulating blood of an individual can be obtained by apheresis or leukapheresis.
• the apheresis product typically contains lymphocytes, including T cells and precursor T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
• Leukapheresis is a laboratory procedure in which white blood cells are separated from a sample of blood.
• Cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. Washing steps can be accomplished by methods known to those in the art, such as by using a semi-automated“flow-through” centrifuge. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg- free PBS. Alternatively, the undesirable components of the apheresis sample can be removed and the cells directly re-suspended in a culture medium.
• precursor T cells can be isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient.
• leukocytes are collected by leukapheresis, and may be subsequently enriched for CD8+ T cells, for example, by depleting the sample of CD4+ cells and/or positively enriching for CD8+ cells.
• other cell types are depleted, such as NK cells.
• the CD8-enriched cells may then be further enriched for antigen-specific T cells.
• the sample comprising the immune cells is contacted with an artificial Antigen Presenting Cell (aAPC) having magnetic properties.
• aAPC Antigen Presenting Cell
• Paramagnetic materials have a small, positive susceptibility to magnetic fields. These materials are attracted by a magnetic field and the material does not retain the magnetic properties when the external field is removed.
• Exemplary paramagnetic materials include, without limitation, magnesium, molybdenum, lithium, tantalum, and iron oxide.
• Paramagnetic beads suitable for magnetic enrichment are commercially available (DYNABEADSTM, MACS MICROBEADSTM, Miltenyi Biotec).
• the aAPC particle is an iron dextran bead (e.g., dextran-coated iron-oxide bead).
• Antigen presenting complexes comprise an antigen binding cleft, and are generally MHC class I, which can be linked or tethered to provide dimeric or multimeric MHC.
• the MHC are monomeric, but their close association on the nano-particle is sufficient for avidity and activation.
• the MHC are dimeric.
• Dimeric MHC class I ligands can be constructed by fusion to immunoglobulin heavy chain sequences, which are then associated through one or more disulfide bonds (with or without associated light chains).
• MHC multimers can be created by direct tethering through peptide or chemical linkers, or can be multimeric via association with streptavidin through biotin moieties.
• the antigen presenting complexes are MHC class I complexes involving fusions with immunoglobulin sequences.
• MHC class I molecular complexes having immunoglobulin sequences are described in U.S. Patent 6,268,411, which is hereby incorporated by reference in its entirety. These MHC class I molecular complexes may be formed in a conformationally intact fashion at the ends of immunoglobulin heavy chains. MHC class I molecular complexes to which antigenic peptides are bound can stably bind to antigen-specific lymphocyte receptors (e.g., T cell receptors).
• the immunoglobulin heavy chain sequence is not full length, but comprises an Ig hinge region, and one or more of CH1, CH2, and/or CH3 domains.
• the Ig sequence may or may not comprise a variable region, but where variable region sequences are present, the variable region may be full or partial.
• the complex may further comprise immunoglobulin light chains.
• MHC class I ligands e.g., HLA-Ig
• Exemplary MHC class I molecular complexes comprise at least two fusion proteins.
• a first fusion protein comprises a first MHC class I a chain and a first immunoglobulin heavy chain (or portion thereof comprising the hinge region), and a second fusion protein comprises a second MHC class I a chain and a second immunoglobulin heavy chain (or portion thereof comprising the hinge region).
• the first and second immunoglobulin heavy chains associate to form the MHC class I molecular complex, which comprises two MHC class I peptide-binding clefts.
• the immunoglobulin heavy chain can be the heavy chain of an IgM, IgD, IgGl, IgG3, 3 ⁇ 4 ⁇ 2b, IgG2a, IgG4, IgE, or IgA.
• an IgG heavy chain is used to form MHC class I molecular complexes. If multivalent MHC class I molecular complexes are desired, IgM or IgA heavy chains can be used to provide pentavalent or tetravalent molecules, respectively.
• Exemplary class I molecules include HLA-A, HLA-B, HLA-C, HLA-E, and these may be employed individually or in any combination.
• the antigen presenting complex is an HLA-A2 ligand.
• MHC as used herein, can be replaced by HLA in each instance.
• Immunoglobulin sequences in some embodiments are humanized monoclonal antibody sequences.
• the aAPCs may contain a“Signal 2”, such as an anti-CD28 ligand.
• Signal 2 is generally a T cell affecting molecule, that is, a molecule that has a biological effect on a precursor T cell or on an antigen-specific T cell.
• signal 2 is a T cell costimulatory molecule. T cell costimulatory molecules contribute to the activation of antigen-specific T cells.
• Such molecules include, but are not limited to, molecules that specifically bind to CD28 (including antibodies), CD80 (B7-1), CD86 (B7-2), B7-H3, 4- 1BB, 4-1BBL, CD27, CD30, CD134 (OX-40L), B7h (B7RP-1), CD40, LIGHT, antibodies that specifically bind to HVEM, antibodies that specifically bind to CD40L, and antibodies that specifically bind to 0X40.
