Methods for enhancing cellular immunotherapy

Abstract

Provided are methods and compositions directed to treating an individual with 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, e.g., a long-acting interleukin-15 receptor agonist.

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 April 5, 2019, U.S. Provisional Patent Application No. 62 / 861,858, filed June 14, 2019, U.S. Provisional Patent Application No. 62 / 898,473, filed September 10, 2019, and U.S. Provisional Patent Application No. 62 / 944,955, filed December 6, 2019, the contents of each of which are incorporated herein by reference.

[0002] The present application relates (among other things) to the field of immunotherapy, including 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 technology]

[0003] Novel therapeutic approaches are continually being developed to improve 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 therapeutic strategies designed to tailor immune responses to induce the patient's own immune system to fight cancer. Among current immunotherapeutic approaches, adoptive cell transfer therapy (also called ACT) has shown promise in treating certain types of cancer patients. Adoptive cell therapy involves the isolation and ex vivo expansion of tumor-specific lymphocytes to obtain a larger number of tumor-reactive effector T cells than can be achieved by simple vaccination. Tumor-specific T cells are infused into cancer patients to induce the patient's immune system to initiate tumor cell killing. Adoptive cell transfer has shown effective clinical outcomes, especially in metastatic melanoma (Dudley, ME, JR Underlich et al., J Clin Oncol 23(10):2346-2357(2005); Dudley, ME, JC Yang et al., J Clin Oncol 26(32):5233-5239(2008)). Adoptive cell transfer can 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). CARs are a type of synthetic receptor that can reprogram lymphocyte specificity and function (Sadelain, M., et al., Nature, 545, 25 May 2017, p. 423-431). CAR T cell therapy uses ex vivo engineered T cells transduced to express artificial receptors that redirect the specificity of T cells to targeted tumor-associated antigens (TAA) expressed on the tumor cell surface (June, CH, et al., Sci Transl Med 2015; 7(280): 280 ps7). Unlike naturally occurring T cells and T cell receptor engineered T cells, which recognize their cognate antigens in terms of specific major histocompatibility complexes (MHC), antigen recognition by CAR T cells is MHC-independent, thereby expanding the applicability of this cell-based mode of immunotherapy. The first generation of CAR T cells contained the single chain variable region (scFv) of a monoclonal antibody, a T cell receptor transmembrane domain, and the intracellular signaling domain of the CD3 zeta (CD3ζ) chain. Later iterations also utilized one or more costimulatory domains and / or a controllable on-off switch. CAR T cell therapy has advanced over time to provide engineered T cells with improved specificity and safety profiles.

[0005] Although CAR T cell-based therapy has been approved in the United States to treat diffuse large B-cell lymphoma, not all patients respond to CAR T cells, and the durability of the response remains limited. An additional challenge for CAR T cell therapy is the poor survival of transplanted cells. In relapsed or refractory large B-cell lymphoma, nearly 60% of patients relapse or fail and progress, with poor prognosis after failure (Nair, J., et al., Best Practice & Research Clinical Haematology 31 (2018) 293-298). Successful CAR T cell therapy outcomes, i.e., favorable and durable responses with complete remission rates at 6 months, depend at least in part 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 remarkable efficacy for many patients who initially responded to the treatment, but durability of response remains limited (Shah, N., et al., Frontiers in Oncology, 9 (March 2019) Art. 146). In addition, acute toxicities reported after CAR T cell therapy include cytokine release syndrome (CRS) and neurotoxicity (referred to as CAR-associated encephalopathy syndrome) (Neelapu, SS, et al., Nat Rev Clin Oncol 2018; 15 (10: 47-62). Other less frequently observed adverse side effects include hemophagocytic lymphohistiocytosis (HLH) / macrophage activation syndrome (MAS), anaphylaxis, and tumor lysis syndrome. Although considerable efforts have been expended to date in developing effective CAR T cell-based therapies, there remains a need to provide new and more effective immunotherapeutic CAR T cell strategies and associated treatment regimens that address one or more of the shortcomings of current therapies. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Dudley, ME, JR Wunderlich et al., J Clin Oncol 23(10):2346-2357(2005) [Non-Patent Document 2] Dudley, ME, JCYang et al., J Clin Oncol 26(32):5233-5239(2008) [Non-Patent Document 3] Sadelain,M.,et al.,Nature,545,25 May 2017,p.423-431 [Non-Patent Document 4] June,CH,et al.,Sci Transl Med 2015;7(280):280 ps7 [Non-Patent Document 5] Shah, N., et al.,Frontiers in Oncology,9(March 2019)Art.146 [Non-Patent Document 6] Neelapu,SS,et al.,Nat Rev Clin Oncol 2018;15(10:47-62) Summary of the Invention [Means for solving the problem]

[0007] The present disclosure therefore seeks to address these shortcomings and other needs by providing novel and efficient CAR T cell-based immunotherapies, such as immunotherapies that induce CAR T cell populations to adopt tumor-killing traits, that have, among other advantages, better survival and improved efficacy (to be described in more detail below).

[0008] In a first aspect, provided herein is a method comprising adoptive cell transfer into a subject with cancer in combination with administration of a long-acting IL-15 receptor agonist, as will be described in more detail herein. The present disclosure stems, at least in part, from the recognition that a combination therapeutic regimen comprising one or more cycles of adoptive cell therapy (e.g., by infusion of CAR T cells) and administration of a long-acting IL-15 receptor agonist as described herein, administered sequentially in any order or substantially simultaneously, can be particularly effective in treating cancer in certain subjects and / or can augment, enhance, or prolong the activity and / or number of the transplanted cells, or result in a measurable beneficial response to the cancer cells (e.g., stabilization, regression, shrinkage, necrosis, etc., as applicable), to an extent that is enhanced, preferably significantly enhanced, over any single immunotherapy approach alone.

[0009] In a second aspect, provided herein is a combination immunotherapy for treating a subject having cancer, comprising administering to a subject an adoptive cellular immunotherapy composition comprising T cells that have been modified to express a chimeric antigen receptor (CAR T cell); 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, e.g., CAR T cell therapy, for treating a subject having cancer, the method comprising providing to a subject having cancer an adoptive cell immunotherapy composition comprising T cells that have been modified to express a chimeric antigen receptor; and administering to the subject a long-acting IL-15 receptor agonist, where 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, should be considered both alone and in combination unless otherwise indicated.

[0012] In some embodiments, the adoptive cell transfer comprises administering an adoptive cellular immunotherapy composition comprising T cells that have been 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 expand natural killer (NK) cells. In yet one or more additional embodiments, the long-acting IL-15 receptor agonist supports CD8+ T cell survival and memory formation, e.g., without substantially inducing inhibitory regulatory T cells (T regs). In yet 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 aforementioned characteristics, namely (i) being effective to preferentially stimulate and expand NK cells, (ii) supporting CD8+ T cell survival and memory formation, e.g., without substantially inducing inhibitory regulatory T cells (T regs), and (iii) having IL-15 receptor alpha specificity.

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

[0015] In one or more embodiments relating to a 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 particular embodiments, (n) has a value of about 907 or about 909, on average.

[0016] For purposes of clarity, with respect to the order of administration (wherein the term "administering" is used herein to refer to delivery of the adoptive cellular immunotherapy composition or the 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. Furthermore, treatment of any component of the combination can include a single cycle of therapy or can include multiple cycles. That is, after an initial cycle of therapy comprising administration of an adoptive cellular immunotherapy composition comprising T cells that have been modified to express a chimeric antigen receptor, e.g., CD19-directed CAR T cells, and administration of a long-acting IL-15 agonist, additional rounds of therapy can comprise adoptive cell transfer, 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 transfer (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 hematological cancer, for example a recurrent or refractory malignancy.

[0019] In one or more related non-limiting embodiments, the cancer is lymphoma or leukemia. In one or more related embodiments, the cancer is selected from Hodgkin's and non-Hodgkin's 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 still some further embodiments, the cancer is multiple myeloma.

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

[0023] In some further embodiments of the methods provided herein, the methods result in a beneficial response to the treatment that is enhanced over the response to the treatment observed when administration is performed according to administration of an adoptive cellular 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.

[0025] Additional aspects and embodiments are described below and in the claims. [Brief description of the drawings]

