Patent Application
20200268795
In recent years, immune checkpoint inhibitors are being rapidly approved for the management of advanced malignancies, including melanoma, non-small cell lung cancer, renal cell carcinoma, urothelial carcinoma, and head and neck cancer (M. K. Callahan et al., Immunity 44, 1069-1078 (2016)). However, only a small subset (10-30%) of patients responds to single agent immune checkpoint therapy (C. Robert et al., The New England journal of medicine 372, 2521-2532 (2015)), and a myriad of combination strategies are currently being actively investigated in clinical trials to enhance the potency of this approach.
Co-targeting of CTLA-4 and PD-1 immune checkpoint pathways is one strategy that has demonstrated significant clinical outcomes in melanoma (J. Larkin et al., The New England journal of medicine 373, 23-34 (2015)). Encouraging data combining ipilimumab and nivolumab has also been reported for non-small cell lung cancer (M. D. Hellmann et al., The lancet oncology 18, 31-41 (2017)). Despite these advances, a significant fraction of patients still does not achieve objective responses to checkpoint inhibitors. In multiple large randomized trials (E. D. Kwon et al., The lancet oncology 15, 700-712 (2014); H. Borghaei et al., The New England journal of medicine 373, 1627-1639 (2015); J. Bellmunt et al., The New England journal of medicine 376, 1015-1026 (2017)), patients receiving immune checkpoint inhibitors actually have worse survival outcomes compared to control arms during the initial months of treatment, at a time before immune-related toxicities fully manifest. In addition, the phenomena of “tumor hyper-progression” has been recently described where 9% of cancer patients receiving immune checkpoint inhibitors have accelerated tumor growth (S. Champiat et al., Clinical cancer research: an official journal of the American Association for Cancer Research 23, 1920-1928 (2017)).
Several studies have contributed to the understanding of mechanisms underlying differential responses and mechanisms of resistance to immune checkpoint strategies. This includes the role of pre-existing CD8+ T cells in the tumor invasive margins in melanoma patients treated with PD-1 blockade (P. C. Tumeh et al., Nature 515, 568-571 (2014)), and interferon-dependent expression of inhibitory ligands on tumor cells that mediate therapy resistance (J. L. Benci et al., Cell 167, 1540-1554 e1512 (2016)). However, the influence of tumor burden on the efficacy of immune checkpoint therapies has not been carefully investigated.
In some aspects, a human tumor-specific T-cell is provided that does not express a functional Interferon-γ (IFN-γ) receptor. In some embodiments, the T-cell comprises a mutation compared to wildtype that blocks IFN-γ receptor expression. In some embodiments, the mutation is a mutation in an IFN-γ receptor promoter or IFN-γ receptor coding sequence. In some embodiments, part or all of a coding sequence for IFN-γ receptor has been deleted. In some embodiments, the T-cell comprises an siRNA or antisense polynucleotide that inhibits expression of IFN-γ receptor. In some embodiments, the T-cell comprises a tumor-specific T-cell receptor. In some embodiments, the tumor-specific T-cell receptor is heterologous to the T-cell. In some embodiments, the T-cell is bound by a bispecific binding reagent that binds CD-3 and a tumor antigen. In some embodiments, the tumor antigen is CD-20. In some embodiments, the bispecific binding reagent is a bispecific antibody. In some embodiments, the T-cell comprises a heterologous chimeric antigen receptor (CAR).
In some aspects, a method of killing cancer cells in a human is provided. In some embodiments, the method comprises, administering to the human a sufficient number of the T-cell as described above o elsewhere herein to kill cancer cells in the human. In some embodiments, the method further comprises administering to the human (one or two or more immune pathway inhibitors (e.g., a CTL-4 inhibitor and a PD-1 inhibitor). In some embodiments, the T-cells have been obtained from the human and then altered to inhibit expression of the functional Interferon-γ (IFN-γ) receptor. In some embodiments, the human has melanoma. In some embodiments, the method further comprises administering to the human an antibody that binds to IFN-γ (e.g., a sufficient amount of an antibody that binds to IFN-γ to promote survival of the administered T-cells).
Also provided is a method of killing cancer cells in a human, the method comprising, administering to the human an effective amount of a JAK inhibitor, a CTL-4 inhibitor and a PD-1 inhibitor, and optionally a further agent that is toxic to cancer cells, thereby killing cancer cells in the human. In some embodiments, the human has melanoma. In some embodiments, the method further comprises administering to the human a sufficient number of the T-cell as described above or elsewhere herein.
A polynucleotide or polypeptide sequence is “heterologous” to a cell if it originates from a different cell, or, if from the same cell, is modified from its original form. For example, when a T-cell receptor is said to be heterologous to a T-cell, it means that the receptor or coding sequence thereof is from a first T-cell whereas the receptor is present or expressed in a second T-cell or the T-cell receptor is modified from its original form. For example the heterologous receptor can be expressed in the second T-cell by cloning or otherwise obtaining the coding sequence of the receptor from the first T-cell and inserting the coding sequence into the second T-cell such that the receptor is expressed in the second T-cell.
“Tumor-specific” means that the T-cell targets to tumor (cancer) cells. For example a tumor-specific T-cell can have a receptor that has binding affinity for a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA associated antigen is not unique to a tumor cell and instead is also expressed on a normal cell (e.g., under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells.
