Method for enhancing cellular immunotherapy

Abstract

To provide a method for enhancing cellular immunotherapy.SOLUTION: Provided are methods and compositions directed at the treatment of an individual having cancer by (i) administering to the individual an adoptive cellular immunotherapy composition comprising CAR T cells and (ii) administering to the individual an interleukin-15 receptor agonist, such as a long-acting interleukin-15 receptor agonist. The present application relates to (among other things) the field of immunotherapy and involves the treatment of an individual having a condition such as cancer, by administering, to the individual, chimeric antigen receptor (CAR) modified T cells and an interleukin-15 receptor agonist.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 830,212, filed on April 5, 2019; U.S. Provisional Patent Application No. 62 / 861,858, filed on June 14, 2019; U.S. Provisional Patent Application No. 62 / 898,473, filed on September 10, 2019; and U.S. Provisional Patent Application No. 62 / 944,955, filed on December 6, 2019, the contents of these provisional patent applications are hereby incorporated by reference into this specification, respectively.

[0002] This application relates to (in particular) the field of immunotherapy and includes the treatment of an individual having a condition such as cancer by administering to the individual chimeric antigen receptor (CAR) - modified T cells and an interleukin - 15 receptor agonist.

Background Art

[0003] New therapies are continuously being developed for the improvement of cancer treatment. One of the most promising areas of cancer treatment is cancer immunology (i.e., cancer immunotherapy). Cancer immunotherapy refers to a diverse set of treatment strategies designed to modulate the immune response in order to induce the patient's own immune system to fight cancer. Among current immunotherapeutic approaches, adoptive cell transfer therapy (also called ACT) has been shown to be promising in treating certain types of cancer patients. Adoptive cell therapy involves the isolation and ex vivo expansion of tumor-specific lymphocytes to obtain a greater number of tumor-reactive effector T cells than can be achieved by simple vaccination. Tumor-specific T cells are infused into cancer patients to initiate the killing of tumor cells by the patient's immune system. Adoptive cell transfer has shown effective clinical outcomes, particularly in metastatic melanoma (Dudley, M.E., J.R. Wunderlich et al., J Clin Oncol 23(10):2346-2357(2005); Dudley, M.E., J.C. Yang et al., J Clin Oncol 26(32):5233-5239(2008)). Adoptive cell transfer may be autologous, as is common in adoptive T cell therapy, or allogeneic.

[0004] One form of adoptive T cell therapy is CAR T cell therapy (chimeric antigen receptor-modified T cell therapy). A CAR is a type of synthetic receptor that can reprogram the specificity and function of lymphocytes (Sadelain, M., et al., Nature, 545, 25 May 2017, p. 423 - 431). CAR T cell therapy uses ex vivo genetically engineered T cells that are transduced to express an artificial receptor that redirects the specificity of the T cells against target tumor - associated antigens (TAAs) expressed on the surface of tumor cells (June, C. H., et al., Sci Transl Med 2015; 7(280):280ps7). Antigen recognition by CAR T cells is MHC - independent, differing from naturally occurring T cells and T - cell receptor - engineered T cells that recognize cognate antigens from the perspective of a specific major histocompatibility complex (MHC), thereby expanding the applicability of this cell - based mode of immunotherapy. First - generation CAR T cells contained the single - chain variable region (scFv) of a monoclonal antibody, the transmembrane domain of the T - cell receptor, and the intracellular signaling domain of the CD3 zeta (CD3ζ) chain. Subsequent iterations have also utilized one or more co - stimulatory domains and / or a controllable on - off switch. CAR T cell therapy has evolved over time to provide genetically engineered T cells with improved specificity and safety profiles.

[0005] Therapies based on CAR T cells are approved in the United States for the treatment of diffuse large B - cell lymphoma, but not all patients respond to CAR T cells, and the durability of the response remains limited. A further challenge with CAR T cell therapy is the inadequate survival of the transplanted cells. In relapsed or refractory diffuse large B - cell lymphoma, approximately 60% of patients relapse or fail to progress, and the prognosis after failure is poor (Nair, J., et al., Best Practice & Research Clinical Haematology 31(2018)293 - 298). Successful CAR The outcome of T cell therapy, i.e., a favorable and durable response with a complete remission rate at 6 months, depends at least to some extent on the long-term persistence of CAR T cells. Clinical outcomes for phase II trials of CAR T cell therapy in treating patients with lymphoma have reported significant efficacy for many patients who initially responded to the treatment, but the durability of the response has remained limited (Shah, N., et al., Frontiers in Oncology, 9 (March 2019) Art. 146). Furthermore, the acute toxicities reported after CAR T cell therapy include cytokine release syndrome (CRS) and neurotoxicity (termed CAR-related encephalopathy syndrome) (Neelapu, S.S., et al., Nat Rev Clin Oncol 2018;15(10:47-62). Other less frequently observed but reported adverse side effects include hemophagocytic lymphohistiocytosis (HLH) / macrophage activation syndrome (MAS), anaphylaxis, and tumor lysis syndrome. Considerable efforts have been made to date to develop effective CAR T cell-based therapies, but there remains a need to provide new and more effective immunotherapy CAR T cell strategies and related treatment regimens that address one or more of the drawbacks of current therapies.

Prior Art Documents

Non-Patent Documents

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Summary of the Invention

Means for Solving the Problems

[0007] Accordingly, the present disclosure has other advantages, but in particular, a novel and efficient CAR T cell-based immunotherapy having better persistence and improved efficacy (to be described in more detail below), for example, by providing an immunotherapy that induces a tumor-killing trait in a CAR T cell population, endeavors to address these drawbacks and other needs.

[0008] In a first aspect, provided herein is a method comprising adoptive cell transfer to a subject having cancer, in combination with administration of a long-acting IL-15 receptor agonist, which will be described in more detail herein. The present disclosure recognizes that a combination treatment regimen comprising one or more cycles of adoptive cell therapy (e.g., by infusion of CAR T cells), administered sequentially in any order or substantially simultaneously, and administration of a long-acting IL-15 receptor agonist as described herein, is particularly effective in treating cancer in certain subjects to a degree that is enhanced, preferably significantly enhanced, compared to any single immunotherapy approach alone, and / or increases, enhances, or extends the activity and / or number of the transplanted cells, or can result in a measurable beneficial response (e.g., stabilization, regression, shrinkage, necrosis, etc., if applicable) against cancer cells.

[0009] In a second aspect, provided herein is a combination immunotherapy for the treatment of a subject having cancer, comprising administering to the subject an adoptive cell therapy composition comprising T cells modified to express a chimeric antigen receptor (CAR T cells); and administering to the subject a long-acting IL-15 receptor agonist.

[0010] In a third aspect, provided is a method of improving the therapeutic efficacy of adoptive cell therapy, such as CAR T cell therapy, for treating a subject having cancer, the method comprising providing to the subject having cancer an adoptive cell therapy composition comprising T cells modified to express a chimeric antigen receptor; and administering to the subject a long-acting IL-15 receptor agonist (wherein administration of the long-acting IL-15 receptor agonist is effective to improve the subject's response to the adoptive cell therapy).

[0011] The following embodiments are intended to apply equally to each of the aspects described above, and, where applicable, both alone and in combination should be considered unless otherwise indicated.

[0012] In some embodiments, adoptive cell transfer comprises administering an adoptive cell therapy composition comprising T cells modified to express a CD19-directed chimeric antigen receptor.

[0013] In one or more further embodiments, the long-acting IL-15 receptor agonist is effective to preferentially stimulate and proliferate natural killer (NK) cells. In one or more additional further embodiments, the long-acting IL-15 receptor agonist aids CD8+ T cell survival and memory formation, for example, without substantially inducing inhibitory regulatory T cells (Tregs). In some further embodiments, the long-acting IL-15 receptor agonist has IL-15 receptor alpha specificity. In some further embodiments, the long-acting IL-15 receptor agonist has one or more of the foregoing characteristics, namely, (i) being effective to preferentially stimulate and proliferate NK cells, (ii) aiding CD8+ T cell survival and memory formation, for example, without substantially inducing inhibitory regulatory T cells (Tregs), and (iii) having IL-15 receptor alpha specificity.

[0014] In some further embodiments, the long-acting IL-15 receptor agonist has the structure: [Chemical formula] (wherein IL-15 is an interleukin-15 moiety, (n) is an integer from about 150 to about 3,000, and ~NH~ represents the amino group of the IL-15 moiety) and has.

[0015] In one or more embodiments related to the long-acting IL-15 receptor agonist of formula (I), (n) ranges from about 795 to about 1068. In some additional embodiments, (n) ranges from about 840 to about 1023. In one or more specific embodiments, (n) has an average value of about 907 or about 909.

[0016] For clarity purposes, with respect to the order of administration (wherein the term "administering" is used when referring to the delivery of an adoptive cell immunotherapy composition or a long-acting IL-15 receptor agonist), the adoptive cells and the long-acting IL-15 receptor agonist can be administered simultaneously or sequentially, and in any order. Further, the treatment of any component of the combination can include a single cycle of treatment or multiple cycles. That is, for example, after the first cycle of treatment comprising administration of an adoptive cell immunotherapy composition comprising T cells modified to express a chimeric antigen receptor such as a CD19-directed CAR T cell and administration of a long-acting IL-15 agonist, additional rounds of treatment can include adoptive cell transplantation, e.g., administration of CAR T cells in combination with administration of a long-acting IL-15 receptor agonist, or adoptive cell therapy, e.g., CAR T cell therapy without administration of a long-acting IL-15 receptor agonist, or administration of a long-acting IL-15 receptor agonist without adoptive cell transplantation (e.g., administration of CAR T cells such as CD19 CAR T cells).

[0017] In one or more embodiments, the subject is a human subject.

[0018] In one or more non-limiting embodiments, the cancer is a liquid cancer such as a blood cancer, e.g., a recurrent or refractory malignancy.

[0019] In one or more related non-limiting embodiments, the cancer is a lymphoma or leukemia. In one or more related embodiments, the cancer is selected from Hodgkin and non-Hodgkin lymphoma.

[0020] In some additional non-limiting embodiments, the cancer is a B cell malignancy. In some further embodiments, the cancer is a B cell lymphoma.

[0021] In some further additional embodiments, the cancer is multiple myeloma.

[0022] In one or more alternative embodiments, the cancer is a solid cancer.

[0023] In some further embodiments of the methods provided herein, the method provides a beneficial response to treatment that is enhanced over the response to treatment observed when administration is carried out according to administration of an adoptive cell immunotherapy composition or administration of a long-acting IL-15 receptor agonist alone.

[0024] In some embodiments related to the foregoing, the beneficial response to treatment is based on a suitable animal model, such as an in vivo xenogenetic B cell lymphoma model and the like.

[0025] Additional aspects and embodiments are described in the following description and claims.

Brief Description of the Drawings

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Best Mode for Carrying Out the Invention

[0027] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0028] In describing and claiming certain features of the present disclosure, the following terms are used in accordance with the definitions set forth below unless otherwise indicated.

[0029] "PEG" or "polyethylene glycol" as used herein is intended to encompass any water-soluble poly(ethylene oxide). Unless otherwise indicated, "PEG polymer" or polyethylene glycol is one in which substantially all (preferably all) of the monomer subunits are ethylene oxide subunits, however the polymer may contain individual end-capping moieties or functional groups, for example for conjugation. The PEG polymers used in the present disclosure can include one of the following two structures depending on whether one or more terminal oxygens have been substituted, for example during synthetic transformation: "-(CH 2 CH 2 O) n -" or "-(CH 2 CH 2 O) n-1 CH 2 CH 2 -". For PEG polymers, the variable (n) typically ranges from about 3 to 4000, and the end groups and the overall structure of the PEG can vary. Exemplary or preferred molecules containing PEG can include one or more specific PEG structures and / or linkers, and / or molecular weight ranges.

[0030] The molecular weight in the context of water-soluble polymers such as PEG can be expressed as the number average molecular weight or the weight average molecular weight. Unless otherwise indicated, all references to molecular weight in this specification refer to the weight average molecular weight. Both number average and weight average molecular weight determinations can be measured using gel permeation chromatography or other liquid chromatography techniques (e.g., gel filtration chromatography). The most commonly used ones are gel permeation chromatography and gel filtration chromatography. Other methods for determining molecular weight include end group analysis or measurement of colligative properties (e.g., freezing point depression, boiling point elevation, or osmotic pressure) for determining the number average molecular weight, or light scattering techniques, ultracentrifugation, MALDI TOF, or the use of viscometers for determining the weight average molecular weight. PEG polymers are typically polydisperse (i.e., the number average molecular weight and the weight average molecular weight of the polymer are not equal). PEG polymers used for covalent attachment to target molecules such as IL-15 and as described herein generally have a low polydispersity value, preferably less than about 1.2, more preferably less than about 1.15, even more preferably less than about 1.10, e.g., less than about 1.05, or less than about 1.03.

[0031] A "physiologically cleavable" or "hydrolyzable" or "degradable" bond is a relatively labile bond that generally reacts with water (i.e., is hydrolyzed) under physiological conditions and under any suitable method of hydrolysis. The tendency of a bond to hydrolyze in water can depend not only on the general type of linkage connecting two atoms within a given molecule, but also on the substituents attached to these atoms and the overall molecular structure. Bonds that are labile or weak to hydrolysis typically include, but are not limited to, carboxylic acid esters, phosphate esters, anhydrides, acetals, ketals, acyloxyalkyl ethers, imines, orthoesters, peptides, oligonucleotides, thioesters, and carbonates.

[0032] An "enzymatically degradable bond" means a bond that is degraded by one or more enzymes.

[0033] A "stable" linkage or bond refers to a chemical bond that is substantially stable in water, i.e., a bond that undergoes no appreciable hydrolysis under physiological conditions over an extended period of time. Examples of bonds that are stable to hydrolysis include, but are not limited to, generally the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, amines, etc. Generally, a stable bond exhibits a hydrolysis rate of less than about 1-2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found by referring to many standard chemical textbooks.

[0034] As used herein, the term "IL-15 moiety" refers to a peptide or protein moiety having human IL-15 activity. Further, the term "IL-15 moiety" encompasses both the IL-15 moiety prior to conjugate formation with a PEG moiety and the IL-15 moiety after conjugate formation (i.e., covalent bonding) (e.g., reaction) with a reactive PEG moiety such as mPEG-succinimidyl butanoate. As will be explained in more detail below, one of ordinary skill in the art can determine whether a given moiety has IL-15 activity. Proteins containing an amino acid sequence corresponding to any one of SEQ ID NOs: 1 to 3, and likewise any protein or polypeptide that is substantially homologous thereto, are exemplary IL-15 moieties. As used herein, the term "IL-15 moiety" includes, for example, peptides and proteins that have been deliberately or incidentally modified via site-directed mutagenesis or via mutations. Included with these are IL-15 sequences having from 1 to 6 additional glycosylation sites, sequences having at least one additional amino acid at the carboxy terminus of a peptide or protein (wherein the additional amino acid includes at least one glycosylation site), and sequences having an amino acid sequence that includes at least one glycosylation site. This term is intended to include IL-15 moieties produced naturally, recombinantly, and synthetically. References to long-acting IL-15 receptor agonists are intended to encompass their pharmaceutically acceptable salt forms.