[0026] [Figure 1] Provided are the amino acid sequence of an exemplary recombinant human IL-15 from E. coli (SEQ ID NO:1); an exemplary recombinant human IL-15 (SEQ ID NO:2) that includes a methionine at the beginning of the sequence for initiation of translation in E. coli, i.e., a single non-glycosylated polypeptide chain containing 115 amino acids with a molecular weight of 12.9 kDa; and an exemplary precursor form of IL-15 (SEQ ID NO:3). [Diagram 2] FIG. 1 is an illustration of a suitable CD19 CAR lentiviral construct used to transduce CD4 and CD8 T cells isolated from healthy donors as described in the Examples. [Figure 3-1] Figure 3 shows expression of IL-15Rα by human CD19 CAR T cells as measured by flow cytometry, as further described in Example 2. Expression by CD8 CAR T cells is shown in Figure 3A (solid line); IL-15Rα expression by CD4 CAR T cells is shown in Figure 3B (solid line). Both figures include FMO control (grey fill) and isotype control (dashed line). [Figure 3-2]Graph showing STAT5 phosphorylation (percent) for CD8 CAR T cells (Figure 3C) or CD4 CAR T cells (Figure 3D) versus the logarithm of concentration (ng / mL) after 20 minutes of stimulation with different concentrations of MPBA-IL15 (●) or IL-15 (■), as further described in Example 2, as determined by flow cytometry. [Figure 3-3] Graph showing proliferation (division (%)) of CD8 CAR T cells (Figure 3E) and CD4 CAR T cells (Figure 3F) labeled with CFSE and incubated for 20 minutes with various concentrations of MPBA-IL15 (●) or IL-15 (■), as further described in Example 2, as determined by flow cytometry. [Figure 4] (FIG. 4A) Graph of mean tumor brightness (p / s / cm2 / sr) versus time (days after CAR T cell infusion) as assessed in an in vivo xenogeneic B cell lymphoma model as described in Example 3 for tumor-bearing mice treated with CAR T cells alone (■) and in combination with different dosages of mono(methoxyPEG-N-butanamide) interleukin-15 (MPBA-IL15): 0.030 mg / kg (green ◇), 0.10 mg / kg (purple ◇), and 0.30 mg / kg (▼). PBS control is shown as a circle (●). (FIG. 4B) Illustrative treatment protocol for evaluating the efficacy of CD19 CAR T cell combination immunotherapy in a preclinical mouse lymphoma model as described in Example 3 (Study A). [Diagram 5]Graphs showing the number of CD8 CAR T cells (FIG. 5A) or CD4 CAR T cells (FIG. 5B) in the blood of lymphoma cell-bearing mice injected with CAR T cells (DO) followed by MPBA-IL15 (0.3 mg / kg) treatment (starting on day -1, 7, or 14, and weekly thereafter). Mice were bled weekly and CD8 and CD4 CAR T cells were identified by flow cytometry. The plot provides the number of cells / μl (blood) versus days after CAR T cell administration. Study groups included: tumor-bearing mice injected with MPBA-IL15 alone (●), tumor-bearing mice injected with CAR T cells alone (■), tumor-bearing mice treated with CAR T cells in combination with MPBA-IL15 starting on day -1 (▲), 7 (▼), or 14 (◇) (for up to 28 days after CAR T cell injection, as described in Example 3). [Figure 6] FIG. 13 is a graph assessing tumor burden by providing the mean tumor intensity (p / s / cm2 / sr) over time (days after CAR T cell infusion) as assessed in an in vivo xenograft B cell lymphoma model in tumor-bearing mice following infusion of MPBA-IL15 alone (●), CAR T cells alone (■), or CAR T cells in combination with MPBA-IL15 starting on day -1 (▲), day 7 (▼), or day 14 (◇) and weekly thereafter, as described in Example 3. [Figure 7-1] FIG. 13 is a graph showing percent survival of tumor-bearing mice treated with CAR T cells alone (blue line), MPBA-IL15 alone (black line), or CAR T cells in combination with MPBA-IL15 (0.3 mg / kg) starting on day -1 (purple line), day 7 (red line), or day 14 (green line) following CAR T cell infusion (day 0) as assessed in an in vivo xenograft B cell lymphoma model as described in Example 3. [Figure 7-2] An illustrative treatment protocol is provided to evaluate the efficacy of CD19 CAR T cell combination immunotherapy in a preclinical mouse lymphoma model, as described in Example 3 (Study B). [Figure 8-1] Figure 8A is a plot of the total number of CAR T cells, Ki67 expression, and PD1 and TIM3 expression, respectively, of CD8 CAR T cells from bone marrow of NSG mice bearing Raji lymphoma cells that were infused with CAR T cells on DO (CAR T cell monotherapy) or that received weekly injections of MPBA-IL15 starting on D7 in addition to infusion of CAR T cells, as described in Example 3. Figure 8A is a graph showing the total number of CD8 CAR T cells in bone marrow of tumor-bearing mice after infusion of CAR T cells alone (●) and in tumor-bearing mice that received CAR T cell therapy in combination with MPBA-IL15 (■). Figure 8B is a graph showing Ki67 expression (Ki67+(%)) in CD8 CAR T cells from bone marrow of tumor-bearing mice after CAR T cell monotherapy (●) and from tumor-bearing mice that received CAR T cells in combination with MPBA-IL15 (■). Figure 8C is a graph showing PD1 and TIM3 expression (PD1+TIM3+ (%)) in CD8 CAR T cells from bone marrow of tumor-bearing mice following CAR T cell infusion alone (●) (monotherapy) and from tumor-bearing mice receiving CAR T cell therapy in combination with MPBA-IL15 (■). [Figure 8-2] Same as above. [Figure 9-1]9A and 9B are plots of total number of CAR T cells, Ki67 expression, and PD1 and TM3 expression, respectively, of CD4 CAR T cells from bone marrow of NSG mice bearing Raji lymphoma cells that were infused with CAR T cells on DO (CAR T cell monotherapy) or further receiving weekly injections of MPBA-IL15 starting on D7, as described in Example 3. FIG. 9A is a graph showing total number of CD4 CAR T cells in bone marrow of tumor-bearing mice after infusion of CAR T cells alone (●) and in tumor-bearing mice that received CAR T cell therapy in combination with MPBA-IL15 (■). FIG. 9B is a graph showing Ki67 expression (Ki67+(%)) in CD4 CAR T cells from bone marrow of tumor-bearing mice after CAR T cell monotherapy (●) and from tumor-bearing mice that received CAR T cells in combination with MPBA-IL15 (■). Figure 9C is a graph showing PD1 and TIM3 expression (PD1+TIM3+ (%)) in CD4 CAR T cells from bone marrow of tumor-bearing mice following CAR T cell infusion alone (●) (monotherapy) and from tumor-bearing mice receiving CAR T cell therapy in combination with MPBA-IL15 (■). [Figure 9-2] Same as above. [Figure 10] Bioluminescence images are provided for representative time points for tumor-free mice previously treated with MPBA-IL-15 and CAR T cells, re-challenged with Raji tumor cells at D38, followed by weekly imaging to assess tumor burden. This figure shows that mice previously treated with an exemplary long-acting IL-15 receptor agonist, i.e., MPBA-IL-15, and CAR T cells, are able to reject Raji tumor re-challenge when assessed in a mouse lymphoma model. [Figure 11] An illustrative treatment protocol is provided to evaluate the efficacy of ROR1 CAR T cell combination immunotherapy with an exemplary long-acting IL-15 receptor agonist, i.e. MPBA-IL15, in a preclinical mouse lymphoma model as described in Example 4. [Figure 12-1]Figure 12A is a graph of the percent change in tumor volume for mice in different treatment groups in a mouse lymphoma model described in Example 4: control T cells (rectangles), control T cells with MPBA-IL15 (▲), ROR1 CAR T cells (▼), and ROR1 CAR T cells with MPBA-IL15 (◇); Figure 12B is a graph of the percent change in tumor volume versus weeks post-infection for individual mice treated with ROR1 CAR T cell monotherapy (18.5% regression for the group). Figure 12C is a graph of the percent change in tumor volume versus weeks post-infection for individual mice treated with ROR1 CAR T cell and MPBA-IL-15 dual combination therapy (44.4% regression for the group). [Figure 12-2] Same as above. [Figure 13] Figure 13A is a plot of CD8 CAR T cell frequency expressed as a percentage of viable cells in the spleen and tumor, respectively, for the treatment groups described in Example 4: control T cells (black), ROR1 CAR T cells (red), and ROR1 CAR T cells with MPBA-IL15 (blue); Figure 13B is a plot of total CD8 cell frequency expressed as a percentage of viable cells in the spleen and tumor, respectively, for the various treatment groups described in Example 4. [Figure 14] Images of lung tissue from mice in different treatment groups in the ROR1 lung model showing that MPBA-IL-15, an illustrative long-acting IL-15 agonist, enhances ROR1 CAR T cell trafficking and persistence in the lung. [Figure 15]Figure 15 shows protein expression in CD8 CAR T cells after treatment with MPBA-IL15 in vitro as described in Example 5. Figure 15A provides expression of IFNγ in pg / ml 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. Figure 15B provides expression of TNFα in pg / ml 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. [Figure 16-1] Figure 1 shows CAR T cell proliferation (as fold expansion) for 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, as described in Example 5. [Figure 16-2] Figure 2 provides expression of bcl-2 (in bcl-2 MFI) and activated (as caspase 3+ (%)) 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, respectively, as described in Example 5. [Figure 17]Figure 17 shows bcl-2 expression in CAR T cells from lymphoma-bearing mice treated with CAR T cells alone or in combination with MPBA-IL15, as described in detail in Example 6. Histograms show Bcl-2 expression in CAR T cells determined at D8 post-infusion for both CD8 (Figure 17A) and CD4 (Figure 17B) CAR T cells (grey = mice treated with CAR T cells only; red and blue = mice administered CAR T cells and mono(methoxyPEG-N-butanamide) interleukin-15 (MPBA-IL15)). [Figure 18] Also shown is bcl-2 expression in CAR T cells from mice with lymphoma treated with CAR T cells alone or in combination with MPBA-IL15, as detailed in Example 6. Bar graphs show Bcl-2 expression in CAR T cells determined at D8 post-infusion for both CD8 (Figure 18A) and CD4 (Figure 18B) CAR T cells (black = mice with CAR T cells only, red = mice receiving CAR T cells and mono(methoxyPEG-N-butanamide) interleukin-15 (MPBA-IL15)). [Figure 19] Expression of memory markers CD45RA and CCR7 in CAR T cells as described in Example 6. Figure 19A shows protein expression in CD8 CAR T cells of mice administered CAR T cells alone; Figure 19B shows protein expression in CD8 CAR T cells of mice administered CAR T cells and mono(methoxyPEG-N-butanamide) interleukin-15 (MPBA-IL15); Figure 19C shows protein expression in CD4 CAR T cells of mice administered CAR T cells alone; Figure 19D relates to protein expression in CD4 CAR T cells of mice administered CAR T cells and mono(methoxyPEG-N-butanamide) interleukin-15 (MPBA-IL15), where expression data is provided for CD5RA-CCR7- (orange), CD5RA+CCR7- (green), and CD5RA-CCR7+ (red). Graphs show mean ± SEM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 terminology will be used in accordance with the definitions set out 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, a "PEG polymer" or polyethylene glycol is one in which substantially all (preferably all) of the monomeric subunits are ethylene oxide subunits; however, the polymer may contain separate end-capping moieties or functional groups, e.g., for conjugation. The PEG polymers used in this disclosure may include one of the following two structures, depending on whether one or more terminal oxygens have been replaced, e.g., during synthetic transformations: "-(CH2CHO) n -" or "-(CH2CH2O) n-1 For PEG polymers, the variable (n) typically ranges from about 3 to 4000, and the end groups and overall PEG structure can vary. Exemplary or preferred molecules comprising PEG can include one or more specific PEG structures and / or linkers, and / or molecular weight ranges.

[0030] Molecular weight in the context of water-soluble polymers such as PEG can be expressed as number average molecular weight or weight average molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to 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 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) to determine number average molecular weight, or the use of light scattering techniques, ultracentrifugation, MALDI TOF, or viscometers to determine weight average molecular weight. PEG polymers are typically polydisperse (i.e., the number average and weight average molecular weights 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 low polydispersity values, preferably less than about 1.2, more preferably less than about 1.15, even more preferably less than about 1.10, such as less than about 1.05, or less than about 1.03.

[0031] A "physiologically cleavable" or "hydrolyzable" or "degradable" bond is a relatively unstable bond that typically 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 may depend not only on the general type of linkage connecting two atoms in a given molecule, but also on the substituents attached to those atoms and the overall molecular structure. Hydrolytically unstable or weak bonds typically include, but are not limited to, carboxylate esters, phosphate esters, anhydrides, acetals, ketals, acyloxyalkyl ethers, imines, orthoesters, peptides, oligonucleotides, thioesters, and carbonates.

[0032] "Enzymatically degradable bond" means a bond that is subject to degradation by one or more enzymes.

[0033] A "stable" linkage or bond refers to a chemical bond that is substantially stable in water, i.e., that does not undergo any appreciable degree of hydrolysis under physiological conditions over an extended period of time. Examples of hydrolytically stable bonds generally include, but are not limited to, the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, amines, and the like. In general, a stable linkage is one that exhibits a hydrolysis rate of less than about 1-2% per day under physiological conditions. Hydrolysis rates for representative chemical bonds can be found in many standard chemistry textbooks.

[0034] The term "IL-15 moiety" as used herein refers to a peptide or protein moiety that has human IL-15 activity. Moreover, the term "IL-15 moiety" encompasses both IL-15 moieties before conjugation with a PEG moiety and IL-15 moieties after conjugation (i.e., covalent attachment) (e.g., reaction) with a reactive PEG moiety, such as, for example, mPEG-succinimidyl butanoate. As described in more detail below, one of skill in the art can determine whether a given moiety has IL-15 activity. Proteins that include an amino acid sequence corresponding to any one of SEQ ID NOs: 1 to 3, as well as any protein or polypeptide substantially homologous thereto, are exemplary IL-15 moieties. As used herein, the term "IL-15 moiety" includes peptides and proteins that have been modified, for example, by site-directed mutagenesis or accidentally through mutation. Included with these are IL-15 sequences having one to six additional glycosylation sites, sequences having at least one additional amino acid at the carboxy terminus of the peptide or protein, where the additional amino acid comprises at least one glycosylation site, and sequences having an amino acid sequence that includes at least one glycosylation site. The term is intended to include naturally, recombinantly, and synthetically produced IL-15 moieties. Reference to a long-acting IL-15 receptor agonist is intended to encompass pharma- ceutically acceptable salt forms thereof.

[0035] The terms "substantially homologous" or "substantially identical" mean that a particular subject sequence, e.g., a mutant sequence, differs from a reference sequence by one or more substitutions, deletions, or additions whose net effect is not to result in adverse functional differences between the reference and subject sequences. For purposes of the present invention, sequences having greater than 95 percent homology (identity) under stringent conditions, equivalent biological activity (although not necessarily equivalent strength of biological activity), and equivalent expression characteristics to a given sequence are considered to be substantially homologous (identical). For purposes of determining homology, truncations of the mature sequence should be disregarded. Exemplary IL-15 polypeptides for use herein include sequences that are substantially homologous to SEQ ID NO:1. SEQ ID NO:2 is nearly 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" refers to any protein or polypeptide that has the amino acid sequence of a portion or fragment of a protein or polypeptide, e.g., an IL-15 portion, and has the biological activity, or substantially the biological activity, of the protein or polypeptide, e.g., 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 can include, for example, reducing tumor size or reducing the number of cancer cells, putting the cancer into remission, or preventing the growth in size or number of cancer cells, etc. In some situations, treatment according to the present disclosure leads to improved prognosis.

[0038] As used herein in the methods for treating a subject having cancer, the phrase "subject in need of treatment" refers to an individual or subject who has been diagnosed with cancer.

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

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

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

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

[0043] The phrases "therapeutically effective," "therapeutically effective amount," "effective amount," or "effective amount" 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 that is sufficient to promote an enhanced response to administration of an adoptive cellular immunotherapy composition comprising, for example, CAR T cells. The precise amount will depend on a number of factors, such as, for example, the particular condition being treated, the patient population, individual patient considerations, the components and material properties of the therapeutic composition and the particular combination to be administered, the particular adoptive cell transfer therapy being administered (e.g., the particular composition of cells included in the CAR T cell composition and / or the chimeric antigen receptor expressed by the CAR T cells), and can be determined by one of skill in the art.

[0044] "Substantially" or "essentially" means almost completely or entirely, e.g., 95% or more 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 circumstance described thereafter does not necessarily have to occur, such that the description includes cases where the circumstance occurs and cases where it does not occur.

[0047] A "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a component that may be included in the compositions described herein and that causes no significant adverse toxicological effects in a subject.

[0048] The term "patient", or "subject", as used herein, refers to an organism suffering from or susceptible to a condition that can be prevented or treated by administration of a compound or composition or combination as provided herein, such as cancer, and includes both humans and animals. Subjects include, but are not limited to, mammals (e.g., murine, simian, equine, bovine, porcine, canine, feline, etc.), and are preferably humans (including pediatric and adult subjects).

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

[0050] Adoptive chimeric antigen receptor cell transfer therapy and compositions The therapeutic methods provided herein include administering genetically engineered T cells transduced to express an artificial target tumor-associated antigen (TAA) binding domain, i.e., expanded ex vivo, to stimulate cancer-specific immune responses. The compositions and methods provided herein are particularly useful in both clinical and research applications. Without being bound by theory, it is believed that improved outcomes of anti-tumor drugs can be achieved via the IL-15 pathway (i.e., by administering a long-acting IL-15 receptor agonist together with adoptive cell transfer) to stimulate desired T cell responses due to the complementary mechanisms of immune activation of adoptive cell transfer, e.g., CAR T cell transfer, and long-acting IL-15 receptor agonists as provided herein.