The term “interfering RNA” or “RNAi” or “interfering RNA sequence” refers to double-stranded RNA (i.e., duplex RNA) that targets (i.e., silences, reduces, or inhibits) expression of a target gene (i.e., by mediating the degradation of mRNAs which are complementary to the sequence of the interfering RNA) when the interfering RNA is in the same cell as the target gene. Interfering RNA thus refers to the double stranded RNA formed by two complementary strands or by a single, self-complementary strand. Interfering RNA typically has substantial (e.g., at least 70%, 80%, 90%, or 95%) or complete identity to the target gene. The sequence of the interfering RNA can correspond to the full length target gene, or a subsequence thereof. Interfering RNA includes small-interfering RNA″ or “siRNA,” i.e., interfering RNA of about 15-60, 15-50, 15-50, or 15-40 (duplex) nucleotides in length, more typically about, 15-30, 15-25 or 19-25 (duplex) nucleotides in length, e.g., about 20-24 or about 21-22 or 21-23 (duplex) nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-60, 15-50, 15-50, 15-40, 15-30, 15-25 or 19-25 nucleotides in length, e.g. about 20-24 or about 21-22 or 21-23 nucleotides in length, and the double stranded siRNA is about 15-60, 15-50, 15-50, 15-40, 15-30, 15-25 or 19-25 e.g., about 20-24 or about 21-22 or 21-23 base pairs in length). siRNA duplexes may comprise 3′ overhangs of about 1 to about 4 nucleotides, e.g., of about 2 to about 3 nucleotides and 5′ phosphate termini. The siRNA can be chemically synthesized or may be encoded by a plasmid (e.g., transcribed as sequences that automatically fold into duplexes with hairpin loops). siRNA can also be generated by cleavage of longer dsRNA (e.g., dsRNA greater than about 25 nucleotides in length) with the E. coli RNase III or Dicer. These enzymes process the dsRNA into biologically active siRNA (see, e.g., Yang et al., PNAS USA 99: 9942-7 (2002); Calegari et al., PNAS USA 99: 14236 (2002); Byrom et al., Ambion TechNotes 10(1): 4-6 (2003); Kawasaki et al., Nucleic Acids Res. 31: 981-7 (2003); Knight and Bass, Science 293: 2269-71 (2001); and Robertson et al., J. Biol. Chem. 243: 82 (1968)). In some embodiments, dsRNA are at least 50 nucleotides to about 100, 200, 300, 400 or 500 nucleotides in length. A dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotides in length, or longer. The dsRNA can encode for an entire gene transcript or a partial gene transcript.
The term “siRNA” refers to a short inhibitory RNA that can be used to silence gene expression of a specific gene. The siRNA can be a short RNA hairpin (e.g. shRNA) that activates a cellular degradation pathway directed at mRNAs corresponding to the siRNA.
The term “antisense nucleic acid” as used herein means a nucleotide sequence that is complementary to its target e.g. a IFN-γ receptor transcription product. The nucleic acid can comprise DNA, RNA or a chemical analog, that binds to the messenger RNA produced by the target gene. Binding of the antisense nucleic acid prevents translation and thereby inhibits or reduces target protein expression. Antisense nucleic acid molecules may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with mRNA or the native gene e.g. phosphorothioate derivatives and acridine substituted nucleotides. The antisense sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.
The terms “treating” and “treatment” as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage. Thus, “treating” and “treatment includes: (i) inhibiting the disease or condition, i.e., arresting its development; (ii) relieving the disease or condition, i.e., causing regression of the disease or condition; or (iii) relieving the symptoms resulting from the disease or condition, i.e., relieving pain without addressing the underlying disease or condition.
It has been discovered that treatment of cancer patients with checkpoint inhibitors can result in deletion of tumor-specific T-cells and that this effect is mediated by interferon-gamma (IFNγ). It has been further discovered that the deleterious effect of IFNγ on T-cells can be countered by knocking out the IFNγ receptor in the T-cells, rendering the T-cells immune from the negative effects of IFNγ and thus remaining available to target cancer cells.
Accordingly, in some aspects, tumor-specific T-cells are provided that do not express a functional IFNγ receptor. Such cells can be administered to treat cancer in a human. In some embodiments, the T-cells can be T-cells obtained from the human and then altered to block expression of a functional IFNγ receptor. In some embodiments, the T-cells can be further modified, for example to express a heterologous protein that targets the T-cells to cancer cells. Such heterologous proteins can include, for example chimeric antigen receptors or T-cell receptors that are heterologous to the T-cells. The T-cells can be administered with, or as a complementary treatment with, checkpoint inhibitors. Exemplary check point inhibitors include but are not limited to CTLA-4 inhibitors, PD-1 inhibitors or combinations thereof.
As JAK functions downstream of the IFNγ receptor, the effect of JAK inhibitors was also determined in the context of checkpoint inhibitors. It was discovered that combination of a JAK inhibitor with anti-CTLA-4 and anti-PD-1 checkpoint inhibitors were effective in reducing incidence of tumors in a mouse cancer model. Accordingly, an alternative to (or possible combination with) knock out of a functional IFNγ receptor in T-cells is to administer to a human having cancer an effective amount of a JAK inhibitor in combination with a CTLA-4 inhibitor and a PD-1 inhibitor.
Any type of T-cells can be modified to block expression of a functional IFNγ receptor. In some embodiments, the T-cell will be a CD8+ T-cell or a CD4+ T cell. These can include T-cells that have been transduced with chimeric antigen receptors (CAR) or T cell receptors (TCR) that target the relevant antigen in tumors both on the tumor cell or within the tumor microenvironment.
T cells can be obtained from a number of sources, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. For example, in some embodiments, cells from the circulating blood of an individual are obtained by apheresis, optionally followed by enrichment for T-cells, for example by affinity (e.g., antibody)-based cell sorting. In some embodiments, the T-cells can be cryopreserved and/or expanded before use. Exemplary details of T-cell manipulation can be found in, e.g., U.S. Pat. No. 9,394,368.
In some embodiments, the T-cells are obtained from the individual, modified as described herein, and returned to the same individual. Alternatively, T-cells can be obtained from one individual and administered to a different individual. In some embodiments, major HLA loci will be matched between the donor individual and the recipient to avoid rejection of the T-cells by the recipient.
T-cells do that not express a functional Interferon-γ (IFN-γ) receptor can be generated in a number of ways. In some embodiments, point mutations or deletions can be induced in the coding sequence of the IFN-γ receptor or in promoter or other transcriptional or translational regulator regions in DNA to result in a non-functional IFN-γ receptor. Thus in some embodiments the DNA of the T-cell can include a coding sequence of a functional IFN-γ receptor that is not expressed. Alternatively, the DNA of the T-cell can encode a non-functional IFN-γ receptor that includes one or more amino acid change, deletion or addition that impairs or eliminates IFN-γ receptor function (e.g., the ability to bind IFN-γ or to trigger downstream signaling based on IFN-γ binding). In yet other embodiments, all or part of the IFN-γ receptor gene can be deleted, thereby preventing expression of a functional IFN-γ receptor.
In yet other embodiments, expression of a native T-cell IFN-γ receptor can be inhibited by introduction of one or more agent that inhibits expression of the IFN-γ receptor. Exemplary agents include, but are not limited to, RNAi, siRNA, or antisense polynucleotides that are complementary or substantially (e.g., at least 75%, 80%, 85%, 90%, or 95%) complementary to all or a subsequence of at least 15, 20, 25, 30, 50, or more nucleotides of native RNA encoding the IFN-γ receptor.
