EA041322B1 - TOXICITY CONTROL IN THE EVENT OF ANTITUMOR ACTIVITY OF CHIMERIC ANTIGEN RECEPTORS (CAR) - Google Patents

Description

Cross-reference to related applications

This application claims priority to U.S. Provisional Application No. 61/671,482, filed July 13, 2012, and U.S. Provisional Application No. 61/782,982, filed March 14, 2013, each of which is incorporated herein by reference in its entirety. volume.

Background of the Invention

Patients with relapsed and chemotherapy-refractory acute lymphocytic leukemia (ALL) have a poor prognosis despite the use of invasive treatments such as allogeneic hematopoietic stem cell transplantation (Barrett et al., 1994, N Engl J Med 331:1253-8; Gokbuget et al., 2012, Blood 120:2032-41) and CD19 bispecific antibody fragments (Bargou et al., 2008, Science 321:974-7). T cells modified with a chimeric antigen receptor targeting the CD19 and CD20 lineage specific antigens have been reported to be effective in adults with CLL and B cell lymphomas (Till et al., 2008, Blood 112:2261-71; Kochenderfer et al. , 2010, Blood 116:4099-102; Brentjens et al., 2011, Blood 118:4817-28; Porter et al., 2011, N Engl J Med 365:725-33; Kalos et al., 2011, Science Translational Medicine 3:95ra73; Savoldo et al., 2011, J Clin Invest 121:1822-5). However, the effects of CAR-T cells on blast cells in ALL, a more immature leukemia with more rapid progression, have not been fully investigated.

Delayed onset of tumor lysis syndrome and cytokine secretion have been reported, coupled with extensive in vivo expansion of chimeric antigen receptor T cells (Porter et al., 2011, N Engl J Med 365:725-33; Kalos et al., 2011, Science Translational Medicine 3:95ra73). However, the effects of cytokine secretion, and the disorders associated with in vivo expansion of T cells with a chimeric antigen receptor, have not been fully investigated.

Thus, there is a need in the present field for compositions and methods for treating cancer using Chimeric Antigenic

Receptors (CAR) and resolution of CAR toxicity problems. The present invention meets this need.

Summary of the essence of the invention

The present invention relates to a method of treating a patient suffering from a disease, disorder or condition associated with increased expression of a tumor antigen. In one embodiment, the method involves administering first line therapy and second line therapy to a patient, where first line therapy comprises administering to the patient an effective amount of cells genetically modified to express a CAR, where the CAR contains an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

In one embodiment, following first line therapy, the patient's cytokine levels are monitored to determine the appropriate type of second line therapy to administer to the patient, and the appropriate second line therapy is administered to the patient.

In one embodiment, an increase in the level of a cytokine identifies the type of cytokinin inhibitory therapy to be administered to the patient.

In one embodiment, the cytokine is selected from the group consisting of IL-1ep, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL- 15, IL-17, IL-1Ra, IL-2R, IFN-a, IFN-γ, MIP-1a, MJP-1p, MCP-1, TNFa, GM-CSF, G-CSF, CXCL9, CXCL10, CXCR factors , VEGF, RANTES, EOTAXIN, EGF, HGF, FGF-β, CD40, CD40L, ferritin, and any combination thereof.

In one embodiment, the cytokinin inhibitory therapy is selected from the group consisting of small interfering RNA (siRNA), miRNA, antisense nucleic acid, ribozyme, expression vector encoding a transdominant negative mutant, antibody, peptide, small molecule, cytokinin inhibitory drug, and any combination thereof. .

In one embodiment, cytokine levels are monitored by detecting the protein level of the cytokine in a patient biological sample.

In one embodiment, cytokine levels are monitored by detecting the level of nucleic acid for the cytokine in a patient biological sample.

The present invention relates to a method for reducing or preventing an adverse event associated with the administration of cells genetically modified to express a CAR, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, a method comprising monitoring cytokine levels in a patient to determine the appropriate type of cytokine therapy to administer to the patient and administer the appropriate cytokine therapy to the patient.

In one embodiment, an increase in the level of a cytokine identifies the type of cytokinin inhibitory therapy to be administered to the patient.

In one embodiment, the cytokine is selected from the group consisting of IL-1e, IL-2, IL-4, IL5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN-a, IFN-γ, MIP-1a, MIP-1e, MCP-1, TNFa, GM-CSF, G-CSF, CXCL9, CXCL10, CXCR factors, VEGF , RANTES, EOTAXIN, EGF, HGF, FGF-β,

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CD40, CD40L, ferritin, and any combination thereof.

In one embodiment, the cytokinin inhibitory therapy is selected from the group consisting of small interfering RNA (siRNA), microRNA, antisense nucleic acid, ribozyme, expression vector encoding a transdominant negative mutant, intracellular antibody, peptide, small molecule, cytokinin inhibitory drug, and any of them. combinations.

In one embodiment, cytokine levels are monitored by detecting the level of protein for the cytokine in a patient biological sample.

In one embodiment, cytokine levels are monitored by detecting the level of nucleic acid for the cytokine in a patient biological sample.

Brief description of the drawings

The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying figures. To illustrate the invention, the figures show embodiments that are currently preferred. It should be understood, however, that the invention is not limited to the exact spacings and tools of the embodiments shown in the figures.

Fig. 1 is an image showing serum levels of cytokines in four different patients. All patients showed the release of cytokines, including IL-6.

Fig. 2 is an image showing serum levels of cytokines plotted for a representative patient. The patient was in a critical condition for 5-7 days, and the improvement of the condition began only after the introduction of tocilizumab.

Fig. 3 is an image demonstrating that antibody interventions have no effect on CART19 cellular functionality as measured by markers of T cell activity (perforin and IFN-γ).

Fig. 4 containing FIG. 4A-4C is a series of images showing clinical responses. Fig. 4A shows results for two children with multiple relapse chemotherapy-refractory acute lymphoblastic leukemia with CD19+ B cell progenitors who were treated with CTL019 cells infused on Day 0. Changes in serum lactate dehydrogenase (LDH) and body temperature after CTL019 infusion, with a maximum temperature for a 24-hour period are indicated by circles. CHOP-100 received methylprednisolone starting on day 5 at 2 mg/kg/day, tapering off on day 12. On the morning of day 7, etanercept was taken, 0.8 mg/kg×1. At 6 pm on day 7, tocilizumab, 8 mg/kg×1, was administered. Temporary improvement in pyrexia occurred with the administration of corticosteroids on day 5 of CHOP-100, with complete relief of the high temperatures experienced after administration of cytokine-targeted therapy consisting of etanercept and tocilizumab on day 8. FIG. 4B shows serum cytokines and inflammatory markers measured at multiple time points after CTL019 infusion. Values for cytokines are shown using a semi-log plot with fold change from baseline. Baseline values (Day 0, pre-infusion) (pg/mL serum) for each analyte were (CHOP-100, CHOP-101): ILt-β: (0.9, 0.2); IL-6: (4.3, 1.9); TNF-α: (1.5, 0.4); IL2Ra: (418.8, 205.7); IL-2: (0.7, 0.4); IL-10 (9.9, 2.3); IL1Ra: (43.9, 27.9). Both patients developed marked increases in cytokines and cytokine receptors, including soluble interleukin 1A and 2 receptor (IL-1RA and IL-2R), interleukins 2, 6 and 10 (IL-2, IL-6 and IL-10), factor tumor necrosis-α (TNF-α), and interferon-γ (INF-γ). Fig. 4C shows circulating changes in absolute neutrophil count (ANC), absolute lymphocyte count (ALC) and absolute white blood cell count (WBC). Notably, the increase in ALC primarily consisted of activated CT019 T lymphocytes.

Fig. 5 containing FIG. 5A-5D are a series of images showing the expansion and visualization of CTL019 cells in peripheral blood, bone marrow, and CSF. Fig. 5A shows the results of a flow cytometric analysis of peripheral blood stained with antibodies to detect CD3 and anti-CD19 with CAR. The percentage of CD3 cells expressing CARs in CHOP100 and CHOP-101 is shown. Fig. 5B shows the presence of CTL019 T cells in peripheral blood, bone marrow, and CSF by quantitative real-time PCR. Genomic DNA was isolated from whole blood, bone marrow and CSF aspirates collected at subsequent time points before and after CTL019 infusion. Fig. 5C shows flow cytometry detection of CTL019 cells in CSF harvested from CHOP-100 and CHOP-101. Fig. 5D shows images of activated large granular lymphocytes in Wright-stained peripheral blood smears and CSF cytospin smears.

Fig. 6 is an image showing CD19 expression at baseline and at relapse in CHOP-101. Bone marrow samples from CHOP-101 were obtained before CTL019 infusion and at the time of relapse 2 months later. Mononuclear cells isolated from bone marrow samples were stained for CD45, CD34 and CD19 and analyzed on a flow cytometer.

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Accuri C6. After setting the live cell discrimination window, the blast cell (CD45+ SSC low) window was adjusted for CD34+ cells and histograms were generated for CD19 expression.

The split line represents the threshold for the same discrimination window for isotype controls. Pre-treatment power cells have a CD19 distribution range, with a small population of very dimly stained cells visible as the tail of the left histogram at 10 ² on the x-axis. The relapse sample did not contain any CD19 positive blast cells. Analysis of CD19 expression in the blast cell population before treatment showed the presence of a small population of CD19-dull or -negative cells. The mean fluorescence intensity (MFI) of this small cell population was 187 (left panel), similar to the MFI for anti-CD19-stained recurrent blast cells (201, right panel). The pre-treatment bone marrow sample was hypocellular with 10% blasts, and the relapse bone marrow sample was normocellular with 68% blasts, allowing for differences in events available for data collection.

Fig. 7 is an image showing the induction of bone marrow remission in CHOP-101 at day +23 after CTL019 infusion. Clinical immunophenotyping report for CHOP101 at baseline (Upper panel) and day +23 (Lower panel). Cells were stained to detect CD10, CD19, CD20, CD34, CD38 and CD58. Flow cytometry was performed after erythrocyte lysis. The report on day +23 showed that leukocytes consisted of 42.0% lymphocytes, 6.0% monocytes, 50.3% myeloid forms, 0.17% myeloid blast cells, and did not contain viable lymphoid precursor cells. According to flow cytometry, there was no convincing immunophenotypic confirmation of residual lymphoblastic progenitors of leukemia/lymphoma B cells. As such, no viable B cells have been identified.

Fig. 8 is an image showing in vivo propagation and persistence of CTL019 cells in blood. The number of leukocytes (WBC), CD3+ T cells, and CTL019 cells in the blood is shown for CHOP-100 and CHOP-101. The number of cells is shown on the graph on a semi-logarithmic scale.

Fig. 9 containing FIG. 9A and 9B are a series of images demonstrating that individuals had depletion of CD19 positive cells in bone marrow and blood within 1 month of CTL019 infusion. Fig. 9A shows persistent B cell aplasia in CHOP-100. The top panel shows the predominant population of leukemic blast cells in the bone marrow aspirated from CHOP-100 expressing CD19 and CD20 at day +6. This population is absent on day +23 and after 6 months. Fig. 9B shows B cell aplasia and the shedding of escaped CD19 variant cells in CHOP-101. Flow cytometric analysis of bone marrow aspirates from CHOP-101 stained for anti-CD45, CD34 and CD19. In the bottom row, side scatter and CD45 weakly positive cells were used to identify leukemic cells that express variable amounts of CD34 and CD19 at baseline. Only CD19-negative blasts were detected at day 64. The numbers in the upper panel represent the fraction of total leukocytes represented in each quadrant. The numerical values in the lower panel represent the percentage of total leukocytes present in the low CD45 weak/33 channel.

Fig. 10 is a graph showing the levels of ferritin present in a patient after ingestion of CAR-T cells.

Fig. 11 is a graph showing the levels of myoglobin present in a patient after ingestion of CAR-T cells.

Fig. 12 is a graph depicting plasminogen activator inhibitor-1 (PAI-1) levels present in a patient after ingestion of CAR-T cells.

Detailed description of the invention

The present invention relates to compositions and methods for the treatment of malignant neoplasms, including, but not limited to, hemoblastoses and solid tumors. The invention also encompasses methods of treating and preventing certain types of cancers, including primary and metastatic tumors, as well as cancers that are untreatable or resistant to conventional chemotherapy. The methods include administering to a patient a therapeutically or prophylactically effective amount of T cells transduced to express a chimeric antigen receptor (CAR). CARs are molecules that combine the specificity of an antibody for a desired antigen (eg, tumor antigen) with the receptor-activating intracellular domain of T cells to generate a chimeric protein that exhibits specific antitumor cellular immune activity.