• the costimulatory molecule is an antibody (e.g., a monoclonal antibody) or portion thereof, such as F(ab’)2, Fab, scFv, or single chain antibody, or other antigen binding fragment.
• the antibody is a humanized monoclonal antibody or portion thereof having antigen-binding activity, or is a fully human antibody or portion thereof having antigen-binding activity.
• Combinations of co-stimulatory ligands that may be employed (on the same or separate nanoparticles) include anti-CD28/anti-CD27 and anti-CD28/anti-4lBB. The ratios of these co-stimulatory ligands can be varied to effect expansion.
• Exemplary signal 1 and signal 2 ligands are described in WO 2014/209868, which describe ligands having a free sulfhydryl (e.g., unpaired cysteine), such that the constant region may be coupled to nanoparticle supports having the appropriate chemical functionality.
• a free sulfhydryl e.g., unpaired cysteine
• Adhesion molecules useful for nano-aAPC can be used to mediate adhesion of the nano-aAPC to a T cell or to a T cell precursor.
• Useful adhesion molecules include, for example, ICAM-l and LFA-3.
• signal 1 is provided by peptide-HLA-A2 complexes
• signal 2 is provided by B7. l-Ig or anti-CD28.
• An exemplary anti-CD28 monoclonal antibody is 9.3 mAb (Tan et al, J. Exp. Med. 1993 177: 165), which may be humanized in certain embodiments and/or conjugated to the bead as a fully intact antibody or an antigen-binding fragment thereof.
• Magnetic activation may take place for from 2 minutes to 5 hours, or from 5 minutes to 2 hours, followed by expansion in culture for at least 5 days, and up to 2 weeks or up to 3 weeks in some embodiments. In some embodiments, magnetic activation occurs for at least 2 minutes, but less than 30 minutes or less than 15 minutes (e.g., about 5 or 10 minutes).
• Resulting CD8+ T cells may be phenotypically characterized to confirm the presence of T memory stem cells (T S cm), as well as high central and effector memory phenotype.
• T cell growth factors during expansion, which affect proliferation and/or differentiation of T cells.
• T cell growth factors include cytokines (e.g., interleukins, interferons) and superantigens.
• cytokines can be present in molecular complexes comprising fusion proteins, or can be encapsulated by the aAPC, or provided in soluble form.
• Particularly useful cytokines include MIR-Ib, IL- 1b, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, IFN-g, and CXCL10.
• the growth factors include 3, 4, 5, or 6 from MIR-Ib, IE-1b, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL-21, and INF-g.
• the cells are expanded in culture in the presence cytokines including one, two, three cytokines selected from MIR-Ib, I ⁇ - ⁇ b, IL-6, and IL-10.
• the cells are not cultured in the presence of IL-7 and/or IL-21 and/or IL-15.
• Cells can be expanded in culture from 1 to 4 weeks, such as about 2 weeks (about 14 days), or about 3 weeks.
• the cells are expanded in culture in the presence of from 4 to 8 cytokines, to achieve a balance between T cell expansion (including antigen- specific T cell expansion), activation, and memory phenotype.
• the cells are expanded in the presence of IL-4.
• the cells are expanded in the presence of IL-4 and IL-6.
• the cells are expanded in the presence of IL-4 and I ⁇ -1b.
• the cells are expanded in the presence of IL-4, IL-6, and I ⁇ -1b.
• the cells are expanded in the presence of IL-2, IL-4, and IL-6.
• the cells are expanded in culture in the presence of IL-2, IL-4, IL-6, INF-g, and I ⁇ -1b. In some embodiments, the cells are further expanded in the presence of IL-10. In various embodiments, these cytokines are used in conjunction with artificial or natural antigen presenting cells to expand antigen specific T cells.
• the growth factors consist, or consist essentially of, IL-2, IL-4, IL-6, INF-g, I ⁇ - ⁇ b, and optionally IL-10.
• IL-2 is present at the start of culture at 10 to 200 International Units (IU) per ml, such as from about 20 to about 100 IU/ml, or about 20 to about 60 IU/ml. In some embodiments, IL-2 is present at the start of culture at about 30 to about 50 IU/ml (e.g., about 40 IU/ml). IL-2 IU (86/500 NIBSC) can be determined using a proliferation assay (e.g., using CTLL-2 cell line), as described for example by Gearing and Bird (1987) in Lymphokines and Interferons A Practical Approach. Clemens, MJ et al. (eds): IRL Press. 295. In some embodiments, IL-2 is present at the start of culture at about 2 to about 25 ng/ml, or at about 2 to about 15 ng/ml, such as from about 5 to about 15 ng/ml.
• IU International Units
• IL-4 is present at the start of culture at 0.2 to 25 International Units (IU) per ml, such as from about 0.5 to about 10 IU/ml, or from about 0.5 to about 5 IU/ml. In some embodiments, IL-4 is present at the start of culture at about 1 IU/ml.