[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 respect. 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 is understood that any suitable CAR T cell known in the art can be used in the methods and treatments described herein. Non-limiting examples of suitable CAR T cells and therapies for use herein include those described, for example, in U.S. Patent Application Publication Nos. 2017 / 0209492 and 2019 / 091308, and 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 cell comprises an antigen binding domain that binds to a tumor antigen. In yet some further embodiments, the tumor antigen is selected from the group consisting of CD19, CD20, CD22, and ROR1, and combinations of the foregoing. In yet some further embodiments, the CAR T cell is a CD19-targeted T cell that comprises an antigen binding domain that binds to CD19. See, e.g., Turtle, CJ, et al., Clinical Pharmacology & Therapeutics, 12 May 2016 (online). In addition to the exemplary publications provided in the preceding paragraphs, further illustrative CD19 CAR T cells, e.g., CD19 CAR T cells of defined CD4+:CD8+ composition, are described, e.g., in Turtle, CJ, J. Clin Invest. 2016;126(6):2133-2138. Additional CAR T cells suitable for use in the methods and treatments described herein include, for example, CD19-directed tisagenlecleucel (KYMRIAH®) and CD19-directed axicabtageneciloleucel (YESCARTA®), both of which are USFDA approved, for use in treating B cell malignancies. In yet some other embodiments, the CAR T cells express receptor tyrosine kinase-like orphan receptor 1 (ROR1), a tumor-associated molecule that is expressed, for example, in the majority of B lymphocyte and epithelial cancers, and in a subgroup 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 treatments 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, JM, 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 also contains the 4-1BB / CD3ζ intracellular signaling domain. In some embodiments, the ROR1 CAR T production process utilizes autologous peripheral blood lymphocytes, separated into CD4 and CD8 subpopulations, which are cultured independently with anti-CD3 / anti-CD28 beads and IL-2, and then transduced with a lentiviral vector encoding the ROR1 CAR. In yet some further embodiments, the CAR T cell product is formulated in a 1:1 ratio of CD4+ and CD8+ CAR T cells.

[0053] CAR T cell therapy generally involves the administration of CAR T cells to treat patients suffering from cancer, particularly cancers whose tumor cells express the subject tumor antigen. In some embodiments, CAR T cells are prepared as described herein or by methods known in the art. For example, after isolation, the host T cells are transduced to express the target tumor-associated antigen recognition domain, expanded, and reinfused into the subject. Prior to infusion, the patient may also be preconditioned using, for example, non-myeloablative chemotherapy (NMC) to deplete lymphocytes in order to suppress endogenous regulatory T cells and provide an optimized environment for the infused CAR T cells; alternatively, cyclophosphamide or any other suitable conditioning agent may be used. Such preconditioning is useful to eliminate or substantially reduce the number of Tregs (regulatory T cells) and lymphocytes that compete with the transplanted cells for homeostatic cytokines. The host cells can be isolated from various sources, such as lymph nodes, e.g., inguinal, mesenteric, superficial distal auxiliary, etc.; bone marrow; spleen; or peripheral blood, as well as from tumors, e.g., tumor-infiltrating lymphocytes. The cells can be allogeneic or, preferably, autologous. For ex vivo stimulation, the host cells are aseptically removed and suspended in any suitable medium, as known in the art. After transduction, the cells are stimulated and expanded using various protocols, in particular using any combination of 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., 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) administering non-myeloablative lymphodepleting chemotherapy (NMC) to the subject; and (iv) administering the expanded CAR T cells after administration of NMC. Autologous cells can be obtained from blood or cloned using autologous antigen-presenting cells and tumor-derived peptides.

[0055] CAR T cells can be prepared as described herein or by any means known in the art. Methods for generating CARs and / or CAR T cells are described herein and also in U.S. Pat. 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 / Publication Nos. WO 2010 / 065818, WO 2010 / 025177, and WO 2007 / 059298, the methods of which are incorporated herein by reference. Further 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 provide targeting of any TAA such that when the CAR T cells bind to their cognate antigen on the surface of the tumor cell, the tumor cell is affected, resulting in a reduction, attenuation, or elimination of the tumor burden in the patient. T cells can be engineered to express one or more chimeric antigen receptors (CARs) as described herein or using any method known in the art. In one embodiment, isolated T cells are engineered to express a CAR construct by introducing the T cells with an expression vector encoding the CAR construct. Methods for transducing a T cell population to express a selected CAR construct are known in the art and are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 4, incorporated herein by reference. th Edition, Cold Spring Harbor Laboratory Press (2012).

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

[0058] Generally, the CARs described herein are directed to molecules (e.g., proteins) expressed on the cell surface of cancer or tumor cells. Several TAAs are known in the art, and non-limiting examples include phosphorylated proteins, transmembrane proteins, glycoproteins, glycolipids, and growth factors. The assay 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 by those skilled in the art through routine experimentation. Any CAR as described herein or known in the art can be used in the methods and cell 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 a B cell. In some embodiments, the CAR comprises an anti-CD19 recognition or binding domain. Illustrative CD19-CAR constructs include: (i) CD19-directed chimeric antigen receptor (CTL019) lentiviral vector (CAR antigen recognition portion derived from SCFv:FMC63; costimulatory domain: 4-1BB), Maude,SL.,et al. N Engl J Med 2014;371(16):1507-1517; (ii) CD19-directed chimeric antigen receptor, gamma-retroviral vector incorporating anti-CD19 single chain variable fragment plus TCR zeta and CD28 signaling domains (CAR antigen recognition portion derived from SCFv:FMC63: FMC63; costimulatory domain: CD28), Lee,DW et al.,Lancet 2015;385(9967)517-528; mouse step cell virus-based splice-gag vector, (iii) Kochenderfer JN,et al.,J Immunother 2009;32(7):689-702; (iv) the CD-19-specific CD28 / CD3ζ dual signaling CAR, 19-28z (Park, JH, et al., Blood, 30 June 2016, 127(26), p. 3312-3320, in addition to those described in Table 1, Brentjens, RJ., et al., Sci Trans Med, 20 Mar 2013:5(177):177).

[0060] The human CD19 antigen is a 95 kDa glycoprotein that belongs to the immunoglobulin superfamily. CD19 is used as a biomarker for normal and neoplastic B cells and for follicular dendritic cells. CD19 is expressed from early stages of pre-B cell development until terminal differentiation and regulates B lymphocyte development and function. CD19 expression is highly conserved on most B cell tumors, including B cell lymphomas such as non-Hodgkin's lymphoma. CD19 is also expressed in most types of leukemia, including B cell leukemia, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and Waldenstrom's macroglobulinemia (WM). The majority of B cell malignancies (lymphomas and leukemias) express CD19 at normal to high levels. In some embodiments, the CAR comprises an anti-CD19 binding moiety. In still further embodiments, 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 but not limited to non-Hodgkin's lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and Waldenstrom's macroglobulinemia (WM). In some preferred embodiments, CD19 CAR T cell therapy comprises genetically modified autologous T cells directed against CD-19.

[0061] An impediment to the durability of CD19 CAR T cell therapy responses has 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-targeted CAR T cell therapy that includes targeting to CD19 and one or more additional TAAs. In some embodiments, the CAR T cell therapy includes at least a portion of a cell population that expresses a CD19 recognition or binding domain and one or more additional TAAs. In some embodiments, the CAR T cell therapy includes targeting to a TAA selected from CD20, CD22, CD38, CD123, CD70, or CD30. In some embodiments, the CAR T cell therapy includes two or more cell populations, each population expressing a different CAR. The cell populations can be administered as a mixture or can be co-administered sequentially. Preferably, the multi-targeted CAR T cell therapy comprises 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 comprises targeting to a TAA that is ROR1.

[0062] In some embodiments, the CAR comprises one or more intracellular costimulatory signaling domains. Exemplary costimulatory domains include, but are not limited to, CD28 with CD3zeta, CD123, or 4-1BB.

[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 a combination thereof. Non-specific T cell receptor stimuli can include, for example, a stimulatory amount of a mouse monoclonal anti-CD3 antibody (available, for example, from LS Bio, Seattle WA). Alternatively, T cells can be rapidly expanded by stimulation of peripheral blood mononuclear cells (PBMCs) in vitro with one or more antigens (including antigenic portions thereof, such as epitopes), optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, optionally in the presence of a T cell growth factor, such as interleukin-2 or interleukin-15, with interleukin-2 being preferred. In vitro induced T cells rapidly proliferate 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 with 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 can include enriching the cultured T cells for CD8+ T cells before 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, for example, using CD8 microbead separation. In some embodiments, a T cell growth factor that promotes the proliferation and activation of autologous T cells is administered to the subject either simultaneously with the autologous T cells or subsequently to the autologous T cells. The T cell growth factor can be any suitable growth factor that promotes the proliferation 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] and HLA-A2 by fluorescence-activated cell sorting analysis - HLA-A2 but not 888 melanoma line +The expression of chimeric antigen receptors in CAR T cells is compared in untransduced (UnTd) and transduced (Td) cells by in vitro recognition of the 526 melanoma line (Rosenberg, S., et al., Nat Rev. Cancer, 2008 Apr; 84(4): 299-308). Universal type T cells, such as those described in Qasim, W., et al., Sci. Transl. Med. 9, eaaj2013 (2017), can also be used. For example, universal CAR19 T cells can be generated using TALEN-mediated cell engineering in combination with lentiviral transduction and used in adoptive cell therapy. The cells are generated by lentiviral transduction of non-human leukocyte antigen-matched donor cells and simultaneous transcription activator-like effector nuclease (TALEN)-mediated gene integration of the T cell receptor alpha chain and CD52 locus.

[0066] Prior to administration of CAR T cells, the patient can be preconditioned with chemotherapy (e.g., cyclophosphamide and fludarabine, e.g., as described in U.S. Pat. No. 9,855,298) to improve the efficacy of the therapy. Without being limited by theory, chemotherapy preconditioning can create space for CAR T cell proliferation by removing normal lymphocytes, and / or can eliminate cytokine sinks to increase the availability of homeostatic cytokines that promote CAR T cell proliferation, and / or can reduce the number of immunosuppressive cells, such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (Nair et al.). It is understood that any suitable chemotherapeutic method can be used as known in the art.

[0067] The expanded cells are then administered to the host by infusion, for example, by intravenous or intra-arterial infusion or other suitable form of delivery, which generally lasts from about 30 to about 60 minutes, although shorter or longer durations may be utilized. Other routes of administration include intraperitoneal, intrathecal, and intralymphatic injection. The expanded cells are provided in a suitable medium, which may optionally include any of a variety of pharma- ceutically acceptable additives, binders, fillers, carriers, preservatives, stabilizers, emulsifiers, buffers, and the like. Diluents and excipients include water, saline, and glucose. Exemplary media include, but are not limited to, Multiple Electrolytes Injection, Type 1, USP, having a normal pH range of, for example, 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, for example, PRIME-XV T Cell Expansion XSFM (Irvine Scientific). Commercially available media include, for example, RPMI 1640 (Thermo Fisher Scientific, Waltham, Mass.), AIM V cell culture medium (Thermo Fisher Scientific, Waltham Mass.), and X-VIVO 15 (Lonza, Basel, Switzerland).

[0068] IL-15 receptor agonists The methods described herein, in one or more embodiments, include administration of a long-acting IL-15 receptor agonist. A compound is considered to be a long-acting IL-15 receptor agonist according to the present disclosure so long as, after administration to a subject, the agonist exhibits IL-15 agonism in vivo for a longer period of time than administration of the same interleukin-15 receptor agonist moiety in unmodified form. Conventional techniques, such as those involving radiolabeling a compound, administering the compound in vivo, and determining its clearance, can be used to assess whether a compound is a long-acting IL-15 receptor agonist (i.e., has a longer clearance than unmodified 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 times after administration of the agonist in mice. For reference, the signal fades in approximately 24 hours for IL-15, but persists for longer periods for the long-acting IL-15 agonists described herein.

[0069] The long-acting IL-15 receptor agonist may be in the form of a pharma- ceutically acceptable salt, and reference to a long-acting IL-15 receptor agonist is intended to include pharma- ceutically acceptable salts thereof. Typically, such salts are formed by reaction with a pharma- ceutically acceptable acid or acid equivalent. In this context, the term "pharmaceutically acceptable salt" may generally refer to relatively non-toxic inorganic and organic acid addition salts. These salts may be prepared in situ during the administration vehicle or dosage form manufacturing process, or by reacting a long-acting interleukin-15 receptor as otherwise described herein with a suitable organic or inorganic acid and isolating the salt so 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 laurylsulfonate (see, e.g., Berge et al. (1977) "Pharmaceutical Salts," J. Pharm. Sci. 66:1-19). Thus, the salts as described may be derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric acid, and the like; or may be prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

[0070] An exemplary long-acting IL-15 receptor agonist has the structure: [ka] (wherein IL-15 is an interleukin-15 moiety, (n) is an integer from about 150 to about 3,000, and -NH- represents an amino group in the IL-15 moiety.) The above-mentioned IL-15 receptor agonist is referred to herein as mono(methoxy PEG-N-butanamide) interleukin-15 (i.e., MPBA-IL15). An illustrative preparation of mono(methoxy PEG-N-butanamide) interleukin-15 is shown in Example 1.

[0071] In one or more embodiments relating to the long-acting IL-15 receptor agonists 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) averages a 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 class of primarily mono-PEGylated IL-15 (i.e., having a single methoxyPEG-N-butanamide moiety covalently attached to an amino group of IL-15, i.e., to a lysine or to the N-terminal alpha amine of IL-15), with minor amounts of di-PEGylated and higher PEGylated IL-15 species. Further characteristics of mono(methoxyPEG-N-butanamide) interleukin-15 are described, for example, in WO 2018 / 213341.