As part of a complete treatment regimen, the invention encompasses methods for controlling certain cancers (eg, preventing or prolonging their recurrence or prolonging remission time) by evaluating the soluble factor profile in patients after T cell infusion. Preferably, the soluble factor profile includes an assessment of the cytokine profile. When the cytokine profile indicates an increase in a particular cytokine after T cell infusion compared to before T cell infusion, the person skilled in the art

- 3 041322 may choose to administer to a patient in need of such control an effective amount of a cytokinin inhibitory compound, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrug thereof, to control elevated cytokine levels following T cell infusion.

The present invention is based in part on the discovery that the identification of a unique combination of factors whose modulation from baseline or pre-existing levels at baseline can help track T cell activation, target activity, and potential deleterious side effects following CAR-T cell infusion in order to to help control cancer treatment. Example factors include, but are not limited to, IL-1e, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL -17, IL-1Ra, IL-2R, IFN-a, IFN-γ, MIP1a, MIP-1e, MCP-1, TNFa, GM-CSF, G-CSF, CXCL9, CXCL10, CXCR factors, VEGF, RANTES, EOTAXIN, EGF, HGF, FGF-β, CD40, CD40L, ferritin, etc.

The present invention relates to an adoptive cell transfer strategy of T cells transduced to express a chimeric antigen receptor (CAR) in combination with toxicity control, where a soluble factor profile is generated from a patient after T cell infusion, and a therapy directed against an increased soluble factor is administered. for the treatment of malignant neoplasm. For example, real-time soluble factor profiling allows intervention in elevated soluble factors with an appropriate inhibitor to bring levels down to normal levels.

In one embodiment, the CAR of the invention comprises an extracellular domain having an antigen recognition domain that targets the desired antigen, a transmembrane domain, and a cytoplasmic domain. The present invention is not limited to a specific CAR. Moreover, any CAR that targets the desired antigen can be used in accordance with the present invention. Compositions and methods for producing CAR are described in PCT/US11/64191, which is incorporated herein by reference in its entirety.

In some embodiments of any of the methods above, the methods result in a measurable reduction in tumor size or confirmation of disease or disease progression, complete remission, partial remission, stabilization of the disease, increase or prolongation of progression-free survival, increase or prolongation of the overall survival or reduced toxicity.

Definitions

Unless otherwise defined, all technical and scientific terms used in the present description have the same meaning, which is usually understood by an ordinary specialist in the field to which the invention pertains. While any methods and materials similar or equivalent to those described herein may be practiced to test the present invention, preferred materials and methods are described herein. In describing and formulating the claims of the present invention, the following terminology will be used.

It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting.

Approximately, as used herein, when referring to a measurable value such as an amount, duration of time, etc., means that it covers variations equal to ±20% or ±10%, in some cases ±5%, in in some cases ±1%, and in some cases ±0.1% of the indicated value, as such variations are appropriate for the implementation of the disclosed methods.

The term activation, as used herein, refers to the state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector functions. The term activated T cells refers, inter alia, to T cells that go through cell division.

The terms soluble factor activators or agonists are used herein to refer to agent molecules capable of activating or increasing levels of soluble factor. Activators are compounds that increase, initiate, induce activation, activate or upregulate the activity or expression of a soluble factor, such as an agonist.

Assays for detecting activators include, for example, expression of a soluble factor in vitro, in cells, or cell membranes, using putative agonist compounds, and then determining the functional effects on the activity of the soluble factor, as described elsewhere herein.

The term antibody as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. The antibodies may be intact immunoglobulins obtained from natural sources or from recombinant sources and may be immunoreactive portions of intact immunoglobulins. Antibodies are often tetramers of immunoglobulin molecules. The antibodies of the present invention may exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab) ₂ , as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, B: Using Antibodies: Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85 :5879-5883; Bird et al., 1988, Science 242:423-426).

The term antibody fragment refers to a portion of an intact antibody and refers to the antigen-determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

The term antigen or Ag, as used herein, defines a molecule that elicits an immune response. This immune response may involve either the production of antibodies or the activation of specific immunocompetent cells, or both. One skilled in the art will appreciate that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. In addition, antigens can be obtained from recombinant or genomic DNA. One of skill in the art will appreciate that any DNA that contains nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an antigen as the term is used herein. In addition, one of skill in the art will appreciate that an antigen need not be encoded solely by the full nucleotide sequence of a gene. Obviously expressed that the present invention includes, but is not limited to, the use of partial nucleotide sequences from more than one gene, and that these nucleotide sequences are located in various combinations to cause the desired immune response. Moreover, one skilled in the art will appreciate that an antigen does not need to be encoded by the genome at all. Obviously expressed that the antigen can be generated synthesized or can be obtained from a biological sample. Such a biological sample may include, but is not limited to, a tissue sample, a tumor sample, a cell, or a biological fluid.

The term self-antigen means, according to the present invention, any self-antigen that is recognized by the immune system as if it were foreign. Self antigens include, but are not limited to, cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

The term autoimmune disease, as used herein, is defined as a disorder that results from an autoimmune response. Autoimmune disease is the result of an inadequate and excessive response to self antigen. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Segren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, among the others.

As used herein, the term autologous is intended to mean any material derived from an individual that is later to be administered to that same individual.

Allogeneic refers to a transplant obtained from another animal of the same species.

Xenogeneic refers to a transplant obtained from an animal of a different species.

The term malignant neoplasm, as used herein, is defined as a disease characterized by the rapid uncontrolled growth of aberrant cells.

Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia , lung cancer, etc.

As used herein, the term combination therapy means that the first agent is administered in conjunction with yet another agent. The term in conjunction with refers to the administration of one treatment in addition to yet another other treatment. As such, the expression in conjunction with refers to the administration of one treatment before, during, or after the delivery of another treatment to an individual. Such combinations are considered as part of a single treatment regimen or regimen.

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As used herein, the term co-administration means that the administration of the first therapy and the administration of the second therapy in combination therapy overlap with each other.

The term co-stimulatory ligand as used herein includes a molecule on an antigen presenting cell (e.g., aARC, dendritic cell, B cell, etc.) that specifically binds to a cognate co-stimulatory molecule on a T cell, thereby providing a signal that, in addition to the primary signal provided by, for example, binding of the TCR/CD3 complex to the peptide-loaded MHC molecule, mediates a T cell response including, but not limited to, proliferation, activation, differentiation, and the like. The costimulatory ligand may include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PDL1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin receptor beta, 3/TR6, ILT3, ILT4, HVEM, agonist or antibody that binds to Toll ligand receptor, and ligand , which specifically binds to B7-H3. A costimulatory ligand also encompasses, among others, an antibody that specifically binds to a costimulatory molecule present on a T cell, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, Cd4o, PD-1, ICOS , operably linked lymphocyte antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-I3, and a ligand that specifically binds to CD83.

The term costimulatory molecule refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response from the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, the MHC class I molecule, BTLA, and the Toll ligand receptor.

The term co-stimulatory signal, as used herein, refers to a signal that, in combination with a primary signal, such as TCR/CD3 ligation, results in T cell proliferation and/or up- or down-regulation of key molecules.

A disease is a state of health in an animal where the animal cannot maintain homeostasis, and where, if the disease is not alleviated, then the health of the animal continues to deteriorate. In contrast, a disorder in an animal is a health condition in which the animal is able to maintain homeostasis, but in which the animal's health is less favorable than it would be in the absence of the disorder. Left untreated, the disorder does not necessarily cause further deterioration in the health of the animal.

An effective amount, as used herein, means an amount that provides a therapeutic or prophylactic benefit.

As used herein, endogenous refers to any substance from or produced within an organism, cell, tissue, or system.

As used herein, the term exogenous refers to any substance introduced into an organism, cell, tissue, or system that has been produced outside the organism, cell, tissue, or system.

The term expression, as used herein, is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

An expression vector refers to a vector containing a recombinant polynucleotide containing expression control sequences operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, such as cosmids, plasmids (eg, isolated or contained in liposomes), and viruses (eg, lentiviruses, retroviruses, adenoviruses, and adeno-related viruses), which include a recombinant polynucleotide.

The term homologous refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position at both of the two compared sequences is occupied by the same base or the same monomeric amino acid subunit, for example, if a position in each of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions that are the same in the two sequences divided by the number of positions compared x100. For example, if 6 out of 10 positions in two sequences are identical or homologous, then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC have 50% homology. Typically, the comparison is made when the two sequences are aligned to ensure maximum homology.

The term immunoglobulin or Ig, as used herein, is defined as a class

- 6 041322 proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD and IgE. IgA is a primary antibody that is present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucosal secretions from the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response in most individuals. It is the most effective immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in protection against bacteria and viruses. IgD is an immunoglobulin that has no known antibody function, but can serve as an antigen receptor. IgE is an immunoglobulin that mediates immediate-type hypersensitivity by causing the release of mediators from mast cells and basophils when exposed to an allergen.

As used herein, the term immune response includes T cell mediated and/or B cell mediated immune responses. Examples of immune responses include T cell responses such as cytokine production and cellular cytotoxicity. In addition, the term immune response includes immune responses that are indirectly influenced by T cell activation, such as antibody production (humoral responses) and activation of cytokine-responsive cells, such as macrophages. Immune cells involved in the immune response include lymphocytes such as B cells and T cells (CD4+, CD8+, Th1 and Th2 cells); antigen-presenting cells (eg professional antigen-presenting cells such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen-presenting cells such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer cells; myeloid cells such as macrophages, eosinophils, mast cells, basophils and granulocytes.

The terms soluble factor inhibitors or antagonists are used herein to refer to agent molecules capable of inhibiting, inactivating or reducing soluble factor levels. Inhibitors are compounds which, for example, bind to a soluble factor, partially or completely block activity, reduce, prevent, delay activation, inactivate, desensitize or down-regulate the activity or expression of a soluble factor, for example antagonists. Inhibitors include polypeptide inhibitors such as antibodies, soluble receptors, and the like, as well as nucleic acid inhibitors such as siRNAs or antisense RNAs, genetically modified versions of a soluble factor, such as versions with altered activity, and natural and synthetic soluble factor antagonists. factors, small chemical molecules, etc. Assays for the detection of inhibitors include, for example, expression of a soluble factor in vitro, in cells or cell membranes, using putative antagonist compounds, and then determining the functional effects on the activity of the soluble factor, as described elsewhere herein.

As used herein, educational material includes a publication, record, chart, or any other form of expression that can be used to communicate the applicability of the compositions and methods of the invention. The training material of the kit of the invention may, for example, be attached to a container that contains the nucleic acid, peptide and/or composition of the invention, or be supplied with a container that contains the nucleic acid, peptide and/or composition. Alternatively, the training material may be supplied separately from the container with the intention that the training material and the connection are to be shared by the recipient.

Distinguished means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not isolated, but the same nucleic acid or peptide partially or completely separated from co-existing substances in its natural state is isolated.

An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-native environment such as, for example, a host cell.

Lentivirus, as used herein, belongs to the genus of the Retroviridae family. Lentiviruses are unique among retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so that they are one of the most efficient gene delivery vector methods. HIV, SIV and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer a means to achieve significant levels of gene transfer in vivo.

The phrase level of soluble factor in a biological sample, as used herein, generally refers to the amount of protein, protein fragment, or peptide levels of the soluble factor that is present in the biological sample. The level of soluble factor does not have to be quantified, but can simply be detected, e.g. subjectively,

- 7 041322 by human visual detection, with or without comparison with the level from the control sample or the level expected for the control sample.

The term modulation, as used herein, is meant to mediate a detectable increase or decrease in the level of response in an individual compared to the level of response in an individual with no treatment or compound, and/or compared with the level of response in an otherwise identical but untreated individual. individual. The term encompasses perturbing a native signal or response and/or influencing a native signal or response, thereby mediating a favorable therapeutic response in an individual, preferably a human.

Parenteral administration of an immunogenic composition includes, for example, subcutaneous (SC), intravenous (IV), intramuscular (IM), or intrasternal injection or infusion techniques.

The terms patient, individual, etc. are used interchangeably herein and refer to any animal or its cells, either in vitro or in situ, subjected to the methods described herein. In some non-limiting embodiments, the patient or individual is a human.

The term simultaneous administration, as used herein, means that the first therapy and the second therapy in combination therapy are administered with a time separation of no more than about 15 minutes, such as no more than about any interval of 10.5 or 1 min. When the first and second therapies are administered simultaneously, the first and second therapies may be contained in the same composition (e.g., a composition containing both the first and second therapy) or in separate compositions (e.g., the first therapy in one composition and the second therapy contained in yet another composition).

