HK40078882A - Chimeric antigen receptor t cell therapy - Google Patents

Description

Chimeric antigen receptor T cell therapy

Technical Field

The present application relates to CAR-T cells, methods of making the same, and methods of using the same to treat cancer.

Background

Human cancers are essentially composed of normal cells that have undergone genetic or epigenetic transformation to become abnormal cancer cells. Cancer cells express proteins and other antigens that are different from those expressed by normal cells. The body's innate immune system can use these abnormal tumor antigens to specifically target and kill cancer cells. However, cancer cells employ various mechanisms to prevent immune cells (such as T and B lymphocytes) from successfully targeting cancer cells. Human T cell therapy relies on ex vivo enriched or modified human T cells to target and kill cancer cells in a subject (e.g., a patient). The preparation of a population of T cells with an enriched concentration of naturally occurring T cells capable of targeting a tumor antigen has been developed; removing circulating tumor cells; and/or various techniques to genetically modify T cells to specifically target known cancer antigens, thereby generating populations of Chimeric Antigen Receptor (CAR) -T cells for cancer therapy. Some of these therapies have shown promising effects on tumor size and patient survival.

Disclosure of Invention

Any aspect or embodiment described herein may be combined with any other aspect or embodiment as disclosed herein. While the invention has been described in connection with specific embodiments thereof, the description is intended to be illustrative, and not to limit the scope of the invention, which is defined, in part, by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following embodiments/claims.

Embodiment 1: a method for treating Mantle Cell Lymphoma (MCL) or B-cell ALL in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a T cell product comprising autologous T cells expressing an anti-CD 19 Chimeric Antigen Receptor (CAR).

Embodiment 2: the method of embodiment 1, wherein the MCL and B cell ALL are relapsed or refractory MCL and B cell ALL, optionally wherein the MCL is a classical, blast-like (blastoid) and polymorphic MCL.

Embodiment 3: the method of any one of embodiments 1 and 2, wherein the MCL and B-cell ALL are refractory to, or have relapsed after, one or more therapies of chemotherapy, radiation therapy, immunotherapy (including T-cell therapy and/or treatment with antibodies or antibody-drug conjugates), autologous stem cell transplantation, or any combination thereof.

Embodiment 4: the method according to any one of embodiments 1 to 3, wherein the subject has received 1 to 5 prior treatments, optionally wherein at least one of the prior treatments is selected from autologous SCT, anti-CD 20 antibody, anthracycline-containing or bendamustine-containing chemotherapy and/or Bruton's Tyrosine Kinase Inhibitor (BTKi).

Embodiment 5: the method of embodiment 4, wherein the BTKi is ibrutinib (ibrutinib) or acarpinib (acalaburtinib).

Embodiment 6: the method of any one of embodiments 1-5, wherein R/R B cellular ALL is defined as refractory to first-line therapy (i.e., primary refractory), relapsed ≦ 12 months after first remission, relapsed or refractory after ≧ 2 previous lines of systemic therapy, or relapsed after allogeneic Stem Cell Transplantation (SCT), optionally wherein the subject is in need of Eastern Cooperative tumor Group performance status (Eastern Cooperative Oncology Group performance status) with bone medulloblasts, 0, or 1 of ≧ 5% and/or adequate kidney, liver, and heart function.

Embodiment 7: the method of any one of embodiments 1-6, wherein if the B-cell ALL subject has received prior bornatumumab (blinatumomab), the subject is in need of leukemic blast cells with CD19 expression ≧ 90%.

Embodiment 8: the method according to any one of embodiments 1 to 7, wherein the subject receives bridging therapy after leukapheresis and before opsonization/lymphodepletion chemotherapy.

Embodiment 9: the method of any one of embodiments 1 to 8, wherein the MCL subject receives a lymphodepleting chemotherapy regimen of both 500mg/m2 cyclophosphamide and 30mg/m2 fludarabine administered intravenously on each of the fifth, fourth and third days prior to T cell infusion.

Embodiment 10: the method of any one of embodiments 1 to 9, wherein the B-cell ALL subject receives a lymphodepletion regimen of 25mg/m2 fludarabine per day administered Intravenously (IV) on each of the fourth, third, and second days prior to T-cell infusion and 900mg/m2 cyclophosphamide per day IV administered on the second day prior to infusion.

Embodiment 11: the method according to any one of embodiments 8 to 10, wherein the MCL bridging therapy is selected from dexamethasone (e.g., 20 to 40mg per PO or IV administered daily, or equivalent, for 1 to 4 days); methylprednisolone, ibrutinib (e.g., 560mg PO daily) and/or acatinib (e.g., 100mg PO twice daily); an immunomodulator; R-CHOP, bendamustine; an alkylating agent; and/or a platinum-based agent, wherein the bridging therapy is administered after leukopheresis and is completed within, for example, 5 days or less prior to opsonic chemotherapy.

Embodiment 12: the method according to any one of embodiments 8 to 10, wherein the B-cell ALL subject may receive any one or more of the following bridging chemotherapy regimens:

embodiment 13: the method according to any one of embodiments 1 to 12, wherein the T cell product comprises CD4+ and CD8+ CAR T cells prepared from Peripheral Blood Mononuclear Cells (PBMCs) by positive enrichment and subsequent partial or complete depletion of circulating cancer cells.

Embodiment 14: the method of embodiment 13, wherein said PBMCs are enriched for T cells by positive selection of CD4+ and CD8+ cells, activated with anti-CD 3 and anti-CD 28 antibodies in the presence of IL-2, and then transduced with a replication-defective viral vector containing FMC63-28Z CAR, a Chimeric Antigen Receptor (CAR) comprising an anti-CD 19 single-chain variable fragment (scFv), CD28, and CD 3-zeta domain.

Embodiment 15: the method of any one of embodiments 13 and 14, wherein the T cell product comprises fewer cancer cells than a T cell product comprising T cells from a leukapheresis product that have not been positively selected for CD4+ and CD8+ T cells.

Embodiment 16: the method according to any one of embodiments 13 to 15, wherein the T cell product has other superior product attributes relative to T cell products comprising T cells from leukapheresis products that have not been positively selected/enriched for CD4+ and CD8+ T cells.

Embodiment 17: the method of embodiment 16, wherein the superior product attributes are selected from the group consisting of increased percentage of CDRA45+ CCR7+ (naive-like) T cells, decreased percentage of differentiated T cells, increased percentage of CD3+ cells, decreased IFN- γ production, decreased percentage of CD 3-cells.

Embodiment 18: the method according to any one of embodiments 1 to 17, wherein the MCL subject is administered one or more doses of 1.8 x 10 ⁶ 1.9 x 10 ⁶ 2 or 2X 10 ⁶ Individual CAR-positive live T cells/kg body weight with a maximum of 2 x 10 ⁸ (iii) individual CAR-positive viable T-cells (for 100kg and above patients), and administering to the B-cell ALL subject 0.5X 10 ⁶ 、1×10 ⁶ 2 or 2X 10 ⁶ Individual CAR-positive live T cells/kg body weight with a maximum of 2 x 10 ⁸ Individual CAR-positive live T cells (for patients of 100kg and above).

Embodiment 19: the method according to any one of embodiments 1 to 17, wherein the subject may receive a second infusion of anti-CD 19 CAR T cells if the subject has achieved a complete response to the first infusion, provided CD19 expression has been retained and neutralizing antibodies against the CAR are not suspect if progressed >3 months after remission, wherein response is assessed using the Lugano classification.

Embodiment 20: the method according to any one of embodiments 1 to 19, wherein the subject is monitored for signs and symptoms of Cytokine Release Syndrome (CRS) and neurotoxicity following T cell administration.

Embodiment 21: the method according to embodiment 20, wherein the subject is monitored daily for at least seven days, preferably for four weeks, after infusion for signs and symptoms of CRS and neurotoxicity.

Embodiment 22: the method according to any one of embodiments 20 and 21, wherein the signs or symptoms associated with CRS comprise fever, chills, fatigue, tachycardia, nausea, hypoxia and hypotension, and the signs or symptoms associated with neurological events comprise encephalopathy, seizures, changes in consciousness level, speech impairment, tremors and confusion.

Embodiment 23: the method according to any one of embodiments 20 to 22, wherein cytokine release syndrome in an MCL subject is managed according to the following protocol:

embodiment 24: the method according to any one of embodiments 20 to 23, wherein neurotoxicity in an MCL subject is managed according to the following protocol:

embodiment 25: the method according to any one of embodiments 1 to 24, wherein the MCL subject is a high risk patient determined by a Ki-67 tumor proliferation index ≧ 50% and/or the presence of the TP53 mutation.

Embodiment 26: the method according to any one of embodiments 20 to 22, wherein CRS in B-cell ALL subjects is managed according to the following protocol:

embodiment 27: the method according to any one of embodiments 20-22 and 26, wherein neurotoxicity in a B-cell ALL subject is managed according to one of two regimens:

embodiment 28: the method according to any one of embodiments 1-27, wherein the B-cell ALL subject may receive any one or more of the following bridging chemotherapy regimens:

embodiment 29: an autologous T cell expressing an anti-CD 19 CAR for use in a method for treating Mantle Cell Lymphoma (MCL) or B-cell ALL according to any one of embodiments 1 to 28.

Embodiment 30: use of an autologous T cell expressing an anti-CD 19 CAR in the manufacture of a medicament for the treatment of Mantle Cell Lymphoma (MCL) or B-cell ALL according to any one of embodiments 1 to 28.

Embodiment 31: a method of predicting:

(i) an objective response of a subject to CAR T cell therapy (optionally, the method of any one of embodiments 1 to 28), the predictive method comprising measuring peak CAR T cell levels and comparing them to a reference standard, wherein the objective response is positively correlated with the peak CAR T cell levels, wherein the objective response comprises both a complete response and a partial response, and wherein all responses are assessed using the Lugano classification;

(ii) (ii) minimal residual disease (e.g., at week 4) in response to CAR T cell therapy (optionally, according to the method of any one of embodiments 1 to 28), the predictive method comprising measuring peak CAR T cell levels and comparing them to a reference standard, wherein negative minimal residual disease is associated with higher peak CAR T cell levels;

(iii) (ii) CRS class 3 and/or Neurological Events (NEs) class 3 in a subject receiving CAR T cell therapy (optionally, according to the method of any one of embodiments 1 to 28), the predictive method comprising measuring peak CAR T cell expansion after treatment and comparing the level to a reference value, wherein the more CAR T cell expansion, the greater the chance of CRS class 3 and/or NE events class 3;

(iv) CRS grade ≧ 3 and/or NE grade ≧ 3, the predictive method comprising measuring peak levels of GM-CSF and IL-6 after CAR T-cell therapy (optionally according to the method of any one of embodiments 1-28) and comparing them to reference levels, wherein the higher the peak levels of these cytokines, the greater the chance of CRS grade ≧ 3 and/or NE grade 3;

(v) grade 3 CRS or more in a subject receiving CAR T cell therapy (optionally, according to the method of any one of embodiments 1 to 28), the predictive method comprising measuring the peak level of serum ferritin after CAR T cell therapy and comparing it to a reference level, wherein the higher the peak level of ferritin the greater the chance of grade 3 CRS;

(vi) grade ≧ 3 CRS, the prediction method comprising measuring peak levels of serum IL-2 and IFN- γ following CAR T-cell therapy (optionally, as described in any of embodiments 1-28) and comparing them to reference levels, wherein the higher the peak levels of IL-2 and IFN- γ, the greater the chance of grade ≧ 3 NE;

(vii) grade ≧ 3 CRS, the predictive method comprising measuring cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8, and/or Vascular Cell Adhesion Molecule (VCAM) after CAR T-cell treatment (optionally, described in any of embodiments 1-28) and comparing them to reference levels, wherein the higher the cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8, and/or Vascular Cell Adhesion Molecule (VCAM), the greater the chance of grade ≧ 3 NE;

(viii) ≧ 3-grade CRS after CAR T cell therapy (optionally, the method according to any one of embodiments 1-28), the predictive method comprising measuring peak serum levels of IL-15, IL-2 Ra, IL-6, TNF α, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1B, granzyme A, granzyme B and/or perforin after anti-CD 19 CART therapy, and comparing the levels to reference levels, wherein said peak serum level of IL-15, IL-2 Ra, IL-6, TNF α, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1B, granzyme A, granzyme B, and/or perforin is positively correlated with CRS grade 3 or higher;

(ix) CRS grade 3 or greater following CAR T cell therapy on B-cell ALL (optionally, the method of any one of embodiments 1-28), the predictive method comprising measuring the peak serum level of IL-15 following anti-CD 19 CAR T therapy and comparing said level to a reference level, wherein said peak serum level of IL-15 is negatively correlated with CRS grade 3 or greater;

(x) CRS grade 3 and/or NE grade 3 following CAR T cell therapy (optionally, a method according to any one of embodiments 1 to 28), the predictive method comprising measuring peak serum levels of IL-6, TNF α, GM-CSF, IL-10, MIP-1B, and granzyme B following anti-CD 19 CAR T therapy and comparing the levels to reference levels, wherein the peak serum levels of IL-6, TNF α, GM-CSF, IL-10, MIP-1B, and granzyme B are positively correlated with CRS grade 3 and NE grade 3;

(xi) Whether a patient will be MRD (10) at 4 weeks/month after CAR T cell treatment (optionally, according to any one of embodiments 1 to 28) ^-5 Sensitivity of (b) negative, the prediction method comprising measuring a peak serum level of IFN- γ, IL-6 and/or IL-2 after treatment and comparing said level to a reference standard, wherein said peak serum level of IFN- γ, IL-6 and/or IL-2 positively correlates with MRD negative at one month.

Embodiment 32: the method according to any one of embodiments 20 to 24, 26, 27 and 30 to 31, wherein the CRS and NE are obtained by Lee et al, Blood, 2014, 124: 188-.

Embodiment 33: the method of embodiment 31, wherein the reference standard is established by any method commonly used in the biomarker art, such as a quartile analysis of a patient population with known responses, toxicity ratings, and MRD levels.

Embodiment 34: the method of embodiment 31, wherein CAR T cell levels are measured by CAR gene copies per microgram DNA in the blood.

Embodiment 35: the method of any of embodiments 1-43, further comprising reducing the level/activity of a cytokine positively correlated with CRS ≧ 3 and/or NE ≧ 3 after the CAR T-cell infusion to reduce CRS ≧ 3 and/or NE ≧ 3.

Embodiment 36: a method of improving the effectiveness of CAR T cell therapy (e.g., classical, blast-like and polymorphic MCL and B cell ALL) in a subject in need thereof, the method comprising manipulating a T cell phenotype of the T cell product administered to the subject, optionally wherein the manipulating comprises increasing the number of CD3+ T cells, decreasing the number of CD 3-cells, increasing the number/percentage of CDRA45+ CCR7+ (naive-like) T cells and/or decreasing the number/percentage of differentiated cells in the T cell product, decreasing the level of IFN- γ production by the T cells during production, wherein the improvement is observed relative to the effectiveness of a T cell product prepared without any deliberate manipulation of the number/percentage of CDRA45+ CCR7+ (naive like) T cells and/or the number/percentage of differentiated cells in the T cell product.

Drawings

FIG. 1A-FIG. 1F: comparable pharmacodynamic profiles in the prognostic set defined by the Ki-67 proliferation index and a trend of increased cytokine levels in patients with mutant TP 53.

Fig. 2A-fig. 2I: peak levels of selected cytokines in serum are increased in patients achieving MRD negative status.

FIG. 3: ZUMA-3 study design. CAR, chimeric antigen receptor; DLT, dose limiting toxicity.

FIG. 4: ZUMA-3CONSORT diagram. AE is grade 3 lung mass (n ═ 1), grade 1 subdural hematoma (n ═ 1), and grade 3 febrile neutropenia (n ═ 1);AE is grade 4 sepsis (n ═ 1) and grade 5 sepsis (n ═ 1);one patient did receive KTE-X19 on the same occasion due to deep vein thrombosis (study exclusion criteria). AE, adverse event.

FIG. 5 is a schematic view of: subgroup analysis of complete response rates. BM, bone marrow; ORR, overall remission rate; SCT, stem cell transplantation.

FIG. 6: duration of response, relapse-free survival and overall survival at the dose level.

FIG. 7: peak CAR T cell expansion and association with response, minimal residual disease and toxicity.

FIG. 8: CAR T cell area under the curve and correlation with response, minimal residual disease and toxicity. AE, adverse event; AUC, area under the curve; CAR, chimeric antigen receptor; CRS, cytokine release syndrome; MRD, minimal residual disease.

FIG. 9: peak cytokine levels over time.

FIG. 10: inflammatory markers in blood serum samples at baseline and post-infusion peak. Values represent the lower quantitative limit of the assay used. The values represent the upper limit of quantitation for the assays used. AE, adverse event; CAR, chimeric antigen receptor; CCL, C-C motif ligands; CRP, C-reactive protein; CXCL, C-X-C motif chemokine ligand; FGFBF, fibroblast growth factor basic form; FLT-1, fms-related receptor tyrosine kinase 1GM-CSF, granulocyte-macrophage colony stimulating factor; ICAM-1, intercellular adhesion molecule 1; IFN, interferon; IL, interleukin; MCP, monocyte chemoattractant protein 1; MDC, macrophage-derived chemokine; MIP, macrophage inflammatory protein; PDL1, programmed death ligand 1; PLGF, placental growth factor; r α, receptor α; RA, receptor antagonists; SAA, serum amyloid a; SFASL, soluble Fas ligand; TARC, thymus and activation regulatory cytokines; TNF, tumor necrosis factor; VCAM, vascular cell adhesion protein; VEGF, vascular endothelial growth factor; VEGFC, vascular endothelial growth factor C; VEGFD, vascular endothelial growth factor D.

FIG. 11: correlation of serum biomarkers with cytokine release syndrome and neurological events. Values represent the lower quantitative limit of the assay used.The values represent the upper limit of quantitation for the assays used. CRP, C-reactive protein; CXCL, C-X-C motif chemokine ligand; GM-CSF, granulocyte-macrophage colony stimulating factor; IFN γ, interferon γ; IL, interleukin; IP, interferon gamma inducible protein; MCP, monocyte attractant protein; r α, receptor α; RA, receptor antagonists; SAA, serum amyloid a.

FIG. 12: pharmacodynamic profile of KTE-X19 between the MCL morphological subgroups. AUC, area under the curve; CAR, chimeric antigen receptor; CXCL10, C-X-C motif chemokine ligand 10; IFN-g, interferon gamma; IL, interleukin; MCL, mantle cell lymphoma; MCP-1, monocyte chemoattractant protein 1; MIP-1 β, macrophage inflammatory protein 1 β; PD-L1, programmed death ligand 1; PRF, perforin; r α, receptor α; TNF- α, tumor necrosis factor α.

FIG. 13: pharmacological profile of KTE-X19 across the MCL morphological subgroup.

FIG. 14: pharmacodynamic profile of KTE-X19 between the previous BTKi subgroups. AUC, area under curve; CAR, chimeric antigen receptor; CXCL10, C-X-C motif chemokine ligand 10; IFN-g, interferon gamma; IL, interleukin; MCL, mantle cell lymphoma; MCP-1, monocyte chemoattractant protein 1; MIP-1 β, macrophage inflammatory protein 1 β; PD-L1, programmed death ligand 1; PRF, perforin; r α, receptor α; TNF- α, tumor necrosis factor α.

FIG. 15: pharmacological profile of KTE-X19 among the previous BTKi subgroups.

FIG. 16: progressive response rates between subgroups.

Detailed Description

Each of the following terms shall have the meaning set forth below, unless the context clearly provides otherwise. Additional definitions are set forth throughout this application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, "The simple Dictionary of biomedical and Molecular Biology (The contact Dictionary of Biomedicine and Molecular Biology)", Juo, Pei-Show, 2 nd edition, 2002, CRC Press (CRC Press); "Dictionary of Cell and Molecular Biology (The Dictionary of Cell and Molecular Biology)", 3 rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology (Oxford Dictionary of Biochemistry and Molecular Biology), revised edition, 2000, Oxford University Press provides the skilled artisan with a general Dictionary of many of the terms used in this application.

Units, prefixes, and symbols are expressed in their international system of units (SI) accepted form. Numerical ranges include the numbers that define the range. The disclosure provided herein is not limiting to the various aspects of the present application, which can be referred to in the present specification as a whole. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Juo, "The Concise Dictionary of Biomedicine and Molecular Biology (The circumcise Dictionary of Biomedicine and Molecular Biology)", 2 nd edition, 2001, CRC Press (CRC Press); "Dictionary of Cell & Molecular Biology (The Dictionary of Cell & Molecular Biology)", 5 th edition, 2013, Academic Press; and "The Oxford Biochemistry And Molecular Biology Dictionary (The Oxford Dictionary Of Biochemistry And Molecular Biology)", edited by Cammacack et al, 2 nd edition, 2006, Oxford University Press, provides one Of skill in The art with a general Dictionary Of many Of The terms used in this disclosure.

The article "a/an" refers to "one or more" of any recited or listed component.

The term "about" or "consisting essentially of … refers to a value or composition that is within an acceptable error range for that value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "consisting essentially of …" can mean within 1 or more than 1 standard deviation, according to practice in the art. Alternatively, "about" or "consisting essentially of …" may mean a range of up to 10% (i.e., ± 10%). For example, about 3mg may include any amount between 2.7mg and 3.3mg (for 10%). With respect to biological systems or processes, the term can mean values of up to an order of magnitude or up to 5-fold. When certain values or compositions are provided in the present application and claims, unless otherwise indicated, the meaning of "about" or "consisting essentially of …" is inclusive of acceptable error ranges for the value or composition. Any concentration range, percentage range, ratio range, or integer range is inclusive of the value of any integer within the recited range and, where appropriate, fractions thereof (such as tenths and hundredths of integers), unless otherwise specified.

As used herein, the term "or" is understood to be inclusive and to encompass both "or" and "unless specifically stated or apparent from the context. The term "and/or" means that each of the two specified features or components is with or without the other. Thus, the term "and/or" as used in phrases such as "a and/or B" herein is intended to include "a and B," "a or B," "a" (alone) and "B" (alone); similarly, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

The terms "such as" and "i.e.," are used by way of example only, are not intended to be limiting, and are not to be construed as referring only to those items explicitly recited in the specification.

The term "or more", "at least", "over", etc., such as "at least one" include, but are not limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 105, 104, 106, 102, 107, 111, 112, 109, 111, 112, 103, 112, 45, 25, 40, 75, 25, 40, 75, 72, 75, 72, 7, 72, 7, 72, 7, 76, 72, 76, 72, 7, 77, 7, 72, 7, 76, 7, 25, 72, 76, 25, 72, 76, 25, 7, 76, 7, 76, 7, 76, 7, 9, 7, 9, 7, 9, 7, 9, 7, 9, 76, 7, 9, 76, 9, 7, and/80, 76, 7, 76, 7, 113. 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than the stated values. But also any larger number or fraction therebetween. The term "not more than" includes every value that is less than the stated value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 and 0 nucleotides. But also any smaller number or fraction therebetween.

The terms "plurality", "at least two", "two or more", "at least a second", etc., include, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 103, 105, 104, 106, 107, 111, 109, 111, 110, 109, 111, 110, 109, 110, 45, 46, 47, 60, 61, 60, 61, 25, 40, 25, 40, 63, 40, 63, 65, 76, 77, 76, 77, 76, 68, 76, 77, 68, 76, 68, 7, 80, 7, 25, and so on, 112. 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more. But also any larger number or fraction therebetween.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. It should be understood that wherever aspects are described herein in the language "comprising," other similar aspects are also provided that are described in terms of "consisting of …" and/or "consisting essentially of …. The term "consisting of …" excludes any element, step, or ingredient not specified in the claims. For Gray,53F.2d 520,11USPQ 255(CCPA 1931); davis,80USPQ 448,450(bd. app.1948) ("consisting of …" is defined as "the closed claim to include materials other than those recited except for impurities normally associated therewith"). The term "consisting essentially of …" limits the scope of the claims to the specified materials or steps and those materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

Unless otherwise indicated or apparent from the context, the term "about," as used herein, refers to a value or composition that is within an acceptable error range for the particular value or composition, as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "approximately" can mean within one or more than one standard deviation, according to practice in the art. "about" or "approximately" may mean a range of up to 10% (i.e., ± 10%). Thus, "about" may be understood as being within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5mg may include any amount between 4.5mg and 5.5 mg. Furthermore, especially for biological systems or processes, these terms may mean at most an order of magnitude or at most 5 times a certain value. When a particular value or composition is provided in the present disclosure, unless otherwise stated, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range for that particular value or composition.

As used herein, any concentration range, percentage range, ratio range, or integer range is to be understood as including the value of any integer within the range, and fractions thereof (such as tenths and hundredths of integers) as appropriate, unless otherwise specified.

The terms "activate," "activated," and the like refer to the state of a cell, including but not limited to an immune cell (e.g., a T cell), that is sufficiently stimulated to induce detectable cell proliferation. Activation may be associated with induced cytokine production and detectable effector function. The term "activated T cell" primarily refers to a T cell that is undergoing cell division. T cell activation can be characterized by increased T cell expression of one or more biomarkers, including but not limited to CD57, PD1, CD107a, CD25, CD137, CD69, and/or CD 71. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. patents 6,905,874, 6,867,041; and 6,797,514; and PCT publication WO 2012/079000, the contents of which are hereby incorporated by reference in their entirety. Typically, such methods comprise contacting cells (such as T cells) in a solution (such as a feed, culture, and/or growth medium) in the presence of certain cytokines (such as IL-2, IL-7, and/or IL-15) with an activator, stimulator, or co-stimulator (such as an anti-CD 3 antibody and/or an anti-CD 28 antibody) that can attach, coat, or bind to beads or other surfaces. Activators (such as anti-CD 3 antibody and anti-CD 28 antibody) attached to the same bead act as "surrogate" Antigen Presenting Cells (APCs). One example is The system, a CD3/CD28 activator/stimulator system for physiological activation of human T cells. In one embodiment, T cells are activated and stimulated to proliferate with certain antibodies and/or cytokines using the methods described in U.S. patents 6,040,177 and 5,827,642 and PCT publication WO2012/129514, the contents of which are hereby incorporated by reference in their entirety.

The term "Administering" (administration, etc.) refers to physically introducing an agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration for immune cells prepared by the methods disclosed herein include intravenous (i.v. or IV), intramuscular, subcutaneous, intraperitoneal, spinal cord, or other parenteral routes of administration (e.g., by injection or infusion). Parenteral routes of administration refer to modes of administration other than enteral and topical administration (typically by injection) and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. In one embodiment, the immune cells (e.g., T cells) prepared by the methods of the invention are administered by injection or infusion. Non-parenteral routes include topical, epidermal or mucosal routes of administration, e.g. intranasal, vaginal, rectal, sublingual or topical. Administration may also be performed once, twice, or multiple times over one or more extended periods of time. In the case of administration of one or more therapeutic agents (e.g., cells), the administration can be performed concomitantly or sequentially. Sequential administration includes administering one agent only after administration of another agent or agents has been completed.

The term "antibody" (Ab) includes, but is not limited to, an immunoglobulin that specifically binds an antigen. In general, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region may comprise three or four constant domains CH1, CH2, CH3 and/or CH 4. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region may comprise one constant domain CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The immunoglobulin may be derived from any commonly known isotype, including, but not limited to, IgA, secretory IgA, IgG, and IgM. The IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG 4. "isotype" refers to the Ab class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. For example, the term "antibody" includes both naturally occurring antibodies and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric antibodies and humanized antibodies; a human or non-human antibody; fully synthesizing an antibody; and single chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Where not explicitly stated, and unless the context indicates otherwise, the term "antibody" also includes antigen-binding fragments or portions of any of the foregoing immunoglobulins, monovalent and bivalent fragments or portions, and single chain antibodies.

"antigen binding molecule," "antibody fragment," and the like refer to any antibody portion that is less than the entire antibody. The antigen binding molecule may comprise antigen Complementarity Determining Regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab ', F (ab')2, and Fv fragments, dabs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. In one aspect, the CD19 CAR construct comprises an anti-CD 19 single chain FV. "Single chain Fv" or "scFv" antibody binding fragments comprise the heavy chain variable (V) of an antibody _H ) Domain and light chain variable (V) _L ) Domains, wherein the domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises V _H Domains and V _L A polypeptide linker between the domains, which enables the scFv to form the desired structure for antigen binding. All antibody-related terms used herein take their customary meanings in the art and are well understood by those of ordinary skill in the art.

"antigen" refers to any molecule that elicits an immune response or is capable of being bound by an antibody or antigen binding molecule. The immune response may involve antibody production, or activation of specific immunocompetent cells, or both. One skilled in the art will readily appreciate that any macromolecule, including virtually all proteins or peptides, can be used as an antigen. The antigen may be expressed endogenously, i.e. from genomic DNA, or may be expressed recombinantly. The antigen may be specific for a certain tissue (such as a cancer cell), or it may be expressed broadly. In addition, larger molecules fragments may serve as antigens. In some embodiments, the antigen is a tumor antigen.

The term "neutralizing" refers to an antigen binding molecule, scFv, antibody or fragment thereof that binds to a ligand and prevents or reduces the biological effect of the ligand. In some embodiments, the antigen binding molecule, scFv, antibody or fragment thereof directly blocks a binding site on the ligand, or alters the binding capacity of the ligand by an indirect means (e.g., a structural or energetic change in the ligand). In some embodiments, the antigen binding molecule, scFv, antibody or fragment thereof prevents the protein to which it binds from performing a biological function.

The term "autologous" refers to any material that is derived from the same individual and is later reintroduced into the individual. For example, the engineered autologous cell therapy methods described herein involve harvesting lymphocytes from an individual (such as a donor or patient) which are then engineered to express a CAR construct and subsequently administered back into the same individual.

The term "allogeneic" refers to any material that is derived from one individual and subsequently introduced into another individual of the same species, such as allogeneic T cell transplantation.

"cancer" refers to a broad group of diseases characterized by uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade adjacent tissues and may also metastasize to distal parts of the body through the lymphatic system or blood stream. "cancer" or "cancerous tissue" can include tumors at various stages. In one embodiment, the cancer or tumor is in stage 0, such that, for example, the cancer or tumor is developing at a very early stage and has not metastasized. In another embodiment, the cancer or tumor is in stage I, such that, for example, the cancer or tumor is relatively small in size, does not spread into nearby tissue, and has not metastasized. In other embodiments, the cancer or tumor is in stage II or III, such that, for example, the cancer or tumor is greater than stage 0 or stage I, and it has grown into adjacent tissue, but it has not metastasized, except potentially to lymph nodes. In additional embodiments, the cancer or tumor is in stage IV, such that, for example, the cancer or tumor has metastasized. Stage IV may also be referred to as advanced or metastatic cancer.

As used herein, "anti-tumor effect" refers to a biological effect that can manifest as, but is not limited to, a reduction in tumor volume, inhibition of tumor growth, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, a reduction in the number/extent of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with a tumor. Anti-tumor effects may also refer to the prevention of tumorigenesis, e.g. vaccines.

The term "progression-free survival" (PFS) refers to the time from the date of treatment to the date of disease progression (according to general guidelines such as revised IWG Malignant Lymphoma Response Criteria (revised IWG Response Criteria for Malignant Lymphoma)) or death for any reason. The term "disease progression" can be assessed by measurement of malignant lesions on radiographs, or other methods should not be reported as an adverse event. Death due to disease progression without signs and symptoms can be reported as a primary tumor type (e.g., DLBCL). The term "duration of response" (DOR) refers to the time period between the subject's first objective response to the date of confirmation of disease progression (according to general guidelines such as revised IWG malignant lymphoma response criteria) or death. The term "overall survival" (OS) refers to the time from the date of treatment to the date of death.

"cytokine" refers to a non-antibody protein that is released by immune cells (including macrophages, B cells, T cells, and mast cells) to transmit an immune response. In one embodiment, one or more cytokines are released in response to therapy. In other embodiments, those cytokines secreted in response to therapy may indicate or suggest an effective therapy. In one embodiment, "cytokine" refers to a non-antibody protein released by one cell in response to contact with a specific antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell. As used herein, "cytokine" refers to a protein released by one cell population that acts on another cell as an intercellular mediator. The cytokine may be expressed endogenously by the cell or administered to the subject. Cytokines can be released by immune cells (including macrophages, B cells, T cells, and mast cells) to spread the immune response. Cytokines can induce a variety of responses in recipient cells. Cytokines may include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute phase proteins. For example, homeostatic cytokines, including Interleukin (IL)7 and IL-15, promote immune cell survival and proliferation, and pro-inflammatory cytokines may promote inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and Interferon (IFN) γ. Examples of proinflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, Tumor Necrosis Factor (TNF) - α, TNF- β, Fibroblast Growth Factor (FGF)2, granulocyte macrophage colony stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1(sICAM-1), soluble vascular cell adhesion molecule 1(sVCAM-1), Vascular Endothelial Growth Factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme a, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid a (saa).

A "chemokine" is a cytokine that mediates chemotaxis or directed movement of cells. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemotactic protein 1(MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1 alpha (MIP-1 alpha, MIP-1a), MIP-1 beta (MIP-1b), gamma-inducing protein 10(IP-10), and thymus activation-modulating chemokine (TARC or CCL 17).

"therapeutically effective amount," "therapeutically effective dose," and the like, refer to an amount produced by the methods of the invention (resulting in a T cell product) and when used alone or in combination with another therapeutic agent, protects or treats a subject from the onset of disease or promotes disease regression (e.g., by decreasing the severity of disease symptoms, increasing the frequency and duration of disease asymptomatic phases, and/or preventing injury or loss due to disease affliction)Demonstrable) of cells (such as immune cells or engineered T cells). The ability to promote disease regression can be assessed using a variety of methods known to the skilled artisan, such as in human subjects during clinical trials, in animal model systems predicting efficacy in humans, or by measuring the activity of the agent in vitro assays. In some embodiments, donor T cells for T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells for T cell therapy are obtained from a subject that is not a patient. The T cells may be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells can be at least about 10 ⁴ At least about 10 cells ⁵ At least about 10 cells ⁶ At least about 10 cells ⁷ At least about 10 cells ⁸ At least about 10 cells ⁹ Or at least about 10 ¹⁰ And (4) respectively. In another embodiment, the therapeutically effective amount of T cells is about 10 ⁴ One cell, about 10 ⁵ One cell, about 10 ⁶ One cell, about 10 ⁷ One cell or about 10 ⁸ And (4) cells. In some embodiments, the therapeutically effective amount of CAR T cells is about 2 x 10 ⁶ Individual cell/kg, about 3X 10 ⁶ Individual cell/kg, about 4X 10 ⁶ Individual cell/kg, about 5X 10 ⁶ Individual cell/kg, about 6X 10 ⁶ Individual cell/kg, about 7X 10 ⁶ Individual cell/kg, about 8X 10 ⁶ Individual cell/kg, about 9X 10 ⁶ Individual cell/kg, about 1X 10 ⁷ Individual cell/kg, about 2X 10 ⁷ Individual cell/kg, about 3X 10 ⁷ Individual cell/kg, about 4X 10 ⁷ Individual cell/kg, about 5X 10 ⁷ Individual cell/kg, about 6X 10 ⁷ Individual cell/kg, about 7X 10 ⁷ Individual cell/kg, about 8X 10 ⁷ Individual cell/kg or about 9X 10 ⁷ Individual cells/kg. In some embodiments, the therapeutically effective amount of CAR-positive living T cells is between about 1 x 10 per kg body weight ⁶ And about 2X 10 ⁶ Between CAR-positive living T cells up to about 1X 10 ⁸ Maximum dose of individual CAR-positive live T cells. In some embodiments, the therapeutically effective amount of CAR-positive living T cells is between about 0.4 x 10 ⁸ And about 2X 10 ⁸ Individual CAR positive activityBetween T cells. In some embodiments, the therapeutically effective amount of CAR-positive living T cells is about 0.4 x 10 ⁸ About 0.5X 10 ⁸ About 0.6X 10 ⁸ About 0.7X 10 ⁸ About 0.8X 10 ⁸ About 0.9X 10 ⁸ About 1.0X 10 ⁸ About 1.1X 10 ⁸ About 1.2X 10 ⁸ About 1.3X 10 ⁸ About 1.4X 10 ⁸ About 1.5X 10 ⁸ About 1.6X 10 ⁸ About 1.7X 10 ⁸ About 1.8X 10 ⁸ About 1.9X 10 ⁸ Or about 2.0X 10 ⁸ Individual CAR-positive live T cells.

As used herein, the term "lymphocyte" can include a Natural Killer (NK) cell, a T cell, an NK-T cell, or a B cell. NK cells are a cytotoxic (to the cell) lymphocyte that represents a major component of the innate immune system. NK cells reject virus-infected tumors and cells through apoptosis or programmed cell death processes. They are called "natural killers" because they do not require activation to kill the cells. T cells play a major role in cell-mediated immunity (no participation of antibodies). T Cell Receptors (TCRs) distinguish themselves from other lymphocyte types. The thymus is a specialized organ of the immune system, primarily responsible for the maturation of T cells.

There are several types of "immune cells," including, but not limited to, macrophages (e.g., tumor-associated macrophages), neutrophils, basophils, eosinophils, granulocytes, natural killer cells (NK cells), B cells, T cells, NK-T cells, mast cells, Tumor Infiltrating Lymphocytes (TILs), bone marrow-derived suppressor cells (MDSCs), and dendritic cells. The term also includes precursors of these immune cells. Hematopoietic stem and/or progenitor cells can be derived from bone marrow, cord blood, adult peripheral blood after cytokine mobilization, and the like, by methods known in the art. Some precursor cells are those that can differentiate into lymphoid lineages, such as hematopoietic stem or progenitor cells of lymphoid lineages. Examples of additional immune cells that can be used in immunotherapy are described in U.S. publication 20180273601, which is incorporated by reference herein in its entirety.

T cell reductionThere are several types, namely: helper T cells (e.g., CD4+ cells, effector T cells) _EFF Cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells ((i) stem memory T cells _SCM Cells (e.g., naive cells) are CD45RO-, CCR7+, CD45RA +, CD62L + (L-selectin), CD27+, CD28+, and IL-7 Ra +, but they also express large amounts of CD95, IL-2R β, CXCR3, and LFA-1, and exhibit many of the functional attributes characteristic of memory cells); (ii) central memory T _CM The cells express L-selectin and are CCR7 ⁺ And CD45RO ⁺ (ii) they secrete IL-2 but do not secrete IFN γ or IL-4, whereas (iii) effector memory T _EM The cells do not express L-selectin or CCR7, but do express CD45RO and produce effector cytokines such as IFN γ and IL-4), regulatory T cells (Treg, suppressor T cells, or CD4 ⁺ CD25 ⁺ Regulatory T cells), natural killer T cells (NKTs), and γ δ T cells. T cells found within tumors are called "tumor infiltrating lymphocytes" (TILs). On the other hand, B cells play a major role in humoral immunity (with antibody involvement). It produces antibodies and antigens and functions as an Antigen Presenting Cell (APC), and is transformed into a memory B cell after being activated by antigen interaction. In mammals, immature B cells are formed in the bone marrow from which they are named.

By "naive" T cells is meant mature T cells that remain immunologically undifferentiated. After positive and negative selection in the thymus, T cells served as CD4 ⁺ Or CD8 ⁺ Naive T cells appeared. In their naive state, T cells express L-selectin (CD 62L) ⁺ ) IL-7 receptor alpha (IL-7R-alpha) and CD132, but they do not express CD25, CD44, CD69 or CD45 RO. As used herein, "immature" may also refer to exhibiting naive T cells or immature T cells (such as T cells) _SCM Cells or T _CM-- Cells) are selected. For example, immature T cells may express L-selectin (CD 62L) ⁺ ) One or more of IL-7R α, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2R β, CXCR3 and LFA-1. Naive or immature T cells can differentiate efficiently from the terminalT-responsive cells (such as T) _EM Cells and T _EFF Cells) were compared.

"T cell function" as referred to herein refers to the normal characteristics of a healthy T cell. T cell function may include T cell proliferation, T cell activity, and/or cytolytic activity. In one embodiment, the methods of the present application of producing T cells under certain oxygen and/or pressure conditions will increase one or more T cell functions, thereby making the T cells more suitable and/or more effective for therapeutic purposes. In some embodiments, T cells prepared according to the methods of the invention have increased T cell function compared to those in the absence of certain oxygen and/or pressure conditions. In other embodiments, T cells prepared according to the methods of the invention will have increased T cell proliferation compared to T cells cultured in the absence of certain oxygen and/or pressure. In additional embodiments, T cells prepared according to the methods of the invention have increased T cell activity compared to T cells cultured in the absence of certain oxygen and/or pressure. In additional embodiments, T cells prepared according to the methods of the invention have increased cytolytic activity compared to T cells cultured in the absence of certain oxygen and/or pressure.

The term "proliferation" of a cell refers to the ability of a cell to grow digitally by cell division. Proliferation can be measured by staining cells with carboxyfluorescein succinimidyl ester (CFSE). Cell proliferation may occur in vitro (e.g., during T cell culture) or in vivo (e.g., after administration of an immune cell therapy (e.g., T cell therapy)). Cell proliferation can be measured or determined by methods described herein or known in the art. For example, cell proliferation can be measured or determined by Viable Cell Density (VCD) or Total Viable Cells (TVC). The VCD or TVC may be theoretical (an aliquot or sample is taken from the culture at a certain point in time to determine the number of cells, which is then multiplied by the culture volume at the beginning of the study) or actual (an aliquot or sample is taken from the culture at a certain point in time to determine the number of cells, which is then multiplied by the actual culture volume at a certain point in time). The term "T cell activity" refers to any activity common to healthy T cells. In one embodiment, T cell activity includes cytokine production (such as INF γ, IL-2, and/or TNF α). In other embodiments, T cell activity comprises production of one or more cytokines selected from the group consisting of interferon gamma (IFN γ or IFN- γ), tissue necrosis factor alpha (TNF α or IFN α), and both. The terms "cytolytic activity", "cytotoxicity", and the like, refer to the ability of a T cell to destroy a target cell. In one embodiment, the target cell is a cancer cell, such as a tumor cell. In other embodiments, the T cell expresses a Chimeric Antigen Receptor (CAR) or a T Cell Receptor (TCR), and the target cell expresses a target antigen.

The terms "genetically engineered," "gene editing," or "engineered" refer to methods of modifying the genome of a cell, including, but not limited to, deleting a coding region or a non-coding region or a portion thereof, or inserting a coding region or a portion thereof. In one embodiment, the modified cell is a lymphocyte (e.g., a T cell) obtainable from a patient or donor. The cells can be modified to express exogenous constructs, such as Chimeric Antigen Receptors (CARs) or T Cell Receptors (TCRs), that are incorporated into the genome of the cells.

The terms "transduction" and "transduced" refer to the process of introducing foreign DNA into cells by viral vectors (see Jones et al, "Genetics: principles and analysis", Boston: Jones & Bartlett publication (Boston: Jones & Bartlett publication.), (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr viral vector, a papillomavirus vector, a vaccinia viral vector, a herpes simplex viral vector, an adeno-associated vector, a lentiviral vector, or any combination thereof.