• IL-4 IU (88/656 NIBSC) can be defined using a proliferation assay (e.g., using TF-l cell line), as described for example, by Kitamura T. et al, (1991) IL-l up-regulates the expression of cytokine receptors on a factor-dependent human hemopoietic cell line TF-l. Int. Immunol.
• IL-4 is present at the start of culture at about 0.2 to about 2 ng/ml, such as from about 0.2 to about 1 ng/ml (e.g., about 0.5 ng/ml).
• IL-6 may be present at the start of culture at 10 to 200 International Units (IU) per ml, such as from about 25 to about 100 IU/ml, such as from 25 to 75 IU/ml. In some embodiments, IL-6 is present at the start of culture at about 40 to about 60 IU/ml (e.g., about 50 IU/ml).
• IL-6 IU 89/548 NIBSC
• a proliferation assay e.g., using B9 cell line
• IL-6 is present at the start of culture at about 0.2 to about 10 ng/ml, such as from about 0.2 to about 5 ng/ml (e.g., about 0.2 to 1 ng/ml, or about 0.5 to 2 ng/ml).
• Interferon gamma may be present at the start of culture at from 10 to 200 International Units (IU) per ml, such as from about 20 to about 100 IU/ml, such as from 20 to 60 IU/ml. In some embodiments, INF-g is present at the start of culture at about 30 to about 50 IU/ml (e.g., about 40 IU/ml). INF- g IU (87/586 NIBSC) can be defined using an antiviral assay (e.g., with Hela cells infected with EMC), as described for example in Meager A. (1987) in Lvmphokines and interferons a Practical Approach.
• an antiviral assay e.g., with Hela cells infected with EMC
• INF-g is present at the start of culture at about 0.5 to about 20 ng/ml, such as from about 0.5 to about 10 ng/ml, or from about 0.5 to about 5 ng/ml, or from about 1 to about 10 ng/ml (e.g., from 1 to 5 ng/ml).
• IE-1b may be present at the start of culture at 5 to 100 International Units (IU) per ml, such as from about 10 to about 50 IU/ml, such as from about 10 to about 30 IU/ml. In some embodiments, IL- I b is present at the start of culture at about 10 to about 20 IU/ml (e.g., about 15 IU/ml).
• IE-1b IU (86/680 NIBSC) can be defined using a proliferation assay (e.g., using D.10.G4.1 cells), as described for example by Poole, S. and Gaines-Das, RE (1991) The international standards for interleukin- 1 alpha and interleukin- 1 beta. Evaluation in an international collaborative study. J. Immunol.
• P b is present at the start of culture at about 0.1 to 5 ng/ml, or at about 0.2 to about 5 ng/ml, such as from about 0.2 to about 2 ng/ml, or from about 0.2 to about 1 ng/ml.
• the cells are cultured in the presence of a growth factor cocktail comprising or consisting of IL-2, IL-4, IL-6, INF-g, and IL- 1 b.
• a growth factor cocktail comprising or consisting of IL-2, IL-4, IL-6, INF-g, and IL- 1 b.
• the relative activity (defined by the respective IU) of IL-2 and INF-g is about 0.5: 1 to about 1 :0.5 (e.g., about 1: 1).
• the relative activity (defined by respective IU) of IL-2 and IL-6 is about 0.5: 1 to 1 :0.5.
• the relative activity of I ⁇ - ⁇ b with respect to IL-2, IL- 6, and/or IFN-g is from 1:4 to 1:2 (e.g., about 1:3).
• the relative activity of IL-4 with respect to IL-2, IL-6, and/or IFN-g is from 1:30 to 1:60.
• the relative activity of IL-4 with respect to I ⁇ -1b is from about 1:5 to about 1 :25, such as from about 1 : 10 to about 1 :20.
• the specific activity of each growth factor (IL-2, IL-4, IL- 6, INF-g, and I L- 1 b ) at the start of culture (in IUs) can be shown as a percentage when the total IUs of all the growth factors in the culture is considered as 100%.
• the percentage of each growth factor in the culture can be as follows:
• IL-2 20% to 40% IL-2 (e.g., 20 to 30% IL-2);
• IL-4 0.5% to 5% IL-4 (e.g., 1 to 3% IL-4);
• IL-6 25% to 50% IL-6 (e.g., 30 to 40% IL-6);
• IFN-g 20% to 40% IFN-g (e.g., 20 to 30% IFN-g);
• I ⁇ - ⁇ b 5% to 20% I ⁇ - ⁇ b (e.g., 5 to 15% I ⁇ - ⁇ b).
• the aAPC nanoparticles can be made of any material, and materials can be appropriately selected for the desired magnetic property, and may comprise, for example, metals such as iron, nickel, cobalt, or alloy of rare earth metal.
• Paramagnetic materials also include magnesium, molybdenum, lithium, tantalum, and iron oxide.
• Paramagnetic beads suitable for enrichment of materials (including cells) are commercially available, and include iron dextran beads, such as dextran-coated iron oxide beads.