The term simultaneous administration, as used herein, means that the first therapy and the second therapy in combination therapy are administered with a time separation of no more than about 15 minutes, such as no more than about any interval of 10.5 or 1 min. When the first and second therapies are administered simultaneously, the first and second therapies may be contained in the same composition (e.g., a composition containing both the first and second therapy) or in separate compositions (e.g., the first therapy in one composition and the second therapy contained in yet another composition).

By specifically binds, as used herein in relation to an antibody, is meant an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in the sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But such interspecies reactivity does not in itself change the classification of an antibody as specific. In yet another example, an antibody that specifically binds to an antigen may also bind to various allelic forms of the antigen. However, such cross-reactivity does not in itself alter the classification of an antibody as specific. In some cases, the terms specific binding or specific binding may be used when referring to the interaction of an antibody, protein, or peptide with a second chemical species, to indicate that the interaction is dependent on the presence of a particular structure (eg, antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a particular protein structure more than to proteins in general. If the antibody is specific for an A epitope, the presence of a molecule containing an A epitope (or a free, unlabeled A) in a reaction containing a labeled A and an antibody will reduce the amount of labeled A bound to the antibody.

By the term stimulation is meant a primary response induced by the binding of a stimulatory molecule (e.g., TCR/CD3 complex) to its cognate ligand, thereby mediating a signaling event, such as, but not limited to, signaling through the TCR/CD3 complex. Stimulation may mediate altered expression of certain molecules, such as down-regulation of TGF-β and/or reorganization of cytoskeletal structures, and the like.

Stimulating molecule, as used herein, means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell.

A stimulatory ligand, as used herein, means a ligand that, when present on an antigen presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner (referred to herein as stimulatory molecule) on the T cell, thereby mediating the T cell's primary response, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulating ligands are well known in the art and include, but are not limited to, a peptide-loaded MHC Class I molecule, an anti-CD3 antibody, an anti-CD28 superagonist antibody, and an anti-CD2 superagonist antibody.

The term individual is intended to include living organisms in which an immune response can be elicited (eg, mammals). Examples of individuals include humans, dogs, cats, mice, rats and their transgenic species.

As used herein, a substantially purified cell is one that is substantially free of other cell types. The term substantially purified cell also refers to a cell that has been separated from other cell types with which it is normally associated in its natural state. In some instances, a population of substantially purified cells refers to a homogeneous population of cells. In other cases, the term simply refers to a cell that has been separated from the cells with which it was naturally associated in its natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

The term therapeutic, as used herein, means treatment and/or prophylaxis. The therapeutic effect is obtained by suppression, remission or elimination of the disease state.

The term "therapeutically effective amount" refers to the amount of a compound of interest that will elicit a biological or medical response in a tissue, system, or individual that is being sought by a researcher, veterinarian, medical doctor, or other clinician. The term "therapeutically effective amount" includes that amount of a compound which, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. A therapeutically effective amount will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the individual being treated.

A transplant, as used herein, refers to cells, tissue, or an organ that is introduced into an individual. The source of transplanted material may be cultured cells, cells from another individual, or cells from the same individual (eg, after in vitro culture of the cells). Examples of organ transplants are kidney, liver, heart, lung and pancreas.

As used herein, the term to treat a disease means to reduce the incidence or severity of at least one sign or symptom of a disease or disorder experienced by an individual.

The term transfected or transformed or transduced as used herein refers to the process by which an exogenous nucleic acid is transferred or introduced into a host cell. A transfected or transformed or transduced cell is a cell that has been transfected, transformed or transduced using an exogenous nucleic acid. A cell includes a primary subject cell and its progeny.

Spacing: Throughout the specification, various aspects of the invention may be presented in spacing format. It should be understood that the description in interval format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, a description of an interval should be considered as specifically disclosing all possible subranges as well as individual numerical values within that interval. For example, a range description such as 1 to 6 should be considered as specifically disclosing subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6 etc., as well as individual numbers within a given interval, for example, 1, 2, 2.7, 3, 4, 5, 5.3 and 6. This approach is applied regardless of the latitude of the interval.

Description

The present invention relates to compositions and methods for treating cancer in a patient. In one embodiment, the method of treatment comprises a first line therapy comprising administering a CAR of the invention to a patient to induce an antitumor immune response and monitoring the patient's soluble factor levels following T cell infusion to determine the type of second line therapy appropriate to treat the patient as a result. first line therapy.

In one embodiment, second-line therapy comprises evaluating a patient's soluble factor profile after receiving an infusion of appropriate T cells with CAR (referred to elsewhere in this specification as post T cell infusion), where, when the soluble factor profile indicates an increase in a particular soluble factor after T cell infusion versus before T cell infusion, one of skill in the art may choose to administer to a patient in need an effective amount of a soluble factor inhibitory compound to control elevated levels of soluble factor after T cell infusion. Accordingly, second line therapy in one embodiment comprises administering a type of soluble factor inhibitory therapy to control elevated levels of certain soluble factors resulting from first line therapy using CAR T cells.

In yet another embodiment, a second line therapy relating to the administration to a patient of a soluble factor inhibitory compound may be combined with other conventional therapies used to treat, prevent, or control diseases or disorders associated with or characterized by unwanted angiogenesis. Examples of such conventional therapies include, but are not limited to, surgery, chemotherapy, radiation therapy, hormonal therapy, biological therapy, and immunotherapy.

In one embodiment, a CAR of the invention can be engineered to contain an extracellular domain having an antigen-binding domain that targets a tumor antigen fused to an intracellular T-cell antigen receptor complex chain zeta signaling domain (e.g., CD3 zeta). An example of a B cell antigen tumor antigen is CD19 because this antigen is expressed on malignant B cells. However, the invention is not limited to targeting CD19. Rather, the invention includes any moiety that binds a tumor antigen. The antigen binding fragment is preferably fused to the intracellular domain of one or more of the co-stimulatory molecule and the zeta chain. Preferably, the antigen-binding fragment is fused to one or more intracellular domains selected from the group of CD137 signal domain (4-1BB), CD28 signal domain, CD3-zeta signal domain, and any combination thereof.

In one embodiment, the CAR of the invention contains the CD137 (41BB) signaling domain. The reason for this is that the present invention is based in part on the discovery that CAR-mediated T cell responses can be further enhanced by the addition of co-stimulatory domains. For example, incorporation of the CD137 signaling domain (4-1BB) significantly increased CAR-mediated activity and in vivo viability of CAR-T cells compared to identical CAR-T cells not engineered to express CD137 (4-1BB). However, the invention is not limited to a particular CAR. Rather, any CAR that targets a tumor antigen can be used in the present invention. Compositions and methods for the preparation and use of CARs are described in PCT/US 11/64191, which is incorporated herein by reference in its entirety.

Ways

The treatment regimen of the invention results in a measurable reduction in tumor size or confirmation of disease or disease progression, complete remission, partial remission, stabilization of the disease, increase or prolongation of progression-free survival, increase or prolongation of overall survival, or decrease in toxicity.

As part of a complete treatment regimen, the invention encompasses first line and second line therapy, where the first line therapy comprises administering CAR-T cells of the invention to the patient. The treatment regimen of the invention provides cancer control and treatment by evaluating the soluble factor profile in patients following T cell infusion. Appropriate second line therapy involves administering to the patient an appropriate soluble factor inhibitor to reduce elevated levels of soluble factor resulting from first line therapy. In some cases, appropriate second line therapy involves administering to the patient an appropriate soluble factor activator to increase suppressed levels of soluble factor resulting from first line therapy.

In one embodiment, the appropriate second line therapy comprises administering to the patient an appropriate cytokine inhibitor to reduce elevated cytokine levels resulting from first line therapy. In some cases, appropriate second line therapy involves administering to the patient an appropriate cytokine activator to increase suppressed cytokine levels resulting from first line therapy.

In one embodiment, the differential levels are above expression (high expression) or below expression (low expression) when compared to the expression level of a normal or control cell, a given patient population, or an internal control. In some embodiments, the level comparison is between a patient and a normal individual, between a post-T cell infusion patient and a pre-T cell infusion, or between a post-T cell infusion patient at the first time stamp and the second time stamp.

In one embodiment, the invention includes assessing differential levels of one or more cytokines to obtain a cytokine profile in a patient following T cell infusion to determine the type of cytokine therapy to be administered to the patient to downregulate the cytokine to normal levels. The invention can therefore be used to identify elevated levels of a cytokine as a result of the presence of the CAR T cells of the invention in a patient, which allows targeted treatment of the patient with cytokine inhibitors to lower the elevated levels of the cytokine. In yet another embodiment, the invention can be used to identify cytokine levels reduced as a result of the presence of the CAR-T cells of the invention in a patient, which allows specialized treatment of the patient with cytokine activators to increase the reduced levels of the cytokine.

In one embodiment, the levels of cytokines that are elevated as a result of infusion of CAR T cells include, but are not limited to, IL-Ιβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN-α, IFN-γ, MIP-1a, MJP-1p, MCP- 1, TNFa, GM-CSF, GCSF, CXCL9, CXCL10, CXCR factors, VEGF, RANTES, EOTAXIN, EGF, HGF, FGF-β, CD40, CD40L, ferritin, etc. However, the invention should not be limited to these listed cytokines. Rather, the invention includes any cytokine identified as elevated in a patient as a result of receiving an infusion of CAR T cells.

In one embodiment, levels of cytokines that are reduced by receiving CAR-T cell infusion include, but are not limited to, IL-Τβ, IL-2, IL-4, IL-5, IL-6, IL-8 , IL10, IL-12, IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN-a, IFN-γ, MIP-1a, MIP-1P, MCP-1, TNFa, GM -CSF, GCSF, CXCL9, CXCL10, CXCR factors, VEGF, RANTES, EOTAXIN, EGF, HGF, FGF-β, CD40, CD40L, ferritin, etc. However, the invention should not be limited to these listed cytokines. Rather, the invention includes any cytokine identified as decreased in a patient as a result of receiving an infusion of CAR-T cells.

Cytokine detection and treatment

While this section describes cytokine detection and treatment as part of second line therapy, the invention encompasses detection of any soluble factor and its treatment as part of second line therapy. Therefore, the description in the context of a cytokine can equally apply to a soluble factor.

In one embodiment, as part of a second line therapy, the invention includes methods for detecting cytokine levels in a patient who has received an infusion of CAR-T cells of the invention. In some embodiments, the presence or level of a cytokine may be used to select a candidate treatment. In some other embodiments, the presence or levels of a cytokine can be used to determine success during or after a course of first line, second line, or both first and second line therapy.

Biological samples in which the cytokine can be detected include, for example, serum. In some embodiments, the biological samples include a biopsy of tissue, which may or may not have a fluid component.

Immunological assays can be used to qualitatively or quantitatively analyze cytokine levels in a biological sample. A general overview of the technology involved can be found in a number of readily available manuals, such as Harlow & Lane, Cold Spring Harbor Laboratory Press, Using Antibodies: A Laboratory Manual (1999).

In addition to using immunological assays to detect levels of cytokines in a biological sample of a patient, an assessment of cytokine expression and levels can be made based on the level of a particular cytokine expression gene. RNA hybridization techniques to determine the presence and/or level of mRNA expression are well known to those skilled in the art and can be used to assess the presence or level of expression of a cytokine gene of interest.

In some embodiments, the methods of the present invention use cytokine selective binding partners to identify the presence or determine levels of a cytokine in a biological sample. The selective binding partner for use with the methods and kits of the present invention may be, for example, an antibody. In some aspects, monoclonal antibodies to a particular cytokine may be used. In some other aspects, polyclonal antibodies to a particular cytokine can be used to practice the methods and kits of the present invention.

Commercially available antibodies against cytokines are available and can be used in conjunction with the methods and kits of the present invention. It is well known to those skilled in the art that the type, source, and other aspects of the antibody to be used should be considered in light of the assay in which the antibody is used. In some cases, antibodies that will recognize their target antigen on a Western blot may not be usable in an ELISA or ELISpot assay and vice versa.

In some embodiments, antibodies for use in the assays of the present invention may be prepared using methods for producing monoclonal or polyclonal antibodies that are well known in the art (see, for example, Coligan, Current Protocols in Immunology (1991); Harlow & Lane , supra, Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986), and Kohler & Milstein, Nature 256:495-497 (1975).Such methods include generating antibodies by selecting antibodies from recombinant antibody libraries in phage or similar vectors. , as well as the production of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, for example, Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature 341:544546 (1989)). can be used in therapeutic and diagnostic applications, for example, in the treatment and/or detection of any of the specific cytokine-related diseases or conditions described nyh in the present description.