The chimeric antigen receptors (CARs or CAR-T) and T Cell Receptors (TCRs) of the present application are genetically engineered receptors. These engineered receptors can be readily inserted into and expressed by immune cells, including T cells, according to techniques known in the art. Using CARs, a single receptor can be programmed to both recognize a specific antigen and, upon binding the antigen, activate immune cells to attack and destroy cells carrying or expressing the antigen. When these antigens are present on tumor cells, the CAR-expressing immune cells can target and kill the tumor cells. In one embodiment, the cell prepared according to the present application is a cell having a Chimeric Antigen Receptor (CAR) or T cell receptor comprising an antigen binding molecule, a co-stimulatory domain and an activation domain. The co-stimulatory domain may comprise an extracellular domain, a transmembrane domain, and an intracellular domain. In one embodiment, the extracellular domain comprises a hinge or a truncated hinge domain.

By "immune response" is meant the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, Natural Killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules produced by any of these cells or the liver, including abs, cytokines, and complements, resulting in the selective targeting, binding, damage, destruction, and/or elimination of invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or normal human cells or tissues in the context of autoimmune or pathological inflammation, from the vertebrate body.

The term "immunotherapy" (immunological or immunological therapy, etc.) refers to the treatment of a subject suffering from a disease or at risk of developing a disease or suffering from a relapse of a disease by a method that includes inducing, enhancing, inhibiting or otherwise altering an immune response. Examples of immunotherapy include, but are not limited to, T cell and NK cell therapy. T cell therapy may include adoptive T cell therapy, Tumor Infiltrating Lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy, and allogeneic T cell transplantation. One skilled in the art will recognize that the methods of preparing immune cells disclosed herein will enhance the efficacy of any cancer or transplanted T cell therapy. Examples of T cell therapies are described in U.S. patent publication nos. 2014/0154228 and 2002/0006409; U.S. patent nos. 7,741,465; 6,319,494, respectively; and 5,728,388; and PCT publication No. WO 2008/081035, which are incorporated by reference in their entirety.

The term "engineered autologous cell therapy" (abbreviated as "eACT ^TM ", also known as adoptive cell transfer) is the process by which a patient's own T cells are collected and these cells are subsequently genetically engineered to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. T cells can be engineered to express, for example, a Chimeric Antigen Receptor (CAR) or a T Cell Receptor (TCR). CAR positive (+) T cells are engineered to express an extracellular single chain variable fragment (scFv) specific for a tumor antigen, linked to an intracellular signaling moiety comprising a costimulatory domain and an activation domain. The co-stimulatory domain may be a signaling region derived from: CD28, CTLA4, CD16, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death protein 1(PD-1), programmed death ligand 1(PD-L1), inducible T cell costimulatory factor (ICOS), ICOS-L, lymphocyte function-associated antigen 1(LFA-1(CDl la/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276(B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD 57379 5), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signal transduction lymphocyte activating molecule (SLAM protein), activating cell receptor, BTLA, ligand, ICAM-1, B-24-IL-3, CDS-59h 3, ICAM-5, and ICAM, GITR, BAFFR, LIGHT, HVEM (LIGHT TR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19 alpha, CD19 beta, IL2 19 gamma, IL7 19 alpha, ITGA 19, VLA 19, CD49 19, ITGA 19, VLA-6, CD49 19, ITGAD, CDl, ITGAE, CD103, ITGAL, CDLAL, LFA-1, ITGAM, CDlb, ITGAX, CDlc, ITGBL, CD19, IT6854, CD19, LFA-1, ITGB 19, TNGF 19, TNFR 19, TNGFR 19, CD 6857 CD19, 6857 CD 6857-19, 6857-19, 6857-19, 6857-19, 6857-19, CD19, 6857-19, CD19, CD 6857-19, CD 6857B, CD19, CD 6857B, CD19, CD 6857B, CD19, CD 6857B, CD19, 6857, CD 6857, 19, CD 6857, CD19, CD 6857, CD 19. Activation The domains may be derived, for example, from CD3, such as CD3 ζ, epsilon, delta, gamma, and the like. In one embodiment, the CAR is designed to have two, three, four, or more co-stimulatory domains. CAR scFv can be designed to target, for example, CD19, which CD19 is a transmembrane protein expressed by cells in the B cell lineage (including ALL normal B cell and B cell malignancies, including but not limited to NHL, CLL and non-T cell ALL). Exemplary CAR + T cell therapies and constructs are described in U.S. patent publications 2013/0287748, 2014/0227237, 2014/0099309, and 2014/0050708, which are hereby incorporated by reference in their entirety.

As used herein, "co-stimulatory signal" refers to a signal that elicits a T cell response (such as, but not limited to, proliferation and/or up-or down-regulation of key molecules) in combination with a primary signal such as a TCR/CD3 linkage.

As used herein, "co-stimulatory ligand" includes molecules on antigen presenting cells that specifically bind to cognate co-stimulatory molecules on T cells. Binding of the co-stimulatory ligand provides a signal that mediates T cell responses including, but not limited to, proliferation, activation, differentiation, etc. The co-stimulatory ligand induces a signal in addition to the primary signal provided by the stimulatory molecule, for example, through the binding of the T Cell Receptor (TCR)/CD3 complex to a peptide-loaded Major Histocompatibility Complex (MHC) molecule. Costimulatory ligands can include, but are not limited to, 3/TR6, 4-1BB ligand, agonists or antibodies that bind Toll ligand receptors, B7-1(CD80), B7-2(CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpes virus invasion mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT)3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), ligand that specifically binds to B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or Programmed Death (PD) L1. Costimulatory ligands include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, ligands that specifically bind to CD83, lymphocyte function-associated antigen 1(LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14(TNFSF14 or LIGHT).

A "co-stimulatory molecule" is a cognate binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, "costimulatory molecules" are cognate binding partners on T cells that specifically bind to costimulatory ligands, thereby mediating costimulatory responses of T cells, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, 4-1BB/CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 33, CD 45, CD100(SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160(BY55), CD 55, CD19 55, CD247, CD 55, CD 685276 (B55-H55), CD 55 (alpha; beta; delta; epsilon; gamma; zeta), CD 55, CD 68549, CD 55, CDI-55, CDI 55, GAI-55, CDI-55, CDI-55, CDI-55, CDI-6857-55, CDI-6857-55, CDI-6857, CDI-55, CDI-6857, CDI-55, CDI-6857-55, CDI-6857, CDI-55, CDI-685, CDI-6857, CDI-55, CDI-685, CDI-55, CDI-685, CDI-6857, CDI-55, CDI-6852-6857, CDI-II, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9(CD229), lymphocyte function-associated antigen-1 (LFA-1(CDl la/CD18), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), OX 2, PAG/82bp, PD-1, PSGL1, SELPLG (CD162), signal transducing lymphocyte activating molecules, SLAM (SLAMF 1; CD 150; IPO-3), SLAMF4(CD 244; 2B4), SLAMF6 (VLC-A; VLSLPF 87452), SLF 9376, SLNF 9376, TNFR2, TNFR 3646, TNFR-7, or their combinations.

In some aspects, the cells of the present application can be obtained by T cells obtained from a subject. In one aspect, the T cells can be obtained from, for example, Peripheral Blood Mononuclear Cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusionFluid, spleen tissue and tumors. Additionally, the T cells may be derived from one or more T cell lines available in the art. Various techniques known to the skilled person (such as FICOLL) may also be used ^TM Isolation and/or apheresis) to obtain T cells from a blood unit collected from a subject. In some aspects, cells collected by apheresis are washed to remove plasma fractions and placed in an appropriate buffer or culture medium for subsequent processing. In some aspects, the cells are washed with any solution (e.g., a solution with a neutralizing PH or PBS) or culture medium. It will be appreciated that a washing step may be used, such as by using a semi-automatic flow-through centrifuge, e.g. Cobe ^TM 2991 cell processor, Baxter CytoMate ^TM And the like. In some aspects, the washed cells are resuspended in one or more biocompatible buffers or other salt solutions with or without buffers. In some aspects, unwanted components of the blood apheresis sample are removed. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. patent publication 2013/0287748, which is hereby incorporated by reference in its entirety.

In some embodiments, the red blood cells are lysed and monocytes are depleted (e.g., by using a red blood cell lysate by PERCOLL) ^TM Centrifugation of the gradient) to separate T cells from PBMCs. In some embodiments, specific subpopulations of T cells, such as CD4+, CD8+, CD28+, CD45RA +, and CD45RO + T cells, are further isolated by positive or negative selection techniques known in the art. For example, enrichment of a population of T cells by negative selection can be accomplished using a combination of antibodies directed against surface markers specific to the negatively selected cells. In some embodiments, cell sorting and/or selection via negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells may be used. For example, to enrich for CD4+ cells by negative selection, monoclonal antibody cocktails typically comprise antibodies against CD8, CD11b, CD14, CD16, CD20, and HLA-DR. In some embodiments, flow cytometry and cell sorting are used to isolate cell populations of interest for use in the present disclosure.

In one embodiment, CD3+ T cells are isolated from PBMCs using Dynabeads coated with anti-CD 3 antibody. CD8+ and CD4+ T cells were further isolated separately by positive selection using CD8 microbeads (e.g., Miltenyi Biotec, germany) or CD4 microbeads (e.g., Miltenyi Biotec, germany).

In some embodiments, PBMCs are used directly for genetic modification of immune cells (such as CARs) using methods as described herein. In some embodiments, after PBMC isolation, T lymphocytes are further isolated, and both cytotoxic and helper T lymphocytes are sorted into naive, memory and effector T cell subpopulations, either before or after genetic modification and/or expansion.

One or more of the immune cells described herein can be obtained from any source, including, for example, a human donor. The donor may be a subject in need of an anti-cancer treatment (e.g., treatment with one of the immune cells produced by the methods described herein) (i.e., an autologous donor), or may be an individual who donates a lymphocyte sample (i.e., an allogeneic donor) to be used to treat a different individual or cancer patient after the population of cells produced by the methods described herein is produced. The immune cells may be differentiated from a population of hematopoietic stem cells in vitro or the immune cells may be obtained from a donor. The immune cell population can be obtained from a donor by any suitable method used in the art. For example, the lymphocyte population can be obtained by any suitable in vitro method, venipuncture, or other blood collection method by which a blood sample is obtained with or without lymphocytes. The lymphocyte population was obtained by apheresis. One or more immune cells can be collected from any tissue comprising one or more immune cells, including but not limited to a tumor. The tumor or portion thereof is collected from the subject and one or more immune cells are isolated from the tumor tissue. Any T cell can be used in the methods disclosed herein, including any immune cell suitable for use in T cell therapy. For example, one or more cells useful in the present application may be selected from the group consisting of: tumor Infiltrating Lymphocytes (TILs), cytotoxic T cells, CAR T cells, engineered TCs R T cells, natural killer T cells, dendritic cells, and peripheral blood lymphocytes. T cells can be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. Additionally, the T cells may be derived from one or more T cell lines available in the art. Various techniques known to the skilled person (such as FICOLL) may also be used ^TM Isolation and/or apheresis) to obtain T cells from a blood unit collected from a subject. T cells are also available from Artificial Thymus Organoid (ATO) cell culture systems, which replicate the human thymus environment to support efficient ex vivo differentiation of T cells from primary and reprogrammed pluripotent stem cells. Additional methods of isolating T cells for T cell therapy are disclosed in U.S. patent publication nos. 2013/0287748, PCT publication nos. WO2015/120096, and WO2017/070395, all of which are incorporated herein by reference in their entirety for the purpose of describing these methods. In one embodiment, the T cell is a tumor infiltrating leukocyte. In certain embodiments, the one or more T cells express CD8, e.g., is CD8 ⁺ T cells. In other embodiments, the one or more T cells express CD4, e.g., is CD4 ⁺ T cells. Additional methods of isolating T cells for T cell therapy are disclosed in U.S. patent publication nos. 2013/0287748, PCT publication nos. WO2015/120096, and WO2017/070395, all of which are incorporated herein by reference in their entirety for the purpose of describing these methods.

Immune cells and their precursor cells can be isolated by available methods (see, for example, Rowland-Jones et al, "Lymphocytes: Practical applications (Lymphocytes: A Practical Approach)," Oxford University Press, "New York (New York), 1999). Sources of immune cells or their precursors include, but are not limited to, peripheral blood, cord blood, bone marrow, or other hematopoietic cell sources. Negative selection methods can be used to remove cells that are not the desired immune cells. In addition, positive selection methods may isolate or enrich for desired immune cells or their precursors, or a combination of positive and negative selection methods may be employed. Monoclonal antibodies (mabs) can be used to identify markers associated with certain cell lineages and/or differentiation stages of both positive and negative selections. If certain types of cells (e.g., certain types of T cells) are to be isolated, various Cell surface markers or combinations of markers (including, but not limited to CD3, CD4, CD8, CD34 (for hematopoietic stem and progenitor cells), etc.) can be used to isolate the cells, as is well known in the art (see Kearse, T Cell Protocols: Development and Activation (T Cell Protocols: Development and Activation), Humana Press, Totwa Totta (Totowa N.J..) 2000, U.S.A.; De Libero, T Cell Protocols (T Cell Protocols), "Methods in Molecular Biology Methods (Methods in Molecular Biology), Vol 514, Humana Press, Totta Va, N.J. (2009)).

PBMCs can be used directly for genetic modification of immune cells (such as CARs). After PBMC isolation, T lymphocytes are further isolated, and both cytotoxic and helper T lymphocytes are sorted into naive, memory and effector T cell subpopulations, either before or after genetic modification and/or expansion. In one embodiment, CD8+ cells may be further classified as naive, central memory and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In other embodiments, expression of phenotypic markers of central memory T cells includes CCR7, CD3, CD28, CD45RO, CD62L, and CD127 and is negative for granzyme B. In some embodiments, the central memory T cell is a CD8+, CD45RO +, and CD62L + T cell. In a certain embodiment, the effector T cells are negative for CCR7, CD28, CD62L, and CD127 and positive for granzyme B and perforin. In additional embodiments, the CD4+ T cells may be further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory and effector cells by identifying cell populations with cell surface antigens.

The methods described herein further include enriching or preparing a population of immune cells obtained from a donor between harvesting from the donor and exposing one or more cells obtained from a donor subject. Enrichment of the immune cell (e.g., the one or more T cells) population can be achieved by any suitable separation The separation method includes, but is not limited to, using a separation medium (e.g., FICOLL-PAQUE) ^TM 、ROSETTESEP ^TM HLA total lymphocyte enrichment mixtures, lymphocyte isolation media (LSA) (MP Biomedical) catalog No. 0850494X), etc.), cell size, shape or density separation by filtration or panning, immunomagnetic separation (e.g., magnetically activated cell sorting system, MACS), fluorescent separation (e.g., fluorescence activated cell sorting system, FACS), or bead-based column separation.

In one embodiment, the T cells are obtained from a donor subject. In other embodiments, the donor subject is a human patient suffering from a cancer or tumor. In additional embodiments, the donor subject is a human patient not suffering from a cancer or tumor. Also provided are compositions or formulations comprising a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative and/or adjuvant. In certain embodiments, the composition or formulation comprises an excipient. The terms composition or formulation are used interchangeably herein. The terms composition, therapeutic composition, therapeutically effective composition, pharmaceutical composition, pharmaceutically effective composition, and pharmaceutically acceptable composition are used interchangeably herein. The composition may be selected for parenteral delivery, for inhalation, or for delivery through the digestive tract, such as oral administration. The compositions may be prepared by known methods by those skilled in the art. Buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically in the pH range of about 5 to about 8. When parenteral administration is contemplated, the compositions are in the form of a pyrogen-free, parenterally acceptable aqueous solution in a pharmaceutically acceptable vehicle, which aqueous solution comprises the compositions described herein, with or without an additional therapeutic agent. For example, the vehicle for parenteral injection is sterile distilled water, wherein the compositions described herein are formulated as a sterile isotonic solution with or without at least one additional therapeutic agent, which is suitably preserved. Preparation involves formulating the desired agent with a polymeric compound (such as polylactic or polyglycolic acid), beads, or liposomes to provide controlled or sustained release of the product, which is then delivered by depot injection. In addition, implantable drug delivery devices can be used to introduce desired therapeutic agents.

In some embodiments, donor T cells for T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells for T cell therapy are obtained from a subject that is not a patient. The T cells may be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells can be at least about 10 ⁴ At least about 10 cells ⁵ At least about 10 cells ⁶ At least about 10 cells ⁷ At least about 10 cells ⁸ At least about 10 cells ⁹ Or at least about 10 ¹⁰ And (4) respectively. In another embodiment, the therapeutically effective amount of T cells is about 10 ⁴ One cell, about 10 ⁵ One cell, about 10 ⁶ One cell, about 10 ⁷ One cell or about 10 ⁸ And (4) cells. In some embodiments, the therapeutically effective amount of CAR T cells is about 2 x 10 ⁶ Individual cell/kg, about 3X 10 ⁶ Individual cell/kg, about 4X 10 ⁶ Individual cell/kg, about 5X 10 ⁶ Individual cell/kg, about 6X 10 ⁶ Individual cell/kg, about 7X 10 ⁶ Individual cell/kg, about 8X 10 ⁶ Individual cell/kg, about 9X 10 ⁶ Individual cell/kg, about 1X 10 ⁷ Individual cell/kg, about 2X 10 ⁷ Individual cell/kg, about 3X 10 ⁷ Individual cell/kg, about 4X 10 ⁷ Individual cell/kg, about 5X 10 ⁷ Individual cell/kg, about 6X 10 ⁷ Individual cell/kg, about 7X 10 ⁷ Individual cell/kg, about 8X 10 ⁷ Individual cell/kg or about 9X 10 ⁷ Individual cells/kg. In some embodiments, the therapeutically effective amount of CAR-positive living T cells is between about 1 x 10 per kg body weight ⁶ And about 2X 10 ⁶ Between CAR-positive living T cells up to about 1X 10 ⁸ Maximum dose of individual CAR-positive live T cells.

As used herein, "patient" includes any person who suffers from a disease or disorder, including cancer (e.g., lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein. The term "donor subject" refers to a subject whose cells have been obtained for further in vitro engineering. The donor subject can be a cancer patient to be treated with a population of cells produced by the methods described herein (i.e., an autologous donor), or can be an individual who donates a lymphocyte sample (i.e., an allogeneic donor) to be used to treat a different individual or cancer patient after the population of cells produced by the methods described herein is produced. Those subjects that receive cells prepared by the methods of the invention may be referred to as "recipient subjects".

The term "stimulation" or "stimulating", etc., refers to a primary response induced by the binding of a stimulating molecule to its cognate ligand, wherein the binding mediates a signaling event. A "stimulatory molecule" is a molecule on a T cell (e.g., the T Cell Receptor (TCR)/CD3 complex) that specifically binds to a cognate stimulatory ligand presented on an antigen presenting cell. A "stimulatory ligand" is a ligand that, when presented on an antigen presenting cell (e.g., an artificial antigen presenting cell (aAPC), dendritic cell, B cell, etc.), can specifically bind to a stimulatory molecule on the T cell, thereby mediating a primary response (including but not limited to activation, initiation of an immune response, proliferation, etc.) of the T cell. Stimulatory ligands include, but are not limited to, MHC class I molecules loaded with peptides, anti-CD 3 antibodies, superagonist anti-CD 28 antibodies, and superagonist anti-CD 2 antibodies. As used herein, "activated" or "active" refers to T cells that have been stimulated. Active T cells can be characterized by expression of one or more markers selected from the group consisting of CD137, CD25, CD71, CD26, CD27, CD28, CD30, CD154, CD40L, and CD 134.

The term "exogenous activation material" refers to any activation substance derived from an external source. For example, exogenous anti-CD 3 antibodies, anti-CD 28 antibodies, IL-2, exogenous IL-7, or exogenous IL-15 can be obtained commercially or produced recombinantly. "exogenous IL-2", "exogenous IL-7" or "exogenous IL-15" when added to or contacted with one or more T cells means that the T cells do not produce such IL-2, IL-7 and/or IL-15. The T cells prior to mixing with "exogenous" IL-2, IL-7, or IL-15 may contain trace amounts (i.e., endogenous "exogenous" IL-2, IL-7, or IL-15) produced by the T cells or isolated from the subject with the T cells. One or more of the T cells described herein can be contacted with an exogenous anti-CD 3 antibody, an anti-CD 28 antibody, an "exogenous" IL-2, IL-7, and/or IL-15 by any means known in the art, including adding isolated "exogenous" IL-2, IL-7, and/or IL-15 to the culture medium, including an anti-CD 3 antibody, an anti-CD 28 antibody, an "exogenous" IL-2, IL-7, and/or IL-15 in the culture medium, or expressing "exogenous" IL-2, IL-7, and/or IL-15 by one or more cells in culture other than the one or more T cells, such as by a feeder layer.

As used herein, the term "in vitro cell" refers to any cell cultured ex vivo. In one embodiment, the in vitro cell comprises a T cell.

The term "persistence" refers, for example, to the ability of one or more transplanted immune cells or their progenitors (e.g., differentiated or mature T cells) administered to a subject to remain in the subject at a detectable level for a period of time. As used herein, increasing the persistence of one or more transplanted immune cells or their progenitors (e.g., differentiated or mature T cells) refers to increasing the amount of time that the transplanted immune cells can be detected in a subject following administration. For example, the in vivo persistence of the one or more transplanted immune cells may be increased by at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months. As such, the in vivo persistence of the one or more transplanted immune cells may be increased by at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 4.5 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, or at least about 10 fold as compared to the one or more transplanted immune cells not prepared by the inventive methods disclosed herein.

The terms "reduce" and "decrease" are used interchangeably herein and indicate any change that is less than the original value. ' subtract"Low" and "reduced" are relative terms requiring a comparison between before and after measurement. "reduce" and "reducing" include complete depletion. As used herein, the term "modulating" T cell maturation refers to controlling the maturation and/or differentiation of one or more cells (such as T cells) using any of the interventions described herein. For example, modulation refers to inactivation, delay, or inhibition of T cell maturation. In another example, modulation refers to accelerating or promoting T cell maturation. The term "delay or inhibition of T cell maturation" refers to the maintenance of one or more T cells in an immature or undifferentiated state. For example, "delaying or inhibiting T cell maturation" may refer to maintaining T cells in naive or T _CM State and progress to T _EM Or T _EFF- The state is reversed. Furthermore, "delaying or inhibiting T cell maturation" may refer to increasing or enriching immature or undifferentiated T cells (e.g., naive T cells and/or T cells) in a mixed population of T cells _CM-- Cells) in the sample. The status of T cells (e.g., as mature or immature) can be determined, for example, by screening for the expression of various genes and the presence of various proteins expressed on the surface of the T cells. For example, the presence of one or more markers selected from the group consisting of L-selectin (CD62L +), IL-7R- α, CD132, CR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2R β, CXCR3, LFA-1, and any combination thereof may indicate a less mature undifferentiated T cell.

By "treatment" or "treating" of a subject/patient is meant any type of intervention or process performed on the subject/patient, or the administration of one or more T cells prepared by the present application to the subject/patient, with the purpose of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, severity, or recurrence of symptoms, complications, or disorders associated with the disease, or biochemical indicators. In one aspect, "treating" or "treatment" includes partial remission. In another aspect, "treating" or "treatment" includes complete remission.

Various aspects of the present application are described in further detail in the following subsections.

Patients with B cell malignancies with high levels of circulating tumor cells expressing CD19 represent a very high unmet population. For example, Mantle Cell Lymphoma (MCL) is challenging to treat in a relapsed or refractory setting, and remains incurable. Second-line and higher-level chemotherapy has no standard of care. Treatment options include cytotoxic chemotherapy, proteasome inhibitors, immunomodulatory drugs, tyrosine kinase inhibitors, and stem cell transplantation (both autologous [ ASCT ] and allogeneic stem cell transplantation [ allo-SCT ]). The choice of protocol is influenced by previous therapy, co-morbidities and tumor chemosensitivity. Although a high initial response rate was observed with Bruton's tyrosine kinase inhibitor (BTK inhibitor), most patients will eventually develop progressive disease. New therapeutic strategies are needed to ameliorate the poor prognosis of patients with r/r MCL whose disease has not been effectively controlled with chemo-immunotherapy, stem cell transplantation, and BTK inhibitors.

The anti-CD 19 CAR T cell therapy or product used in CD19 CAR-T can be made from the patient's own T cells, minimizing CD 19-expressing tumor cells in the final product by leukapheresis suitable for B cell malignancies with circulating tumor cell burden. T cells from harvested leukocytes from a leukocyte apheresis product can be enriched by selecting CD4+/CD8+ T cells, activated with anti-CD 3 and anti-CD 28 antibodies, and/or transduced with a viral vector comprising an anti-CD 19 CAR gene. Further details of the method can be found in PCT/US2015/014520 published as WO2015/120096 and PCT/US2016/057983 published as WO 2017/070395. In one embodiment, the cells are not treated with an AKT inhibitor, IL-7 and IL-15. These engineered T cells can be propagated to produce sufficient numbers of cells to achieve a therapeutic effect. Such processes remove both malignant and normal B cells expressing CD19, which can reduce activation, expansion, and depletion of anti-CD 19 CAR T cells.

Activation, transduction, and/or expansion of immune cells may be performed at any suitable time that allows (i) a sufficient number of cells to be produced in the engineered immune cell population for at least one dose administered to a patient, (ii) an engineered immune cell population having a favorable proportion of juvenile cells compared to a typical longer course, or (iii) both (i) and (ii). Suitable times may involve several parameters, including the population of one or more cells, the cell surface receptor expressed by the immune cells, the vector used, the dose required to have a therapeutic effect, and/or other variables. The activation time may be 0 day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or more than 21 days. The activation time of the method according to the present application will be reduced compared to amplification methods known in the art. For example, the activation time may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or may be 75% shorter. In addition, the amplification time may be 0 day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or more than 21 days. The amplification time of the method according to the present application will be reduced compared to amplification methods known in the art. For example, the amplification time may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or may be 75% shorter. In one embodiment, the time for cell expansion is about 3 days and the time from enrichment of the cell population that produces the engineered immune cells is about 6 days.

The delay or inhibition of maturation or differentiation of one or more T cells or DC cells can be measured by any method known in the art. For example, the delay or inhibition of maturation or differentiation of one or more T cells or DC cells can be measured by detecting the presence of one or more biomarkers. The presence of one or more biomarkers can be detected by any method known in the art including, but not limited to, immunohistochemistry and/or Fluorescence Activated Cell Sorting (FACS). One or more kinds of raw materialsThe marker is selected from the group consisting of: l-selectin (CD 62L) ⁺ ) IL-7R α, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2R β, CXCR3, LFA-1, or any combination thereof. In certain aspects, the delay or inhibition of maturation or differentiation of one or more T cells or DC cells can be determined by detecting L-selectin (CD 62L) ⁺ ) The presence of one or more of IL-7 ra and CD 132. One skilled in the art will recognize that while the methods of the invention can increase the relative proportion of immature and undifferentiated T cells or DC cells in the collected cell population, some mature and differentiated cells may still be present. Thus, the delay or inhibition of maturation or differentiation of one or more T cells or DC cells can be measured by calculating the total percentage of immature and undifferentiated cells in a population of cells before and after exposing one or more cells obtained from a donor subject to hypoxic culture conditions with or without a pressure above atmospheric pressure. The methods disclosed herein can increase the percentage of immature and undifferentiated T cells in a T cell population.

The methods described herein also include stimulating a population of cells (such as lymphocytes) with one or more T cell stimulators to produce a population of activated T cells under suitable conditions. Any combination of one or more suitable T cell stimulatory agents including, but not limited to, antibodies or functional fragments thereof that target T cell stimulatory or co-stimulatory molecules (e.g., anti-CD 2 antibodies, anti-CD 3 antibodies such as OKT-3, anti-CD 28 antibodies or functional fragments thereof) or any other suitable mitogen (e.g., Tetradecanoyl Phorbol Acetate (TPA), Phytohemagglutinin (PHA), concanavalin a (cona)), Lipopolysaccharide (LPS), pokeweed mitogen (PWM), or natural ligands of T cell stimulatory or co-stimulatory molecules may be used to generate a population of activated T cells.

Suitable conditions for stimulating or activating an immune cell population as described herein also include temperature, duration of time, and/or presence of CO ₂ In the presence of horizontal. The temperature of the stimulus can be about 34 ℃, about 35 ℃, about 36 ℃, about 37 ℃ or about 38 ℃, about 34 ℃ to 38 ℃, about 35 ℃ to 37 ℃, about 36 ℃ to 38 ℃, about 36 ℃ to 37 ℃ or about 37 ℃.

Another condition for stimulating or activating an immune cell population as described herein can also include the time of stimulation or activation. The time of stimulation is about 24-72 hours, about 24-36 hours, about 30-42 hours, about 36-48 hours, about 40-52 hours, about 42-54 hours, about 44-56 hours, about 46-58 hours, about 48-60 hours, about 54-66 hours, or about 60-72 hours, about 44-52 hours, about 40-44 hours, about 40-48 hours, about 40-52 hours, or about 40-56 hours. In one embodiment, the time of stimulation is about 48 hours or at least about 48 hours.

Other conditions for stimulating or activating an immune cell population as described herein may further include CO ₂ And (4) horizontal. CO for stimulation ₂ At a level of about 1.0-10% CO ₂ About 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO ₂ About 3-7% CO ₂ About 4-6% CO ₂ About 4.5-5.5% CO ₂ . In one embodiment, CO for stimulation ₂ At a level of about 5% CO ₂ 。

Conditions for stimulating or activating the immune cell population can also include temperature, an amount of time for stimulation, and/or at CO ₂ Any combination in the presence of levels. For example, the step of stimulating the population of immune cells can include stimulating the population of immune cells at a temperature of about 36-38 ℃ and at about 4.5% -5.5% CO ₂ CO of ₂ Stimulating the population of immune cells with one or more immune cell stimulating agents in the presence of the levels for an amount of time of about 44-52 hours. One or more immune cells of the present application can be administered to a subject for immune or cellular therapy. Thus, one or more immune cells can be collected from a subject in need of immune or cellular therapy. Once collected, the one or more immune cells can be treated for any suitable period of time prior to administration to a subject.

The concentration, amount, or population of lymphocytes or resulting products prepared by the methods herein is about 1.0-10.0 x 10 ⁶ Individual cells/mL. In certain aspects, the concentration is about 1.0 to 2.0X 10 ⁶ Individual cell/mL, about 1.0 to 3.0X 10 ⁶ About 1.0-4.0X 10 cells/mL ⁶ About 1.0-5.0X 10 cells/mL ⁶ About 1.0-6.0X 10 cells/mL ⁶ About 1.0-7.0X 10 cells/mL ⁶ About 1.0-8.0X 10 cells/mL ⁶ 1.0-9.0X 10 cells/mL ⁶ About 1.0-10.0X 10 cells/mL ⁶ About 1.0-1.2X 10 cells/mL ⁶ About 1.0-1.4X 10 cells/mL ⁶ About 1.0-1.6X 10 cells/mL ⁶ About 1.0-1.8X 10 cells/mL ⁶ About 1.0-2.0X 10 cells/mL ⁶ At least about 1.0X 10 cells/mL ⁶ At least about 1.1X 10 cells/mL ⁶ At least about 1.2X 10 cells/mL ⁶ At least about 1.3X 10 cells/mL ⁶ At least about 1.4X 10 cells/mL ⁶ At least about 1.5X 10 cells/mL ⁶ At least about 1.6X 10 cells/mL ⁶ At least about 1.7X 10 cells/mL ⁶ At least about 1.8X 10 cells/mL ⁶ At least about 1.9X 10 cells/mL ⁶ At least about 2.0X 10 cells/mL ⁶ At least about 4.0X 10 cells/mL ⁶ At least about 6.0X 10 cells/mL ⁶ At least about 8.0X 10 cells/mL ⁶ Individual cells/mL or at least about 10.0X 10 ⁶ Individual cells/mL.

An anti-CD 3 antibody (or a functional fragment thereof), an anti-CD 28 antibody (or a functional fragment thereof), or a combination of an anti-CD 3 antibody and an anti-CD 28 antibody may be used according to the step of stimulating a lymphocyte population in conjunction with or independent of exposing one or more cells obtained from a donor subject to hypoxic culture conditions with or without a pressure above atmospheric pressure. Any soluble or immobilized anti-CD 2, anti-CD 3, and/or anti-CD 28 antibody or functional fragment thereof can be used (e.g., clone OKT3 (anti-CD 3), clone 145-2C11 (anti-CD 3), clone UCHT1 (anti-CD 3), clone L293 (anti-CD 28), clone 15E8 (anti-CD 28)). In some aspects, the antibodies can be purchased commercially from suppliers known in the art including, but not limited to, Tian Ma whirlwind biotech, BD Biosciences (BD Biosciences), in Germany (e.g., 1mg/mL pure MACS GMP CD3, part number 170-. Furthermore, the skilled person will understand how to generate anti-CD 3 antibodies and/or anti-CD 28 antibodies by standard methods. In some aspects, the one or more T cell stimulating agents used according to the step of stimulating a lymphocyte population comprise an antibody or functional fragment thereof that targets a T cell stimulating or co-stimulating molecule in the presence of a T cell cytokine. In one embodiment, the one or more T cell stimulating agents include an anti-CD 3 antibody and IL-2. In certain embodiments, the T cell stimulating agent comprises an anti-CD 3 antibody at a concentration of 50 ng/mL. The concentration of the anti-CD 3 antibody is about 20ng/mL-100ng/mL, about 20ng/mL, about 30ng/mL, about 40ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90ng/mL, or about 100 ng/mL. In an alternative aspect, T cell activation is not required.

The methods described herein further comprise transducing the activated population of immune cells with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor using a single or multiple viral transduction cycles to produce a transduced population of immune cells. Several recombinant viruses have been used as viral vectors to deliver genetic material to cells. The viral vector that may be used in accordance with the transduction procedure may be any parental or bidirectional viral vector, including but not limited to recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, and recombinant adeno-associated virus (AAV) vectors. The method further comprises transducing the one or more immune cells with a retrovirus. In one aspect, the viral vector used to transduce the activated immune cell population is a MSGV1 gamma retroviral vector. In one embodiment, the viral vector used to transduce the activated immune cell population is the PG13-CD19-H3 vector described by: kochenderfer, J.Immunotherer 32(7) 689-702 (2009). According to one aspect of this aspect, the viral vector is grown in suspension culture in a medium specific for viral vector production (referred to herein as a viral vector inoculum). Any suitable growth medium and/or supplement for growing viral vectors may be used in the viral vector inocula according to the methods described herein. According to some aspects, the viral vector inoculum is then added during the transduction step to the serum-free culture described below In the nutrient medium. In some aspects, the one or more immune cells can be transduced with a retrovirus. In one embodiment, the retrovirus contains a heterologous gene encoding a cell surface receptor. In another embodiment, the cell surface receptor can bind to an antigen on the surface of a target cell (e.g., on the surface of a tumor cell). In addition to optionally exposing one or more cells obtained from a donor subject to hypoxic culture conditions with or without a pressure above atmospheric pressure, conditions for transducing an activated immune cell population as described herein can include a specific time, at a specific temperature, and/or at a specific CO ₂ In the presence of horizontal. The temperature of transduction is about 34 ℃, about 35 ℃, about 36 ℃, about 37 ℃ or about 38 ℃, about 34 ℃ to 38 ℃, about 35 ℃ to 37 ℃, about 36 ℃ to 38 ℃, about 36 ℃ to 37 ℃. In one embodiment, the transduction temperature is about 37 ℃. The predetermined transduction temperature may be about 34 ℃, about 35 ℃, about 36 ℃, about 37 ℃, about 38 ℃ or about 39 ℃, about 34 ℃ to 39 ℃, about 35 ℃ to 37 ℃. In one embodiment, the predetermined transduction temperature may be about 36 ℃ to 38 ℃, about 36 ℃ to 37 ℃, or about 37 ℃. The transduction time is about 12 hours to 36 hours, about 12 hours to 16 hours, about 12 hours to 20 hours, about 12 hours to 24 hours, about 12 hours to 28 hours, about 12 hours to 32 hours, about 20 hours, or at least about 20 hours, about 16 hours to 24 hours, about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, or at least about 26 hours. CO for transduction ₂ At a level of about 1.0-10% CO ₂ About 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0% CO ₂ About 3-7% CO ₂ About 4-6% CO ₂ About 4.5-5.5% CO ₂ Or about 5% CO ₂ 。

Transduction of an activated immune cell population as described herein can be at a certain time period, at a certain temperature, and/or at a specific CO ₂ In the presence of levels in any combination: a temperature of about 36 ℃ to 38 ℃, for an amount of time of about 16 hours to 24 hours, and at about 4.5% to 5.5% CO ₂ CO of ₂ In the presence of horizontal. Exempt fromThe immune cells may be prepared by a combination of any one of the methods of the present application with any manufacturing method for preparing T cells for immunotherapy, including but not limited to those described in PCT publications WO2015/120096 and WO2017/070395, which are incorporated herein by reference in their entirety for the purpose of describing these methods; for preparing aliskiren orAny and all methods of (a); for the preparation of tesserai/Kymriah ^TM Any and all methods of (a); any and all methods for preparing "off-the-shelf" T cells for immunotherapy; and any other method of preparing lymphocytes for administration to a human. The manufacturing method may be adapted to remove circulating tumor cells from cells obtained from a patient.

CAR-T cells can be engineered to express other molecules, and these CAR-T cells can be of any of the following exemplary types or other types available in the art: a first, second, third, fourth, fifth, or more CAR-T cells; armored CAR-T cells, sports CAR-T cells, TRUCK T cells, switch receptor CAR-T cells; a gene-edited CAR T cell; a dual receptor CAR T cell; suicide CAR T cells, drug-induced CAR-T cells, synNotch-induced CAR T cells; and an inhibitory CAR T cell. In one aspect, the T cell is an autologous T cell. In one aspect, the T cells are autologous stem cells (for autologous stem cell therapy or ASCT). In one aspect, the T cells are non-autologous T cells.

Cells (such as immune cells or T cells) are genetically modified after isolation or selection using known methods, or are activated and/or expanded (or differentiated in the case of progenitor cells) in vitro prior to genetic modification. Immune cells (e.g., T cells) are genetically modified (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) with the chimeric antigen receptors described herein and activated and/or amplified in vitro. Methods for activating and expanding T cells can be found in U.S. patents 6,905,874, 6,867,041; and 6,797,514 and PCT publication W Found in O2012/079000, which are hereby incorporated by reference in their entirety. Generally, such methods may comprise contacting PBMCs or isolated T cells with stimulating and co-stimulating agents (such as anti-CD 3 antibodies and/or anti-CD 28 antibodies) that may be attached to beads or other surfaces in media with certain cytokines (such as IL-2). Can be appliedThe system, a CD3/CD28 activator/stimulator system for physiological activation of human T cells. T cells can be activated and stimulated to proliferate with suitable feeder cells, antibodies, and/or cytokines as described in U.S. patents 6,040,177 and 5,827,642 and PCT publication WO 2012/129514, which are hereby incorporated by reference in their entirety.

The cell surface receptor expressed by the engineered immune cells may be any antigen or molecule targeted by a CAR, such as an anti-CD 19 CAR, FMC63-28Z CAR, or FMC63-CD828BBZ CAR (Kochenderfer et al, journal of immunotherapy (J immunology) 2009,32(7): 689; Locke et al, Blood, 2010, 116(20):4099, the subject matter of both documents being hereby incorporated by reference At least about 2 million to less than about 3 million transduced engineered T cells per kilogram body weight (cells/kg). In one embodiment, the predetermined dose of engineered T cells may be about 2 million transduced engineered T cells/kg. In another embodiment, the predetermined dose of engineered T cells may be at least about 2 million transduced engineered T cells/kg. Examples of the predetermined dose of engineered T cells may be about 2.0 million, about 2.1 million, about 2.2 million, about 2.3 million, about 2.4 million, about 2.5 million, about 2.6 million, about 2.7 million, about 2.8 million, or about 2.9 million transduced engineered T cells per kg.

The methods described herein comprise increasing or enriching one or more transduced immune cell populations for a period of time to produce an engineered immune cell population. The expansion time can be any suitable time that allows (i) a sufficient number of cells to be produced in the engineered immune cell population for at least one dose administered to a patient, (ii) an engineered immune cell population that produces a favorable proportion of naive cells compared to a typical longer course, or (iii) both (i) and (ii). This time will depend on the cell surface receptor expressed by the immune cell, the vector used, the dose required to have a therapeutic effect, and other variables. The predetermined amplification time may be 0 day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or more than 21 days. In one embodiment, the amplification time of the methods of the invention is reduced compared to methods known in the art. For example, the predetermined amplification time may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or may be 75% shorter. In one example, the expansion time is about 3 days, and the time from enrichment of the lymphocyte population that produces the engineered immune cells is about 6 days.

Conditions for expanding the transduced immune cell population may include temperature and/or at a certain CO ₂ In the presence of horizontal. In certain aspects, the temperature is about 34 ℃, about 35 ℃, about 36 ℃, about 37 ℃ or about 38 ℃, about 35 ℃ to 37 ℃, about 36 ℃ to 37 ℃, or about 37 ℃. CO 2 ₂ At a level of 1.0-10% CO ₂ About 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0% CO ₂ About 4.5-5.5% CO ₂ About 5% CO ₂ About 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5% or about 6.5% CO ₂ 。

Each step of the methods described herein may be performed in a closed system. The closed system may be any suitable cell culture bag (e.g., Tian Mei and whirly Biotech, Germany)GMP Cell differentiation bag, air-permeable life Cell Culture bag (PermaLife Cell Culture bags) by orlik biomedicine (origin) system. Cell culture bags used in closed bag culture systems may be coated with recombinant human fibronectin fragments during the transduction step. Recombinant human fibronectin fragments can include three functional domains: central cell binding domain, heparin binding domain II and CS1 sequences. Recombinant human fibronectin fragments can increase the gene transfer efficiency of retrovirus-transduced immune cells by aiding the co-localization of the target cell and the viral vector. In one embodiment, the recombinant human fibronectin fragment is (Nippon Bao bioengineering Co., Ltd. (Takara Bio, Japan)). The cell culture bag is coated with a recombinant human fibronectin fragment at a concentration of about 1 μ g/mL to 60 μ g/mL or about 1 μ g/mL to 40 μ g/mL, about 1 μ g/mL to 20 μ g/mL, 20 μ g/mL to 40 μ g/mL, 40 μ g/mL to 60 μ g/mL, about 1 μ g/mL, about 2 μ g/mL, about 3 μ g/mL, about 4 μ g/mL, about 5 μ g/mL, about 6 μ g/mL, about 7 μ g/mL, about 8 μ g/mL, about 9 μ g/mL, about 10 μ g/mL, about 11 μ g/mL, about 12 μ g/mL, about 13 μ g/mL, about 14 μ g/mL, about 15 μ g/mL, about 16 μ g/mL, about, About 17. mu.g/mL, about 18. mu.g/mL, about 19. mu.g/mL, about 20. mu.g/mL, about 2. mu.g/mL-5. mu.g/mL, about 2. mu.g/mL-10. mu.g/mL, about 2. mu.g/mL-20. mu.g/mL, about 2. mu.g/mL-25. mu.g/mL, about 2. mu.g/mL-30. mu.g/mL, about 2. mu.g/mL-35. mu.g/mL, about 2. mu.g/mL-40. mu.g/mL, about 2. mu.g/mL-50. mu.g/mL, about 2. mu.g/mL-60. mu.g/mL, at least about 2. mu.g/mL, at least about 5. mu.g/mL, at least about 10. mu.g/mL, at least about 15. mu.g/mL, at least about 20. mu.g/mL, At least about 25 μ g/mL, at least about 30 μ g/mL, at least about 40 μ g/mL, at least about 50 μ g/mL, or at least about 60 μ g/mL of recombinant human fibronectin fragments. In one embodiment, the cell culture bag is coated with at leastAbout 10. mu.g/mL recombinant human fibronectin fragment. The cell culture bag used in the closed bag culture system may optionally be closed with human albumin serum (HSA) during the transduction step. In another embodiment, the cell culture bag is not closed with HSA during the transduction step.