Any method of genetic manipulation can be used to introduce the above-described mutations to block IFN-γ receptor expression. In some embodiments, a double-strand break (DSB) or nick for can be created by a site-specific nuclease in or near the target gene (e.g., the IFN-γ receptor gene). Exemplary targeted nucleases include but are not limited to zinc-finger nuclease (ZFN) or TAL effector domain nuclease (TALEN), or the CRISPR/Cas9 system with an engineered crRNA/tract RNA (single guide RNA) to guide specific cleavage. See, for example, Burgess (2013) Nature Reviews Genetics 14:80-81, Urnov et al. (2010) Nature 435(7042):646-51; United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20090263900; 20090117617; 20100047805; 20110207221; 20110301073 20110301073;20130177983; 20130177960 and International Publication WO 2007/014275, WO2003087341; WO2000041566; WO2003080809. Nucleases specific for targeted genes can be utilized such that a transgene construct is inserted by either homology directed repair (HDR) or by end capture during non-homologous end joining (NHEJ) driven processes.
In some embodiments, the T-cells are tumor-specific. Tumor-specific T-cells include one or more receptor that targets the T-cell to a cancer cell. In some embodiments, the T-cell has a naturally-occurring T-cell receptor that targets the T-cell to a cancer epitope. In other embodiments, the T-cell can be modified by introduction of a heterologous T-cell receptor specific for a cancer epitope. In yet other embodiments, the T-cell can be modified to express a chimeric antigen receptor that targets a cancer epitope. In other embodiments, the T-cell can be bound to a bispecific binding agent where the binding agent binds to the T-cell (e.g., at a surface protein on the T-cell) and separately has a binding affinity for a cancer epitope.
T-cells having a naturally-occurring T-cell receptor that targets the T-cell to a cancer epitope can be isolated from T-cell populations. In some embodiments, the T-cells are from an individual having a cancer that expresses the cancer epitope. T-cells that target the cancer epitope can be enriched using affinity selection in vitro using the cancer epitope or active fragment thereof.
Alternatively, T-cells can be modified by introduction of a heterologous T-cell receptor specific for a cancer epitope. Methods of doing so are described, in, e.g., Rapoport et al., Nature Medicine 21:914-921 (2015).
In some embodiments, the cancer epitope targeted by the tumor-specific T-cell is CD19, CD20, CD22, ROR1, mesothelin, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR. In some embodiments, the cancer epitope is one of: Differentiation antigens such as MART-1/MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.291\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\Pl, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.
In some embodiments, the T-cell is further modified to lack a naturally-occurring T-cell receptor (this can also occur when the T-cell is modified to express a chimeric antigen receptor). See, e.g., U.S. Pat. No. 9,181,527 describing methods of knocking out endogenous T-cell receptors.
In yet other embodiments, the T-cell can be modified to express a chimeric antigen receptor (CAR) that targets a cancer epitope. Exemplary CAR receptors are reviewed in Jackson et al., Nat Rev Clin Oncol. 2016 Jun;13(6):370-83. In some embodiments, the CAR comprises an antibody or antibody fragment that includes a cancer epitope (e.g., as listed above)-binding domain (e.g., a humanized antibody or antibody fragment that specifically binds to the cancer epitope), a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain). A number of CAR designs have been described including but not limited to those described in U.S. Pat. Nos. 9,522,955; 9,511,092; 9,499,629; 9,499,589; 9,447,194; and 9,394,368. In some embodiments, the binding domain of CAR comprises a scFv, comprising the light (VL) and heavy (VH) variable fragments of a cancer epitope-specific monoclonal antibody joined by a flexible linker. The intracellular signaling portions of the CAR can differ. In some embodiments (e.g., first generation CARs), the CAR only has the signal transduction domain of the CD3-zeta chain (CD3ζ) or Fc receptor γ (FcRγ). In other embodiments, the CAR further comprises one or more co-stimulatory domains (including but not limited to one or more of CD28, 4-1BB, or OX40), which in some embodiments lead to the enhanced cytotoxicity and cytokine secretion along with prolonged T cell persistence.
In some embodiments, viral or non-viral based gene transfer methods can be used to introduce nucleic acids encoding a heterologous T-cell receptor or a CAR T-cell receptor into T-cells as desired. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome, lipid nanoparticle or poloxamer Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Methods of non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, lipid nanoparticles, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, mRNA, artificial virions, and agent-enhanced uptake of DNA. In some embodiment, one or more nucleic acids are delivered as mRNA. In some embodiments, capped mRNAs are used to increase translational efficiency and/or mRNA stability. See, e.g., U.S. Pat. Nos. 7,074,596 and 8,153,773.
In other embodiments, the T-cell can be bound to a bispecific binding agent where the binding agent binds to the T-cell (e.g., at a surface protein on the T-cell, for example including but not limited to CD3) and separately has a binding affinity for a cancer epitope (for example, but not limited to CD20). Bi-specific antibodies capable of targeting T cells to tumor cells have been identified and tested for their efficacy in the treatment of cancers. Blinatumomab is an example of a bispecific anti-CD3-CD19 antibody in a format called BiTE™ (Bi-specific T-cell Engager) that has been identified for the treatment of B-cell diseases such as relapsed B-cell non-Hodgkin lymphoma and chronic lymphocytic leukemia (Baeuerle et al (2009)12:4941-4944). The BiTE™ format is a bi-specific single chain antibody construct that links variable domains derived from two different antibodies. Additional bi-specific antibodies include but are not limited to those described in WO 2015006749 and WO 2016110576. Such bi-specific binding agents can be mixed with a T-cell that does not express a functional IFN-γ receptor, thereby rendering the T-cell tumor-specific by way of the affinity of the bi-specific agent for a cancer epitope.
Treatment and Combination with Checkpoint Inhibitors
The tumor-specific T-cells lacking expression of a functional IFN-γ receptor can be used to treat cancer in a human. Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may comprise non-solid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors. Types of cancers to be treated with the T-cells described herein include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.
Hematologic cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.
Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases). In one embodiment, the epitope-binding portion of the CAR is designed to treat a particular cancer. In some embodiments, a CAR or T-cell receptor targeting CD19 can be used to treat cancers and disorders including but are not limited to pre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, salvage post allogenic bone marrow transplantation, and the like. In another embodiment, a CAR or T-cell receptor targeting CD22 to treat diffuse large B-cell lymphoma. In one embodiment, cancers and disorders include but are not limited to pre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, salvage post allogenic bone marrow transplantation, and the like can be treated using a combination of CAR or T-cell receptors targeting CD19, CD20, CD22, and ROR1. In one embodiment, a CAR or T-cell receptor targeting mesothelin can be used to treat mesothelioma, pancreatic cancer, and ovarian cancer. In one embodiment, a CAR or T-cell receptor targeting CD33/IL3Ra can be used to treat acute myelogenous leukemia. In one embodiment, a CAR or T-cell receptor targeting c-Met can be used to treat triple negative breast cancer, and non-small cell lung cancer. In one embodiment, a CAR or T-cell receptor targeting PSMA can be used to treat prostate cancer. In one embodiment, a CAR or T-cell receptor targeting Glycolipid F77 can be used to treat prostate cancer. In one embodiment, a CAR or T-cell receptor targeting EGFRvIII can be used to treat glioblastoma. In one embodiment, a CAR or T-cell receptor targeting GD-2 can be used to treat neuroblastoma, and melanoma. In one embodiment, a CAR or T-cell receptor targeting NY-ESO-1 TCR can be used to treat myeloma, sarcoma, and melanoma. In one embodiment, a CAR or T-cell receptor targeting MAGE A3 TCR can be used to treat myeloma, sarcoma, and melanoma.
The T cells described herein may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations. Briefly, pharmaceutical compositions may comprise a T-cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present invention can be formulated for intravenous administration.
Pharmaceutical compositions as described herein may be administered in a manner appropriate to the disease to be treated. The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
When “an immunologically effective amount”, “an anti-tumor effective amount”, “an tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions as described herein to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). In some embodiments, a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 104 to 109 cells/kg body weight, e.g., 105 to 106 cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell compositions are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions are administered by i.v. injection. The compositions of T cells may be injected directly into a tumor, lymph node, or site of infection.
In certain embodiments, the T-cells as described herein are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, Cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efalizumab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p′7056 kinase that is important for growth factor induced signaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). In a further embodiment, the cell compositions as described herein are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell compositions are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in one embodiment, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the T-cells as described herein. In an additional embodiment, expanded cells are administered before or following surgery.
As noted herein, inhibiting immune checkpoint pathways, while useful in treating various cancers, can result in an increase in IFN-γ. Thus, in some embodiments in T-cells described herein are used in combination with one or more immune pathway checkpoint inhibitor. In some embodiments the immune pathway checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a Lag-3 inhibitor, a TIM-3 inhibitor, or a combination thereof. Exemplary inhibitors can include but are not limited to antibodies that bind to the immune pathway checkpoint protein in question (e.g., PD-1 or CTLA-4). PD-1 and CTLA-4 inhibition is discussed in, e.g., Buchbinder, and Desai, Am J Clin Oncol. 2016 February; 39(1): 98-106. Exemplary CTLA-4 antibodies include but are not limited to Ipilimumab (trade name Yervoy™) as well as those described in, e.g., WO 2001/014424, U.S. Pat. No. US 7,452,535; 5,811,097. Exemplary PD-1 antibodies include but are not limited to Pembrolizumab (formerly MK-3475 and lambrolizumab, trade name Keytruda™) as well as those described in, e.g., U.S. Pat.t No. 8,008,449 and Zarganes-Tzitzikas, et al., Journal Expert Opinion on Therapeutic Patents Volume 26, 2016, Issue 9. Inhibitors of the immune pathway checkpoints can be administered before, after or simultaneously with administration of the T-cells as described herein. The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices.
In alternate embodiments, instead of providing T-cells that do not express a functional IFN-γ receptor, an antibody or other binding agent that binds to IFN-γ can be administered to the human. This will inactivate or eliminate IFN-γ sufficiently to interfere with IFN-γ′s negative effect on T-cells in the human. Thus in some embodiments, an antibody or other binding agent that binds to IFN-γ is administered to the human in conjunction with (e.g., simultaneously with or before or after within 1-30 days) one or more immune checkpoint inhibitors as described herein (including but not limited to, for example anti-PD-1 and anti-CTLA4 agents). In some embodiments, a T-cell is also administered to the human as described herein albeit the T-cells can be, but need not be, blocked for IFN-γ include a receptor (heterologous or native, and optionally CAR) that targets a cancer epitope as described herein.
As noted herein, in some embodiments, an effective amount of one or more Janus kinase (JAK) inhibitor is administered to a human having cancer, optionally in combination with an anti-PD-1 agent and an anti-CTLA4 agent. Agents administered in combination can be administered simultaneously or in a mixture together, or can be administered in series. For administration in series (not simultaneous) agents should be administered within a time frame such that an initially administered agent still has the desired effect (e.g., has not been significantly degraded or excreted from the body) by the time later agents in the combination are administered to have the desired effect, e.g., cancer cell killing or reduction of tumor incidence.
Exemplary JAK inhibitors include but are not limited to Ruxolitinib (trade names Jakafi/Jakavi), Tofacitinib (trade names Xeljanz/Jakvinus, formerly known as tasocitinib and CP-690550, Oclacitinib (trade name Apoquel), Baricitinib (trade name Olumiant), Filgotinib (G-146034, GLPG-0634), Gandotinib (LY-2784544), Lestaurtinib (CEP-701), Momelotinib (GS-0387, CYT-387), Pacritinib (SB1518), PF-04965842, Upadacitinib (ABT-494), Peficitinib (ASP015K, JNJ-54781532), or Fedratinib (SAR302503).
The following examples are offered to illustrate, but not to limit the claimed invention.