• nanoparticles can also be made of nonmetal or organic (e.g., polymeric) materials such as cellulose, ceramics, glass, nylon, polystyrene, rubber, plastic, or latex.
• exemplary material for preparation of nanoparticles is poly(lactic-co-glycolic acid) (PLGA) or PLA and copolymers thereof, which may be employed in connection with these embodiments.
• PLGA poly(lactic-co-glycolic acid)
• PLA poly(lactic-co-glycolic acid)
• copolymers thereof which may be employed in connection with these embodiments.
• Other materials including polymers and co-polymers that may be employed include those described in PCT/US2014/25889, which is hereby incorporated by reference in its entirety.
• the particle has a size (e.g., average diameter) within about 10 to about 500 nm, or within about 40 to about 400 nm, or within about 40 nm to 200 nm.
• the nanoparticles have a size (mean diameter) in the range of 10 to 250 nm, or 50 to 200 nm, or 80 to 200 nm, or 20 to 100 nm in some embodiments.
• Receptor-ligand interactions at the cell-nanoparticle interface are not well understood.
• nanoparticle binding and cellular activation are sensitive to membrane spatial organization, which is particularly important during T cell activation, and magnetic fields can be used to manipulate cluster-bound nanoparticles to enhance activation. For example, T cell activation induces a state of persistently enhanced nanoscale TCR clustering and nanoparticles are sensitive to this clustering in a way that larger particles are not.
• T cell activation is mediated by aggregation of signaling proteins, with“signaling clusters” hundreds of nanometers across, initially forming at the periphery of the T cell-APC contact site and migrating inward.
• an external magnetic field can be used to enrich antigen-specific T cells (including rare naive cells) and to drive aggregation of magnetic nano-aAPC bound to TCR, resulting in aggregation of TCR clusters and enhanced activation of naive T cells.
• Magnetic fields can exert appropriately strong forces on paramagnetic particles, but are otherwise biologically inert, making them a powerful tool to control particle behavior.
• T cells bound to paramagnetic nano-aAPC are activated in the presence of an externally applied magnetic field.
• Nano-aAPC are themselves magnetized, and attracted to both the field source and to nearby nanoparticles in the field, inducing bead and thus TCR aggregation to boost aAPC-mediated activation.
• Activation chemistries can be used to allow the specific, stable attachment of molecules to the surface of nanoparticles.
• proteins to functional groups There are numerous methods that can be used to attach proteins to functional groups.
• the common cross-linker glutaraldehyde can be used to attach protein amine groups to an aminated nanoparticle surface in a two-step process. The resultant linkage is hydrolytically stable.
• cross-linkers containing n-hydrosuccinimido (NHS) esters which react with amines on proteins cross-linkers containing active halogens that react with amine-, sulfhydryl-, or histidine-containing proteins, cross-linkers containing epoxides that react with amines or sulfhydryl groups, conjugation between maleimide groups and sulfhydryl groups, and the formation of protein aldehyde groups by periodate oxidation of pendant sugar moieties followed by reductive amination.
• NHS n-hydrosuccinimido
• nanoparticles can be coupled with HLA- A2-Ig and anti-CD28 (or other signal 2 ligands) at a variety of ratios, such as about 30: 1, about 25: 1, about 20: 1, about 15: 1, about 10: 1, about 5: 1, about 3: 1, about 2: 1, about 1: 1, about 0.5: 1, about 0.3: 1; about 0.2: 1, about 0.1 : 1, or about 0.03: 1. In some embodiments, the ratio is from 2: 1 to 1:2.
• the total amount of protein coupled to the supports may be, for example, about 250 mg/ml, about 200 mg/ml, about 150 mg/ml, about 100 mg/ml, or about 50 mg/ml of particles. Because effector functions such as cytokine release and growth may have differing requirements for Signal 1 versus Signal 2 than T cell activation and differentiation, these functions can be determined separately.
• the aAPCs are paramagnetic particles in the range of 50 to 150 nm, with a PDI (size distribution) of less than 0.2, or in some embodiments less than 0.1.
• the aAPCs may have a surface charge of from 0 to -10 mV, such as from about -2 to -6 mV.
• aAPCs may have from 10 to 120 ligands per particle, such as from about 25 to about 100 ligands per particle, with ligands conjugated to the particle through a free cysteine introduced in the Fc region of the immunoglobulin sequences.
• the particles may contain about 1: 1 ratio of HLA dimer: anti-CD28, which may be present on the same or different populations of particles.
• the nanoparticles provide potent expansion of cognate T cells, while exhibiting no stimulation of non-cognate TCRs, even with passive loading of peptide antigen. Particles are stable in lyophilized form for at least two or three years.
• the antigen-specific T cell component of the sample will be at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25% antigen specific T cells.
• these T cells comprise T memory stem cells, and can also comprise central and effector memory T cells.