[0073] Illustrative compositions of MPBA-IL15 comprise primarily mono-PEGylated species, with less than about 10 mol % of PEG dimers (i.e., those having two methoxy PEG-N-butanamide moieties attached to IL-15) and even lesser amounts of more PEGylated species (i.e., those having three or more methoxy PEG-N-butanamide moieties attached to IL-15) attached to IL-15. A composition of mono(methoxy PEG-N-butanamide) interleukin-15 will generally have at least about 80 mol % of mono-PEGylated IL-15 species (based on all interleukin-15 species in the composition, including unmodified IL-15 (if present) and other IL-15-containing species, e.g., di-PEGylated IL-15 and greater). A composition of MPBA-IL15 may preferably have at least about 90 mol % of mono-PEGylated IL-15 species, with less than about 10 mol % of other IL-15 species. In some embodiments, the MPBA-IL15 compositions contain less than about 5 mol% PEG dimer (di-PEGylated IL-15 having two methoxy PEG-N-butanamide moieties covalently attached to IL-15) and less than about 5 mol% of all other higher PEGylated species. In one or more embodiments, the MPBA-IL15 compositions contain at least about 85 mol%, 90 mol%, 95 mol%, 98 mol%, or 99 mol% mono-PEGylated species of formula (I).

[0074] For example, in some preferred embodiments, the long-acting IL-15 receptor agonist compositions have the formula, taken collectively: [ka] (wherein the value of n is as described above for all embodiments), or when collectively considered according to formula (II), contains at most about 15 molar percent (mol %) of a long-acting IL-15 receptor agonist (of the IL-15 containing molecules in the composition); or when collectively considered according to formula (II), contains at most about 10 molar percent (of the IL-15 containing molecules in the composition) of a long-acting IL-15 receptor agonist.

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

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

[0077] With respect to the above formula, "n" corresponds to the average number of (OCH2CH2) monomer subunits. MPBA-IL15 can be prepared using a suitably activated mPEG-butanoate ester reagent having, for example, an average molecular weight of about 6600 Daltons to about 132,000 Daltons. For example, MPBA-IL15 can be prepared using a suitably activated mPEG-butanoate ester reagent having, for example, an average molecular weight selected from 10 kD, 15 kD, 20 kD, 25 kD, 30 kD, 45 kD, 50 kD, or 60 kD. The activated polymeric reagent, when reacted with an amino group (e.g., lysine or N-terminus) of IL-15, is effective to form a stable amide bond between the IL-15 moiety and the polyethylene glycol moiety.

[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), or about 15,000 daltons (where n is about 340), or about 20,000 daltons (where n is about 454), or about 25,000 daltons (where n is about 568), or about 30,000 daltons (where n is about 681), or about 40,000 daltons (where n is about 909), or about 50,000 daltons (where n is about 1136), or about 60,000 daltons (where n is about 1364) or greater.

[0079] Illustrative PEG reagents used to prepare MPBA-IL15 will typically have a polydispersity value of less than about 1.1, e.g., about 1.05. Thus, for a PEG reagent such as mPEG-succinimidyl butanoate, which has 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 kilodaltons to about 47 kilodaltons, or about 37 kilodaltons to about 45 kilodaltons (41 kD±4 kDa). In some preferred embodiments, the mPEG butanoic acid activating ester reagent has a nominal average molecular weight of about 40 kilodaltons, i.e., where, on average, n is about 907-909.

[0080] When considering an IL-15 moiety, the term "IL-15 moiety" refers to the IL-15 moiety pre-conjugation and to the IL-15 moiety post-conjugation, however, it will be understood that when the original IL-15 moiety is attached to the polyethylene glycol moiety, the IL-15 moiety will be slightly altered due to the presence of one or more covalent bonds associated with the bond to the polymer (e.g., in the case of an amide bond formed during preparation of MPBA-IL15).

[0081] The IL-15 moiety can be derived from non-recombinant and recombinant methods, and the disclosure is not limited in this respect. Additionally, the IL-15 moiety can be derived from human, animal (including insect), fungal (including yeast), and plant sources.

[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 commercially, for example, from GenScript USA Inc. (Piscataway NJ) and Peprotech (Rockyhill, NJ).

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

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

[0085] Depending on the system used to express a 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, to provide for easy attachment of the polymer to atoms in the side chains of the amino acids. Examples of substitutions of the IL-15 moiety are described in U.S. Patent No. 6,177,079. Additionally, the IL-15 moiety can be modified to include non-naturally occurring amino acid residues. Amino acid residues and techniques for adding 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, e.g., U.S. Patent 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 / WO 2018 / 213341. Preferred IL-15 moieties include those having an amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 1-3 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: 1-3. In some embodiments, the IL-15 portion is a functional homolog having at least about 95%, 98%, or 99% identity to any one of SEQ ID NOs:1-3.

[0088] In certain cases, the IL-15 moiety will be in a "monomeric" form, where a single expression of the peptide of interest is organized into a separate unit, whereas in other cases, the IL-15 moiety will be in a "dimer" form, where two monomeric forms of the protein are associated with each other (e.g., a dimer of recombinant IL-15).

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

[0090] Truncations, hybrid variants, and peptidomimetics of any of the foregoing sequences can also serve as IL-15 moieties. Biologically active fragments, deletion variants, substitution variants, or addition variants of any of the foregoing that maintain at least some IL-15 activity can also serve as IL-15 moieties.

[0091] For any given peptide, protein moiety, or conjugate, it is possible to determine whether the peptide, protein moiety, or conjugate has some degree of IL-15 activity. Various methods for determining IL-15 activity in vitro have been 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 with IL-15 activity, initiation of a signal cascade occurs, including phosphorylation of STAT5 at tyrosine residue 694 (Tyr694), which can be quantitatively measured. Assay protocols and kits are known, such as the MSD Phospho(Tyr694) / Total STATa,b Whole Cell Lysate Kit (Meso Scal Diagnostics, LLC, Gaithersburg, MD). For example, using this approach, a pSTAT5 EC of at most about 300 ng / mL (more preferably at most about 150 ng / mL) was detected in at least one of 5 or 10 minutes. 50A proposed IL-15 moiety exhibiting a pSTAT5 EC value of less than 150 ng / mL for at least one of 5 or 10 minutes is typically considered to be an "IL-15 moiety" for the purposes of this disclosure. However, the IL-15 moiety used may be more potent (e.g., a pSTAT5 EC value of less than about 1 ng / mL, more preferably less than 0.5 ng / mL for at least one of 5 or 10 minutes). 50 It is preferred that the .lambda.-based .DELTA..times ...

[0092] Other methodologies known in the art can also be used to assess IL-15 function, including electrometric, spectrophotometric, chromatographic, and radiometric methodologies. See, e.g., Ring et al. (2012) Nat. Immunol. 13(12):1187-1195 for further types of such assays.

[0093] As described above, the amino group on the IL-15 moiety provides a site of attachment for reaction with an mPEG-succinimidyl butanoate reagent to provide an IL-15 receptor agonist encompassed by formula (I). In view of the exemplary IL-15 amino acid sequence provided herein, it is apparent that there are seven lysine residues, each with an ε-amino acid that may be available for conjugation. Additionally, the N-terminal amine of methionine may also serve as an attachment point for the PEG moiety. It is understood that the polyethylene glycol moiety may be attached 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 using SEQ ID NO:1, Lys 11 and Lys 12 In some embodiments, the polyethylene glycol moiety is attached to the N-terminal amine. 37 Or Lys 42It will be appreciated that any of the positions 100 to 150 of the MPBA-IL15 may be suitable as an attachment site for the PEG moiety. In some embodiments, MPBA-IL15 comprises a mixture of positional isomers in which the covalent attachment of the polyethylene glycol moiety is predominantly at the N-terminus (i.e., within the collection of positional isomers, the isomer having the PEG moiety attached at the N-terminus is present in the greatest amount when compared to the other positional isomers.

[0094] MPBA-IL15 is an immunotherapeutic 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 and maintains the full range of IL-15 biology, including pharmacodynamic (PD) effects on both NK cells and CD8+ memory T cells, while 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 the inventors deem particularly advantageous when combined with CAR T cell therapy. For example, MPBA-IL-15 has been shown to (i) stimulate and expand NK cell proliferation, (ii) support CD8 T cell survival and memory formation without substantially inducing suppressive regulatory T cells, (iii) enhance the formation of long-term immune memory, and furthermore retains receptor binding to 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 mutated 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 (heterodimeric IL-15, an IL-15 / soluble IL-15Rα dimer, see, e.g., AACR; Cancer Res 2019; 79(13):211-212), and / or NIZ985 (a fusion protein that is a fusion protein that is a fusion protein that is a 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). Suppl)), AM0015 (polyethylene glycol modified IL-15, see, e.g., WO 2017 / 112528), and OXS-3550 (single chain, trispecific scFv recombinant fusion protein conjugate consisting of the 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).

[0097] method Based on at least one or more characteristics of the long-acting IL-15 receptor agonist, MPBA-IL15, in one aspect, provided herein is an effective method for inducing an immune response in a cancer patient prior to administration of MPBA-IL15 by administering an adoptive cellular immunotherapy composition comprising autologous or allogeneic (preferably autologous) anti-tumor T cells, such as CD19 CAR T cells as described above and having anti-tumor activity, which have been genetically transformed to express a chimeric antigen receptor targeting tumor cells, and which is accompanied / followed by administration of a long-acting IL-15 receptor agonist, i.e., MPBA-IL15, thereby achieving enhanced therapeutic efficacy. In a preferred embodiment, the T cell immunotherapy comprises genetically modified autologous T cells directed against CD19. An illustrative adoptive cellular immunotherapy composition includes tumor-reactive T cells engineered to express a chimeric antigen receptor that includes the extracellular variable domain, hinge and transmembrane domain of an antibody directed to an antigen associated with a cancer (e.g., CD19), and an intracellular signaling domain (such as a costimulatory domain) of a T cell or other receptor. For example, tumor-reactive T cells can be engineered with a chimeric antigen receptor derived from a single-chain antibody directed to a CD19 molecule. In some embodiments, the cell composition includes CAR-engineered cytotoxic tumor cells, e.g., CD8+ T lymphocytes engineered with a CD19 CAR, and can further include other types of T lymphocytes (e.g., helper T lymphocytes), e.g., CD4+ or other T cells that have been genetically engineered to have a chimeric antigen receptor directed to an antigen associated with a cancer (e.g., CD19), and the disclosure is not limited in this respect (i.e., to the specific configuration of an adoptive cellular immunotherapy composition that includes CAR T cells, e.g., CD19-directed CAR T cells). Exemplary CAR T cell compositions suitable for use in the methods provided herein are described in the preceding sections.

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

[0099] Adoptive cellular immunotherapy compositions comprising CAR T cells, e.g., CD19 CAR T cells, are typically administered in a therapeutically effective amount, i.e., an amount effective to immunize the subject. Immunization means to alleviate one or more physical symptoms associated with the tumor or cancer in which the lymphocyte response is induced. Adoptive cellular immunotherapy compositions are typically administered by injection, with each injection containing at least 2 cells to at least 10 6 ~10 10 cells / kg, preferably at least 10 7 ~about 10 9 in the range of cells / kg, or preferably at least 10 6 ~about 10 8 In some specific, but non-limiting, embodiments, each injection contains at least about 0.2×10 cells / kg. 6 cells / kg ~ approx. 6.0×10 8 Cells / kg, at least about 2.0 x 10 6 cells / kg ~ approx. 2.0×10 8 At least about 0.2 x 10 cells / kg 6 cells / kg ~ approx. 5.0×10 6 At least about 0.1 x 10 cells / kg 8 cells / kg ~ approx. 2.5×10 8 cells / kg, or at least about 0.6×10 8 cells / kg ~ approx. 6.0×10 8The T cell immunotherapy is administered in a dose of 100 mg / kg, including 100 mg / kg of cells / kg. The cells can be administered by a single infusion or multiple infusions. In some specific embodiments, the cells are administered as a single dose infusion. Since different individuals respond differently, the number of cells to be infused, as well as the number of infusions and the time range over which multiple infusions are administered, can be determined by a medical professional, as determined by routine investigation. 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 administered in an amount effective to enhance the results of CAR T cell immunotherapy. To confirm, with respect to the long-acting IL-15 receptor agonist, MPBA-IL15, the amount and degree of activation can vary widely and can still be effective when combined with the administration of an 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 of time can still be a long-acting IL-15 receptor agonist only when administered in combination with CAR T cell therapy, and the methods described herein allow for a clinically meaningful response. In certain cases, due to (for example) synergistic interactions and responses, only minimal IL-15 receptor agonist activity may be required when associated with CAR T cell therapy, e.g., CD19 CAR T cell therapy. It is understood that the therapeutic amount of one or both of the long-acting IL-15 receptor agonists and the number of CAR T cells administered can be lower than the therapeutically effective amount of either component when administered alone.