- 11 041322

Detection methods using immunological assays are particularly suitable for practical use in a patient monitoring site. Such methods allow immediate diagnosis and/or prognostic assessment of the patient. Patient monitoring site diagnostic systems are described, for example, in US Pat. No. 6,267,722, which is incorporated herein by reference. Other immunoassay formats are also available so that evaluation of a biological sample can be performed without having to send the sample to a laboratory for evaluation. Typically, these assays are formatted as solid phase assays where a reagent, such as an antibody, is used to detect the cytokine. Examples of test devices suitable for use in conjunction with immunoassays such as the assays of the present invention are described, for example, in US Pat. Nos. 7,189,522; 6818455 and 6656745.

In some aspects, the present invention provides methods for detecting polynucleotide sequences that encode for a cytokine in a biological sample. As noted above, a biological sample refers to a cell or population of cells or an amount of tissue or fluid from a patient. Most commonly, a sample is removed from a patient, but the term biological sample can also refer to cells or tissue analyzed in vivo, i.e., without being removed from the patient. Typically, a biological sample will contain the patient's cells, but the term can also refer to non-cellular biological material.

In one embodiment, amplification-based assays are used to measure the level of a desired cytokine. In this analysis, the nucleic acid sequence of the desired cytokine acts as a template in an amplification reaction (eg, polymerase chain reaction or PCR). With quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison with appropriate controls provides a measure of the copy number of the cytokine-related gene. Quantitative amplification techniques are well known to those skilled in the art. Detailed protocols for quantitative PCR are provided, for example, in Innis et al. (1990) PCR Protocols, Guide to Methods and Applications, Academic Press, Inc. N.Y.). RT-PCR methods are well known in the art (see, for example, Ausubel et al., supra). In some embodiments, a quantitative RT-PCR, such as the TaqMan™ assay, is used, thereby allowing comparison of the mRNA level in the sample with a control sample or value. Known nucleic acid sequences for the desired cytokine are sufficient to enable one of skill in the art to routinely select primers to amplify any portion of the gene. Suitable primers for amplifying specific sequences can be designed using principles well known in the art (see, for example, Dieffenfach & Dveksler, PCR Primer: Laboratory Manual (1995)).

In some embodiments, hybridization-based assays may be used to detect the amount of a desired cytokine in cells of a biological sample. Such assays include RNA dot blot as well as other assays such as fluorescence in situ hybridization, which is performed on samples that contain cells. Other hybridization assays are readily available in the art.

In numerous embodiments of the present invention, the level and/or presence of a cytokine polynucleotide or polypeptide will be detected in a biological sample, thereby allowing differential expression of the cytokine to be detected to obtain a cytokine profile from a biological sample obtained from a patient infused with CAR-T cells of the invention. , in comparison with the control biological sample.

The amount of a cytokine polynucleotide or polypeptide found in a biological sample is indicative of the presence of a cytokine to obtain a cytokine profile in order to classify a patient for appropriate cytokine treatment. For example, when the cytokine profile indicates an increase in a particular cytokine after T cell infusion compared to a control (e.g., prior to T cell infusion), one of skill in the art may choose to administer to a patient in need of such control an effective amount of the cytokine. - inhibitory compound. Alternatively, when the cytokine profile indicates a decrease in a particular cytokine after T cell infusion compared to a control (e.g., prior to T cell infusion), one of skill in the art may choose to administer to a patient in need of such control an effective amount of a cytokine activating agent. connections.

In some embodiments, the difference in cytokine levels between the T cell infusion sample and the control sample is at least about 0.5, 1.0, 1.5, 2.5, 10, 100, 200, 500, 1000 times.

The methods of the present invention may also be used to evaluate the effectiveness of a course of treatment. For example, in a patient following an infusion of T cells containing an increased amount of the cytokine IL-6, the efficacy of an anti-IL-6 treatment can be assessed by monitoring IL-6 over time. For example, lowering levels of a polynucleotide or polypeptide to

- 12 041322

IL-6 in a biological sample taken from a patient after treatment, compared with the level in a sample taken from a mammal before, or in the early stages of treatment, indicates effective treatment.

In one embodiment, the treatment regimen may be based on the neutralization of an elevated cytokine. For example, cytokine antagonists may be chosen for treatment. Antibodies are an example of a suitable antagonist and include murine antibodies, chimeric antibodies, humanized antibodies, and human antibodies or fragments thereof. Chimeric antibodies are antibodies whose light and heavy chain genes have been engineered, usually through genetic engineering, from immunoglobulin gene segments belonging to different species (see, e.g., Wause et al., Annals of Oncology 14:520-535 (2003) ) . For example, variable (V) gene segments from mouse monoclonal antibodies can be combined with human constant (C) segments. A typical chimeric antibody is thus a fusion protein consisting of the V or antigen-binding domain from a mouse antibody and the C or effector regions from a human antibody.

Humanized antibodies have variable region framework residues largely from a human antibody (referred to as an acceptor antibody) and complementarity-determining regions largely from a mouse antibody (referred to as a donor immunoglobulin). See Queen et al., Proc. Natl. Acad. sci. USA 86: 10029-10033 (1989) and WO 90/07861, US Patent No. 5,693,762, US Patent No. 5,693,761, US Patent No. 5,585,089, US Patent No. 5,530,101 and Winter, US Patent No. 5,225,539. , are also largely or wholly derived from human immunoglobulin. Antibodies can be generated by conventional hybridoma approaches, phage display (see e.g. Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047), use of transgenic mice with human immune systems (Lonberg et al. , WO93/12227 (1993)), among other sources. Nucleic acids encoding immunoglobulin chains can be derived from antibody-producing hybridomas or cell lines, or based on immunoglobulin nucleic acid or amino acid sequences in the published literature.

Other desired cytokine antagonists may also be used for therapeutic purposes. For example, a class of antagonists that can be used for the purposes of the present invention are soluble forms of cytokine receptors. For merely illustrative purposes, an IL-6 antagonist is an anti-IL-6 antibody that specifically binds to IL-6. A specific antibody has the ability to inhibit or antagonize the action of IL-6 in a systemic manner. In some embodiments, the antibody binds IL-6 and prevents it from interacting with or activating its receptors (eg, IL-6Ra or IL-6Re). In some embodiments, IL-6 activity can be antagonized through the use of an interleukin-6 receptor (IL-6R) antagonist. US Patent Application No. 2006251653 describes methods of treating a disease related to interleukin-6 and discloses a number of interleukin-6 antagonists, including, for example, humanized anti-IL-6R antibodies and chimeric anti-IL-6R antibodies. In some embodiments, an IL-6 or IL-6R derivative may be used to block and antagonize the interaction between IL-6/IL-6R.

The present invention is not limited to the cytokines and their respective activators and inhibitors described herein. Rather, the invention includes the use of any cytokine activator and/or inhibitor that is used in the art to modulate cytokines. The reason for this is that the invention is based on the control of cancer treatment in a patient receiving an infusion of the CAR-T cells of the invention, where the infused CAR-T cells lead to an increase and decrease in the levels of various cytokines. One of skill in the art, based on the disclosure provided herein, will be able to appreciate that the differential levels of cytokine expression in a T cell infusion sample compared to a control sample can be targeted for treatment to have the cytokine level elevated or reduced to normal levels.

Therapeutic use

The present invention encompasses a cell (eg, T cell) transduced using a lentiviral vector (LV). For example, LV encodes a CAR that combines the antigen recognition domain of a specific antibody with the intracellular domain of CD3-zeta, CD28, 4-1BB, or any combination thereof. Therefore, in some cases, a transduced T cell can elicit a CAR-mediated T cell response.

The invention provides the use of CAR to redirect the specificity of a primary T cell to a tumor antigen. Thus, the present invention also provides a method for stimulating a T cell-mediated immune response to a target T cell population or tissue in a mammal, comprising the step of administering to the mammal T cells that express CAR, wherein the CAR contains a binding moiety that specifically interacts with pre- a given target, a portion of the zeta chain containing, for example, the intracellular domain of human CD3 zeta, and a costimulatory signaling region.

In one embodiment, the present invention includes a type of cell therapy, where

- 13 041322

The T cells are genetically modified to express CAR and the CAR T cells are infused into the recipient. The infused cells are capable of destroying tumor cells in the recipient. In contrast to antibody therapies, CAR-T cells are able to replicate in vivo, resulting in long-term viability that can lead to prolonged tumor control.

In one embodiment, the CAR-T cells of the invention can undergo sustained T cell expansion in vivo and can survive for an extended amount of time. In yet another embodiment, the CAR-T cells of the invention are converted into specific memory T cells that can be reactivated to inhibit any additional tumor formation or growth. For example, it was surprising that the CART19 cells of the invention can undergo sustained T cell expansion in vivo and survive at high levels for extended periods of time in the blood and bone marrow and form memory-specific T cells. Without wishing to be bound by a particular theory, the authors believe that CAR-T cells can differentiate in vivo into a central memory-like state upon contact, followed by elimination of target cells expressing the surrogate antigen.

Without wishing to be bound by a particular theory, the authors believe that the antitumor immune response elicited by CAR-modified T cells may be an active or passive immune response. In addition, a CAR-mediated immune response may be part of an adoptive immunotherapy approach in which CAR-modified T cells induce an immune response specific for an antigen-binding fragment in CAR. For example, CART19 cells elicit an immune response specific against cells expressing CD19.

While the data disclosed herein specifically discloses a lentiviral vector containing anti-CD19 scFv derived from mouse monoclonal antibody FMC63, human CD8a hinge and transmembrane domain, and human 4-1BB and CD3-zeta signaling domains, the invention follows considered as including any number of variations for each of the components of the design, as described elsewhere in the present description. In other words, the invention includes the use of any antigen-binding fragment in a CAR to generate a CAR-mediated T cell response specific for the antigen-binding fragment. For example, an antigen-binding fragment in a CAR of the invention may be targeted to a tumor antigen for the purpose of treating cancer.

Malignancies that may be treated include tumors that are not vascularized, or only slightly vascularized, as well as vascularized tumors. Malignancies may include non-solid tumors (such as hematologic tumors such as leukemias and lymphomas) or may include solid tumors. The types of malignancies to be treated with the CAR of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignancies, e.g., sarcomas, carcinomas, and melanomas. Adult tumors/malignancies and pediatric tumors/malignancies are also included.

Hematologic malignancies are malignant neoplasms of the blood or bone marrow. Examples of hematologic (or hematogenous) malignancies include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemias), chronic leukemias (such as chronic granmyelocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (low and high grade), multiple myeloma, Waldenström'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 fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after 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, lymphoneoplasia, pancreatic cancer, breast cancer, cancer lung, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma sebaceous carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular cancer, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as glioma (such

- 14 041322 as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannomas, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neurinoma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases.

In one embodiment, a CAR antigen-binding fragment of the invention is designed to treat a specific cancer. For example, a CAR designed to target CD19 can be used to treat malignancies and disorders including, but not limited to, pre-B ALL (children), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, back-up treatment after allogeneic bone marrow transplant, etc.

In yet another embodiment, a CAR can be engineered to target CD22 for the treatment of diffuse large B cell lymphoma.

In one embodiment, malignancies and disorders include, but are not limited to, pre-B ALL (children), adult ALL, mantle cell lymphoma, diffuse large B cell lymphoma, back-up treatment after allogeneic bone marrow transplantation, etc. that can be treated with a combination of CARs that target CD19, CD20, CD22 and ROR1.

In one embodiment, a CAR can be engineered to target mesothelin for the treatment of mesothelioma, pancreatic cancer, ovarian cancer, and the like.

In one embodiment, a CAR can be engineered to target CD33/IL3Ra for the treatment of acute myelogenous leukemia and the like.

In one embodiment, a CAR can be engineered to target c-Met for the treatment of triple negative breast cancer, non-small cell lung cancer, and the like.

In one embodiment, the CAR may be engineered to target PSMA for the treatment of prostate cancer and the like.

In one embodiment, the CAR may be engineered to target the F77 glycolipid for the treatment of prostate cancer and the like.

In one embodiment, the CAR may be engineered to target EGFRvIII for the treatment of glioblastoma and the like.

In one embodiment, a CAR can be engineered to target GD-2 for the treatment of neuroblastoma, melanoma, and the like.

In one embodiment, the CAR can be engineered to target the NYESO-1 TCR for the treatment of myeloma, sarcoma, melanoma, and the like.

In one embodiment, the CAR can be engineered to target the MAGE A3 TCR for the treatment of myeloma, sarcoma, melanoma, and the like.