[0035] The terms "substantially identical" or "substantially the same" mean that a particular target sequence, such as a mutant sequence, differs from a reference sequence by one or more substitutions, deletions, or additions (the net effect of which does not result in an adverse functional difference between the reference sequence and the target sequence). For the purposes of the present invention, sequences having greater than 95 percent homology (identity) under stringent conditions, equivalent biological activity (not necessarily equivalent in strength of biological activity), and equivalent expression characteristics with respect to a given sequence are considered to be substantially identical. For the purpose of determining homology, truncations of the mature sequence should be ignored. Exemplary IL-15 polypeptides for use herein include sequences that are substantially identical to SEQ ID NO: 1. SEQ ID NO: 2 is almost identical to SEQ ID NO: 1 except that SEQ ID NO: 2 has a methionine at the beginning of the sequence that is required for translation in E. coli.

[0036] The term "fragment" means any protein or polypeptide having a portion or fragment of the amino acid sequence of a protein or polypeptide, such as an IL-15 portion, and having the biological activity, or substantially the biological activity, of the protein or polypeptide, such as IL-15. Fragments include proteins or polypeptides produced by proteolysis, as well as proteins or polypeptides produced by chemical synthesis by methods conventional in the art.

[0037] As used herein, the term "treating cancer" is not intended to be an absolute term and may include, for example, reducing tumor size or the number of cancer cells, putting cancer into remission, or preventing the growth of the size or number of cancer cells. In some situations, treatment according to the present disclosure leads to an improvement in prognosis.

[0038] In a method for treating a subject having cancer, as used herein, the expression "subject in need of treatment" refers to an individual or subject diagnosed with cancer.

[0039] As used herein, the term "facilitate" refers to an improvement in the ability of a subject, or of tumor cells, to respond to treatment, such as that disclosed herein, when compared to a certain baseline or reference therapy, for example, in the context of response facilitation. For example, response facilitation can include an improvement in responsiveness of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more, based on one or more indicators of responsiveness to treatment. As used herein, "facilitate" can also refer to facilitating the number of subjects who respond favorably to treatment, for example, when compared to a certain basis for such comparison.

[0040] As used herein, "refractory" refers to a disease that does not respond to treatment, such as cancer. Refractory cancer can be resistant to treatment before or at the start of treatment, or refractory cancer can become resistant during treatment. Refractory cancer is also referred to as resistant cancer.

[0041] As used herein, "relapsed" or "relapses" refers to the reappearance of a disease (such as cancer), or the signs and symptoms of a disease such as cancer, after a period of improvement or responsiveness, for example, before and after a prior treatment with a therapy (such as cancer treatment).

[0042] As used herein, the term "CD19" refers to surface antigen classification 19 protein, an antigenic determinant detectable on preleukemic cells. Human and mouse amino acid and nucleic acid sequences can be referenced in public databases such as GenBank, UniProt, and Swiss-Prot, for example. As used herein, "CD19" includes proteins that contain mutations, such as point mutations, fragmentation, insertions, deletions, and splice variants of full-length wild-type CD19. CD19 is expressed on most B-lineage cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma.

[0043] The expressions "therapeutically effective", "therapeutically effective amount", "effective amount", or "effective amount of" refer to an amount or dosage sufficient to promote a desired physiological response, such as in the case of administration of a long-acting IL-15 receptor agonist, i.e., an amount sufficient to promote an enhanced response to administration of an adoptive cell immunotherapy composition, such as a composition containing CAR T cells. The specific amount will depend on numerous factors such as the particular condition being treated, the patient population, the considerations of each patient, the therapeutic composition to be administered and the components and physical properties of the particular combination, the particular adoptive cell transplantation therapy being performed (e.g., the specific composition of the cells contained in the CAR T cell composition and / or the chimeric antigen receptor expressed by the CAR T cells), and can be determined by those skilled in the art.

[0044] "Substantially" or "essentially" means almost completely or completely, e.g., more than 95% of a given amount.

[0045] Similarly, "about" or "approximately" as used herein means within plus or minus 5% of a given amount.

[0046] "Optional" or "optionally" means that the circumstances described thereafter need not necessarily occur, and as a result, the description includes both the case where the circumstances occur and the case where they do not occur.

[0047] "Pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a component that can be included in the compositions described herein and that does not cause a significant adverse toxic effect on the subject.

[0048] As used herein, the terms "patient" or "subject" refer to an organism suffering from or susceptible to a condition that can be prevented or treated by administration of a compound, composition, or combination as provided herein, such as cancer, and include both humans and animals. Subjects include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), preferably humans (including pediatric and adult subjects).

[0049] Summary Although adoptive cell therapy using CAR T cells is a promising treatment for inducing an effective antitumor response, for example, in hematological malignancies, patients often relapse after an initial favorable response to the treatment. Thus, in an attempt to address at least some of the drawbacks associated with current CAR T cell strategies by, for example, improving persistence, improving response durability, overcoming or addressing resistance, improving safety, and / or improving patient outcome (efficacy), provided herein is a method comprising administering to a subject having cancer an adoptive cell immunotherapy composition comprising T cells modified to express a chimeric antigen receptor such as a CD19-directed CAR T cell, and a long-acting IL-15 receptor agonist having the characteristics as described herein. Given the drawbacks associated with current CAR T cell immunotherapy, further enhancements are needed to provide a durable and effective response to treatment. Accordingly, the present disclosure is at least in part based on the discovery of a particularly beneficial therapeutic cancer immunocomination comprising CAR T cell therapy and the administration of a long-acting IL-15 agonist, more specifically one that preferably retains receptor binding to IL-15 receptor α, as will be apparent from the present disclosure and as shown in exemplary in vivo models that support the examples.

[0050] Adoptive Chimeric Antigen Receptor Cell Transplantation Therapy and Compositions The treatment methods provided herein include administering ex vivo expanded, genetically engineered T cells that have been transduced to express an artificial target tumor-associated antigen (TAA) binding domain, i.e., to stimulate a cancer-specific immune response. The compositions and methods provided herein are utilized in both clinical and research applications, in particular. Without being bound by theory, due to the complementary mechanisms of adoptive cell transfer, e.g., CAR T cell transfer and immune activation of long-acting IL-15 receptor agonists as provided herein, it is contemplated that improvement in the outcome of anti-tumor agents can be achieved via the IL-15 pathway (i.e., by administering a long-acting IL-15 receptor agonist in conjunction with adoptive cell transfer) to stimulate the desired T cell response.

[0051] Any suitable chimeric antigen receptor T cell (CAR T cell) therapy can be used in the methods provided herein, and the disclosure is not limited in this regard. See, for example, Rosenberg, S., et al., Adoptive Cell Transfer: A clinical path to effective cancer immunotherapy. Nat Rev Cancer. 2008 Apr;8(4):299-308 and Sadelain, M., et al, Current Opinion in Immunology, Vol 21(2), 215-223 (2009); also see Kalos, M., et al., Sci Transl Med 2011;3:95ra73; and Grupp SA, et al, N Engl J Med 2013;368:1509-1518. It should be understood that any suitable CAR T cells known in the art can be used in the methods and therapies described herein. Non-limiting examples of suitable CAR T cells and therapies for use herein are described, for example, in U.S. Patent Application Publication No. 2017 / 0209492, and No. 2019 / 091308, and also in U.S. Patent Nos. 8,911,993; 8,975,071; 9,328,156; 9,987,308, and 10,253,086.

[0052] In one or more embodiments, the CAR T cells comprise an antigen-binding domain that binds to a tumor antigen. In some further embodiments, the tumor antigen is selected from the group consisting of CD19, CD20, CD22, and ROR1, and combinations thereof. In some further embodiments, the CAR T cells are CD19-targeted T cells that comprise an antigen-binding domain that binds to CD19. See, e.g., Turtle, C.J., et al., Clinical Pharmacology & Therapeutics, 12 May 2016 (online). In addition to the exemplary publications provided in the preceding paragraph, additional examples of CD19 CAR T cells, such as CD19 CAR T cells of defined CD4+:CD8+ compositions, are described, e.g., in Turtle, C.J., J. Clin Invest. 2016;126(6):2133-2138. Further CAR T cells suitable for use in the methods and therapies described herein include, for example, CD19-directed tisagenlecleucel (KYMRIAH®) and CD19-directed axicabtagene ciloleucel (YESCARTA®), both of which are approved by the U.S. FDA, for use in treating B cell malignancies. In some other further embodiments, the CAR T cells express receptor tyrosine kinase-like orphan receptor 1 (ROR1), a tumor-associated molecule that is expressed, for example, in aggressive B lymphocytes and epithelial cancers, and in subsets of non-small cell lung cancer and triple-negative breast cancer, but not in normal B cells. ROR1-specific CAR T cells useful in the methods and therapies described herein are described, for example, in Hudecek, M., Clinical Cancer Research, June 2013, 19(12), 3153-3164; Hudecek, M., et al., Blood 2010, 116:4532-4541; and Sprecht, J.M., et al., Cancer Research, 78(13 Supplement):CT131, July 2018.In some embodiments, the cell construct targets the Ig / Fz portion of the extracellular domain of ROR1 and contains the 4-1BB / CD3ζ intracellular signaling domain. In some embodiments, the ROR1 CAR T production process utilizes autologous peripheral blood lymphocytes, separates them into CD4 and CD8 subsets, independently cultures them with anti-CD3 / anti-CD28 beads and IL-2, and then transduces them using a lentiviral vector encoding the ROR1 CAR. In some further embodiments, the CAR T cell product is CD4+ and CD8+ CAR. They are formulated at a 1:1 ratio of T cells.

[0053] CAR T cell therapy generally involves the administration of CAR T cells to treat patients suffering from cancer, particularly cancer whose tumor cells express the tumor antigen of interest. In some embodiments, the CAR T cells are prepared by methods as described herein or known in the art. For example, after isolation, host T cells are transduced to express a target tumor-associated antigen recognition domain, expanded, and reinfused into the subject. Prior to infusion, the patient may also be preconditioned using lymphodepleting non-myeloablative chemotherapy (NMC) to suppress endogenous regulatory T cells, for example, and to provide an environment optimized for the infused CART cells; or cyclophosphamide or some other appropriate conditioning agent may be used. Such preconditioning is useful for eliminating or substantially reducing the number of Tregs (regulatory T cells) and lymphocytes that compete with the transplanted cells for homeostatic cytokines. The host cells are lymph nodes, such as inguinal, mesenteric, superficial distal axillary etc., such as bone marrow, spleen, or peripheral blood, and can be isolated from various sources, as well as from tumors, such as tumor-infiltrating lymphocytes. The cells may be allogeneic or, preferably, autologous. For ex vivo stimulation, host cells are aseptically removed and suspended in some suitable medium, as is known in the art. After transduction, the cells are stimulated and grown using various protocols, in particular using any of the combinations such as anti-CD3, B7, anti-CD28, etc. Suitable protocols for ex vivo expansion of host T cells are described in “Focus on Adoptive T Cell Transfer Trials in Melanoma”, Clinical and Developmental Immunology, Vol 2010, Art.ID 260267.

[0054] For example, adoptive cell transfer of CAR T cells can be performed by (i) obtaining autologous lymphocytes from a mammalian subject such as a human, (ii) genetically engineering the autologous lymphocytes to express a target tumor-associated antigen (TAA) recognition or binding domain, (iii) culturing the genetically engineered lymphocytes to produce expanded CAR T cells, and (iv) administering the expanded CAR T cells to the subject (i.e., the patient). Autologous adoptive cell therapy can also be performed by (i) genetically engineering autologous lymphocytes to express a TAA binding domain, (ii) culturing the genetically engineered lymphocytes to produce expanded CAR T cells; (iii) subjecting the subject to non-myeloablative lymphocyte depletion chemotherapy (NMC); and (iv) administering the expanded CAR T cells after NMC. Autologous cells can be obtained from blood or can also be cloned using autologous antigen-presenting cells and tumor-derived peptides.

[0055] CAR T cells can be prepared by any means as described herein or known in the art. Methods for generating CARs and / or CAR T cells are described herein and are incorporated herein by reference in U.S. Patent Nos. 6,319,494; 6,410,319; 7,446,179; 7,446,191; 7,514,537; 7,741,465; and 9,987,308; U.S. Patent Application Publication Nos. 2016 / 0185861, 2017 / 0137783, and 2019 / 0091308; and PCT Application / International Publication Nos. 2010 / 065818, 2010 / 025177, and 2007 / 059298. Additional methods for generating CAR T cells are described by Berger C. et al., J. Clinical Investigation, 118:1 294-308 (2008) and Wang et al. (Molecular Therapy-Oncolytics (2016) 3, 16015), which are incorporated herein by reference.

[0056] CAR T cells can redirect antigen recognition based on the binding specificity of the CAR. The CAR can target any TAA such that when the CAR T cell binds to its cognate antigen on the tumor cell surface, the tumor cell is affected and as a result the tumor burden in the patient is reduced, regresses, or is eliminated. T cells can be engineered to express one or more chimeric antigen receptors (CARs) using any method as described herein or known in the art. In one embodiment, isolated T cells are genetically engineered to express a CAR construct by introducing the T cells with an expression vector encoding the CAR construct. Methods for transducing a population of T cells to express a selected CAR construct are known in the art and are incorporated herein by reference Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 4 th Edition, Cold Spring Harbor Laboratory Press (2012).

[0057] Generally, a CAR comprises an extracellular recognition or binding region / domain or extracellular domain that binds to a TAA on a tumor cell (e.g., single-chain variable fragment of an antibody (scFV)), a transmembrane domain, and any intracellular domain capable of providing a signal for T cell activation to attack the tumor cell.

[0058] Generally, the CARs described herein are induced by molecules (e.g., proteins) expressed on the cell surface of cancer or tumor cells. Some TAAs are known in the art and non-limiting examples include phosphorylated proteins, transmembrane proteins, glycoproteins, glycolipids, and growth factors. Assays for determining whether a given compound is suitable for use as a CAR recognition region for any of the antigens or targets described herein can be determined via routine experimentation by one of ordinary skill in the art. Any CAR as described herein or known in the art can be used in the methods, cells, and adoptive cell immunotherapy compositions used herein. Exemplary CARs include those described in U.S. Patent Nos. 7,446,190; 7,741,465; 9,499,629; 9,987,308; and 10,253,086.

[0059] In some embodiments, the CAR recognition domain targets an antigen expressed on the cell surface of B cells. In some embodiments, the CAR comprises an anti-CD19 recognition or binding domain. Exemplary CD19-CAR constructs include: (i) a CD19-directed chimeric antigen receptor (CTL019) lentiviral vector (CAR antigen recognition portion derived from SCFv:FMC63; co-stimulatory domain: 4-1BB), Maude, S.L., et al. N Engl J Med 2014;371(16):1507-1517; (ii) a CD19-directed chimeric antigen receptor incorporating an anti-CD-19 single-chain variable fragment + TCR zeta and CD28 signaling domains, a gamma-retroviral vector (CAR antigen recognition portion derived from SCFv:FMC63: FMC63; co-stimulatory domain: CD28), Lee, D.W et al., Lancet 2015;385(9967)517-528; a murine step cell virus-based splice-gag vector, (iii) MSG-FMC63-28Z encoding an anti-CD19 CAR as described in Kochenderfer J.N, et al., J Immunother 2009;32(7):689-702; (iv) a CD-19 specific CD28 / CD3ζ dual signaling CAR, 19-28z (in addition to that described in Park, J.H, et al., Blood, 30 June 2016, 127(26), p.3312-3320, Table 1, Brentjens, R.J., et al., Sci Trans Med, 20 Mar 2013:5(177):177).