Combination checkpoint inhibition with high tumor burden
Ipilimumab is a humanized IgG1 antibody targeting CTLA-4, and one of its immunomodulatory mechanisms is engagement with FcγRIIIA to potentially antagonize or deplete regulatory T cells (E. Romano et al., Proceedings of the National Academy of Sciences of the United States of America 112, 6140-6145 (2015); M. J. Selby et al., Cancer Immunol Res 1, 32-42 (2013); T. R. Simpson et al., The Journal of experimental medicine 210, 1695-1710 (2013)). In contrast, anti-PD-1 antibodies such as nivolumab have been engineered to avoid FcγR binding to prevent depletion of activated T cells through antibody dependent cellular cytotoxicity (ADCC) (C. Wang et al., Cancer Immunol Res 2, 846-856 (2014); R. Dahan et al., Cancer cell 28, 285-295 (2015)). To mimic the anti-PD-1 antibodies used clinically, we generated an anti-PD-1 antibody without ADCC (anti-PD-1 DANA) for use in our preclinical experiments (
We first investigated the anti-tumor activity of single agent and combined immune checkpoint blockade in the setting of established tumors. Mice were inoculated with either MC-38 (
To investigate immune infiltration within the tumor microenvironment after treatment, we first evaluated pathologic changes of tumor samples harvested three days after the last treatment. In H&E staining, there were peri-tumor lymphocytic aggregates with prominent perivascular localization and intra-tumor lymphocytic penetration in combo and monotherapy treated group (
To evaluate the exhaustion status of the infiltrating CD8+ T cells, we gated on CD8+ subsets and assessed Tim-3 expression (
Next, we evaluated whether the combo could enhance anti-tumor responses in the setting of low tumor burdens. TRAMP-C2 has a relatively slow tumor growth rate and tumors do not become palpable until approximately 30 days post implantation. For these experiments, mice were treated on day 3, 6 and 9 (
To determine whether similar outcomes can also be observed in patients treated with dual checkpoint blockade, we analyzed 152 melanoma patients and stratified patient cohorts based on tumor burden. Patients treated with anti-PD-1 monotherapy (n=101), or the combination of anti-CTLA-4 and anti-PD-1 (n=51) were assessed for best objective response rate as determined by RECIST 1.1. Baseline tumor size was calculated per RECIST 1.1, and the baseline tumor groups were stratified into size ≤6 cm, >6 to ≤11 cm, or >11 cm. Patients with a complete or partial response were categorized as responders, and those with stable disease or progressive disease as their best response were categorized as non-responders. The responder fraction was calculated by dividing responders/all patients. Consistent with our findings in mice, patients treated with dual checkpoint blockade demonstrated significantly worse response rates compared to those treated with monotherapy in the low, but not medium or high, tumor burden settings (
Loss of Antigen-Specific T Cells with Combination Checkpoint Blockade with Low Tumor Burden
To examine the mechanism underlying this paradoxical effect, we investigated changes in the number of antigen-specific T cells at different time points in mice (
To investigate the loss of antigen-specific T cells, we sorted Spas-1 specific CD8+ T cells from draining lymph nodes of checkpoint inhibitors treated TRAMP-C2 tumor-bearing mice at Day 28 (
PD-1 blockade has been shown to prevent terminal exhaustion of antigen-specific T cells rather than promote apoptosis. We hypothesized that the contraction of antigen-specific T cells observed could result from cytokines mediating T cell contraction (T. Yajima et al., J Immunol 176, 507-515 (2006)) and homeostasis (C. D. Surh, J. Sprent, Immunity 29, 848-862 (2008)). We first investigated changes in cytokine levels after treatment during early tumor development. Analysis of serum samples two days after the last dose of checkpoint antibodies showed that IFN-γ, IL-5 and IL-15 were increased in the combination group compared to the other treatment groups (
We hypothesized that the loss of Spas-1 specific CD8+ T cells may be related to increased IFN-γ signaling. We first investigated T cells subsets two days after antibody treatment, and found that CD4+ T cells from combination-treated mice secreted higher amounts of IFN-γ compared to monotherapy or isotype (
It has been shown that T cell homeostasis can be achieved by down-modulation of antigen specific T cell receptor (TCR) signaling (A. M. Gallegos et al., Nature immunology 17, 379-386 (2016)). To investigate whether combination treatment eliminated different T cell clones depended on the strength of TCR binding to cognate cancer epitope, we gated on CD8 subsets and investigated Spas-1hi versus Spas-11o CD8+ T cell clones (
The generation of long-term T cell memory responses is important for an effective and durable anti-tumor response. Because the persistence of antigen specific T cells is important in the formation of memory responses (C. A. Klebanoff, L. Gattinoni, N. P. Restifo, Immunological reviews 211, 214-224 (2006)), we evaluated the effect of combination treatment during early tumor growth on memory response formation. Mice challenged with TRAMP-C2 were treated and observed for three months. 20-30% of mice treated with combination treatment and 80-90% of mice treated with anti-CTLA-4 alone were tumor-free at 90 days. These protected mice were rechallenged with either TRAMP-C2 or MC-38 (control) in the contralateral flank (
Deficiency of the IFN-γ Receptor in Immune Cells Rescues Anti-Tumor Activity after Combination Therapy
IFN-γ is essential in triggering potent anti-tumor responses by inducing MHC I expression and enhancing antigen presenting capabilities (H. Ikeda, L. J. Old, R. D. Schreiber, Cytokine & growth factor reviews 13, 95-109 (2002)). Although neutralization of IFN-γ can potentially prevent antigen specific T cells loss, neutralization may also abrogate anti-tumor responses. To evaluate whether IFN-γ signaling is important for antigen-specific T cells, we used IFN-γ receptor knockout (RKO) mice (
To determine whether IFN-γ signaling in immune cells or non-immune cells (e.g. stromal cells) are responsible for this difference, we performed experiments in bone marrow chimera mice. WT mice underwent myeloablative conditioning and were adoptively reconstituted with bone marrow cells from CD45.2 RKO mice and CD45.1 congenic mice in a 1:1 ratio (
Finally, we sought to investigate the differential effects of checkpoint inhibition in the setting of low tumor burden (LTB) versus high tumor burden (HTB). During tumor development, antigen-specific T cells undergo a dynamic exhaustion process that correlates with tumor progression and tumor burden (A. Schietinger et al., Immunity 45, 389-401 (2016)). To investigate the effects of tumor burden on T cell function, mice were challenged with TRAMP-C2 tumors. T cells were later isolated from spleens on either Day 11 (LTB setting) or Day 50 (HTB setting) post tumor injection (
In addition, human primary CD8 T cells were isolated from health patients. CD8 T Cells were then transfected with CAR-19 structure to become CAR-19 T cells. Untransfected CD8 T cells were used as control. Both untransfeted CD8 T cells and CAR-19 T cells were cultured in vitro with supplement of 30 IU/ml of hIL-2. For stimulation of T cells, K-562 cancer cells express CD-19 ligand were co-cultured with CAR-19 T cells. See
Current cancer immunotherapy strategies aim to counteract the suppressive tumor environment by enhancing antigen recognition of T cell receptors (H. Torikai et al., Blood 119, 5697-5705 (2012)), increasing anti-tumor cytotoxicity capabilities via cytokines (C. A. Klebanoff et al., Proceedings of the National Academy of Sciences of the United States of America 101, 1969-1974 (2004); S. A. Rosenberg, J Immunol 192, 5451-5458 (2014)), or unleashing the “brakes” in the immune system and preventing terminal T cell exhaustion by blocking different immune checkpoint inhibitors (A. Schietinger et al., Immunity 45, 389-401 (2016); P. Sharma, J. P. Allison, Cell 161, 205-214 (2015)). The clinical success of CTLA-4 and PD-1 blockade in melanoma (J. Larkin et al., The New England journal of medicine 373, 23-34 (2015)) has shown combination immunotherapy to be a viable strategy in improving anti-tumor response. It has been shown that T cells isolated from patients treated with dual checkpoint blockade demonstrated a significant increase in IFN-γ levels compared to pre-treatment samples at baseline (R. Das et al., J Immunol 194, 950-959 (2015)), and various combination therapies to enhance IFN-γ production are the subject of ongoing clinical investigation. We found, however, that potent combination therapy with CTLA-4 and PD-1 blockade can actually be detrimental, and we report here on the negative impact of IFN-γ on anti-tumor immunity.