The engineered immune cell population produced by the above methods can optionally be cryopreserved so that the cells can be used later. Also provided herein is a method for cryopreserving an engineered immune cell population. Such methods may include the steps of washing and concentrating the engineered immune cell population with a diluent solution. For example, the diluent solution is physiological saline, 0.9% saline, PlasmaLyte a (PL), 5% glucose/0.45% NaCl saline solution (D5), Human Serum Albumin (HSA), or a combination thereof. In addition, HSA can be added to the washed and concentrated cells to improve cell viability and cell recovery after thawing. In another aspect, the wash solution is normal saline and the washed and concentrated cells are supplemented with HSA (5%). The method may further comprise the step of generating a cryopreservation mixture, wherein the cryopreservation mixture comprises the diluted cell population in the diluent solution and a suitable cryopreservation solution. The cryopreservation Solution can be any suitable cryopreservation Solution, including but not limited to CryoStor10 (BioLife Solution) mixed with a diluent Solution of engineered immune cells in a 1:1 or 2:1 ratio. HSA can be added to provide a final concentration in the cryopreservation mixture of about 1.0-10%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0%, about 1-3% HSA, about 1-4% HSA, about 1-5% HSA, about 1-7% HSA, about 2-4% HSA, about 2-5% HSA, about 2-6% HSA, about 2-7% HAS, or about 2.5% HSA. Cryopreservation of the engineered immune cell population can include washing the cells with 0.9% saline, adding HSA at a final concentration of 5% to the washed cells, and diluting the cells 1:1 with cryostor CS10 (for a final concentration of 2.5% HSA in the final cryopreservation mixture). In some aspects, the method further comprises the step of freezing the cryopreservation mixture. In addition, it is cold Freeze preservation mixtures using a defined freezing cycle in a controlled rate freezer to between about 1X 10 ⁶ To about 1.5X 10 ⁷ Cell concentrations between individual cells/mL of the cryopreservation mixture were frozen. The method may further comprise the step of storing the cryopreservation mixture in gaseous phase liquid nitrogen.

Engineered immune cell populations produced by the methods described herein can be cryopreserved at a predetermined dose. The predetermined dose may be a therapeutically effective dose, which may be any therapeutically effective dose as provided below. The predetermined dose of engineered immune cells may depend on the cell surface receptors expressed by the immune cells (e.g., the affinity and density of the cell surface receptors expressed on the cells), the type of target cells, the nature of the disease, or the pathological condition being treated, or a combination thereof.

In one embodiment, the population of engineered T cells may be cryopreserved at a predetermined dose of about 1 million engineered T cells per kilogram body weight (cells/kg). In certain embodiments, the population of engineered T cells may be cryopreserved at a predetermined dose of about 500,000 to about 1 million engineered T cells/kg. In certain embodiments, the population of engineered T cells may be cryopreserved at a predetermined dose of at least about 1 million, at least about 2 million, at least about 3 million, at least about 4 million, at least about 5 million, at least about 6 million, at least about 7 million, at least about 8 million, at least about 9 million, at least about 1000 million engineered T cells/kg. In other aspects, the population of engineered T cells can be administered at a dose of less than 1 million cells/kg, 2 million cells/kg, 3 million cells/kg, 4 million cells/kg, 5 million cells/kg, 6 million cells/kg, 7 million cells/kg, 8 million cells/kg, 9 million cells/kg, a predetermined dose of 1000, more than 2000, more than 3000, more than 4000, more than 5000, more than 6000, more than 7000, more than 8000, more than 9000 or more than 1 million cells/kg. In certain aspects, the population of engineered T cells can be cryopreserved at a predetermined dose of about 1 million to about 2 million engineered T cells/kg. The engineered T cell population may be cryopreserved at a predetermined dose of between about 1 million cells to about 2 million cells/kg, between about 1 million cells to about 3 million cells/kg, between about 1 million cells to about 4 million cells/kg, between about 1 million cells to about 5 million cells/kg, between about 1 million cells to about 6 million cells/kg, between about 1 million cells to about 7 million cells/kg, between about 1 million cells to about 8 million cells/kg, between about 1 million cells to about 9 million cells/kg, between about 1 million cells to about 1000 million cells/kg. The predetermined dose of the engineered T cell population can be calculated based on the body weight of the subject. In one example, the engineered T cell population can be cryopreserved in about 0.5-200mL of cryopreservation media. In addition, the engineered T cell population can be cryopreserved in about 0.5mL, about 1.0mL, about 5.0mL, about 10.0mL, about 20mL, about 30mL, about 40mL, about 50mL, about 60mL, about 70mL, about 80mL, about 90mL, or about 100mL, about 10-30mL, about 10-50mL, about 10-70mL, about 10-90mL, about 50-70mL, about 50-90mL, about 50-110mL, about 50-150mL, or about 100-200mL of cryopreservation medium. In certain aspects, the engineered T cell population may preferably be cryopreserved in about 50-70mL of cryopreservation media.

In one embodiment, serum-free media without added serum is used to perform at least one of: (a) contacting a population of immune cells with exogenous IL-2, exogenous IL-7, exogenous IL-15, and/or other cytokines, (b) stimulating a population of immune cells, (c) transducing a population of activated immune cells, and (d) expanding the transduced population of immune cells. In some aspects, each of (a) through (d) is performed using a serum-free medium without added serum. As referred to herein, the term "serum-free medium" or "serum-free medium" means that the growth medium used is not supplemented with serum (e.g., human serum or bovine serum). In other words, serum is not added to the culture as a separate isolated and distinct component for the purpose of supporting viability, activation and growth of the cultured cellsIn the formula (I). Any suitable immune cell growth medium may be used to culture the cells in suspension according to the methods described herein. For example, the immune cell growth medium may include, but is not limited to, a sterile low glucose solution comprising suitable amounts of buffer, magnesium pyruvate, calcium pyruvate, sodium pyruvate, and sodium bicarbonate. In one aspect, the T cell growth medium is OPTMIZER ^TM (Life Technologies). In contrast to typical methods for producing engineered immune cells, the methods described herein can use media that is not supplemented with serum (e.g., human or bovine).

The present application provides various methods of treating cancer with T cells. In one aspect, the T cells are CAR-T cells directed to CD19, which can be prepared by any one of the methods of the present application in combination with any step of the manufacturing process of preparing T cells for immunotherapy, including but not limited to those described in PCT publications WO2015/120096 and WO2017/070395, which are incorporated herein by reference in their entirety for the purpose of describing these methods; for preparing aliskiren orAny and all methods of (a); for the preparation of tesserai/Kymriah ^TM Any and all methods of (a); any and all methods for preparing "off-the-shelf" T cells for immunotherapy; and any other method of preparing lymphocytes for administration to a human. In some aspects, the manufacturing method is adapted to specifically remove circulating tumor cells from cells obtained from a patient.

In one aspect, the T cells are CD19 CAR-T cells prepared by the methods described in PCT/US 2016/057983. In one embodiment, a population of T cells depleted of circulating tumor cells is prepared from a leukapheresis product. These cells can be prepared as described in PCT/US2016/057983, and are further described herein as CD19 CAR-T cells. Briefly, CD19 CAR-T is an autologous CAR T cell product in which a subject's T cells are engineered to express a target linked by an activation domain linked to CD28 and CD3 zeta The receptor consisting of a single chain antibody fragment of CD19, results in the clearance of cells expressing CD 19. In CAR and CD19 ⁺ Upon target cell engagement, the CD3 zeta domain activates a downstream signaling cascade that leads to T cell activation, proliferation, and acquisition of effector functions, such as cytotoxicity. The intracellular signaling domain of CD28 provides a costimulatory signal that works with the primary CD3 zeta signal to enhance T cell function, including Interleukin (IL) -2 production. Together, these signals can stimulate proliferation of CAR T cells and directly kill the target cells. In addition, activated T cells can secrete cytokines, chemokines, and other molecules that can recruit and activate additional anti-tumor immune cells. anti-CD 19 CARs in CD19 CAR-T cells can comprise FMC 63-28Z.

The manufacture of CD19 CAR-T cells includes CD4 due to the presence of circulating tumor cells in certain cancers ⁺ And CD8 ⁺ And (4) a T cell enrichment step. The T cell enrichment or isolation step can reduce circulating tumor cells of CD19 expressed in leukopheresis material, and can involve activation, expansion, and depletion of anti-CD 19 CAR T cells during manufacturing.

The methods described herein may enhance the therapeutic outcome or efficacy of an immune or cell therapy, which may be an adoptive T cell therapy selected from the group consisting of: tumor Infiltrating Lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT) ^TM ) Allogeneic T cell transplantation, non-T cell transplantation and any combination thereof. Adoptive T cell therapy broadly includes any selection, in vitro enrichment, and administration to a patient of autologous or allogeneic T cells that recognize and are capable of binding tumor cells. TIL immunotherapy is a type of adoptive T cell therapy in which lymphocytes capable of infiltrating tumor tissue are isolated, enriched in vitro, and administered to a patient. TIL cells may be autologous or allogeneic. Autologous cell therapy is adoptive T cell therapy, which involves isolating T cells capable of targeting tumor cells of a patient, enriching the T cells in vitro and administering the T cells back to the same patient. Allogeneic T cell transplantation may include transplantation of ex vivo expanded naturally occurring T cells or genetically engineered T cells. As aboveThe engineered autologous cell therapy described in detail is an adoptive T cell therapy in which the patient's own lymphocytes are isolated, genetically modified to express tumor targeting molecules, expanded in vitro and administered back to the patient. non-T cell transplantation may include autologous or allogeneic therapy with non-T cells, such as but not limited to Natural Killer (NK) cells.

The immune cell therapy of the present application is an engineered autologous cell therapy (eACT) ^TM ). According to this aspect, the method may comprise collecting immune cells from a donor. The isolated immune cells can then be contacted with an exogenous activating agent (e.g., a cytokine), expanded, and engineered to express a chimeric antigen receptor ("engineered CAR T cell") or a T cell receptor ("engineered TCR T cell"). In some aspects, the engineered immune cells treat a tumor in a subject. For example, the one or more immune cells are transduced with a retrovirus that includes a heterologous gene encoding a cell surface receptor. In one embodiment, the cell surface receptor is capable of binding an antigen on the surface of a target cell (e.g., on the surface of a tumor cell). In some embodiments, the cell surface receptor is a chimeric antigen receptor or a T cell receptor. In another embodiment, the one or more immune cells can be engineered to express a chimeric antigen receptor. The chimeric antigen receptor may comprise a binding molecule to a tumor antigen. The binding molecule may be an antibody or an antigen binding molecule thereof. For example, the antigen binding molecule may be selected from scFv, Fab ', Fv, F (ab')2, and dAb, and any fragment or combination thereof. The chimeric antigen receptor may also include a hinge region. The hinge region may be derived from the hinge region of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8 α. In one embodiment, the hinge region is derived from the hinge region of IgG 4. The chimeric antigen receptor may also comprise a transmembrane domain. The transmembrane domain may be that of any transmembrane molecule which is a co-receptor on an immune cell or that of a member of the immunoglobulin superfamily. In certain embodiments, the transmembrane domain is derived from the transmembrane domains of: CD28, CD28T, CD8 α, CD4, or CD 19. In another embodiment In one embodiment, the transmembrane domain comprises a domain derived from the transmembrane domain of CD 28. In another embodiment, the transmembrane domain comprises a domain derived from the transmembrane domain of CD 28T. The chimeric antigen receptor may also comprise one or more costimulatory signaling regions. For example, the costimulatory signaling region can be the signaling region of CD28, CD28T, OX-40, 41BB, CD27, inducible T cell costimulation (ICOS), CD3 γ, CD3 δ, CD3 ε, CD247, Ig α (CD79a), or Fc γ receptors. In further embodiments, the co-stimulatory signaling region is a CD28 signaling region. In another embodiment, the costimulatory signaling region is the CD28T signaling region. In additional embodiments, the chimeric antigen receptor further comprises a CD3 zeta signaling domain.

In some aspects, the tumor antigen is selected from 707-AP (707 alanine proline), AFP (alpha (a) -alpha fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen; B-catenin/m, B-catenin/mutated), BCMA (B-cell maturation antigen), Bcr-abl (cluster of cleavage-Abelson), CAIX (carbonic anhydrase IX), CD19 (cluster of differentiation 19), CD20 (cluster of differentiation 20), CD22 (cluster of differentiation 22), CD30 (cluster of differentiation 30), CD33 (cluster of differentiation 33), CD44v7/8 (cluster of differentiation 44, exon 7/8), CAMEL (antigen recognized by CTL on melanomas), CAP-1 (carcinoembryonic antigen peptide 1), CASP-8 (caspase 8), CDC27m (mutated cell division cyclin 27), CDK4/m (mutated cyclin-dependent kinase 4), CDK4/m (mutated cyclin dependent kinase 4), CEA (carcinoembryonic antigen), CT (cancer/testis (antigen)), Cyp-B (cyclophilin B), DAM (differentiation antigen on melanoma), EGFR (epidermal growth factor receptor), EGFRvIII (epidermal growth factor receptor variant III), EGP-2 (epidermal glycoprotein 2), EGP-40 (epidermal glycoprotein 40), Erbb2, 3, 4 (erythroblastic leukemia virus oncogene homolog-2, -3, 4), ELF2M (mutant elongation factor 2), ETV6-AML1(Ets variant gene 6/acute myeloid leukemia 1 gene ETS), FBP (folate binding protein), fAchR (fetal acetylcholine receptor), G250 (glycoprotein 250), GAGE sialic acid (G antigen), GD2 (disialoganglioside 2), GD3 (disoganglioside 3), GnT-V (N-acetylglucosamine transferase V), Gp100 (glycoprotein 100kD), HAGE (helicase antigen), HER-2/neu (human epidermal receptor 2/neural; also known as EGFR2), HLA-A (human leukocyte antigen A), HPV (human papilloma virus), HSP70-2M (mutated heat shock protein 70-2), HST-2 (human annular body tumor factor 2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxyesterase), IL-13R-a2 (interleukin 13 receptor subunit alpha-2), KIAA0205, KDR (kinase insert region receptor), kappa light chain, LAGE (L antigen), LDLR/FUT (low density lipid receptor/GDP-L-fucose: b-D-galactosidase 2-a-L fucosyltransferase), LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), MAGE-A3, MAGE-A6, mesothelin, murine CMV-infected cells, MART-1/Melan-A (melanoma antigen 1/melanoma antigen A recognized by T cells), MC1R (melanocortin 1 receptor), myosin/M (mutated myosin), MUC1 (mucin 1), MUM-1, -2, -3 (melanoma ubiquitous mutant 1, 2, 3), NA88-A (NA cDNA clone of patient M88), NKG2D (Natural killer group 2, member D) ligand, NY-BR-1 (New York mammary differentiation antigen 1), NY-ESO-1 (New York squamous cell carcinoma-1), carcinoembryonic antigen (h5T4), P15 (protein 15), P190 smaller bcr-bcl (190KD protein), Pml/RARa (promyelocytic leukemia/retinoic acid receptor a), PRAME (preferentially expressed antigen of melanoma), PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate specific membrane antigen), RAGE (kidney antigen), RU1 or RU2 (renal ubiquitin 1 or 2), SAGE (sarcoma antigen), SART-1 or SART-3 (squamous antigen 1 or 3 that rejects tumors), SSX1, -2, -3, 4 (synovial sarcoma X1, -2, -3, -4), TAA (tumor-associated antigen), TAG-72 (tumor-associated glycoprotein 72), TEL/AML1 (translocation Ets family leukemia/acute myeloid leukemia 1), TPI/m (mutated triose phosphate isomerase), TRP-1 (tyrosinase-related protein 1 or 75), TRP-2 (tyrosinase related protein 2), TRP-2/INT2 (TRP-2/Intron 2), VEGF-R2 (vascular endothelial growth factor receptor 2), WT1(Wilms tumor gene), and any combination thereof. In one embodiment, the tumor antigen is CD 19.

T cell therapy involves administering to a patient engineered T cells that express a T cell receptor ("engineered TCR T cells"). The T Cell Receptor (TCR) may comprise a binding molecule to a tumor antigen. In some aspects, the tumor antigen is selected from the group consisting of: 707-AP, AFP, ART-4, BAGE, BCMA, Bcr-abl, CAIX, CD19, CD20, CD22, CD30, CD33, CD44v7/8, CAMEL, CAP-1, CASP-8, CDC27m, CDK4/m, CEA, CT, Cyp-B, DAM, EGFR, EGFRvIII, EGP-2, EGP-40, Erbb2, 3, 4, ELF2M, ETV6-AML1, FBP, fAchR, G250, GAGE, GD2, HSP 3, GnT-V, Gp, HAGE, HER-2/neu, HLA-A, HPV, 70-2-M, HST, hTRT or hTRT, iCE, IL-13R-a 56, kappa 0205, KIAA0205, KILR, LAGE, LACK-1/863, MUERTY, MUERT-863, MUERT-1/863, MUGE, MUERT-1/863, MUERT-MA, MUERT-1/863, MUERT-7, MUERT-I, MAG-I, NA88-A, NKG2D ligand, NY-BR-1, NY-ESO-1, carcinoembryonic antigen, P15, P190 minor bcr-abl, Pml/RARa, PRAME, PSA, PSCA, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SSX1, -2, -3, 4, TAA, TAG-72, TEL/AML1, TPI/m, TRP-1, TRP-2/INT2, VEGF-R2, WT1, and any combination thereof.

"CD 19-directed genetically modified autologous T cell immunotherapy" refers to a suspension of Chimeric Antigen Receptor (CAR) positive immune cells. An example of such immunotherapy is Clear CAR-T therapy, which uses CAR-T cells that are free of circulating tumor cells and enriched for CD4+/CD8+ T cells. Another example is aliskiren (axicabagene ciloleucel) (also known as Axi-cel) ^TM ，). See Kochenderfer et al, journal of immunotherapy (jimmunether), 2009; 32:689-702. Other non-limiting examples include JCAR017, JCAR015, JCAR014, kymeriah (tisagenlecucel), Uppsala u. anti-CD 19CAR (NCT02132624) and UCART19 (Celectis). See, Sadelain et al, for a review of Nature: cancer (Nature rev. cancer), volume 3, 2003; ruella et al, current hematologic malignancy report (Curr Hematol Malig Rep), press publication of schprings, new york, NY, 2016; and Sadelain et al, Cancer Discovery (2013, 4 months). To prepare CD 19-directed genetically modified autologous T cell immunotherapy, the patient's own T cells may be collected and ex vivoGenetically modified by reverse transcription transduction to express a Chimeric Antigen Receptor (CAR) comprising an anti-CD 19 single chain variable fragment (scFv) linked to CD28 and a CD 3-zeta costimulatory domain. In some embodiments, the CAR comprises a murine anti-CD 19 single chain variable fragment (scFv) linked to a 4-1BB and CD 3-zeta costimulatory domain. anti-CD 19CAR T cells can be expanded and infused back into the patient, where they can recognize and eliminate target cells expressing CD 19.

In one aspect, the TCR comprises a binding molecule to a viral oncogene. In one embodiment, the viral oncogene is selected from the group consisting of Human Papilloma Virus (HPV), Epstein-Barr virus (EBV) and human T-lymphotropic virus (HTLV). In other embodiments, the TCR comprises a binding molecule to a testicular, placental, or fetal tumor antigen. In one embodiment, the testicular, placental, or fetal tumor antigen is selected from the group consisting of: NY-ESO-1, synovial sarcoma X breakpoint 2(SSX2), Melanoma Antigen (MAGE), and any combination thereof. In another embodiment, the TCR comprises a binding molecule to a lineage specific antigen. In additional embodiments, the lineage specific antigen is selected from the group consisting of: melanoma antigen 1 recognized by T cells (MART-1), gp100, Prostate Specific Antigen (PSA), Prostate Specific Membrane Antigen (PSMA), Prostate Stem Cell Antigen (PSCA), and any combination thereof. In certain embodiments, the T cell therapy comprises administering to the patient an engineered CAR T cell expressing a chimeric antigen receptor that binds CD19 and further comprises a CD28 co-stimulatory domain and a CD 3-zeta signaling region. In additional embodiments, the T cell therapy comprises administering KTE-C19 to the patient. In one aspect, antigenic portions also include, but are not limited to, Epstein-Barr virus (EBV) antigens (e.g., EBNA-1, EBNA-2, EBNA-3, LMP-1, LMP-2), hepatitis a virus antigens (e.g., VP1, VP2, VP3), hepatitis b virus antigens (e.g., HBsAg, HBcAg, HBeAg), hepatitis c virus antigens (e.g., envelope glycoproteins E1 and E2), herpes simplex virus type 1, type 2 or type 8 (HSV1, HSV2 or HSV8) virus antigens (e.g., glycoproteins gB, gC, gE, gG, gH, gI, gJ, gK, gL, gM, UL20, UL32, US43, UL45, UL49A), Cytomegalovirus (CMV) virus antigens (e.g., glycoproteins gB, gC, gH, gig, HIV, gig, HIV virus (g), HIV) viral antigens), other immune envelope antigens (gp) antigens, or human virus antigens (gp) antigens, human envelope antigens (E) antigens, gp41 or p24), an influenza virus antigen (e.g., Hemagglutinin (HA) or Neuraminidase (NA)), a measles or mumps virus antigen, a Human Papilloma Virus (HPV) virus antigen (e.g., L1, L2), a parainfluenza virus antigen, a rubella virus antigen, a Respiratory Syncytial Virus (RSV) virus antigen, or a varicella-zoster virus antigen. In such aspects, the cell surface receptor can be any TCR, or any CAR that recognizes any of the aforementioned viral antigens on a virally infected target cell. In other aspects, the antigenic moiety is associated with a cell having an immune or inflammatory dysfunction. Such antigenic moieties may include, but are not limited to, Myelin Basic Protein (MBP), myelin proteolipid protein (PLP), Myelin Oligodendrocyte Glycoprotein (MOG), carcinoembryonic antigen (CEA), proinsulin, glutamine decarboxylase (GAD65, GAD67), Heat Shock Protein (HSP), or any other tissue-specific antigen involved in or associated with pathogenic autoimmune processes.

The methods disclosed herein may involve a T cell therapy comprising transferring one or more T cells to a patient. The T cells may be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells (e.g., engineered CAR + T cells or engineered TCR + T cells) can be at least about 10 ⁴ At least about 10 cells ⁵ At least about 10 cells ⁶ At least about 10 cells ⁷ At least about 10 cells ⁸ At least about 10 cells ⁹ Or at least about 10 ¹⁰ And (4) respectively. In another aspect, the therapeutically effective amount of T cells (e.g., engineered CAR + T cells or engineered TCR + T cells) is about 10 ⁴ One cell, about 10 ⁵ One cell, about 10 ⁶ One cell, about 10 ⁷ One cell or about 10 ⁸ And (4) cells. In one embodiment, the therapeutically effective amount of T cells (e.g., engineered CAR + T cells or engineered TCR + T cells) is about 2 x 10 ⁶ Individual cell/kg, about 3X 10 ⁶ Individual cell/kg, about 4X 10 ⁶ Individual cell/kg, about 5X 10 ⁶ Individual cell/kg, about 6X 10 ⁶ Individual cell/kg, about 7X 10 ⁶ Individual cell/kg, about 8X 10 ⁶ Individual cell/kg, about 9X 10 ⁶ Individual cell/kg, about 1X 10 ⁷ Individual cell/kg, about 2X 10 ⁷ Individual cell/kg, about 3X 10 ⁷ Individual cell/kg, about 4X 10 ⁷ Individual cell/kg, about 5X 10 ⁷ Individual cell/kg, about 6X 10 ⁷ Individual cell/kg, about 7X 10 ⁷ Individual cell/kg, about 8X 10 ⁷ Individual cell/kg or about 9X 10 ⁷ Individual cells/kg. In one embodiment, the amount of CD19 CAR-T cells is 2X 10 ⁶ The maximum dose per kg of individual cells for a subject of ≥ 100kg is 2X 10 ⁸ And (4) cells. In another embodiment, the amount of CD19 CAR-T cells is 0.5X 10 ⁶ The maximum dose per kg of individual cells is 0.5X 10 for a subject of more than or equal to 100kg ⁸ And (4) cells.

The patient may be preconditioned or lymphodepleted prior to administration of the T cell therapy. The patient may be preconditioned according to any method known in the art, including but not limited to treatment with one or more chemotherapy drugs and/or radiation therapy. In some aspects, preconditioning can include any treatment that reduces the number of endogenous lymphocytes, removes cytokine levels, increases serum levels of one or more homeostatic cytokines or pro-inflammatory factors, enhances effector function of T cells administered after conditioning, enhances antigen presenting cell activation and/or availability prior to T cell therapy, or any combination thereof. Preconditioning can include increasing the serum level of one or more cytokines in the subject. The method further comprises administering a chemotherapeutic agent. The chemotherapeutic agent may be a lymphodepleting (preconditioning) chemotherapeutic agent. Beneficial preconditioning treatment regimens are described in U.S. patent 9,855,298, which is hereby incorporated by reference in its entirety, along with associated beneficial biomarkers. These provisional patent applications describe, for example, methods of conditioning a patient in need of a T cell therapy comprising administering to the patient a specified beneficial dose of cyclophosphamide (200 mg/m) ² Daily and 2000mg/m ² Between/day) and the indicated dose of fludarabine (20 mg/m) ² Daily and 900mg/m ² Between/day). One such dosage regimen involves treating a patient comprising administering to the patient about 500mg/m per day for three days before administering to the patient a therapeutically effective amount of engineered T cells ² Cyclophosphamide per day and about 60mg/m ² Fludarabine/day. In one aspect, the conditioning regimen comprises 500mg/m for 3 days ² Cyclophosphamide +30mg/m ² Fludarabine. They may be administered on days-4, 3 and 2 or on days-5, 4 and 3 (day 0 is the day on which the cells are administered). In one embodiment, the conditioning regimen comprises 200mg/m per day ² 、250mg/m ² 、300mg/m ² 、400v、500mg/m ² Cyclophosphamide lasts 2, 3 or 4 days and 20mg/m ² 、25mg/m ² Or 30mg/m ² Fludarabine lasts for 2, 3 or 4 days. In one embodiment, and following leukapheresis, opsonic chemotherapy (30 mg/m per day) ² Fludarabine and 500mg/m per day ² Cyclophosphamide) was administered on days-5, 4, and 3 prior to intravenous infusion of the suspension of CD19 CAR-T cells. In some embodiments, the intravenous infusion time is between 15 and 120 minutes. In one embodiment, the intravenous infusion time is between 1 and 240 minutes. In some embodiments, the intravenous infusion time is up to 30 minutes. In some embodiments, the intravenous infusion time is at most 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at most 100 minutes. In some embodiments, the infusion amount is between 50 and 100 mL. In some embodiments, the infusion amount is between 20 and 100 ml. In some embodiments, the infusion volume is about 30mL, 35mL, 40mL, 45mL, 50mL, 55mL, 60mL, or about 65 mL. In some embodiments, the infusion amount is about 68 mL. In some embodiments, the suspension has been frozen and used within 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour of thawing. In some embodiments, the suspension has not been frozen. In some embodiments, the immunotherapy is infused from an infusion bag. In some embodiments, the infusion bag is stirred during infusion. In some embodiments, administration is within 3 hours after thawing The immunotherapy is used. In some embodiments, the suspension further comprises albumin. In some embodiments, albumin is present in an amount of about 2% to 3% (v/v). In some embodiments, albumin is present in an amount of about 2.5% (v/v). In some embodiments, albumin is present in an amount of about 1%, 2%, 3%, 4%, or 5% (volume/volume). In some embodiments, the albumin is human albumin. In some embodiments, the suspension further comprises DMSO. In some embodiments, DMSO is present in an amount of about 4% to 6% (volume/volume). In some embodiments, DMSO is present in an amount of about 5% (volume/volume). In some embodiments, DMSO is present in an amount of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (v/v).

The methods disclosed herein can be used to treat cancer in a subject, reduce the size of a tumor, kill tumor cells, prevent tumor cell proliferation, prevent growth of a tumor, eliminate a tumor in a patient, prevent recurrence of a tumor, prevent metastasis of a tumor, induce remission in a patient, or any combination thereof. In certain aspects, the methods can induce a complete response. In other aspects, the methods can induce a partial response.

Treatable cancers include tumors that are not vascularized, not sufficiently vascularized, or vascularized. Cancer may also include solid tumors or non-solid tumors.

In one embodiment, the method may be used to treat B cell malignancies that carry high levels of circulating tumor cells expressing CD19, and will be indicated for different patient populations with high unmet needs.

Exemplary treatment of MCL

In some embodiments, the malignant tumor can be Mantle Cell Lymphoma (MCL). MCL is an aggressive subtype of non-hodgkin's lymphoma (NHL). MCL accounts for approximately 6% of all new NHL cases in the United States (US) and 5% to 7% of malignant lymphomas in Western Europe (Western Europe), with an estimated annual incidence of MCL in the united states and Europe of approximately 1 to 2 per 100,000 individuals. MCL affects men more likely than women, and the median age at diagnosis is 68 years. In some embodiments, relapsed or refractory (r/r) MCL is relapsed or refractory to treatment with allogeneic stem cell transplantation (allo-SCT), which itself may lead to persistent remission for approximately 25% of patients with r/r MCL if the patient's disease is shown to be chemosensitive prior to transplantation, but allo-SCT is also associated with up to 40% treatment-related mortality.

In some embodiments, the r/r MCL is relapsed or refractory to treatment with bortezomib, lenalidomide and temsirolimus, which itself results in an ORR in the range of 22% to 32%. Bruton's Tyrosine Kinase (BTK) inhibitors such as ibrutinib and acatinib resulted in an ORR of 68% and 81% respectively in patients with r/r MCL. However, most patients progress after BTK inhibitor treatment and have poor results in response to rescue therapy, with ORR ranging from 20% to 42%, median duration of response (DOR) ranging from 3 to 5.4 months, and median OS ranging from 2.5 to 9 months. In some embodiments, the present disclosure provides methods that can be used to treat cancers with poor prognostic factors, such as high Ki67 tumor proliferation index expression (. gtoreq.30% or. gtoreq.50%) and mutated TP 53. In some embodiments, the cancer is MCL. In some embodiments, the MCL morphology is classical, polymorphic, or blast-like. In some embodiments, the Ki-67 index may be between 5% and 80%. In some embodiments, the Ki-67 index is about 38%. In some embodiments, the high risk patient has a Ki-67 of ≧ 50% and/or a TP53 mutation as determined by next generation sequencing. In some embodiments, the patient is aged ≧ 18 years. In some embodiments, MCL is pathologically confirmed by the record of cyclin D1 overexpression and/or the presence of t (11: 14).

In some embodiments, the CAR T cell intervention comprises T cells that expand from a population of T cells that deplete circulating lymphoma cells and are enriched for CD4+/CD8+ T cells by positive selection of monocytes from a leukapheresis sample with an anti-CD 3 antibody and an anti-CD 28 anti-CD 3832 antibody in the presence of IL-2In vivo activation, followed by transduction with a replication-defective viral vector containing the anti-CD 19 CAR construct. In some embodiments, the CAR construct is FMC63-28 ZCAR. CAR T cells produced using this method may be referred to as KTE-X19. In some embodiments, the cells are autologous. In some embodiments, the cell is heterologous. In some embodiments, the dose of CAR-positive T cells is 2 x 10 ⁶ anti-CD 19 CAR T cells/kg. In some embodiments, the dose of CAR-positive T cells is 1 x 10 ⁶ anti-CD 19 CAR T cells/kg. In some embodiments, the dose of CAR-positive T cells is 1.6 x 10 ⁶ 1.8X 10 anti-CD 19 CAR T cells/kg ⁶ anti-CD 19 CAR T cells/kg or 1.9X 10 ⁶ anti-CD 19 CAR T cells/kg. In some embodiments, the CD19 CAR construct contains a CD3 ζ T cell activation domain and a CD28 signaling domain.

In some embodiments, 25mg/m per day on days-5, 4, and 3 after leukapheresis ² Fludarabine and 900mg/m daily on day-2 ² CAR T cells were administered as a single infusion on day 0 after opsonization therapy of cyclophosphamide. In some embodiments, the conditioning therapy comprises 300mg/m per day for 3 days ² Cyclophosphamide and 30mg/m daily ² Fludarabine. In some embodiments, the conditioning chemotherapy comprises 30mg/m per day on days-5, 4, and 3 ² Fludarabine and 500mg/m per day ² Cyclophosphamide. In some embodiments, the patient may also receive acetaminophen and diphenhydramine or another H1 antihistamine about 30 to 60 minutes prior to infusion of the anti-CD 19 CAR T cells. In some embodiments, the patient receives one or more additional doses of anti-CD 19 CAR T cells.

In some embodiments, the MCL cancer is relapsed/refractory MCL (r/r MCL). In some embodiments, the patient has received one or more prior treatments. In some embodiments, the patient has received 1 to 5 past treatments. In some embodiments, the prior treatment may include autologous SCT, anti-CD 20 antibodies, anthracycline-or bendamustine-containing chemotherapy, and/or Bruton's Tyrosine Kinase Inhibitor (BTKi). In some embodiments, the BTKi is ibrutinib (Ibr). In some embodiments, BTKi is acatinib (Acala). In some embodiments, the disclosure demonstrates that MCL patients previously treated with ibrutinib have a more pronounced response to anti-CD 19 CAR T cell therapy compared to patients previously treated with acartinib. Accordingly, the present disclosure provides a method of treating r/r MCL with an anti-CD 19 CAR T cell therapy, wherein the patient has been previously treated with ibrutinib or acartinib and the cancer thereof is preferably relapsed/refractory to ibrutinib or acartinib. In some embodiments, the BTKi is tiraprutinib (ONO-4059), Zebrintinib (BGB-3111), CGI-1746, or spebrutinib (AVL-292, CC-292).

In some embodiments, the disclosure demonstrates a median (range) peak CAR T cell level of 95.9 (0.4-2589.5), 13.7 (0.2-182.4), or 115.9 (17.2-1753.6), respectively, for patients receiving previous ibrutinib, acatinib, or both. In some embodiments, the ORR/CR rate of anti-CD 19 CAR T cell therapy in patients with MCL is 94%/65% in patients receiving past ibrutinib, 80%/40% in patients receiving past alcatinib, and 100%/100% in patients receiving both BTKi. In some embodiments, the 12-month survival rate in patients receiving prior ibrutinib, acatinib, or both is 81%, 80%, or 100%, respectively. In some embodiments, CAR T cell expansion correlates with ORR/CR rates in patients previously treated with ibrutinib and/or acatinib. Thus, in one embodiment, the patient is treated with both ibrutinib and acartinib. In one embodiment, the present disclosure provides a method of predicting ORR/CR in MCL patients previously treated with ibrutinib and/or acatinib by measuring peak CAR T-cell levels and comparing them to reference standards. In one embodiment, the disclosure provides a method of predicting a progressive response based on a measure of CAR T cell peak level/baseline tumor burden (CEN and INV). In one embodiment, the higher the ratio, the higher the likelihood of a progressive response at/to 12 months. In one embodiment, a ratio between 0.00001 and 0.005 predicts no response at/to 12 months. In one embodiment, a ratio between 0.006 and 0.3 predicts recurrence at/to 12 months. In one embodiment, a ratio between 0.4 and 1 predicts a progressive response at/to 12 months. In one embodiment, the ratio may be determined from the average population by one of ordinary skill in the art.

In some embodiments, the additional inclusion criteria include those listed in example 2. In some embodiments, the additional exclusion criteria include those listed in example 2.

In some embodiments, the patient may have received bridging therapy with dexamethasone (e.g., 20mg to 40mg PO or IV daily or equivalent for 1 day to 4 days), methylprednisolone, ibrutinib (e.g., 560mg PO daily), and/or acartinib (e.g., 100mg PO twice daily) after leukapheresis and within, for example, 5 days or less prior to opsonic chemotherapy. In some embodiments, such patients may have a high disease burden. In some embodiments, the bridging therapy is selected from an immunomodulator, R-CHOP, bendamustine, an alkylating agent, and/or a platinum-based agent.

In some embodiments, the disclosure demonstrates that all MCL patients who respond to CAR T cell infusion achieve T cell expansion, whereas no expansion is observed in unreacted patients. In some embodiments, the response is an objective response (complete response + partial response). The disclosure demonstrates that CAR T cell levels are associated with ORR in the first 28 days, with area under the curve (AUC) from day 0 to day 28 _0-28 ) The sum peak level is higher in responders than in non-responders>200-fold, indicating that higher amplification leads to a better and possibly deeper response, also at minimal residual disease (MRD, 10) as compared to MRD positive patients (at week 4) ^-5 Sensitivity of) in negative patients the peak/AUC CAR T cell levels were high>As indicated by 80 times. Accordingly, the present disclosure provides a method of predicting patient response and MRD to CAR T cell treatment of MCL, the method comprising measuring peak/AUC CAR T cell levels and comparingWhich is compared to a reference standard. In some embodiments, peak CAR T cell expansion is observed between day 8 and day 15 after CAR T cell administration. In some embodiments, CAR T cell levels are measured by qPCR. In some embodiments, peak CAR T cell levels, AUC, are monitored by next generation sequencing _0-28 And/or MRD. In some examples, CAR T cell numbers are measured in cells/microliter of blood. In some examples, CAR T cell number is measured by CAR gene copy number/μ g host DNA. In some examples, CAR T cell numbers are measured as described in the following documents: kochenderfer J.N et al journal of clinical oncology (j.clin.oncol.) 2015; 33:540-549. In one embodiment, CAR T cell levels are measured as described in: locke FL et al, molecular therapeutics (Mol Ther.), 2017; 25(1):285-295.

In some embodiments, the present disclosure demonstrates a difference between T cell expansion in responders and non-responders. In some embodiments, the present disclosure demonstrates a median peak anti-CD 19 CAR T cell level in responders (those with complete and partial remission) of 102.4 cells/μ L (range: 0.2 to 2589.5 cells/μ L; n ═ 51) and 12.0 cells/μ L in non-responders (range: 0.2 to 1364.0 cells/μ L, n ═ 8). In some embodiments, the disclosure demonstrates a median AUC from day 0 to day 28 (AUC) _0-28 ) In patients with objective responses 1487.0 cells/μ L day (range: 3.8 to 2.77 x 10 ⁴ Individual cells/. mu.L.day; n-51), 169.5 cells/μ L day in non-responders (range: 1.8 to 1.1710X 10 ⁴ Individual cells/. mu.L.day; n-8). Median peak (24.7 cells/. mu.L) anti-CD 19 CAR T cells (peak: and AUC) in patients not receiving either corticosteroid or tollizumab (n ═ 18) _0-28 Levels (360.4 cells/. mu.L.day) were similar to those of patients receiving corticosteroid only (n ═ 2) (peak: 24.2 cells/. mu.L; AUC) _0-28 : 367.8 cells/. mu.L.day). In patients receiving tositumumab only (n ═ 10), the mean peak anti-CD 19 CAR T cells was 86.5 cells/μ L, AUC _0-28 The concentration was 1188.9 cells/. mu.L.day. Upon receiving both corticosteroid and toslizumabThe mean peak value of the patient (n-37) was 167.2 cells/. mu.L, AUC _0-28 The concentration was 1996.0 cells/. mu.L.day. Median peak anti-CD 19CAR T cell values were 74.1 cells/μ L in patients aged 65 years (n 39) at age ≧ 65, at age<Patients 65 years old (n-28) had 112.5 cells/μ L. Median anti-CD 19CAR T cell AUC _0-28 The value was 876.5 cells/. mu.L.day in patients aged 65 or older, at age<1640.2 cells/μ L-day in 65 years old patients. AUC of gender versus CD19CAR T cells _0-28 And C _max There was no significant effect. Accordingly, the present disclosure provides a method of predicting response in MCL comprising measuring T cell expansion following anti-CD 19CAR T therapy and comparing the level to a reference standard.

In some embodiments, the disclosure demonstrates that CAR T cell expansion is higher in patients with grade 3 MCL than in patients with grade 3 CRS and NE events. Accordingly, the present disclosure provides a method of predicting CRS & gt, grade 3 and NE events, the method comprising measuring CAR T cell expansion following CAR T cell therapy and comparing the level to a reference value, wherein the higher the CAR T cell expansion, the greater the chance of CRS & gt, grade 3 and NE events.

In some embodiments, cytokine levels are measured by (one of) protein or mRNA levels. In some embodiments, cytokine levels are measured as described in the following references: locke FL et al, molecular therapeutics (Mol Ther.), 2017; 25(1):285-295.

In some embodiments, the disclosure demonstrates that peak serum GM-CSF and IL-6 levels (reached about 8 days after CAR T cell administration) are positively correlated with grade 3 CRS and grade 3 NE in MCL patients. Thus, the present disclosure provides a method of predicting CRS ≧ 3 and NE ≧ 3, the method comprising measuring peak levels of GM-CSF and IL-6 after CAR T-cell administration and comparing them to reference levels, wherein the higher the peak levels of these cytokines, the greater the chance of CRS ≧ 3 and NE.

In some embodiments, the disclosure demonstrates a positive correlation of serum ferritin with CRS grade 3 or greater in MCL patients. Accordingly, the present disclosure provides a method of predicting CRS grade 3 or greater comprising measuring peak levels of serum ferritin following CAR T cell administration and comparing them to reference levels, wherein the higher the peak level of ferritin, the greater the chance of CRS grade 3 or greater.

In some embodiments, the disclosure demonstrates that serum IL-2 and IFN- γ are positively correlated with grade 3 or more NE in MCL patients. Accordingly, the present disclosure provides a method of predicting grade 3 or greater CRS comprising measuring peak levels of IL-2 and IFN- γ in serum after CAR T cell administration and comparing them to reference levels, wherein the higher the peak levels of IL-2 and IFN- γ, the greater the chance of grade 3 NE.