[0060] Human CD19 antigen is a 95 kDa glycoprotein belonging to the immunoglobulin superfamily. CD19 is used as a biomarker for normal and neoplastic B cells, as well as for follicular dendritic cells. CD19 is expressed from the early stages of pre-B cell development to terminal differentiation and regulates the development and function of B lymphocytes. The expression of CD19 is highly conserved on most B cell tumors, including B cell lymphomas such as non-Hodgkin lymphoma. CD19 is also expressed in most types of leukemia, including B cell leukemia, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and Waldenström macroglobulinemia (WM). Most B cell malignancies (lymphomas and leukemias) express CD19 at normal to high levels. In some embodiments, the CAR comprises an anti-CD19 binding portion. In even further embodiments, although not limited, a combination of a long-acting IL-15 receptor agonist and CD19-directed CAR T cells is used in the treatment of B cell malignancies, including non-Hodgkin lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and Waldenström macroglobulinemia (WM). In some preferred embodiments, the CD19 CAR T cell therapy comprises CD-19-directed genetically modified autologous T cells.

[0061] Impairments to the durability of response to CD19 CAR T cell therapy have been identified as downregulation of the target antigen CD19 from the tumor cell surface (Shah, et al., Frontiers in Oncology (2019) Vol. 9, Article 146). In some embodiments, the CAR T cell therapy is a multi-target CAR T cell therapy that includes targeting of CD19 and one or more additional TAAs. In some embodiments, the CAR T cell therapy comprises at least a portion of a cell population that expresses a CD19 recognition or binding domain, as well as one or more additional TAAs. In some embodiments, the CAR T cell therapy includes targeting of a TAA selected from CD20, CD22, CD38, CD123, CD70, or CD30. In some embodiments, the CAR The T cell therapy involves two or more cell populations, each population expressing a different CAR. The cell populations can be administered as a mixture or sequentially simultaneously. Preferably, the multi-target CAR T cell therapy involves administration of anti-CD19 CAR T cells and CAR T cells directed to one or more additional TAAs selected from CD20, CD22, CD38, CD123, CD70, or CD30. In some other embodiments, the CAR T cell therapy involves targeting a TAA that is ROR1.

[0062] In some embodiments, the CAR includes one or more intracellular co-stimulatory signaling domains. Exemplary co-stimulatory domains include, but are not limited to, CD28, CD123, or 4-1BB in combination with CD3ζ.

[0063] Proliferation of lymphocytes such as T cells can be accomplished by any of a number of methods known in the art. For example, T cells can be expanded using non-specific T cell receptor stimulation in the presence of feeder lymphocytes and interleukin-2 (IL-2), IL-7, IL-15, IL-21, or combinations thereof. The non-specific T cell receptor stimulant can include a stimulatory amount of, for example, a murine monoclonal anti-CD3 antibody (e.g., available from LS Bio, Seattle WA). Alternatively, T cells can be rapidly expanded by in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with one or more antigens (including its antigenic portion such as an epitope), which can optionally be expressed from a vector in the presence of a T cell growth factor such as interleukin-2 or interleukin-15, with interleukin-2 being preferred. In vitro-derived T cells rapidly expand upon restimulation with the same antigen of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, T cells can be restimulated with irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and interleukin-2.

[0064] The specific tumor reactivity of the expanded T cells can be tested by any method known in the art, for example by measuring cytokine (e.g., interferon-gamma) release after co-culture with tumor cells. For example, adoptive cell transfer may involve enriching the cultured T cells for CD8+ T cells prior to rapid expansion of the cells. After culturing the T cells in a medium containing interleukin-2, the T cells are depleted of CD4+ cells and enriched for CD8+ cells using, for example, CD8 microbead separation. In some embodiments, the T cell growth factor that promotes the expansion and activation of autologous T cells is administered to the subject either simultaneously with or subsequent to the autologous T cells. The T cell growth factor can be any suitable growth factor that promotes the expansion and activation of autologous T cells. Examples of suitable T cell growth factors include interleukin (IL)-2, IL-7, IL-15, IL-12, and IL-21, which may be used alone or in various combinations such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7, and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2.

[0065] by fluorescence-activated cell sorting analysis and HLA-A2 - not the 888 melanoma strain but HLA-A2 +By in vitro recognition of melanoma strains, the expression of chimeric antigen receptors in CAR T cells is compared in untransduced (UnTd) and transduced (Td) cells (Rosenberg, S., et al., Nat Rev. Cancer, 2008 Apr;84(4):299 - 308). Universal types of T cells can also be used, such as those described in Qasim, W., et al., Sci. Transl. Med. 9, eaaj2013 (2017). For example, TALEN - mediated cell manipulation can be used in combination with lentiviral transduction to generate universal CAR19 T cells, which can be used in adoptive cell therapy. These cells are generated by lentiviral transduction of non - human leukocyte antigen - compatible donor cells and transcription activator - like effector nuclease (TALEN) - mediated gene editing of the T - cell receptor alpha chain and CD52 locus.

[0066] Before administering CAR T cells, to improve the effectiveness of the treatment, the patient's condition can be pre - conditioned with chemotherapy (e.g., cyclophosphamide and fludarabine as described, for example, in U.S. Patent No. 9,855,298). Without being limited by theory, pre - conditioning with chemotherapy can create space for the proliferation of CAR T cells by normal lymphocyte depletion and / or exclude cytokine sinks to increase the availability of homeostatic cytokines that promote CAR T cell proliferation and / or decrease the number of immunosuppressive cells such as regulatory T cells (Tregs) and myeloid - derived suppressor cells (Nair et al.). It should be understood that any suitable chemotherapy method can be used as known in the art.

[0067] Next, the expanded cells are administered to the host by infusion, for example, intravenously or intraarterially or by other suitable delivery modalities, which generally lasts from about 30 to about 60 minutes, although shorter or longer durations can be utilized. Other routes of administration include intraperitoneal, intrathecal, and intralymphatic injections. Optionally, cells expanded in a suitable medium that may contain any of various pharmaceutically acceptable additives, binders, fillers, carriers, preservatives, stabilizers, emulsifiers, buffers, etc. are provided. Diluents and excipients include water, saline, and glucose. Representative media include, for example, Multiple Electrolytes Injection, Type 1, USP, which has a normal pH range of about 5.5 to 8.0; tissue culture media containing human serum or fetal bovine serum; or xeno-free and serum-free media such as PRIME-XV T Cell Expansion XSFM (Irvine Scientific), among others. Commercially available media include, for example, RPMI 1640 (Thermo Fisher Scientific, Waltham, MA), AIM V cell culture medium (Thermo Fisher Scientific, Waltham MA), and X-VIVO 15 (Lonza, Basel, Switzerland).

[0068] IL-15 receptor agonist The methods described herein, in one or more embodiments, include administration of a long-acting IL-15 receptor agonist. A compound is considered a long-acting IL-15 receptor agonist according to the present disclosure as long as, after administration to a subject, the agonist exhibits IL-15 agonism in vivo for a longer period of time than in the case of administration of the same interleukin-15 receptor agonist moiety in non-modified form. Conventional techniques such as those involving radiolabeling the compound, administering the compound in vivo, and determining its clearance can be used to evaluate whether the compound is a long-acting IL-15 receptor agonist (i.e., has a longer clearance than non-modified IL-15 administered in the same in vivo system). For example, the long-acting nature of an IL-15 receptor agonist can be determined using flow cytometry to measure STAT5 phosphorylation in lymphocytes at various time points after administration of the agonist in mice. By way of reference, the signal disappears in approximately 24 hours for IL-15, but persists for a longer period of time for the long-acting IL-15 agonists described herein.

[0069] The long-acting IL-15 receptor agonist can be in the form of a pharmaceutically acceptable salt, and the reference to a long-acting IL-15 receptor agonist is intended to include its pharmaceutically acceptable salts. Typically, such salts are formed by reaction with a pharmaceutically acceptable acid or acid equivalent. In this context, the term "pharmaceutically acceptable salt" can generally refer to relatively non-toxic inorganic acid and organic acid addition salts. These salts can be prepared in situ in the dosage medium or during the manufacture of the dosage form, or by reacting the long-acting interleukin-15 receptor as described herein with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, oxalate, mesylate, glucoheptonate, lactobionate, and lauryl sulfonate, etc. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19). Accordingly, the salts as described may be derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc.; or prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicyclic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isothionic acid, etc.

[0070] Exemplary long-acting IL-15 receptor agonists have the structure:

Chemical formula

[0071] In one or more embodiments related to the long-acting IL-15 receptor agonist of formula (I), (n) ranges from about 795 to about 1068. In some additional embodiments, (n) ranges from about 840 to about 1023. In one or more specific embodiments, (n) has an average value of about 907 or about 909, such that the average molecular weight of the polyethylene glycol chain is about 40,000 daltons.

[0072] Mono(methoxyPEG-N-butanamide)interleukin-15 (MPBA-IL15) is typically prepared as a classification of mainly monomethoxypolyethylene glycolated IL-15 (i.e., having a single methoxyPEG-N-butanamide moiety covalently attached to the amino group of IL-15, i.e., to the lysine of IL-15 or the N-terminal alpha amine), with trace amounts of dimethoxypolyethylene glycolated and higher methoxypolyethylene glycolated IL-15 species. Further characteristics of mono(methoxyPEG-N-butanamide)interleukin-15 are described, for example, in WO 2018 / 213341 pamphlet.

[0073] Compositions that are examples of MPBA-IL15 mainly contain mono-PEGylated species, less than about 10 mole % of PEG dimers (i.e., those having two methoxy PEG-N-butanamide moieties attached to IL-15), and even smaller amounts of additional PEGylated species attached to IL-15 (i.e., those having three or more methoxy PEG-N-butanamide moieties attached to IL-15). Compositions of mono-(methoxy PEG-N-butanamide) interleukin-15 will generally have at least about 80 mole % mono-PEGylated IL-15 species (based on all interleukin-15 species in the composition, including non-modified IL-15 (if present) and other IL-15-containing species such as di-PEGylated IL-15 and larger). Compositions of MPBA-IL15 can preferably have at least about 90 mole % mono-PEGylated IL-15 species, along with less than about 10 mole % of other IL-15 species. In some embodiments, the MPBA-IL15 composition contains less than about 5 mole % of PEG dimers (di-PEGylated IL-15 having two methoxy PEG-N-butanamide moieties covalently attached to IL-15) and less than about 5 mole % of all other additional PEGylated species. In one or more embodiments, the MPBA-IL15 composition contains at least about 85 mole %, 90 mole %, 95 mole %, 98 mole %, or 99 mole % of the mono-PEGylated species of formula (I).

[0074] For example, in some preferred embodiments, the long-acting IL-15 receptor agonist composition, when considered collectively, has the formula:

Chemical formula

[0075] In some embodiments, the MPBA-IL15 composition comprises from about 0.1 to 15, 0.1 to 10, 0.1 to 5, 0.1 to 1, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or about 15 to 20 mol% of the compound of formula (II).

[0076] In some additional embodiments, the long-acting IL-15 receptor agonist composition, when considered collectively, comprises at most about 1 to 5 mol% of free IL-15 protein of (the IL-15-containing molecule in the composition).

[0077] Regarding the above formula, "n" corresponds to the average number of (OCH 2 CH 2 ) monomer subunits. MPBA-IL15 can be prepared using a suitably activated mPEG-butanoic acid ester reagent having an average molecular weight of, for example, from about 6600 daltons to about 132,000 daltons. For example, MPBA-IL15 can be prepared using a suitably activated mPEG-butanoic acid ester reagent having an average molecular weight selected from, for example, 10 kD, 15 kD, 20 kD, 25 kD, 30 kD, 45 kD, 50 kD, or 60 kD. The activated polymer reagent is effective to form a stable amide bond between the IL-15 moiety and the polyethylene glycol moiety when reacted with the amino groups (e.g., lysine or N-terminus) of IL-15.

[0078] In one or more embodiments, n is an integer having a value corresponding to a polyethylene glycol moiety having a weight average molecular weight selected from the group consisting of about 10,000 daltons (where n is about 227 here), or about 15,000 daltons (where n is about 340 here), or about 20,000 daltons (where n is about 454 here), or about 25,000 daltons (where n is about 568 here), or about 30,000 daltons (where n is about 681 here), or about 40,000 daltons (where n is about 909 here), or about 50,000 daltons (where n is about 1136 here), or about 60,000 daltons (where n is about 1364 here) or greater.

[0079] Exemplary PEG reagents used to prepare MPBA-IL15 will typically have a polydispersity value of less than about 1.1, for example, about 1.05. Thus, for a PEG reagent such as mPEG-succinimidyl butanoate having a nominal average weight of about 40,000 daltons, the PEG reagent (and the resulting IL-15 conjugate) will have a covalently attached PEG moiety in the molecular weight range of about 35 kDa to about 47 kDa, or about 37 kDa to about 45 kDa (41kD ± 4kDa). In some preferred embodiments, the ester reagent that activates mPEG butanoic acid has a nominal average molecular weight of about 40,000 daltons, i.e., here, on average, n is about 907 - 909.

[0080] When considering the IL-15 moiety, the term "IL-15 moiety" refers to the IL-15 moiety before conjugate formation and the IL-15 moiety after conjugate formation. However, it will be understood that when the original IL-15 moiety is attached to the polyethylene glycol moiety, the IL-15 moiety may change slightly due to the presence of one or more covalent bonds associated with the attachment to the polymer (e.g., in the case of the amide bond formed during the preparation of MPBA-IL15).

[0081] The IL-15 moiety can be obtained from non-recombinant and recombinant methods, and the present disclosure is not limited in this regard. Further, the IL-15 moiety can be obtained from human origin, animal origin (including insects), fungal origin (including yeast), and plant origin.

[0082] The IL-15 moiety can be obtained, for example, according to the procedures described by Grabstein et al. (Grabstein et al. (1994) Science 264:965-968). The IL-15 moiety can also be prepared using recombinant methods such as those described in European Patent No. 0772624 B2 to Immunex Corporation. Alternatively, the IL-15 moiety can be purchased as a commercial product from, for example, GenScript USA Inc. (Piscataway NJ) and Peprotech (Rockyhill, NJ).

[0083] More specifically, the IL-15 moiety can be expressed in bacterial [e.g., Escherichia coli (E. coli), see, for example, Fischer et al. (1995) Biotechnol. Appl. Biotechnol. 21(3):295-311], mammalian [see, for example, Kronman et al. (1992) Gene 121:295-304], yeast [e.g., Pichia pastoris, see, for example, Morel et al. (1997) Biochem. J. 328(1):121-129], and plant [see, for example, Mor et al. (2001) Biotechnol. Bioeng. 75(3):259-266] expression systems. Expression can occur via exogenous expression (when the host cell naturally contains the desired genetic code) or via endogenous expression.

[0084] Further methods for the preparation and / or purification of the IL-15 moiety are described in PCT Application / US Patent Application Publication No. 2018 / 032817.