While combination therapy in mice with established tumors achieved improved tumor control, combination treatment in the context of low tumor burden compromised anti-tumor effects in both mice and metastatic melanoma patients. Mechanistically, we found that combination treatment during early tumor development leads to heightened IFN-γ production, which in turn results in apoptosis of the dominant tumor-specific T cells via AICD. In addition to dampening of anti-tumor response, the loss of antigen specific T cells also negatively impacts long-term T cell memory responses. Overall, our results underscored a new role of IFN-γ signaling in the regulation of anti-tumor responses after immune checkpoint therapies.
IFN-γ has conventionally been demonstrated to have immune stimulatory roles that mediate anti-tumor effects. The secretion of IFN-γ from tumor infiltrating lymphocytes can activate both dendritic cells and macrophages to enhance antigen presentation (A. J. Minn, Trends in immunology 36, 725-737 (2015)). IFN-γ signaling on cancer cells can also activate MHC I expression and Stat-1—dependent genes, including P21 and cyclin kinase that inhibit cell cycle progression, resulting in apoptosis of tumor cells (H. Ikeda, L. J. Old, R. D. Schreiber, Cytokine & growth factor reviews 13, 95-109 (2002); J. Gao et al., Cell 167, 397-404 e399 (2016)). However, there is also evidence showing the paradoxical role of IFN-γ in cancer immunotherapies, in particular, its association with acquired resistance (M. R. Zaidi, G. Merlino, Clinical cancer research: an official journal of the American Association for Cancer Research 17, 6118-6124 (2011)). IFN-γ has been shown to promote therapy resistance to immune checkpoint blockade by upregulation of IDO and PD-L1 (S. Spranger et al., Sci Transl Med 5, 200ra116 (2013)) and other co-inhibitory receptors, including Tim-3 and Lag-3 (J. L. Benci et al., Cell 167, 1540-1554 e1512 (2016); S. Koyama et al., Nature communications 7, 10501 (2016)). Here, we demonstrated that IFN-γ signaling can be immunosuppressive, mediating therapy resistance through a PD-L1 independent pathway. Induction of IFN-γ secretion following dual blockade treatments can promote apoptosis of tumor-reactive CD8+ T cells while limiting the formation of effector memory anti-tumor responses. We also found that T cells isolated from melanoma patients respond to IFN-γ induced apoptosis. Overall, our study highlights the importance of type II interferon that not only accounts for cytotoxic effects against cancer cells, but can also mediate the loss of tumor-specific T cells. These results provide a potential mechanism that underlies accelerated tumor growth seen clinically in some checkpoint inhibitor treated patients (S. Champiat et al., Clinical cancer research: an official journal of the American Association for Cancer Research 23, 1920-1928 (2017)), as well as explain why more frequent dosing of combination checkpoint inhibitors is associated with a lower overall response rate in lung cancer patients (M. D. Hellmann et al., The lancet oncology 18, 31-41 (2017)).
Taken together, these results indicate that there exists a potential window within which the immune system can optimally respond to cancer. In the setting of low tumor burden, optimal immunotherapy, such as CTLA-4 or PD-1 blockade alone, can induce invigoration of T cells and provide substantial benefits to cancer patients (A. C. Huang et al., Nature 545, 60-65 (2017)). The blunted IFN-γ responses in high tumor burden create a different threshold where combination checkpoint blockade may be necessary for therapeutic benefit.
8-10 week-old aged control male C57BL/6j, Ifngr KO and CD45.1 congenic (C57BL/6j background) mice were obtained from Jackson Laboratory and used in the experiments. Mice were implanted subcutaneously (S.C.) with either TRAMP-C2 or MC-38 cell line at a dosage of 1×106 per mouse at the right flank on day 0, and were treated with different antibodies intraperitoneally (I.P.) on day 3, 6, and 9. In the late intervention TRAMP-C2 group, 1×106 TRAMP-C2 cells were similarly implanted S.C. at the right flank on day 0, but allowed to grow for 30-45 days prior to treatment. Mice with tumor volumes within 50-200 mm3 were selected into different treatment groups before treatment. The average tumor sizes among different treatment groups were checked and ensured to be similar before treatment. Mice were injected with different antibodies I.P. on day 3, 6, and 9. In memory re-challenge experiments, mice were implanted with TRAMP-C2 tumors at a dose of 1×106 per mouse at the right flank on day 0. Mice were then treated with different immune checkpoint antibodies on day 3, 6, and 9. Tumors were measured twice a week, every 3-4 days. Ninety days after the initial tumor implantations (day 90), tumor-free mice from either anti-CTLA-4 or anti-CTLA-4 and anti-PD-1 DANA combination treatment groups were rechallenged with TRAMP-C2 tumors at the left flank at a dosage of 1×106. There were no tumor-free mice treated with anti-PD-1 DANA antibody alone or isotype control. Sibling WT mice without prior tumor challenge or treatment were aged together in the same vivarium and used later as controls for rechallenge experiments. Tumor measurement=L (length)×W (width)×W/2 (mm3); whereas the longer diameter was defined as length and the shorted diameter was defined as width. All mice were maintained at UCSF vivarium in accordance with Institutional Animal Care and Use Committee (IACUC) standards.