• the antigen-specific T cells in various embodiments are expanded (in about 7 days) from about lOO-fold to about 10,000 fold, such as at least about lOO-fold, or at least about 200-fold. After 2 weeks, antigen-specific T cells are expanded at least 1000-fold, or at least about 2000-fold, at least about 3,000 fold, at least about 4,000-fold, or at least about 5,000-fold in various embodiments. In some embodiments, antigen-specific T cells are expanded by greater than 5000-fold or greater than 10,000 fold after two weeks. After one or two weeks of expansion, at least about 10 6 , or at least about 10 7 , or at least about 10 8 , or at least about 10 9 antigen-specific T cells are obtained.
• Suitable incubation conditions include those used to culture T cells or T cell precursors, as well as those known in the art for inducing formation of antigen-specific T cells using DC or artificial antigen presenting cells.
• the cell composition can be administered to patients by any appropriate routes, including intravenous infusion, intra-arterial administration, intralymphatic administration, and intratumoral administration.
• the patient receives or initiates immunotherapy with one or more checkpoint inhibitors, prior to (or optionally after) receiving the cell composition by adoptive transfer.
• the checkpoint inhibitor(s) target one or more of CTLA-4 or PD-1/PD-L1, which may include antibodies against such targets, such as monoclonal antibodies, or portions thereof, or humanized or fully human versions thereof.
• the checkpoint inhibitor therapy comprises ipilimumab or Keytruda (pembrolizumab), or comparable monoclonal antibody.
• the patient previously received PD1 blockade therapy, and was refractory or only partially responsive to that treatment.
• the cell composition described herein can restore a robust T cell response, optionally in combination with a second round of immunotherapy (e.g., anti-CTLA4 or PD-l blockade therapy).
• a second round of immunotherapy e.g., anti-CTLA4 or PD-l blockade therapy.
• the patient receives about 1 to 5 rounds of adoptive immunotherapy (e.g., one, two, three, four or five rounds).
• each administration of adoptive immunotherapy is conducted simultaneously with, or after (e.g., from about 1 day to about 1 week after), a round of checkpoint inhibitor therapy.
• adoptive immunotherapy is provided about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 1 week after a checkpoint inhibitor dose.
• the patient receives only a single administration of the cell composition.
• the invention provides methods for personalized cancer immunotherapy.
• the methods are accomplished using the aAPCs to identify antigens to which the patient will respond, followed by administration of the appropriate peptide- loaded aAPC to the patient, or followed by enrichment and expansion of the antigen specific T cells ex vivo.
• Genome-wide sequencing also has the potential to revolutionize our approach to cancer immunotherapy. Sequencing data can provide information about both shared as well as personalized targets for cancer immunotherapy. In principle, mutant proteins are foreign to the immune system and are putative tumor-specific antigens. Indeed, sequencing efforts have defined hundred if not thousands of potentially relevant immune targets. Limited studies have shown that T cell responses against these neo-epitopes can be found in cancer patients or induced by cancer vaccines. However, the frequency of such responses against a particular cancer and the extent to which such responses are shared between patients are not well known. One of the main reasons for our limited understanding of tumor-specific immune responses is that current approaches for validating potential immunologically relevant targets are cumbersome and time consuming.
• the nano-aAPC system is used to screen for neo-epitopes that induce a T cell response in a variety of cancers, or in a particular patient’s cancer.
• Cancers may be genetically analyzed, for example, by whole exome-sequencing.
• a list of candidate peptides can be generated from overlapping nine amino acid windows in mutated proteins. All nine-AA windows that contain a mutated amino acid, and 2 non-mutated“controls” from each protein will be selected. These candidate peptides will be assessed computationally for MHC binding using a consensus of MHC binding prediction algorithms, including Net MHC and stabilized matrix method (SMM). Nano-aAPC and MHC binding algorithms have been developed primarily for HLA-A2 allele. The sensitivity cut-off of the consensus prediction can be adjusted until a tractable number of mutation containing peptides (-500) and non-mutated control peptides (-50) are identified.
• the cell composition comprises, in a pharmaceutically acceptable carrier: at least 70%, at least 80% or at least 90% CD8+ or CD4- T cells and less than 5% CD4+ T cells; and at least 5% TSCM cells, where the CD8+ cells comprise at least 10 6 T cells specific for from 1 to 10 target peptide antigens.
• the CD8+ or CD4- T cells may comprise T cells specific for bacterial, viral, fungal and/or parasitic pathogens.
• the CD8+ or CD4- T cells are TSCM, central, and effector memory T cells, with less than 10% of the CD8+ or CD4- T cells being terminally differentiated T cells and less than 10% of the CD8+ or CD4- cells being naive cells.
• at least 50% of the CD8+ or CD4- T cells specific for the target peptide antigens are TSCM, central and effector memory T cells.
• the cell composition comprises from about 5% at about 25% T memory stem cells (Tscm), or from about 5% to about 20% T memory stem cells.
• the cell composition further comprises gd T cells.
• the cell composition may comprise at least about 2% gd T cells, or at least about 5% gd T cells.
• the cell composition comprises at least about 10% gd T cells, or at least about 20% gd T cells.