[0085] Depending on the system used to express the protein having IL-15 activity, the IL-15 moiety can be non-glycosylated or glycosylated, and either can be used. That is, the IL-15 moiety can be non-glycosylated or the IL-15 moiety can be glycosylated. In one or more embodiments, the IL-15 moiety is non-glycosylated.

[0086] The IL-15 moiety can be advantageously modified to include and / or substitute one or more amino acid residues such as, for example, lysine, cysteine and / or arginine in order to provide for easy attachment of the polymer to the atoms within the side chain of the amino acid. Examples of substitutions of the IL-15 moiety are described in U.S. Patent No. 6,177,079. Further, the IL-15 moiety can be modified to include non-naturally occurring amino acid residues. Techniques for adding amino acid residues and non-naturally occurring amino acid residues are well known to those of skill in the art.

[0087] Exemplary IL-15 moieties are described herein as well as in the literature, for example, in U.S. Patent Application Publication No. 2006 / 0104945, Pettit et al. (1997) J. Biol. Chem. 272(4):2312-2318, Wong et al., (2013) OncoImmunology 2(11), e26442:1-3, and PCT Application / International Publication No. 2018 / 213341 Pamphlet. Preferred IL-15 moieties include those having an amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 3 and sequences substantially homologous thereto. A preferred IL-15 moiety has an amino acid sequence corresponding to SEQ ID NO: 1. In some embodiments, the IL-15 moiety is a functional homolog having at least about 85% or at least about 90% identity to any one of SEQ ID NOs: 1 to 3. In some embodiments, the IL-15 moiety is a functional homolog having at least about 95%, 98%, or 99% identity to any one of SEQ ID NOs: 1 to 3.

[0088] In some cases, the IL-15 moiety assumes a "monomeric" form in which the single expression of the corresponding peptide is organized into separate units. In other cases, the IL-15 moiety assumes a "dimeric" form (e.g., a dimer of recombinant IL-15) in which two monomeric forms of the protein associate with each other.

[0089] Furthermore, can be used as the IL-15 moiety. An exemplary precursor form of IL-15 has the sequence of SEQ ID NO: 3.

[0090] Cleaved forms, hybrid variants, and peptidomimetics of any of the foregoing sequences can also function as the IL-15 moiety. Any biologically active fragment, deletion variant, substitution variant, or addition variant of the foregoing that maintains at least some degree of IL-15 activity can also function as the IL-15 moiety.

[0091] For any given peptide, protein moiety, or conjugate, it is possible to determine whether that peptide, protein moiety, or conjugate has some degree of IL-15 activity. Various methods for determining IL-15 activity in vitro are described in the art. An exemplary approach is based on the pSTAT assay. Briefly, if IL-15-dependent CTLL-2 cells are exposed to a test substance having IL-15 activity, initiation of a signaling cascade involving phosphorylation of STAT5 at tyrosine residue 694 (Tyr694) occurs, which can be measured quantitatively. Assay protocols and kits are known, for example, the MSD Phospho(Tyr694) / Total STATa,b Whole Cell Lysate Kit (Meso Scal Diagnostics, LLC, Gaithersburg, MD). For example, using this approach, at least one of 5 minutes or 10 minutes, and at most about 300 ng / mL (more preferably at most about 150 ng / mL) of pSTAT5 EC 50The proposed IL-15 moieties that exhibit a value are typically considered to be "IL-15 moieties" within the scope of the present disclosure. However, the IL-15 moieties used are preferably more potent (e.g., less than 150 ng / mL at at least one of 5 minutes or 10 minutes, e.g., less than about 1 ng / mL, more preferably less than 0.5 ng / mL at at least one of 5 minutes or 10 minutes, of pSTAT5 EC 50 value).

[0092] To evaluate IL-15 function, other methodologies known in the art can also be used, including potentiometry, spectrophotometry, chromatography, and radiometric methodologies. For example, see Ring et al. (2012) Nat. Immunol. 13(12):1187-1195 for such additional types of assays.

[0093] As described above, the amino groups on the IL-15 moiety provide sites of attachment for reaction with an mPEG-butyric acid succinimidyl reagent to provide an IL-15 receptor agonist encompassed by formula (I). Considering the exemplary IL-15 amino acid sequences provided herein, it is clear that there are seven lysine residues, each having an ε-amino acid that may be available for conjugate formation. Additionally, the N-terminal amine of methionine can also serve as an attachment point for the PEG moiety. It should be understood that the polyethylene glycol moiety can attach to any one or more of the lysine or N-terminal amine positions. In some embodiments, the polyethylene glycol moiety attachment site is Lys 10 and Lys 11 (using the numbering as shown in SEQ ID NO: 2 as an example) or SEQ ID NO: 1 to Lys 11 and Lys 12 in one or more of. In some embodiments, the polyethylene glycol moiety is attached to the N-terminal amine. Any of the lysine sites (e.g., Lys of SEQ ID NO: 1 37 or Lys 42) should be understood to be suitable as an attachment site for the PEG moiety. In some embodiments, MPBA-IL15 comprises a mixture of positional isomers, wherein the covalent attachment of the polyethylene glycol moiety is primarily at the N-terminus (i.e., among the collected positional isomers, the isomer with the PEG moiety attached to the N-terminus is present in the largest amount when compared to the other positional isomers).

[0094] MPBA-IL15 is an immunotherapeutic agent that provides sustained IL-15 bioactivity without the need for daily dosing through binding to all IL-15 receptor subunits (IL-15α, β, and γ subunits). More specifically, MPBA-IL15 binds to the IL-15 receptor α and interleukin-2 (IL-2) / IL-15 βγ subunits, maintaining the entire spectrum of IL-15 biology, including the pharmacodynamic (PD) effects on both NK cells and CD8+ memory T cells. On the other hand, the polyethylene glycol moiety expands the hydrodynamic volume of the molecule, which serves to extend the effective half-life compared to unmodified rhIL-15.

[0095] Preclinical studies in rodents and non-human primates have shown that MPBA-IL15 has characteristics that are particularly advantageous to the inventors when combined with CAR T cell therapy. For example, MPBA-IL-15 (i) stimulates and expands NK cell proliferation, (ii) aids in CD8 T cell survival and memory formation without substantially inducing inhibitory regulatory T cells, (iii) enhances the formation of long-term immune memory, and further, has been shown to retain receptor binding to the IL-R but with a lower affinity than unmodified IL-15.

[0096] Additional IL-15 receptor agonists that may be suitable for use in the methods provided herein include, for example, N-803 (formerly ALT-803, a mutant IL-15 / IL-15Rα fusion protein, see, e.g., Han, K., et al., Cytokine, 2011;56(3):804-810; Zhu, X., et al., J Immunol. 2009;183(6):3598-3607, and Xu, W., et al., Cancer Res. 2013;73(10):3075-3086), NIZ985 (a heterodimeric IL-15, IL-15 / soluble IL-15Rα dimer, see, e.g., AACR; Cancer Res 2019;79(13 Suppl)), AM0015 (a polyethylene glycol-modified IL-15, see, e.g., WO 2017 / 112528 pamphlet), and OXS-3550 (a triple-specific scFv recombinant fusion protein conjugate consisting of single-chain, variable regions of the heavy and light chains of anti-CD16 and anti-CD33 antibodies and a modified form of IL-15, CAS Registry No. 2094086-30-1, UNI: E5GE91Q5FX), and the like.

[0097] Method Based on at least one or more characteristics of a long-acting IL-15 receptor agonist, MPBA-IL15, in one aspect, provided herein is a method effective to induce an immune response in a cancer patient prior to administration of MPBA-IL15, and concomitantly / consequently followed by administration of the long-acting IL-15 receptor agonist, namely MPBA-IL15, by administering an adoptive cell immunotherapy composition comprising autologous or allogeneic (preferably autologous) anti-tumor T cells genetically transformed to express a chimeric antigen receptor that targets tumor cells, such as CD19 CAR T cells as described previously and having anti-tumor activity. In a preferred embodiment, the T cell immunotherapy comprises CD19-directed genetically modified autologous T cells. An exemplary adoptive cell immunotherapy composition comprises tumor-reactive T cells modified to express a chimeric antigen receptor comprising an extracellular variable domain of an antibody induced by a cancer-associated antigen (such as CD19), a hinge and transmembrane domain, and an intracellular signaling domain (such as a co-stimulatory domain) of a T cell or other receptor. For example, the tumor-reactive T cells can be modified with a chimeric antigen receptor derived from a single-chain antibody induced by the CD19 molecule. In some embodiments, the cell composition comprises cytotoxic tumor cells modified with a CAR, such as CD8+ T lymphocytes modified with a CD19 CAR, and further can comprise other types of T lymphocytes (such as helper T lymphocytes), such as CD4+ or other T cells genetically modified to have a chimeric antigen receptor induced by a cancer-associated antigen (such as CD19), and the present disclosure is not limited in this regard (i.e., to a particular configuration of an adoptive cell immunotherapy composition comprising CAR T cells, such as CD19-directed CAR T cells). Representative CAR T cell compositions suitable for use in the methods provided herein are described in the preceding section.

[0098] In some embodiments, the cells included in the adoptive cell immunotherapy composition are first recovered from their culture medium, subsequently washed, and formulated by concentrating the cells in a medium and container system suitable for administration in a therapeutically effective amount. Suitable infusion media can be, for example, any isotonic media formulation such as, in particular, normal saline, RPMI 1640 (ThermoFisher), AIM V serum-free medium (ThermoFisher), and X-VIVO™ medium (Lonza Walkersville), 5% aqueous dextrose solution, or lactated Ringer's solution.

[0099] An adoptive cell immunotherapy composition containing CAR T cells, such as CD19 CAR T cells, is typically administered in a therapeutically effective amount, i.e., an amount effective to confer immunity to a subject. Immunity means alleviating one or more physical symptoms associated with a tumor or cancer in which a lymphocyte response is induced. The adoptive cell immunotherapy composition is typically administered by infusion, and each infusion contains at least 2 cells to at least 10 6 ~10 10 cells / kg, preferably at least 10 7 ~ about 10 9 cells / kg, or preferably at least 10 6 ~ about 10 8 cells / kg. In some specific, but non-limiting, embodiments, each infusion contains at least about 0.2×10 6 cells / kg to about 6.0×10 8 cells / kg, at least about 2.0×10 6 cells / kg to about 2.0×10 8 cells / kg, at least about 0.2×10 6 cells / kg to about 5.0×10 6 cells / kg, at least about 0.1×10 8 cells / kg to about 2.5×10 8 cells / kg, or at least about 0.6×10 8 cells / kg to about 6.0×10 8It contains cells / kg. The cells can be administered by single injection or multiple injections. In some specific embodiments, the cells are administered as a single-dose injection. Since different individuals have different responses, the number of cells injected, as well as the number of injections and the time range over which multiple injections are given, can be determined by medical personnel when determined by conventional investigations. In some embodiments, the administration of T cell immunotherapy is preceded by the administration of a lymphodepleting chemotherapy regimen, for example, the administration of cyclophosphamide (typically intravenously) and fludarabine (typically intravenously).

[0100] According to the methods described herein, the long-acting IL-15 receptor agonist is a CAR It is administered in an amount effective to enhance the results of T cell immunotherapy. For confirmation, with respect to the long-acting IL-15 receptor agonist, MPBA-IL15, the amount and degree of activation can vary widely and may still be effective when combined with the administration of the adoptive cell immunotherapy composition. That is, an amount of MPBA-IL15 that exhibits only minimal IL-15 receptor agonist activity for a sufficiently long period can still be a long-acting IL-15 receptor agonist when administered in combination with CAR T cell therapy, and the methods described herein enable a clinically meaningful response. In certain cases, for example, due to synergistic interactions and responses, when accompanied by CAR T cell therapy, such as CD19 CAR T cell therapy, only minimal IL-15 receptor agonist activity may be required. It should be understood that the therapeutic doses of one or both of the long-acting IL-15 receptor agonists administered and the number of CAR T cells can be lower than the therapeutically effective amount of either component when administered alone.

[0101] As described herein, one aspect of the disclosure provides a method useful for treating a patient suffering from a condition (such as cancer) that is responsive to treatment with either or both of an adoptive cell immunotherapy composition and a long-acting IL-15 receptor agonist. For example, a patient may be responsive to each agent alone, as well as in combination, but may be more responsive to the combination. As a further example, a patient may be non-responsive to one of the individual immunotherapy agents, but responsive to the combination. As yet a further example, a patient may be non-responsive to either of the individual agents alone, but responsive to the combination.

[0102] The long-acting IL-15 receptor agonist, MPBA-IL15, can be administered by any suitable means known in the art. In some embodiments, the long-acting IL-15 receptor agonist is administered parenterally. As used herein, the term "parenteral" includes subcutaneous, intravenous, intraarterial, intratumoral, intralymphatic, intraperitoneal, intracardiac, intrathecal, and intramuscular injection, as well as infusion.

[0103] MPBA-IL15 can be combined with one or more suitable excipients or diluents to form a composition suitable for administration or further use. The long-acting IL-15 receptor agonist can be included in a single-dose composition optionally accompanied by one or more pharmaceutically acceptable excipients. Suitable pharmaceutically acceptable excipients include, for example, those described in Handbook of Pharmaceutical Excipients, 7 th ed., Rowe, R.C., Ed., Pharmaceutical Press, 2012. Such exemplary formulations include those in which MPGA-IL15 is placed in a solution containing phosphate buffer and trehalose (pH 6.8). For example, exemplary formulations include those in which MPBA-IL15 is formulated in a solution of potassium phosphate buffer, trehalose, and polysorbate 20 (pH ~6).

[0104] Suitable dosage forms for parenteral administration include, in particular, immediately injectable solutions, dry powders for combination with a solvent before use, immediately injectable suspensions, dry insoluble compositions for combination with a vehicle before use, and emulsions and liquid concentrates for dilution before administration. In some specific embodiments, the long-acting IL-15 receptor agonist is provided in a formulation suitable for intravenous administration and is administered intravenously. In some other embodiments, the long-acting IL-15 receptor agonist is provided in a formulation suitable for subcutaneous administration and is administered subcutaneously. In some additional embodiments, the long-acting IL-15 receptor agonist is administered intratumorally. Other modes of administration are contemplated, such as intrapulmonary, nasal, buccal, intrarectal, sublingual, and transdermal.

[0105] Generally, the therapeutically effective amount of the long-acting IL-15 receptor agonist will range from about 5 mcg (μg) to about 10 mg, based on the dose of the protein (IL-15 equivalent) being administered. A given dose can be administered periodically, for example, until a clinician determines that an appropriate endpoint (e.g., cure, regression, partial regression, etc.) has been achieved.