In some embodiments, the present disclosure demonstrates that cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8, and Vascular Cell Adhesion Molecule (VCAM) are positively correlated with ≧ grade 3 NE in MCL patients. Accordingly, the present disclosure provides a method of predicting grade 3 or greater CRS comprising measuring cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8 and/or Vascular Cell Adhesion Molecule (VCAM) after CAR T cell administration and comparing them to reference levels, wherein the higher the cerebrospinal fluid levels of the C-reactive protein, ferritin, IL-6, IL-8 and/or Vascular Cell Adhesion Molecule (VCAM), the greater the chance of grade 3 or greater NE. In some embodiments, one or more adverse events are managed according to table 13 and/or table 14.

In some embodiments, the present disclosure demonstrates that peak serum levels of cytokines positively correlated with CRS grade ≧ 3 include IL-15, IL-2R α, IL-6, TNF α, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1B, granzyme A, granzyme B, and perforin. In some embodiments, the present disclosure demonstrates that peak serum levels of cytokines associated with grade 3 NE or greater include IL-2, IL-1Ra, IL-6, TNF α, GM-CSF, IL-12p40, IFN- γ, IL-10, MCP-4, MIP-1B, and granzyme B. In some embodiments, the present disclosure demonstrates that cytokines associated with both CRS and NE grades 3 or greater include IL-6, TNF α, GM-CSF, IL-10, MIP-1B, and granzyme B. In some embodiments, the cytokine serum level peaks within 7 days of CAR T cell administration. Accordingly, the present disclosure provides a method of predicting ≧ 3-grade CRS following CAR T cell administration, the method comprising measuring peak serum levels of IL-15, IL-2 ra, IL-6, TNF α, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1B, granzyme a, granzyme B, and/or perforin following anti-CD 19 CAR T treatment and comparing the levels to a reference standard. Accordingly, the present disclosure also provides a method of predicting grade 3 CRS and grade 3 NE in MCL comprising measuring peak serum levels of IL-6, TNF α, GM-CSF, IL-10, MIP-1B, and granzyme B following anti-CD 19 CAR T treatment and comparing the levels to a reference standard.

In some embodiments, the present disclosure demonstrates a trend of increased peak cytokine levels of proliferation (IL-15, IL-2) and inflammation (IL-6, IL-2R α, sPD-L1, and VCAM-1) in patients with MCL of mutant TP53 relative to wild-type TP 53. Thus, in some embodiments, the disclosure provides a method of improving response to CAR T cell therapy in MCL, the method comprising manipulating the level of proliferative and/or inflammatory cytokines after administration of the CAR T cells.

In some embodiments, the disclosure demonstrates a trend of increased peak levels of IFN- γ and IL-6 and increased IL-2 for patients that are MRD negative one month after CAR T cell administration relative to patients that are MRD positive one month. Accordingly, the present disclosure provides a method of predicting whether a patient is MRD negative in MCL, the method comprising measuring peak serum levels of IFN- γ, IL-6 and/or IL-2 following anti-CD 19 CAR T treatment and comparing the levels to a reference standard.

In some embodiments, the disclosure demonstrates that T cell product phenotypes vary between MCL types. In some embodiments, the disclosure demonstrates that in the manufactured anti-CD 19 CAR T cell product, the median (range) CD4+/CD8+ T cell ratios for patients with classical, blast-like, or polymorphic MCL are 0.7 (0.04-2.8), 0.6 (0.2-1.1), or 0.7 (0.5-2.0), respectively. The product T cell phenotype (median [ range ]) included less differentiated CCR7+ T cells (classically 40.0% [ 2.6-88.8 ]; blast-like 35.3% [ 14.3-73.4 ]; polymorphism 80.8% [ 57.3-88.8 ]) and effector memory CCR7-T cells (classically 59.9% [ 11.1-97.4 ]; blast-like 64.8% [ 26.6-85.7 ]; polymorphism 19.2% [ 11.1-42.7 ]). In some embodiments, the disclosure demonstrates that 12-month survival of patients with classical, blast-like, or polymorphic MCL is 86.7%, 67.9%, or 100%, respectively. Accordingly, the present disclosure provides a method of improving treatment of classical, blast-like or polymorphic MCL by manipulating the T cell product phenotype administered to a patient.

Exemplary treatment of B cell ALL

B-ALL cells typically express CD19, and CAR T cell therapy targeting CD19 is a therapeutic approach to R/RB-ALL. Pehlivan k.c. et al, "recent hematological malignancy report (Curr hematosol Malig Rep.) 2018; 13(5):396-406. anti-CD 19 CAR T-cell therapy containing CD3 ζ and CD28 costimulatory domains was developed at the National Cancer Institute (Kochenderfer JN et al, J Immunoth., 2009; 32(7): 689-fold 702; Kochenderfer JN et al, Blood (Blood), 2010; 116(19): 3875-fold 3886) and showed an overall remission rate of 70% after median 10-month follow-up in phase 1 trials in children with R/R B-ALL and adults aged ≦ 30 years. Lee DW et al "Lancet" (Lancet.) 2015; 385(9967):517-528. Similar CAR constructs evaluated in phase 1 trials in adults with R/R B-ALL provided a Complete Remission (CR) rate of 83% at a median 29-month follow-up and an Overall Survival (OS) of 12.9 months. Park JH et al, new england journal of medicine (N Engl J Med.), 2018; 378(5):449-459. In these studies, CAR T cells were prepared from leukapheresis samples that were not enriched for CD4+/CD8+ T cells.

In some embodiments, the invention relates to a T cell product wherein the T cells are expanded from a population of T cells depleted of circulating lymphoma cells and enriched for CD4+/CD8+ T cells by positive selection of monocytes from a leukapheresis sample activated with an anti-CD 3 antibody and an anti-CD 28 antibody in the presence of IL-2 and then transduced with a replication-deficient viral vector containing an anti-CD 19CAR construct. In some embodiments, this isThe T-like cell products can be used for treating ALL, CLL, AML. In some embodiments, the CAR construct is a FMC63-28Z CAR. In some embodiments, the cells are autologous. In some embodiments, the cell is heterologous. In some embodiments, the dose of CAR-positive T cells is 2 x 10 ⁶ Individual anti-CD 19CAR T cells/kg. In some embodiments, the dose of CAR-positive T cells is 1 x 10 ⁶ anti-CD 19CAR T cells/kg. In some embodiments, the dose of CAR-positive T cells is 1.6 x 10 ⁶ 1.8X 10 anti-CD 19CAR T cells/kg ⁶ anti-CD 19CAR T cells/kg or 1.9X 10 ⁶ anti-CD 19CAR T cells/kg. In some embodiments, the CD19CAR construct contains a CD3 ζ T cell activation domain and a CD28 signaling domain. In some embodiments, the T cell product is KTE-X19. In some embodiments, the disclosure demonstrates that anti-CAR T cell products prepared as described in the preceding paragraphs can be used in B cell ALL and B cell NHL. In one embodiment, the T cell product has the characteristics of the products of table 23. In some embodiments, the product characteristics may be selected from the percentage of T cells of a particular subpopulation (naive, central memory, effector and effector memory), the percentage of CD4+ cells, the percentage of CD8+ cells and the CD4/CD8 ratio. In some embodiments, the product characteristic is the level of IFN γ production (pg/mL) in a co-culture of CD 19-expressing target cancer cells (e.g., Toledo) cells mixed with anti-CD 19CAR T product cells at a 1:1 ratio. In one embodiment, IFN γ can be measured in cell culture media 24 hours after incubation using a qualified ELISA. In some embodiments, one or more of these product characteristics are superior to the product characteristics of anti-CAR T cells prepared by leukapheresis without enrichment of CD4+/CD8+ positive cells. In some embodiments, the superior product characteristics may be selected from an increase in the percentage of cells with a naive phenotype (CD45RA + CCR7+), a decrease in the percentage of cells with a differentiated phenotype (CCR7-), a decrease in the level of IFN γ -producing cells, and an increase in the level of CD8+ cells. In some embodiments, the anti-CD 19T cell product comprises T _CM Central memory T cells (CD45RA-CCR7 +); t is _EFF Effector T cells (CD45RA + CCR 7-); t is _EM Effector memory T cells (CD45RA-CCR 7-); and/or T _N Naive-like T cells (CD45RA + CCR7 +). In some embodiments, the product comprises T _N Naive-like T cells (meaning T cells that are CD45RA + CCR7+) and contain stem-like memory cells. In some embodiments, the T cell product is KTE-X19. In some embodiments, KTE-X19 has IFN- γ production of ≧ 190 pg/mL. In certain embodiments, KTE-X19 has ≧ 90% CD3+ cells. In some other embodiments, the percentage of NK cells in KTE-X19 is 0.1% (ranging from 0.0% to 2.8%). In some additional embodiments, CD3 in KTE-X19 ^- The percentage of cellular impurities was 0.5% (range 0.3% -3.9%).

In some embodiments, the cancer is relapsed/refractory B-cell ALL. In some embodiments, the patient is ≦ 21 years old. In some embodiments, the patient is < 21 years old, weighs > 10kg, and has B-cell ALL that is primary refractory, relapses within 18 months of first diagnosis, is relapsed/refractory after > 2-line systemic therapy, or is relapsed/refractory after allogeneic stem cell transplantation for at least 100 days prior to enrollment. In one embodiment, the cancer is a slow-progressing lymphoma or leukemia. In one embodiment, the cancer is an aggressive B-cell lymphoma, which includes diffuse large B-cell lymphoma (DLBCL), Burkitt's Lymphoma (BL), mantle cell lymphoma and its blast-like variants, and many types, subtypes, and variants of B lymphoblastic lymphoma. DLBCL can be DLBCL NOS, large B cell lymphoma enriched for T cells/tissue cells, primary DLBCL of CNS, primary skin DLBCL, old person leg type EBV positive DLBCL. Other lymphomas of large B cells include primary mediastinal (thymic) LBCL, DLBCL, lymphomatoid granulomatosis, ALK-positive LBCL, plasmablast lymphoma, large B cell lymphoma produced in HHV 8-associated multicenter castleman disease, and primary effusion lymphoma associated with chronic inflammation. Other types of lymphomas include non-classifiable B-cell lymphomas with characteristics between DLBCL and burkitt's lymphoma and non-classifiable B-cell lymphomas between DLBCL and classical hodgkin's lymphoma, splenic marginal zone B-cell lymphomas, extranodal marginal zone B-cell lymphomas of MALT, nodal marginal zone B-cell lymphomas, hairy cell leukemia, lymphoplasmacytic lymphoma (fahrenheit macroglobulinemia), and primary effusion lymphoma. The cancer may be at any stage from stage 1 to stage 4.

ALL is a common childhood malignancy, constituting about 80% of childhood leukemias and about 25% of ALL childhood cancers. About 20% of pediatric patients do not achieve long-term remission after initial therapy, and the 5-year overall survival rate is about 55%. Hunger SP et al, N Engl J Med (N Engl J Med.), 2015; 373: 1541-; sun W et al, Leukemia (Leukemia) 2018; 2316 and 2325; rheinglod SR et al, J Clin Oncol 2019; 10008 (supplement, abstract); and Oskarsson T et al, hematology (Haematologica.) 2016; 101:68-76. The results were poor for the following patients: patients who relapse early or have primary refractory disease after initial treatment; patients with refractory/relapsed disease after stem cell transplantation; and multiple relapsing patients. Sun W et al, Leukemia (Leukemia) 2018; 2316 and 2325; rheinglod SR et al, J Clin Oncol 2019; 10008 for 37 (supplement, abstract); oskarsson T et al, hematology (Haematologica.) 2016; 101: 68-76; nguyen K et al, Leukemia (Leukemia) 2008; 22: 2142-; crotta A et al, recent medical research opinions (Curr Med Res Opin.) 2018; 435: 440; schrappe M et al, New England journal of medicine (N Engl J Med.), 2012; 366:1371-1381. Patients who relapse within 18 months of initial diagnosis typically have an overall 5-year survival rate of 21% -28%. Rheinglod SR et al, J Clin Oncol 2019; 10008 for 37 (supplement, abstract); nguyen K et al, Leukemia (Leukemia) 2008; 22:2142-2150. The likelihood of achieving remission and the duration of EFS decreases with salvage therapy for each subsequent line. Sun W et al, Leukemia (Leukemia) 2018; 32:2316-2325. Pediatric and adolescent patients with R/R ALL still have poor outcomes after treatment with the novel therapies bornauzumab and oxuntolizumab (inotuzumab ozogamicin), with an overall 1-year survival rate of about 36%, highlighting the need for more effective treatment options. von Stackelberg A et al, J.Clin Oncol. (J Clin Oncol.) 2016; 34:4381-4389. 10; bhojwani D et al, Leukemia (Leukemia.) 2019; 33:884-892.

In some embodiments, the cancer is B-cell NHL, and the key registration criteria include age <18 years, body weight ≧ 10kg, histologically confirmed diffuse large B-cell lymphoma (DLBCL NOS), primary mediastinal large B-cell lymphoma, Burkitt Lymphoma (BL), burkitt-like lymphoma, or unclassified B-cell lymphoma between DLBCL and BL with ≧ 1 measurable lesion. In one embodiment, for NHL treatment, the disease may be primary refractory, relapsed/refractory after ≧ 2-line systemic therapy, or relapsed/refractory after autologous or allogeneic stem cell transplantation before ≧ 100 days of enrollment. Patients with acute graft versus host disease or chronic graft versus host disease requiring treatment within 4 weeks of enrollment may not be eligible.

In some embodiments, these B cell ALL and/or B cell NHL patients received 25mg/m daily on days-4, 3 and 2 ² Fludarabine and 900mg/m daily on day-2 ² Opsonic chemotherapy of cyclophosphamide followed by 1X 10 on day 0 ⁶ Target dose of individual anti-CD 19 CAR T cells/kg a single infusion of anti-CD 19 CAR T cells enriched for CD4+/CD8+ (prepared as described immediately above).

In some embodiments, the disclosure provides the use of CD4+/CD8+ -enriched/cancer cell-depleted anti-CD 19 CAR T cells to successfully treat B-cell ALL, wherein the patient is aged 18 or older, has R/R B-cell ALL, is defined as refractory to first-line therapy (i.e., primary refractory), relapses no more than 12 months after first remission, relapses or refractory after 2 previous-line systemic therapies, or relapses after allogeneic Stem Cell Transplantation (SCT). In some embodiments, the patient in need thereof has ≧ 5% medulloblasts, 0 or 1 beautyThe eastern national tumor cooperative group of physical performance status and adequate kidney, liver and heart function. For patients receiving prior bornaemetic, leukemic blast cells with > 90% expression of CD19 are required. Central Nervous System (CNS) -2 diseases with philadelphia chromosome positive (Ph +) disease, coexisting extramedullary disease, no neurological changes (cerebrospinal fluid [ CSF ]]The mother cell has<5 leukocytes/mm ³ ) Patients and patients with down syndrome are eligible. CNS-3 diseases independent of neurological changes (CSF blasts with > 5 leukocytes/mm) ³ ) And history of CNS disorders was excluded. In some embodiments, additional inclusion and exclusion criteria are described in example 9.

In some embodiments, the patient may have a primary refractory cancer. In some embodiments, the patient may have cancer that recurs after SCT. In some embodiments, the patient may have received prior bornauzumab, which may have been the last therapy used prior to anti-CD 19 CAR T cell therapy. In some embodiments, the patient baseline characteristic is a patient baseline characteristic of any one of the patients set forth in table 18.

In some embodiments, by 2 × 10 ⁶ 、1×10 ⁶ Or 0.5X 10 ⁶ Individual CAR T cells/kg were administered to these B-cell ALL patients. In some embodiments, 0.5 × 10 ⁶ Individual CAR T cells/kg were administered in a total volume of 40mL of formulation. In another embodiment, 0.5X 10 ⁶ Individual CAR T cells/kg were administered in a total volume of 68mL of formulation. In some embodiments, the CAR T cell product is formulated in a total volume of 20mL, 25mL, 30mL, 35mL, 40mL, 45mL, 50mL, 55mL, 60mL, 65mL, 70mL, 75mL, 80mL, 85mL, 90mL, 95mL, 100mL, 200mL, 300mL, 400mL, 500mL, 700mL, 800mL, 900mL, or 1000 mL. In some embodiments, the 40mL formulation is intended to maintain cell density and cell viability during the freeze/thaw process.

In some embodiments, the treatment is associated with an adverse event. In some embodiments, the one or more adverse events are managed according to any of table 13, table 14, table 16, or a combination thereof. In some embodiments, the one or more adverse events are managed according to the Original Management Guidelines (Original Management Guidelines) of table 16. In some embodiments, one or more adverse events are managed according to the revised management guidelines of table 16. In some embodiments, a boosting agent may be administered to treat CRS. In some embodiments, the signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia, and hypotension. In some embodiments, the signs or symptoms associated with a neurological event include encephalopathy, seizures, changes in consciousness level, speech impairment, tremors, and confusion.

In some embodiments, the patient may have a high disease burden at baseline, which is defined as having a high disease burden in the bone marrow by local viewing>25% of leukemic blast cells or more than 1,000 blast cells/mm in the peripheral circulation ³ ). In some embodiments, the patient may receive bridging chemotherapy after leukapheresis and before opsonic chemotherapy. In some embodiments, the bridging chemotherapy follows one of the predetermined bridging chemotherapy regimens of table 17.

In some embodiments, the opsonization chemotherapy/lymph depletion regimen is administered after ≧ 7 days or 5 half-lives (if shorter) clearance from the bridging chemotherapy. In some embodiments, the opsonic chemotherapy/lymph depletion regimen consists of daily Intravenous (IV) administration of 25mg/m on days-4, 3, and 2 ² Fludarabine and daily IV 900mg/m on day-2 ² Cyclophosphamide. On day 0, a single infusion of anti-CD 19 CAR T cells may be administered. In some embodiments, additional infusions of anti-CD 19 CAR T cells may be administered at a later time. In some embodiments, patients who achieve a complete response to the first infusion may receive a second infusion of anti-CD 19 CAR T cells if in remission>Progressing after 3 months, the provided CD19 expression has been retained and it is not suspect to neutralize antibodies against the CAR.

In some embodiments, droplet digital polymerase chain reaction may be used to measure transduced anti-CD 19 CAR + T thin lines in bloodPresence, amplification and persistence of cells. In some embodiments, the procedure is performed as described in the following documents: locke f.l. et al, molecular therapeutics (Mol Ther), 2017; 25(1):285-295. In some embodiments, the present disclosure provides a method of treatment, wherein the CAR T cell level is as described in table 22. In some embodiments, the disclosure demonstrates that CAR T cells may not be detectable at relapse. Median peak CAR T cell levels may be at 1 × 10 ⁶ Individual CAR T cells/kg are highest in case and may be similar between patients receiving original and revised AE management. In some embodiments, patients who achieve CR/CRi have a greater median peak amplification than non-responders, as do patients with undetectable MRD versus detectable MRD. Higher median peak amplification was also observed in patients with grade 3 NE compared to those with grade 2 NE. Some patients who relapse may have detectable CD19 positive cells or may not have detectable CD19 positive cells at the time of relapse. In some embodiments, flow cytometry (NeoGenomics, Fort Myers, FL) can be used to assess undetected MRD (defined as MRD) according to the methods described in the following references<1 leukemia cells/10,000 viable cells): borowitz MJ, Wood BL, Dedevidas M et al, Blood (Blood), 2015; 126(8) 964-971; bruggemann m et al, Blood progression (Blood Adv.) 2017; 2456-2466; or Gupta s. et al, Leukemia (leukamia) 2018; 32(6):1370-1379.

In some embodiments, the disclosure demonstrates that peak levels of some cytokines, chemokines, and pro-inflammatory markers occur on day 7. In some embodiments, 1X 10 ⁶ 2X 10 administration of individual CAR T cells/kg ⁶ Some of these tended to be higher in individual CAR T cell/kg patients (IL-15, CRP, SAA, CXCL10, IFN γ) or lower in patients managed with revised AEs than in patients managed with original AEs (IL-6, ferritin, IL-1RA, IFN γ, IL-8, CXCL10, MCP-1). In some embodiments, the levels of these proteins/biomarkers are varied as described in figure 9, figure 10, and figure 11. Thus, in some embodiments, the first and second electrodes are,the present disclosure provides methods for using these protein levels as biomarkers for CRS grade 3 and/or grade 0-2. In some embodiments, the present disclosure provides methods of using these protein levels as biomarkers for CRS grades 3 and/or 0-2 according to their values in FIG. 11.

In some embodiments, the disclosure demonstrates that peak IL-15 serum levels are lower in patients with CRS grade ≧ 3. In some embodiments, the present disclosure provides that the median peak levels of several proinflammatory markers tend to be higher in patients with CRS grade ≧ 3 and patients with NE grade ≧ 3 (IFN γ, IL-8, GM-CSF, IL-1RA, CXCL10, MCP-1, granzyme B), as depicted in FIG. 11. Thus, in some embodiments, the present disclosure provides a method for predicting whether a patient will have CRS grade ≧ 3 by measuring peak levels of serum IL-15 and comparing to reference standards. In some embodiments, the present disclosure provides a method for predicting whether a patient will have grade 3 CRS and/or grade 3 NE by measuring the peak levels of IFN γ, IL-8, GM-CSF, IL-1RA, CXCL10, MCP-1, and/or granzyme B and comparing to reference standards. In some embodiments, the present disclosure provides a method of improving anti-CD 19 CAR T cell therapy by administering an agent that reduces the level of one or more of these biomarkers.

The reference level/standard may be established by any method known to one of ordinary skill in the art. They are used to identify a threshold or set of values (e.g., quartile) that can be compared to determine which set the measured value (cytokine level, CAR T cell number, etc.) for each subject falls within or is above or below the threshold. These groups are established by comparison of different populations selected, as is typical in the art. Depending on where the measurement falls, one can predict many treatment characteristics, such as objective response, CRS level, NE level, etc.

In certain embodiments, the cancer may be selected from tumors derived from: acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), adenoid cystic carcinoma, adrenocortical carcinoma, cancer, AIDS-related carcinoma, anal carcinoma, appendiceal carcinoma, astrocytoma, atypical teratoid/rhabdoid tumors, central nervous system carcinoma, B-cell leukemia, lymphoma or other B-cell malignancies, basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, bone carcinoma, osteosarcoma and malignant fibrous histiocytoma, brain stem glioma, brain tumor, breast carcinoma, bronchial tumors, Burkitt's lymphoma, carcinoid tumors, central nervous system carcinoma, cervical carcinoma, chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), chronic myeloproliferative disorders, colon carcinoma, colorectal carcinoma, craniopharyngioma, cutaneous t-cell lymphoma, embryonic tumors, central nervous system carcinoma, endometrial carcinoma, cervical carcinoma, bladder carcinoma, carcinoma of the head of the body, and neck, Ependymoma, esophageal cancer, sensoroblastoma, ewing's sarcoma family of tumors, extracranial germ cell tumors, extrahepatic bile duct cancer, eye cancer, bone fibroblastic cell tumors, malignancies, osteosarcoma, gallbladder cancer, stomach (gastic/stomamach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST), soft tissue sarcoma, germ cell tumors, gestational trophoblastic tumors, gliomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors (endocrine pancreas), kaposi's sarcoma, kidney cancer, langerhans cell histiocytosis, larynx cancer, leukemia, lip cancer and oral cancer, liver cancer (primary), Lobular Carcinoma In Situ (LCIS), lung cancer, lymphoma, macroglobulinemia, liver cancer (primary), and bladder cancer, Male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, medulloblastoma, melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary mesogenic carcinomas involving the NUT gene, oral cancer, multiple endocrine tumor syndrome, multiple myeloma/plasma cell tumor, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative tumors, Chronic Myelogenous Leukemia (CML), Acute Myeloid Leukemia (AML), multiple myeloma, myeloproliferative disorders, cancers of the nasal and sinus cavities, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, cancer of the sinuses and nasal cavities, Parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate differentiated pinealoma, pinealoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumors, plasma cell tumors/multiple myeloma, pleuropulmonablastoma, pregnancy and breast cancer, primary Central Nervous System (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma, sezary syndrome, small cell lung cancer, small cell carcinoma of the small intestine, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, gastric (gastic/stomach) cancer, supratentorial primitive neuroectodermal tumors, t-cell lymphoma, skin cancer, testicular cancer, laryngeal, thymoma and thymus cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic cell tumor, Ureter and renal pelvis cancer, cancer of the urethra, cancer of the uterus, uterine sarcoma, vaginal cancer, cancer of the vulva, Waldenstrom's macroglobulinemia, Wilms' tumor. In certain embodiments, the cancer is treated with KTE-X19.

In one embodiment, the method can be used to treat a tumor, wherein the tumor is a lymphoma or leukemia. Lymphomas and leukemias are hematological cancers that specifically affect lymphocytes. All leukocytes in the blood originate from a single type of pluripotent hematopoietic stem cell found in the bone marrow. Such stem cells produce both myeloid and lymphoid progenitor cells, which then produce the various types of leukocytes found in vivo. Leukocytes produced by myeloid progenitor cells include T lymphocytes (T cells), B lymphocytes (B cells), natural killer cells, and plasma cells. Leukocytes produced by lymphoid progenitor cells include megakaryocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, and macrophages. Lymphomas and leukemias may affect one or more of these cell types in a patient. In certain embodiments, the tumor is treated with KTE-X19.

Generally, lymphomas can be divided into at least two subgroups: hodgkin lymphoma and non-hodgkin lymphoma. Non-hodgkin's lymphoma (NHL) is a heterogeneous group of cancers derived from B lymphocytes, T lymphocytes or natural killer cells. B-cell lymphomas represent 80-85% of reported cases in the United states. In 2013, approximately 69,740 new cases of NHL associated with disease and more than 19,000 deaths were estimated to occur. Non-hodgkin's lymphoma is the most prevalent hematologic malignancy and is the seventh major site of new cancer in men and women, and accounts for 4% of all new cancer cases and 3% of cancer-related deaths. In certain embodiments, the lymphoma is treated with KTE-X19.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of NHL, accounting for approximately 30% of NHL cases. There are approximately 22,000 new diagnosed DLBCLs in the united states each year. It is classified as aggressive lymphoma, with the majority of patients cured with conventional chemotherapy (NCCN guideline NHL 2014). First-line therapy of DLBCL typically includes anthracycline-containing regimens with rituximab, such as R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), which has an objective response rate of about 80% and a complete response rate of about 50%, with about one-third of patients suffering from refractory disease to initial therapy or relapsing after R-CHOP. For those patients who relapse after responding to first-line therapy, a second response to additional chemotherapy may be achieved in approximately 40-60% of patients. The standard of care for second line therapy in eligible patients for Autologous Stem Cell Transplantation (ASCT) includes rituximab and combination chemotherapies such as R-ICE (rituximab, ifosfamide, carboplatin, and etoposide) and R-DHAP (rituximab, dexamethasone, cytarabine, and cisplatin), each with an objective response rate of about 63% and a complete response rate of about 26%. Patients who respond to second-line therapy and are considered sufficiently suitable for transplantation receive a combination with high-dose chemotherapy and ASCT, which is cured in about half of the transplanted patients. Patients who fail ASCT have a very poor prognosis and no cure option. Primary mediastinal large B-cell lymphoma (PMBCL) has different clinical, pathological, and molecular characteristics compared to DLBCL. PMBCL is thought to be produced by thymic (medullary) B cells and represents approximately 3% of patients diagnosed with DLBCL. PMBCL is usually identified in the younger adult population in the fortieth year of life, with women being somewhat dominant. Gene expression profiles indicate deregulated pathways where PMBCL overlaps with hodgkin lymphoma. Initial therapy for PMBCL typically includes anthracycline-containing regimens with rituximab, such as etoposide, doxorubicin, and cyclophosphamide with adjustable infusion doses, as well as vincristine, prednisone, and rituximab (DA-EPOCH-R), with or without radiation therapy of the affected area. Follicular Lymphoma (FL), a B-cell lymphoma, is the most common indolent (slow-growing) form of NHL, accounting for approximately 20% to 30% of all NHLs. Some patients with FL will be histologically Transformed (TFL) to DLBCL, which is more aggressive and associated with poor outcome. The histological transformation to DLBCL was carried out for 15 years at an annual rate of approximately 3%, with the risk of transformation continuing to decline in subsequent years. The biological mechanism of histological transformation is unknown. Initial treatment of TFL is affected by previous therapy of follicular lymphoma, but typically involves an anthracycline-containing regimen with rituximab to eliminate the aggressive components of the disease. The treatment options for relapsed/refractory PMBCL and TFL are similar to those in DLBCL. Given the low prevalence of these diseases, a number of prospective randomization studies have not been performed in these patient populations. Patients with chemotherapy-refractory disease have a similar or worse prognosis than patients with refractory DLBCL. For example, subjects with refractory, aggressive NHL (e.g., DLBCL, PMBCL, and TFL) have major unmet medical needs, and further studies with novel treatments are needed in these populations. In certain embodiments, the DLBCL is treated with KTE-X19.

The CAR T cell therapy of the present disclosure can be administered as a first line therapy or a second or later line of therapy. In some embodiments, CAR T cell therapy is administered as a third line, fourth line, fifth line, and the like. The previous line of therapy can be any previous anti-cancer therapy, including, but not limited to, Bruton's Tyrosine Kinase Inhibitor (BTKi), checkpoint inhibitors (e.g., anti-PD 1 antibody, Pabollizumab (Keytruda), cimetiprizumab (Libtayo), nivolumitumumab (Opdivo); anti-PD-L1 antibody, cetilizumab (Tecntriq), Avermectimab (Bavencio), Duvaliuzumab (Imfinizi); anti-CTLA-4 antibody, ipilimumab (Yervoy)), anti-CD 19 antibody (e.g., Boratezumab), anti-CD 52 antibody (e.g., alemtuzumab); allogeneic stem cell transplantation, anti-CD 20 antibodies (e.g., rituximab), systemic chemotherapy, rituximab, anthracyclines, ofatumumab, and combinations thereof. Previous therapies may also be used in combination with the CD19 CAR T therapy of the present application. In one aspect, eligible patients may have a disease refractory to recent therapy or relapse within 1 year after autologous hematopoietic stem cell transplantation (HSCT/ASCT). CAR T cell therapy can be administered to a patient having or suspected of having cancer that is refractory and/or relapsed from a previous therapy to one or more lines. The cancer may be refractory to first line therapy (i.e., primary refractory) or refractory to therapy of one or more lines. Cancer may recur twelve months after first remission, recur or be refractory after two or more lines of prior therapy, or recur after HSCT/ASCT. In some embodiments, the cancer is refractory to ibrutinib or acatinib. In some embodiments, the cancer is NHL and the disease must be primary refractory, relapsed/refractory after systemic therapy of two or more lines, or relapsed/refractory after autologous or allogeneic stem cell transplantation for > 100 days prior to enrollment into CAR T cell therapy and > 4 weeks with cessation of immunosuppressive drugs. In certain embodiments, the CAR T cell therapy is KTE-X19.

Thus, the method may be used to treat a lymphoma or leukemia, wherein the lymphoma or leukemia is a B-cell malignancy. Examples of B cell malignancies include, but are not limited to, non-hodgkin's lymphoma (NHL), small lymphocytic lymphoma (SLL/CLL), Mantle Cell Lymphoma (MCL), FL, Marginal Zone Lymphoma (MZL), extranodal (MALT lymphoma), nodal (monocyte-like B cell lymphoma), spleen diffuse large cell lymphoma, B cell chronic lymphocytic leukemia/lymphoma, burkitt's lymphoma, and lymphoblastic lymphoma. In some aspects, the lymphoma or leukemia is selected from B-cell chronic lymphocytic leukemia/small cell lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (e.g., waldenstrom's macroglobulinemia), splenic marginal zone lymphoma, hairy cell leukemia, plasma cell tumors (e.g., plasma cell myeloma (i.e., multiple myeloma, or plasmacytoma), extranodal marginal zone B cell lymphoma (e.g., MALT lymphoma), nodal marginal zone B cell lymphoma, Follicular Lymphoma (FL), Transformed Follicular Lymphoma (TFL), primary cutaneous follicular central lymphoma, mantle cell lymphoma, Diffuse Large B Cell Lymphoma (DLBCL), Epstein-Barr virus positive bcl, lymphoblastoid granulomatosis, primary mediastinal (thymus) large B cell lymphoma (PMBCL), intravascular large B cell lymphoma, lymphoblastic leukemia, and lymphoma, ALK + large B cell lymphoma, plasmacytoma lymphoma, primary effusion lymphoma, large B cell lymphoma arising from HHV 8-associated multicenter Castleman disease, Burkitt's lymphoma/leukemia, T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T-cell leukemia/lymphoma, extranodal NK/T cell lymphoma, enteropathy-associated T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides/sezary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, peripheral T cell lymphoma, angioimmunoblastic T cell lymphoma, anaplastic large cell lymphoma, B lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities, T lymphoblastic leukemia/lymphoma and hodgkin's lymphoma. In some aspects, the cancer is refractory to one or more previous treatments, and/or the cancer has relapsed after one or more previous treatments. In certain embodiments, the leukemia or lymphoma is treated with KTE-X19.

In one embodiment, the cancer is selected from follicular lymphoma, transformed follicular lymphoma, diffuse large B-cell lymphoma, and primary mediastinal (thymic) large B-cell lymphoma. In another embodiment, the cancer is diffuse large B-cell lymphoma. In some embodiments, the cancer is refractory to, or has relapsed after, one or more therapies of chemotherapy, radiation therapy, immunotherapy (including T cell therapy and/or treatment with antibodies or antibody-drug conjugates), autologous stem cell transplantation, or any combination thereof. In one embodiment, the cancer is refractory diffuse large B-cell lymphoma. In certain embodiments, the cancer is treated with KTE-X19.

In some embodiments, the CAR T cell therapy is KTE-X19, and the cancer is selected from MCL, ALL, CLL, and SLL. In some embodiments, the CAR T cell therapy is KTE-X19 and the cancer is NHL. In some embodiments, the cancer is selected from diffuse large B-cell lymphoma (DLBCL NOS), primary mediastinal large B-cell lymphoma, Burkitt's Lymphoma (BL), burkitt-like lymphoma, or unclassified B-cell lymphoma between DLBCL and BL, not otherwise specified. In some embodiments, the cancer is relapsed/refractory. In some embodiments, KTE-X19 treatment is administered as first line, second line therapy, or after 1 or more existing line therapies. In some embodiments, the patient is a pediatric patient, an adolescent patient, an adult patient, a patient less than 65 years old, over 65 years old, or any other age group.

In some embodiments, the compositions comprising immune cells disclosed herein can be administered in combination with any number of additional therapeutic agents. In one embodiment, the additional therapeutic agent is administered concurrently with the T cell therapy. In one embodiment, the additional therapeutic agent is administered before, during, and/or after the T cell therapy. In one embodiment, one or more additional therapeutic agents are administered prophylactically. In one aspect, a composition comprising immune cells is administered in combination with an agent for managing adverse events, many of which are described elsewhere in the application, including the examples section. These agents may manage one or more signs and symptoms of adverse reactions such as fever, hypotension, tachycardia, hypoxia and chills, including cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal insufficiency, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphoblastic cell proliferation/macrophage activation syndrome (HLH/MAS), epilepsy, encephalopathy, headache, tremor, vertigo, aphasia, delirium, insomnia anxiety, anaphylaxis, fever neutropenia, thrombocytopenia, neutropenia and anemia.

Examples of such agents include, but are not limited to, tollizumab, steroids (e.g., methylprednisolone), rabbit anti-thymocyte globulin. In some aspects, vancomycin and aztreonam (1 mg given twice daily IV each) may be administered for non-neutropenic fever. In some aspects, the method further comprises administering a non-sedating antiepileptic drug for epilepsy prevention; administering at least one of erythropoietin, alfabepiptin, platelet infusion, filgrastim or pefilgrastim; and/or administering tollizumab, cetuximab. In one aspect, the agent is a member of the CSF family such as GM-CSF (granulocyte-macrophage colony stimulating factor, also known as CSF 2). GM-CSF can be produced by a variety of hematopoietic and non-hematopoietic cell types when stimulated, and it can activate/"prime" the myeloid cell population to produce inflammatory mediators, such as TNF and interleukin 1 β (IL1 β). In some embodiments, the GM-CSF inhibitor is an antibody that binds to and neutralizes circulating GM-CSF. In some embodiments, the antibody is selected from the group consisting of ranibizumab; natalizumab (AMG 203); GSK3196165/MOR 103/otilizumab (GSK/MorphoSys), KB002 and KB003(KaloBios), MT203(Micromet and Nycomed) and MORAB-022/nivolumab (Morphotek). In some embodiments, the antibody is a biosimilar of the antibody. In some embodiments, the antagonist is E21R, a modified form of GM-CSF that antagonizes the function of GM-CSF. In some embodiments, the inhibitor/antagonist is a small molecule. In one embodiment, the CSF family member is M-CSF (also known as macrophage colony stimulating factor or CSF 1). Non-limiting examples of agents that inhibit or antagonize CSF1 include small molecules, antibodies, chimeric antigen receptors, fusion proteins, and other agents. In one embodiment, the CSF1 inhibitor or antagonist is an anti-CSF 1 antibody. In one embodiment, the anti-CSF 1 antibody is selected from those prepared by roche (e.g., RG7155), Pfizer (PD-0360324), novartis (MCS 110/trastuzumab), or A biosimilar version of any one of the antibodies. In some embodiments, the inhibitor or antagonist inactivates the activity of the GM-CSF-R- α (also known as CSF2R) or CSF1R receptor. In some embodiments, the inhibitor is selected from mavrilimumab (formerly CAM-3001), a fully human GM-CSF receptor alpha monoclonal antibody currently being developed by medical immunology companies; cabepratumab (Five Prime Therapeutics); LY3022855(IMC-CS4) (lilac), emmetruzumab, also known as RG7155 or RO 5509554; FPA008, a humanized monoclonal antibody (Five Prime/BMS); AMG820 (ann corporation); ARRY-382(Array Biopharma); MCS110 (nova corporation); PLX3397 (Plexxikon); ELB041/AFS98/TG3003(ElsaLys Bio, Transgene), SNDX-6352 (Syndax). In some embodiments, the inhibitor or antagonist is expressed in a CAR-T cell. In some embodiments, the inhibitor is a small molecule (e.g., a heteroaryl amide, a quinolinone series, a pyridopyrimidine series); BLZ945 (Nowa), PLX7486, ARRY-382, Pexidrtinib (also known as PLX3397) or 5- ((5-chloro-1H-pyrrolo [2, 3-b)]Pyridin-3-yl) methyl) -N-06- (trifluoromethyl) pyridin-3-yl) methyl) pyridin-2-amine; GW 2580(CAS 870483-87-7) available from Ambit Silocinenes Inc, (CAS 623142-96-1), AC708, or any CSF1R inhibitor listed in: cannarile et al, Journal of ImmunoTherapy of Cancer 2017, 5:53 and US20180371093, which are incorporated herein by reference for their purpose of disclosing inhibitors. Additional neutralizing antibodies to GM-CSF or its receptor have been described in the art, including, for example, "GM-CSF as a target for inflammatory/autoimmune diseases: evidence for prior and future therapeutic potential (GM-CSF a target in inflammation/autoimmunity disease), "Hamilton, J.A., review of clinical immunology experts (Expert Rev.Clin. Immunol.), 2015; and "Targeting GM-CSF in inflammatory diseases", Wicks, i.p., Roberts, a.w., for natural reviews: rheumatology (nat. Rev. Rhe)umatol.), 2016. In other embodiments, the agent is an anti-IL 6 or anti-IL 6 receptor blocker, including tocilizumab and cetuximab.

In one aspect, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and Cyclophosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzotepa, carboquone, metotepipa, and uretepa; ethyleneimine and methylmelamine, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine (trimethlomelamine) resin; nitrogen mustards such as chlorambucil, naphazel, cholorfamide, estramustine, ifosfamide, dichloromethyl diethylamine, mechlorethamine hydrochloride, melphalan, neomustard, benzene mustard cholesterol, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorourethrin, fotemustine, lomustine, nimustine, ramustine; antibiotics, such as aclacinomycin, actinomycin, anthracycline, azaserine, bleomycin, actinomycin C, calicheamicin, carminomycin, carzinophilin, chromomycin, actinomycin D, daunorubicin, mitomycin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, isosbacin, idarubicin, sisomicin, mitomycin, mycophenolic acid, norubicin, olivomycin, pelomycin, pofiomycin, puromycin, trirubicin, roxobicin, streptonigromycin, streptozotocin, tubercidin, ubenimex, setastin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as carpoterone, drostandrosterone propionate, epitioandrostanol, meindroxane, testolactone; anti-adrenaline, e.g. aminoglutethimide Mitotane, trostane; folic acid replenisher such as folinic acid; acetic acid glucurolactone; an aldehydic phosphoramide glycoside; (ii) aminolevulinic acid; amsacrine; busamustine; a bisantrene group; edatrexae; desphosphamide; colchicine; diazaquinone; iloxanel (elformithine); ammonium etiolate; etoglut; gallium nitrate; a hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidanol; diamine nitracridine; pentostatin; methionine; pirarubicin; podophyllinic acid; 2-ethyl hydrazide; procarbazine;lezoxan; a texaphyrin; a germanium spiroamine; alternarionic acid; a tri-imine quinone; 2,2' -trichlorotriethylamine; uratan; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; gatifloxacin (gacytosine); arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (TAXOLTM, Bristol-Myers Squibb) and docetaxel (R)Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; novier; noscapine; (ii) teniposide; daunomycin; aminopterin; (ii) Hirodar; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoic acid derivatives such as targretin (bexarotene), panretin (aliskiren acid); ONTAKTM (Deny interleukin); esperamicin (esperamicin); capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. In some aspects, a composition comprising an immune effector cell expressing a CAR and/or TCR disclosed herein can be administered in combination with an anti-hormonal agent, such as an anti-estrogen agent, for modulating or inhibiting hormonal effects on a tumor Elements including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5) -imidazole, 4-hydroxyttamoxifen, trovaxifen, raloxifene hydrochloride, LY117018, onapristone, and toremifene (faretone); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Where appropriate, a combination of chemotherapeutic agents is also administered, including but not limited to CHOP, i.e., cyclophosphamideDoxorubicin (hydroxydoxorubicin), vincristineAnd prednisone.

These chemotherapeutic agents may be administered at the same time as or within one week after the engineered cell or nucleic acid is administered. In other aspects, the chemotherapeutic agent is administered 1 to 4 weeks or 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after administration of the engineered cell or nucleic acid. In some aspects, the chemotherapeutic agent is administered at least 1 month prior to administration of the cell or nucleic acid. In some aspects, the method further comprises administering two or more chemotherapeutic agents.