8-10 week-old C57BL/6j mice (H2b) were used as recipient mice and underwent lethal total body irradiation (1050 cGy;137Cs source) followed by transplantation from donor CD45.2 Ifngr KO mice and CD45.1 congenic mice. T cell-replete bone marrows were mixed in 1:1 ratio (5×105 cells total) and injected intravenously (I.V.) through the tail vein per recipient mouse. Chimera mice were reconstituted for 30 days and checked for chimerism by tail bleeding. Chimera mice were implanted subcutaneously with 1×106 TRAMP-C2 cells at the right flanks on day 30. Mice were subsequently injected with different checkpoint inhibitors (10 mg/kg/injection/mouse) on day 33, 36, 39. On day 58, mice were sacrificed and cells were harvested from tumor draining lymph nodes. The ratio of CD45.2+/CD45.1+ cells in CD8+Spas1 cells was calculated by dividing the total number of CD45.2+CD8+Spas-1 cells by the number of CD45.1+CD8+Spas-1 cells. The proportion of CD8+Spas-2 and CD8+ double negative subsets were similarly derived. All mice were maintained at the UCSF vivarium in accordance with IACUC standards.
CD45+CD3+CD8+Spas-1 T cells were sorted from draining lymph nodes from TRAMP-C2 bearing mice on day 28 after treatment. RNA was extracted from sorted CD8+Spas-1 T cells using an Ambion micro RNA isolation kit (AM1931) according to the manufacturer's protocol, and genomic DNA was eliminated using DNase kit purchased from Qiagen. RNA quality was checked by the A260/A280 ratio using NanoDrop Lite (Thermal Scientific). lOng RNA from each sample was used for subsequent cDNA synthesis. cDNA was synthesized and cDNA templates pre-amplified were using RT2 PreAMP cDNA synthesis kit (Qiagen Cat 330451) according the manufacturer's protocol and ProFlex PCR machine (Applied Biosystems). cDNA samples derived were evaluated for apoptotic gene expression arrays using RT2 Profiler™ PCR Array kits purchased from Qiagen (PAMM-012Zc-12, Cat 330231) with SYBR Green qPCR Mastermix (Qiagen Cat 330522). Quantitative real-time RT-PCR arrays was performed using Applied Biosystems Cycler (AB Step-ONE Plus). PCR arrays were analyzed and gene expression heat map generated using software provided on Qiagen website under Data Analysis Center. All samples passed quality control (QC). Expression level for each gene is presented as fold change in comparison to internal control of house keeping genes (beta-actin, Gus and Hsp90ab1) in each group. Array data are available in GEO database under accession number: GSE95433
Clinical Outcomes with Immune Checkpoint Inhibition
Patients were treated with either PD-1 monotherapy with pembrolizumab 2 mg/kg or 10 mg/kg (n=101), or PD-1/CTLA-4 combination therapy with ipilimumab 3 mg/kg plus nivolumab 1 mg/kg (n=51). Best objective response rate was determined by RECIST 1.1. Baseline tumor size was calculated by summing the largest diameter of the target lesions per RECIST 1.1. Patients were stratified according to the baseline tumor size into ≤6 cm, >6-≤11 cm, or >11 cm. Patients with a complete or partial response were categorized as responders, and those with stable disease or progressive disease as their best response were categorized as non-responders. The responder fraction was calculated by dividing responders/all patients. The error bars represent SEM. Significance was calculated by the Mann-Whitney test, p value is 2 sided, NS, p<0.05.
Cryopreserved PBMCs were collected from an HLA-A*0201 patient with metastatic melanoma receiving 4 cycles of ipilimumab and nivolumab followed by nivolumab monotherapy maintenance. PBMCs were thawed and counted by Vi-Cell to determine cell numbers and stained with HLA-A*0201 MHC/peptide dextramers specific for different melanoma antigens (Melanoma Dextramer Collection 1, Cat. No. RX01; Immudex). The dilution rate and staining procedures followed manufacturer's protocols. Flow samples were run on a BD X-50 and analyzed by Flow-Jo software. The clinical trial were approved and under supervision of IRB at University of California, San Francisco.
Antibodies against mouse PD-1 were generated by immunizing HSD rats with recombinant mouse PD-1 protein (R&D Cat: 1021-PD). Hybridomas were generated by fusing IgG producing cells from immunized mice with myeloma cells (NS0-Mouse Myeloma, PTA-4796), and screened for binding to PD-1. The UC10-4F10-11 hybridoma expressing mouse anti-CTLA-4 antibody was purchased from ATCC (HB-304). The antibody variable domains were cloned from the hybridomas and expressed as murine IgG2a WT or with mutations to inactivate FcR binding (D265A; N297A; DANA) (Baudino, L., et al. (2008). Journal of immunology 181, 6664-6669; Chao, D.T., et al. (2009) Immunological investigations 38, 76-92). PD-1 and CTLA-4 antibodies were additionally screened for neutralization of the PD-1—PD-L1/L2 or CTLA-4—CD86 interactions, respectively.
Anti-CTLA-4, anti-PD-1, anti-PD-1 DANA and IgG2a antibodies were obtained from Abbvie or Bioxell (Supplemental Table 1). All antibodies were stored in −80° C. in small working aliquots to avoid repeated freeze-thaw cycles before use. Antibodies were dissolved in phosphate buffer saline (PBS) and injected I.P. at indicated time points. For combination treatments with anti-CTLA-4 and GVAX, 106 irradiated (10,000 rads) GVAX cells were injected S.C. into the skin over the neck on day 3, 6 and 9, at the same time as I.P. antibody injection.
In Vitro T Cell Cultures and Stimulation with Different Concentrations of Recombinant IFN-γ
Splenocytes were harvested from TRAMP-C2 bearing C57BL/6j mice on day 50 and T cells were purified by magnetic beads according to the manufacturer's protocol (Miltenyi Biotec Cat 130-095-130). Cells were checked for over 90% purity. Purified T cells were suspend in DMEM (UCSF cell culture core) +10% fetal bovine serum (Lonza Cat 14-501F) +1% penicillin/streptomycin (UCSF cell culture core)+murine 20 IU IL-2 (Peprotech Cat 212-12), and seeded in 96 wells at 2×105 cells per well. Recombinant murine IFN-γ (Peprotech Cat 212-12) was added into the wells at indicated concentrations. Cells were cultured for 12-72 hours and analyzed by flow cytometry. For the patient sample, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Sigma Cat F4375) and PBMCs were seeded in 96 wells at 2×105 cells per well supplemented with 20 IU human IL-2 (Peprotech Cat 200-02). Recombinant human IFN-γ (Peprotech Cat 212-12) was added to the wells. Cells were harvested and analyzed by flow cytometry 72 hours after incubation.