[0106] In some embodiments, the therapeutically effective dose of the long-acting IL-15 receptor agonist, MPBA-IL15, is in the range of about 0.10 to 70 mcg / kg (micrograms per kilogram, μg / kg (IL-15 equivalent)). In other embodiments, the therapeutically effective dose is in the range of about 0.10 mcg / kg to about 50 mcg / kg, or about 0.30 to about 45 mcg / kg, or about 0.25 mcg / kg to about 0.1 mg / kg, about 0.01 mg / kg to about 0.1 mg / kg / day, or about 0.03 mg / kg to about 0.1 mg / kg / day. In other embodiments, the therapeutically effective dose is in the range of about 1 to 10 mcg / kg, about 0.03 mg / kg to about 0.1 mg / kg. In some specific, but non-limiting, embodiments, the therapeutically effective dose is about 0.25 mcg / kg, 0.3 mcg / kg, 0.5 mcg / kg, 1 mcg / kg, 2 mcg / kg, 3 mcg / kg, 5 mcg / kg, 6 mcg / kg, 7 mcg / kg, 10 mcg / kg, 15 mcg / kg, 20 mcg / kg, 25 mcg / kg, 0.01 mg / kg, 0.03 mg / kg, 0.05 mg / kg, or 0.1 mg / kg.

[0107] In some further embodiments, the therapeutically effective dose of the long-acting IL-15R agonist ranges from about 0.25 to 25 μg / kg. In other embodiments, the therapeutically effective dose ranges from about 0.25 μg / kg to about 0.1 mg / kg (e.g., per day), about 1.0 μg / kg to about 20 μg / kg, about 1.0 μg / kg to about 15 μg / kg, about 1.0 μg / kg to about 10 μg / kg, about 1.0 μg / kg to about 5.0 μg / kg, about 1 μg / kg to about 1.5 μg / kg, about 1.5 μg / kg to about 20 μg / kg, about 1.5 μg / kg to about 15 μg / kg, about 1.5 μg / kg to about 10 μg / kg, about 1.5 μg / kg to about 5.0 μg / kg, about 5.0 μg / kg to about 20 μg / kg, about 5.0 μg / kg to about 15 μg / kg, about 5.0 μg / kg to about 10 μg / kg, about 10 to about 1.5 μg / kg to about 20 μg / kg, about 10 μg / kg to about 20 μg / kg, about 10 μg / kg to about 15 μg / kg, about 15 μg / kg to about 20 μg / kg, about 0.01 mg / kg to about 0.1 mg / kg / day, or about 0.03 mg / kg to about 0.1 mg / kg / day. In other embodiments, the therapeutically effective dose ranges from about 1 to 10 μg / kg, about 0.03 mg / kg to about 0.1 mg / kg. In some specific but non-limiting embodiments, the therapeutically effective dose is about 0.25 μg / kg, 0.3 μg / kg, 0.5 μg / kg, 1 μg / kg, 1.5 μg / kg, 2 μg / kg, 3 μg / kg, 5 μg / kg, 6 μg / kg, 7 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg, 25 μg / kg, 0.01 mg / kg, 0.03 mg / kg, 0.05 mg / kg, or 0.1 mg / kg / day.

[0108] As in the case of the administration of the adoptive cell immunotherapy composition, the dose of the long-acting IL-15 receptor agonist will vary, for example, according to the age, weight, and general condition of the subject, as well as the severity of the condition being treated, the individual adoptive cell immunotherapy composition, and the judgment of the medical practitioner.

[0109] In one or more embodiments, adoptive cell transfer is performed prior to administration of a long-acting IL-15 receptor agonist. For example, cell infusion based on adoptive cell transfer of CAR T cells can be performed immediately, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 1 day, up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days, up to 13 days, up to 14 days, up to 15 days, up to 16 days, up to 17 days, up to 18 days, up to 19 days, up to 20 days, up to 21 days, up to 22 days, up to 23 days, up to 24 days, up to 25 days, up to 26 days, up to 27 days, up to 28 days, up to 29 days, up to 1 month, up to 3 months, up to 6 months, or any combination thereof, before administration of MPBA-IL15.

[0110] Alternatively, in some embodiments, adoptive cell transfer is performed after administration of a long-acting IL-15 receptor agonist. For example, cell injection based on adoptive cell transfer of CAR T cells can be performed immediately, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 1 day, up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days, up to 13 days, up to 14 days, up to 15 days, up to 16 days, up to 17 days, up to 18 days, up to 19 days, up to 20 days, up to 21 days, up to 22 days, up to 23 days, up to 24 days, up to 25 days, up to 26 days, up to 27 days, up to 28 days, up to 29 days, up to 1 month, up to 3 months, up to 6 months, or any combination thereof, after administration of MPBA-IL15.

[0111] As used herein with respect to the treatment of a subject having cancer, the terms "treatment," "treating," and "treatment" are intended to include the full range of interventions for the cancer from which the subject suffers, such as the administration of a combination to reduce, slow down, stop, or reverse one or more symptoms of cancer, or to delay the progression of cancer, even if the cancer is not actually removed. Treatment can include, for example, a decrease in the severity of symptoms, the number of symptoms, or the frequency of recurrence, such as suppression of tumor growth, arrest of tumor growth, or regression of an existing tumor.

[0112] For example, improvement of cancer or a cancer-related disease can be characterized as complete or partial response. "Complete response" refers to the normalization of any previous abnormalities in X-ray examinations, bone marrow, and cerebrospinal fluid (CSF), or monoclonal protein measurements, and the absence of clinically detectable disease. "Partial response" refers to at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decrease in all measurable tumor burden (i.e., the number of malignant T cells present in the subject, or the measured volume of a tumor mass, or the amount of abnormal monoclonal protein) in the absence of new lesions. The term "treatment" contemplates both complete and partial responses.

[0113] Regarding the frequency and schedule of infusion of the adoptive cell immunotherapy composition and administration of the long-acting IL-15 receptor agonist, one of ordinary skill in the art will be able to determine the appropriate frequency. For example, a clinician may perform adoptive cell transfer in a treatment cycle in combination with administration of a long-acting IL-15 receptor agonist either simultaneously with or preferably after adoptive cell transfer of CAR T cells. For example, in some treatment modalities, the long-acting IL-15 receptor agonist is administered within about 7 days of adoptive cell transfer of CAR T cells (e.g., on any one of days 1, 2, 3, 4, 5, 6, or 7). In some examples, the long-acting IL-15 receptor agonist, i.e., MPBA-IL15, is administered within 4 days of adoptive cell transfer, e.g., on any one of days 1, 2, 3, or 4. Based on the long-acting nature of MPBA-IL15, the IL-15 receptor agonist is typically administered at a relatively low frequency (e.g., once every 3 weeks, once every 2 weeks, once every 8 - 10 days, once a week, etc.).

[0114] Exemplary lengths of time associated with the treatment process include about 1 week; about 2 weeks; about 3 weeks; about 4 weeks; about 5 weeks; about 6 weeks; about 7 weeks; about 8 weeks; about 9 weeks; about 10 weeks; about 11 weeks; about 12 weeks; about 13 weeks; about 14 weeks; about 15 weeks; about 16 weeks; about 17 weeks; about 18 weeks; about 19 weeks; about 20 weeks; about 21 weeks; about 22 weeks; about 23 weeks; about 24 weeks; about 7 months; about 8 months; about 9 months; about 10 months; about 11 months; about 12 months; about 13 months; about 14 months; about 15 months; about 16 months; about 17 months; about 18 months; about 19 months; about 20 months; about 21 months; about 22 months; about 23 months; about 24 months; about 30 months; about 3 years; about 4 years; about 5 years. Generally, a patient is provided with one round of adoptive cell transfer, e.g., CD19 CAT T cells, followed by administration of the long-acting IL-15 receptor agonist, MPBA-IL15, one or more times. However, in some examples, one or more additional cycles of adoptive cell transfer may be performed.

[0115] The treatment methods described herein are typically continued as long as the clinician overseeing patient care deems the treatment method to be effective, i.e., the patient is responding to the treatment. Non-limiting parameters that can serve as indicators of the effectiveness of the treatment method can include one or more of the following: tumor shrinkage (in terms of weight and / or volume and / or appearance); decrease in the number of individual tumor colonies; decrease in the number of cancer cells; tumor disappearance; progression-free survival; appropriate response by a suitable tumor marker (where applicable), increase in the number of NK (natural killer) cells, increase in the number of T cells, increase in the number of memory T cells, increase in the number of central memory T cells, decrease in the number of regulatory T cells, such as CD4+Treg, CD25+Treg, and FoxP3+Treg.

[0116] As described above, adoptive cell compositions (e.g., including CAR T cells) and long-acting IL-15 receptor agonists can be administered separately. Alternatively, it may be desired for the provision of adoptive cell transplantation and administration of the long-acting IL-15 receptor agonist to be simultaneous, either as a first dose, or throughout the course of treatment, or at various stages of the dosing regimen (and the CAT T cells and the long-acting IL-15 receptor agonist are compatible together and in a given formulation), and simultaneous administration can be achieved by infusion of a single-dose form / formulation (e.g., intravenous administration of an intravenous formulation containing both immunological components).

[0117] The methods and compositions described herein can be used to treat patients suffering from any condition that can be treated (remedy) or prevented by the methods provided herein, such as cancer. For example, these methods are useful for the treatment of, among other conditions, solid tumors, hematological tumors (liquid cancers), or melanoma. Exemplary conditions are cancer, such as melanoma, renal cancer, non-small cell lung breast cancer (e.g., triple-negative breast cancer), bladder cancer, head and neck cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, brain cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, testicular cancer, lung cancer, small cell lung cancer, brain cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, multiple myeloma, neuroblastoma, retinoblastoma, and leukemia, etc.

[0118] In some particular embodiments, the cancer is a solid cancer.

[0119] Also in some further embodiments, the cancer is selected from, for example, breast cancer, ovarian cancer, colon cancer, prostate cancer, bone cancer, colorectal cancer, gastric cancer, lymphoma, malignant melanoma, liver cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancer, renal cancer, cholangiocarcinoma, brain cancer, cervical cancer, maxillary sinus cancer, bladder cancer, esophageal cancer, and adrenocortical carcinoma.

[0120] Also in one or more further embodiments, the cancer is selected from melanoma, renal cancer, non-small cell lung cancer, breast cancer, bladder cancer, head and neck cancer, and colon cancer.

[0121] In one or more particular embodiments, the breast cancer is triple-negative breast cancer. Triple-negative breast cancer is a very aggressive tumor lacking estrogen receptor, progesterone receptor, and ERBB2 (HER2) gene amplification.

[0122] In some other embodiments, the cancer is lymphoma or leukemia.

[0123] In some further embodiments, the cancer is a B-cell malignancy selected from, but not limited to, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and Waldenström macroglobulinemia (WM), and the patient population includes both pediatric and adult patients.

[0124] The method is useful for enhancing the therapeutic efficacy of adoptive cell transfer of CAR T cells, such as CD19 CAR T cells, by improving the response of the subject, for example, by administration of a long-acting IL-15 receptor agonist, MPBA-IL15. The enhanced response can be evaluated at any suitable time during treatment, after a single round of treatment, after 2-3 cycles of treatment, etc., and by any of several suitable methods, including, for example, reduction of the tumor (partial response), i.e., evaluation of tumor size or volume, disappearance of the tumor, decrease in the number of cancer cells, reduction of disease progression (absence of cancer progression), and, where appropriate, analysis of one or more tumor markers. The comparison can be made in human patients or in suitable animal models such as suitable mouse models of cancer.

[0125] In yet some other embodiments, the methods, kits, compositions, and combinations provided herein are effective to stimulate T cell and / or NK cell activity and / or proliferation in a subject. In some embodiments, the method is effective to increase the number of CD8+ T cells in a subject, for example when evaluated in a cancer mouse model of the corresponding cancer. In yet some other embodiments, the method is effective to increase the number of NK cells in a subject, for example when evaluated in a mouse model of the corresponding cancer.

[0126] To characterize and monitor CAR-T cells and to evaluate the effect of a therapy on the number and activation of immune cell populations, including but not limited to NK cells, CD8+ T cells, and CD8+ memory cells, blood samples can be collected from a subject both before and during treatment. Characterization and continuous monitoring of genetically modified CD19-directed CAR-T cells can be performed in peripheral blood by quantitative polymerase chain reaction (qPCR) both before and during treatment, and the phenotype of CAR-T cells can also be evaluated by flow cytometry. Blood samples can also be collected before and during treatment to determine changes in cytokine levels in response to the therapy and to profile changes in gene expression. Additionally, whole blood samples or PBMCs can be collected and used for the evaluation of other immune functions.

[0127] When possible, fresh bone marrow biopsy material can be collected before, during, and after treatment for the characterization of tumor cells and immune system activation. The evaluation can include the evaluation of changes in tumor-specific protein markers and immune cell populations in the tumor microenvironment. Characterization and monitoring of genetically modified CD19 CAR-T cells in the bone marrow can be performed by quantitative polymerase chain reaction (qPCR) before and after administration of a long-acting IL-15 receptor agonist, such as MPBA-IL15.

[0128] Tumor biopsy materials can also be collected. The biopsy should preferably be performed on lesions that have not been previously exposed to radiation. Biopsy materials can be obtained from non-target lesions if no other lesions suitable for biopsy are present. Tumor tissue biopsy materials before and after treatment should preferably be taken from the same lesion if possible. Additionally, biopsy materials can be taken from non-injected lesions far away to serve as a control biopsy. Immunohistochemistry (IHC) using a panel of markers (including but not limited to CD3, CD4, CD8, and CD56) and / or flow cytometry can be used to characterize the infiltrating immune cell population using tumor tissue biopsies.

[0129] In some embodiments, the combination immunotherapy described herein is effective to increase the extent and persistence (i.e., maintenance) of exogenously delivered CAR T cells at the tumor site or in the blood, as opposed to many ACT-based therapies, thereby providing an increase in anti-tumor efficacy.

[0130] All papers, books, patents, patent publications, and other publications referenced herein are hereby incorporated by reference in their entirety. In the event of a conflict between the teachings herein and the technology incorporated by reference, the meaning of such teachings and the definitions herein shall prevail (especially with respect to the terms used in the claims appended hereto). For example, if the present application and a publication incorporated by reference define the same term differently, the definition of the term shall be maintained within the scope of the teachings of the document in which the definition is set forth.

Examples

[0131] It should be understood that the foregoing description and the following examples are intended to be illustrative and not to limit the scope of the present disclosure. Other aspects, advantages, and modifications within the scope of the present invention will be apparent to those skilled in the art to which the present disclosure pertains.

[0132] Materials and Methods CAR T cells: Human CD19 CAR T cells expressing the CD19 / 4-1BB / CD3ζ CAR were produced from healthy donors as previously described. See, for example, Turtle, C.J., et al. J. Clin Invest. 2016;126(6):2123-2138 and U.S. Patent No. 9,987,308. Briefly, CD4 T cells and CD8 T cells were isolated, separately transduced with the CD19 CAR lentiviral vector (Figure 2), the transduced cells were sorted, and then expanded for 14-16 days using LCL (lymphoblastoid B cell line cells; cells were analyzed on day 15).

[0133] Recombinant IL-15 ("rIL-15") having SEQ ID NO: 1 (as provided in Figure 1), prepared using conventional techniques, was used in the following examples, although any suitable IL-15 moiety can be used as well. SEQ ID NO: 1, a non-glycosylated recombinant human IL-15 expressed and purified from E. coli inclusion bodies, contains 115 amino acids, has two disulfide bridges, and has a molecular weight of approximately 12.9 kDa. The rIL-15 sequence contains a methionine added at the N-terminus that is not present in native secreted human IL-15.