A variety of additional therapeutic agents may be used in combination with the compositions or agents/treatments described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors, such as nivolumab PembrolizumabPembrolizumab, pidilizumab (CureTech), and alemtuzumab (Roche), tosubuzumab (with and without corticosteroids; GM-CSF, CSF1, GM-CSFR, or CSF1R GM-CSF, CSF1, GM-CSFR, or CSF1R inhibitors (anti-CSF 1 antibodies are selected from those made by Roche)(e.g., RG7155), Pfizer (PD-0360324), Novartis (Novartis) (MCS 110/lactotuzumab)), Mavrilimumab (formerly CAM-3001), fully human GM-CSF receptor alpha monoclonal antibody recently developed by medimernini (medimmunene, Inc.); cabepratumab (Five Prime Therapeutics); LY3022855(IMC-CS4) (lilac), emmetruzumab, also known as RG7155 or RO 5509554; FPA008, a humanized monoclonal antibody (Five Prime/BMS); AMG820 (ann corporation); ARRY-382(Array Biopharma); MCS110 (nova corporation); PLX3397 (Plexxikon); ELB041/AFS98/TG3003(ElsaLys Bio, Transgene), SNDX-6352 (Syndax). In some aspects, the inhibitor or antagonist is expressed in a CAR-T cell. In some aspects, the inhibitor is a small molecule (e.g., a heteroaryl amide, a quinolinone series, a pyridopyrimidine series); BLZ945 (Nowa), PLX7486, ARRY-382, Pexidrtinib (also known as PLX3397) or 5- ((5-chloro-1H-pyrrolo [2, 3-b) ]Pyridin-3-yl) methyl) -N-06- (trifluoromethyl) pyridin-3-yl) methyl) pyridin-2-amine; GW 2580(CAS 870483-87-7) available from Ambit Silocinenes Inc,(CAS 623142-96-1), AC708, or any CSF1R inhibitor listed in: cannarile et al, Journal of ImmunoTherapy of Cancer, 2017, 5:53 and US20180371093, which are incorporated herein by reference for their purpose of disclosing inhibitors. Additional neutralizing antibodies against GM-CSF or its receptor have been described in the art. Additional therapeutic agents suitable for use in combination with the compositions or agents/treatments and methods disclosed herein include, but are not limited to, ibrutinibOlympic single antibodyRituximabBevacizumabTrastuzumabEnmetuzumabImatinibCetuximabPanitumumabCartuzumab, ibritumomab, Aframumab, Tusimumab, Bentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, lenatinib, lenalidomide, acitinib, masitinib, pazopanib, sunitinib, sorafenib, tosubulin, taranib, lestatinib, axitinib, cerdinib, lenvatinib, nidanib, nidatinib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tizanib, ritanib, vandetatinib, enretinib, cabertinib, imatinib, dasatinib, nilotinib, ponatinib, pluriptinib, laditinib, bosutinib, lesitinib, lestinib, gefitinib, letitinib, ritinib, erlotinib, gefitinib, ge, Crizotinib, aflibercept, adepside (adiplotide), dinil interleukin, mTOR inhibitors such as everolimus and temsirolimus, hedgehog inhibitors such as sonedgi and vismodegib, CDK inhibitors such as CDK inhibitors (palbociclib).

The composition or medicament/treatment comprising immune cells is or may be administered with an anti-inflammatory agent. Anti-inflammatory agents or anti-inflammatory agents may include, but are not limited to, steroids andglucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, corticosteroids, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone); non-steroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolate mofetil. Exemplary NSAIDs include ibuprofen, naproxen sodium, Cox-2 inhibitors, and sialylating agents. Exemplary analgesics include acetaminophen, oxycodone, tramadol, or propoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept)AdalimumabAnd infliximabChemokine inhibitors and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies as well as recombinant forms of the molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold preparations (oral (auranofin) and intramuscular), and minocycline.

The compositions or agents/treatments described herein may be administered in combination with cytokines and/or cytokine modulators as additional therapeutic agents. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; (ii) prorelaxin; glycoprotein hormones, such as producitonFollicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), and Luteinizing Hormone (LH); hepatocyte Growth Factor (HGF); fibroblast Growth Factor (FGF); prolactin; placental lactogen; a Mullerian tube inhibiting substance; mouse gonadotropin-related peptides; a statin; an activin; vascular endothelial growth factor; an integrin; thrombopoietin (TPO); nerve Growth Factor (NGF), such as NGF-beta; platelet growth factor; transforming Growth Factors (TGF), such as TGF-alpha and TGF-beta; insulin-like growth factors-I and-II; erythropoietin (EPO, and EPO,) (ii) a An osteoinductive factor; interferons such as interferon α, β, and γ; colony Stimulating Factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, tumor necrosis factors such as TNF- α or TNF- β; and other polypeptide factors, including LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, as well as biologically active equivalents of the native sequence cytokines. In one embodiment, the compositions described herein are administered in combination with a steroid or corticosteroid.

Corticosteroid therapy can be used to treat adverse events. Corticosteroids (or any other steroid and any other treatment of adverse events) may be used prophylactically before any symptom of an adverse event is detected and/or after an adverse event is detected. They may be administered prior to T cell administration, on the same day as T cell administration (before, after and/or during T cell administration), and/or one or more days after T cell administration. They may be administered before, during or after the conditioning therapy. Any corticosteroid may be appropriate for this use. In one embodiment, the corticosteroid is dexamethasone. In some embodiments, the corticosteroid is methylprednisolone. In some embodiments, the two agents are administered in combination. In some embodiments, the glucocorticoids include synthetic and non-synthetic glucocorticoids. Exemplary glucocorticoids include, but are not limited to: alclomethasone, aleprogesterone, beclomethasone (e.g., beclomethasone dipropionate), betamethasone (e.g., betamethasone 17 valerate, betamethasone sodium acetate, betamethasone sodium phosphate, betamethasone valerate), budesonide, clobetasol (e.g., clobetasol propionate), clobetasol, clocortolone (e.g., clocortolone pivalate), prednol, corticosterone, cortisone and hydrocortisone (e.g., hydrocortisone acetate), clovazole, deflazacort, desonide, desoximethasone, dexamethasone (e.g., dexamethasone 21-phosphate, dexamethasone acetate, dexamethasone sodium phosphate), diflunisal (e.g., diflunisal), diflucortolone, difluprednate, glycyrrhetinic acid, fluzacort, flurandrenolide, fludrocortisone (e.g., fludrocortisone acetate), Flumethasone (e.g., flumethasone pivalate), flunisolide, fluocinolone (e.g., fluocinonide), fluocinonide, fluocortin, fluocortolone, fluoromethalone (e.g., fluoromethalone acetate), fluperlone (e.g., fluperlone acetate), fluprednidene, flupredlone, fludrocortolone, fluticasone (e.g., fluticasone propionate), formocortat, halcinonide, halobetasol, halomethasone, haloprednisolone, hydrocortisone ester, hydrocortisone (e.g., hydrocortisone 21-butyrate, hydrocortisone aceponate, hydrocortisone acetate, hydrocortisone propionate, hydrocortisone butyrate, hydrocortisone cypionate, hydrocortisone hemisuccinate, hydrocortisone propionate, sodium hydrocortisone phosphate, hydrocortisone succinate, hydrocortisone valerate), loteprednol etanol etabonate, methylprednisolone, Methylosone, methylprednisolone (methylprednisolone aceponate, methylprednisolone acetate, methylprednisolone hemisuccinate, methylprednisolone sodium succinate), mometasone (mometasone furoate, for example), paramethasone (paramethasone acetate, for example), prednisolone carbonate, prednisolone (prednisolone 25-diethylaminoacetate, prednisolone sodium phosphate, prednisolone 21-hemisuccinate, prednisolone acetate; prednisolone farnesoate, prednisolone hemisuccinate, prednisolone-21 (beta-D-glucuronide), prednisolone metasulebenzoate, sparteinisone, prednisolone butyrate, prednisolone tetrahydrophthalate), prednisone, prednisolone valerate, prednisolone, rimexolone, cortisone, triamcinolone (for example, triamcinolone acetonide, prednisolone acetonide, mometasone furoate, prednisolone hemisuccinate, prednisolone acetate, prednisolone hemisuccinate, prednisolone acetate, prednisolone hemisuccinate, prednisolone, and prednisolone acetate, prednisolone, and prednisolone, and prednisolone, and prednisolone derivatives, and water, and, Triamcinolone acetonide, triamcinolone acetonide 21 palmitate, triamcinolone diacetate). These glucocorticoids and their salts are discussed in detail in, for example, the following documents: remington's Pharmaceutical Sciences, edited by A.Osol, Mark publishing company of Iston, Pa. (Mack pub. Co., Easton, 16 th edition 1980) and Remington: pharmaceutical sciences and practices (Remington: The Science and Practice of Pharmacy), 22 nd edition, Lippincott Williams & Wilkins, Philadelphia, Pa., Philadelphia, Philinia, and any other version of The company, Philington, Philin, Philinia, supra, is hereby incorporated by reference. In some embodiments, the glucocorticoid is selected from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, and prednisone. In one embodiment, the glucocorticoid is dexamethasone. In other embodiments, the steroid is a mineralocorticoid. Any other steroid may be used in the methods provided herein.

The one or more corticosteroids may be administered at any administration dose and frequency that may be appropriate for the severity/grade of the adverse event (e.g., CRS and NE). Tables 13, 14, and 16 provide examples of dosing regimens for managing CRS and NE. In another embodiment, corticosteroid administration comprises oral or intravenous administration of 10mg dexamethasone, 1 to 4 times per day. Another embodiment (sometimes referred to as a "high dose" corticosteroid) involves the intravenous administration of 1g of methylprednisolone, alone or in combination with dexamethasone, per day. In some embodiments, the one or more corticosteroids are administered at a dose of 1 to 2mg/kg per day.

The corticosteroid can be administered in any amount effective to ameliorate one or more symptoms associated with an adverse event, such as with CRS or neurotoxicity. Corticosteroids (e.g., glucocorticosteroids) may be administered to a 70kg adult subject, for example, in an amount of between or about 0.1 and 100mg, 0.1 to 80mg, 0.1 to 60mg, 0.1 to 40mg, 0.1 to 30mg, 0.1 to 20mg, 0.1 to 15mg, 0.1 to 10mg, 0.1 to 5mg, 0.2 to 40mg, 0.2 to 30mg, 0.2 to 20mg, 0.2 to 15mg, 0.2 to 10mg, 0.2 to 5mg, 0.4 to 40mg, 0.4 to 30mg, 0.4 to 20mg, 0.4 to 15mg, 0.4 to 10mg, 0.4 to 5mg, 0.4 to 4mg, 1 to 20mg, 1 to 15mg, or 1 to 10mg per dose. Typically, a corticosteroid (such as a glucocorticoid) is administered to an ordinary adult subject in an amount of between or about 0.4 and 20mg per dose, for example at or about 0.4mg, 0.5mg, 0.6mg, 0.7mg, 0.75mg, 0.8mg, 0.9mg, 1mg, 2mg, 3mg, 4mg, 5mg, 6mg, 7mg, 8mg, 9mg, 10mg, 11mg, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, or 20 mg.

In some embodiments, the amount of the compound may be, for example, at or about 0.001mg/kg (subject), 0.002mg/kg, 0.003mg/kg, 0.004mg/kg, 0.005mg/kg, 0.006mg/kg, 0.007mg/kg, 0.008mg/kg, 0.009mg/kg, 0.01mg/kg, 0.015mg/kg, 0.02mg/kg, 0.025mg/kg, 0.03mg/kg, 0.035mg/kg, 0.04mg/kg, 0.045mg/kg, 0.05mg/kg, 0.055mg/kg, 0.06mg/kg, 0.065mg/kg, 0.07mg/kg, 0.025mg/kg, 0.08mg/kg, 0.085mg/kg, 0.09mg/kg, 0.095mg/kg, 0.1mg/kg, 0.15mg/kg, 0.25mg/kg, 0.40mg/kg, 0.35mg/kg, 0.25mg/kg, 0.35mg/kg, 0.25mg/kg, A dose of 0.45mg/kg, 0.50mg/kg, 0.55mg/kg, 0.60mg/kg, 0.65mg/kg, 0.70mg/kg, 0.75mg/kg, 0.80mg/kg, 0.85mg/kg, 0.90mg/kg, 0.95mg/kg, 1mg/kg, 1.05mg/kg, 1.1mg/kg, 1.15mg/kg, 1.20mg/kg, 1.25mg/kg, 1.3mg/kg, 1.35mg/kg or 1.4mg/kg, to an ordinary adult subject, typically weighing about 70 to 75 kg.

Generally, the dose of corticosteroid administered depends on the particular corticosteroid because of differences in efficacy between different corticosteroids. It is generally understood that the potency of the drug varies, and thus the dosage may vary to achieve an equivalent effect. The equivalence in terms of potency of various glucocorticoids and routes of administration is well known. Information relating to the administration of equivalent steroids (in a non-chronotherapeutic manner) can be found in British National Formulary (BNF) 37, month 3 1999.

In some embodiments, the adverse event/reaction may be selected from one or more of the following:

other adverse reactions include: gastrointestinal disorders; drying the mouth; infection and infection disorders: fungal infections; metabolic and nutritional disorders: dehydrating; disorders of the nervous system: ataxia, seizures, increased intracranial pressure; respiratory, thoracic and mediastinal disorders: respiratory failure, pulmonary edema; skin and subcutaneous tissue disorders: rash; vascular disorders: bleeding is caused.

In one embodiment, the symptoms of cytokine release syndrome include, but are not limited to, fever, chills, fatigue, anorexia, myalgia, arthala, nausea, vomiting, headache, rash, diarrhea, tachypnea, hypoxemia, tachycardia, hypotension, pulse width, increased early cardiac output, decreased late cardiac output, hallucinations, tremors, altered gait, seizures, and death. In one embodiment, methods for ranking CRS are described in Neelapu et al, Nature review-clinical oncology (Nat Rev Clin Oncol.), 15(1):47-62, 2018 and Lee et al, Blood (Blood), 2014; 124:188-195. In one embodiment, neurotoxicity/neurological events can be detected by Lee et al, Blood (Blood), 2014; 124: 188-.

In some embodiments, adverse events are managed with toslizumab (or another anti-IL 6/IL6R agent/antagonist), corticosteroid therapy, or anti-epileptic drugs for toxicity prevention. In some embodiments, the adverse event is managed by one or more agents selected from the group consisting of an inhibitor of GM-CSF, CSF1, GM-CSFR or CSF1R, anti-thymocyte globulin, lentizilumab, mavrilimumab, a cytokine, and an anti-inflammatory agent.

In some embodiments, the present disclosure provides methods of preventing or reducing the severity of adverse reactions to T cell therapy of the present disclosure. In some embodiments, the cell therapy is administered with one or more agents that prevent, delay the onset of, reduce the symptoms of, treat, or prevent an adverse event, such that the adverse event comprises cytokine release syndrome and neurotoxicity. In one embodiment, the medicament has been described above. In other embodiments, the agent is described below. In some embodiments, the agent is administered prior to, after, or simultaneously with the administration of the cells by one of the methods and dosages described elsewhere in this specification. In one embodiment, the agent is administered to a subject who may be susceptible to the disease but has not yet been diagnosed with the disease.

In this regard, the disclosed methods can include administering a "prophylactically effective amount" of tollizumab, corticosteroid therapy, and/or an anti-epileptic drug for toxicity prevention. In some embodiments, the method comprises administering an inhibitor of GM-CSF, CSF1, GM-CSFR, or CSF1R, lentizilumab, mavrilimumab, a cytokine, and/or an anti-inflammatory agent. The pharmacological and/or physiological effect may be prophylactic, i.e. the effect completely or partially prevents the disease or a symptom thereof. A "prophylactically effective amount" can refer to an amount (both in dosage and for a period of time necessary) effective to achieve a desired prophylactic result (e.g., to prevent the onset of an adverse reaction).

In some embodiments, the method comprises the management of an adverse reaction in any subject. In some embodiments, the adverse reaction is selected from the group consisting of Cytokine Release Syndrome (CRS), neurotoxicity, hypersensitivity, severe infection, cytopenia, and hypogammaglobulinemia. In some embodiments, the signs and symptoms of adverse reactions are selected from the group consisting of fever, hypotension, tachycardia, hypoxia, and chills, including cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal insufficiency, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS), epilepsy, encephalopathy, headache, tremor, vertigo, aphasia, delirium, insomnia anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia. In some embodiments, the patient is identified and selected based on one or more of the biomarkers of the adverse event. In some embodiments, patients have been identified and selected simply by clinical presentation (e.g., presence and level of toxicity symptoms). In some embodiments, the adverse event is managed by any one of the schemes in table 13, table 14, table 16, and table 17.

In some embodiments, the method comprises preventing or reducing the severity of CRS in chimeric receptor therapy. In some embodiments, the engineered CAR T cells are inactivated upon administration to a patient. In some embodiments, the method comprises identifying the CRS based on clinical presentation. In some embodiments, the method comprises assessing and treating other causes of fever, hypoxia, and hypotension. Patients who develop CRS levels greater than or equal to 2 (e.g., hypotension, hypoxia without response to fluid replacement or need for supplemental oxygen) should be monitored using continuous cardiac telemetry and pulse oximetry. In some embodiments, for patients with severe CRS, performing echocardiography to assess cardiac function is considered. For severe or life-threatening CRS, intensive care support therapy may be considered. In some embodiments, the method comprises monitoring the patient for CRS signs and symptoms at least daily for 7 days at a certified medical facility following the infusion. In some embodiments, the method comprises monitoring the patient for signs or symptoms of CRS for 4 weeks after infusion. In some embodiments, the method comprises advising the patient to go to medical attention immediately if signs or symptoms of CRS are present at any time. In some embodiments, treatment with supportive care, tollizumab, or tollizumab and a corticosteroid occurs when the first signs of CRS are exhibited.

In some embodiments, the method comprises monitoring the patient for signs and symptoms of neurotoxicity. In some embodiments, the method comprises excluding other causes of the neurological condition. Patients with grade 2 or greater neurotoxicity should be monitored using continuous cardiac telemetry and pulse oximetry. Intensive care support therapy is provided for severe or life-threatening neurotoxicity. In some embodiments, the symptoms of neurotoxicity are selected from encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, and anxiety.

In some embodiments, the cell therapy is administered before, during/simultaneously with, and/or after administration of one or more agents (e.g., steroids) or treatments (e.g., atherectomy) that treat and/or prevent (prophylactic) one or more symptoms of an adverse event. A prophylactically effective amount is an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. In one embodiment, the prophylactically effective amount is administered to the subject prior to or at an earlier stage of the disease. In one embodiment, the prophylactically effective amount will be less than the therapeutically effective amount. In one embodiment, adverse event treatment or prevention is administered to any patient who will receive, is symptomatic of, or has received cell therapy. In some embodiments, the method of managing adverse events comprises monitoring the patient for neurotoxic signs and symptoms at least daily for 7 days at a certified medical facility following infusion. In some embodiments, the method comprises monitoring the patient for signs or symptoms of neurotoxicity and/or CRS for 4 weeks after infusion.

In some embodiments, the disclosure provides two methods of managing adverse events in a subject receiving CAR T cell therapy with a steroid and an anti-IL 6/anti-IL-6R antibody. In one embodiment, the present disclosure provides a method of managing adverse events, wherein if there is no improvement after 3 days and for all grade 1 neurological events, corticosteroid therapy is initiated for managing all cases of grade 1 CRS. In one embodiment, if there is no improvement after 3 days and for all grade 2 neurological events, Tolizumab is initiated for all cases of grade 1 CRS. In one embodiment, the disclosure provides a method of reducing overall steroid exposure in a patient receiving adverse event management after CAR T cell administration, the method comprising initiating corticosteroid therapy for managing all cases of grade 1 CRS if there is no improvement after 3 days and for all grade ≧ 1 neurological events, and/or initiating tollizumab if there is no improvement after 3 days and for all grade ≧ 2 neurological events. In one embodiment, the corticosteroid and toslizumab are administered in a regimen selected from those exemplified in the examples section. In one embodiment, the disclosure demonstrates that earlier steroid use is not associated with increased risk of severe infection, reduced CAR T cell expansion, or reduced tumor response.

In one embodiment, the present disclosure supports the safety of levetiracetam prevention in the treatment of CAR T cell cancer. In one embodiment, the cancer is NHL. In one embodiment, the cancer is R/R LBCL and the patient receives KTE-X19. Accordingly, in one embodiment, the present disclosure provides a method of managing an adverse event in a patient treated with CAR T cells, the method comprising administering to the patient a prophylactic dose of an anti-epileptic drug. In some embodiments, if a neurological event occurs after discontinuation of prophylactic levetiracetam, the patient receives levetiracetam starting on day 0 of CAR T cell therapy (post conditioning) and also at > 2 grade neurotoxic episodes (e.g., 750mg given orally or intravenously twice daily). In one embodiment, if the patient does not experience any grade 2 neurotoxicity or greater, levetiracetam is gradually reduced and discontinued as clinically indicated. In one embodiment, levetiracetam prevention is combined with any other adverse event management regimen.

In one embodiment, the patient may receive levetiracetam starting on day 0 (750 mg given orally or intravenously twice daily). At the onset of grade 2 or more, the levetiracetam dose was increased to 1000mg twice daily. In one embodiment, if the patient does not experience any grade 2 or greater neurological events, the levetiracetam is gradually reduced and discontinued as clinically indicated. Patients also received tollizumab on day 2 (8 mg/kg [ no more than 800mg ] IV over 1 hour). Further toslizumab (± corticosteroid) may be recommended at the onset of CRS class 2 or in the case of CRS class ≧ 3 in patients with comorbidity or age. Tolizumab is initiated for patients experiencing grade > 2 neurological events, and corticosteroids are added for patients with comorbidity or older age, or if there is any occurrence of grade > 3 neurological events, when symptoms worsen despite Tolizumab use.

In one embodiment, the present disclosure demonstrates that prophylactic steroid use appears to reduce the rate of severe CRS and NE to a similar extent as the early steroid use administration. Accordingly, the present disclosure provides methods for managing adverse events in CAR T cell therapy, wherein patients receive 10mg of dexamethasone administered PO on days 0 (prior to infusion), 1, and 2. Steroids may also be administered starting from grade 1 NE and grade 1 CRS when no improvement is observed after 3 days of supportive care. Tolizumab may also be administered for management of grade 1 CRS if no improvement is observed after 24 hours of supportive care. In one embodiment, the disclosure demonstrates that adverse event management of CAR T cell therapy with antibodies that neutralize and/or deplete GM-CSF prevents or reduces treatment-related CRS and/or NE in treated patients. In one embodiment, the antibody is lentillumab.

In some embodiments, adverse events are managed by administering an agent/agents that are antagonists or inhibitors of IL-6 or the IL-6 receptor (IL-6R). In some embodiments, the agent is an antibody that neutralizes IL-6 activity, such as an antibody or antigen-binding fragment that binds IL-6 or IL-6R. For example, in some embodiments, the agent is or includes toslizumab (atalizumab) or sarlizumab, an anti-IL-6R antibody. In some embodiments, the agent is an anti-IL-6R antibody described in U.S. Pat. No. 8,562,991. In some cases, the agent targeting IL-6 is an anti-TL-6 antibody, such as cetuximab, aximumab, ALD518/BMS-945429, sirucimumab (CNTO 136), CPSI-2634, ARGX 109, FE301, FM101, or ololizumab (CDP6038), and combinations thereof. In some embodiments, the agent can neutralize IL-6 activity by inhibiting ligand-receptor interactions. In some embodiments, the IL-6/IL-6R antagonist or inhibitor is an IL-6 mutein, such as the IL-6 mutein described in U.S. Pat. No. 5591827. In some embodiments, the agent that is an antagonist or inhibitor of IL-6/IL-6R is a small molecule, protein or peptide, or nucleic acid.

In some embodiments, other agents useful for managing adverse reactions and their symptoms include antagonists or inhibitors of cytokine receptors or cytokines. In some embodiments, the cytokine or receptor is IL-10, TL-6 receptor, IFNy, IFNGR, IL-2R/CD25, MCP-1, CCR2, CCR4, MIP13, CCR5, TNF α, TNFR1 such as TL-6 receptor (IL-6R), IL-2 receptor (IL-2R/CD25), MCP-1(CCL2) receptor (CCR2 or CCR4), TGF- β receptor (TGF- β I, II or III), IFN- γ receptor (IFNGR), MIP1P receptor (e.g., CCR5), TNF α receptor (e.g., TNFR1), IL-1 receptor (IL1-Ra/IL-1RP), or IL-10 receptor (IL-10R), IL-1 and IL-1 Ra/IL-1 β. In some embodiments, the agent comprises cetuximab, sarlizumab, ocluzumab (CDP6038), aximumab, ALD518/BMS-945429, sirupumab (CNTO 136), CPSI-2634, ARGX 109, FE301, or FM 101. In some embodiments, the agent is an antagonist or inhibitor of a cytokine, such as transforming growth factor beta (TGF-beta), interleukin 6(TL-6), interleukin 10(IL-10), IL-2, MIP13(CCL4), TNF α, IL-1, interferon γ (IFN- γ), or monocyte chemoattractant protein-I (MCP-1). In some embodiments, the agent is an agent that targets (e.g., inhibits or is an antagonist of) a cytokine receptor, such as the TL-6 receptor (IL-6R), the IL-2 receptor (IL-2R/CD25), the MCP-1(CCL2) receptor (CCR2 or CCR4), the TGF- β receptor (TGF- β I, II or III), the IFN- γ receptor (IFNGR), the MIP1P receptor (e.g., CCR5), the TNF α receptor (e.g., TNFR1), the IL-1 receptor (IL1-Ra/IL-1RP), or the IL-10 receptor (IL-10R), and combinations thereof. In some embodiments, the agent is administered prior to, after, or simultaneously with the administration of the cells by one of the methods and dosages described elsewhere in this specification.

In some embodiments, the agent is administered at a dose of about 1mg/kg to 10mg/kg, 2mg/kg to 8mg/kg, 2mg/kg to 6mg/kg, 2mg/kg to 4mg/kg, or 6mg/kg to 8mg/kg, inclusive, or at least about 2mg/kg, 4mg/kg, 6mg/kg, or 8 mg/kg. In some embodiments, administration is at a dose of about 1mg/kg to 12mg/kg (such as at or about 10 mg/kg). In some embodiments, the agent is administered by intravenous infusion. In one embodiment, the agent is tositumumab. In some embodiments, the agent (e.g., specific toslizumab) is administered prior to, after, or simultaneously with the administration of the cells by one of the methods and dosages described elsewhere in the specification.

In some embodiments, the method comprises identifying the CRS based on clinical presentation. In some embodiments, the method comprises assessing and treating other causes of fever, hypoxia, and hypotension. If CRS is observed or suspected, it may be managed according to the recommendations in protocol a, which may also be used in combination with other treatments of the present disclosure, including neutralization or reduction of the CSF/CSFR1 axis. Patients who develop CRS levels greater than or equal to 2 (e.g., hypotension, hypoxia without response to fluid replacement or need for supplemental oxygen) should be monitored using continuous cardiac telemetry and pulse oximetry. In some embodiments, for patients with severe CRS, performing echocardiography to assess cardiac function is considered. For severe or life-threatening CRS, intensive care support therapy may be considered. In some embodiments, a biomimetic or equivalent of tositumumab may be used in place of tositumumab in the methods disclosed herein. In other embodiments, another anti-IL 6R may be used in place of truzumab.

In some embodiments, adverse events are managed according to the following scheme (scheme a):

(a) lee DW et al, 2014, the current concept of cytokine release syndrome diagnosis and management (Current concentrations in the diagnosis and management of cytokine release syndrome), "Blood (Blood), 2014, 7, 10 days; vol 124, 2 nd, p 188-195 nd.

(b) For the management of neurotoxicity, see table 2.

(c) For more detailed information, please refer to(Tozumab) prescription information, https:// www.gene.com/download/pdf/actionra _ describing.pdf (last visit time: 2017, 10/18/month). The time of initial approval in the united states is indicated to be 2010.

Neurotoxicity

In some embodiments, the method comprises monitoring the patient for signs and symptoms of neurotoxicity. In some embodiments, the method comprises excluding other causes of the neurological condition. Patients with grade 2 or greater neurotoxicity should be monitored using continuous cardiac telemetry and pulse oximetry. Intensive care support therapy is provided for severe or life-threatening neurotoxicity. For any grade 2 neurotoxicity, non-sedating antiepileptic drugs (e.g., levetiracetam) are contemplated for seizure prevention. The following treatments may be used in combination with other treatments of the present disclosure, including neutralization or reduction of the CSF/CSFR1 axis.

In some embodiments, the adverse events are managed according to the following scheme (scheme B):

additional security management strategies with corticosteroids

Administration of corticosteroids and/or toslizumab at grade 1 may be considered prophylactic. Supportive care may be provided in all scenarios under all CRS and NE severity levels. In one embodiment of the regimen for managing adverse events associated with CRS, tollizumab and/or corticosteroid are administered as follows: level 1 CRS: toxolizumab-free; corticosteroid-free; level 2 CRS: toslizumab (only in cases of co-morbidities or older age); and/or corticosteroids (only in cases of co-morbidities or older age); 3-level CRS: (ii) toclizumab; and/or a corticosteroid; 4-level CRS: (ii) toclizumab; and/or a corticosteroid. In another embodiment of the regimen for managing adverse events associated with CRS, tollizumab and/or corticosteroid are administered as follows: level 1 CRS: toslizumab (if no improvement after 3 days); and/or corticosteroids (if there is no improvement after 3 days); level 2 CRS: (ii) toclizumab; and/or a corticosteroid; 3-level CRS: (ii) toclizumab; and/or a corticosteroid; 4-level CRS: (ii) toclizumab; and/or corticosteroids, high doses.

In one embodiment of the regimen for managing adverse events associated with NE, tollizumab and/or corticosteroid are administered as follows: grade 1 NE: toxolizumab-free; corticosteroid-free; grade 2 NE: toxolizumab-free; corticosteroid-free; grade 3 NE: (ii) toclizumab; and/or corticosteroids (standard doses are only used if tollizumab has not improved); grade 4 NE: (ii) toclizumab; and/or a corticosteroid. In another embodiment of the regimen for managing adverse events associated with NE, tollizumab and/or corticosteroid are administered as follows: grade 1 NE: toxolizumab-free; and/or a corticosteroid; grade 2 NE: (ii) toclizumab; and/or a corticosteroid; grade 3 NE: (ii) toclizumab; and/or corticosteroids, high doses; grade 4 NE: (ii) toclizumab; and/or corticosteroids, high doses. In one embodiment, corticosteroid treatment is initiated at CRS grade ≧ 2 and toslizumab treatment is initiated at CRS grade ≧ 2. In one embodiment, corticosteroid treatment is initiated at CRS grade ≧ 1 and toslizumab treatment is initiated at CRS grade ≧ 1. In one embodiment, corticosteroid treatment is initiated at NE rating ≧ 3 and Tolizumab treatment is initiated at CRS rating ≧ 3. In one embodiment, corticosteroid treatment is initiated at CRS grade ≧ 1 and Tolizumab treatment is initiated at CRS grade ≧ 2. In some embodiments, prophylactic use of tocilizumab administered on day 2 can reduce the rate of CRS grade 3 or more. The one or more corticosteroids may be administered at any administration dose and frequency that may be appropriate for the severity/grade of the adverse event (e.g., CRS and NE). Table 1 and table 2 provide examples of dosing regimens for managing CRS and NE, respectively. In another embodiment Corticosteroid administration includes oral or intravenous administration of 10mg dexamethasone, 1 to 4 times per day. Another embodiment (sometimes referred to as a "high dose" corticosteroid) involves the intravenous administration of 1g of methylprednisolone, alone or in combination with dexamethasone, per day. In some embodiments, the one or more corticosteroids are administered at a dose of 1mg/kg to 2mg/kg per day. Generally, the dose of corticosteroid administered depends on the particular corticosteroid, as there are differences in efficacy between different corticosteroids. It is generally understood that the potency of the drug varies and therefore the dosage may vary to achieve an equivalent effect. The equivalence in terms of potency of various glucocorticoids and routes of administration is well known. Information relating to the administration of equivalent steroids (in a non-chronic therapeutic manner) can be found in the British National Formulary (BNF) 37 (month 3 1999). The present application also provides for dosing and administration of cells prepared by the methods of the present application, e.g., an infusion bag for CD 19-directed genetically modified autologous T cell immunotherapy comprising a Chimeric Antigen Receptor (CAR) positive T cell suspension for infusion in about 68 mL. In some embodiments, the CAR T cells are formulated for infusion in about 40 mL. In some embodiments, the CAR T cell product is formulated in a total volume of 35mL, 40mL, 45mL, 50mL, 55mL, 60mL, 65mL, 70mL, 75mL, 80mL, 85mL, 90mL, 95mL, 100mL, 200mL, 300mL, 400mL, 500mL, 700mL, 800mL, 900mL, 1000 mL. In one aspect, the dosage and administration of cells prepared by the methods of the present application (e.g., infusion bag for CD 19-directed genetically modified autologous T cell immunotherapy) comprises 1 x 10 in about 40mL ⁶ Individual CAR-T positive cell suspensions. The target dose may be between about 1X 10 per kg body weight ⁶ And about 2X 10 ⁶ Maximum of 2X 10 between live CAR-positive T cells ⁸ Individual CAR-positive live T cells.

In some embodiments, the dosage form comprises a cell suspension for infusion in a single-use patient-specific infusion bag; the route of administration is intravenous; the entire contents of each single-use patient-specific bag were infused by gravity or peristaltic pump over 30 minutes. In one embodimentThe dosage schedule is 2.0 × 10 ⁶ Single infusion consisting of anti-CD 19 CAR T cells/kg body weight (+ -20%) with maximum dose of 2 x 10 ⁸ anti-CD 19 CAR T cells (for subjects ≧ 100 kg). In some embodiments, the T cells comprising the dose are CD19 CAR-T cells.

In some embodiments, the CD 19-directed T cell immunotherapy is KTE-X19, which is prepared as described elsewhere in this application. In one embodiment, KTE-X19 can be used to treat MCL, ALL, CLL, SLL, and any other B cell malignancy. In some embodiments, the CD 19-directed genetically modified autologous T cell immunotherapy is Axi-cel prepared by one of the methods of the present application ^TM (Aliskiren). CAR T cell amounts, dosing regimens, methods of administration, subjects, cancers falling within the scope of these methods are described elsewhere in the application, and these methods are administered alone or in combination with another chemotherapeutic agent, with or without preconditioning, to any patient described elsewhere in the application.

The following examples are intended to illustrate various aspects of the present application. Therefore, the specific aspects discussed should not be construed as limiting the scope of the application. For example, although the following examples are directed to T cells transduced with an anti-CD 19 Chimeric Antigen Receptor (CAR), one skilled in the art will appreciate that the methods described herein may be applied to immune cells transduced with any CAR. It will be apparent to those skilled in the art that various equivalent changes and modifications may be made without departing from the scope of the application, and it is to be understood that such equivalent aspects are to be included herein. In addition, all references cited in this application are hereby incorporated by reference in their entirety as if fully set forth herein.

The patent and scientific literature referred to herein establishes knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated herein by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, dictionaries, documents, manuscripts, genomic database sequences, and scientific literature cited herein are hereby incorporated by reference.

Other features and advantages of the present disclosure will be apparent from the accompanying drawings and from the following detailed description, including examples.

Examples

Example 1

In this study, patients with R/R MCL who received 1 to 5 previous therapies, including Bruton's Tyrosine Kinase Inhibitor (BTKi), were treated with autologous anti-CD 19 CAR-T cells.

Eligible patients with R/R MCL (age ≧ 18 years) had an ECOG score of 0-1 and ≦ 5 previous therapies including chemotherapy, anti-CD 20 antibodies, and BTK inhibitors (BTKi). The patient underwent leukophoresis and chemotherapy (300 mg/m per day) ² Cyclophosphamide and 30mg/m daily ² Fludarabine, for 3 days) and then administered at 2 × 10 ⁶ Target doses of individual CAR T cells/kg CD19 CAR-T was infused. Patients may receive a bridging therapy of dexamethasone, ibrutinib or acatinib after leukapheresis and prior to chemotherapy. The primary endpoint was the objective response rate (ORR [ Complete Response (CR) + Partial Response (PR) according to the Lugano classification]). Researchers used revised IWG malignant lymphoma response criteria to assess intermediate efficacy endpoints. The key secondary endpoints are duration of response (DOR), Progression Free Survival (PFS), OS, frequency of Adverse Events (AE), CAR T cell levels in blood and cytokine levels in serum.

28 patients received CD19 CAR-T cells and a follow-up of 1 year or longer (median 13.2 months [ Range, 11.5-18.5 ]). Forty-three percent of patients have an ECOG score of 1, 21% have blast-like morphology, 82% have stage IV disease, 50% have intermediate/high risk MIPI, 86% receive a median of 4 (range, 1 to 5) previous therapies, and 57% are refractory to the last previous therapy. The median Ki-67 index was 38% (range, 5% -80%) in 20 of 28 patients. Eight patients received bridging therapy; all patients had disease after bridge therapy. ORR was 86% (95% CI, 67% -96%), CR was 57% (95% CI, 37% -76%). 75% of responders remain responsive and 64% of treated patients have a progressive response. The 12-month estimates of DOR, PFS, and OS were 83% (95% CI, 60% -93%), 71% (95% CI, 50% -84%), 86% (95% CI, 66% -94%), respectively, and no median was obtained. Grade > 3 AE (> 20% of patients) were anemia (54%), decreased platelet count (39%), decreased neutrophils (36%), decreased neutrophil count (32%), decreased leukocyte count (29%), encephalopathy (25%) and hypertension (21%). Reported in 18% of patients as a result of Lee DW et al, Blood (Blood) 2014; grade 3/4 Cytokine Release Syndrome (CRS) assessed at 124:188, manifested by hypotension (14%), hypoxia (14%) and fever (11%). Grade 3/4 Neurological Events (NEs) were reported in 46% of patients and included encephalopathy (25%), confusion status (14%) and aphasia (11%). No grade 5 CRS or NE occurred. All CRS events and most NEs (15 of 17 patients) were reversible. Median time to onset and CRS regression were 2 days (range, 1-7 days) and 13 days (range, 4-60 days), respectively. Median time to onset of NE was 6 days (range, 1-15 days) and median time to regression was 20 days (range, 9-99 days). Median CAR T cell levels as measured by peak and area under the curve were 99 cells/μ L (range, 0.4-2589) and 1542 cells/μ L (range, 5.5-27239), respectively. Peak CAR T cell expansion was observed between day 8 and day 15 and declined over time.

Example 2

This example provides an additional analysis of the above study. Eligible patients are aged > 18 years, have pathologically confirmed MCL with documented overexpression or presence of cyclin D1 (11; 14), and are relapsed/refractory to 1-5 previous regimens of MCL. Previous therapies must include chemotherapy with anthracyclines or bendamustine, anti-CD 20 monoclonal antibodies, and ibrutinib or acatinib. All patients received past BTKi. Although patients must have past BTKi therapy, they need not be the last line therapy before study entry, and patients need not be refractory to BTKi therapy. Eligible patients had an absolute lymphocyte count of > 100/. mu.L. Patients who underwent autologous SCT or had prior CD19 targeted therapy or allogeneic SCT within 6 weeks of CD19CAR-T cell infusion were excluded.

Additional inclusion criteria include: at least 1 measurable lesion. Lesions that have been previously irradiated are considered measurable only when progress is recorded after completion of radiotherapy; if the only measurable disease is lymph node disease, then at least 1 lymph node should be ≧ 2 cm; magnetic Resonance Imaging (MRI) of the brain showed no evidence of Central Nervous System (CNS) lymphoma; in addition to systemic suppressive/stimulatory immune checkpoint therapy, at the time the patient is scheduled for leukapheresis, at least 2 weeks or 5 half-lives (whichever is shorter) must elapse starting from any previous systemic therapy or BTKi (ibrutinib or akatinib); when a patient is scheduled for leukapheresis (e.g., ipilimumab, nivolumab, pembrolizumab, atlizumab, OX40 agonist, 4-1BB agonist), at least 3 half-lives must elapse starting with any prior systemic inhibitory/stimulatory immune checkpoint molecular therapy; the toxicity resulting from previous therapies must have stabilized and returned to grade 1 or less (except for no clinically relevant toxicities, such as alopecia); 0 or 1, eastern american tumor cooperative group (ECOG) physical performance status; absolute Neutrophil Count (ANC) greater than or equal to 1000/μ L; the platelet count is more than or equal to 75000/mu L; the absolute lymphocyte count is more than or equal to 100/mu L; adequate kidney, liver, lung and heart function was defined as: creatinine clearance (as estimated by Cockcroft Gault) ≧ 60 cc/min; serum alanine aminotransferase/aspartate aminotransferase < 2.5 normal Upper Limit (ULN); total bilirubin is less than or equal to 1.5mg/dL, patients with gilbert syndrome have no evidence of pericardial effusion, as determined by Echocardiography (ECHO), and no clinically relevant Electrocardiogram (ECG) results, except that the epicardial ejection fraction is greater than or equal to 50%; no clinically relevant pleural effusion; baseline oxygen saturation in room air > 92%; and women with fertility potential must have a negative serum or urine pregnancy test. Women who have undergone surgical sterilization or have been menopausal for at least 2 years are not considered to have fertility potential.

Additional exclusion criteria included: history of malignancy except for non-melanoma skin cancer or carcinoma in situ (e.g., cervix, bladder, breast) unless disease-free for at least 3 years; history of allogeneic stem cell transplantation; previous CAR therapy or other genetically modified T-cell therapy; history of severe immediate hypersensitivity due to aminoglycosides; the presence of fungal, bacterial, viral or other infections is uncontrolled or requires Intravenous (IV) antimicrobials for management. If responsive to active treatment and after consultation with a medical monitor, allows for simple Urinary Tract Infection (UTI) and uncomplicated bacterial pharyngitis; a history of Human Immunodeficiency Virus (HIV) infection or acute or chronic active hepatitis B or C infection. Patients with a history of hepatitis infection must clear their infection as determined by standard serological and genetic tests; there is any indwelling wire or tube (e.g., a percutaneous nephrostomy tube, an indwelling Foley catheter, a bile duct, or a spleen/peritoneum/pericardium catheter). Ommaya reservoirs and dedicated central venous access catheters, such as Port-a-Cath or Hickman catheters, are permissible; patients with detectable cerebrospinal fluid malignant cell or brain metastasis or with a history of CNS lymphoma, cerebrospinal malignant cell or brain metastasis; history or presence of CNS disorders (such as seizure disorders, cerebrovascular ischemia/hemorrhage, dementia, cerebellar disease, cerebral edema, reversible posterior encephalopathy syndromes, or any autoimmune disease with CNS involvement); a history of myocardial infarction, angioplasty or stenting, unstable angina, active arrhythmia or other clinically relevant cardiac disease within 12 months of enrollment; patients with atrial or ventricular lymphoma involvement; a history of symptomatic deep vein thrombosis or pulmonary embolism over the last 6 months of enrollment; emergency treatment may be required due to a progressive or imminent oncology emergency (e.g., tumor mass effect, tumor lysis syndrome); primary immunodeficiency; any medical condition that may interfere with the assessment of safety or efficacy of a study treatment; history of severe immediate hypersensitivity to any of the agents used in this study; live vaccine ≤ 6 weeks before scheduling for the regulatory regimen; pregnant or lactating women with fertility potential due to the potentially dangerous effects of preparative chemotherapy on the fetus or infant; patients of both sexes who are unwilling to practice fertility control from the time of consent to 6 months after completion of CD19 CAR-T cell therapy; at the discretion of the investigator, the patient is unlikely to complete the study visits or procedures (including follow-up visits) required for all protocols or to comply with the participation requirements of the study; and a history of autoimmune diseases (e.g., crohn's disease, rheumatoid arthritis, systemic lupus), resulting in end organ damage or a need for systemic immunosuppression/systemic disease modulators over the past 2 years.