[0101] As described herein, one aspect of the present disclosure provides a method that is useful for treating a patient suffering from a condition (such as cancer) that is responsive to treatment with (among other things) an adoptive cellular immunotherapy composition and a long-acting IL-15 receptor agonist or both. For example, a patient may be responsive to each of the individual agents alone as well as the combination, but is more responsive to the combination. As a further example, a patient may be unresponsive to one of the individual immunotherapeutic agents, but is responsive to the combination. As an even further example, a patient may be unresponsive to any of the individual agents alone, but is 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 and 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 pharma- ceutically acceptable excipients. Suitable pharma-ceutically acceptable excipients include, for example, those described in Handbook of Pharmaceutical Excipients, 7 th ed., Rowe, RC, Ed., Pharmaceutical Press, 2012. Such exemplary formulations include MPGA-IL15 in a solution containing phosphate buffer and trehalose (pH of 6.8). For example, an exemplary formulation includes MPBA-IL15 formulated in a solution of potassium phosphate buffer, trehalose, and polysorbate 20 (pH of about 6).

[0104] Suitable formulations for parenteral administration include, among others, ready-to-inject liquids, dry powders for combination with a solvent before use, ready-to-inject suspensions, dry insoluble compositions for combination with a vehicle before use, and emulsions and liquid concentrates for dilution before administration. In some particular 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 also contemplated, such as intrapulmonary, nasal, buccal, rectal, sublingual, and transdermal.

[0105] In general, a therapeutically effective amount of a long-acting IL-15 receptor agonist will range from about 5 mcg (μg) to about 10 mg, based on the dose of protein (IL-15 equivalents) 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 equivalents)). 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 0.25mcg / kg, 0.3mcg / kg, 0.5mcg / kg, 1mcg / kg, 2mcg / kg, 3mcg / kg, 5mcg / kg, 6mcg / kg, 7mcg / kg, 10mcg / kg, 15mcg / kg, 20mcg / kg, 25mcg / kg, 0.01mg / kg, 0.03mg / kg, 0.05mg / kg, or 0.1mg / kg.

[0107] In yet some further embodiments, the therapeutically effective dose of the long-acting IL-15R agonist is in the range of about 0.25 to 25 μg / kg. In other embodiments, the therapeutically effective dose is (e.g., per day) about 0.25 μg / kg to about 0.1 mg / kg, 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. In another embodiment, the therapeutically effective dose is in the range of about 1 to about 10 μg / kg, about 0.03 mg / kg to about 0.1 mg / kg / day, or about 1 to about 10 μg / kg, about 0.03 mg / kg to about 0.1 mg / kg / day. 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 with administration of adoptive cellular immunotherapy compositions, the dosage of the long-acting IL-15 receptor agonist will vary depending, for example, on the age, weight, and general condition of the subject, as well as the severity of the condition being treated, the particular adoptive cellular 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, adoptive cell transfer of CAR T cells based cell infusion can be performed 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 1 day, up to 1 month, up to 3 months, up to 6 months, or any combination thereof, prior to administration of MPBA-IL15.

[0110] Alternatively, in some embodiments, adoptive cell transfer is performed following 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, by 1 hour, by 2 hours, by 3 hours, by 4 hours, by 5 hours, by 6 hours, by 7 hours, by 8 hours, by 9 hours, by 10 hours, by 11 hours, by 12 hours, by 1 day, by 2 days, by 3 days, by 4 days, by 5 days, by 6 days, by 7 days, by 8 days, by 9 days, by 10 days, by 11 days, by 12 days, by 13 days, by 14 days, by 15 days, by 16 days, by 17 days, by 18 days, by 19 days, by 20 days, by 21 days, by 22 days, by 23 days, by 24 days, by 25 days, by 26 days, by 27 days, by 28 days, by 29 days, by 1 month, by 3 months, by 6 months, or any combination thereof, following administration of MPBA-IL15.

[0111] As used herein in reference to the treatment of a subject with cancer, the terms "treatment," "treat," and "treating" are intended to include the full range of interventions against the cancer from which the subject is suffering, such as the administration of a combination to alleviate, slow, stop, or reverse one or more symptoms of the cancer, or to delay the progression of the cancer, even if the cancer is not actually eliminated. Treatment can include, for example, a reduction in the severity of symptoms, the number of symptoms, or the frequency of recurrence, for example, suppression of tumor growth, prevention of tumor growth, or regression of an existing tumor.

[0112] For example, improvement of cancer or cancer-related disease can be characterized as complete response or partial response. "Complete response" refers to the absence of clinically detectable disease, with normalization of any previous radiological, bone marrow, and cerebrospinal fluid (CSF) abnormalities, or abnormal monoclonal protein measurements. "Partial response" refers to at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in all measurable tumor burden (i.e., the number of malignant T cells present in the subject, or the measured volume of 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] With regard to the frequency and schedule of infusion of the adoptive cell immunotherapy composition and administration of the long-acting IL-15 receptor agonist, one skilled in the art will be able to determine the appropriate frequency. For example, the clinician may perform adoptive cell transfer in combination with administration of a long-acting IL-15 receptor agonist either simultaneously with the adoptive cell transfer of CAR T cells or preferably after the adoptive cell transfer in a treatment cycle. For example, in some treatment modalities, the long-acting IL-15 receptor agonist is administered within about 7 days (e.g., on any one of days 1, 2, 3, 4, 5, 6 or 7) of the adoptive cell transfer of CAR T cells. In some examples, the long-acting IL-15 receptor agonist, i.e., MPBA-IL15, is administered within 4 days of the 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 is typically administered relatively infrequently (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 a treatment course 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; and about 5 years. Typically, a patient is provided with one round of adoptive cell transfer, e.g., CD19 CAT T cells, followed by one or more administrations of the long-acting IL-15 receptor agonist, MPBA-IL15, although in some instances, one or more additional cycles of adoptive cell transfer may be performed.

[0115] The therapeutic method described herein is typically continued as long as the clinician supervising the patient's care considers the therapeutic method to be effective, i.e., the patient is responding to the treatment.Non-limiting parameters that indicate that the therapeutic method is effective may include one or more of the following: tumor shrinkage (in terms of weight and / or volume and / or appearance); reduction in the number of individual tumor colonies; reduction in the number of cancer cells; tumor disappearance; progression-free survival; appropriate response by suitable tumor markers (if 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 previously described, the adoptive cell composition (e.g., including CAR T cells) and the long-acting IL-15 receptor agonist can be administered separately. Alternatively, if it is desired to provide simultaneous administration of the adoptive cell transfer and the long-acting IL-15 receptor agonist, either as an initial dose or throughout the course of treatment or at various stages of an administration regimen (and the CAT T cells and the long-acting IL-15 receptor agonist are compatible together and in a given formulation), simultaneous administration can be achieved by infusion of a single dosage 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 remedied or prevented by the methods provided herein, such as cancer. For example, these methods are useful for treating, for example, solid tumors, hematological tumors (liquid cancers), or melanoma, among other conditions. Exemplary conditions include cancer, such as melanoma, kidney 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, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovium, 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 carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, multiple myeloma, neuroblastoma, retinoblastoma, and leukemia.

[0118] In certain embodiments, the cancer is a solid tumor.

[0119] In still some further embodiments, the cancer is selected from, e.g., 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, kidney cancer, bile duct cancer, brain cancer, cervical cancer, maxillary sinus cancer, bladder cancer, esophageal cancer, and adrenal cortical carcinoma.

[0120] In yet 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, which is a highly aggressive tumor that lacks estrogen receptors, progesterone receptors, 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's lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and Waldenstrom's 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, e.g., CD19 CAR T cells, by improving the subject's response, e.g., by administration of a long-acting IL-15 receptor agonist, MPBA-IL15. The enhanced response can be assessed at any suitable time point, such as during treatment, after a single round of treatment, after 2-3 cycles of treatment, and by, e.g., any of a number of suitable methods, including tumor shrinkage (partial response), i.e., evaluation of tumor size or volume, tumor disappearance, reduction in the number of cancer cells, reduced disease progression (cancer not progressing), and, where appropriate, analysis of one or more tumor laboratory markers. Comparisons can be made in human patients or in a suitable animal model, such as a suitable mouse model of cancer.

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

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

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

[0128] Tumor biopsy collection may also be performed. Biopsy should preferably be performed on a lesion that has not been previously exposed to radiation. Biopsy may be obtained from a non-target lesion unless there are no other lesions suitable for biopsy. Pre- and post-treatment tumor tissue biopsies should preferably be taken from the same lesion, if possible. Additionally, biopsies may be taken from a distant non-injected lesion to serve as a control biopsy. Tumor tissue biopsies may be used to characterize infiltrating immune cell populations using immunohistochemistry (IHC) and / or flow cytometry using a panel of markers, including but not limited to CD3, CD4, CD8, and CD56.

[0129] In some embodiments, the combination immunotherapies described herein, in contrast to many ACT-based therapies, are effective in increasing the extent and persistence (i.e., maintenance) of exogenously delivered CAR T cells at the tumor site or in the blood, thereby providing increased anti-tumor efficacy.

[0130] All articles, books, patents, patent publications and other publications referred to in this specification are incorporated by reference in their entirety. In the event of a discrepancy between the teachings of this specification and the art incorporated by reference, the meaning of the teachings and the definitions of this specification shall prevail (especially with respect to the terms used in the claims appended hereto). For example, if this application and the publications incorporated by reference define the same term differently, the definition of the term shall be maintained within the teachings of the document in which the definition is found. EXAMPLES

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

[0132] Materials and Methods CAR T cells: Human CD19 CAR T cells expressing the CD19 / 4-1BB / CD3ζ CAR were generated from healthy donors as previously described. See, e.g., Turtle, CJ, et al. J. Clin Invest. 2016;126(6):2123-2138 and U.S. Patent No. 9,987,308. Briefly, CD4 and CD8 T cells were isolated and transduced separately with the CD19 CAR lentiviral vector (Figure 2), 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") SEQ ID NO:1 (as provided in FIG. 1), prepared using conventional techniques, was used in the following examples, although any suitable IL-15 portion can similarly be used. SEQ ID NO:1, 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 includes an additional methionine at the N-terminus that is not present in native, secreted human IL-15.

[0134] The reactive linear polymer reagent, mPEG-succinimidyl butanoate, 40 kDa ("mPEG-SBA"), CAS No. 187848-51-7, has the following structure: [ka] where 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., where, on average, n is about 907-909. The PEG reagent has a polydispersity value of less than about 1.1, e.g., about 1.05, resulting in a nominal average molecular weight in the range of about 37 kilodaltons to about 45 kilodaltons (41 kD±4 kDa). mPEGSBA is typically in the form of a white to off-white powder. Additional mPEG-succinimidyl butanoate reagents suitable for use include, for example, 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 polymeric reagent is effective to form a stable amide bond between the IL-15 moiety and the polyethylene glycol moiety when reacted with an 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 class of predominantly mono-PEGylated IL-15 with a single mPEG-N-butanamide moiety covalently attached to a lysine or to the N-terminal alpha amine of IL-15, with minor amounts of di-PEGylated and higher PEGylated IL-15 species (CAS Registry No. 2361317-09-9). Additional characteristics of mono(methoxyPEG-N-butanamide) interleukin-15 are described, for example, in WO 2018 / 213341, the contents of which are incorporated herein by reference.

[0136] Potency Bioassay: The potency of MPBA-IL15 was determined by phosphorylation of STAT5 in CTLL-2 cells, a murine T-lymphocyte cell line expressing the IL-15α subunit, using a 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 assessed to measure short-term biological responses.

[0137] Reference materials, assay controls, and test samples were serially diluted to 10-fold final concentrations using assay medium and then applied to a constant number of cells for 10 min incubation at 37° C. / 5% CO2. Phosphorylated and total STAT5 were measured using a phospho-STAT5 / total STAT5 multiplex assay (Meso Scale Discovery, MD). Dose-dependent phosphoprotein response curves (%) were generated by nonlinear regression analysis using a four-parameter model. Parallel line assay (PLA) software was used to assess the parallelism, significance of the regressions, and to calculate the relative potency of samples to reference materials within the same plate.

[0138] The purity of MPBA-IL15 was assessed using reversed-phase HPLC using a HALO Protein C4 analytical column with ultraviolet (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] Size-exclusion HPLC was used to assess the relative purity of MPBA-IL15 using a Shodex Protein KW-803 column operated at a flow rate of 0.5 mL / min at 25° C. 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 assess the relative purity of MPBA-IL15 by separating acidic and basic charge variants using an Agilent PL-SAX column operated at 40° C. with a flow rate of 1.0 mL / min. Elution was performed 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(methoxyPEG-N-butanamide) 40KD Preparation of Interleukin-15 [ka] Preparation 1: 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, pH 8, was added to adjust the pH to pH 8. mPEG-SBA, 40 kDa (nominal average molecular weight), stored at −20° C. under nitrogen, was warmed to ambient temperature. A 10-fold excess (relative to the molar amount of IL-15) of mPEG-SBA-40K was dissolved in 2 mM HCl to form a 10% PEG reagent solution. This 10% PEG reagent solution was added quickly to the IL-15 solution and mixed thoroughly. After the addition of mPEG-SBA-40K, the pH of the reaction mixture was determined and adjusted to pH 8 using conventional techniques. To allow coupling of mPEG-SBA-40K to IL-15 (i.e., via formation of a stable amide bond), the reaction solution was placed on a Slow Speed ​​Lab Rotator for 1.5 hours to facilitate conjugate formation at room temperature. The reaction was stopped by the addition of a solution of glycine.