[0134] The reactive linear polymer reagent, mPEG-butyric acid N-succinimidyl ester, 40 kDa ("mPEG-SBA"), CAS No. 187848-51-7 has the following structure [Chemical formula] (wherein n corresponds to the average number of monomer subunits to provide a polymer having a nominal average molecular weight of about 40 kilodaltons, i.e., here, on average, n is about 907-909) It has. The PEG reagent has a polydispersity value of less than about 1.1, for example, about 1.05. As a result, the nominal average molecular weight ranges from about 37 kilodaltons to about 45 kilodaltons (41 kD ± 4 kDa). mPEGSBA is typically in the form of a white to off-white powder. Examples of additional mPEG-butanic acid succinimidyl reagents suitable for use include those having a weight average molecular weight of about 10 kD, 15 kD, 20 kD, 25 kD, 30 kD, 45 kD, 50 kD, or 60 kD. This activated polymer reagent is effective in forming a stable amide bond between the IL-15 moiety and the polyethylene glycol moiety when reacting with the amino group (e.g., lysine or N-terminus) of IL-15.

[0135] Mono(methoxyPEG-N-butanamide) interleukin-15 can be prepared, for example, as described in Example 1 below. Mono(methoxyPEG-N-butanamide) interleukin-15 (which may be referred to herein as MPBA-IL15) is a mainly mono-PEGylated IL-15 classification having a single mPEG-N-butanamide moiety covalently attached to the lysine of IL-15 or the N-terminal alpha amine, accompanied by trace amounts of di-PEGylated and higher PEGylated IL-15 species (CAS Registry No. 2361317-09-9). Additional features of mono(methoxyPEG-N-butanamide) interleukin-15 are described, for example, in International Publication No. WO 2018 / 213341, the content of which is incorporated herein by reference.

[0136] Potency Bioassay: The potency of MPBA-IL15 was determined by phosphorylation of STAT5 in CTLL-2 cells, a mouse T lymphocyte cell line expressing the IL-15α subunit, using the pSTAT5 / total STAT5 multiplex assay (Meso Scale Discovery, MD). After receptor binding on CTLL-2 cells, downstream cell signaling activates STAT5 via phosphorylation to promote gene expression and induce cell proliferation. For the potency assay, phosphorylation of STAT5 signaling molecules downstream of the receptor upon ligand binding was evaluated to measure the biological response over a short time.

[0137] Reference materials, assay controls, and test samples were serially diluted to a final concentration of 10-fold using assay medium and then applied to a fixed number of cells for a 10-minute incubation at 37°C / 5% CO 2 2. Phosphorylated and total STAT5 were measured using the phosphorylation STAT5 / total STAT5 multiplex assay (Meso Scale Discovery, MD). A dose-dependent phosphorylated protein response curve (%) was generated by non-linear regression analysis using a 4-parameter model. Parallel line assay (PLA) software was used to evaluate the parallelism and significance of the regression and calculate the relative potency of the sample against the reference material within the same plate.

[0138] The purity of MPBA-IL15 was evaluated by reverse-phase HPLC using a HALO Protein C4 analytical column for UV detection at 214 nm, a temperature of 25°C, a flow rate of 1.0 mL / min, and a linear gradient of water / acetonitrile / trifluoroacetic acid (TFA).

[0139] The relative purity of MPBA-IL15 was evaluated by size exclusion HPLC using a Shodex Protein KW-803 column operating at 25°C with a flow rate of 0.5 mL / min. Chromatographic elution was performed using 15 mM sodium phosphate, pH 7.2 (in acetonitrile) with UV detection at 214 nm.

[0140] Ion exchange HPLC was also used to evaluate the relative purity of MPBA-IL15 by separating acidic and basic charge variants using an Agilent PL-SAX column operating at 40 °C at a flow rate of 1.0 mL / min. Elution was carried out using a linear gradient of bis-tris propane (pH 6.8): isopropyl alcohol and bis-tris propane (pH 6.8) (containing a solution of NaCl): isopropyl alcohol, and UV detection at 214 nm.

[0141] Example 1 Long-acting IL-15 receptor agonist, mono (methoxy PEG-N-butanamide) 40KD Preparation of Interleukin-15 [Chemical formula] Preparation 1: A 2.7 ml solution of rIL-15 (1.23 mg / ml, in PBS buffer, pH 7.4) was transferred to a small reaction vial. 300 μl of 0.6 M borate buffer at pH 8 was added to adjust the pH to pH 8. mPEG-SBA, 40 kDa (nominal average molecular weight), stored at -20 °C in nitrogen, was warmed to ambient temperature. A 10-fold excess of mPEG-SBA-40K (relative to the molar amount of IL-15) was dissolved in 2 mM HCl to form a 10% PEG reagent solution. This 10% PEG reagent solution was rapidly added to the IL-15 solution and mixed well. After the addition of mPEG-SBA-40K, the pH of the reaction mixture was determined and adjusted to pH 8 using conventional techniques. To enable the coupling of mPEG-SBA-40K to IL-15 (i.e., via the formation of a stable amide bond), the reaction solution was placed on a Slow Speed Lab Rotator for 1.5 hours to promote conjugate formation at room temperature. The reaction was stopped by the addition of a solution of glycine.

[0142] This reaction resulted in 40% mono-conjugate (i.e., having a single PEG moiety attached to IL-15), 24% di-conjugate (having two PEGs attached to IL-15), and 6% tri-conjugate (having three PEGs attached to IL-15) species. Approximately 30% unreacted IL-15 remained in the reaction mixture, although the reaction conditions were not optimized.

[0143] The mono-conjugate was separated / isolated by anion exchange chromatography using a Q Sepharose High Performance column and sodium phosphate buffer as the elution phase. The purified mono-mPEG-SBA40K-IL-15 conjugate (herein, mono-mPEG-butanamide-40K-IL-15 or mono-(methoxyPEG-N-butanamide) 40kD Interleukin-15, or mono-mPEG 40K -C4-amide-IL-15, also referred to as) was characterized by HPLC and SDS-PAGE. For the remaining examples, the purified mono-mPEG-SBA40K-IL-15 is referred to as conjugate 1.

[0144] As shown by the SDS gel, it was determined that the purified conjugate had a high level of purity and no detectable amount of unreacted IL-15 was present. Based on the HPLC plot, the purified mono-mPEG-SBA-40K-IL-15 composition contained less than about 10% (molar amount) of di- or higher level conjugates.

[0145] Using this synthetic method, conjugates such as mono-mPEG-SBA-10K-IL-15, mono-mPEG-SBA-15K-IL-15, mono-mPEG-SBA-20K-IL-15, mono-mPEG-SBA-25K-IL-15, mono-mPEG-SBA-30K-IL-15; mono-mPEG-SBA-45K-IL-15; mono-mPEG-SBA-50K-IL-15; and mono-mPEG-SBA-60K-IL-15 are prepared using mPEG-SBA having different nominal average molecular weights (e.g., having nominal average molecular weights of about 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 45 kDa, 50 kDa, 60 kDa, etc., respectively).

[0146] Preparation 2: An approximately 2 mg / ml solution of rIL-15 in buffer (50 mM sodium phosphate, 100 mM sodium chloride, 10% sucrose, pH 7.4) was transferred to each of two different reaction vessels (referred to herein as Composition 1 and Composition 2). To adjust the pH to 8.0, boric acid buffer at pH 8 (0.4 M or 0.6 M) was added. A 10-fold excess of mPEG-SBA-40K (mPEG-SBA, 40 kDa) diluted with 2 mM HCl (relative to the molar amount of IL-15) was added to each of the IL-15 solutions and mixed well. After the addition of mPEG-SBA-40K, the pH of the reaction mixture was determined to be pH 8 or adjusted by using additional boric acid buffer if necessary. The final concentration of rIL-15 in the reaction was targeted at 1 g / L using additional diluent if necessary (a buffer containing 50 mM sodium phosphate, 100 mM sodium chloride, 10% sucrose (pH 7.4) was used for Composition 1 and water was used for Composition 2). To enable the coupling of mPEG-SBA-40K to IL-15 (i.e., mainly through the formation of stable amide bonds), the reaction solution was mixed for 45 or 60 minutes for Composition 1 or Composition 2, respectively, to promote conjugate formation at room temperature. The reaction was stopped by the addition of a 71-fold excess solution of glycine at pH 8.0 for 30 minutes (relative to the molar amount of PEG initially added to the reaction). For Composition 1, the pH was adjusted to pH 7.0 by titration using 0.2 M phosphoric acid.

[0147] The resulting compositions were characterized by reverse-phase HPLC (RP-HPLC), SDS-PAGE, and ion-exchange HPLC (IEX-HPLC). The results of the RP-HPLC analysis are provided in Table 1A below.

[0148]

Table 1A

[0149] The results of the SEC-HPLC analysis are provided in Table 1B below.

[0150]

Table 1B

[0151] The results of the IEX-HPLC analysis are provided in Table 1B below.

[0152]

Table 1C

[0153] The prepared composition mainly contained mPEG-SBA-40K monomethoxypolyethylene glycol (mPEG)-ylated species, with less than about 10 mol% of PEG dimers (i.e., those having two PEG moieties attached to IL-15), and even smaller amounts of additional PEGylated species attached to IL-15 (i.e., those having three or more PEG moieties). The composition of mono-(methoxypolyethylene glycol-N-butanamide) interleukin-15, as otherwise described, will generally have at least about 80 mol% of monomethoxypolyethylene glycol (mPEG)-ylated interleukin-15 species (based on all interleukin-15 species in the resulting composition, including non-modified IL-15 and other IL-15-containing species, such as di-PEGylated IL-15 and larger ones), and preferably can have at least about 90 mol% of monomethoxypolyethylene glycol (mPEG)-ylated interleukin-15 species, along with less than about 10 mol% of other IL-15 species. In some embodiments, the mono-(methoxypolyethylene glycol-N-butanamide) interleukin-15 composition contains less than about 5 mol% of PEG dimers (di-PEGylated IL-15 having two methoxypolyethylene glycol-N-butanamide moieties attached to IL-15) and less than about 5 mol% of all other additional PEGylated species.

[0154] Two additional compositions of mono-(methoxypolyethylene glycol-N-butanamide) interleukin-15 were prepared and analyzed. A summary of the analysis results is provided in Table 1D below.

[0155] MPBA-IL15 can be formulated for further use as a solution containing MPBA-IL15 at a concentration of 1 mg / mL (protein-based) in 10 mM potassium phosphate, 260 mM trehalose, and 0.02 w / v% polysorbate 20 (pH 6.8).

[0156]

Table 1D

[0157] Example 2 In Vitro Study of Mono(methoxyPEG-N-butanamide)Interleukin-15 on Phosphorylation and Proliferation of CAR T Cells The in vitro effects of mono(methoxyPEG-N-butanamide)interleukin-15 on human CD19 CAR T cells were investigated as described below.

[0158] For the in vitro study, CAR T cells were incubated with or without CD19 antigen and with mono(methoxyPEG-N-butanamide)interleukin-15 (0 - 100 ng / mL). STAT5 phosphorylation and CFSE dilution were evaluated by flow cytometry.

[0159] IL15Rα expression was measured by flow cytometry as shown in Figure 3A (CD8, green solid line, right end) for CD8 CAR T cells and in Figure 3B (CD4, blue solid line, right end) for CD4 CAR T cells. Also shown in each figure are the FMO (gray-filled) and isotype (dashed line) controls. CD8 and CD4 CAR T cells express IL-15Rα.

[0160] The dose-dependent phosphorylation of STAT5 in response to mono-(methoxyPEG-N-butanamide) interleukin-15 (MPBA-IL15) for both CD8 CAR T cells and CD4 CAR T cells is shown in FIGS. 3C and 3D, respectively. CAR T cells were stimulated with various concentrations of MPBA-IL15 or IL-15 for 20 minutes. The EC 50 values (ng / ml) for CD8 and CD4 CAR T cells in response to IL-15 or mono-(methoxyPEG-N-butanamide) interleukin-15 in the STAT-5 phosphorylation assay are summarized below:

[0161] [Table 2]

[0162] The proliferation of CAR T cells labeled with CFSE and incubated with various concentrations of MPBA-IL15 or IL-15 for 4 days was also analyzed by flow cytometry. The results are shown in FIGS. 3E (CD8 CAR T cells) and 3F (CD4 CAR T cells). The EC50 values are summarized below.

[0163] [Table 3]

[0164] For FIGS. 3C, D, E, and F, squares correspond to IL-15 and circles correspond to MPBA-IL15.

[0165] As described above, in vitro, mono-(methoxyPEG-N-butanamide) interleukin-15 dose-dependently induces STAT5 phosphorylation and antigen-dependent proliferation of both CD8 CD19 CAR T cells and CD4 CD19 CAR T cells.

[0166] Example 3 Study of mono(methoxyPEG-N-butanamide)interleukin-15 on the efficacy of CD19 CAR T cell immunotherapy in a preclinical mouse lymphoma model Mono(methoxyPEG-N-butanamide)interleukin-15 (e.g., as described in Example 1 above) retains binding affinity for IL-15Rα, exhibits reduced clearance, and provides persistence of pharmacodynamic response. The effect of mono(methoxyPEG-N-butanamide)interleukin-15 on human CD19 CAR T cells in an in vivo xenograft B cell lymphoma model was investigated in experiments as described below.

[0167] General method: For in vivo studies, NSG mice were stably transduced intravenously with 5×10 5 individual Raji lymphoma cells expressing firefly luciferase on day -7 (D-7), followed by injection of subtherapeutic doses (0.8×10 6 ) of CAR T cells (1:1 CD4:CD8) on D0, either alone or in combination with mono(methoxyPEG-N-butanamide)interleukin-15 (0.03, 0.10, or 0.30 mg / kg, intravenous injection) initiated on D-1, 7, or 14 and administered weekly. Tumor-free mice were re-challenged with Raji cells on D38. Tumors were evaluated weekly by bioluminescence imaging of the mice. The results are shown in Figure 10.

[0168] As shown in Figure 4A (average tumor luminance versus days after CAR T cell administration for various treatment groups), treatment with 0.10 mg / kg and 0.30 mg / kg of mono-(methoxyPEG-N-butanamide) interleukin-15 in combination with CAR T cells resulted in a decrease in tumor burden and eradication of Raji lymphoma in NSG mice compared to CAR T cell therapy alone. In this study (Study A), NSG mice bearing Raji received a sub-therapeutic dose of CAR T cells on D0, followed by 0.030, 0.10, or 0.30 mg / kg of mono-(methoxyPEG-N-butanamide) interleukin-15 starting on D6 and then weekly (D13, D20, D27, D33, etc.). The treatment regimen is shown in Figure 4B.

[0169] In a further study (Study B), NSG mice bearing Raji received CAR T cell infusion on D0; 0.30 mg / kg of mono-(methoxyPEG-N-butanamide) interleukin-15 was administered on D-1, D7, or D14 and then weekly thereafter (5 mice / group). See Figure 7B. Mice were bled weekly and CD8 and CD4 CAR T cells were identified by flow cytometry (Figure 5A and 5B, respectively). Tumor burden was evaluated by weekly bioluminescence imaging (average tumor luminance versus days after CAR T cell administration) and survival (Figure 7A) as shown in Figure 6.