All patients underwent leukapheresis to obtain cells for CD19 CAR-T cell therapy manufacturing. The manufacturing method was modified with respect to that of aliskiren (axicabtagene ciloleucel) to enrich CD4 by positive ⁺ /CD8 ⁺ The cells eliminate circulating lymphoma cells. Single intravenous infusion of 2X 10 on day 0 ⁶ Fludarabine (30 mg/m/day) was administered on days-5, 4 and 3 before individual CAR T cells/kg of CD19 CAR-T cells ² ) And cyclophosphamide (500 mg/m per day) ² ) The method of (1). Doses were announced from studies of aliskiren in large B-cell lymphomas and CD19 CAR-T cells in acute lymphoblastic leukemia. Neelapu SS et al, New England journal of medicine (The New England journal of medicine) 2017; 377: 2531; locke FL et al, molecular therapy (Mol Ther.) 2017; 25: 285; shah BD et al, Journal of Clinical Oncology (Journal of Clinical Oncology) 2019; 37 (supplement; abstract 7006); and Lee DW et al, annual report in oncology: european Medical Oncology Association/official journal of ESMO (Annals of Oncology: the European Society for Medical Oncology/ESMO) 2017; 28:1008PD, all of which are incorporated herein by reference in their entirety. Following leukapheresis and prior to opsonic therapy, patients with high disease burden are allowed to receive dexamethasone or an equivalent corticosteroid, ibrutinib or acartib at the discretion of the investigator Niemann's bridging therapy followed by repeated baseline positron emission tomography-computed tomography (PET-CT) scans. The goal of bridging therapy is not curative, but to keep the patient stable throughout the manufacturing period. Hospitalization after CD19 CAR-T cell infusion was required until day 7.

The primary endpoint was the objective response rate (ORR [ Complete Response (CR) + Partial Response (PR) ] as assessed by the independent radiology review board (IRRC) using the Lugano classification. Cheson et al, J.Clin Oncol (J Clin Oncol), 2014; 32:3059-68. In order to confirm CR, bone marrow evaluation was required in addition to PET-CT. Secondary endpoints include duration of response (DOR), Progression Free Survival (PFS), OS, investigator-assessed ORR (according to Cheson et al, J Clin Oncol, 2007; 25:579-86), incidence of Adverse Events (AE), CAR T cell levels in blood and cytokines in serum, and fractional changes in European Quality of life-5 Dimensions-5 levels per dimension with 5levels per dimension (EQ-5D-5L)). CAR T cell presence, expansion and persistence and serum cytokines and their association with clinical outcome were assessed as previously reported. Kochenderfer JN et al, J.Clin Oncol. (JClin Oncol.) 2017; 35: 1803-13; locke FL et al, molecular therapy (Mol Ther.) 2017; 25:285-95, both of which are incorporated herein by reference in their entirety.

Changes in EQ-5D-5L score from baseline to month 6 were assessed. Cytokine Release Syndrome (CRS) was graded according to: lee et al, Blood (Blood) 2014; 124:188, which is incorporated herein by reference in its entirety. The severity of AE (including neurological Events) and CRS symptoms was graded using the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.03. Minimal residual disease (MRD; 10) ^-5 Sensitivity) was an exploratory assay evaluated at baseline and at months 1, 3 and 6 in cryopreserved peripheral blood mononuclear cells and by next generation sequencing (adapted biotechnology) using the clonoSeq assayAnalysis WAs performed by companies (Adaptive Biotechnologies, Seattle, WA), Wash.

For all patients, positron emission tomography-computed tomography (PET-CT) scans of disease-specific sites were required at baseline, 4 weeks post-infusion, and at regular intervals during post-treatment. Bone marrow aspiration/biopsy is required to confirm complete response in patients with bone marrow disease involvement at baseline and patients with uncertain bone marrow involvement at baseline, or if a baseline bone marrow biopsy is not completed or results are not available. Patients with symptoms of CNS malignancies undergo lumbar puncture at screening to examine cerebrospinal fluid (CSF). Lumbar puncture was also performed if applicable for patients with new grade 2 neurotoxic episodes following infusion of anti-CD 19 CAR T cells. In addition, for patients who signed an optional portion of the consent, lumbar punctures were performed before infusion of anti-CD 19 CAR T cells at baseline and after infusion of anti-CD 19 CAR T cells (day 5 ± 3 days) to collect CSF; samples were submitted to a central laboratory and analyzed for changes in cytokine levels.

The primary efficacy analysis was performed after enrollment, treatment and assessment of response for 60 patients 6 months after disease assessment at week 4, as required by the protocol. The assay has a potency of > 96% to distinguish between active therapies with a true response rate of 50% and therapies with a response rate of < 25%, with a unilateral alpha level of 0.025. The ORR was analyzed using an exact binomial test. All efficacy endpoints were analyzed in the above 60 efficacy evaluable patients, including time to event endpoint as assessed using Kaplan-Meier estimates. Safety analysis was performed in all treated patients (n-68). Correlation between measurements and CAR T cell and cytokine levels using Wilcoxon rank sum test; the P value was adjusted using the Holm program. Complete analysis set (N ═ 74): consists of all enrolled/leukapheresis patients and is used for a summary of patient treatment. Safety analysis set (n ═ 68): defined as all patients treated with any dose of anti-CD 19 CAR T cells. This analysis set was used for a summary of demographic and baseline characteristics as well as for overall security analysis. Set of inferential analyses (efficacy evaluable) (n 60): consisted of the first 60 treated CD19 CAR-T cell patients. This analysis set was used for hypothesis testing of the primary endpoint of objective response rate at the time of primary analysis, as well as all other efficacy analyses. The primary endpoint hypothesis was that ORR on CD19 CAR-T cells using the central assessment would be greater than the pre-specified 25% historical control rate at a unilateral significance level of 0.025 using the exact binomial test. The hypothesis is tested in an inferential analysis set. The historical control rate of ORR was determined a priori based on 2 retrospective studies published at the time of study protocol development. In these 2 studies, the outcome after rescue therapy was evaluated in patients with relapsed/refractory MCL who progressed on BTKi treatment (previous therapy as required for compliance with study eligibility). These studies indicate that patients with relapsed/refractory MCL with > 3 previous lines before receiving BTKi have an ORR of approximately 25% for rescue therapy. Wang M et al, "lancets" (Lancet) 2018; 391: 659; martin P et al, Blood (Blood) 2016; 127:1559, both of which are incorporated by reference herein in their entirety.

74 patients were registered; CD19 CAR-T cells were prepared for 71 patients and had been administered to 68 patients. A major efficacy analysis performed after treatment of 60 patients showed 93% ORR (67% complete response). At the median follow-up (range, 7.0 months-32.3 months) of 12.3 months, 57% of patients remained in remission and did not reach median duration of response. The estimated 12-month progression-free survival and overall survival were 61% and 83%, respectively. Common grade 3 or more adverse events were cytopenia (94%) and infection (32%). Cytokine release syndrome and neural events of grade 3 or greater occurred in 15% and 31% of patients, respectively; it is not fatal. Two grade 5 infectious adverse events occurred.

CD19 CAR-T cells were prepared for 71 patients (96%) and had been administered to 68 patients (92%). The median time from leukapheresis to CD19 CAR-T cell delivery to the study site was 16 days (range, 11-128 days). One patient with the prepared CD19 CAR-T cells was ineligible for patient entry into the study due to treatment with bendamustine-rituximab for rapid PD following leukapheresis. After later development of PD, the patient's raw product was shipped from the manufacturing facility 127 days after the initial leukapheresis date and 1 day later to the treatment site. Three patients with manufacturing problems did not continue with additional apheresis due to AE (n-1; deep vein thrombosis), death due to progressive disease (PD; n-1), or consent to withdrawal (n-1). Two additional patients discontinued before opsonic chemotherapy due to death from PD. After receiving opsonic chemotherapy, 1 patient with progressive atrial fibrillation (exclusion criteria) was considered ineligible for CD19 CAR-T cell infusion. The median follow-up for patients with evaluable efficacy was 12.3 months (range, 7.0 months-32.3 months); the follow-up visit of 28 patients is more than or equal to 24 months.

The median age was 65 years (range, 38-79 years) and 57 (84%) patients were males. (table 1) 65% of patients had an ECOG fitness status score of 0 and 35% of patients had an ECOG fitness status score of 1. Patients had high risk characteristics at baseline including stage IV disease (85%), blast-like or polymorphic morphology (31%), a Ki-67 proliferation index of > 30% (40/49[ 82% ]) (Wang ML et al, Lancet Oncology 2016; 17:48), and The TP53 mutation (6/36[ 17% ]). Eighty-one percent of patients have received > 3 previous line therapies (median, 3[ range 1-5 ]).

TABLE 1 Baseline patient characteristics

^* Bone marrow and spleen involvement were excluded.At the time of diagnosis.Researchers reported that one patient had kappa light chain restricted MCL at the time of diagnosis. There were 10 patients with unknown morphologic reports. ^§ Ki-67 data was available for 49 patients at diagnosis.Induction and recruitment/maintenance that occurred between sequential complete responses and/or all treatments were counted as 1 regimen. BTKi, Bruton tyrosine kinase inhibitor; ECOG, eastern american tumor cooperative group; MCL, mantle cell lymphoma; MIPI, international prognostic index for mantle cell lymphoma; SCT, stem cell transplantation.

All patients progressed to BTKi (ibrutinib n ═ 58; akatinib n ═ 16; both n ═ 6), and 43% of patients had prior autologous SCT (table 2). The median time from the end of the last BTKi therapy to the exclusion of bridging to CD19 CAR-T cell infusion was 88 days (range, 25 days-1047 days). Forty percent of patients were refractory to the last therapy, including 3 ibrutinib-intolerant patients with confirmed progression after the last therapy. Twenty-five patients (37%) received bridge therapy with ibrutinib (n ═ 14), acatinib (n ═ 5), dexamethasone (n ═ 12), and/or methylprednisolone (n ═ 2). Post-bridging scans showed that the tumor burden was higher than the median at screening in most patients.

Table 2: bridging therapy

Feature(s)	N＝68
		Any bridging therapy, n (%)	25(37)
Ibrutinib	14(21)
		Acatinib	5(7)
Dexamethasone	12(18)
		Methylprednisolone	2(3)
Both BTKi and steroid, n (%)	6(9)
		Ibrutinib + steroid	4(6)
Acatinib + steroid	2(3)

BTKi, Bruton tyrosine kinase inhibitor.

IRRC assessment in 60 patients prescribed with CD19 CAR-T treatment regimen with minimal 7-month follow-up was 93% (95% CI, 84-98), with 67% CR rate and 27% PR rate. A high degree of agreement (95%) was observed between IRRC assessment and investigator assessed ORR (table 3).

Table 3: responses in patients can be assessed based on efficacy assessed by the investigator (according to Cheson BD et al, J Clin Oncol 2007; 25:57) and by IRRC review by Lugano classification (2014) in Intent-to-Treat (Intent-to-Treat) patients.

^* No evaluation was made at the time of analysis.Identity is the percentage of subjects whose IRRC-evaluated reads matched the investigator-evaluated reads. CR, complete response; IRRC, independent radiology review board; N/A, not applicable; ORR, objective response rate.

All enrolled patients (n-74) had an IRRC assessment with an ORR of 85% (95% CI, 75-92), with a CR rate of 59%. ORR is consistent among key subgroups including age, relapsed/refractory subgroup, previous therapy number, MCL morphology, disease stage, extranodal disease, bone marrow involvement, simplified MIPI, CD19 positive, tumor burden, serum lactate dehydrogenase levels, TP53 mutation status, Ki-67 index, AE management using tollizumab or steroids, and use of bridging therapy. Median time to initial response was 1.0 month (range, 0.8-3.1) and median time to CR was 3.0 months (range, 0.9-9.3). Of the 42 patients who initially achieved PR or SD, 24 patients (57%) (including 21 initial responses with PR and 3 initial responses with SD) subsequently converted to CR after a median of 2.2 months (range, 1.8-8.3) after initial response; 18 of these 24 patients remained in remission. MRD analysis was performed in 29/60 patients (48%); 24/29 patients (83% [19 CR; 5PR ]) were MRD negative at week 4, and 15/19 patients (79%) with available data remained MRD negative at week 6. Due to the lack of availability of formalin fixed paraffin embedded tumor biopsy samples for calibration, MRD cannot be assessed in all patients, which is necessary for methodology and for establishing the major rearranged IgH (VDJ or DJ), IgK or IgL receptor gene sequences that track in blood over time. Two patients who progressed after responding to CD19 CAR-T cells received a second infusion approximately 1 year and 2.6 years after the initial infusion; these patients were analyzed.

Median DOR has not been reached after 12.3 months of median follow-up (median (95% CI)); not reached (8.6, NE). Median progression-free survival (95% CI) was not reached (9.2, NE). The median overall survival (95% CI) was also not reached (24.0, NE). Fifty-seven percent of all patients and 78% of CR patients remained in remission. However, the median follow-up for the first 28 patients treated was 27.0 months (range; 25.3-32.3), with 43% continuing remission without additional therapy. Progressive response rates are consistent between key covariates including age, MCL morphology, relapsed/refractory subgroups, Ki-67 index, disease stage, extranodal disease, bone marrow involvement, simplified MIPI, TP53 mutation, CD19 positivity, bridging therapy, tumor burden, and use of tolbizumab or steroids. 3 patients with CD 19-tumors at baseline achieved CR and maintained a progressive response at data cutoff. Median PFS and OS were not achieved, and 12-month rates were estimated to be 61% (95% CI, 45-74) and 83% (95% CI, 71-91), respectively. Although limited in sample size, a subset analysis of PFS showed that the rate of PFS at 6 months was consistent between patients with blast-like or polymorphic morphology, a TP53 mutation, or a Ki-67 index ≧ 50%. At the time of this analysis, 76% of all patients remained alive. Of the patients with response, 14 patients had PD. One patient with PR underwent allogeneic SCT.

This study showed an ORR of 93% in 60 patients prescribed by the protocol with relapsed/refractory MCL, all of which relapsed after BTKi therapy or were refractory to BTKi therapy. After a single infusion, the ORR included a 67% CR rate. Median DOR was not reached after 12.3 months median follow-up; 57% of all patients and 78% of CR patients maintained response. Twenty-eight (28) patients treated with CD19CAR-T cells had a longer median follow-up of 27 months (range, 25.3-32.3), and 43% of patients continued to be in remission without additional therapy. Response rates, including progressive responses, are substantially similar in a key subgroup including patients with high risk characteristics. Ki-67 ≧ 50% of patients, as well as patients with either blast-like/polymorphic morphology or TP53 mutation, have high ORR and a 6-month PFS rate similar to the entire population, suggesting that CD19CAR-T cell therapy is beneficial for patients with generally poorer prognosis.

All patients reacted after CAR T cell infusion achieved T cell expansion. This expansion was not observed in non-responsive patients, suggesting that a response may be associated with sufficient CAR T cell expansion. Similar to previous studies, CAR T cell levels correlated with ORR in the first 28 days, suggesting that higher expansion resulted in better and possibly deeper responses compared to positive patients, as indicated by > 80-fold higher peak/AUC CAR T cell levels in MRD negativity. The response rate was also similar whether or not bridging therapy was administered, and most patients (87%) scanned post-bridging had increased SPD compared to the scan before bridging.

All treated patients experienced grade 1 or more AE, and grade 3 or more AE in 99% of the treated patients (Table 2). The most common AEs at any level were fever (94%), neutropenia (87%), thrombocytopenia (74%) and anemia (68%). The most common class 3 AEs are neutropenia (85%), thrombocytopenia (51%), anemia (50%) and infection (32%). Twenty-six percent of patients had > grade 3 cytopenia, including neutropenia (16%), thrombocytopenia (16%) and anemia (12%), >90 days after CD19 CAR-T cells. CRS occurred in 91% of patients (table 4). No patients died due to CRS. The majority of cases were grade 1/2 (76%), with grade 3 CRS occurring in 15% of patients. The most common grade ≧ 3 CRS symptoms are hypotension (22%), hypoxia (18%), and fever (11%). For CRS management, 59% of patients received toslizumab, 22% received steroids and 16% required angiostatic. Median time to onset of CRS at any grade and ≧ 3 after infusion was 2 days (range, 1-13) and 4 days (range, 1-9), respectively; all events resolved within the 11 day median.

Table 4: adverse events, cytokine release syndrome and neurological events

Included are adverse events occurring in > 30% of patients and symptoms and neurological events of CRS occurring in > 15% of patients.The percentage of 62 patients in the CRS line who experienced CRS was calculated.

Sixty-three percent of patients experienced NE (table 4). None of the patients died due to NE. Grade 1/2 NE occurs in 32% of patients, and > 3 NE occurs in 31% of patients. Common grade 3 NE is encephalopathy (19%), confusion (12%) and aphasia (4%). One patient developed grade 4 cerebral edema and was fully recovered with invasive multimodal therapy including a ventricular ostomy. Tollizumab and steroids were used to treat NE in 26% and 38% of patients, respectively. Median time to onset of NE at any grade and grade 3 were 7 days (range, 1-32) and 8 days (range, 5-24), respectively. Median duration of NE was 12 days, with complete resolution of events in 37/43 patients (86%). As analyzed, 4 patients had progressive events including grade 1 tremor (n ═ 3), grade 2 attention deficit disorder (n ═ 1), and grade 1 dysesthesia (n ═ 1). Severe AE occurred in 68% of patients (table 5).

Table 5: severe adverse events in at least 3 patients

Thirty-two percent of patients experience grade 3 or more infections. Pneumonia (9%) was most common (table 6).

Table 6: the infection occurred in at least 2 patients

^* One patient died from grapeStaphylococcal bacteremia. One patient died of organizing pneumonia (acute kidney injury occurred in the case of infection, and had a previously undiagnosed pulmonary embolism during necropsy in addition to organizing pneumonia).

Grade 2 cytomegalovirus infection occurred in two cases (3%). Grade 3 hypogammaglobulinemia and grade 3 tumor lysis syndrome occurred once (1%) in 1 patient. Twenty-two patients (32%) received intravenous immunoglobulin therapy. There are no cases reported for replication competent retroviruses, EBV-associated lymphoid tissue proliferation, lymphohistiocytosis with haemophilus or CD19 CAR-T cell associated secondary cancer. The EQ-5D score depletion showed a decrease from baseline in the health-related quality of life reported by patients at week 4, with improvements in activity, self-care, daily activities and overall health (EQ-5D visual analog scale) observed by month 3, with overall health returning to baseline or better levels by month 6 in most patients (table 7).

Table 7: EQ-5D summarization at visit

^* The EQ-5D Visual Analog Scale (VAS) evaluates overall health on a scale from 0 to 100, with higher scores indicating better health status. EQ-5D, European quality of life-5D; N/A, not applicable; SD, standard deviation

Sixteen patients (24%) who received CD19 CAR-T cells died primarily due to PD (n-14 [ 21% ]). Two patients had a grade 5 AE (3%), including 1 patient with organized pneumonia associated with opsonic chemotherapy and 1 patient with staphylococcal bacteremia associated with opsonic chemotherapy and CD19 CAR-T cell therapy.

The median time to peak anti-CD 19 CAR T cell levels after CD19 CAR-T cell infusion was 15 days (range, 8-31), and cells were still detectable at 24 months in some patients with evaluable samples at the time of data cutoff (6/10[ 60% ]) in the presence of normal median B cell levels. CAR T cell persistence over time in blood showed time decline in patients with progressive responses and relapses as measured by qPCR.

The rapid expansion, regression to baseline, and clearance over time are consistent with the known mechanism of action of anti-CD 19 CAR T cells with CD28 and CD3 ζ costimulatory domains. All 4 patients who did not respond to CD19 CAR-T cell therapy had detectable B cells at baseline; none of the patients experienced B cell dysplasia at any point of the study. Although there was no correlation with baseline tumor burden, amplification correlated with response (P ═ 0.0036), with area under the curve (AUC) and peak > 200-fold higher in responders than in non-responders, with similar trends between MRD negative and MRD positive patients at week 4. For both CRS and NE, amplification was higher in patients with ≧ 3 events than with ≦ 2 events, and the highest peak and AUC were noted in patients receiving toclizumab + -steroids following CD19 CAR-T cell infusion. Median time to peak was 8 days for the cytokines evaluated; most subsided to baseline levels by day 28. Serum granulocyte-macrophage colony stimulating factor and Interleukin (IL) -6 are associated with CRS and NE grades 3 or more. Serum ferritin is only associated with grade 3 CRS whereas serum IL-2 and interferon-gamma are only associated with grade 3 NE. In addition, cerebrospinal fluid cytokine analysis revealed higher levels of C-reactive protein, ferritin, IL-6, IL-8 and vascular cell adhesion molecule 1 in patients with grade 3 NE. No induction of anti-CAR antibodies was observed in any patient.

Ratios of CRS grade ≧ 3 and NE similar to those reported previously in the case of T-cell therapy with anti-CD 19CAR in aggressive NHL. Neelapu SS et al, New England journal of medicine (The New England journal of medicine) 2017; 377: 2531; schuster SJ et al, New England journal of medicine 2019; 380:45. There was no death due to CRS or NE, and most symptoms occurred early in treatment and were usually reversible, and there was no long-term clinical sequelae impairing activities of daily living. At the peak value of serum cytokines and more than or equal to grade 3The observed association between CRS and/or class 3 neural events indicates a role for CD19CAR-T cells in these toxicities as they were observed to be commensurate with the rising and peak levels of CAR T cells in the blood. The correlation of peak levels of CAR and bone marrow cell-associated serum cytokines, chemokines, and effector molecules with toxicity is consistent with previously published data using similar CAR constructs in the case of NHL. ^10,13 One case of grade 4 cerebral edema occurred, but patients recovered completely and maintained CR at 24 months follow-up with no unremoved neurological sequelae. The results reported by the patients similarly indicate that there is no long-term quality of life deficiency following CD19CAR-T cell therapy.

Example 3

This example provides an additional analysis of the above study. Eligible patients with R/R MCL (age ≧ 18 years) had an ECOG score of 0-1 and ≦ 5 previous therapies including chemotherapy, anti-CD 20 antibodies, and BTKi. Patients underwent leukopheresis and chemotherapy (300 mg/m2 cyclophosphamide per day and 30mg/m2 fludarabine per day on days-5, -4 and-3 for 3 days) and then were treated by single infusion at 2X 10 on day 0 ⁶ Target dose of individual CAR T cells/kg a single infusion of CD19 CAR-T cells. The CD19 CAR construct contained a CD3 ζ T cell activation domain and a CD28 signaling domain. The method of manufacture removes circulating leukemia cells expressing CD19 from the leukapheresis product. Sabatino M et al, Blood (Blood) 2016; 128:1227.

Some patients receive a bridging therapy with dexamethasone (20 mg to 40mg per PO or IV administration per day or equivalent for 1 to 4 days), ibrutinib (560 mg per PO administration per day), or acatinib (100 mg per PO administration twice per day), administered after leukoapheresis and completed < 5 days before initiating opsonic chemotherapy; PET-CT is required after bridging. The primary endpoint was the objective response rate (ORR [ Complete Response (CR) + partial response ]). The key secondary endpoints are duration of response (DOR), Progression Free Survival (PFS), OS, frequency of Adverse Events (AE), CAR T cell levels in blood and cytokine levels in serum. Efficacy and safety assays included all patients receiving CD19 CAR-T cell therapy.

Key inclusion criteria include R/R MCL, defined as disease progression after the last regimen or failure to exhibit CR or PR for the last regimen; chemotherapy with anthracycline or bendamustine and anti-CD 20 monoclonal antibody therapy and one to five prior therapies of ibrutinib or acatinib must be included; more than or equal to 1 measurable lesion; ECOG is 0 or 1 when the age is more than or equal to 18 years old; and adequate bone marrow, kidney, liver, lung, and heart function. Key exclusion criteria included previous autologous stem cell transplantation (alloSCT); previous CD19 targeted therapies; previous CAR T cell therapy; clinically relevant infections; and history or current CNS involvement of MCL or other CNS disorders.

A total of 68 patients received CD19 CAR-T cell therapy. Updated safety (68 patients) and efficacy (60 patients) results are provided herein, with a median follow-up of 12.3 months [ range 7.0-32.3 ]). A total of 28 patients (47%) had a follow-up of 24 months. The median time to initial response was 1.0 month [ range 0.8-3.1] and the median time to complete response was 3.0 months [ range 0.9-9.3 ]. A total of 24 patients (40%) converted from PR/SD to CR, of which 21 patients (35%) converted from PR to CR and 3 patients (5%) converted from SD to CR.

The median age was 65 years (range, 38-79 years), and 39 (57%) patients were male. One hundred percent (100%) of patients have an ECOG score of 0/1, 25% have blast-like morphology, 85% have stage IV disease, 56% have mid/high risk MIPI, 81% receive 3 or more prior therapies, median 3 (range, 1-5) prior therapies, 99% receive prior anthracyclines or bendamustine, 100% receive prior anti-CD 20 monoclonal antibody, and 100% bendamustine receive prior BTKi (both 85% ibrutinib, 24% acatinib, and 9%). Forty-three (43%) patients relapsed after autoSCT, 56% were refractory to ibrutinib, and 12% were refractory to acatinib. 34/49 patients had data available with a Ki-67 index of > 50%. Twenty-five (37%) patients receivedBridge therapy (21% ibrutinib, 7% alcatinib, 18% dexamethasone, 3% methylprednisolone, 9% BTKi and steroid, 6% ibrutinib and steroid, 3% alcatinib and steroid); 23/25 patients had post-bridging PET-CT to record measurable disease prior to CD19 CAR-T cell infusion (20/23 patients had SPD mm increased from screening ² (ii) a 3/23 patients had SPD mm slightly decreased from screening ² )。

High ORR was observed in both evaluable efficacy and ITT patients. 95% identity for ORR; for CR, 90% identity. The ORR assessed by the investigators was 88% (95% CI, 77% -95%) among 60 patients with evaluable efficacy, with a CR rate of 70% (95% CI, 57% -81%) and a PR rate of 18% (95% CI, 10% -30%). The ORR in patients was estimated to be 93% (95% CI, 84% -98%) with a CR rate of 67% (95% CI, 53% -78%) and a PR rate of 27% (95% CI, 16% -40%) by the efficacy of IRRC assessment of 60 patients. ORR is consistent between key subgroups (age, MCL morphology, Ki-67 index, disease stage, simplified MIPI, AE management using steroids, tositumumab use, and bridging therapy use). Investigators among ITT patients evaluated an ORR of 80% (95% CI, 69% -88%), with a CR rate of 59% (95% CI, 47% -71%), and a PR rate of 20% (95% CI, 12% -31%). ORR in ITT patients assessed by IRRC was 85% (95% CI, 75% -92%), with CR rate 59% (95% CI, 47% -71%), PR rate 26% (95% CI, 16% -37%).

Median DOR has not been reached after 12.3 months of median follow-up. Fifty-seven percent (57%) and 78% of all patients with CR remained in remission. The first 28 treated patients had a median follow-up of 27.0 months (range, 25.3-32.3), 43% of which remained in sustained remission without additional therapy. Median PFS and median OS were not reached after 12.3 months of median follow-up. The 12 month PFS rate (95% CI) was 61% (45% -74%). The 12-month OS rate (95% CI) was 83% (71% -91%).

Over 35% of patients had adverse events with treatment (0% grade 1; 1% grade 2; 16% grade 3; 76% grade 4 and 3% grade 5). The most common grade 3 AE ≧ 20% of patients are neutropenia (69%, grade 4), thrombocytopenia (35%, grade 4), anemia (50%, grade 3), hypophosphatemia (22%, grade 3). None of the patients died from Cytokine Release Syndrome (CRS). Grade 3 CRS was reported in 15% of patients (assessed by Lee DW et al, Blood, 2014,124: 188). The most common symptoms of any grade of CRS are hypotension (51%), hypoxia (34%) and fever (91%). Adverse event management included toslizumab (59%) and corticosteroid (22%). Median time to onset was 2 days (range 1-13), median duration was 11 days, and events had resolved in 62/62 (100%) patients with any grade CRS.

Any level of Neurological Events (NE) were reported in 63% of patients (31% with grade. gtoreq.3 NE) and included encephalopathy (31%), confusion status (21%) and tremor (35%). No patient died from a neurological event. One patient had grade 4 cerebral edema that was completely resolved with invasive multimodal therapy including a ventricular ostomy and IV administration to rabbits, anti-thymocyte globulin (ATG). All CRS events and most NEs (37 out of 43 patients) were reversible. Median time to onset and NE duration were 7 days (range, 1-32 days) and 12 days, respectively.

Higher peak levels of CAR T cells were associated with responders (objective responses) compared to non-responders. The higher peak levels of CAR T cells correlated with negative MRD at week 4 rather than positive MRD. The median time to peak anti-CD 19 CAR T cell levels after CD19 CAR-T cell infusion was 15 days (range, 8-31). In most patients with an evaluable sample (6/10[ 60% ]), anti-CD 19 CAR T cells could be detected at 24 months. Amplification correlates with response and MRD status. Amplification in patients with grade 3 or more was greater than in patients with grade 2 CRS and neurological events.

Several associations were observed between peak serum biomarker levels and toxicity. Analytes associated with CRS class 3 or greater include IL-15, IL-2R α, IL-6, TNF α, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1B, granzyme A, granzyme B, and perforin. Analytes associated with grade > 3 neural events include IL-2, IL-1Ra, IL-6, TNF α, GM-CSF, IL-12p40, IFN- γ, IL-10, MCP-4, MIP-1B, and granzyme B. And analytes associated with both CRS grade 3 and neural events include IL-6, TNF α, GM-CSF, IL-10, MIP-1B, and granzyme B.

CD19 CAR-T cell therapy described herein administered in a single infusion showed a high rate of durable response in R/RMCL. 93% ORR (including 67% CR rate) is the highest reported rate of disease control in patients with past BTKi failure. Of the initial 28 patients treated, 43% remained in remission after a follow-up period of 24 months or more. The safety profile is consistent with the results reported in previous studies of anti-CD 19 CAR T cell therapy in aggressive NHL. No death due to CRS or neurological events; most symptoms occur early in treatment and are usually reversible. Efficacy, reliable and rapid manufacturing, and manageable toxicity identify the role of the CD19 CAR-T cell therapy described herein in treating patients with R/R MCL that do not meet medical needs.

Example 4

This example provides additional analysis of the clinical study described above. Eligible patients are aged > 18 years, have pathologically confirmed MCL with documented overexpression or presence of cyclin D1 (11; 14), and are relapsed/refractory to 1-5 previous regimens of MCL. Previous therapies must include chemotherapy with anthracyclines or bendamustine, anti-CD 20 monoclonal antibodies, and ibrutinib or acatinib. All patients received past BTKi. Although patients must have past BTKi therapy, they need not be the last line therapy before study entry, and patients need not be refractory to BTKi therapy. Eligible patients had an absolute lymphocyte count of > 100/. mu.L. Patients who underwent autologous SCT or had prior CD19 targeted therapy or allogeneic SCT within 6 weeks of CD19CAR-T cell infusion were excluded. All patients underwent leukapheresis to obtain cells for CD19CAR-T cell therapy manufacture. The patient receives optional bridging therapy comprising dexamethasone (20 mg to 40mg per day PO or IV or equivalent for 1 day to 4 days), ibrutinib (560 mg per day via oral cavity (PO)) or acatinib (twice daily) PO 100 mg). The manufacturing method was modified with respect to that of aliskiren (axicabtagene ciloleucel) to enrich CD4 by positive ⁺ /CD8 ⁺ The cells eliminate circulating lymphoma cells. This product is referred to herein as a "CAR T cell". This product was also identified as KTE-X19. Single intravenous infusion of 2X 10 on day 0 ⁶ Fludarabine (30 mg/m/day) was administered on days-5, 4 and 3 before individual CAR T cells/kg of CD19 CAR-T cells ² ) And cyclophosphamide (500 mg/m per day) ² ) The method of (1). More details about patient treatment can be found in example 2.

The goal of this study was twofold. First, to compare the pharmacological profiles of CAR T products in lower and higher risk patients defined by the TP53 (tumor protein p53) gene mutation status and Ki-67 tumor proliferation index in clinical trial ZUMA-2. Patients with high risk MCL characteristics, including mutations in the tumor protein p53 gene (TP53) and high Ki-67 proliferation index, often have poor prognosis with current standard therapies. Cheah CY et al, J.Clin Oncol., 2016; 34:1256-1269. Lower risk patients in this assay had Ki-67 proliferation index < 50% (by central assessment) or wild-type TP 53; high risk patients have Ki-67 ≧ 50% or a TP53 mutation determined by next generation sequencing. In the primary efficacy analysis of ZUMA-2 (N ═ 60), after a median follow-up of 12.3 months, the ORR was 93% (67% CR). 57% of all patients and 78% of CR patients with progressive response. ORR is generally comparable between lower and higher risk patients in ZUMA-2 (including in patients with Ki-67 proliferation index < or > 50% and non-mutated versus mutated TP 53). Wang M et al, New Engl JMed, 2020; 382:1331-1342.

The second objective was to characterize the pharmacodynamic profile of patients achieving the Minimal Residual Disease (MRD) negative status at early stage (day 28) and those with grade 4 neurotoxicity. In previous analysis results of ZUMA-2, CAR T cell levels in blood were determined by peak and area under the curve (AUC) on days 0-28 with ORR (including undetectable MRD) and ≧ 3 grade CRS and neurologic eventsThe pieces are related. Wang M et al, New Engl J Med, 2020; 382:1331-1342. In this analysis, CRS and neurological events were mainly reversible (N ═ 68 treated patients): 15% has CRS of grade 3 or more; 31% of patients have grade 3 or more neural events; and two patients had a grade 5 AE (one of which was CAR T product related). As previously reported, MRD was evaluated by next generation sequencing (10) ^-5 Sensitivity). Wang M et al, New Engl J Med, 2020; 382:1331-1342.

This update reported pharmacological data for all 68 patients in ZUMA-2 treated with CAR T cells. Product attributes, CAR T cell levels in blood and cytokine levels in serum and their correlation with clinical outcome were analyzed using the methods described previously. Locke FL et al, molecular therapeutics (Mol Ther.), 2017; 25:285-295. The Wilcoxon rank-sum test is used to measure the association between subgroup results and CAR T cell and cytokine levels. The P value was not adjusted for multiple tests.

CAR T cell product attributes were generally comparable in the prognostic set defined by Ki-67 proliferation index and TP53 mutation status. There was a trend towards more differentiated phenotypes in the high Ki-67 subgroup and CD 4-based phenotypes in patients with the TP53 mutation. (Table 8).

TABLE 8

^a Product characterization data was available for 65 total patients out of all 68 treated patients. Product profile data was available for 48/49 total patients, Ki-67 data was available for all patients, and TP53 mutation data was available for 36 patients. TP53, tumor protein p53 Gene

There was also comparable CAR T cell expansion in the group with different prognostic factors defined by Ki-67 proliferation index and TP53 mutation status. Both the peak level and AUC of CAR T cells in the blood after administration were comparable in patients with wild type and mutant TP53 or Ki-67 proliferation indices < 50% to > 50%, which is in agreement with efficacy in these subgroups. The primary endpoints of the patient's Objective Response Rate (ORR) are shown in table 9. The median time to response was 28 days (range: 24 to 92 days) and the median follow-up time was 12.3 months. The potential follow-up for 28 patients was > 24 months, and 12 of these patients remained in remission. Efficacy was established based on complete response and duration of response (DOR).

ORR of the patients with Ki-67 proliferation index of less than 50% and more than 50% is 100% and 94%, while CR of the patients with Ki-67 proliferation index of less than 50% and more than 50% is 64% and 78%. Table 9: the number of patients for which Ki-67 proliferation index data was available was 49.

TABLE 9

	ORR(95％CI)，％	CR rate (95% CI)%
			Ki-67 PI<50％	100(77–100)	64(35–87)
Ki-67 PI≥50％	94(79–99)	78(60–91)

The ORR was 100% for patients with both wild-type and mutant TP53, whereas the CR rates were 67% and 100% for wild-type and mutant TP53 patients. Table 10: the number of patients for which TP53 data was available was 36. All six patients with the TP53 mutation and all 30 patients without mutation responded. Of six patients with the TP53 mutation, three patients had grade 3 or greater neurotoxicity and two patients had grade 3 or greater CRS

Watch 10

	ORR(95％CI)，％	CR rate (95% CI)%
			TP53 mutation	100(54–100)	100(54–100)
TP53 non-mutant	100(88–100)	67(47–83)

Up to 44 biomarkers including IL (interleukin), INF-gamma (interferon gamma), MCP-1 (monocyte chemoattractant protein 1), IL-2R alpha (IL-2 receptor alpha), sPD-L1 (soluble programmed death ligand 1), and sVCAM (soluble vascular cell adhesion molecule) were measured in serum at various time points before treatment, on day 0, and to day 28 after CAR T-cell infusion. The pharmacodynamic profiles of the two prognostic groups with Ki-67 proliferation index < 50% and > 50% are comparable with respect to proliferation (IL-15, IL-2), inflammation (IL-6, IL-2R α, sPD-L1 and VCAM-1), immunomodulation (IFN-. gamma., IL-10), chemokines (IL-8 and MCP-1) and effector cytokines (granzyme B). In addition there was a tendency to increase the level of proliferative (IL-15, IL-2) and inflammatory (IL-6, IL-2 Ra, sPD-L1 and VCAM-1) cytokines in patients with mutated TP53 relative to wild type TP 53. FIGS. 1A-1F.

Peak levels of selected cytokines in serum are also increased in patients achieving MRD negative status. MRD was analyzed in 29 of 68 patients (43%); 24 of these patients (83% [19 patients with complete response and 5 with partial response ]) were MRD negative one month after CAR T cell administration. One month after CAR T cell administration, MRD negative (n-24/29) and positive patients (n-5/29) had a tendency to increase median peak levels of Interferon (IFN) - γ and Interleukin (IL) -6 and increased IL-2. Cytokine levels in serum peaked within 7 days of treatment. A consistent trend was observed for PD-L1 and granzyme B. Peak CAR T cell levels measured within 14 days post treatment were also observed in patients that were MRD negative at 1 month. Fig. 2A-2I.

Six patients developed grade 4 neurological events, including one with cerebral edema. Three patients had grade 4 CRS at the same time. Patients with a grade 4 neurological event exhibit increased peak levels of proinflammatory serum biomarkers (e.g., IFN γ, MCP-1, TNF- α, IL-2, and IL-6) as compared to patients without a neurological event.

Cerebral edema resolved completely after invasive multimodal therapy. Wang M et al, New Engl J Med, 2020; 382:1331-1342. (ii) CAR T cell expansion and peak serum levels of IL-2 are highest in the patient; the rise in multiple cytokines was several fold higher in this patient compared to the median of other study/ZUMA-2 patients. Table 11:

TABLE 11

^a Among the 66 patients with available data.

CAR T cell pharmacokinetic and pharmacodynamic profiles were comparable between MCL patient groups with different prognostic marker states associated with lower and higher risk (defined by Ki-67 and mutated TP 53), consistent with comparable clinical response rates. There was a higher trend for proinflammatory marker levels in patients with mutated TP 53.

The pharmacodynamic profile of CAR T cell administration correlated with efficacy (MRD status at 1 month) and neurological events with grade 4 treatment appearance. Patients who develop cerebral edema have the highest peak CAR T cell levels and serum IL-2, as well as elevated post-treatment pro-inflammatory markers.

Example 5

Phase 2 one-armed clinical studies were conducted on CD 19-directed genetically modified autologous T cell immunotherapy-treated patients with relapsed or refractory Mantle Cell Lymphoma (MCL) who had received one or more prior treatments, which may include anti-CD 20 antibodies, anthracycline-or bendamustine-containing chemotherapy, and/or Bruton's Tyrosine Kinase Inhibitor (BTKi) (such as ibrutinib or acatinib). Eligible patients also have disease progression after their last treatment or refractory disease for their last treatment. The study excluded patients with active or severe infection, previous allogeneic Hematopoietic Stem Cell Transplantation (HSCT), detectable cerebrospinal fluid malignant cell or brain metastases, and any history of Central Nervous System (CNS) lymphoma or CNS disorders.

Peripheral blood mononuclear cells of the patient were obtained by a leukapheresis procedure. Monocytes were enriched for T cells by selecting CD4+ and CD8+ cells, activated with anti-CD 3 and anti-CD 28 antibodies in the presence of IL-2, and then transduced with a replication-defective viral vector containing FMC63-28Z CAR, a Chimeric Antigen Receptor (CAR) comprising an anti-CD 19 single-chain variable fragment (scFv), CD28, and CD 3-zeta domain. Without being bound by any hypothesis, selection of CD4+ cells and CD8+ cells may have reduced circulating tumor cells potentially expressing CD19 in patient leukapheresis material included during the ex vivo manufacturing process. The T cell product of this process can be identified as KTE-X19. Expanding anti-CD 19 CAR T cellsAnd washing, preparing into a suspension, and freezing and storing. 500mg/m intravenously administered on each of the fifth, fourth and third days prior to infusion of CAR T cells prior to receiving anti-CD 19 CAR T cell therapy ² Cyclophosphamide and 30mg/m ² (ii) treating the patient with a lymphodepleting chemotherapy regimen of fludarabine; the patient may also receive acetaminophen and diphenhydramine or another H1 antihistamine approximately 30 to 60 minutes prior to infusion of the anti-CD 19 CAR T cells. The prophylactic use of systemic corticosteroids is avoided as it may interfere with the activity of CAR T cells.

The target dose is 2X 10 ⁶ Individual CAR positive live T cells or anti-CD 19 CAR T cells/kg body weight, with a maximum dose of 2 x 10 ⁸ anti-CD 19 CAR T cell (for patients of 100kg and above) cells. 68 patients received a single infusion (by gravity or peristaltic pump for approximately 30 minutes) of anti-CD 19 CAR T cells and 60 of these patients were followed at least 6 months after their 4 th week disease assessment and were determined to be evaluable for efficacy. 56 patients received 2X 10 ⁶ anti-CD 19 CAR T cells/kg; 1 patient received 1X 10 ⁶ Dose of anti-CD 19 CAR T cells/kg, 1 patient received 1.6X 10 ⁶ 2 patients received a dose of 1.8X 10 anti-CD 19 CAR T cells/kg ⁶ Dose of anti-CD 19 CAR T cells/kg, and-2 patients received 1.9X 10 ⁶ Dose of anti-CD 19 CAR T cells/kg. Of these 60 patients, the median age was 65 years (range: 38 to 79 years), 51 patients were male, and 56 patients were caucasian. 50 patients had stage IV disease. Based on the simplified international prognostic index (s-MIPI) for mantle cell lymphoma, 25 patients were classified as low risk, 25 patients were classified as intermediate risk, 8 patients were classified as high risk, and 2 patients had an unknown risk status. 20 patients had baseline bone marrow exams performed according to the protocol; of these patients, 10 patients were negative, 8 patients were positive, and 2 patients were inconclusive. All 60 efficacy values assessed the median number of previous therapies in the patients was 3 (range: two to five). 26 patients relapsed after or were refractory to autologous HSCT. 21 patients in the end of their study One MCL treatment was followed by relapse, and 36 patients were refractory to their last MCL treatment. 14 patients had blast-like MCL. Following leukaemotomy and prior to infusion of anti-CD 19 CAR T cells, 21 patients received bridging therapy. 19 patients were treated with BTKi, 14 patients with corticosteroid, and 6 patients with both BTKi and corticosteroid. 53 patients received 500mg/m intravenously administered on each of the fifth, fourth and third days prior to anti-CD 19 CAR T therapy (day 0) ² Cyclophosphamide and 30mg/m ² A lymphodepleting chemotherapy regimen of both fludarabine. The remaining 7 patients received the same dose of lymphodepleting chemotherapy 4 or more days prior to CAR T therapy. The primary endpoints of the patient's Objective Response Rate (ORR) are shown in table 12. The median time to response was 28 days (range: 24 to 92 days) and the median follow-up time was 12.3 months. The potential follow-up of twenty-eight patients was ≧ 24 months, and twelve of these patients remained in remission. Efficacy was established based on complete response and duration of response (DOR).