[0142] The reaction yielded 40% mono-conjugate (i.e., with a single PEG moiety attached to IL-15), 24% di-conjugate (with two PEGs attached to IL-15), and 6% tri-conjugate (with 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 (referred to herein as mono-mPEG-butanamide-40K-IL-15 or mono(methoxyPEG-N-butanamide) 40kD Interleukin-15, or mono-mPEG 40K The purified mono-mPEG-SBA40K-IL-15 (also referred to as -C4-amide-IL-15) 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] The purified conjugate was determined to have a high level of purity as shown by SDS gel, with no detectable amounts of unreacted IL-15. Based on the HPLC plot, the purified mono-mPEG-SBA-40K-IL-15 composition contained less than about 10% (molar amount) of the di- or higher conjugate.

[0145] Using this synthetic approach, mPEG-SBAs having different nominal average molecular weights (e.g., having nominal average molecular weights of about 10 kilodaltons, 15 kilodaltons, 20 kilodaltons, 25 kilodaltons, 30 kilodaltons, 45 kilodaltons, 50 kilodaltons, 60 kilodaltons, etc., respectively) are used to prepare 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.

[0146] Preparation 2: Approximately 2 mg / ml solutions of rIL-15 in buffer (50 mM sodium phosphate, 100 mM sodium chloride, 10% sucrose, pH 7.4) were transferred to each of two different reaction vessels (referred to herein as Composition 1 and Composition 2). pH 8 borate buffer (0.4 M or 0.6 M) was added to adjust the pH to 8.0. A 10-fold excess (relative to the molar amount of IL-15) of mPEG-SBA-40K (mPEG-SBA, 40 kDa), diluted in 2 mM HCl, was added to each of the IL-15 solutions and mixed thoroughly. After the addition of mPEG-SBA-40K, the pH of the reaction mixture was determined to be pH 8 or was adjusted by the use of additional borate buffer if necessary. The final concentration of rIL-15 in the reaction was targeted to 1 g / L using additional diluent if necessary (for composition 1, a buffer containing 50 mM sodium phosphate, 100 mM sodium chloride, 10% sucrose (pH 7.4) was used, and for composition 2, water was used). To allow coupling of mPEG-SBA-40K to IL-15 (i.e., primarily via the formation of a stable amide bond), the reaction solution was mixed for 45 or 60 minutes for composition 1 or composition 2, respectively, to facilitate conjugate formation at room temperature. The reaction was stopped by the addition of a solution of 71-fold excess (relative to the molar amount of PEG initially added to the reaction), pH 8.0, for 30 minutes. 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 compositions prepared contained primarily mPEG-SBA-40K monoPEGylated species, with less than about 10 mol % PEG dimers (i.e., those with two PEG moieties attached to IL-15) and even lesser amounts of higher PEG species (i.e., those with three or more PEG moieties) attached to IL-15. As described elsewhere, compositions of mono(methoxyPEG-N-butanamide) interleukin-15 will generally have at least about 80 mol % monoPEGylated IL-15 species (based on all interleukin-15 species in the resulting composition, including unmodified IL-15 and other IL-15-containing species, e.g., diPEGylated IL-15 and greater), and preferably at least about 90 mol % monoPEGylated IL-15 species, with less than about 10 mol % other IL-15 species. In some embodiments, mono(methoxyPEG-N-butanamide) interleukin-15 compositions contain less than about 5 mol % PEG dimers (diPEGylated IL-15 having two methoxyPEG-N-butanamide moieties attached to IL-15) and less than about 5 mol % of all other higher PEGylated species.

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

[0155] MPBA-IL15 can be formulated for further use as a solution containing MPBA-IL15 at a concentration of 1 mg / mL (on a protein basis) (in 10 mM potassium phosphate), 260 mM trehalose, and 0.02 w / v% polysorbate 20 (pH of 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 effect of mono(methoxyPEG-N-butanamide) interleukin-15 on human CD19 CAR T cells was investigated as described below.

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

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

[0160] Dose-dependent phosphorylation of STAT5 in response to mono(methoxyPEG-N-butanamide) interleukin-15 (MPBA-IL15) for both CD8 and CD4 CAR T cells is shown in Figures 3C and 3D, respectively. CAR T cells were stimulated with various concentrations of MPBA-IL15 or IL-15 for 20 min. EC50 values ​​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. 50 Values ​​(ng / ml) 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 Figures 3E (CD8 CAR T cells) and 3F (CD4 CAR T cells). The EC50 values ​​are summarized below.

[0163] [Table 3]

[0164] For Figures 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 induces STAT5 phosphorylation and antigen-dependent proliferation of both CD8 CD 19 CAR T cells and CD4 CD 19 CAR T cells in a dose-dependent manner.

[0166] Example 3 Study of mono(methoxy PEG-N-butanamide) interleukin-15 on the efficacy of CD19 CAR T cell immunotherapy in preclinical mouse lymphoma models 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 a sustained 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 an experiment as described below.

[0167] General Methods: For in vivo studies, NSG mice were intravenously injected on day -7 (D-7) with 5 × 10 5 Raji lymphoma cells were then administered on D0 at a sub-therapeutic dose (0.8×10 6 ) CAR T cells (1:1 CD4:CD8). Tumor-free mice were re-challenged with Raji cells on D38. Tumors were assessed weekly by bioluminescence imaging of mice. Results are shown in Figure 10.

[0168] As shown in Figure 4A (mean tumor brightness versus days after CAR T cell administration for various treatment groups), treatment with 0.10 mg / kg and 0.30 mg / kg mono(methoxyPEG-N-butanamide) interleukin-15 in combination with CAR T cells results in a reduction in tumor burden and eradication of Raji lymphoma in NSG mice when compared to CAR T cell therapy alone. In this study (Study A), NSG mice with Raji received a subtherapeutic dose of CAR T cells on D0, followed by 0.030, 0.10, or 0.30 mg / kg mono(methoxyPEG-N-butanamide) interleukin-15 starting on D6 and weekly thereafter (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 mono(methoxyPEG-N-butanamide) interleukin-15 was administered on D-1, D7, or D14 and weekly thereafter (5 mice / group). See Figure 7B. Mice were bled weekly and CD8 and CD4 CAR T cells were identified by flow cytometry (Figures 5A and 5B, respectively). Tumor burden was assessed by weekly bioluminescence imaging (mean tumor brightness versus days after CAR T cell administration) and survival (Figure 7A) as shown in Figure 6.

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

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

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

[0173] In vivo infusion of mono(methoxyPEG-N-butanamide) interleukin-15 beginning on D-1, 7, or 14 increased peak CAR T cell numbers in the blood. Raji cells were cleared from the bone marrow by D14 in mice receiving CAR T cells and mono(methoxyPEG-N-butanamide) interleukin-15 on D7 (see especially the 0.30 mg / kg dosing group), but not in mice receiving CAR T cells alone. Mice receiving CAR T cells with mono(methoxyPEG-N-butanamide) interleukin-15 showed superior mono(methoxyPEG-N-butanamide) interleukin-15 dose-dependent tumor control and survival compared to CAR T cells or mono(methoxyPEG-N-butanamide) interleukin-15 alone. Based on this experimental setting, but not intended to be limiting in any way, the benefit of combination therapy in this preclinical model appeared to be greater when mono(methoxyPEG-N-butanamide)interleukin-15 administration was initiated by approximately D7. Residual CAR T cells in mice treated with mono(methoxyPEG-N-butanamide)interleukin-15 rejected rechallenge with Raji tumor cells administered more than 5 weeks after CAR T cell infusion.

[0174] In this lymphoma model, mono(methoxyPEG-N-butanamide) interleukin-15 administration was found to improve the antitumor efficacy and kinetics of administered CD19 CAR T cells. More specifically, demonstrating superior efficacy compared to CAR T cells alone, the combination of CAR T cells with mono(methoxyPEG-N-butanamide) interleukin-15 significantly reduced tumor burden and 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 mice treated with mono(methoxyPEG-N-butanamide) interleukin-15 (0.03 mg / kg) / CAR T cells survived 70 days after tumor injection, compared with none of the mice receiving the vehicle control surviving by day 14 and none of the mice treated with CAR T cells alone surviving by day 59. The results are shown in Figure 4A (bioluminescence imaging results) and for mice treated with the 0.30 mg / kg dose of MPBA-IL15 in Figure 7A (survival). Furthermore, mice previously treated with mono(methoxyPEG-N-butanamide) interleukin-15 and CAR T cells were able to reject Raji tumor rechallenge, supporting CAR T cell survival and potentially long-term memory CAR T formation; a striking result is shown in FIG. 10, which demonstrates that a long-acting interleukin-15 agonist such as MPBA-IL-15, when administered in combination with CAR-T cell therapy, not only significantly reduces tumor burden but also exhibits the ability to eradicate 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 Cohorts of mice (n = 5–6 / group) were treated with 3 × 10 4 The development of ROR1+ lung tumors was induced by intratracheal infection with pfu of Cre-ffluc-hROR1 lentivirus. At 12 and 15 weeks after infection, mice were treated with 100 mg / kg cyclophosphamide for lymphodepletion and 6 × 10 6ROR1 CAR T cells or control T cells (1:1 ratio of CD8:CD4) were adoptively transferred intravenously. ROR1 (receptor tyrosine kinase-like orphan receptor 1) is expressed in many malignancies, including non-small cell lung cancer (NSCLC) and a subgroup of triple-negative breast cancer (TNBC). Mice were inoculated with 5 × 10 CAR T cells every other day for 8 days to support engraftment of the transferred T cells. 4 IU of IL-2 intraperitoneally. Beginning on the day of T cell transfer, a subgroup of mice was treated with 0.33 mg / kg MPBA-IL15 intravenously every 7 days. The preclinical treatment protocol is shown in FIG.

[0177] Tumor burden was quantified by acquiring serial 1 mm images across the entire lung and summing the tumor area across all images to quantify tumor volume. 17 weeks after infection, all mice were euthanized and whole lungs were analyzed by flow cytometry and immunohistochemistry. To distinguish lung tumor-infiltrating cells from contaminating cells in the circulating blood, mice were injected intravenously with PE-conjugated anti-CD45 antibody 5 min prior to euthanasia to label all immune cells in the circulating blood and PE - This allowed the cells to be determined as non-vascular lung parenchymal cells. Lungs were analyzed by IHC staining for CD8a and CD8 infiltration into the tumor; staining was quantified using HALO software.

[0178] The results are shown in Figure 12A (change in tumor volume (%) for mice in the different treatment groups: control T cells (rectangles), control T cells with MPBA-IL15 (▲), ROR1 CAR T cells (▼), and ROR1 CAR T cells with MPBA-IL15 (◇)); Figure 12B (change in tumor volume (percent) versus weeks post-infection for individual mice treated with ROR1 CAR T cell monotherapy (18.5% regression for the group); Figure 12C (change in tumor volume (percent) versus weeks post-infection for individual mice treated with ROR1 CAR T cell and MPBA-IL-15 dual combination therapy (44.4% regression for the group); Figure 13A (change in tumor volume (percent) versus weeks post-infection for the different treatment groups, expressed as percentage of viable cells in the spleen and tumor, respectively) 13B (CD8 cell frequency expressed as percentage of live cells in spleen and tumor, respectively, for different treatment groups), and Figure 14A, B, C, and D (IHC staining showing that MPBA-IL-15, an illustrative long-acting IL-15 agonist, 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 treating subjects with cancer, and enhances the trafficking and persistence of ROR1 CAR T cells in cancerous lung tissue; thus, the present disclosure provides a novel and uniquely advantageous immunotherapeutic approach to treat 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 numbers 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. Cells were either untreated or treated with mono(methoxyPEG-N-butanamide) interleukin-15 (at concentrations of 1 ng / ml, 10 ng / ml, or 30 ng / ml). IFNγ and TNFα production were analyzed by Luminex after 24 hours of co-culture. CAR T cell numbers and intracellular expression of bcl-2 and activated caspase 3 were determined after 5 days of co-culture. Expression of bcl-2 and activated caspase 3 was determined by flow cytometry. Results are shown in Figures 15A, 15B, and 16A-C.

[0181] Figure 15A provides IFNγ expression in pg / ml 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.

[0182] Figure 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 not treated with MPBA-IL15 or treated with MPBA-IL15 at concentrations of 1 ng / ml, 10 ng / ml, or 30 ng / ml.

[0183] Figure 16A shows CAR T cell proliferation (as fold expansion) for 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.

[0184] Figures 16B and 16C provide the expression of bcl-2 (in bcl-2 MFI) and activated (as caspase 3+ (%)) 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, respectively.

[0185] These results demonstrate that treatment of CAR T cells in vitro 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 following administration of CAR T cells in combination with mono(methoxyPEG-N-butanamide) interleukin-15 in a preclinical mouse lymphoma model NSG mice received Raji lymphoma cells on D-7, CAR-T cells on D0, and weekly injections of mono(methoxyPEG-N-butanamide) interleukin-15 (0.3 mg / kg) as described in more detail in Example 3 above starting on D7. Mice were euthanized on D8, 11, 14, 21, and 28 after CAR T cell infusion. 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] Bcl-2 expression in CAR T cells determined at D8 post-infusion for both CD8 (Figure 17A) and CD4 (Figure 17B) CAR T cells is shown in histograms (grey = mice with CAR T cells only, red and blue = mice that received CAR T cells and mono(methoxyPEG-N-butanamide) interleukin-15 (MPBA-IL15)).