However, the invention should not be construed as being limited solely to the antigenic targets and diseases disclosed herein. Rather, the invention should be considered to include any antigenic target that is associated with a disease, where CARs can be used to treat the disease.

The CAR-modified T cells of the invention may also serve as a vaccine for ex vivo immunization and/or in vivo therapy in a mammal. Preferably the mammal is a human.

With respect to ex vivo immunization, at least one of the following events occurs in vitro prior to the introduction of the cells into the mammal: i) expansion of the cells, ii) introduction of the CAR-encoding nucleic acid into the cells, and/or iii) cryopreservation of the cells.

Ex vivo methods are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (ie, transduced or transfected in vitro) using the CAR expression vector disclosed herein. The CAR-modified cells may be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human, and the CAR-modified cells may be autologous to the recipient. Alternatively, the cells may be allogeneic, syngeneic, or xenogeneic with respect to the recipient.

The ex vivo propagation technique for hematopoietic stem and progenitor cells described in US Pat. No. 5,199,942, incorporated herein by reference, can be applied to the cells of the present invention. Other suitable methods are known in the art, therefore the present invention is not limited to any particular method for expanding cells ex vivo. Briefly, ex vivo cultivation and expansion of T cells includes: (1) harvesting mammalian CD34+ hematopoietic stem and progenitor cells from a collection of peripheral blood or bone marrow explants, and (2) ex vivo expansion of such cells. In addition to the cell growth factors described in US Pat. No. 5,199,942, other factors such as

- 15 041322 as flt3-L, IL-1, IL-3 and c-kit ligand can be used for cell culture and expansion.

In addition to using a cell-based vaccine in terms of ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient.

In general, cells activated and propagated as described herein can be used in the treatment and prevention of diseases that occur in individuals who are immunocompromised (immune compromised patients). In particular, the CAR-modified T cells of the invention are useful in the treatment of CCL. In some embodiments, the cells of the invention are used in the treatment of patients at risk of developing CCL. Thus, the present invention provides methods for treating or preventing CCL comprising administering to an individual in need of such treatment a therapeutically effective amount of the CAR-modified T cells of the invention.

The CAR-modified T cells of the present invention may be administered either alone or as a pharmaceutical composition in combination with diluents and/or other components such as IL-2 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention may contain the target population of cells, as described in the present description, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may contain 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 (eg aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

The pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease being treated (or prevented). The amount 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 illness, although appropriate dosages may be determined through clinical trials.

When an immunologically effective amount, an antitumor effective amount, a tumor-inhibiting effective amount, or a therapeutic amount is administered, the precise amount of compositions of the present invention to be administered may be determined by the physician, taking into account individual differences in age, weight, tumor size, degree of infection or metastases, and the condition of the patient (individual). ). In general, it can be stated that pharmaceutical compositions containing T cells described herein can be administered at a dosage of 104-10 ⁹ cells/kg body weight, preferably 105-106 cells/kg body weight, including all whole values within these intervals. The T cell compositions may also be administered multiple times at these dosages. Cells can be administered using infusion techniques that are well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one of skill in the medical field by monitoring the patient for signs of disease and appropriate treatment selection.

In some embodiments, it may be desirable to administer activated T cells to an individual and then subsequently re-bleed (or perform apheresis), activate the harvested T cells in accordance with the present invention, and reinfuse the activated and expanded T cells into the patient. This process can be carried out many times every few weeks. In some embodiments, the implementation of T cells can be activated from the taken blood samples equal to from 10 to 400 cm ³ . In some embodiments, T cells are activated from blood samples taken at 20, 30, 40, 50, 60, 70, 80, 90, or 100 cm ³ . Without being bound by a particular theory, the authors believe that the use of this multiple blood draw/multiple reinfusion protocol may serve to select certain populations of T cells.

Administration of the subject compositions may be by any conventional means, including 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 (IV) injection, or intraperitoneally. In one embodiment, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In yet another embodiment, the T cell compositions of the present invention are preferably administered via iv. injections. T cell compositions can be injected directly into a tumor, lymph node, or site of infection.

In some embodiments of the present invention, cells activated and expanded using the methods described herein, or other methods known in the art, where T cells are expanded to therapeutic levels, are administered to the patient in combination with (for example, before, concurrently, or after) any number of significant treatments, including, but not limited to, treatment using agents such as antiviral therapy, treatment with cidofovir and interleukin-2, Cytarabine (also known as ARA-C), or natalizumab for MS- patients or treatment with efalizumab for patients with psoriasis or other treatments for patients with PML. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunodestructive agents such as CAM PATH, anti-CD3 antibodies, or other therapies. antibodies, cytotoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and radiation. These drugs inhibit either calcium-dependent calcineurin phosphatase (cyclosporine and FK506) or inhibit p70S6 kinase, which 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 of the present invention are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) bone marrow transplantation, T cell ablative therapy using either chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide or antibodies such as OKT3 or CAMPATH. In yet another embodiment, the cell compositions of the present invention are administered following ablative B cell therapy, such as agents that react with CD20, such as Rituxan. For example, in one embodiment, individuals may receive standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after the transplant, individuals receive an infusion of expanded immune cells of the present invention. In a further embodiment, the expanded cells are administered before or after surgery.

The dosage of the above treatments for administration to a patient will vary according to the nature of the condition being treated and the recipient of the treatment. Scaling of dosages for human administration can be carried out in accordance with the practical approaches adopted in this field. The dose for CAMPATH, for example, will typically be in the range of 1 to about 100 mg for an adult patient, typically administered daily for a period of between 1 and 30 days. The preferred daily dose is from 1 to 10 mg per day, although higher doses up to 40 mg per day (described in US patent No. 6120766) may be used in some cases.

Treatment of Cytokine Release Syndrome (CRS)

The present invention is based in part on the discovery that in vivo proliferation of CART19 cells and the high antitumor activity associated with it is also associated with CRS leading to hemophagocytic lymphohistiocytosis (HLH), also referred to as macrophage activation syndrome (MAS). While not wishing to be bound by a particular theory, the authors believe that MAS/HLH is a unique biomarker that is associated with and may be required for the high antitumor activity of CART19.

Accordingly, the invention provides a first line therapy comprising administering a CAR of the invention to a patient and a second line therapy comprising administering a type of therapy to control elevated levels of certain soluble factors resulting from first line therapy using CAR-T cells.

In one embodiment, the implementation of the second line of therapy includes compositions and methods for the treatment of CRS. The symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like. The present invention is based on the observation that CART19 cells induced elevated levels of soluble factors in a patient, including but not limited to IFN-γ, TNFa, IL-2 and IL-6. Therefore, the second line of therapy includes compounds and methods to neutralize the effects against increased cytokines resulting from the introduction of CART19 cells. In one embodiment, neutralizing agents are capable of counteracting an undesirable concerted burst of cytokine expression/activity, and thus are useful in the prevention, amelioration, and treatment of CRS associated with CART19 therapy.

In one embodiment, CRS treatment is carried out approximately 10-12 days after infusion of CART19 cells.

In one embodiment, the implementation of the second line of therapy includes the introduction of a steroid to the patient. In yet another embodiment, the second line of therapy comprises the administration of one or more of a steroid, a TNFa inhibitor, and an IL-6 inhibitor. An example of a TNFa inhibitor is etanercept. An example of an IL-6 inhibitor is tocilizumab (TOC).

Experimental examples

The present invention is further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise stated. Thus, the invention should in no way be construed as limited to the following examples, but rather, should be construed as covering any and all variations that become apparent as a result of the teachings provided herein.

Without further description, the authors believe that one of ordinary skill in the art will be able, using the foregoing description and the following illustrative examples, to make and use the compounds of the present invention and to practice the claimed methods. The following working examples are therefore specifically indicative of preferred embodiments of the present invention and should not be construed as limiting in any way the remainder of the disclosure.

Example 1 Cytokine Therapy in Combination with CAR T Cell Infusion

The results presented herein demonstrate that CAR T cell infusion patients exhibit differential levels of expression of various cytokines. In some cases, elevated levels of certain cytokines result from toxicity of infused CAR T cells (FIG. 1). It was observed that tocilizumab (anti-III6) could reduce CAR toxicity and appear to maintain antitumor effects in 2 out of 2 patients (FIG. 2). Without wishing to be bound by a particular theory, the authors believe that anakinra and other reagents that block IL-1 may also be applicable in this regard. The data presented herein also demonstrates that IL-1 is elevated in patients and this may lead to a later increase in IL-6. Anakinra is a recombinant IL1Ra protein that binds to IL1 receptors and blocks signal transduction from both IL-1 alpha and beta. Anakinra has a short half-life. There is an advantage to using anakinra to start treatment in patients, as both IL-1 alpha and beta will be blocked, and also moderate the cytokine release and retain the antitumor effect.

It was also observed that antibody interventions had no effect on CART19 cellular functionality as measured by perforin and IFN-γ (FIG. 3).

Example 2 CB19 Redirected Chimeric Antigen Receptor (CART19) T Cells Induce Cytokine Release Syndrome (CRS) and Treatable Macrophage Activation Syndrome (MAS), Which Can Be Controlled by the IL-6 Antagonist Tocilizumab (TOC)

Infusion of CART19 cells results in 100-100,000x in vivo proliferation, tumor lysis syndrome accompanied by prolonged antitumor activity and prolonged survival in patients with B cell tumors. The results presented herein demonstrate that the proliferation of CART19 cells in vivo and, as a result, high antitumor activity, is associated with CRS leading to hemophagocytic lymphohistiocytosis (HLH), also referred to as MAS. While not wishing to be bound by a particular theory, the authors believe that MAS/HLH is a unique biomarker that is associated with and may be required for high antitumor activity.

Autologous T cells were transduced with a CAR lentivirus composed of anti-CD19 scFv/4-1BB/CD3-zeta, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into patients with ALL or CLL with persistent disease after 2-8 previous courses of treatment. CART19 activity against ALL was also modeled in a mouse xenograft model with high engraftment of human ALL graft/human T cells and simultaneous detection of CAR and ALL T cells using 2-color bioluminescent imaging.

The results presented herein provide up-to-date results for 10 patients who received CART19 cells, including 9 CLL patients and 1 pediatric patient with relapsed refractory ALL. 6/9 Evaluated patients showed complete recovery (CR) or partial recovery (PR), including 4 cases of delayed CR. While no acute infusion toxicity was observed, all studied patients also developed CRS. All had high temperatures as well as grade 3 or 4 hypotension/hypoxia. CRS was preceded by peak blood expression of CART19 cells and then increased in intensity to peak CART19 cells (Day 10-31 post-infusion). The ALL patient experienced the most significant toxicity, with grade 4 hypotension and respiratory failure. Steroid therapy on Day 6 did not improve. On Day 9, noting high levels of TNFa and IL-6 (peak increases above baseline: IFNy at 6040x; IL-6 at 988x; IL-2R at 56x, IL-2 at 163x and TNFa at 17x), TNFa antagonists and IL-6 (etanercept and tots). This resulted in normalization of temperature and hypotension within 12 hours and a rapid transition from artificial ventilation to room air. These interventions had no clear effect on proliferation or performance of CART19 cells: a peak of T cells with CAR (2539 CAR+ cells/µl; 77% CD3 cells downstream) occurred on Day 11, and on Day 23 the bone marrow showed CR with negative minimal residual disease (MRD), compared to baseline during the study period in the bone marrow, which showed 65% blast cells. Despite no history of ALL in the CNS, cerebrospinal fluid showed detectable CART19 cells (21 lymph/µl; 78% CAR+). At 4 months post-infusion, this patient maintained a CR, with 17 CACT19 cells/µl in blood and 31% CAR+ CD3 cells in bone marrow.

Clinical evaluation of subsequent study patients indicates that all of them had evidence of MAS/HLH, including abrupt increases in ferritin and histological manifestation of HLH. Peak ferritin levels ranged from 44,000 to 605,000, preceded and followed by peak T cell proliferation. Other consistent findings include the rapid onset of non-disease hepatosplenomegaly and mild DIC.

Subsequently, 3 patients with CLL were also treated with tots, also with an immediate and dramatic reduction in high temperatures, hypotension and hypoxia. One patient received tots on Day 10 and achieved a CR followed by CART19 proliferation. Another patient had a rapid decrease in CRS on day 9 post-Toc and the follow-up for response was too short. A third patient with CLL received tots on Day 3 due to early fever and had no proliferation of CART-19 and no response.

To model the temporal pattern of cytokine blockade, xenografts using bioluminescent primary ALL in children were placed and then exposed to excess clinically manufactured cells. CART19 cells proliferated and resulted in prolonged survival. Cytokine blockade prior to T cell infusion using toc and/or etanercept abolished disease control with less in vivo proliferation of infused CART19 cells, confirming the outcome seen in one patient receiving an early dose of toc (Day 3).