• the cell composition comprises at least about 25% gd T cells, or at least about 30%, or at least about 35%, or at least about 40% gd T cells.
• the gd T cells may comprise one or both of Vbl and V52 cells.
• the gd T cells are predominantly V52 (e.g., at least about 60% or at least about 75%).
• a portion of the gd T cells are CD8+.
• the gd T cells are predominately CD28+.
• the cell composition further comprises a pharmaceutically acceptable carrier suitable for intravenous infusion, and which may be suitable as a cryoprotectant.
• a pharmaceutically acceptable carrier suitable for intravenous infusion, and which may be suitable as a cryoprotectant.
• An exemplary carrier is DMSO (e.g., about 10%).
• Cell compositions may be provided in unit vials or bags, and stored frozen until use. Unit doses may comprise from about 5 x 10 5 to about 5 x 10 6 cells per ml, in a volume of from 50 to 200 ml. In certain embodiments, the volume of the composition is ⁇ 100 ml (e.g., from 50 to 100 ml).
• the invention provides a method for treating a patient with cancer, comprising administering the cell composition described herein to a patient in need.
• the patient has a hematological cancer, which in some embodiments has relapsed after allogeneic stem cell transplantation. In some embodiments, the patient has acute myelogenous leukemia (AML) or myelodysplastic syndrome.
• AML acute myelogenous leukemia
• Exemplary cancers include various types of solid tumors, including carcinomas, sarcomas, and lymphomas.
• the cancer is melanoma (including metastatic melanoma), colon cancer, duodenal cancer, prostate cancer, breast cancer, ovarian cancer, ductal cancer, hepatic cancer, pancreatic cancer, renal cancer, endometrial cancer, testicular cancer, stomach cancer, dysplastic oral mucosa, polyposis, head and neck cancer, invasive oral cancer, non-small cell lung carcinoma, small-cell lung cancer, mesothelioma, transitional and squamous cell urinary carcinoma, brain cancer, neuroblastoma, and glioma.
• the cancer is stage I, stage II, stage III, or stage IV.
• the cancer is metastatic and/or recurrent, and/or is nonresectable
• the patient is refractory to chemotherapy and/or checkpoint inhibitor therapy.
• the patient further receives low dose cytokine therapy, which may improve the persistence and in vivo response.
• the cancer is a hematological malignancy, including leukemia, lymphoma, or myeloma.
• the hematological malignancy may be acute myeloid leukemia, chronic myelogenous leukemia, childhood acute leukemia, non- Hodgkin’s lymphomas, acute lymphocytic leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, malignant cutaneous T-cells, mycosis fungoids, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, and T-cell rich cutaneous lymphoid hyperplasia.
• the patient has a hematological cancer such as acute myelogenous leukemia (AML) or myelodysplastic syndrome, and in some embodiments the patient has relapsed after allogeneic stem cell transplantation.
• the therapy does not induce GVHD.
• the patient in addition to allogeneic stem cell transplantation, has also undergoes lympho-deleting therapy, cyto-reductive therapy, or immunomodulatory therapy (prior to administration of the cell therapy).
• the cell therapy may be further provided with or without cytokine support post treatment.
• the patient has an infectious disease or is at risk for an infectious disease.
• patients that have undergone HSCT are at particular risk for infectious disease, given the immunocompromised state.
• Infectious diseases that can be treated or prevented include those caused by bacteria, viruses, prions, fungi, parasites, helminths, etc.
• Such diseases include AIDS, hepatitis B/C, CMV infection, Epstein-Barr virus (EBV) infection, influenza, herpes virus infection (including shingles), and adenovirus infection.
• CMV for example, is the most common viral pathogen found in organ transplant patients and is a major cause of morbidity and mortality in patients undergoing bone marrow or peripheral blood stem cell transplants.
• the patient may receive adoptive immunotherapy comprising T cells specific for pathogen antigens.
• the method can entail generation of virus-specific CTL derived from the patient or from an appropriate donor before initiation of the transplant procedure.
• PTLD occurs in a significant fraction of transplant patients and results from Epstein-Barr virus (EBV) infection.
• EBV infection is believed to be present in approximately 90% of the adult population in the United States. Active viral replication and infection is kept in check by the immune system, but, as in cases of CMV, individuals immunocompromised by transplantation therapies lose the controlling T cell populations, which permits viral reactivation. This represents a serious impediment to transplant protocols.
• EBV may also be involved in tumor promotion in a variety of hematological and non-hematological cancers.
• the invention provides a method for making a population of gd T cells.
• the method comprises expanding a population of T cells in the presence of two or more of IL-2, IL-4, IL-6, INF-g, and IE-1b.
• the population of T cells may be enriched for CD28+ enriched cells, e.g., may be positively selected with anti- CD28 containing beads or particles, including aAPCs as described herein.
• the population of cells is CD4+ depleted or CD8+ selected.
• the starting composition comprises less than about 20% or less than about 10% or less than about 8%, or less than about 5% gd T cells.