[0188] Bcl-2 expression in CAR T cells, determined at D8 post-infusion, for both CD8 (Figure 18A) and CD4 (Figure 18B) CAR T cells, is also shown in bar graphs (black = mice with CAR T cells only, red = mice that received CAR T cells and mono(methoxyPEG-N-butanamide) interleukin-15 (MPBA-IL15)).

[0189] 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 from mice administered CAR T cells alone; Figure 19B relates to protein expression in CD8 CAR T cells from mice administered CAR T cells and mono(methoxyPEG-N-butanamide) interleukin-15 (MPBA-IL15), Figure 19C relates to protein expression in CD4 CAR T cells from mice administered CAR T cells alone; Figure 19D relates to protein expression in CD4 CAR T cells from mice administered CAR T cells and mono(methoxyPEG-N-butanamide) interleukin-15 (MPBA-IL15), where expression data is provided for CD5RA-CCR7- (orange), CD5RA+CCR7- (green), and CD5RA-CCR7+ (red). Graphs show mean ± SEM.

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

[0191] Example 7 clinical research A Phase 1b / 2, Open-Label, Multicenter, Dose-Escalation and Dose-Expansion Study of Mono(MethoxyPEG-N-Butanamide) Interleukin-15 in Combination with CD19-Directed CAR-T Therapy in Patients with B-Cell Non-Hodgkin's Lymphoma This is a phase 1b / 2, open-label, multicenter, dose-escalation and dose-expansion study of mono(methoxyPEG-N-butanamide) interleukin-15 (MPBA-IL15) in combination with CD19+ CAR-T in patients with diffuse lymphocytic B-cell lymphoma (DLBCL). The study is divided into a screening period, a treatment period, an end-of-treatment (eos) period, and a long-term follow-up period.

[0192] The MPBA-IL-15 starting dose in arm 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 recombinant human IL-15 (rhIL-15). Each vial of MPBA-IL-15 drug product contains the equivalent of 1.1 mg rhIL-15. This includes a 0.1 mg overage to ensure consistent withdrawal of the label claiming a 1.0 mg amount after reconstitution.

[0194] Duration of treatment Dose Escalation (Phase 1b): Patients receiving one of two commercially available CD19-directed chimeric antigen receptor T cell (CD19 CAR-T, Kymriah™ (tisagenlecleucel) or Yescarta® (axicabtagene ciloleucel)) therapies who meet safety inclusion criteria will receive intravenous (IV) MPBA-IL-15. During dose escalation, MPBA-IL-15 will be given to patients approximately 14 or 7 days (depending on assigned cohort) after a single dose of CD19 CAR-T infusion. Treatment with MPBA-IL-15 will be 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 Sponsor decision to stop the study. Patients who demonstrate clinical benefit based on the investigator's judgment may continue treatment with approval from the Medical Monitor.

[0195] Dose Expansion (Phase 2): Following determination of the phase 2 recommended dose (RP2D) of MPBA-IL-15 with any of the CD19 CAR-T products, the RP2D dose will be further explored in expansion cohorts during Phase 2. Treatment with MPBA-IL-15 will be 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's discretion, or sponsor's decision to stop the study. Patients showing clinical benefit based on the investigator's judgment may continue treatment with approval of the medical monitor.

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

[0197] Phase 2: To evaluate the efficacy of MPBA-IL-15 after CD19 CAR-T therapy by assessing 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 2: (i) To evaluate the overall response rate (ORR) of MPBA-IL-15 in combination with CD19 CAR-T therapy (ii) To evaluate the progression-free survival (PFS) of MPBA-IL-15 in combination with CD19 CAR-T therapy (iii) To evaluate the overall survival (OS) of MPBA-IL-15 in combination with CD19 CAR-T therapy (Phase 2 only) (iv) To 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) To characterize the pharmacodynamic (PD) effects of NKTR 255 in combination with CD19 CAR-T therapy. (vii) To evaluate the PD efficacy of CD19 CAR-T cells, including the duration of in vivo survival of adoptively transferred T cells and the phenotype of the remaining T cells. (viii) To evaluate the immunogenicity of MPBA-IL-15

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

[0200] Study population: Adults aged 18 years or older receiving CD19 CAR-T cells to treat relapsed / refractory (R / R) B-cell non-Hodgkin lymphoma (B-NHL) after two or more lines of systemic therapy, including diffuse lymphocytic B-cell lymphoma (DLBCL) (unspecified type), primary mediastinal large B-cell lymphoma (PMBCL; Yescarta only), 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 research facilities: Phase 1b: Approximately 5 North American sites Phase 2: Approximately 5 North American sites

[0203] Study design: The study is a Phase 1b / 2, open-label, multicenter study consisting of a dose escalation (Phase 1b) and dose expansion (Phase 2) portion.

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

[0205] [Table 4]

[0206] MPBA-IL-15 will be tested in sequential combinations, initially starting with Yescarta therapy. The study will begin with a starting dose of MPBA-IL-15 of 1.5 μg / kg IV, administered 14 days after Yescarta infusion (Cohort A). After establishing safety and tolerability at the three dose levels of MPBA-IL-15 administered 14 days (± 3 days) after Yescarta (Cohort A), the next cohorts, Kymriah (14 days after CAR-T infusion; Cohort B) and Yescarta (7 days after CAR-T infusion, Cohort C), will be initiated in parallel at confirmed safe dose levels. The doses tested can be tapered to the most recently 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 with the 14-day regimen. Dose escalation will continue exclusively with MPBA-IL-15 on a 7-day regimen until the MTD or RP2D is established. If the 7-day regimen proves to be untolerable, MPBA-IL-15 dose escalation on a 14-day regimen can begin again.

[0207] During the escalation phase of the study for dose level selection and determination of the MTD, a two-parameter Bayesian logistic regression model (BLRM) using the escalation with overdose control (EWOC) principle (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 predicated when at least six 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 may be opened to further explore 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 dose escalation and will be based on a review of all available data on safety, PK, PD, and optimal biological response of MPBA-IL-15. Additional RP2D patients may be enrolled to refine the RP2D, and a minimum of 6 patients (including any patients from dose escalation) dosed at the selected RP2D will be required to determine the RP2D.

[0209] Additional rules regarding dose escalation during Phase 1b are: -Intrapatient dose escalation will not be permitted. - Enrollment into new cohorts with escalating doses of MPBA-IL-15 cannot begin until the dose-limiting toxicity (DLT) time window has elapsed since the last patient in the previous cohort's first dose of MPBA-IL-15. The DLT time window is 21 days after MPBA-IL-15 administration. -Escalation to higher doses will only occur if there is experience with that dose in FIH studies (MPBA-IL-15-002). -For dose escalation cohorts, safety will be jointly evaluated by the sponsor medical monitor and the Safety Review Committee (SRC) prior to opening to the next cohort. - The dose level of MPBA-IL-15 for a given cohort may be reduced depending on the severity, duration, and frequency of toxicity observed at previous dose levels tested.

[0210] The decision to determine the RP2D of MPBA-IL-15 following CD19 CAR-T infusion can be made at any given dose level or starting date based on safety, PK, PD, or optimal biological response without reaching the MTD.

[0211] Phase 2 (dose expansion) In Phase 2 of this study, enrollment into dose expansion cohorts will begin once the RP2D has been established for each individual CD19 CAR-T product. The selection of a specific CD19 CAR-T product and the decision on the schedule for Phase 2 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 Phase 1b. Patients will receive IV MPBA-IL-15 14 or 7 days after CD19 CAR-T infusion at the RP2D and schedule determined in Phase 1b. Treatment with MPBA-IL-15 will be repeated every 21 days (i.e., every 3 weeks) for up to 8 cycles (6 months).

[0212] Key eligibility criteria Eligibility will be determined within 1 month prior to leukapheresis for CD19 CAR-T cell generation. If intensive bridging chemotherapy or radiation therapy is administered, eligibility criteria should be re-evaluated prior to lymphodepletion. Male or female patient, ≥ 18 years of age on the date of signing the Informed Consent Form (ICF) Eligible for commercial CD19 CAR-T cell therapy A confirmed diagnosis of B-NHL, including DLBCL (non-specific), PMBCL (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 with failure of autologous hematopoietic stem cell transplantation (ASCT) or ineligible for or not consenting to ASCT Nodular and / or extranodal disease with measurable fluorodeoxyglucose (FDG) avidity according to the Lugano classification (Cheson, 2014, supra) that can be accurately measured to ≥ 1.5 cm in at least one dimension Life expectancy >30 days Acceptable organ function defined as: Adequate pulmonary function, defined as grade ≤1 dyspnea and oxygen saturation ≥92% (room air). If these parameters are not met, at the treating physician's discretion, patients will be eligible if they have a PFT FEV1 ≥50% predicted and a diffusing capacity for carbon monoxide (DLCO; corrected) ≥40% predicted. Adequate cardiac function, as defined by a cardiologist as a left ventricular ejection fraction (LVEF) ≥ 45%, or an LVEF between 40% and 44%, and clearance Adequate renal function, defined as: Serum creatinine ≤ 1.5 × upper limit of normal (ULN) or eGFR ≥ 60 mL / min / 1.73 m 2 Adequate liver function defined as: - Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 3 x ULN - Bilirubin ≤ 2.0 mg / dL (excluding patients with Gilbert-Meulen-Gracht syndrome); patients with Gilbert-Meulen-Gracht syndrome may be included if their total bilirubin is ≤ 3.0 x ULN and direct bilirubin is ≤ 1.5 x ULN Adequate bone marrow banking (without transfusions) defined as: 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 to be assessed prior to first and subsequent MPBA-IL-15 doses Patients are eligible for MPBA-IL-15 infusion if they meet the following criteria: 1. Received CD19 CAR-T infusion 2.No sustained grade ≥1 cytokine release syndrome (CRS) (body temperature ≥38.0°C) on the day of MPBA-IL-15 infusion 3. No grade 4 CRS within 96 hours prior to MPBA-IL-15 infusion 4. No sustained grade ≥2 neurotoxicity on the day of MPBA-IL-15 infusion 5. No prior grade ≥3 neurotoxicity of a period >48 hours at any time prior to MPBA-IL-15 infusion 6.No intervention with tocilizumab and / or dexamethasone within 48 hours prior to MPBA-IL-15 infusion 7. No active, serious, or uncontrolled infections 8. There are no contraindications as assessed by the investigator. 9. Patient has adequate organ function prior to all administrations of MPBA-IL-15: a) AST and ALT levels ≤ 3 × ULN; b) Total bilirubin level ≤ 3 × ULN c)eGFR>30mL / min d) DLCO>40% e) LVEF>45%

[0214] Test products, doses, and modes of administration 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 or 0.9% normal saline for injection. 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 evaluation of safety will involve a continuing review of: Incidence of adverse events (AEs), including serious AEs (SAEs) and immune-mediated AEs (imAEs) · Clinical testing (blood and urine collection) Vital signs Electrocardiogram (ECG) Physical examination Combination 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 collection 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. Peak plasma concentrations (C max Pharmacokinetic parameters such as plasma concentration-time data, 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, where possible.

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

[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 systemic 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] When possible, fresh bone marrow biopsies will be collected pre-, during, and post-treatment according to a Schedule of Events for characterization of tumor cells and immune system activation. Evaluation will include assessment of changes in tumor-specific protein markers and immune cell populations in the tumor microenvironment. Archived tumor tissue samples will be collected, if available, and can be analyzed as well.

[0221] Efficacy: During screening for baseline evaluation and to establish eligibility for measurable disease, a total body (skull base to mid-thigh) 18F-FDG-positron emission tomography (PET) / computed tomography (CT) will be performed (SUV max Subsequent FDG-PET / CT for efficacy assessment by the Lugano classification (Cheson, 2014, supra) will be performed at week 4, month 3 (just before cycle 5), and then every 12 weeks until the subject discontinues the study, and at disease progression, if possible. Tumor biopsies will be obtained, if possible, at approximately baseline, during the first 4 weeks after CD19 CAR-T infusion, and at week 14; and at the investigator's discretion, at end of treatment (EOT).

[0222] Statistical methods: Safety: Safety assessments will include AEs, clinical laboratories, vital signs, physical examination, and ECG (central review). The incidence of DLTs will be assessed for each dose escalation cohort. All grade ≥3 treatment-emergent adverse events (TEAEs) will be outlined by system organ class and preferred term for each dose cohort separately in Phase 1b and Phase 2 of the study. TEAEs will be outlined by incidence, severity, and relationship to study drug. Immune-mediated AEs (imAEs) will be outlined separately.

[0223] Grade ≧3 laboratory and vital sign abnormalities will be outlined descriptively for each dose cohort during Phase 1b and Phase 2 of the study.

[0224] Efficacy: Efficacy assessments of 6-month CRR and ORR will be calculated with 95% confidence intervals (CI) based on rigorous methodology. Kaplan-Meier methods will be used for analysis of PFS, DOR, and OS. CRR at 6 months based on independent review committee (IRC) assessment will be the primary efficacy outcome and will be outlined using a modified intention-to-treat population. Investigator-assessed CRR at 6 months will also be assessed.