CART19 T cells can produce extensive in-vivo proliferation, long-term viability, and antitumor efficacy, but can also induce significant CRS with features suggestive of MAS/HLH, which responds rapidly to cytokine blockade. Created prior to the initiation of significant CART19 proliferation, blockade of TNFa and/or IL-6 may interfere with proliferation and effector function, but if created at a time when cell proliferation is in the process of change, TOC may reduce symptoms that have been observed and that correlate with sustained clinical responses.

Example 3 Remission of ALL by T Cells Expressing Chimeric Antigen Receptor

The results presented herein demonstrate that CAR-T cells have clinical activity in acute lymphocytic leukemia (ALL). Briefly, two pediatric patients with relapsed refractory pre-B-cell ALL were treated with 106 to 10 ⁷ /kg T cells transduced with anti-CD19 antibody and T-cell signaling molecule (CTL019 CAR-T cells; also referred to as CART19). CTL019 T cells multiplied more than 1000-fold in both patients and migrated to the bone marrow. In addition, CAR-T cells were able to cross the blood-brain barrier and remained at high levels for at least 6 months as measured in cerebrospinal fluid. Eight severe adverse events were noted. Both patients developed cytokine release syndrome (CRS) and B-cell aplasia. In one child, CRS was severe and cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome, and still did not prevent CAR-T cell proliferation and antileukemic efficacy. Complete remission was observed in both patients and continued in one patient 9 months after treatment. Another patient relapsed with blast cells that no longer expressed CD19 approximately 2 months after treatment.

The results presented herein demonstrate that CAR-modified T cells have the ability to kill in vivo even aggressive cells in refractory acute leukemia. The emergence of tumor cells that no longer express the target point to the need to target other molecules in addition to CD19 in some patients with ALL.

In vivo expansion and sustained anti-leukemic effects of CTL019 (CART19) cells have been reported in 3 CLL patients (Porter et al., 2011, N Engl J Med 365:725-33; Kalos et al., 2011, Science Translational Medicine 3:95ra73 ). CTL019 is a CAR that includes the CD137 signaling domain (4-1BB) and is expressed using lentiviral vector technology (Milone et al., 1009, Mol Ther 17: 1453-64). The results presented herein demonstrate the use of CTL019 in 2 pediatric patients with refractory and recurrent ALL. Both patients had leukemia remission accompanied by sustained in vivo replication of CTL019 with migration to the bone marrow and CNS. Antileukemic effects were high as one patient had refractory disease after chemotherapy preventing allogeneic donor stem cell transplantation, and the other patient had a relapse after allogeneic cord blood transplantation and was resistant to blinatumomab therapy (a chimeric bispecific anti-CD3 and anti-CD19 antibody). - 19 041322 scrap).

The following section describes the materials and methods used in these experiments.

Materials and methods CART19

The production of CTL019 (CART19) has been previously reported (Porter et al., 2011, N Engl J Med 365:725-33; Kalos et al., 2011, Science Translational Medicine 3:95ra73). CTL019 was detected and quantified in patient samples as previously reported (Porter et al., 2011, N Engl J Med 365:725-33; Kalos et al., 2011, Science Translational Medicine 3:95ra73).

Collection and processing of samples

Samples (peripheral blood, bone marrow) were collected in purple cap vacuum tubes (K2EDTA) or red cap (no additives) (Becton Dickinson). Purple cap tubes were delivered to the laboratory within 2 hours of collection or delivered overnight at room temperature in insulated containers essentially as described (Olson et al., 2011, J Transl Med 9:26) prior to processing. Samples were processed within 30 minutes of receipt in accordance with established laboratory SOP. Peripheral blood and bone marrow mononuclear cells were purified, processed and stored in liquid nitrogen as described (Kalos et al., 2011, Science Translational Medicine 3:95ra73). Tubes with red caps were processed within 2 hours of collection, including coagulation time; serum isolated by centrifugation was collected in 100 μl aliquots for single use and stored at -80°C. CSF was delivered to the laboratory within 30 minutes of aspiration and cells in the CSF were collected by CSF centrifugation and processed for DNA extraction and analysis by flow cytometry.

Analysis by quantitative PCR

Whole blood or bone marrow samples were collected in purple-capped BD vacuum tubes (K2EDTA) (Becton Dickinson). Genomic DNA was isolated directly from whole blood and qPCR analysis on genomic DNA samples was performed in conjunction with ABI Taqman technology and a validated assay to detect CD19-integrated CAR transgenic sequence as described (Kalos et al., 2011, Science Translational Medicine 3:95ra73) using 200 ng genomic DNA per time point for peripheral blood and bone marrow samples and 18-21.7 ng genomic DNA per time point for CSF (cerebrospinal fluid) samples. To determine the number of copies per DNA unit, an 8-point standard curve was generated consisting of 5x106 copies of the lentiviral plasmid CTL019 introduced into 100 ng of non-transduced control genomic DNA. Each data entry point (sample, standard curve) was evaluated in three replicates with a positive Ct value in 3/3 replicates with a % CV less than 0.95% for all quantifiable values. A parallel amplification reaction to control the quality of the requested DNA was performed using 20 ng of the injected genomic DNA from peripheral blood and bone marrow (2-4.3 ng for CSF samples) and a primer/probe combination specific for the non-transcribed genomic sequence upstream of the CDKN1A gene, as described (Kalos et al., 2011, Science Translational Medicine 3:95ra73). These amplification reactions allowed the generation of a correction factor (CC) to correct the calculated DNA input from the actual DNA input. Transgene copies per microgram of DNA were calculated according to the formula: copies calculated from the standard curve for CTLO19 per introduced DNA (ng)xKKx1000 ng. The accuracy of this assay was determined by the ability to quantify the labeling of the infused cell product by Q-PCR. These blind determinations generated Q-PCR and flow label values of 11.1% and 11.6%, respectively, for CHOP-100 and a label of 20.0 and 14.4%, respectively, for CHOP-101 infusion products.

Soluble factor analysis

Whole blood was collected in BD vacuum tubes with red cap (no additives) (Becton Dickinson), processed to obtain serum using the established laboratory SOP, aliquoted for single use and stored at -80°C. Soluble cytokine factors were quantified using Luminex Bead Matrix Technology and kits supplied by Life technologies (Invitrogen). Analytical tests were performed according to the manufacturer's protocol with a 9-point standard curve generated using serial 3-fold dilutions. 2 points of the external standard were evaluated in two repeated experiments and 5 points of internal standards in one experiment; all samples were evaluated in two repeated experiments at a dilution of 1:2; the calculated % CV for two replicate measurements were less than 15%. Data were acquired on a FlexMAP-3D in percent and analyzed using XPonent 4.0 software and 5-parameter logistic regression analysis. Standard curve quantitation intervals were determined from the 80-120% interval (observed/expected value). Values reported included values within the interval of the standard curve and values calculated by logistic regression analysis.

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Antibody reagents

The following antibodies were used for these studies: MDA-CAR (Jena and Cooper, 2013, L. Anti-idiotypic antibodies for CD19. PlosONE 2013; in press), mouse anti-CD19 CAR antibodies conjugated to Alexa647. Antibodies for multiparametric immunophenotyping: T cell detection panels: anti-CD3-FITC, anti-CD8-PE, anti-CD14-PE-Cy7, anti-CD16-PE-Cy7, anti-CD19-PE-Cy7, anti-CD16-PE -Su7. B cell detection panels: anti-CD20-FITC, anti-CD45-PE, antu-CD45-APC, anti-CD19-PE-Cy7, anti-CD19-PE, anti-CD34-PCP-e710 and anti-CD34- APC was purchased from e-Biosciences.

Multiparametric flow cytometry

Cells were evaluated by flow cytometry immediately after treatment with FicollPak, except for the baseline CHOP-101 sample, which was evaluated immediately after thawing the cryopreserved sample. Multiparametric immunophenotyping for peripheral blood and bone marrow samples was performed using approximately 0.2-0.5x106 total cells per condition (depending on cell yield in the samples), and for CSF samples using trace amounts of cells collected after CSF centrifugation, and using fluorescence minus one (FMO) dyes as described in the text. Cells were stained in 100 µl PBS for 30 min on ice using manufacturer's recommended concentrations of antibody and reagent, washed and resuspended in 0.5% paraformaldehyde and data were obtained using an Accuri C6 cytometer equipped with Blue (488) and Red (633 nm). ) laser. Accuri files were exported in FCS file format and analyzed using FlowJo software (Version 9.5.3, Treestar). Compensation values were set using single antibody stains and BD compensation beads (Becton Dickinson) and calculated by software. The sorting strategy for T cells was Live Cells (FSC/SSC) > Dump Channel (CD14+CD16+CD19PECy7) versus CD3+ > CD3+. The general sorting strategy for B cells was as follows: Live cells (FSC/SSC) > SSC low events > CD19+. Sorting for samples CHOP100 and CHOP-101 is described in more detail in the individual figures.

Molecular analysis MRD

Molecular analysis of MRD was performed by Adaptive Biotechnologies (Seattle, WA) and high throughput next generation sequencing of the IGH CDR3 BCR region using the immunoSEQ analytical assay on the Illumina HiSeq/MiSeq platform (Larimore et al., 2012, J Immunol 189:3221-30). For these assays, 701-6000 ng (approximately 111,000-950,000 genomic equivalents) of genomic DNA isolated from patient-derived whole blood or bone marrow samples were subjected to combined multiplex PCR and sequencing followed by algorithmic analyzes to quantify the individual IGH CDR3 sequences in the samples. . Parallel amplification and sequencing of the TCRB CDR3 region (Robins et al., 2009, Blood 114:4099-107) in each sample was performed to assess the quality of the DNA samples. For each patient, analyzed IGH CDR3 nucleotide sequences from samples at different time points were aligned using the EMBL-EBI multiple sequence alignment tool (Goujon et al., 2010, Nucleic Acids Res 38:W695-9; Sievers et al., 2011, Mol Syst Biol 7:539). The dominant clone from the earliest time point sample was bioinformatically run through the analyzed IGH CDR3 sequences in the next time point samples to identify the presence of sequences with 95% or greater pairwise sequence identity. Total sequencing reads for these sequences, similar to those for the dominant clone, are shown for each time point.

The results of the experiments are described below.

Descriptions of clinical cases

CHOP-100 was a 7 year old girl on her second ALL recurrence. She was diagnosed 2 years earlier and achieved minimal residual disease (MRD) negative remission, with a relapse 17 months after diagnosis. She was in remission again after reinduction chemotherapy, but the disease returned 4 months later, after which she did not respond to furteclofaribine/etoposide/cyclophosphamide. Her baseline karyotype was 48,XX,del(9)(p21.3),+11,del(14)(q2?q24),+16/46,XX[4]. Peripheral blood mononuclear cells (PBMCs) were harvested by apheresis prior to intensive chemotherapy, suggesting that there may not be enough circulating T cells capable of reproducing cells after such intensive treatment. This patient was infused with CTL019 cells that had been expanded under anti-CD3/CD28 conditions and lentivirally transduced to express the antiCD19 CAR at a total dose of 3.8x108 cells/kg (1.2x10 ⁷ CTL019 cells/kg) administered every 3 consecutive days as previously described (Porter et al., 2011, N Engl J Med 365:725-33; Kalos et al., 2011, Science Translational Medicine 3:95ra73). She did not receive lymph-depleting chemotherapy prior to her CTL019 infusions, with most recent cytotoxic therapy given 6 weeks prior to her CTL019 infusion. No immediate infusion toxicity was noted, but she was hospitalized for low grade body temperature, which progressed to high fever on day 4, and on day 5 the patient was transferred to the pediatric intensive care unit (CHOP-100 , Fig. 4A). This was accompanied by a rapid increase in significant respiratory and cardiovascular insufficiency, requiring mechanical ventilation and maintenance of blood pressure.