• source cells are from peripheral blood.
• the population of T cells are expanded in the presence of IL-4, or are expanded in the presence of IL-4 and IL-6. In some embodiments, the cells are expanded in the presence of IL-4 and IE-1b. In some embodiments, the cells are expanded in the presence of IL-4, IL-6, and IL- 1 b. In some embodiments, the cells are expanded in the presence of IL-2, IL-4, and IL-6. In some embodiments, the cells are expanded in culture in the presence of IL-2, IL-4, IL-6, INF-g, and IL- I b. Expansion of cells in culture can take place as described herein, such as for 1 to 4 weeks.
• the percent cells that are gd may be between about 5% and about 60%, such as between about 10% and about 60%, or from about 15% to about 60%, with the numbers of gd T cells expanded by at least about 100 or at least about 1000, or at least about 10,000, compared to the starting population of cells.
• gd T cells can be separated from other cells using known methods, such as FACS or magnetic cell sorting
• gd T cells can be provided as a cell composition for adoptive transfer or research use, and alternatively may be engineered to express one or more heterologous genes, such as a T cell receptor, which is an optionally an ab TCR.
• the gd T cells are engineered to heterologously express a chimeric antigen receptor (CAR).
• CAR chimeric antigen receptor
• PBMCs fresh PBMCs were obtained from a donor by leukapheresis, as schematically shown in FIGURE 2.
• Cells were depleted of CD4+ cells by negative selection with anti-CD4 microbeads.
• Resulting cells were enriched for antigen-specific T cells by incubating with paramagnetic nanoparticles (i.e., dextran-coated iron oxide nanoparticles or PLGA-PEG nanoparticles, ranging in size from about 80-200 nm in diameter).
• the nanoparticles have dimeric HLA ligands conjugated to the surface (presenting the target peptide antigen) that can incorporate multiple tumor specific antigenic peptides.
• the dimeric HLA ligand contains two HLA-A2 domains, comprising the peptide binding clefts, each fused to an arm of the Ig hinge region.
• Dimeric HLA-Ig are co-expressed with b 2 microglobulin.
• a dimeric HLA ligand, such as an HLA-IgG4 hinge dimer, can be readily modified for multiple HLA-subtypes and provide direct engagement with target T cells.
• Co stimulatory or inhibitory ligands, such as an anti-CD28 monoclonal antibody, are also conjugated to the nanoparticle, as shown in FIGURE 1.
• the co-stimulatory or inhibitory ligands provide specific instructions (e.g activation, suppression) to target T cells (i.e., naive T cells or memory T cells) relative to the therapeutic goal.
• Target T cells i.e., naive T cells or memory T cells
• Ligands and aAPC constructs are disclosed in WO 2016/044530 and WO 2016/105542, which are hereby incorporated by reference in their entirety.
• the enrichment and expansion process can be performed in an enclosed, automated cellular expansion system. Such a system can provide for simple, scalable, and cost-efficient manufacturing, as well as consistent and rapid generation of antigen-specific CD8+ T cells using different antigen peptide cocktails (i.e., sourced from patient or donor PBMCs).
• PCT/US2018/051971 titled CELL COMPOSITIONS COMPRISING ANTIGEN-SPECIFIC T CELLS FOR ADOPTIVE THERAPY.
• PCT/US2018/051971 is hereby incorporated by reference in its entirety.
• FIGURE 3 Enrichment and expansion of acute myeloid leukemia (AML)-specific T cells using the methods of the present disclosure are shown in FIGURE 3.
• the graph on the left in FIGURE 3 shows the total number of CD8+ T cells generated from fresh PBMCs of four healthy donors after the T cells were enriched and expanded ex vivo for AML- specific antigens WTI37-45, WTI126-134, PRAME425, Cyclin AI227-235, and Cyclin AI341-351.
• the graph on the right in FIGURE 3 shows the percentage total of the acute myeloid leukemia (AML) specific antigens after the CD8+ T cells were enriched and expanded ex vivo.
• the results in FIGURE 3 demonstrate that a significantly higher proportion of AML-specific CD8+ T cells are generated by the methods of the present disclosure compared to other Endogenous T cell therapy derived cellular compositions.
• TN naive T cells
• TCM central memory T cells
• TEM effector memory T cells
• TEMRA effector memory RA+ T cells
• TSCM T memory stem cells
• FIGURE 4B Greater than 60% of the E+E generated T cells were of the T memory stem cell phenotype and central memory T cell phenotype. Also, as demonstrated in FIGURE 4B, the E+E system generated consistent memory T cell phenotypes across all donors. The E+E process also generated significant amounts of multiple myeloma-specific T memory stem cells from two different healthy donor leucopaks. (See, FIGURE 8 and FIGURE 9A) In FIGURE 9A and FIGURE 9B, the multiple myeloma-specific antigenic T cells were enriched and expanded in batch to about ⁇ l.6xl0 9 CD8+ T cells, based on hinge dimer staining.