TABLE 12

CI, confidence interval; NE, not estimable; NR, not reached; PR, partial remission.

a. The results were obtained among all respondents. DOR is measured from the date of first objective response to the date of progression or death.

b. The checked value.

CRS (cytokine release syndrome) was observed in 75 of 82 patients, including ≧ 3 (Lee rating System 1) CRS in 15 of 82 patients. Median time to onset of CRS was 3 days (range: 1 to 13 days) and median duration of CRS was 10 days (range: 1 to 50 days). Among patients with CRS, key manifestations (i.e. those occurring in > 10% of patients) include fever (99% of patients), hypotension (60% of patients), hypoxia (37% of patients), chills (33% of patients), tachycardia (37% of patients), headache (24% of patients), fatigue (19% of patients), nausea (13% of patients), increased alanine aminotransferase (13% of patients), increased aspartate aminotransferase (12% of patients), and diarrhea (11% of patients). Severe events associated with CRS include hypotension, fever, hypoxia, acute kidney injury, and tachycardia. In response to CRS, the patient may have received tositumumab and/or corticosteroids as indicated in table 13.

Watch 13

Lee DW et al, 2014. The Current concept of cytokine release syndrome diagnosis and management (Current receptors in the diagnosis and management of cytokine release syndrome), "Blood", 2014, month 7 and 10; 124(2):188-195.

b. For the management of neurotoxicity, see table 14.

c. For detailed information, please refer to the prescription information of Tolizumab

Neurological events were observed in 53 patients, 20 of whom experienced grade 3 or higher (severe or life threatening) adverse reactions. The median time to onset of the neural event was 6 days (range: 1 to 32 days). In 52 of 66 patients, the neurological events resolved with a median duration of 21 days (range: 2 to 454 days). 3 patients had progressive neurological events at the time of death, including 1 patient with severe encephalopathy. The remaining non-resolved neurological events were grade 1 or grade 2. Patient 54 experienced CRS by the onset of a neurological event. 5 patients did not experience CRS with a neurological event, and 8 patients developed a neurological event after CRS subsided. 56 patients experienced the first CRS or neurological event within 7 (seven) days after infusion of anti-CD 19 CAR T cells.

The most common neurological events (occurring in > 10% of patients) include encephalopathy (51% of patients), headache (35% of patients), tremor (38% of patients), aphasia (23% of patients), and delirium (16% of patients). Serious events including encephalopathy, aphasia and seizures occurred after treatment. Some of the adverse reactions observed in at least ten percent of treated patients include: disorders of the blood and lymphatic system (coagulopathy, heart disease, tachycardia, bradycardia, non-ventricular arrhythmias); gastrointestinal disorders (nausea, constipation, diarrhea, abdominal pain, oral pain, vomiting, dysphagia); general disorders and conditions at the site of administration (fever, fatigue, chills, edema, pain); immune system disorders (cytokine release syndrome, hypogammaglobulinemia); infections and infestations (infection by pathogens unspecified, viral infection, bacterial infection); metabolic and nutritional disorders (reduced appetite), musculoskeletal and connective tissue disorders (musculoskeletal pain, motor dysfunction); nervous system disorders (encephalopathy, tremor; headache, aphasia, dizziness, neuropathy); psychotic disorder (insomnia, delirium, anxiety); renal and urinary disorders (renal insufficiency, decreased urine output); respiratory, thoracic and mediastinal disorders (hypoxia, cough, dyspnea, pleural effusion); skin and subcutaneous tissue disorders (rashes); and vascular disorders (hypotension, hypertension, thrombosis). Patients who experienced grade 2 or higher neurotoxicity can be treated according to the indications shown in table 14.

TABLE 14

a. The severity of the criteria is based on the common adverse event terminology.

By measuring cytokines, chemokines and other molecules in blood after infusion of anti-CD 19 CAR T cellsTransient elevation of cells, pharmacodynamic response was assessed over four week intervals. The levels of IL-6, IL-8, IL-10, IL-15, TNF- α, IFN- γ and/or sIL2R α were analyzed. Peak elevations in these cytokine levels are typically observed between 4 and 8 days post-infusion, and levels typically return to baseline within 28 days. Periods of B cell dysplasia are expected. After infusion, the initial expansion of anti-CD 19 CAR T cells subsequently dropped to near baseline levels by 3 months. Peak levels of anti-CD 19 CAR T cells occurred within the first 7 to 15 days after infusion. The results indicate that levels of anti-CD 19 CAR T cells in the blood correlate with objective responses, i.e., Complete Remission (CR) or Partial Remission (PR). Median peak anti-CD 19 CAR T cell levels in responders (those with complete and partial remission) were 102.4 cells/μ L (range: 0.2 to 2589.5 cells/μ L; n-51) and 12.0 cells/μ L in non-responders (range: 0.2 to 1364.0 cells/μ L, n-8). Median AUC days 0-28 (AUC) _0-28 ) In patients with objective responses 1487.0 cells/μ L day (range: 3.8 to 2.77X 10 ⁴ Individual cells/. mu.L.day; n-51), 169.5 cells/μ L day in non-responders (range: 1.8 to 1.1710X 10 ⁴ Individual cells/. mu.L.day; n-8). Median peak (24.7 cells/. mu.L) anti-CD 19 CAR T cells (peak: and AUC) in patients not receiving either corticosteroid or tollizumab (n ═ 18) _0-28 Levels (360.4 cells/. mu.L.day) were similar to those of patients receiving corticosteroid only (n ═ 2) (peak: 24.2 cells/. mu.L; AUC) _0-28 : 367.8 cells/. mu.L.day). In patients receiving tositumumab only (n ═ 10), the mean peak anti-CD 19 CAR T cells was 86.5 cells/μ L, AUC _0-28 The concentration was 1188.9 cells/. mu.L.day. In patients receiving both corticosteroid and toslizumab (n ═ 37), the mean peak was 167.2 cells/μ L, AUC _0-28 The concentration was 1996.0 cells/. mu.L.day. Median peak anti-CD 19 CAR T cell value was 74.1 cells/μ L in patients aged 65 years (n 39) at age ≥ age, at age<Patients 65 years old (n-28) had 112.5 cells/μ L. Median anti-CD 19 CAR T cell AUC _0-28 The value was 876.5 cells/. mu.L.day in patients aged 65 or older, at age <1640.2 patients of 65 years oldCells/. mu.L.day. AUC of gender versus CD19 CAR T cells _0-28 And C _max There was no significant effect.

Example 6

MCL patients who progressed following BTKi therapy had only a median overall survival of 5.8 months with rescue therapy. ZUMA-2(clinical trials. gov identifier: NCT02601313) is a phase 2 registered multicenter study of R/R MCL patients following 1 to 5 prior therapies, including BTKi. Autologous anti-CD 19 Chimeric Antigen Receptor (CAR) T cell therapy was administered to the patient, prepared and administered as described in example 5. This anti-CD 19 CAR T cell product may be referred to as KTE-X19. In the primary analysis of ZUMA-2 (N ═ 60), the Objective Response Rate (ORR) of treatment with anti-CD 19 CAR T cells (median follow-up 12.3 months) was 93% (67% complete response [ CR ] rate). This example describes a comparative analysis of the pharmacological profile of anti-CD 19 CAR T cell therapy prepared as described in example 5 and characterization of concomitant basal product attributes by MCL morphology and results of past BTKi exposures (ibrutinib and/or alcaniib).

Eligible patients with R/R MCL underwent leukopheresis and opsonic chemotherapy followed by a single infusion of 2X 10 ⁶ anti-CD 19 CAR T cells/kg. Product attributes (e.g., IFN γ production by anti-CD 19 CAR T cells after co-culture with CD19+ cells), CAR T cell levels in blood, and cytokine levels in serum were assessed using the methods described previously (see previous examples). Clinical results were reported for 60 patients with evaluable efficacy; product attributes and pharmacological data were reported for all 68 treated patients.

At baseline, 40 patients (59%) had classical MCL, 17 patients (25%) had blast-like MCL, and 4 patients (6%) had polymorphic MCL, as assessed by the investigator. Prior to study entry, 52 patients (76%) had previous ibrutinib, 10 patients (15%) had previous acatinib, and 6 patients (9%) had both; 88% of patients suffer from BTKi refractory disease. The median (range) CD4+/CD8+ T cell ratios in manufactured anti-CD 19 CAR T cell products for patients with classical, blast-like, or polymorphic MCL were 0.7 (0.04-2.8), 0.6 (0.2-1.1), or 0.7 (0.5-2.0), respectively. The product T cell phenotype (median [ range ]) included less differentiated CCR7+ T cells (classically 40.0% [ 2.6-88.8 ]; blast-like 35.3% [ 14.3-73.4 ]; polymorphism 80.8% [ 57.3-88.8 ]) and effector memory CCR7-T cells (classically 59.9% [ 11.1-97.4 ]; blast-like 64.8% [ 26.6-85.7 ]; polymorphism 19.2% [ 11.1-42.7 ]). The median (range) Interferon (IFN) - γ levels in co-cultures in patients with classical, blast-like or polymorphic MCL were 6309.5pg/mL (424.0-20,000), 6510.0pg/mL (2709.0-18,000) or 7687.5pg/mL (424.0-12,000), respectively. In patients with classical, blast-like or polymorphic MCL, the median (range) peak CAR T cell levels were 77.6 cells/μ L (0.2-2241.6), 35.0 cells/μ L (0.2-2589.5) or 144.9 cells/μ L (39.2-431.3), respectively. The ORR/CR rate was 93%/65% in patients with classical MCL, 88%/53% in patients with blast-like MCL, and 100%/75% in patients with polymorphic MCL. The 12-month survival rate of patients with classical, blast-like or polymorphic MCL is 86.7%, 67.9% or 100%, respectively. Grade 3 Cytokine Release Syndrome (CRS) and neurological events occurred in 15% and 38% of patients with classical MCL, 6% and 8% of patients with blast-like MCL and 25% and 50% of patients with polymorphic MCL.

For patients receiving previous ibrutinib, acartinib, or both, the median CD4+/CD8+ T cell ratios in the manufactured anti-CD 19CAR T cell products were 0.7 (range, 0.04-3.7), 0.6 (range, 0.3-1.2), or 1.0 (range, 0.7-1.9), respectively. The product T cell phenotype (median [ range ]) includes less differentiated CCR7+ T cells (ibrutinib 39.3% [ 2.6-86.4 ]; acatinib 42.7% [16.3-88.8 ]; both 49.5% [ 14.3-83.0 ]) and CCR 7-responsive and responsive memory T cells (ibrutinib 60.6[13.7-97.4 ]; acatinib 57.3% [11.1-83.8 ]; both 50.6% [17.0-85.7 ]). The median (range) IFN- γ levels in co-cultures in patients receiving previous ibrutinib, acatinib or both were 6496.0pg/mL (424.0-20,000), 5972.5pg/mL (2502.0-18,000) or 7985.5pg/mL (2709.0-12,000), respectively. For patients receiving previous ibrutinib, acartinib, or both, the median (range) peak CAR T cell levels were 95.9 (0.4-2589.5), 13.7 (0.2-182.4), or 115.9 (17.2-1753.6), respectively. The ORR/CR rate was 94%/65% in patients receiving past ibrutinib, 80%/40% in patients receiving past acatinib, and 100%/100% in patients receiving both BTKi. The 12-month survival rate in patients receiving previous ibrutinib, acatinib or both is 81%, 80% or 100%, respectively. Grade 3 CRS and neurological events occurred in 17% and 31% of patients receiving past ibrutinib, 10% and 10% of patients receiving past acatinib and 0% and 67% of patients receiving both BTKi. Although CAR T cell levels after treatment were lower in patients with blast-like morphology or previously treated with acatinib alone, all subgroups defined by MCL morphology or past BTKi gained clinical benefit from anti-CD 19CAR T cell treatment as reflected by a similar trend in clinical outcome.

Example 7

This example provides a renewed analysis of efficacy, safety and pharmacology for all patients in ZUMA-2 with a minimum follow-up of 1 year. Eligible patients with R/R MCL underwent leukapheresis and opsonization chemotherapy followed by a single infusion of anti-CD 19 CAR T cell therapy (2X 10) ⁶ Individual CAR T cells/kg) as described in the previous examples. The primary endpoint was ORR (CR + partial response) assessed by the independent review board according to the Lugano classification. Efficacy data for 60 treated patients with a follow-up of more than or equal to 1 year is reported; safety data was presented for all 68 treated patients.

Median follow-up was 17.5 months (range, 12.3-37.6); ORR 92% (95% CI, 81.6-97.2), CR 67% (95% CI, 53.3-78.3). Of all patients with evaluable efficacy, 48% had a progressive response at the data cutoff. Response duration, Progression Free Survival (PFS), or overall survival not reaching median; estimates at 15 months were 58.6% (95% CI, 42.5-71.7), 59.2% (95% CI, 44.6-71.2), or 76.0% (95% CI, 62.8-85.1), respectively. In patients who achieved CR, median PFS was not achieved (PFS rate of 15 months, 75.1% [ 95% CI, 56.8-86.5 ]); of those that achieved partial responses, the median PFS was 3.1 months (95% CI, 2.3-5.2). Median PFS was 1.1 months (95% CI, 0.9-3.0) in non-responsive patients. The first 28 treated patients had a median follow-up of 32.3 months (range, 30.6-37.6); 39.3% of these patients remained in sustained remission without further therapy.

Common grade 3 or more adverse events were neutropenia (85%), thrombocytopenia (53%), anemia (53%) and infection (34%). Grade 3 or more cytopenia was reported in 60% of patients at 30 days post infusion. Cytokine release syndrome grade 3 or greater occurs in 15% of patients; 59% of patients received toclizumab for CRS management. Grade 3 Neurological Events (NEs) were reported in 31% of patients and 38% of patients received steroids for NE management. All CRS events and most NEs (37/43) subside. There are no level 5 CRS events or NEs and a new level 5 event occurs with additional follow-up. There were 2 cases of grade 2 cytomegalovirus infection, 1 case of each of grade 3 or more hypogammaglobulinemia and grade 3 or more oncolytic syndrome, and no cases of Epstein-Barr virus-associated lymphoproliferation, replication competent retrovirus, Haemophilus lymphohistiocytosis, or anti-CD 19 CAR T cell associated secondary cancer.

Median peak CAR T cell levels and area under the median curve (days 0-28) were 98.9 cells/μ L (range, 0.2-2565.8) and 1394.9 cells/μ L (range, 3.8-27,700) in patients with a progressive response at 12 months, 202.6 cells/μ L (range, 1.6-2589.5) and 2312.3 cells/μ L (range, 19.0-27,200) in patients who relapsed at 12 months, and 0.4 cells/μ L (range, 0.2-95.9) and 5.5 cells/μ L (range, 1.8-1089.1) in non-responders. Of the 57 patients evaluable for efficacy with available data, 84% had B cells at baseline that could be detected by flow cytometry. Of those patients in a progressive response at 12 months, 10 of 26 patients with evaluable samples (38%) had detectable B cells at 3 months, 10 of 18 patients (56%) had detectable B cells at 12 months; genetically labeled CAR T cells were no longer detectable at 12 months in 5 of 28 evaluable patients (17%). The ZUMA-2 study continues to show significant and persistent clinical benefit for anti-CD 19 CAR T cell therapy with manageable safety in patients with R/R MCL. Within this patient population, which lacks curative treatment options, most patients achieve persistent CR and no new safety signals are reported. Although early CAR T cell expansion was higher in patients achieving objective responses, those patients who relapsed later showed elevated CAR T cell levels, which points to an alternative mechanism of failure of the second treatment in MCL.

Example 8

Although approximately 80-85% of patients with ALL achieve durable Complete Remission (CR) after initial treatment, the remaining 15-20% of patients with relapsed or refractory (R/R) ALL have adverse outcomes with 2-year event-free survival in ≦ 40% of patients with relapsed disease. anti-CD 19 CAR T cell therapy prepared as described above showed high complete response rates with a manageable safety profile for adult patients with R/R B cell lymphoma (see previous examples). See, for example, example 5, among others). ZUMA-4(clinical trials. gov identifier: NCT02625480) is a 1/2 phase study in which this anti-CD 19 CAR T cell therapy was evaluated in pediatric and adolescent patients with R/R B cell ALL or NHL. The end-of-phase 1 intermittent analysis of ZUMA-4 shows the feasibility of anti-CD 19 CAR T-cell therapy to treat pediatric patients with R/R ALL with an optimized dosing and Adverse Event (AE) management strategy. The phase 2 ZUMA-4 protocol has been modified to include a broader B-cell ALL enrollment criteria, focusing on patients with early relapses associated with poor outcome, and adding a NHL cohort.

Key B cell ALL registration criteria include age ≤ 21 years, weight ≥ 10kg, and having B cell ALL which is primary refractory, relapsing within 18 months of first diagnosis, relapsing/refractory following ≥ 2-line systemic therapy, or at registration At least 100 days prior to allogeneic stem cell transplantation is relapsed/refractory. B cell ALL is also the B precursor cell ALL R/R after autologous stem cell transplantation for > 100 days prior to enrollment and > 4 weeks after cessation of immunosuppressive drugs. Lansky (age)<16 years old) or Karnofsky (age 16 years old) has a physical performance state of PS 80 or more and a body weight of 6kg or more. Eligible patients include patients with CNS-1 disorders, patients with CNS-2 disorders without clinically significant neurological changes, and patients with CNS-2 disorders>5% of patients with BM blasts or MRD-positive diseases (threshold 10 determined by flow cytometry or PCR) ^-4 ). CNS-1 diseases are defined by the absence of detectable lymphoblasts in the CSF; CNS-2 diseases are counted by detectable disease and leukocytes in CSF<5/. mu.L. CNS-3 disease is defined by WBC ≧ 5/. mu.L in CSF. The disease burden criteria have been modified to also include patients with minimal residual disease-positive disease at the time of enrollment. Patients with Philadelphia chromosome positive ALL qualify if they are intolerant to tyrosine kinase inhibitor therapy or if they are relapsed/refractory after ≧ 2 tyrosine kinase inhibitor therapies. Also included are patients receiving prior bornaemetic mab. Patients with lymphoid blast crisis or clinically significant infection of chronic myelogenous leukemia do not qualify. Patients with burkitt's leukemia/lymphoma also do not qualify.

For B cell NHL, key registration criteria included age <18 years, body weight ≧ 10kg, histologically confirmed diffuse large B-cell lymphoma not otherwise specified (DLBCL NOS), primary mediastinal large B-cell lymphoma, Burkitt's Lymphoma (BL), Burkitt's-like lymphoma, or unclassified B-cell lymphoma between DLBCL and BL with ≧ 1 measurable lesion. For NHL, the disease must have been primary refractory, relapsed/refractory after ≧ 2-line systemic therapy, or relapsed/refractory after autologous or allogeneic stem cell transplantation before registration for ≧ 100 days. The patient must have stopped the immunosuppressant drug for 4 weeks or more. Lansky (age <16 years) or Karnofsky (age > 16 years) physical performance status is PS > 80 and body weight > 6 kg. Also included are patients receiving prior bornaemetic mab. The patient must receive sufficient prior therapy with minimal anti-CD 20 mAb and anthracycline-containing chemotherapy and have one or more measurable lesions. Patients with acute graft versus host disease or chronic graft versus host disease in need of treatment did not qualify within 4 weeks of enrollment. Although patients receiving KTE-X19 were eligible for retreatment in this study, patients with prior CAR T cell therapy or other genetically modified T cell therapy were excluded. Patients with cardiac lymphoma involvement or patients in need of urgent treatment due to tumor mass effects are also excluded. Additional exclusions for ALL and NHL groups included: patients with clinically significant infection; patients with acute or chronic GVHD requiring treatment within 4 weeks of enrollment, patients receiving alemtuzumab (or other anti-CD 52 antibody) within the past 6 months, clofarabine or cladribine within the past 3 months, PEG-asparaginase within the past 3 weeks, or Donor Leukocyte Infusion (DLI) within the past 28 days.

Patients with CNS involvement and certain abnormalities were excluded. Patients with central nervous system 1 disease (no detectable lymphoblasts in cerebrospinal fluid), the presence of lymphoblasts, and central nervous system 2 disease with neurological symptoms and no clinically significant neurological changes (detectable disease, but white blood cell count <5/μ L in cerebrospinal fluid) who have received prior bornauzumab therapy may be included in the ALL and NHL groups. Excluding patients with lymphoblasts and neurological symptoms, central nervous system 3 disease (WBC ≧ 5/μ L in CSF) with or without neurological symptoms with lymphoblasts, patients with any CNS tumor mass and/or meningeal mass determined by imaging, history or presence of any CNS disorder such as cerebral ischemia/hemorrhage, dementia, cerebellar disease, or any autoimmune disease with CNS involvement, reversible posterior encephalopathy syndrome or cerebral edema with structural defects, history of stroke or transient ischemic attacks within the past 12 months, and epileptic attacks requiring active anticonvulsants. Patients receiving prior CD 19-directed therapy other than bornaemezumab were excluded.

The patient received 25mg/m given on days-4, 3 and 2 ² Fludarabine and 900mg/m given on day-2 ² Opsonic chemotherapy of cyclophosphamide followed by 1X 10 on day 0 ⁶ Target dose of individual anti-CD 19 CAR T cells/kg single infusion of anti-CD 19 CAR T cells. For ALL, the primary objective of phase 2 was to assess anti-CD 19 CAR T cell efficacy as assessed by overall CR rate (CR and CR with incomplete blood recovery). For NHL, the primary objective of phase 2 was to assess the efficacy of anti-CD 19 CAR T cell therapy by objective response rate (CR + partial response). Secondary stage 2 objectives for ALL and NHL cohorts included changes in safety and tolerability, additional efficacy endpoints, and patient reported outcome scores.

The CAR T cell therapy used in this study, which is an autologous anti-CD 19 CAR T cell therapy for the treatment of relapsed/refractory mantle cell lymphoma and other relapsed/refractory hematologic malignancies, is described in previous examples, such as example 5 (also known as KTE-X19). PBMCs from the apheresis products were enriched for T cells by CD4+/CD8+ positive selection, which resulted in the elimination of malignant cells. The resulting T cells were activated with anti-CD 3 antibody/anti-CD 28 antibody in the presence of IL-2, retroviral transduced to introduce the anti-CAR gene construct (FMC63-28Z CAR) and expanded to the required dose. The expanded T cells may be frozen for transport and transported back to the patient for infusion. Aliskiren is prepared by different methods, such as, for example, Park j.h. et al, new england journal of medicine (NEngl J Med.), 2018; 378(5) 449-; and Lee d.w. et al, "lancets" (Lancet.) 2015; 385(9967) 517-. KTE-X19 treatment improved CR rates, CRi rates or safety profiles in stage 1 in adult patients with R/R B-ALL. Shah BD et al, J Clin Oncol 2019; 7006 is added to 37 (supplement, abstract).

During DLT assessment in phase 1, the initial dose was 2X 10 ⁶ Individual anti-CD 19 CAR T cells/kg. DLT is defined as persistent>7 days of grade 3 non-hematological AE and grade 4 non-hematological AE (regardless of duration, exception specified by protocol) or persistence>Grade 4 hematological AE for 30 days. 1X 10 in a 68mL volume or 40mL volume was also examined ⁶ Individual CAR-T cell doses. Receive 40mL of 1X 10 ⁶ The patients in the group received the repairImproved AE management. Based on available data, 1 × 10 in 40mL was used in 2-phase ⁶ Individual cells/kg. The results of the phase 1 study showed 94% MRD negativity, and 73% CR + Cri was observed in pediatric and adolescent patients with R/R B-ALL. The results also show a manageable AE profile consistent with known toxicity, and with a lower incidence and severity of NE when administered with optimized dosage formulations and modified safety. Wayne AS et al, pediatric hematology and Cancer (Pediatr Blood Cancer) 2019; 66 (supplement) S24.

In phase 2, patients were screened and leukopheresis performed and then conditioning chemotherapy was initiated on day-4. Bridging therapy can be administered after leukapheresis at the discretion of the investigator and must complete ≧ 7 days or 5 half-lives prior to opsonic chemotherapy. KTE-X19 was infused on day 0. The first disease assessment occurred on day 28. Post-treatment safety and efficacy assessments occurred at weeks 2, 4, 2 and 3. Patients were followed every 3 months up to 18 months and every 6 months between 24 months and 60 months. From year 6 onwards, patients return once a year for up to 15 years. A total of 50 patients with R/R ALL and 16 patients with R/RNHL 1X 10 ⁶ A40 mL preparation of KTE-X19 cells/kg was registered. Patients in phase 2 of the current study also included the NHL cohort and expanded enrollment criteria for R/R B-ALL to include patients with early first relapses associated with poor outcome as well as patients with MRD-positive disease. The main objective was efficacy as assessed by overall CR rates (CR and Cri) for ALL and by ORR (CR + PR) for NHL. Secondary goals include assessment of safety, tolerability, DOR, OS, Relapse Free Survival (RFS)/Progression Free Survival (PFS), and Patient Reported Outcome (PRO). For ALL, additional secondary objectives include the assessment of MRD negative rate and allo-SCT rate. For overall CR rates (ALL group only), incidence and exact bilateral 95% CI will be determined. Using an exact binomial test, 35% response rates will be compared at a unilateral alpha level of 0.025. For MRD negative rates (ALL group only), incidence and exact bilateral 95% CI will be determined. Use essence if statistical test of overall CR rate is significantA definitive binomial test compares the MRD negative rate to the rate of 30% at a unilateral alpha level of 0.025. For DOR and OS, the Kaplan-Meier estimate and the two-sided 95% CI will be determined. For AlloSCT rates (ALL group only), the incidence in the mITT set and the exact two-sided 95% CI will be determined. In terms of safety, the incidence of AEs (including all, severe, fatal, CTCAE version 4.03 ≧ 3), and treatment-related AEs that occurred on the day of infusion or after. For the NHL group, no specific hypothesis will be tested. In the case of the planned sample sizes in this cohort, assuming observed ORR of 63% (10/16 patients), 69% (11/16), 75% (12/16 patients), and 81% (13/16 patients), the lower limits of the estimated ORR 95% accurate CI would be 35%, 41%, 48%, and 54%, respectively.

Example 9

This example reports phase 1 results for ZUMA-3(clinical trials. gov identifier: NCT02614066), a 1/2 study evaluating autologous anti-CD 19 Chimeric Antigen Receptor (CAR) T cell therapy comprising CD3 ζ and CD28 co-stimulatory domains and prepared as described in the previous example (CD4+/CD8+ enrichment/elimination of malignant cells) in adults with relapsed/refractory (R/R) B cell ALL. This protocol for making anti-CD 19 CAR T cells that have been depleted of cancer cells reduces the likelihood of activation and depletion of anti-CD 19 CAR T cells during ex vivo manufacturing. The presence of leukemic blast cells in peripheral blood may limit the number of T cells available for the manufacture of CAR T cell products, potentially leading to manufacturing failures. Sabatino m, et al, Blood (Blood), 2016; 128(22):1227. Has been described in Wang m et al, new england journal of medicine (N Engl J Med.), 2020; 382(14) 1331-1342 describe anti-CD 19 CAR T cell products used in this study for MCL. Which is different from the methods described in Sabatino m. et al, Blood, 2016; 128(22) 1227, Park j.h. et al, new england journal of medicine (N Engl J Med.), 2018; 378(5) 449-; and Lee d.w. et al, "lancets" (Lancet.) 2015; 385(9967):517-528. This anti-CD 19 CAR T cell product has different product characteristics in terms of T cell phenotype than that made by the methods described previously. This anti-CD 19 CAR is also referred to in this example and elsewhere in this application as KTE-X19.

Following fludarabine/cyclophosphamide lymphocyte depletion, the patients were treated with 2, 1 or 0.5 × 10 ⁶ Individual cells/kg received anti-CD 19 CAR T cells. Dose-limiting toxicity (DLT) ratio within 28 days after CAR T cell infusion was the primary endpoint. anti-CD 19 CAR T cells were made for 54 enrolled patients and administered to 45 patients (median age 46 years [ range, 18-77)]). DLT may evaluate that no DLT occurred in the group. Grade 3 Cytokine Release Syndrome (CRS) and Neural Events (NE) occur in 31% and 38% of patients, respectively; to optimize benefit to risk ratio, 1 × 10 ⁶ Cells/kg anti-CD 19 CAR T cells evaluated for revised Adverse Event (AE) management of CRS and NE (early steroids were used for NE and tosublizumab only for CRS). Of the 9 patients treated under revised AE management, 33% had grade 3 CRS and 11% had grade 3 NE, no 4/5 grade NE. The overall complete remission rate was correlated with CAR T cell expansion and was 1 × 10 ⁶ 83% of the patients treated per kg and 69% of all patients. Minimal residual disease was not detectable in all responding patients. At the median follow-up of 22.1 months (range, 7.1-36.1), the median DOR was at 1X 10 ⁶ 17.6 months (range, 5.8-17.6) in individual patients treated per kg and 14.5 months (range, 5.8-18.1) in all patients. anti-CD 19 CAR T cell therapy provides high response rates and tolerable safety in adults with R/R B-ALL. Stage 2 at 1X 10 ⁶ Individual cells/kg were performed with revised AE management.

Patient(s) is/are

Eligible patients are aged > 18 years, have R/R B cellular ALL, and are defined as refractory to first-line therapy (i.e., primary refractory), relapse < 12 months after first remission, relapse or refractory after > 2 previous lines of systemic therapy, or relapse after allogeneic Stem Cell Transplantation (SCT). Patients were required to have > 5% of medulloblasts, 0 or 1 in the eastern U.S. tumor cooperative body status with adequate renal, hepatic and cardiac function.The first six patients requiring enrollment had > 25% blast cells in bone marrow. For patients receiving prior bornaemetic, leukemic blast cells with > 90% expression of CD19 are required. Central Nervous System (CNS) -2 diseases with philadelphia chromosome positive (Ph +) disease, coexisting extramedullary disease, no neurological changes (cerebrospinal fluid [ CSF ] ]The mother cell has<5 white blood cells/mm ³ ) Patients and patients with down syndrome are eligible. CNS-3 diseases independent of neurological changes (CSF blasts with > 5 leukocytes/mm) ³ ) And history of CNS disorders was excluded.

Additional qualifying criteria include: a subject with Philadelphia chromosome (Ph) + disease is eligible if they have a disease that is intolerant to Tyrosine Kinase Inhibitor (TKI) therapy, or if they have relapsed/refractory disease despite being treated with ≧ 2 different TKIs; absolute neutrophil count ≧ 500/μ L, unless according to the investigator's opinion cytopenia is due to underlying leukemia and may be reversible with leukemia therapy; platelet count number > 50,000/μ L unless according to the investigator's opinion cytopenia is due to a potential leukemia and may be reversible with leukemia therapy; the absolute lymphocyte count is more than or equal to 100/mu L; sufficient kidney, liver, lung and heart function are defined [ creatinine clearance (as estimated by Cockcroft Gault) > 60 cc/min; serum alanine aminotransferase/aspartate aminotransferase is less than or equal to 2.5 x the upper normal limit; total bilirubin is less than or equal to 1.5mg/dL, excluding subjects with Gilbert syndrome; left ventricular ejection fraction > 50%, no evidence of pericardial effusion as determined by echocardiography, no new york heart association class III or IV functional classification, and no clinically significant arrhythmia. No clinically significant pleural effusion; baseline oxygen saturation in indoor air > 92% ]; women with fertility potential must have a negative serum or urine pregnancy test; women with fertility potential must have a negative serum or urine pregnancy test.

Additional exclusion criteria included: burkitt's leukemia/lymphoma or chronic myelogenous leukemia lymphoid classified according to the world health organizationDiagnosis of blast crisis; history of malignancy except for non-melanoma skin cancer or carcinoma in situ (e.g., cervix, bladder, breast) unless no disease for 3 years; history of severe hypersensitivity to aminoglycosides or any agent used in this study; central Nervous System (CNS) abnormalities [ the presence of CNS-3 disease is defined as detectable cerebrospinal fluid cells in a cerebrospinal fluid (CSF) sample with ≥ 5 White Blood Cells (WBC)/mm ³ With or without neural changes; and the presence of CNS-2 disease is defined as a detectable cerebrospinal blast in a CSF sample having<5 WBC/mm ³ There are neural changes. Note that: subjects with CNS-1 (undetectable leukemia in CSF) and with CNS-2 without clinically significant neural changes qualify for study participation; history or presence of any CNS disorder, such as seizure disorder, cerebral vascular ischemia/hemorrhage, dementia, cerebellar disease, any autoimmune disease with CNS involvement, reversible back-brain disease syndrome or cerebral edema ](ii) a History of severe hypersensitivity to aminoglycosides or any agent used in this study; a history of concomitant genetic syndromes associated with bone marrow failure; a history of clinically significant heart disease within 12 months of enrollment; a history of symptomatic deep vein thrombosis or pulmonary embolism within 6 months of enrollment; primary immunodeficiency; infection with HIV, hepatitis b or hepatitis c virus is known. Allowing a history of hepatitis b or hepatitis c if viral load is not detectable according to quantitative polymerase chain reaction and/or nucleic acid testing; if responsive to active therapy and after consultation with a Kite medical monitor, allows for simple Urinary Tract Infection (UTI) and uncomplicated bacterial pharyngitis; grade II-IV acute Graft Versus Host Disease (GVHD) by International Bone Marrow Transplant Registry index (International Bone Marrow Transplant Registry index) according to the Gluckberg criteria or severity B-D; acute or chronic GVHD requiring systemic treatment within 4 weeks prior to enrollment; previous drugs [ systemic rescue therapy (including chemotherapy, TKI for Ph + disease and bornaemetic mab) ≦ 1 bornaemetic mab); term standard history of common adverse events (grade 4 neurological events or grade 4 cytokine release in the case of targeted therapy with CD19 Syndrome of relaxation); (ii) alemtuzumab for ≤ 6 months prior to enrollment, clofarabine or cladribine for ≤ 3 months prior to enrollment, and PEG-asparaginase for ≤ 3 months prior to enrollment; donor lymphocyte infusions were performed no more than 4 weeks prior to enrollment; treatment with any drug against GVHD and any immunosuppressive antibodies 4 weeks prior to enrollment; at least 3 half-lives must recur from any prior systemic suppressive/stimulatory immune checkpoint molecular therapy prior to enrollment; corticosteroid treatment at pharmacological doses must be avoided 1 week prior to enrollment: (>Prednisone at 5 mg/day or an equivalent dose of another corticosteroid) and other immunosuppressive drugs; any indwelling wires or tubes are present. Ommaya reservoir and dedicated central venous access catheter are allowed; live vaccine no more than 4 weeks prior to enrollment; pregnant or lactating women with fertility potential due to the potentially dangerous effects of preparative chemotherapy on the fetus or infant; two sex sexed subjects unwilling to practice fertility control from the time of consent to 6 months after completion of anti-CD 19 CAR-T cell therapy; subjects are unlikely to complete all protocol-required study visits or procedures at the discretion of the investigator, or to comply with study requirements for participation [ resulting in terminal organ damage or autoimmune disease history requiring systemic immunosuppression or systemic disease modulators over the past 2 years ]。

Study design and treatment

The phase 1 objective was to assess the safety of anti-CD 19 CAR T cell therapy and determine the optimal phase 2 dose based on the incidence of dose-limiting toxicity (DLT) and the overall safety profile. DLT was defined as anti-CD 19 CAR T cell-related Adverse Events (AEs) that occurred within the first 28 days after anti-CD 19 CAR T cell infusion, including grade 3 non-blood AEs that lasted >7 days, grade 4 non-blood AEs regardless of duration (except for pre-specified expected events (e.g., tumor lysis syndrome)), and grade 4 blood AEs that lasted >30 days (except for lymphopenia) (table 15).

Table 15: dose limiting toxicity

CRS, cytokine release syndrome; DLT, dose-limiting toxicity; TLS, tumor lysis syndrome

The initial patient is at 2X 10 ⁶ Initial doses of individual CAR T cells/kg were enrolled (figure 3). Based on the overall safety profile, subsequent patients received 2 × 10 ⁶ 、1×10 ⁶ Or 0.5X 10 ⁶ Individual CAR T cells/kg. At 0.5X 10 ⁶ Individual CAR T cells/kg. The search for receiving lower doses of 0.5X 10 ⁶ Two formulations of individual CAR T cells/kg patients, one with a total volume of 40mL and the other with a volume of 68 mL. The 40mL formulation was intended to maintain cell density and cell viability during the freeze/thaw process.

To mitigate the risk of Cytokine Release Syndrome (CRS) and Neurological Events (NE), the AE management guidelines were revised to limit toslizumab to treatment of CRS (rather than isolated neurotoxicity) and corticosteroid treatment was initiated at the onset of grade 2 rather than grade 3 NE (table 16).

Table 16: original and revised guidelines for neurotoxicity management

In use at 1X 10 ⁶ Revised AE management guidelines were implemented in an additional patient group of CAR T cells/kg treatment. Safety examinationThe team consults (SRT) in a progressive manner for review of safety and efficacy data and gives recommendations on further phase 1 enrollment and suggested phase 2 doses (RP2D) on milestones defined in the agreement and SRT franchise.

Patients underwent leukapheresis at enrollment to obtain 5-10X 10 ⁹ Individual monocytes were targeted for anti-CD 19 CAR T cell manufacture. Predefined bridging chemotherapy (table 17) was suggested after leukapheresis, particularly for patients with high disease burden at baseline (in bone marrow)>25% leukemic blast cells or > 1,000 blast cells/mm in the peripheral circulation by local examination ³ )。

Table 17: bridging chemotherapy

BID, twice daily; CVAD, cyclophosphamide, vincristine, doxorubicin and dexamethasone; DOMP, dexamethasone, 6-mercaptopurine, methotrexate, and vincristine; FLAG, fludarabine, high dose cytarabine and G-CSF; G-CSF, granulocyte colony stimulating factor; IDA, idarubicin; IV, intravenous; MP, 6-mercaptopurine; PO, oral; SC, subcutaneous; VAD, vincristine, doxorubicin and dexamethasone.

After ≧ 7 days or 5 half-lives (if shorter) of clearance from bridging chemotherapy, the patient received 25mg/m per day of Intravenous (IV) administration on days-4, -3, and-2 ² Fludarabine and 900mg/m daily IV administration on day-2 ² Lymphodepletion regimen of cyclophosphamide. A single infusion of anti-CD 19 CAR T cells was administered on day 0.

Results and evaluation

The primary phase 1 endpoint was the incidence of DLT in DLT evaluable patients. Secondary endpoints included safety, overall remission rate assessed by the investigator (CR + CR [ CRi ] with incomplete hematologic recovery), duration of remission (DOR), relapse free survival, OS, and the rate of undetectable Minimal Residual Disease (MRD) in the bone marrow. CAR T cell and cytokine levels in the blood are exploratory endpoints. AEs including symptoms of CRS and NE were ranked according to the common AE term standard version 4.03. CRS was determined according to Lee et al, Blood, 2014; 124(2):188-195. For patients with extranodal disease, responses were assessed according to revised extramedullary and CNS disease response criteria in the international working group malignant lymphoma criteria. Cheson BD et al, J Clin Oncol, 2007; 25(5):579-586. Undetectable MRD was assessed centrally using flow cytometry (NeoGenomics, milsburg, florida, usa), which was defined as <1 leukemia cell per 10,000 viable cells. Borowitz MJ et al, Blood (Blood), 2015; 126(8), 964-971; bruggemann m, et al, Blood progress (Blood Adv.) 2017; 2456-2466; and Gupta s. et al, Leukemia (leukamia) 2018; 32(6):1370-1379.

Hospitalization was required to be > 7 days post infusion. Patients were evaluated on days 14 and 28 and months 2 and 3 by physical examination, vital sign measurements and neurological and laboratory assessments. Bone marrow assessment and response assessment was performed on days 7-14 (optional) and 28, as well as months 2 and 3. For patients with SCT after T-cell infusion of anti-CD 19 CAR, bone marrow assessment was not required during the first 100 days post SCT. For patients with baseline CNS-2 disease, CSF needs to be collected and analyzed to confirm CR. Survival and disease status of patients assessed after completion of month 3 treatment were followed every 3 months up to month 18 and every 6 months between months 24-60 and for up to 15 years each year. Patients achieving CR may receive a second infusion of anti-CD 19 CAR T cells if, following >3 months remission, provided CD19 expression has been retained and neutralizing antibodies to the CAR is not suspect.

Biomarker analyses were performed on blood and serum samples to assess predictive pharmacokinetic and pharmacodynamic markers of anti-CD 19 CAR T cells. As previously described, droplet digital polymerase chain reaction was used to measure the presence, amplification and persistence of transduced CD19 CAR + T cells in blood. Locke FL et al, molecular therapeutics (Mol Ther.), 2017; 25(1):285-295. Serum is assessed for markers of cytokines, chemokines, immune effector molecules and macrophage activation syndrome using previously reported methods. Locke FL et al, molecular therapeutics (Mol Ther.), 2017; 25(1):285-295.

Statistical analysis

The DLT evaluable group includes 2 × 10 ⁶ Dose level treatment of the first 3 patients. Safety and efficacy assays included all patients treated with any dose of anti-CD 19CAR T cells. A Kaplan-Meier estimate of time to event endpoint and a two-sided 95% confidence interval were generated. DOR is defined as the time from CR to death with or without recorded relapse. Patients undergoing allogeneic SCT while in remission were examined for DOR on the day of transplantation. OS was defined as the time from infusion of anti-CD 19CAR T cells to the date of death for any reason. Data are presented to 2019, 4 months and 1 day. All statistical analyses were performed in SAS (version 9.4).