Tumor cell lines, TRAMP-C2 and MC-38, were cultured for cell injection into C57BL/6j male mice. TRAMP-C2 cell medium composed of DMEM (UCSF cell culture core), 5% fetal bovine serum (FBS; Lonza Cat. 14-501F), 5% Nu-serum IV (Corning Cat. 355504), 0.005 mg/ml bovine insulin (Sigma Cat. 10516), 10nM dehydroisoandrosterone (Sigma Cat. D5297), and 1% penicillin/streptomycin (UCSF cell culture core). MC-38 medium composed of DMEM (UCSF cell culture core), 5% fetal bovine serum (Lonza Cat. 14-501F), 5% Nu-serum IV (Corning Cat. 355504), and 1% penicillin/streptomycin (UCSF cell culture core). Frozen cell lines were thawed in the water bath at 37° C. before transfer into corresponding pre-warmed media. After wash, cells were then pelleted and resuspended in fresh media before passage into culture flasks. Once every two days, culture flasks were checked for confluence with a light microscope. Before cells overcrowd the culture flask (>90% of confluency), old media from the flask was decanted into waste and 10 mL of PBS added as a rinse. After cells were rinsed with PBS, the solution was removed and 5 mL of 0.05% trypsin with EDTA (UCSF cell culture core) was introduced into the flask. Subsequently the flask was placed in a CO2 incubator at 37° C. for 5 minutes for trypsinization of adherent cells. After incubation, the trypsin was neutralized with plentiful media, pelleted, and then resuspended in new media before a fractional transfer into new culture flasks. For cell injection into mice, instead of the fractional transfer step, cells were washed and pelleted with PBS twice to remove the presence of FBS. Prior to injection, cells were adjusted with PBS to a concentration of 107 cells per mL with each needle containing 1×106 cells in 100 uL.
Two days after the final checkpoint inhibitor treatment, mouse sera were collected and sent to Eve Technologies (Calgary, Alberta, Canada) for analysis with mouse cytokine 31-plex discovery assay (Cat No: MD31). Serum cytokine levels from treatment groups were each divided by the serum cytokine level of the IgG2a control group to calculate as fold changes. These fold changes were graphed with the Prism 7 software.
Spleens were surgical removed with sterilized surgical equipment and crushed with the blunt end of a 10 mL syringe on petri dishes containing 5 mL of PBS. The spleen mixtures were separately filtered through a 70 μM filter into a 50 mL conical tube, centrifuged at 1500rpm for 5 minutes at 4° C. After wash, cell pellets were resuspended in 5 mL of red blood cell lysis solution (Santa Cruz Biotechnology; Cat sc-296258) on ice for 5 minutes and stopped with the addition of 30 mL of PBS. After wash, cells were reconstituted for counting by Vi-Cell (Beckman Coulter, U.S.A.). Draining lymph nodes were extracted with sterilized surgical equipment and crushed between the frosted surfaces of super-frosted microscope slides into wells containing PBS. Cell mixtures were then filtered through a 70 μM filter into 15 mL conical tubes. Cells were then washed and counted. Tumors were removed from mice with sterile surgical instruments followed by sectioning for paraformaldehyde fixation or flow cytometry analysis. Tumor tissues for flow analysis were kept moist with 1 mL collagenase IV digest media (DMEM+10%FCS+1% penicillin/streptomycin+Collagenase IV+DNase) and minced with scalpel blades. Tumor cell mixtures were then transferred into 15 mL conical tubes and filled with additional 9 mL of collagenase digest media. Tumor samples were subsequently placed on a 37° C. shaker for 1 hour. Samples were filtered through a 100 μM filter into a 50 mL conical tube and washed with PBS before centrifugation. Finally, tumor cell pellets were resuspended and counted before subsequent flow staining.
The flow cytometry protocols were previously described (Sckisel et al., 2015). Single cell suspensions (1 million cells) were first incubated with Fc Block (BD Pharmingen. San Diego, Calif.) for 10 minutes, then co-incubated with antibodies for 20 minutes at 4° C. followed by washing with staining buffer (PBS +1% FBS). Foxp3 and intracellular staining were performed using eBioscience intracellular kit (Cat#00-5523-00) according to the manufacturer's protocol. Active Caspase-3 staining was performed by using BD Caspase-3 apoptosis kit (BD Cat 550480), and Annexin V staining was performed using BD Annexin V apoptosis detection kit (BD Cat 556547) according to the manufacturer's protocol. Flow cytometry was performed on Fortessa X20 Dual, and data analyzed by FlowJo software (TreeStar). Details on flow cytometry antibodies used in this study can be found in Supplemental Table 2.
Tissues harvested from mice were placed in 4% formalin, followed by 70% alcohol and PBS before embedding. Tissues were then embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Tissue sections were evaluated by a board certified pathologist (M.C.). Images were visualized using an Olympus Vanox AHBS3 microscope with an Olympus SPlan Apo x 20/0.70 NA objective (Olympus, Woodbury, N.Y.). A diagnostic instrument spot RT color digital camera utilizing Spot software version 4.0.2 was used to acquire the images (Diagnostic Instruments, Sterling Heights, Mich.) Immunohistochemistry were performed as previous described (DuPage, M., et al, (2011). Cancer cell 19, 72-85). Tumor tissues were fixed in 4% paraformaldehyde, processed, embedded in paraffin, then cut into 5 μm sections. Paraffin sections were blocked with 3% hydrogen peroxide solution (Sigma Cat H1009), vector streptavidin/biotin (Vector Laboratories cat. SP-2002), and CAS-Block protein block (ThermoFisher Cat. 008120), then stained with CD8 antibody (Biorbyt Cat orb10325).
Data shown in this manuscript were presented as mean±SE. Tumor growth curves at different time points were plotted by using Prism 7 and analyzed by two-way ANOVA with a Tukey post hoc test comparison among groups. Flow cytometry data were analyzed by one-way ANOVA with a Tukey post hoc test. P-values less than 0.05 were considered statistically significant. Gene expression arrays were analyzed by software provided by Qiagen website under Data Analysis Center.
C57B1/6j mice were implanted with TRAMP-C2 tumors (106 cells per mouse) on day 0 and treated with combination therapies (anti-CTLA-4+anti-PD-1 DANA) at day 3, 6, and 9. For group 3 and group 4, mice were additionally treated with a JAK inhibitor twice a week from day 11 for total five doses. Table 1 demonstrated the tumor incidence rate at day 55.
As shown above, the combination of a JAK inhibitor with anti-PD-1 and anti-CTLA-4 therapy resulted in a much lower rate of tumor incidence,
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
The present application claims benefit of priority to U.S. Provisional Patent Application No. 62/561,849, filed Sep. 22, 2017, which is incorporated by reference for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/052416 | 9/24/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62561849 | Sep 2017 | US |