[0170] The results of this preclinical study further showed that mono-(methoxyPEG-N-butanamide) interleukin-15 in combination with CAR T cells resulted in an increase in CAR T cells in the blood, a decrease in tumor burden, and an increase in survival of NSG mice bearing Raji lymphoma.

[0171] For the mice in the 0.30 mg / kg mono(methoxyPEG-N-butanamide) interleukin-15 dosing group of Study A, the mice were euthanized at D8, 11, 14, 21, and 28 after CAR T cell infusion. Single cell suspensions were prepared from the bone marrow and CAR T cells; for the CD8 CAR T cell and CD4 CAR T cell suspensions, the total cell number (Figures 8A, 9A), Ki67 expression (Figures 8B, 9B), and PD1 and TIM3 expression (Figures 8C, 9C) were evaluated by flow cytometry, respectively. The graphs show the mean ± SEM.

[0172] As shown in Figures 8A and 9A, mice treated with the exemplary combination immunotherapy had an increased absolute number of CAR T cells in the bone marrow. CAR T cell therapy in combination with MPBA-IL-15 resulted in an increased accumulation and proliferation of CAR T cells in the bone marrow of mice bearing Raji, and a decrease in the extended dual expression of PD1 and TIM3 (Figures 8C, 9C).

[0173] In vivo injection of mono(methoxy PEG-N-butanamide) interleukin-15 starting at D-1, 7, or 14 increased the peak number of CAR T cells in the blood. Raji cells were eliminated from the bone marrow by D14 in mice receiving CAR T cells and D7 mono(methoxy PEG-N-butanamide) interleukin-15 (see, in particular, the 0.30 mg / kg dosing group), but not in mice receiving CAR T cells alone. In mice receiving CAR T cells together with mono(methoxy PEG-N-butanamide) interleukin-15, superior mono(methoxy PEG-N-butanamide) interleukin-15 dose-dependent tumor control and survival were observed compared to CAR T cells or mono(methoxy PEG-N-butanamide) interleukin-15 alone. Without intending to be limiting in any way, based on this experimental setting, the benefit of combination therapy in this preclinical model appeared to be greater when administration of mono(methoxy PEG-N-butanamide) interleukin-15 was initiated around D7. Residual CAR T cells in mice treated with mono(methoxy PEG-N-butanamide) interleukin-15 rejected re-challenge with Raji tumor cells administered after 5 weeks from CAR T cell injection and later.

[0174] In this lymphoma model, administration of mono(methoxy PEG-N-butanamide) interleukin-15 was found to improve the anti-tumor efficacy and kinetics of the administered CD19 CAR T cells. More specifically, it showed superior efficacy compared to CAR T cells alone, and the combination of CAR T cells and mono(methoxy PEG-N-butanamide) interleukin-15 significantly reduced the tumor burden, exerted sustained tumor control, and in some cases eradicated Raji lymphoma in NSG mice. In contrast, tumor progression was observed in the CAR T cell monotherapy group. See Figure 4A.

[0175] More specifically, 100% of the mice treated with mono-(methoxyPEG-N-butanamide) interleukin-15 (0.03 mg / kg) / CAR T cells survived 70 days after tumor injection, compared to none of the mice receiving the vehicle control surviving beyond day 14 and none of the mice treated with CAR T cells alone surviving beyond day 59. The results are shown in Figure 4A (results of bioluminescence imaging) and also in Figure 7A (survival) for mice treated with the 0.30 mg / kg dose of MPBA-IL15. Furthermore, mice previously treated with mono-(methoxyPEG-N-butanamide) interleukin-15 and CAR T cells were able to reject Raji tumor re-challenge, which supported CAR T cell persistence and potentially long-term memory CAR T formation; the prominent results are shown in Figure 10, which shows that long-acting interleukin-15 agonists such as MPBA-IL-15, when administered in combination with CAR-T cell therapy, not only significantly reduce the tumor burden but also demonstrate eradication ability against Raji-lymphoma.

[0176] Example 4 Study of mono-(methoxyPEG-N-butanamide) interleukin-15 on the efficacy of ROR1 CAR T cell immunotherapy in a preclinical murine ROR1 lung tumor model Kras LSL-G12D / + p53 f / f A cohort of mice (n = 5 - 6 / group) was intratracheally infected with 3 × 10 4 pfu of Cre-ffluc-hROR1 lentivirus to induce the development of ROR1+ lung tumors. At 12 and 15 weeks post-infection, the mice were treated with 100 mg / kg cyclophosphamide for lymphodepletion and 6 × 10 6Individual ROR1 CAR T cells or control T cells (CD8:CD4 at a 1:1 ratio) were adoptively transferred intravenously. ROR1 (receptor tyrosine kinase-like orphan receptor 1) is expressed in a number of malignancies, including subsets of non-small cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC). Mice were given 5×10 4 IU of IL-2 intraperitoneally every other day for 8 days to assist in the engraftment of the transplanted T cells. Starting on the day of T cell transplantation, a subset of mice was treated intravenously with 0.33 mg / kg MPBA-IL15 every 7 days. The preclinical treatment protocol is shown in Figure 11.

[0177] Tumor burden was quantified by obtaining serial 1-mm images across the entire lung and summing the tumor area across all images to quantify tumor volume. Seventeen weeks after infection, all mice were euthanized and the entire lungs were analyzed by flow cytometry and immunohistochemistry. To distinguish lung tumor-infiltrating cells from contaminating cells in the circulating blood, mice were intravenously injected with a PE-conjugated anti-CD45 antibody 5 minutes prior to euthanasia to label all immune cells in the circulating blood and enable the determination of PE - cells as non-vascular lung parenchymal cells. Lungs were analyzed by IHC staining for CD8a and CD8 infiltration into tumors; staining was quantified using HALO software

[0178] Results are shown in Figure 12A (change in tumor volume (%): control T cells (rectangles), control T cells and MPBA-IL15 (▲), ROR1 CAR T cells (▼), and ROR1 CAR T cells and MPBA-IL15 (◇)); Figure 12B (ROR1 Change in tumor volume (percent) over weeks post-infection for individual mice treated with CAR T cell monotherapy (18.5% regression for the group); Figure 12C (Change in tumor volume (percent) over weeks post-infection for individual mice treated with dual combination therapy of ROR1 CAR T cells and MPBA-IL-15 (44.4% regression for the group); Figure 13A (CD8 CAR T cell frequency expressed as the percentage of viable cells in the spleen and tumor for various treatment groups, respectively), Figure 13B (CD8 cell frequency expressed as the percentage of viable cells in the spleen and tumor for various treatment groups, respectively), and Figures 14A, B, C, and D (IHC staining showing that MPBA-IL-15, a long-acting IL-15 agonist as an example, enhances ROR1 CAR T cell trafficking in the lung in this preclinical ROR1 lung cancer model).

[0179] The foregoing examples show that administration of MPBA-IL15 significantly improves the anti-tumor efficacy and kinetics of CD19 CAR T cells in the treatment of subjects with cancer and also enhances the trafficking and persistence of ROR1 CAR T cells in cancerous lung tissue; thus, the present disclosure provides a novel and uniquely advantageous immunotherapy approach for treating patients with cancer by administering CAR T cell therapy in combination with a long-acting IL-15 agonist such as MPBA-IL15.

[0180] Example 5 CAR-T cell number and intracellular protein expression in CD8 CAR T cells treated in vitro with mono(methoxyPEG-N-butanamide)interleukin-15 CD8 CAR T cells were generated from healthy donors. On day 15, CAR T cells were co-cultured with irradiated K562-CD19+ or K562-CD19- cells. The cells were either untreated or treated with mono(methoxyPEG-N-butanamide) interleukin-15 at a concentration of (1 ng / ml, 10 ng / ml, or 30 ng / ml). IFNγ and TNFα production was analyzed by Luminex 24 hours after co-culture. The CAR T cell number, as well as the intracellular expression of bcl-2 and activated caspase 3, was determined after 5 days of co-culture. The expression of bcl-2 and activated caspase 3 was determined by flow cytometry. The results are shown in FIGS. 15A, 15B, and FIGS. 16A-C.

[0181] FIG. 15A provides the expression of IFNγ in pg / ml in CD8 CAR T cells co-cultured with irradiated K562-CD19+ or K562-CD19- cells and untreated or treated with MPBA-IL15 at a concentration of 1 ng / ml, 10 ng / ml, or 30 ng / ml.

[0182] FIG. 15B provides the expression of TNFα in pg / ml in CD8 CAR T cells co-cultured with irradiated K562-CD19+ or K562-CD19- cells and untreated or treated with MPBA-IL15 at a concentration of 1 ng / ml, 10 ng / ml, or 30 ng / ml.

[0183] FIG. 16A shows the CAR T cell proliferation (as fold proliferation) for CD8 CAR T cells co-cultured with irradiated K562-CD19+ or K562-CD19- cells and untreated or treated with MPBA-IL15 at a concentration of 1 ng / ml, 10 ng / ml, or 30 ng / ml.

[0184] Figures 16B and 16C provide the expression of bcl-2 (in bcl-2 MFI) and caspase 3 (as activated caspase 3 (%)), respectively, in CD8 CAR T cells co-cultured with irradiated K562-CD19+ or K562-CD19- cells and not treated with MPBA-IL15 or treated with MPBA-IL15 at concentrations of 1 ng / ml, 10 ng / ml, or 30 ng / ml.

[0185] From these results, it can be seen that in vitro treatment of CAR T cells with mono(methoxyPEG-N-butanamide)interleukin-15 increases antigen-specific CD8 CAR T cell production and proliferation and also enhances survival.

[0186] Example 6 Protein expression in CAR T cells after administration of a combination of CAR T cells and mono(methoxyPEG-N-butanamide)interleukin-15 in a preclinical mouse lymphoma model NSG mice were injected weekly with Raji lymphoma cells on D-7, CAR-T cells on D0, and mono(methoxyPEG-N-butanamide)interleukin-15 (0.3 mg / kg) as described in detail in Example 3 above starting on D7. Mice were euthanized on D8, 11, 14, 21, and 28 after CAR T cell injection. Single cell suspensions were made from bone marrow (2 femurs and 2 tibias per mouse). Protein expression (bcl-2, CD45RA, and CCR7) was analyzed by flow cytometry.

[0187] For both CD8 CAR T cells (Figure 17A) and CD4 CAR T cells (Figure 17B), the Bcl-2 expression in CAR T cells determined at D8 after injection is shown in a histogram (gray = mice with CAR T cells only, red and blue = mice administered CAR T cells and mono(methoxyPEG-N-butanamide)interleukin-15 (MPBA-IL15)).

[0188] For both CD8 CAR T cells (Figure 18A) and CD4 CAR T cells (Figure 18B), Bcl-2 expression in CAR T cells, determined at D8 after injection, is also shown in a bar graph (black = mice with CAR T cells only, red = mice administered with CAR T cells and mono(methoxy PEG-N-butanamide) interleukin-15 (MPBA-IL15)).

[0189] The expression of memory markers CD45RA and CCR7 in CAR T cells is shown in Figures 19A - 19D, where Figure 19A relates to protein expression in CD8 CAR T cells of mice administered with CAR T cells only; Figure 19B relates to protein expression in CD8 CAR T cells of mice administered with CAR T cells and mono(methoxy PEG-N-butanamide) interleukin-15 (MPBA-IL15), Figure 19C relates to protein expression in CD4 CAR T cells of mice administered with CAR T cells only; Figure 19D relates to protein expression in CD4 CAR T cells of mice administered with CAR T cells and mono(methoxy PEG-N-butanamide) interleukin-15 (MPBA-IL15), where expression data are provided for CD5RA-CCR7- (orange), CD5RA+CCR7- (green), and CD5RA-CCR7+ (red). The graphs show mean ± SEM.

[0190] CAR-T cells treated with mono(methoxy PEG-N-butanamide) interleukin-15 (MPBA-IL15), and CAR-T cells recovered from mice treated with a combination of CAR-T cell therapy and MPBA-IL15, show increased proliferation and survival rates both in vitro and in vivo, which may be due in part to increased expression of bcl-2.

[0191] Example 7 Clinical Study A Phase 1b / 2 Open-Label, Multicenter Dose Escalation and Expansion Study of Mono(MethoxyPEG-N-Butanamide)Interleukin-15 in Combination with CD19-Directed CAR-T Therapy in Patients with B-Cell Non-Hodgkin Lymphoma This is a Phase 1b / 2 open-label, multicenter dose escalation and expansion study of mono(methoxyPEG-N-butanamide)interleukin-15 (MPBA-IL15) in combination with CD19+ CAR-T in patients with diffuse large B-cell lymphoma (DLBCL). This study is divided into a screening period, a treatment period, an end-of-treatment (eot) period, and a long-term follow-up period.

[0192] The starting dose of MPBA-IL-15 for cohort 1 will be 1.5 μg / kg. Patients will receive IV MPBA-IL-15 in 21-day cycles starting on day 1 of cycle 1.

[0193] The MPBA-IL-15 drug product is provided as a sterile white to off-white lyophilized powder. The MPBA-IL-15 drug product is formulated in 10 mM potassium phosphate, 260 mM trehalose, 0.02% (w / v) polysorbate 20 (pH 6.8) with approximately 1.0 mg / mL of recombinant human IL-15 (rhIL-15). Each vial of the MPBA-IL-15 drug product contains 1.1 mg of rhIL-15 equivalent. This includes an excess of 0.1 mg to ensure a certain withdrawal of the label claiming an amount of 1.0 mg after reconstitution.

[0194] Treatment Period Dose Escalation (Phase 1b): Patients who have received one of two commercially available CD19-directed chimeric antigen receptor T-cell (CD19 CAR-T) therapies (Kymriah™ (tisagenlecleucel) or Yescarta® (axicabtagene ciloleucel)) that meet the safety selection criteria will receive intravenous (IV) MPBA-IL-15. During dose escalation, MPBA-IL-15 will be administered to patients approximately 14 days or 7 days after a single administration of CD19 CAR-T infusion (depending on the assigned cohort). Treatment with MPBA-IL-15 will be given every 21 days (i.e., every 3 weeks [q3w]) for up to 8 cycles (6 months), or until evidence of disease progression, unacceptable toxicity, patient withdrawal, investigator discretion, or a decision by the study sponsor to stop the study. Patients who demonstrate clinical benefit based on the investigator's judgment may continue treatment with the approval of the Medical Monitor.

[0195] Dose Expansion (Phase 2): After determination of the recommended Phase 2 dose (RP2D) of MPBA-IL-15 with either CD19 CAR-T product, the RP2D dose will be further investigated in an expanded cohort during Phase 2. Treatment with MPBA-IL-15 will be given every 21 days (i.e., every 3 weeks) for up to 8 cycles (6 months), or until evidence of disease progression, unacceptable toxicity, patient withdrawal, investigator discretion, or a decision by the study sponsor to stop the study. Patients who demonstrate clinical benefit based on the investigator's judgment may continue treatment with the approval of the Medical Monitor.

[0196] Primary Objectives: Phase 1b: (i) To evaluate the safety and tolerability of MPBA-IL-15 after CD19 CAR-T therapy. (ii) To define the maximum tolerated dose (MTD) or RP2D and optimal dosing period of MPBA-IL-15 after CD19 CAR-T administration.