[0225] The primary analyses for all efficacy endpoints will be based on patients from the dose expansion part of the study and patients treated at the RP2D from the dose escalation part of the study.

[0226] Pharmacokinetics and Biomarkers: Pharmacokinetic parameters will be tabulated and summarized using descriptive statistics. Descriptive profiles for biomarkers will be estimated at each observation. Changes in biomarkers from pre-dose to each observation will also be assessed using descriptive profiles. The present invention provides, for example, the following items. (Item 1) 1. A method for treating a subject having cancer, comprising: (i) administering to a subject an adoptive cellular immunotherapy composition comprising T cells that have been modified to express a chimeric antigen receptor (CAR-T cell); (ii) Structure: [ka] (wherein IL-15 is an interleukin-15 moiety, (n) is an integer from about 150 to about 3,000, and -NH- represents an amino group in the IL-15 moiety.) and administering to the subject an IL-15 receptor agonist having The method includes: (Item 2) 2. The method of claim 1, wherein the adoptive cellular immunotherapy composition comprises CAR T cells that have been modified to express a CD19-directed chimeric antigen receptor. (Item 3) 3. The method according to claim 1 or 2, wherein step (i) and step (ii) are carried out sequentially in any order or substantially simultaneously. (Item 4) The method according to claim 1 or 2, wherein step (i) is carried out before step (ii). (Item 5) The method according to claim 1 or 2, wherein step (ii) is carried out before step (i). (Item 6) 3. The method according to claim 1 or 2, wherein both steps (i) and (ii) are carried out substantially simultaneously. (Item 7) 3. The method according to claim 1 or 2, wherein steps (i) and (ii) are both carried out on the same day. (Item 8) Step (ii) (a) performed on any one of days 1 to 7 after step (i) (e.g., on the 1st, 2nd, 3rd, 4th, 5th, 6th, or 7th day after step (i)); or (b) performed on any one of days 8 to 14 after step (i) (e.g., on the 8th, 9th, 10th, 11th, 12th, 13th, or 14th day after step (i)); or (c) performed on the 7th or 14th day after step (i); The method according to item 4. (Item 9) 9. The method of any one of items 1 to 8, comprising a single administration to the subject of an adoptive cellular immunotherapy composition comprising T cells that have been modified to express a chimeric antigen receptor during the course of treatment. (Item 10) 10. The method of any one of items 1 to 9, comprising multiple administrations of the IL-15 receptor agonist to the subject during the course of treatment. (Item 11) 11. The method of any one of items 1 to 10, wherein the adoptive cellular immunotherapy composition is administered by infusion and / or the IL-15 receptor agonist is administered by infusion. (Item 12) 12. The method according to any one of items 1 to 11, wherein the subject is a human. (Item 13) 13. The method according to any one of items 1 to 12, wherein the cancer is a blood cancer. (Item 14) 13. The method according to any one of items 1 to 12, wherein the cancer is a solid tumor. (Item 15) 14. The method of claim 13, wherein the cancer is lymphoma or leukemia. (Item 16) 16. The method of claim 15, wherein the cancer is a B-cell lymphoma. (Item 17) 17. The method of any one of items 1 to 16, wherein the IL-15 receptor agonist has the structure of formula (I), wherein (n) ranges from about 795 to about 1068. (Item 18) Item 18. The method of item 17, wherein (n) is in the range of about 840 to about 1023. (Item 19) 18. The method of claim 17, wherein, on average, (n) has a value of about 907 (e.g., the poly(ethylene) glycol portion of the molecule has a weight average molecular weight of about 40,000 daltons). (Item 20) 20. The method according to any one of items 1 to 19, which results in a beneficial response treatment that is enhanced beyond the response to treatment observed when administration is performed according to either step (i) or step (ii) alone. (Item 21) 21. The method of claim 20, wherein said beneficial response to treatment is based on an appropriate animal model. (Item 22) 22. The method of claim 21, wherein the model is an in vivo xenograft B cell lymphoma model. (Item 23) 23. The method of any one of items 21 or 22, wherein the beneficial response to said treatment is selected from a change (percent) in tumor volume (i.e., reduction in tumor burden) and total number of CAR-T cells in bone marrow or tumor tissue when assessed 45 days after tumor cell injection. (Item 24) 24. The method of any one of items 1 to 23, wherein the adoptive cellular immunotherapy composition comprises CD-19-directed genetically modified autologous T cells. (Item 25) In step (i), about 10 7 ~about 10 9 25. The method of any one of items 1 to 24, comprising administering to the subject 100 cells / kg of CAR-T cells. (Item 26) Approximately 0.2×10 6 cells / kg ~ approx. 6.0×10 8 Cells / kg, at least about 2.0 x 10 6 cells / kg ~ approx. 2.0×10 8 At least about 0.2 x 10 cells / kg 6 cells / kg ~ approx. 5.0×10 6 At least about 0.1 x 10 cells / kg 8 cells / kg ~ approx. 2.5×10 8 cells / kg, or at least about 0.6×10 8 cells / kg ~ approx. 6.0×10 8 26. The method of claim 25, comprising administering to the subject an amount of CAR-T cells selected from cells / kg. (Item 27) 27. The method according to any one of items 1 to 26, comprising administering about 0.10 to 50 μg / kg of the IL-15 receptor agonist to the subject. (Item 28) 28. The method according to item 27, comprising administering about 0.25 to 25 μg / kg of the IL-15 receptor agonist to the subject. (Item 29) 29. The method according to item 28, comprising administering about 0.25 to 15 μg / kg of the IL-15 receptor agonist to the subject. (Item 30) 30. The method of any one of items 1 to 29, comprising in step (i) a single administration of the adoptive cellular immunotherapy composition, and in step (ii) an initial administration of an IL-15 receptor agonist on any one of days 1 to 14 after step (i), followed by administration of an IL-15 receptor agonist every 21 days throughout the course of treatment. (Item 31) 31. The method of item 30, wherein the course of treatment comprises 1 to 24 cycles of administration of the IL-15 receptor agonist, or 3 to 20 cycles of administration of the IL-15 receptor agonist, or 4 to 15 cycles of administration of the IL-15 receptor agonist. (Item 32) A method of treating a subject having cancer by administering to the subject an adoptive cellular immunotherapy composition comprising T cells that have been modified to express a chimeric antigen receptor (CAR-T cell), comprising the compositions and methods provided in any one of items 1-31, and / or by the methods recited in any one of items 1-31, the improvement further comprising administering to the subject an IL-15 receptor agonist as described in any one of items 1-31, thereby providing an enhanced response to the treatment compared to a treatment of the subject comprising administration of the adoptive cellular immunotherapy composition alone (i.e., in the absence of an IL-15 receptor agonist).

Claims

1. 1. A combination for use in a method of treating a subject having cancer, the combination comprising an adoptive cellular immunotherapy composition comprising a T cell that has been modified to express a chimeric antigen receptor (CAR-T cell), and a T cell having the structure: 【Chemistry 6】 wherein IL-15 is an interleukin-15 moiety, (n) is an integer from about 150 to about 3,000, and ~NH~ represents an amino group of the IL-15 moiety. and wherein the method comprises administering to the patient an interleukin-15 (IL-15) receptor agonist having the formula: (i) administering the adoptive cellular immunotherapy composition to the subject; (ii) administering the IL-15 receptor agonist to the subject. A combination comprising:

2. 2. The combination of claim 1, wherein the adoptive cellular immunotherapy composition comprises CAR T cells that have been modified to express a CD19-directed chimeric antigen receptor.

3. 3. The combination of claim 1 or claim 2, wherein steps (i) and (ii) are carried out sequentially in any order or substantially simultaneously.

4. 3. A combination according to claim 1 or claim 2, wherein step (i) is carried out before step (ii).

5. 3. A combination according to claim 1 or claim 2, wherein step (ii) is carried out before step (i).

6. 3. The combination of claim 1 or claim 2, wherein both steps (i) and (ii) are carried out substantially simultaneously.

7. 3. The combination of claim 1 or claim 2, wherein steps (i) and (ii) are both performed on the same day.

8. Step (ii) is (a) performed on any one of days 1 to 7 after step (i); or (b) performed on any one of days 8 to 14 after step (i); or (c) performed on the 7th or 14th day after step (i); A combination according to claim 4.

9. 9. The combination of any one of claims 1 to 8, wherein the method comprises a single administration to the subject of the adoptive cellular immunotherapy composition comprising T cells that have been modified to express a chimeric antigen receptor during the course of treatment.

10. The combination according to any one of claims 1 to 9, wherein the method comprises multiple administrations of the IL-15 receptor agonist to the subject during the course of treatment.

11. The combination according to any one of claims 1 to 10, wherein said adoptive cellular immunotherapy composition is administered by infusion and / or said IL-15 receptor agonist is administered by infusion.

12. The combination according to any one of claims 1 to 11, wherein the subject is a human.

13. The combination according to any one of claims 1 to 12, wherein the cancer is a blood cancer.

14. The combination according to any one of claims 1 to 12, wherein the cancer is a solid tumor.

15. The combination according to claim 13, wherein the cancer is lymphoma or leukemia.

16. The method of claim 15, wherein the cancer is a B-cell lymphoma.

17. The combination according to any one of claims 1 to 16, wherein the IL-15 receptor agonist has the structure of formula (I), wherein (n) ranges from about 795 to about 1068.

18. 18. The combination of claim 17, wherein (n) is in the range of about 840 to about 1023.

19. 20. The combination of claim 17, wherein, on average, (n) has a value of about 907.

20. 20. A combination according to any one of claims 1 to 19, characterized in that the use of said combination for said method results in an enhanced beneficial response to the treatment beyond that observed when administration is carried out according to either step (i) or step (ii) alone.

21. The combination of claim 20, wherein the beneficial response to treatment is based on administration in an appropriate animal model.

22. The combination according to claim 21 , wherein the model is an in vivo xenograft B cell lymphoma model.

23. The combination of any one of claims 21 or 22, wherein the beneficial response to said treatment is selected from a decrease in tumor volume (i.e., a decrease in tumor burden) and a change (percentage) in the total number of CAR-T cells in bone marrow or tumor tissue when assessed 45 days after tumor cell injection.

24. The combination according to any one of claims 1 to 23, wherein the adoptive cellular immunotherapy composition comprises genetically modified autologous T cells directed against CD-19.

25. The method further comprises the step (i) of: ７ ~about 10 ９ The combination of any one of claims 1 to 24, comprising administering to said subject 100% of CAR-T cells / kg.

26. The method comprises: ６ cells / kg ~ approx. 6.0×10 ８ Cells / kg, at least about 2.0 x 10 ６ cells / kg ~ approx. 2.0×10 ８ At least about 0.2 x 10 cells / kg ６ cells / kg ~ approx. 5.0×10 ６ At least about 0.1 x 10 cells / kg ８ cells / kg ~ approx. 2.5×10 ８ cells / kg, or at least about 0.6×10 ８ cells / kg ~ approx. 6.0×10 ８ 26. The combination of claim 25, comprising administering to the subject an amount of CAR-T cells selected from the group consisting of cells / kg.

27. The combination according to any one of claims 1 to 26, wherein the method comprises administering to the subject about 0.10 to 50 μg / kg of the IL-15 receptor agonist.

28. 28. The combination of claim 27, wherein the method comprises administering to the subject about 0.25 to 25 μg / kg of the IL-15 receptor agonist.

29. 29. The combination of claim 28, wherein the method comprises administering to the subject about 0.25 to 15 μg / kg of the IL-15 receptor agonist.

30. 1. A combination for use in a method of treating a subject having cancer, the combination comprising an adoptive cellular immunotherapy composition comprising a T cell that has been modified to express a chimeric antigen receptor (CAR-T cell), and a T cell having the structure: 【Chemistry 6A】 wherein IL-15 is an interleukin-15 moiety, (n) is an integer from about 150 to about 3,000, and ~NH~ represents an amino group of the IL-15 moiety. and an IL-15 receptor agonist having the formula: The method further comprising: (i) administering a single dose of the adoptive cellular immunotherapy composition to the subject; and (ii) administering an initial dose of the IL-15 receptor agonist to the subject on any one of days 1 to 14 after step (i), followed by administration of the IL-15 receptor agonist every 21 days throughout the course of treatment. A combination comprising:

31. 31. The combination of claim 30, wherein the course of treatment comprises 1 to 24 cycles of administration of the IL-15 receptor agonist, or 3 to 20 cycles of administration of the IL-15 receptor agonist, or 4 to 15 cycles of administration of the IL-15 receptor agonist.

32. 32. An adoptive cellular immunotherapy composition for use in a method of treating a subject having cancer by administering the adoptive cellular immunotherapy composition to the subject, the adoptive cellular immunotherapy composition comprising T cells that have been modified to express a chimeric antigen receptor (CAR-T cells), the adoptive cellular immunotherapy composition being the adoptive cellular immunotherapy composition of any one of claims 1 to 31, the method further comprising administering to the subject an IL-15 receptor agonist by the method described in any one of claims 1 to 31, thereby providing a response to the treatment that is enhanced over a treatment of the subject comprising administration of the adoptive cellular immunotherapy composition alone.

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