The second patient with ALL was a 10-year-old girl (CHOP-101) who underwent her second recurrence after 4/6 cord blood transplants from a matched unrelated donor 28 months after diagnosis and 10 months before CTL019 infusion. She suffered graft-versus-host disease (GVHD) after transplantation, which was abolished by treatment; she was not immunosuppressed at the time of her relapse. She subsequently did not experience remission despite multiple cytotoxic and biological therapies. Her initial karyotype was 46 XX, del(1) (p13), t (2;9) (q?21;q?21), t (3;17) (p24;q23), del (6) (q16q21 ), del(9) (q13q22), der(16)t(1;?;16) (p13;?p13.3) [9],//46, Xy[1]. She underwent two cycles of blinatumomab (Bargou et al., 2008, Science 321:974-7) before PBMC collection without any response. Her peripheral blood cells were 68% of donor origin at the time of PBMC collection. CTL019 T cells are obtained and infused at a total dose of 10 ⁷ cells/kg (1.4 x ^{10 6} CT019 cells/kg) in a single dose following etoposide/cyclophosphamide chemotherapy given for lymphatic depletion one week earlier. Her bone marrow the day before CTL019 infusion was replaced with a population of CD19+/CD34+ ALL cells, with variable CD19 expression by standard clinical flow cytometry (FIG. 7). She did not experience any immediate infusion toxicity, but developed a high fever on Day 6 and was admitted to the hospital. She did not experience any cardiopulmonary toxicity and did not receive glucocorticoids or anti-cytokine therapy. CHOP-101 experienced a high temperature of unknown origin, thought to be due to cytokine release (FIG. 4B), myalgia, and two days of confusion (grade 3), which ended spontaneously. She had no obvious signs of GVHD after infusion of CTL019 cells. Although these cells were collected from a patient, they were mostly of donor origin (cord blood).

Remission induction in both individuals

Both individuals had an increase in circulating lymphocytes and neutrophils 2 weeks after CTL019 infusion, as shown in graphs depicting total WBC, ALC, and ANC versus time of CTL019 infusion (Fig. 4C). Most of the lymphocytes consisted of T cells that expressed the chimeric antigen receptor (FIG. 8), shown in more detail in FIG. 5. Both individuals documented high fever of non-infectious origin, followed by an increase in LDH (Fig. 4A). LDH elevations and high temperatures were similar to those previously described in CLL patients after CTL019 infusion (Porter et al., 2011, N Engl J Med 365:725-33; Kalos et al., 2011, Science Translational Medicine 3:95ra73) . Approximately one month after the infusion, a negative (<0.01%) morphological remission of the MRD of leukemia was achieved in both individuals (Table 1).

Clinical remission in CHOP-100 was associated with deep molecular remission, which was sustained for at least 9 months from January 2013 (Table 1). High-throughput DNA sequencing of the IGH locus showed a marked decrease in total IGH readings at Day +23 in CHOP-100 blood and bone marrow. The malignant clone was not detected in the blood or bone marrow of more than 1 million cell equivalents that were sequenced on Day +180. In contrast, T-cell receptor sequences were readily detectable in blood and bone marrow, indicating DNA integrity tested at all time points.

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Table 1. Induction of molecular remission in blood and bone marrow CHOP-100 and 101

Patient Textile Temporary Number Are common Are common Are common readouts Frequency dot introduced readings readings unique dominant clone (day) genomes ΤΟΗβ IGH readings clone tumors (cellular IGH (%) equivalents) CHP959- Blood -1 111340 525717 189 6 185 97.8θ 100 23 218210 1651129 0 0 0 0.00 87 288152 1416378 0 0 0 0.00 180 420571 1276098 6 2 0 0.00 Bone -1 317460 348687 59791 318 59774 99, 97 brain 23 362819 1712507 37 2 33 89, 19 87 645333 425128 10 1 10 100.00 180 952381 800670 45 7 0 0.00 CHP959- Blood -1 152584 1873116 38170 52 30425 79.71 101 23 417371 1462911 92 5 18 19, 60 Bone -1 158730 2417992 68368 65 50887 74.43 brain 23 305067 1978600 1414 eleven 946 66, 90 60 916571 But 530833 206 363736 68, 90

Molecular analysis of minimal residual disease was performed on DNA isolated from whole blood or bone marrow.

Toxicity CTL019

Grade 3 and 4 adverse events are summarized in Table 1. 2. Acute toxicity was observed in both patients, which consisted of fever and cytokine release syndrome (CRS), which progressed to macrophage activation syndrome (MAS). Both patients were under current follow-up and received prophylactic treatment for tumor lysis syndrome. Both experienced a significant increase in LDH, the causes of which were likely multifactorial but could include tumor lysis syndrome. Each CHOP-100 uric acid value was either below normal or in the normal range, and she only received allopurinol on days 5-6. CHOP-101 received prophylactic allopurinol on days 0-14 and had abnormal uric acid values of 4.8-5.7 on days 8-10, indicating a mild tumor lysis syndrome.

Table 2. Adverse events (grade 3 and 4) for CHOP-100 and CHOP-101

SNR595-100 Category of AE AE toxicity Degree of AE Description of AE Duration INFECTION Febrile neutropenia 3 Febrile neutropenia. Temperature: Peak temperature 40.7°C, decreasing on day 7 after tocilizumab administration 7 days General state hypotension 4 Shock requiring high blood pressure to be maintained. Exclusion of all means of maintaining high blood pressure, except for dobutamine. 4 days at degree 4, exception vasopressors on day 12 VASCULAR MANIFESTATIONS acute syndrome blood loss 4 Life-threatening condition indicated for use to increase blood pressure or support respiratory function see above PULMONARY/ RESPIRATION acute syndrome respiratory failure (ARDS) 4 Presence, intubation is indicated. The chest x-ray becomes clear on day 8. 12 days

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SNR595-101 Category of AE AE toxicity Degree AE Description of AE Duration INFECTION Febrile neutropenia 3 Febrile neutropenia. Temperature: Peak temperature 40.3°C, decline on day 6 6 days NEUROLOGY hypotension 3 Parents noted confusion. MRI is normal. 3 days METABOLIC/ LABORATORY elevated AST 4 Peak AST value: 1060 (degree 4) 1 day at grade 4 METABOLIC/ LABORATORY elevated ALT 4 Peak ALT: 748 (degree 4) 1 day at grade 4

The severity of adverse events was assigned in accordance with the General Terminology Criteria for Adverse Events 3.0.

At CHOP-100, glucocorticoids were administered on Day +5 with a short response on the temperature curve but no remission of hypotension. A single course of anti-cytokine therapy, consisting of etanercept and tocilizumab, was given on Day +8 and was followed by rapid clinical effects: within a few hours, the temperature decreased, the dose of vasoactive drugs and assisted breathing were gradually reduced, as clinical and radiological ARDS was eliminated. She had no laboratory confirmation of tumor lysis syndrome; however, biochemical confirmation of MAS was marked by an increase in ferritin to 45529 ng/dl on Day + 11, coagulopathy with elevated d-dimer and hypofibrinogenemia, hepatosplenomegaly, elevated transaminases, elevated LDH (Fig. 4C), and elevated triglycerides, and a cytokine profile consistent with M.A.S. Her ferritin dropped to 2368 on Day +26 and the clinical and laboratory MAS abnormalities ended.

CHOP-101, despite no direct confirmation of tumor lysis syndrome other than fever and LDH changes (Fig. 4C), also developed MAS features with ferritin elevations up to 33360 on Day+, peaking at 74899 on day 11, transaminases , which reached grade 4 within 1 day, and elevated serum d-dimer. These biochemical changes were reversible as transaminases improved to grade 1 and ferritin decreased to 3894 on Day +21. She was released from the hospital on Day +16.

Both individuals developed significant increases in serum cytokines and cytokine receptors (FIG. 1B). In both patients, increases in interferon-γ and IL-6 were the most significant. These observations were similar to those seen previously in CLL patients who also experienced leukemia remission after CTL019 infusion (Kalos et al., 2011, Science Translational Medicine 3:95ra73). Peak cytokine elevations correlated temporally with systemic inflammation as measured by changes in core body temperature (Fig. 4C).

In vivo propagation of CTL019 in patients with ALL

The circulating CTL019 T cell fraction increased progressively in vivo to 72% (CHOP100) and 34% (CHOP-101) T cells (FIG. 5A). Initial transduction efficiency was 11.6% and 14.4% for T cells infused into CHOP-100 and -101, respectively. Considering that total ALC increased significantly in both patients (Fig. 4C) and that CTL019 cell frequency progressively increased in vivo from baseline frequency (Fig. 8), there was robust and selective expansion of CTL019 cells in both patients. patients. The selective increase in CTL019-expressing T cells in both patients is consistent with an anti-leukemic mechanism involving CD19-driven proliferation and subsequent elimination of cells that express CD19 in both patients (FIGS. 6 and 9).

Molecular deep sequencing analysis of TCR in peripheral blood and bone marrow samples from CHOP-100 obtained on Day +23, when >65% of CD3+ cells in peripheral blood and bone marrow were shown by flow cytometry to be CTL019+, showed no T -cells of the dominant TCR clonotype in both locations, with 10% of the most abundant T cells present at frequencies between 0.18-0.7% in bone marrow and 0.19-0.8% in peripheral blood. Six out of 10 dominant clones were distributed between two locations. In addition, both CD4 and CD8 CAR-T cells are present. Thus, CART cells appear to proliferate after CD19-stimulated reproduction, and not under the action of TCR signals or lineage-specific events such as activation through lentivirus integration.

Targeted migration and morphology of CTL019 CAR-T cells in the bone marrow and CNS

CTL019 cells proliferated more than 1000-fold in peripheral blood and bone marrow (FIG. 5). CTL019 cell frequency increased by more than 10% of circulating T cells at Day + 20 in both individuals (Fig. 8), with an absolute magnitude of CTL019 proliferation similar to that observed in CLL patients (Kalos et al., 2011, Science Translational Medicine 3 :95ra73). Unexpectedly, the cells in the CSF

- 24 041322 also showed a high degree of CTL019 gene marking and also survived at a high rate over 6 months (Fig. 5B). Targeted migration of CTL019 cells into the CSF was unexpected, given that no patient had detectable CNS leukemia on cytospin smear at the time of infusion or assessment 1 month post-treatment. In addition, prior reports of CAR therapy for malignant B cells have not noted targeted migration of CAR-T cells into the CNS (Till et al., 2008, Blood 112:2261-71; Brentjens et al., 2011, Blood 118:4817 -28; Savoldo et al., 2011, J Clin Invest 121:1822-5; Jensen et al., 2010, Biol Blood Marrow Transplant 16:1245-56; Till et al., 2012, Blood 119:394050; Kochenderfer et al. al., 2012, Blood 119:2709-20). Morphology of lymphocytes in blood and CSF is shown for CHOP-100 and 101 in FIG. 5D. Because >70% of the lymphocytes in circulation at Day +10 were CTL019 cells (v 5A and 5B), most of the large granular lymphocytes shown in the left panel of FIG. 5D are likely CTL019 cells. Similarly, since many of the lymphocytes in the CSF obtained from CHOP-101 at Day +23 were CTL019 cells (FIGS. 5B and 5C), the cytospin smear of CSF lymphocytes in FIG. 5D most likely represents the in vivo morphology of CTL019 cells that have migrated directionally into the CNS.

Induction of B cell aplasia

Both individuals had elimination of CD19-positive cells in bone marrow and blood within 1 month of CTL019 infusion (FIG. 6 and FIG. 9). In CHOP-100, a high proportion of cells remaining in the bone marrow at Day+6 post-infusion were CD19+CD20+ leukemic blast cells. This cell population was undetectable at Day+23, with this effect being maintained for over 9 months in this patient (FIG. 9A). Given that CHOP-100 had not received chemotherapy in the 6 weeks prior to the CTL019 infusion, this indicates that there were sufficient CTL019 cells to eliminate normal and leukemic B cells in this case.

Emergence of CD19 extinction variant in CHOP-101

CHOP-101 experienced a clinical relapse, with a clear manifestation in the peripheral blood 2 months after CTL019 infusion, as evidenced by the reappearance of blast cells in the circulation. These cells were CD45 dym-positive, CD34-positive and did not express CD19 (FIG. 6). The absence of original dominant CD34dim+CD34+CD19dim+ cells is consistent with strong antileukemic selective pressure of CD19-targeting CTL019 CAR-T cells (FIG. 9B). Deep sequencing of IGH showed the presence of a malignant clone in peripheral blood and bone marrow as early as Day+23 (Table 1), despite the clinical assessment of MRD negativity by flow cytometry at this time point (Figure 7). In addition, deep sequencing of material from clinical relapse showed that CD45dimCD34+CD19 cells are clonally related to the original dominant CD45dim+CD34+CD19dim+ cells because they share the same IGH sequence.