[0197] Phase 2: Evaluate the efficacy of MPBA-IL-15 after CD19 CAR-T therapy by assessing the complete response rate (CRR) at 6 months, based on the Lugano classification (Cheson BD, Fisher RI, Barrington SF, et al. Recommendations for initial evaluation, staging, and response assessment of Hodgkin and non-Hodgkin lymphoma: the Lugano classification. J Clin Oncol. 2014;32(27):3059).

[0198] Secondary objectives: Phase 1b and Phase 2: (i) Evaluate the overall response rate (ORR) of MPBA-IL-15 in combination with CD19 CAR-T therapy (ii) Evaluate the progression-free survival (PFS) of MPBA-IL-15 in combination with CD19 CAR-T therapy (iii) Evaluate the overall survival (OS) of MPBA-IL-15 in combination with CD19 CAR-T therapy (Phase 2 only) (iv) Evaluate the duration of response (DOR) of MPBA-IL-15 in combination with CD19 CAR-T therapy (v) Characterize the pharmacokinetics (PK) of NKTR-255 in combination with CD19 CAR-T therapy (vi) Characterize the pharmacodynamic (PD) effects of NKTR 255 in combination with CD19 CAR-T therapy (vii) Evaluate the PD effects of CD19 CAR-T cells, including the duration of in vivo persistence of adoptively transferred T cells and the phenotype of remaining T cells (viii) Evaluate the immunogenicity of MPBA-IL-15

[0199] Exploratory objectives: (i) Evaluate the event-free survival (EFS) of MPBA-IL-15 in combination with CD19 CAR-T therapy (ii) Evaluate the association between anti-tumor activity in tumors and blood and immune cells. (iii) Evaluate the transport of adoptively transferred T cells to the bone marrow or other tumor sites and their in vivo function. (iv) Characterize changes from baseline in cytokine levels and immune cell populations.

[0200] Study population: Adults aged 18 years or older who are receiving CD19 CAR-T cells for the treatment of relapsed / refractory (R / R) B-cell non-Hodgkin lymphoma (B-NHL) after two or more lines of systemic therapy, including diffuse large B-cell lymphoma (DLBCL) (non-specific), primary mediastinal large B-cell lymphoma (PMBCL; only Yescarta), high-grade B-cell lymphoma, and DLBCL arising from follicular lymphoma.

[0201] Number of patients (planned): Phase 1b: Approximately 55 patients will be enrolled. Phase 2: Approximately 60 patients will be enrolled.

[0202] Number of study sites: Phase 1b: Approximately 5 North American sites Phase 2: Approximately 5 North American sites

[0203] Study design: This study is a non-blind, multi-center Phase 1b / 2 study consisting of a dose-escalation (Phase 1b) and dose-expansion (Phase 2) part.

[0204] Phase 1b (dose escalation) Patients who have received a US FDA-approved CD19 CAR-T cell (Yescarta or Kymriah) that meets the safety selection criteria will receive IV MPBA-IL-15 q3w starting approximately 14 days or 7 days after CD19 CAR-T infusion (depending on the assigned cohort). During dose escalation (Phase 1b), at least 3 patients in each of up to 5 cohorts will be given escalating doses of MPBA-IL-15 after CD19 CAR-T cell infusion. A sample dose escalation plan for MPBA-IL-15 with CD19 CAR-T is provided in the table below. The first patient in each escalating MPBA-IL-15 dose cohort (sentinel patient) will be monitored for safety and tolerability for 21 days after the first administration of MPBA-IL-15, after which other patients in the same cohort will be dosed.

[0205]

Table 4

[0206] MPBA-IL-15 will be tested in a sequential combination that initially starts with Yescarta therapy. This study will start with a starting dose of 1.5 μg / kg IV of MPBA-IL-15 administered 14 days after Yescarta infusion (cohort A). After establishing safety and tolerability at three dose levels of MPBA-IL-15 administered 14 days (±3 days) after Yescarta, the next cohorts, Kymriah (CAR-T infusion 14 days later; cohort B) and Yescarta (CAR-T infusion 7 days later, cohort C) will be started in parallel at the confirmed safe dose levels. The doses to be tested can be tapered down to the previously tested dose if the 7-day regimen has safety or tolerability issues observed at the starting dose level. The 7-day regimen will be based on the observed safety and tolerability of each individual product in the 14-day regimen. Dose escalation will consist solely of MPBA-IL-15 in the 7-day regimen until the MTD or RP2D is established. If the 7-day regimen is found to be intolerable, dose escalation of MPBA-IL-15 in the 14-day regimen can be started again.

[0207] During the escalation period of the study regarding dose level selection and determination of the MTD, a two-parameter Bayesian logistic regression model (BLRM) using the principle of escalation with overdose control (EWOC) (Neuenschwander B, Branson M, Gsponer T. Critical aspects of the Bayesian approach to phase I cancer trials. Stat Med. 2008 Jun 15;27(13):2420-39) will be used. The MTD will be determined when at least 6 patients are evaluated at a dose and the posterior probability of the targeted toxicity for that dose is at least 70%. The MTD will be determined based on the criteria outlined in Section 5.8. Additional cohorts can also be opened to further investigate the MTD.

[0208] The RP2D of MPBA-IL-15 in combination with CD19 CAR-T will be selected at a dose not exceeding the final recommendation from the dose escalation, and will be based on a review of all available data on the safety, PK, PD, and optimal biological response of MPBA-IL-15. To refine the RP2D, additional RP2D patients can be enrolled, and to determine the RP2D, at least six patients (including any patients from the dose escalation) administered at the selected RP2D will be required.

[0209] Additional rules regarding dose escalation in the 1b phase are as follows: - Dose escalation within a patient will not be permitted. - Enrollment into a new cohort using escalating doses of MPBA-IL-15 cannot be initiated until the dose-limiting toxicity (DLT) time frame has elapsed after the first administration of MPBA-IL-15 to the last patient in the previous cohort. The DLT time frame is 21 days after MPBA-IL-15 administration. - Escalation to a higher dose will only be done if there is experience at that dose in the FIH study (MPBA-IL-15-002). - For the dose escalation cohort, before opening the dose escalation to the next cohort, the sponsor medical monitor and the Safety Review Committee (SRC) will jointly evaluate the safety. - The dose level of MPBA-IL-15 for a given cohort can be decreased according to the severity, duration, and frequency of toxicity observed at the previously tested dose levels.

[0210] The decision to determine the RP2D of MPBA-IL-15 after CD19 CAR-T infusion can be made at any given dose level or start date that does not reach the MTD, based on safety, PK, PD, or optimal biological response.

[0211] Phase 2 (dose expansion) In the second phase of this study, after the RP2D is established for each individual CD19 CAR-T product, enrollment into the dose escalation cohort will be initiated. The specific selection of CD19 CAR-T products and the determination of the schedule for the second phase will be based on a review of all available data on the safety, PK, PD, and optimal biological response of MPBA-IL-15 after CAR-T infusion in the 1b phase. Patients will receive IV MPBA-IL-15 at the RP2D and schedule determined in the 1b phase, 14 or 7 days after CD19 CAR-T infusion. Treatment with MPBA-IL-15 will be repeated every 21 days (i.e., every 3 weeks) for up to 8 cycles (6 months).

[0212] Major eligibility criteria Eligibility will be determined within 1 month prior to leukapheresis for CD19 CAR-T cell production. If intensive bridging chemotherapy or radiotherapy is administered, eligibility criteria should be re-evaluated prior to lymphodepletion. · Male or female patients, ≥ 18 years old on the date of signing the informed consent form (ICF) · Eligible for CD19 CAR-T cell therapy as a product · Definitive diagnosis of B-NHL, including DLBCL (non-specific), PMBCL (for Yescarta only), high-grade B-cell lymphoma; and DLBCL arising from follicular lymphoma · Relapsed / refractory (R / R) disease defined as detectable disease after ≥ 2 lines of therapy including anthracycline, and having failed autologous hematopoietic stem cell transplantation (ASCT) or being ineligible for or not consenting to ASCT · Measurable fluorodeoxyglucose (FDG)-avid nodular and / or extranodal disease by Lugano classification (Cheson, 2014, ibid.) accurately measurable at ≥ 1.5 cm in at least 1 dimension · Median life expectancy > 30 days · Acceptable organ function as defined below: Adequate lung function defined as dyspnea grade ≤ 1 and oxygen saturation ≥ 92% (in room air). If these parameters are not met, patients with FEV1 ≥ 50% of predicted value and diffusing capacity of the lung for carbon monoxide (DLCO; corrected) ≥ 40% of predicted value in pulmonary function test (PFT) are eligible at the discretion of the treating physician. Adequate cardiac function defined as left ventricular ejection fraction (LVEF) ≥ 45% by a cardiologist, or LVEF 40 - 44% and clearance Adequate renal function defined as follows: - Serum creatinine ≤ 1.5 × upper limit of normal (ULN), or eGFR ≥ 60 mL / min / 1.73m 2 Adequate liver function defined as follows - Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 3 × ULN - Bilirubin ≤ 2.0 mg / dL (except for patients with Dubin - Johnson syndrome); patients with Dubin - Johnson syndrome can be included if their total bilirubin is ≤ 3.0 × ULN and direct bilirubin is ≤ 1.5 × ULN Adequate bone marrow reserve (without transfusion) defined as follows: - Absolute neutrophil count (ANC) > 1000 / mm 3 - Absolute lymphocyte count (ALC) ≥ 300 / mm 3 - Platelets ≥ 50,000 / mm 3 Hemoglobin > 8.0 g / dL

[0213] Safety eligibility evaluated before the first and subsequent MPBA - IL - 15 administrations Patients are eligible for MPBA - IL - 15 infusion if they meet the following criteria: 1. Have received CD19 CAR - T infusion 2. Do not have grade ≥ 1 cytokine release syndrome (CRS) (body temperature ≥ 38.0°C) persisting on the day of MPBA - IL - 15 infusion There is no grade 4 CRS within 96 hours before MPBA-IL-15 injection. There is no persistent grade ≥2 neurotoxicity on the day of MPBA-IL-15 injection. There is no previous grade ≥3 neurotoxicity during any period of >48 hours at any time before MPBA-IL-15 injection. There is no intervention with tocilizumab and / or dexamethasone within 48 hours before MPBA-IL-15 injection. There is no active, severe, and uncontrolled infection. There is no contraindication according to the investigator's assessment. The patient has appropriate organ function before all administrations of MPBA-IL-15: a) AST and ALT levels ≤3×ULN; b) Total bilirubin level ≤3×ULN c) eGFR >30 mL / min d) DLCO >40% e) LVEF >45%

[0214] Test Product, Dosage, and Administration Route Reconstituted MPBA-IL-15 will be administered IV every 21 days (i.e., q3w). Reconstituted MPBA-IL-15 should be further diluted with commercially available injectable product or 0.9% normal saline. The final diluted solution will be infused over 30 ± 5 minutes.

[0215] The starting dose of MPBA-IL-15 will be 1.5 μg / kg (every 21 days).

[0216] Safety The safety assessment will be conducted by continuous re-evaluation of the following: · Incidence of adverse events (AE), including serious AEs (SAE) and immune-mediated AEs (imAE) · Clinical tests (blood and urine collection) · Vital signs · Electrocardiogram (ECG) · Physical examination ·Combined drug therapy DLT - Phase 1b only

[0217] Pharmacokinetics Blood samples for MPBA - IL - 15 PK analysis will be collected from all patients. Serial PK samples will be collected at multiple planned time points after each cycle of MPBA - IL - 15. Plasma concentrations of MPBA - IL - 15 will be measured for each PK sample using a validated method. Pharmacokinetic parameters such as the maximum plasma concentration (C max max), area under the concentration - time curve (AUC), clearance (CL), volume of distribution (Vd), and half - life (t1 / 2) will be estimated from plasma concentration - time data if possible.

[0218] Biomarkers The PD effects of MPBA - IL - 15 in combination with CD19 CAR - T based on whole body and tumor tissue (blood and bone marrow) will be investigated.

[0219] Characterization and monitoring of genetically modified CD19 CAR - T cells will be performed in peripheral blood and bone marrow samples by quantitative polymerase chain reaction (qPCR) and flow cytometry before and during treatment with NKTR 255. Blood samples for whole body PD analysis will be collected from all patients before and during treatment to evaluate the effect of MPBA - IL - 15 on the number and activation of immune cell populations including, but not limited to, NK cells, CD8+ T cells, and CD8+ memory cells. Blood samples will also be collected before and during treatment to determine changes in cytokine levels and to profile changes in gene expression in response to MPBA - IL - 15.

[0220] If possible, fresh bone marrow biopsy material will be collected before, during, and after treatment according to the Schedule of Events for the characterization of tumor cells and immune system activation. The evaluation will include the evaluation of changes in tumor-specific protein markers and immune cell populations in the tumor microenvironment. If available, stored tumor tissue samples will be collected and can be analyzed similarly.

[0221] Efficacy: During screening for baseline evaluation and also to establish eligibility for measurable disease, whole body (from the base of the skull to the mid-thigh) 18F-FDG-positron emission tomography (PET) / computed tomography (CT) will be performed (SUV max ). Subsequent FDG-PET / CT for efficacy assessment according to the Lugano classification (Cheson, 2014, ibid.) will be performed at week 4, month 3 (immediately before cycle 5), then every 12 weeks until the subject discontinues the study, and also at the time of disease progression if possible. Tumor biopsy material will be obtained, if possible, at approximately baseline, during the first 4 weeks after CD19 CAR-T infusion, and at week 14; also at the discretion of the investigator at the end of treatment (EOT).

[0222] Statistical methods: Safety: Safety evaluations will include AEs, clinical laboratory tests, vital signs, physical examinations, and ECG (central adjudication). The incidence of DLT will be evaluated for each dose escalation cohort. All grade ≥3 treatment-emergent adverse events (TEAEs) will be summarized separately for each dose cohort by organ major category and preferred term in phases 1b and 2 of the study. TEAEs will be summarized by incidence, severity, and relationship to the study drug. Immune-mediated AEs (imAEs) will be summarized separately.

[0223] Clinical tests with a grade of ≧ 3 and vital sign abnormalities will be outlined by description for each dose cohort in the 1b and 2 phases of the study.

[0224] Efficacy: The efficacy evaluations of CRR and ORR at 6 months will be calculated with a 95% confidence interval (CI) based on a rigorous method. The Kaplan-Meier method will be used for the analysis of PFS, DOR, and OS. The CRR at 6 months based on the independent review committee (IRC) evaluation will be the primary efficacy evaluation item and will be outlined using the modified intention-to-treat analysis population. The CRR at 6 months based on the researcher's evaluation will also be evaluated.

[0225] The primary analysis for all efficacy evaluation items will be based on patients from the dose escalation part of this study and patients treated at the RP2D from the dose ramp-up part of this study.

[0226] Pharmacokinetics and Biomarkers: Pharmacokinetic parameters will be tabulated and outlined using descriptive statistics. Descriptive summaries for biomarkers will be inferred at each observation time. Changes in biomarkers from pre-dose to each observation time will also be evaluated using descriptive summaries. The present invention provides, for example, the following items. (Item 1) A method for treating a subject having cancer, (i) administering to the subject an adoptive cell immunotherapy composition comprising T cells modified to express a chimeric antigen receptor (CAR-T cells); (ii) Structure:

Chemical formula

Claims

[Claim 1] The invention described in the specification.

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