• FIGURE 5A and FIGURE 5B show that AML specific T cells enriched and expanded ex vivo have a high degree of poly functional phenotype, including intracellular staining for IL-2 (proliferation and memory), IFN-g (activating other T cells, memory, upregulation of MHC), TNF-a (pro-inflammatory), and CD107A (granzyme release, cytotoxic activity).
• the majority of AML-specific T cells i.e., about 62%) demonstrated 3-4 effector functions upon non-specific stimulation (FIGURE 5A, top).
• FIGURE 5A (bottom) the graph shows the percentage of T cells expressing IL-2, TNF-a, IFN-g, and CD 107 A.
• FIGURE 5 A the T cells were generated by non-specific stimulation of peptide-pulsed T2 cells.
• the results of this experiment show that for the majority of the E+E generated AML-specific CD8+ T cells, at least 3 or 4 cytokine effector functions were observed.
• FIGURE 5B T cell-mediated tumor specific killing of AML cell line U266 is shown at two effector to target (E:T) ratios, 10: 1 (left bar) and 20: 1 (right bar), from fresh PBMCs of healthy donors for the AML specific antigens.
• E:T effector to target
• the results of this experiment show that E+E generated CD8+ T cell compositions from healthy donors have a robust killing activity across multiple E:T ratios.
• FIGURE 6 consists of four graphs comparing the specificity of Mart- 1 specific T cells generated by the enrichment and expansion process disclosed herein between melanoma patient derived PBMCs (top) and healthy donor derived PBMCs (bottom).
• the enrichment and expansion process produces a consistent cellular composition regardless of the donor source.
• the data in this experiment was generated from frozen PBMCs.
• FIGURE 7 is a graph showing that the AIM ACT based E+E process generated a TCR repertoire that mimics the natural immune response, thereby providing a robust adoptive therapy from a natural T cell repertoire that has undergone natural selection.
• the breadth of the polyclonal TCR repertoire enables a natural and durable immune response.
• FIGURE 8 shows that the E+E process generated significant amounts of multiple myeloma antigen-specific T memory stem (TSCM) cells ((CD62L+, CD45RA+, CD95+).
• the graphs show the phenotype of multiple myeloma-specific antigenic T cells pre and post expansion from a healthy donor leucopak.
• FIGURE 9A shows the phenotype of T cells enriched and expanded ex vivo in batch for multiple myeloma antigen-specific T cells from a healthy donor leucopak.
• the graphs show that the E+E process generated significant amounts of antigen-specific CD8+ T cells ( ⁇ l.6xl0 9 CD8+ T cells based on hinge dimer staining) that comprise T memory stem (TSCM) cells, central memory T cells TCM, and effector memory T (TEM) cells.
• FIGURE 9B shows the phenotype of T cells enriched and expanded ex vivo in batch for multiple myeloma antigen-specific T cells from four different multiple myeloma patients.
• the graphs show that the E+E process generated significant amounts of antigen-specific CD8+ T cells that comprise T memory stem (TSCM) cells, central memory T cells TCM, and effector memory T (TEM) cells.
• T memory cells e.g T memory stem (TSCM) cells, central memory T cells TCM, and effector memory T (TEM) cells.
• FIGURE 10 shows production of gd T cells using the E+E process. Both V51 and V52 TCR subtypes were observed.
• the clinical significance of gd T cells in the context of hematopoietic stem cell transplantation (HSCT) has been reported, and in particular, where higher frequencies of gd T cells after transplantation are associated with favorable outcomes.
• HSCT hematopoietic stem cell transplantation
• the % gd T cells at Day 14 varied, with the average being from about 15% to about 50% gd T cells.
• the number of gd T cells at Day 14 broadly correlates with the number of gd T cells at Day 0.
• CD3 + CD8 + and TCR subtype analysis were generated from different samples. Differences between CD3 + CD8 + and ab T cells are due to different samples and different FCM gating strategies.
• phenotype is the measure of the total % of memory T cells relative to CD3 + cells.
• the memory T Cell populations characterized include T stem cell memory (Tscm) and T central memory (Tcm) populations, both of which retain the ability to proliferate and self-renew as well as T effector memory (Tern) cells.
• the remaining populations characterized include Temra and Tnaive cells.
• the CD8+ T cell compositions disclosed herein directly engage with T cell receptors on naive and memory T cells to trigger a desired immune response.
• the CD8+ T cell compositions which are generated by the enrichment and expansion process are composed of a multi-antigen specific, CD8+ restricted, T cells from an endogenous repertoire.
• the antigen-specific CD8+ T cell composition includes T memory stem cells. T memory stem cells and central memory T cells are instrumental and critical for both initial and long-term clinical responses.
• the antigen-specific CD8+ T cell composition have a polyfunctional phenotype, as assessed by effector cytokine production and target cell killing.
• the E+E process also generated a natural immune response driven by a diverse TCR repertoire.
• gd T cells are believed to provide additional mechanisms of pathogen or cancer cell killing, which are not HLA dependent.

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