ALL remission by T cells expressing chimeric antigen receptor

The results presented herein demonstrate the induction of remission of relapsed and refractory leukemia in the first two patients treated under this protocol. Remission was sustained in one individual and was followed by a relapse due to the appearance of CD19-negative blast cells in another individual. Genetically modified CTL019 cells targeted migrated into the CNS at high levels in both patients. Elevations of cytokines were observed that were targeted, reversible, and transiently accompanied by elimination of blast cells that expressed CD19 in both individuals. The induction of complete remission in refractory CD19-positive ALL after CAR-T cell infusion is encouraging, especially given the low remission rate after infusion of allogeneic donor lymphocytic infusions that do not express CAR (Kolb et al., 1995, Blood 86:2041- 50; Collins et al., 1997, J Clin Oncol 15:433-44; Collins et al., 2000, Bone Marrow Transplant 26(5):511-6). Deep sequencing technology showed that infusion of CTL019 with CAR was associated with a robust 5-log reduction in the incidence of malignant B cells in CHOP-100, further indicating strong antitumor effects in chemotherapy-refractory leukemia.

The unfortunate appearance of CD19-negative blast cells in one individual is consistent with earlier reports that document the existence of CD19-negative progenitor cells in some cases of ALL (Hotfilder et al., 2005, Cancer Research 65: 1442-9; le Viseur et al., 2008, Cancer Cell 14:47-58). Possibly co-infusion of CAR-T cells redirected to novel features in addition to CD19 may reduce the likelihood of this event. So far, relapse with CD19-negative escape cells has not been observed in adults with CLL after treatment with CTL019 cells (Kalos et al., 2011, Science Translational Medicine 3:95ra73), suggesting that this case may be specific to the acute leukemia subtype. CHOP-100 remission induction did not require concomitant chemotherapy, and is consistent with an earlier report showing that remissions in CLL may be delayed for several weeks after chemotherapy (Porter et al., 2011, N Engl J Med 365:725-33) . Thus, concomitant administration of cytotoxic chemotherapy may not be necessary for CAR-mediated antitumor effects.

- 25 041322

Both pediatric patients with ALL experienced significant post-infusion toxicity.

CTL019. An induction of B cell aplasia was observed, which indicates that CAR-T cells can function against the backdrop of relapsing acute leukemia. Both patients also developed clinical and demonstrable evidence of cytokine release syndrome and macrophage activation syndrome within a week of infusion.

The cytokine profile observed in these patients is similar to earlier reports of cytokine distribution patterns in children with hemophagocytosis and macrophage activation syndrome or hemophagocytic lymphohistiocytosis (Tang et al., 2008, Br J Haematol 143:84-91; Behrens et al., 2011, J Clin Invest 121(6):2264-77). Macrophage activation syndrome is characterized by hyperinflammation with prolonged fever, hepatosplenomegaly, and cytopenias. Laboratory findings characteristic of this syndrome are elevated ferritin, triglycerides, transaminases, bilirubin (mostly conjugated) and soluble interleukin-2 receptor achain, and reduced fibrinogen (Janka et al., 2012, Annu Rev Med 63:233- 46). Recent studies have shown that tocilizumab (anti-[L6) is promising for the treatment of glucocorticoid-resistant GVHD (Drobyski et al., 2011, Biol Blood Marrow Transplant 17(12): 18628; Le Huu et al., 2012, J Invest Dermatol 132(12):2752-61; Tawara et al., 2011, Clinical Cancer Research 17:7788), and the results presented here are consistent with this data.

Extensive CTL019 proliferation in vivo, persistent B-cell aplasia, and significant anti-leukemic activity suggest substantial and persistent effector functions of CTL019 cells in pediatric patients with progressive ALL. The high efficiency of targeted migration of CAR-T cells into the CNS is encouraging as a surveillance mechanism to prevent recurrence at a sanctuary site such as the CNS (Pullen et al., 1993, J Clin Oncol 11 (5): 839-49), and supports the need to test targeted therapies with CAR-T cells for CNS lymphomas and primary CNS malignancies. With the exception of B cell aplasia, the duration of which is not currently defined, it is believed that the use of immunotherapies such as CTL019 may have a favorable long-term adverse event profile compared to the high doses of chemotherapy and radiation that are used as the current standard of clinical practice for most cases of childhood leukemia (Garcia-Manero and Thomas, 2001, Hematol Oncol Clin North Am 15(1):163-205).

Induction of complete remissions of ALL by T cells expressing a chimeric antigen receptor

Tocilizumab (anti-IL6) has promise for the treatment of glucocorticoid-resistant GVHD, and the results presented here are consistent with these data. Additionally, it is interesting to note that in CHOP 100, CRS manifestations of high fever, hypotension and multi-organ failure were resistant to high doses of glucocorticoids administered during the previous 2 days prior to cytokine-targeted therapy. Finally, in CHOP-100, the biphasic changes in IL-1e, IL-IRA, and IL-2 shown in FIG. 4B may be relevant to cytokine-targeted therapy using etanercept and tocilizumab.

The induction of remission in a patient refractory to blinatumomab therapy further highlights the activity of CTL019 cells. The high efficiency of targeted migration of CAR-T cells into the CNS is encouraging as a mechanism for surveillance to prevent recurrence at a sanctuary site such as the CNS and confirms the need for testing targeted therapies with CAR-T cells for the treatment of CNS lymphomas and primary malignancies. in the CNS. None of the patients experienced cognitive effects that could be attributed to directed T cell migration into the CNS.

Example 4. Summary information

Various markers were measured in patients receiving CAR-T cells. As a non-limiting example, ferritin, myoglobin, and plasminogen activator inhibitor-1 (PAI-1) were measured; see fig. 10, 11 and 12 respectively. Elevated levels of these markers correlated with outcome. Patients designated as -01, -03, -09, -100 and -101 were classified as patients with complete clinical response. Patients designated as -02, -05, -10 (second infusion and response around Day 70) and -12 were classified as patients with a partial clinical response. Patients designated as -06, -07 and -14 were classified as non-responders.

The disclosures of each and every patent, patent application and publication cited in the present specification are incorporated herein by reference in their entirety. While the present invention has been disclosed with reference to specific embodiments, it is obvious that other embodiments and variations of the present invention may be developed by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be considered as including such embodiments and equivalent variations.

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Claims (28)

CLAIM 1. A method of treating a patient suffering from a cancer associated with increased expression of a tumor antigen, wherein the method comprises administering first-line therapy and second-line therapy to the patient, wherein the first-line therapy comprises administering to the patient an effective amount of T cells genetically modified to express a chimeric antigen. receptor (CAR T-cells), where the chimeric antigen receptor (CAR) contains an antigen-binding domain, a transmembrane domain and a signaling domain, where the cytokine level increases after first-line therapy compared to the level before first-line therapy, where the cytokine is selected from the group, consisting of IL-1 and IL-6, and any combination thereof, where the second line of therapy includes the administration of a cytokine inhibitor selected from the group consisting of anakinra, tocilizumab, and any combination thereof. 2. A method of treating a patient suffering from a cancer associated with increased expression of a tumor antigen, wherein the method comprises administering to the patient an effective amount of T cells genetically modified to express a chimeric antigen receptor (CAR T cell), wherein the chimeric antigen receptor (CAR) contains an antigen-binding domain, a transmembrane domain and a signaling domain, wherein the method further comprises administering a cytokine inhibitor to control toxicity caused by the administration of said T cells, wherein the cytokine inhibitor inhibits a cytokine selected from the group consisting of IL-1, IL-6, and any combination thereof . 3. A method of treating a patient suffering from a cancer associated with increased expression of a tumor antigen, wherein the method comprises administering to the patient an effective amount of T cells genetically modified to express a chimeric antigen receptor (CAR T cell), wherein the chimeric antigen receptor (CAR) contains an antigen-binding domain, a transmembrane domain, and a signaling domain, wherein administration of said CAR-modified T cells results in cytokine release syndrome (CRS), and further comprises the use of a cytokine inhibitor to treat said CRS, wherein the cytokine inhibitor inhibits a cytokine selected from the group consisting of IL-1, IL-6 and any combination thereof. 4. A method of treating cytokine release syndrome (CRS) resulting from the administration of T cells transduced to express a chimeric antigen receptor (CAR T cell) to a patient, comprising administering a cytokine inhibitor to the patient, wherein the chimeric antigen receptor (CAR) contains an antigen-binding domain transmembrane a domain and an intercellular signaling domain, where CAR T cells are used to treat a malignant neoplasm in a patient, where the malignant neoplasm is associated with increased expression of a tumor antigen, further, where said cytokine inhibitor is an IL-6 inhibitor. 5. A method of treating cytokine release syndrome (CRS) resulting from the administration of T cells transduced to express a chimeric antigen receptor (CAR T cells) to a patient, comprising administering a cytokine inhibitor to the patient, where the chimeric antigen receptor (CAR) contains an antigen-binding domain transmembrane a domain and an intercellular signaling domain, where CAR T cells are used to treat a malignant neoplasm in a patient, where the malignant neoplasm is associated with increased expression of a tumor antigen, further, where said cytokine inhibitor is an IL-1 inhibitor. 6. The method according to any one of claims 1 to 3, wherein after administration of CAR T cells, cytokine levels in patients are monitored to determine the appropriate type of cytokine inhibitor. 7. The method of claim 6 wherein the level of the cytokine is monitored by detecting the level of the cytokine protein in the biological sample of the patient. 8. The method of claim 6 wherein the level of the cytokine is monitored by detecting the level of a nucleic acid encoding the cytokine in a biological sample of the patient. 9. The method according to any one of claims 1-4 and 6-8, wherein the cytokine is IL-6 and the cytokine inhibitor is tocilizumab. 10. The method according to any one of claims 1-3 and 5-8, wherein the cytokine is IL-1, IL-1a or ΓΕ-1β and the cytokine inhibitor is anakinra. - 27 041322 11. The method according to any one of claims 1-3 and 5-8, wherein the cytokine is IL-1a or IL-Ιβ and the cytokine inhibitor is anakinra. 12. The method according to any one of claims 1-3 and 5-8, wherein the cytokine is IL-1e and the cytokine inhibitor is anakinra. 13. The method of any one of claims 1-12, wherein the antigen-binding domain is targeted to a tumor antigen. 14. The method of any one of claims 1-13, wherein the antigen binding fragment is fused to one or more intracellular domains selected from the group consisting of the CD137 signal domain (41BB), the CD28 signal domain, the CD3 zeta signal domain, and any combination thereof. 15. The method according to any one of claims 1-14, wherein the tumor antigen is selected from the group consisting of one or more of CD19, CD20, CD22, EGFRvIII, and IL3Ra. 16. The method according to any one of claims 1 to 15, wherein the tumor antigen is CD19. 17. The method of any one of claims 1-16, wherein the CAR T cell induces elevated levels of IFN-γ or IL2 in the patient. 18. The method according to any one of claims 3, 4 and 5, wherein the CRS results in hemophagocytic lymphohistiocytosis and/or macrophage activation syndrome. 19. The method according to any one of claims 1 to 18, wherein the malignancy is a hematological malignancy selected from the group consisting of acute leukemia, acute myelocytic leukemia, acute myelogenous leukemia, myeloid leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia , chronic myelocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia, pre-B ALL (in pediatrics) , ALL in adults, mantle cell lymphoma, and diffuse large B-cell lymphoma. 20. The method of claim 19, wherein the cancer is selected from the group consisting of pre-B ALL (in pediatrics), adult ALL, mantle cell lymphoma, or diffuse large B-cell lymphoma. 21. The method of any one of claims 1-20, wherein the cancer is refractory or resistant to conventional chemotherapy. 22. The method of any one of claims 1 to 21, wherein the CAR T cells are human T cells transduced in vitro with a vector encoding a CAR and the CAR modified T cells are autologous to the recipient. 23. The method according to any one of claims 1 to 22, wherein the CAR-modified T cells are administered as a pharmaceutical composition in combination with a combination of diluents and/or other excipients. 24. The method of claim 9 wherein tocilizumab is administered at a dose of 8 mg/kg. 25. The method of any one of claims 1-24, further comprising administering a corticosteroid to the patient. 26. The method of claim 25 wherein the corticosteroid is methylprednisolone. 27. A method of treating cytokine release syndrome (CRS) resulting from the administration of T cells transduced to express a chimeric antigen receptor (CAR T cell) to a patient, comprising administering tocilizumab to the patient, where the chimeric antigen receptor (CAR) contains an antigen-binding domain, a transmembrane domain and an intercellular signaling domain where CAR T cells are used to treat cancer in a patient. 28. A method of treating cytokine release syndrome (CRS) resulting from the administration of T cells transduced to express a chimeric antigen receptor (CAR T cell) to a patient, comprising administering anakinra to the patient, where the chimeric antigen receptor (CAR) contains an antigen-binding domain, a transmembrane domain and an intercellular signaling domain where CAR T cells are used to treat cancer in a patient. -