Results

Patient's health

Between 2016 at 9 and 2018 at 7 at 12, 54 patients were enrolled and leukopheresis was performed during phase 1 (fig. 4). All 54 patients successfully made anti-CD 19CAR T cell products; 1 patient required 2 leukapheresis procedures and 1 patient required 3 product manufacturing procedures. The time from leukapheresis to delivery of anti-CD 19CAR T cells to the study site was 15 days. Five patients were discontinued prior to lymphocyte depletion due to AE (n-3; fig. 4), withdrawal consent (n-1), or non-eligibility after leukapheresis (n-1). Four additional patients were discontinued after lymphocyte depletion. Three patients did not receive anti-CD 19CAR T cells due to grade 4 sepsis (n-1), initiation of new therapy (n-1), and death due to grade 5 sepsis (n-1). One patient had deep vein thrombosis (exclusion mark) Quasi) but discontinued prior to infusion, but received anti-CD 19CAR T cells under isosexual medication. Forty-five of 54 patients (83%) received anti-CD 19CAR T cells at these dose levels: 2X 10 ⁶ (n＝6)、1×10 ⁶ (n-23) or 0.5X 10 ⁶ CAR T cells/kg (n ═ 16). At 1X 10 ⁶ Nine of 23 patients in each CAR T cell/kg cohort were treated under revised AE management guidelines, requiring earlier NE management with steroids and retaining only toslizumab for treatment of CRS. Forty-four patients received their target dose of anti-CD 19CAR T cells; 1 patient enrolled to receive 1X 10 ⁶ Individual anti-CD 19CAR T cells/kg and revised AE management 0.5X 10 ⁶ Individual cells/kg, but at 1X 10 ⁶ In the analysis of dose levels.

The median age for all treated patients was 46 years (range, 18-77) and 67% of patients received ≧ 3 previous lines of therapy (Table 18). Prior to enrollment, 16 patients (40%) were primary refractory, 13 patients (29%) relapsed after SCT, and 21 patients (47%) received prior bornaemetic. Bornaemezumab was the last therapy used prior to study entry in 8 patients (18%), of which only 1 patient achieved a response (CR) to bornaemezumab.

Table 18: patient baseline characteristics

BM, bone marrow; CNS, central nervous system; ECOG, eastern american tumor cooperative group; SCT, Stem cell transplantation

Safety feature

No DLT was observed among the DLT evaluable set (n ═ 3). Ninety-eight percent of patients experienced grade 3 AE (Table 19). The most common of any grade AE are fever (89%), hypotension (69%), diarrhea (42%) and chills (42%). Common grade > 3 AEs (> 20% of patients) were fever (42%), hypotension (40%), decreased platelet count (33%), anemia (31%), hypophosphatemia (31%), hypoxia (24%), encephalopathy (22%), febrile neutropenia (22%) and decreased neutrophil count (22%). Any grade of severe AE that occurs in 84% of patients.

Table 19: adverse events

Table comprising any grade of adverse events occurring in all patients ≧ 25%

CRS was reported in 42 patients (93%); 14 patients (31%) experienced grade 3 CRS ≧ 3 (Table 19). Common CRS ≧ 3 are fever (45%), hypotension (36%), and hypoxia (17%). Boosters were used to treat CRS in 12 patients (27%). Median time to CRS onset after infusion was 2 days (range, 1-12); median durations for any grade and ≧ 3 grade CRS were 9 days and 4.5 days, respectively. CRS-related events resolved in all patients, but 2 patients experienced a grade 5 anti-CD 19 CAR T cell-related AE. By 2X 10 ⁶ Patients treated with gcar T cells/kg had multiple organ failure secondary to CRS (day 6). By 0.5X 10 ⁶ One patient treated with ge cells/kg developed a cerebrovascular accident (stroke) in the background of CRS and NE (day 7). No other anti-CD 19 CAR T cell-associated grade 5 AEs were reported.

NE was reported in 35 patients (78%); grade ≧ 3 events occurred in 17 patients (38%; (Table 19). Grade 3 NE occurring in > 5% of patients are encephalopathy (22%), aphasia (16%) and confusion status (9%). There were no cases of cerebral edema, nor grade 5 NE. Median time to NE onset after infusion was 6 days (range, 1-31); the median duration for any grade and grade 3 NEs were 12 days and 9 days, respectively. NE regression in 31 of 35 patients (89%); before resolution of the neurological event, 1 patient died from progressive disease and 3 patients died from consideration of AE unrelated to anti-CD 19 CAR T cells (sepsis [ n ═ 1], cerebrovascular accident [ n ═ 1], herpes simplex viremia [ n ═ 1 ]).

Fifty-three percent of all patients received toslizumab, and 36% of patients also received steroids for CRS management; for NE, 31% and 44% of patients received toslizumab and steroids, respectively. Improved overall safety was observed for 9 patients treated under the revised AE management guidelines relative to 14 patients treated at the same dose under the original guideline (table 20). Under the original guide at 1 × 10 ⁶ Four of 14 patients treated with CAR T cells/kg had grade 3 or grade 4 CRS. With revised AE management at 1 × 10 ⁶ 3 of 9 patients treated with CAR T cells/kg had grade 3 CRS, and no grade 4 CRS was reported. These patients also had 1X 10 acceptance than under the original AE guidelines ⁶ Patients with CAR T cells/kg had shorter median CRS durations at grade 3 or greater (4 days and 7 days), and longer times to onset of grade 3 symptoms (6 days and 4.5 days, respectively). Notably, 1 × 10 under the management of original guide ⁶ 9 of 14 patients in the CAR T cell/kg dose cohort experienced 3/4 grade NE, in contrast to one 3-grade event and no 4-grade event in patients receiving the same dose under revised regulatory guidelines (table 20). Based on an examination of all available safety and efficacy data, the benefit/risk ratio is considered to be at 1 × 10 ⁶ The dose of individual CAR T cells/kg was most favorable, resulting in the dose being RP 2D. All phase 2 patients were treated under revised AE management guidelines.

Table 20: cytokine release syndrome and neural events included with revised AE management guidelines

AE, adverse event; CRS, cytokine release syndrome; NE, neural event

Twenty-six treated patients (58%) died for reasons including: disease progression in 19 patients (42%) and AE in 7 patients (16%), including 2 deaths related to the above treatment. The remaining 5 AE-related deaths occurred at the median day 63 after infusion of anti-CD 19 CAR T cells (range, 48-579) and were considered unrelated to anti-CD 19 CAR T cells. They include sepsis (n ═ 2), cerebrovascular accident (n ═ 1), herpes simplex viremia (n ═ 1), and bacteremia (n ═ 1).

Efficacy of

All 45 treated patients were eligible for efficacy analysis. At a median follow-up visit (range, 7.1-36.1) of 22.1 months, the Overall Remission Rate (ORR) was 69%, with 51% of patients achieving CR and 18% of patients achieving CRi (table 21). In use at 1X 10 ⁶ Of 23 patients treated with CAR T cells/kg, ORR was 83%, with 14 patients achieving CR (61%) and 5 patients (22%) achieving CRi. Six of 9 patients receiving revised AE management achieved CR/CRi (4 CR, 2 CRi). Median time to CR/CRi was 30 days (range, 26-192) at dose level, including 1 patient with anucleated myelodysplasia/myelodysplasia at day 28, which did not meet CR criteria until month 6. ORR is generally consistent among key covariates, including refractory patients (56%), previous transplants (77%), previous bornauzumab (57%) or obinutuzumab (50%), and Ph + disease patients (100%) (fig. 5). Undetectable bone marrow MRD was achieved on day 28 in 100% of responders, including 31 patients with CR/CRi, 1 patient with partial response, and 1 patient with BFBM. Residual disease assessment was not available in 1 patient with BFBM. Two of 6 patients undergoing optional bone marrow assessment on days 7-14 had undetectable MRD; 5 patients with available data on day 30 One has undetectable MRD.

Table 21: against CD19 CAR T cell response

Patients had extramedullary disease at response assessment.

Patients died on day 6 due to multiple organ failure secondary to CRS.

Patients died on day 7 due to cerebrovascular accident (stroke) in the context of CRS and neurological events.

In use at 1X 10 ⁶ Of the CAR T cells/kg treated patients, the median DOR was 14.5 months (95% CI, 5.8-18.1; fig. 6A) and 17.6 months (95% CI, 5.8-17.6) for 31 patients achieving CR/CRi. The median DOR was similar regardless of the examination of SCT after T-cells against CD19 CAR (fig. 6B). At the data cutoff, 8 patients (26%) had progressive CR, including receiving 0.5 × 10 ⁶ 2 patients per CAR T cell/kg and receiving 1X 10 ⁶ 6 patients per kg of CAR T cells, with a median follow-up of 6.3 months (range, 5.9-18.2). Six patients (1X 10 for use) ⁶ 2 patients CR and 1 patient partial response per CAR T cell/kg treatment; by 0.5X 10 ⁶ CR) of 3 patients treated per kg) underwent SCT at the median (range, 1.7-4.3) of 2.7 months post infusion. At the time of this analysis, 3 of these patients maintained CR (1X 10 for 2 patients) ⁶ One CAR T cell/kg treatment and 0.5X 10 for 1 patient ⁶ Individual cells/kg treatment). Median duration of relapse free survival was 7.3 months (95% CI, 2.7-18.7) among all dose levels, while receiving 1X 10 ⁶ 7.7 months (95% CI, 3.2-18.7) in one patient per kg of CAR T cells (FIG. 6C). At all dosage levelsHas a median OS of 12.1 months (95% CI, 6.1-19.1) across all dose levels and 1 × 10 ⁶ At 16.1 months (95% CI, 10.2-not-estimable) for each CAR T cell/kg (fig. 6D).

At the end of the data, 1 patient (2%) withdrawn consent, 1 patient (2%) lost follow-up, and 17 patients (38%) survived, including with 1 × 10 ⁶ 11 of 23 patients treated per kg of cells (50%). Four patients received a second infusion of anti-CD 19 CAR T cells; one patient was CR at 15 months post-re-dosing, 2 patients relapsed by month 3 assessment, and 1 patient withdrawn consent prior to first response assessment.

Clinical pharmacology

CAR T cell levels measured by CAR gene copies/μ g DNA in blood for most patients peaked 7-14 days post anti-CD 19 CAR T cell infusion and remained detectable in 2 of 12 evaluable patients at 12 months, both CR (figure 7A; table 22).

Table 22: CAR gene copies in blood over time

AE, adverse event; CAR, chimeric antigen receptor

CAR T cells were undetectable in 5 patients for which data was available at relapse. Median peak CAR T cell levels 1 × 10 ⁶ Individual CAR T cells/kg were highest and similar between patients receiving both original and revised AE management (fig. 7B; fig. 8). Patients who achieved CR/CRi had a greater median peak amplification than non-responders, as did patients with undetectable MRD versus detectable MRD (FIGS. 7C-D; FIGS. 84B-C). Compared with the patients with grade 2 NEHigher median peak amplifications were also observed in patients with grade 3 NE (FIGS. 7E-F; FIGS. 8D-E). Of the 13 patients who had relapsed, 7 patients had detectable CD19 positive cells at the time of relapse, 3 patients had no detectable CD19 positive cells, and 3 patients had no data available.

By day 7, peak levels of key cytokines, chemokines and proinflammatory markers occurred, 1X 10 ⁶ 2X 10 administration of one CAR T cell/kg ⁶ These tended to be higher in individual CAR T cell/kg patients (IL-15, CRP, SAA, CXCL10, IFN γ) or lower in patients managed with revised AEs than in patients managed with original AEs (IL-6, ferritin, IL-1RA, IFN γ, IL-8, CXCL10, MCP-1) (figure 9; figure 10). Although the peak IL-15 serum levels were unexpectedly lower in patients with CRS grade 3 or greater, the median peak levels of several proinflammatory markers tended to be higher in patients with CRS grade 3 or greater and in patients with NE grade 3 or greater (IFN γ, IL-8, GM-CSF, IL-1RA, CXCL10, MCP-1, granzyme B; FIG. 11).

Four patients tested positive during the screening assay for anti-CAR antibodies, but all were negative in the confirmation assay for leukopheresis. The characteristics of the CAR T cell products produced were as expected and previously reported (table 23).

Table 23: product characteristics

Co-culture experiments were performed using Toledo cells mixed with anti-CD 19 CAR T cell product at a 1:1 ratio. IFN γ was measured in cell culture media 24 hours after incubation using a qualified ELISA.

AE, adverse event; IFN gamma, interferon gamma

ZUMA-3 is an adult R/R B-ALL at stage 1 that will be completedFirst multicenter study to evaluate CAR T cell therapy. In phase 1 part, no protocol-defined DLT was observed with anti-CD 19 CAR T cells, and the reported AEs were consistent with previous studies of anti-CD 19 CAR T cell therapy. Neelapu SS et al, New England journal of medicine (N Engl J Med.), 2017; 377(26) 2531 and 2544; maude SL et al, New England journal of medicine (N Engl JMed.), 2018; 378(5):439-448. Paired 1 x 10 with revised AE management guidelines ⁶ The individual CAR T cell/kg doses had the most favorable risk/benefit ratio without affecting activity. Despite the high disease load of patients and extensive pre-treatment, high remission rates and undetectable bone marrow MRD were achieved, particularly at 1 x 10 ⁶ Those patients treated at dosage levels; ORR was 83%, including 61% CR and 22% CRi, all of which had undetectable MRD. Based on these results, showing that anti-CD 19 CAR T cells are safe and have promising efficacy, 1 × 10 was chosen ⁶ Individual CAR T cell/kg doses were used for further evaluation at stage 2 of ZUMA-3.

Treatment of adult R/R B-ALL with anti-CD 19 CAR T cells has proven difficult due to the highly proliferative nature of this disease and intolerance of treatment-related AEs. Past CAR T cell tests in this population ended early due to lethal NE (including 5 cerebral edemas). DeAngelo DJ, Ghobadi a, Park JH et al, Journal of Cancer ImmunoTherapy (Journal for ImmunoTherapy of Cancer.) 2017; 5 (supplement 2) and P217. Under the original AE management guidelines in ZUMA-3, 2 patients died from grade 5 AEs considered associated with anti-CD 19 CAR T cells in the background of CRS and NE secondary to CRS or outside the DLT assessment period. In addition to evaluating multiple doses to identify the dose with the most manageable toxicity, the dose is 1 × 10 ⁶ The revised AE management guidelines requiring earlier steroid intervention for neurotoxicity and using toslizumab only for CRS were implemented in 9 patients enrolled at CAR T cell/kg dose levels. This resulted in a shorter duration of CRS events and a lower incidence, severity and duration of NEs than 14 patients treated at the same dose in the original south of the fingers.

Median follow-up at 22.1 monthsAt the same time, the response was progressive in 26% of patients, who mostly received 1 × 10 ⁶ Individual CAR T cells/kg (32% progressive CR/CRi). The response tends to occur early after treatment. Most occur within the first month, but 1 patient with extramedullary disease achieves CR at month 6. High response rates were observed in all pre-designated subgroups, including 100% CR rates in patients with Ph + disease. The response (CR/CRi) correlated with higher expansion of CAR T cells measured within 2 weeks after treatment. Similarly, in a single-center phase 1 study using anti-CD 19 CAR T cell therapy that also contained CD3 ζ and CD28 costimulatory domains (Park JH et al, N Engl J Med., 2018; 378(5): 449) 459), the overall CR rate was 83%, but after bridge therapy, only half of the patients had > 5% parent cells in the bone marrow, 28% had MRD, and 11% had undetectable MRD. However, these experimental results largely paralleled those of the present study, further supporting the potential utility of anti-CD 19 CAR T-cell therapy using CD3 ζ and CD28 costimulatory domains in adult R/R B-ALL.

Tisangegeleucel (an anti-CD 19 CAR T cell therapy containing a CD3 ζ T cell activation domain and a 4-1BB co-stimulatory domain) was approved for the treatment of R/R B-ALL in children and young adults (25 years). Maude SL et al, New England journal of medicine (N Engl J Med.), 2018; 378(5) 439-; KYMRIAH (tisagenlecucel) [ Package Specification ], Nowa corporation (Novartis), eastern Hannover, N.J., USA; 2018. However, the dosing regimen of tisagenlecucel in younger patients resulted in substantial toxicity and CRS-related death in adults with R/R B-ALL. Frey NV. et al, J.Clin Oncol., 2020; 38(5):415-422. In a single-center study in adult R/R B-ALL among the two clinical trials, dose by fraction resulted in manageable CRS and 90% CR rates. Frey NV et al, J Clin Oncol, 2020; 38(5):415-422. Similar to the ZUMA-3 observations, optimized dosing and toxicity management strategies may enable patients to be vulnerable to life-threatening treatment-related toxicity to benefit from CAR T cell therapy.

Despite the differences in trial design, patient population and OS approach, 1X 10 was used in this study ⁶ The median OS for individual CAR T cells/kg was 16.1 months, whereas the median OS previously reported with bornaemezumab also targeting CD19 was 6.1-7.7 months in adult R/R B-ALL. Topp MS et al, "Lancet Oncol.", 2015; 57-66 parts in (1); kantarjian h, et al, new england journal of medicine (N Engl J Med), 2017; 376(9):836-847. Of the 10 patients whose blast expression of CD19 could be assessed at relapse, 3 patients showed a lack of CD19 expression, reminiscent of additional reports of target loss due to selection of exon splice variants and mutations. Sotillo E et al, Cancer discovery 2015; 5(12):1282-1295. In this study, only 1 of 8 patients (13%) who used bornaemezumab as the last prior therapy responded to bornaemezumab. This may suggest that unmanipulated T cells are not immunologically active in some patients with R/R ALL, potentially limiting the utility of bispecific T cell adaptor therapy. Of the 21 patients with the previous bornaemezumab in any line, 12 patients (57%) achieved CR/CRi following anti-CD 19 CAR T cell therapy. As previously reported (Shah BD. et al, journal of clinical tumors (J Clin Oncol.), 2018; 36 (suppl.): abstract 7006), the response against CD19 CAR T cells was similar regardless of prior bornaemetic exposure in patients with persistent CD19 positivity. Furthermore, 6 patients who achieved CR underwent SCT and were examined at a later time than SCT; the 3 patients remained in remission.

Adults with R/R B-ALL achieved high CR rates and undetectable bone marrow MRD with tolerable safety after treatment with anti-CD 19 CAR T cells. The successful manufacture and relatively fast turnaround time for all enrolled patients supports the feasibility of providing such cell therapy treatment to patients with rapidly progressing diseases that require rapid treatment. It is possible to shift the study from the phase 1 study to the state by carefully evaluating a range of doses and employing safety strategies including the use of tollizumab or a steroid and the conditions under which tollizumab or a steroid should be administered to manage AEPhase 2 study. There were no fatal cerebral edema cases in stage 1 (which were limited to previous studies in this population). Stage 2 of ZUMA-3 at 1X 10 ⁶ Individual CAR T cell/kg doses were performed with revised AE management guidelines.

Example 10

This example describes the results of CD19 Δ Tyr260 in CD19 in B-ALL associated with resistance to CAR T cell therapy treatment. B-ALL patients received 1X 10 following failure of several therapies including bornaemezumab prior to KTE-X19 ⁶ Target dose of individual CAR T cells/kg. The patient does not respond clinically; there were no detectable CAR T and CD19 expressing lymphocytes on day 28. Peripheral Blood Mononuclear Cells (PBMC) were collected at various time points in patients with B-ALL before and after KTE-X19 infusion. Multicolor flow cytometry was used to examine CD19 (clone FMC63, HIB19, SJ25C1) surface expression on patient PBMC and Jurkat cell lines engineered to CD19 Wild Type (WT) or to express CD19 Δ Tyr 260. The presence of genetic variants was assessed using enhanced whole genome and RNA sequencing (TruSeq Stranded Total RNA). Western blots were used to assess the location of cellular protein expression with and without deglycosylation enzyme.

Although local pathology concluded that pre-infused B lymphoblasts were consistently CD19 ^dim Additional analysis of the same samples with FMC63 (a single-chain variable fragment of KTE-X19) revealed that CD19 could not be detected in pre-infused B lymphoblasts. The results of RNA sequencing showed an in-frame deletion at Tyr260 within the intracellular domain of CD19 in circulating leukemic blast cells (CD19 Δ Tyr 260). Additional analysis using flow cytometry showed that no CD19 expression was detected on Jurkat CD19 Δ Tyr260 cells, but was present on Jurkat CD19-WT cells, indicating the lack of visualization of cells carrying this point mutation and their resistance to CAR T cell therapy. Longitudinal RNA and DNA sequencing analysis indicated that mutations had occurred prior to infusion of CAR-T therapy. The fractionated cell lysate showed WT CD19 with high and lower molecular weight bands in the cell membrane, and CD19 Δ Tyr260 with a single low molecular weight band expressed on the surface. Under deglycosylation conditions, in WT CD19 and CDOnly 1 band was present in each of the 19 Δ Tyr260 cell fractions. Without being bound by any scientific theory or hypothesis, it is likely that the CD19 Δ Tyr260 mutation may result in a lack of suitable or functional CD19 glycosylation and/or inhibition detection. Mutations in B-ALL malignant cells may have potential implications for other anti-CD 19 CAR or non-CD 19 CAR cell therapies.

Example 11

MCL patients who progress after BTKi therapy often have poor prognosis with only 5.8 months overall survival with rescue therapy. Martin P et al, Blood (Blood), 2016; 127:1559-1563. KTE-X19 was evaluated in a phase 2 ZUMA-2 study in MCL patients who were relapsed/refractory to 1 to 5 previous therapies, including BTKi. Wang M et al, New England journal of medicine (N Engl J Med.), 2020; 382:1331-1342. At a median follow-up of 12.3 months, ORR was 93% (67% complete response) in the primary efficacy assay of ZUMA-2 (N ═ 60). Aggressive disease variants (including blast-like or polymorphic MCL) are often associated with poor clinical outcome, but ORR is comparable in patients with various histologies in ZUMA-2. Wang M et al, New England journal of medicine (N Engl J Med.), 2020; 382: 1331-; jain P and Wang M, journal of hematology USA (Am J Hematol.), 2019; 94:710-725. In this study, pharmacological profiles and clinical outcomes in patient subgroups defined by MCL morphology and prior BTKi exposure in ZUMA-2 were compared, with characterization of product attributes and other pretreatment factors. Patients underwent leukopheresis and chemotherapy, then were treated on day 0 with a single infusion at 2X 10 ⁶ Target dose of individual CAR T cells/kg a single infusion of CD19 CAR-T cells. Some patients receive a bridging therapy with dexamethasone (20 mg to 40mg per PO or IV administration per day or equivalent for 1 to 4 days), ibrutinib (560 mg per PO administration per day), or acatinib (100 mg per PO administration twice per day), administered after leukoapheresis and completed < 5 days before initiating opsonic chemotherapy; PET-CT is required after bridging. The primary endpoint was the objective response rate (ORR [ Complete Response (CR) + partial response]). Secondary endpoints are duration of response (DOR), Progression Free Survival (PFS), OS, adverse eventsFrequency of elements (AE), CAR T cell levels in blood and cytokine levels in serum. Efficacy and safety assays included all patients receiving CD19 CAR-T cell therapy. The first tumor assessment was completed on day 28. Bone marrow biopsy is done at screening time and if positive, incomplete or uncertain, biopsy is required to confirm CR.

In 60 patients treated for MCL with KTE-X19 with a median follow-up of 12.3 months in ZUMA-2, there was an ORR of 93%, a CR rate of 67%, and 57% of all patients and 78% of CR patients had a progressive response. CRS and neurological events were mostly reversible (N ═ 68 patients treated). About 15% of patients had grade 3 CRS ≧ 31% of patients had grade 3 neurological events ≧ 2 cases of grade 5 AE (1 case KTE-X19 related). Patient subgroups were defined by morphological characteristics (classical, blast-like or polymorphic MCL) and prior exposure to ibrutinib only, alcatinib only, or both ibrutinib and alcatinib. Table 24: the baseline characteristics are generally comparable in these groups. There was a tendency for higher pre-treatment tumor burden in patients previously treated with ibrutinib. Product attributes, CAR T cell levels and cytokine levels in serum were analyzed in blood using the methods described previously. Locke FL et al, molecular therapeutics (Mol Ther.), 2017; 25:285-295. Product T cell attributes are generally comparable between MCL morphological subgroups. There is a tendency for increased percentages of product coculture IFN- γ and CCR7+ cells in products from patients with polymorphic morphology. Table 25: the product T cell attributes are also generally comparable between the previous BTKi subgroups. There was a trend towards increased product co-culture IFN- γ in patients previously treated with ibrutinib. Table 26:

Table 24: patient baseline characteristics。

^a As measured by the sum of the product sizes of all target lesions at baseline. For subjects with bridging therapy, measurements after bridging therapy were used as baseline.

Table 25: cell characterization and MCL morphology。

^a Based on the available data: classical, n is 38; like mother cell, n ═ 16

Table 26: cell characterization and BTKi subgroups。

^a Based on the available data: ibrutinib, n ═ 49

High response rates were achieved between MCL morphologies and the past BTKi subgroup. Table 27: clinical benefit of KTE-X19 treatment was observed in all subgroups defined by MCL morphology or past BTKi. A trend of higher progressive response rate at 6 months was observed in patients previously treated with ibrutinib. Table 27: CRS and neurological events were generally comparable between MCL morphology and the past BTKi subgroup. Table 28: a trend of increasing grade 3 neural event rates was observed in patients with non-blast-like morphology or previously treated with ibrutinib. Table 28:

table 27: response rate

¹ Wang M et al, New England medical journal (N Engl J)Med.), 2020; 382:1331-1342. CR, complete response; MCL, mantle cell lymphoma; NE, not evaluable; ORR, objective response rate; OS, Total Life cycle

Table 28: adverse events

Comparisons between subgroups were performed using the Kruskal-Wallis test; dunn post test was used to make the comparisons between groups. KTE-X19 (2X 10) for report ⁶ Individual cells/kg) pharmacological profile, product attributes and safety data for all 68 patients treated). The pharmacological and pharmacodynamic profile of KTE-X19 among the MCL morphological subgroups showed increased CAR T cell expansion and selection of pro-inflammatory cytokines in patients with classical morphology (figures 12 and 13) compared to patients with blast-like morphology or in patients previously treated with ibrutinib (figures 14 and 15) compared to acatinib alone. Pre-treatment patient and product characteristics are often comparable between MCL morphology and subgroups with different previous therapies. Patients with blast-like morphology showed reduced CAR T cell expansion, circulating bone marrow-associated cytokines and chemokines, and rates of CRS grade 3 and neural events ≧ however, clinical efficacy comparable to that of patients with classical morphology. The trend of improved safety profiles in patients with blast-like morphology is commensurate with lower peak CAR T cell expansion and reduced peak levels of cytokines associated with bone marrow-related inflammation. Patients previously treated with ibrutinib exhibited increased CAR T cell expansion, circulating inflammatory cytokines and chemokines and a rate of grade > 3 neurological events; as well as an increased progressive response rate at 6 months and ORR comparable to patients previously treated with acatinib alone. Patients previously treated with acatinib showed reduced CAR T cell expansion and circulating T1-associated cytokines and chemokines, consistent with an improved safety profile.

Example 12

This example characterizes two anti-CD19 CAR T therapy, KTE-X19 prepared according to example 5 and aliskiren. Cells were labeled with fluorescently conjugated antibodies to CD3 (pan T cell marker), CD14, CD19(B cell marker), CD45 (leukocyte marker), and CD56 (activation and NK marker) and evaluated by flow cytometry. Cell viability was assessed using negative staining with viability dye (SYTOX near infrared). The lower limit of quantitation (LLOQ) of the assay was 0.2% and 5% for NK cells and monocytes. Determination of the percentage of NK cells (NK cells are CD 45) ⁺ 、CD14 ^- 、CD3 ^- And CD56 ⁺ (ii) a The T cell is CD45 ⁺ 、CD14 ^- And CD3 ^- ). Median percentages of NK cells from aliskiren lot 23 and KTE-X19 lot 97 were 1.9% (range 0.8% -3.2%) and 0.1% (range 0.0% -2.8%), respectively. CD3 from the same lot of aliskiren and KTE-X19 ^- The median percentages of cellular impurities were 2.4% (ranging from 0.9% to 4.6%) and 0.5% (ranging from 0.3% to 3.9%), respectively. The results of KTE-X19 and aliskiren in terms of cell viability were equal to or greater than 72% and equal to or greater than 80%, respectively; results in terms of anti-CD 19 CAR expression were ≥ 24% and ≥ 15%, respectively; the results in terms of IFN-gamma production were 190pg/mL or more and 520pg/mL or more, respectively; and in CD3 ⁺ The results in terms of percentage of cells were equal to or greater than 90% and equal to or greater than 85%, respectively.

Example 13

Provides for receiving 2 x 10 in a single infusion in previous examples including example 2 and example 7 ⁶ Additional results for individual KTE-X19 cells/kg patients. ORR assessed by IRRC was 92% (95% CI, 82-97) and CR rate was 67% (95% CI, 53-78). At the median follow-up (range, 12.3-37.6) of 17.5 months, 29 patients maintained a progressive response. The progressive response rate is largely consistent between patients with high risk disease characteristics. The first 28 treated patients had a median follow-up of 32.3 months (range, 30.6-37.6). 39% of patients remain in sustained remission without further therapy. Among all enrolled patients (N ═ 74), ORR was 84% (59% CR rate). After a median follow-up of 17.5 months, the median values for DOR, PFS and OS were not reached. Table 29: poor progressive response rateThe prognosis groups were consistent. Figure 16 at the median follow-up of 17.5 months, the ZUMA-2 study continued to show significant and persistent clinical benefit of KTE-X19 therapy in patients with R/R MCL. No new security signal was observed in the case of additional follow-up. No new CRS or new level 5 events occurred since the previous report. Table 30: the AE rate decreases with time. KTE-X19 therapy showed a manageable safety profile with prolonged follow-up.

Table 29: response duration, progression-free survival and overall survival。

^a Among 55 total response patients.

Table 30: security analysis

^a Any level of AE occurring in ≧ 10% of patients is included.

Of the 57 patients with available data for which efficacy was evaluable, 48 patients (84%) had detectable B cells at baseline. Of patients with progressive responses at 12 months, more than 50% of evaluable patients had detectable B-cells and genetically labeled CAR T-cells at months 6, 12, 15 and 24. The percentage of patients with genetically labeled CAR T cells in patients who were in a progressive response at 12 months generally decreased over time, 100%, 93%, 82%, 89%, 80% and 56% at 3 months, 6 months, 12 months, 15 months, 18 months and 24 months, respectively. There was a reduced peak CAR T cell expansion in patients who failed to respond to KTE-X19. Peak CAR T cell expansion is increased in patients who have a progressive response at 12 months or who relapse at 12 months compared to non-responsive patients. Initially elevated CAR T cell levels were observed in patients who later relapsed, possibly pointing to alternative mechanisms for secondary treatment failure. CAR T cell peak levels normalized by baseline tumor burden and progressive response at 12 month data cutoff are shown in figure 17a (inv) and figure 17b (cen).

All publications, patents, patent applications, and other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent application, or other document were individually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the disclosure.

Claims (36)

1. A method for treating Mantle Cell Lymphoma (MCL) or B-cell ALL in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a T cell product comprising autologous T cells expressing an anti-CD 19 Chimeric Antigen Receptor (CAR).

2. The method of claim 1, wherein the MCL and B-cell ALL are relapsed or refractory MCL and B-cell ALL, optionally wherein the MCL is classical, blast-like, and polymorphic MCL.

3. The method of any one of claims 1 and 2, wherein the MCL and B-cell ALL are refractory to, or have relapsed after, one or more therapies of chemotherapy, radiation therapy, immunotherapy (including T-cell therapy and/or treatment with antibodies or antibody-drug conjugates), autologous stem cell transplantation, or any combination thereof.

4. The method of any one of claims 1 to 3, wherein the subject has received 1 to 5 prior treatments, optionally wherein at least one of the prior treatments is selected from autologous SCT, anti-CD 20 antibodies, anthracycline-containing or bendamustine-containing chemotherapy, and/or Bruton's Tyrosine Kinase Inhibitor (BTKi).

5. The method of claim 4, wherein the BTKi is ibrutinib or acatinib.

6. The method of any one of claims 1-5, wherein R/R B cellular ALL is defined as refractory to first-line therapy (i.e., primary refractory), relapsed ≦ 12 months after first remission, relapsed ≦ 2 past lines of systemic therapy or refractory, or relapsed following allogeneic Stem Cell Transplantation (SCT), optionally wherein the subject is in need of US eastern cooperative histocompatibility with bone medulloblasts, 0, or 1 ≦ 5% and/or adequate kidney, liver, and heart function.

7. The method of any one of claims 1-6, wherein if the B-cell ALL subject has received prior bornauzumab, the subject is in need of leukemia blast cells (leukaemic blast) with CD19 expression ≧ 90%.

8. The method of any one of claims 1 to 7, wherein the subject receives a bridging therapy after leukapheresis and before opsonization/lymphodepletion chemotherapy.

9. The method of any one of claims 1-8, wherein the MCL subject receives 500mg/m intravenously administered on each of the fifth, fourth, and third days prior to T cell infusion ² Cyclophosphamide and intravenous administration at 30mg/m ² A lymphodepleting chemotherapy regimen of both fludarabine.

10. The method of any one of claims 1-9, wherein the B-cell ALL subject receives 25mg/m per day of Intravenous (IV) administration on each of the fourth, third, and second days prior to T-cell infusion ² Fludarabine and 900mg/m daily IV administered the second day before infusion ² Lymphodepletion regimen of cyclophosphamide.

11. The method of any one of claims 8 to 10, wherein the MCL bridge therapy is selected from dexamethasone (e.g., 20 to 40mg per day PO or IV or equivalent for 1 to 4 days); methylprednisolone, ibrutinib (e.g., 560mg PO daily) and/or acatinib (e.g., 100mg PO twice daily); an immunomodulator; R-CHOP, bendamustine; an alkylating agent; and/or a platinum-based agent, wherein the bridging therapy is administered after leukopheresis and is completed within, for example, 5 days or less prior to opsonic chemotherapy.

12. The method of any one of claims 8 to 10, wherein the B-cell ALL subject can receive any one or more of the following bridging chemotherapy regimens:

13. the method of any one of claims 1 to 12, wherein the T cell product comprises CD4+ and CD8+ CAR T cells prepared from Peripheral Blood Mononuclear Cells (PBMCs) by positive enrichment and subsequent partial or complete depletion of circulating cancer cells.

14. The method of claim 13, wherein the PBMCs are enriched for T cells by positively selecting CD4+ and CD8+ cells, activated with an anti-CD 3 antibody and an anti-CD 28 antibody in the presence of IL-2, and then transduced with a replication defective viral vector containing FMC63-28Z CAR, which is a Chimeric Antigen Receptor (CAR) comprising an anti-CD 19 single chain variable fragment (scFv), CD28, and CD 3-zeta domain.

15. The method of any one of claims 13 and 14, wherein the T cell product comprises fewer cancer cells than T cell products comprising T cells from a leukapheresis product that have not been positively selected for CD4+ and CD8+ T cells.

16. The method of any one of claims 13 to 15, wherein the T cell product has other superior product attributes relative to T cell products comprising T cells from leukocyte apheresis products that have not been positively selected/enriched for CD4+ and CD8+ T cells.

17. The method of claim 16, wherein the superior product attributes are selected from increased percentage of CDRA45+ CCR7+ (naive like) T cells, decreased percentage of differentiated T cells, increased percentage of CD3+ cells, decreased IFN- γ production, decreased percentage of CD 3-cells.

18. The method of any one of claims 1-17, wherein the MCL subject is administered one or more doses of 1.8 x 10 ⁶ 1.9X 10 ⁶ 2 or 2X 10 ⁶ Individual CAR-positive live T cells/kg body weight with a maximum of 2 x 10 ⁸ (iii) individual CAR-positive viable T-cells (for 100kg and above patients), and administering to the B-cell ALL subject 0.5X 10 ⁶ 1, 1 × 10 ⁶ 2 or 2X 10 ⁶ Individual CAR-positive live T cells/kg body weight with a maximum of 2 x 10 ⁸ Individual CAR-positive live T cells (for patients of 100kg and above).

19. The method of any one of claims 1 to 17, wherein if the subject has achieved a complete response to the first infusion, the subject may receive a second infusion of anti-CD 19CAR T cells, if progressing after >3 months of remission, the provided CD19 expression has been retained and neutralizing antibodies against the CAR is not suspect, wherein response is assessed using the Lugano classification.

20. The method of any one of claims 1 to 19, wherein the subject is monitored for signs and symptoms of Cytokine Release Syndrome (CRS) and neurotoxicity following T cell administration.

21. The method according to claim 20, wherein the subject is monitored daily for at least seven days, preferably for four weeks, for signs and symptoms of CRS and neurotoxicity following infusion.

22. The method of any one of claims 20 and 21, wherein the signs or symptoms associated with CRS comprise fever, chills, fatigue, tachycardia, nausea, hypoxia, and hypotension, and the signs or symptoms associated with neurological events comprise encephalopathy, seizures, changes in consciousness level, speech impairment, tremors, and confusion.

23. The method according to any one of claims 20 to 22, wherein cytokine release syndrome in an MCL subject is managed according to the following protocol:

24. the method of any one of claims 20 to 23, wherein neurotoxicity in an MCL subject is managed according to the following protocol:

25. the method of any one of claims 1 to 24, wherein the MCL subject is a high risk patient determined by a Ki-67 tumor proliferation index ≥ 50% and/or the presence of the TP53 mutation.

26. The method of any one of claims 20 to 22, wherein CRS in a B-cell ALL subject is managed according to the following protocol:

27. the method of any one of claims 20-22 and 26, wherein neurotoxicity in a B-cell ALL subject is managed according to one of two regimens:

28. the method of any one of claims 1 to 27, wherein the B-cell ALL subject can receive any one or more of the following bridging chemotherapy regimens:

29. an autologous T cell expressing an anti-CD 19 CAR for use in a method for the treatment of Mantle Cell Lymphoma (MCL) or B-cell ALL according to any one of claims 1 to 28.

30. Use of an autologous T cell expressing an anti-CD 19 CAR in the manufacture of a medicament according to any one of claims 1 to 28 for the treatment of Mantle Cell Lymphoma (MCL) or B-cell ALL.

31. A method of predicting:

(i) an objective response of a subject to CAR T cell therapy (optionally, the method of any one of claims 1 to 28), the predictive method comprising measuring peak CAR T cell levels and comparing them to a reference standard, wherein the objective response is positively correlated with the peak CAR T cell levels, wherein the objective response comprises both a complete response and a partial response, and wherein all responses are assessed using the Lugano classification;

(xii) Minimal residual disease (e.g. at week 4) in response to CAR T cell therapy (optionally, according to the method of any one of claims 1 to 28), the predictive method comprising measuring peak CAR T cell levels and comparing them to a reference standard, wherein negative minimal residual disease is associated with higher peak CAR T cell levels;

(xiii) (ii) CRS ≥ 3 and/or neurological event ≥ 3 (NE) in a subject receiving CAR T cell therapy (optionally, according to the method of any one of claims 1 to 28), the predictive method comprising measuring peak CAR T cell expansion following therapy and comparing the level to a reference value, wherein the more CAR T cell expansion, the greater the chance of CRS ≥ 3 and/or NE event ≥ 3;

(xiv) CRS grade ≧ 3 and/or NE grade ≧ 3, the predictive method comprising measuring peak levels of GM-CSF and IL-6 after CART cell therapy (optionally, according to the method of any one of claims 1-28) and comparing them to reference levels, wherein the higher the peak levels of these cytokines, the greater the chance of CRS grade ≧ 3 and/or NE grade 3;

(xv) CRS grade ≥ 3 in a subject receiving CAR T cell therapy (optionally, the method according to any one of claims 1 to 28), the predictive method comprising measuring peak level of serum ferritin after CAR T cell therapy and comparing it to a reference level, wherein the higher the peak level of ferritin the greater the chance of CRS grade ≥ 3;

(xvi) Grade ≧ 3 CRS, the prediction method comprising measuring peak levels of serum IL-2 and IFN- γ following CAR T-cell treatment (optionally according to any one of claims 1 to 28) and comparing them to reference levels, wherein the higher the peak levels of IL-2 and IFN- γ, the greater the chance of grade ≧ 3 NE;

(xvii) Grade ≧ 3 CRS, the predictive method comprising measuring cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8 and/or Vascular Cell Adhesion Molecule (VCAM) after CAR T-cell treatment (optionally according to any of claims 1 to 28) and comparing them to reference levels, wherein the higher the cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8 and/or Vascular Cell Adhesion Molecule (VCAM), the greater the chance of grade ≧ 3 NE;

(xviii) ≧ grade 3 CRS following CAR T cell therapy (optionally, the method of any one of claims 1-28), the predictive method comprising measuring peak serum levels of IL-15, IL-2 Ra, IL-6, TNF α, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1B, granzyme A, granzyme B and/or perforin following anti-CD 19 CAR T therapy and comparing said levels to reference levels, wherein the peak serum level of IL-15, IL-2 Ra, IL-6, TNF alpha, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1B, granzyme A, granzyme B and/or perforin is positively correlated with CRS grade 3 or higher;

(xix) CRS grade 3 or more following CAR T cell therapy on B-cell ALL (optionally, the method of any one of claims 1 to 28), the predictive method comprising measuring the peak serum level of IL-15 following anti-CD 19 CAR T therapy and comparing said level to a reference level, wherein the peak serum level of IL-15 is negatively correlated with CRS grade 3 or more;

(xx) CRS grade 3 and/or NE grade 3 following CAR T cell therapy (optionally, the method of any one of claims 1 to 28), the predictive method comprising measuring peak serum levels of IL-6, TNF α, GM-CSF, IL-10, MIP-1B and granzyme B following anti-CD 19 CAR T therapy and comparing said levels to reference levels, wherein the peak serum levels of IL-6, TNF α, GM-CSF, IL-10, MIP-1B and granzyme B are positively correlated with CRS grade 3 and NE grade 3;

(xxi) Whether a patient will be MRD at 4 weeks/month after CAR T cell therapy (optionally, according to any one of claims 1 to 28) (10) ^-5 Sensitivity of (a) to (b) negative, the prediction method comprising measuring peak serum levels of IFN- γ, IL-6 and/or IL-2 after treatment and comparing the levels to a reference standard, wherein the peak serum levels of IFN- γ, IL-6 and/or IL-2 positively correlate with MRD negativity at one month.

32. A method according to any one of claims 20 to 24, 26, 27 and 30 to 31, wherein CRS and NE are obtained by Lee et al, Blood 2014; the method described in 124: 188-195.

33. The method of claim 31, wherein the reference standard is established by any method commonly used in the biomarker art, such as a quartile (quartile) analysis of a patient population with known responses, toxicity levels, and MRD levels.

34. The method of claim 31, wherein CAR T cell levels are measured by CAR gene copies per microgram DNA in the blood.

35. The method of any one of claims 1-34, further comprising reducing the level/activity of a cytokine positively correlated with CRS grade No. > 3 and/or NE grade No. > 3 following CAR T cell infusion to reduce CRS grade No. > 3 and/or NE grade No. > 3.

36. A method of improving the effectiveness of CAR T cell therapy (e.g., classical, blast-like, and polymorphic MCL and B cell ALL) in a subject in need thereof, the method comprising manipulating the T cell phenotype of the T cell product administered to the subject, optionally wherein the manipulating comprises increasing the number of CD3+ T cells, decreasing the number of CD 3-cells, increasing the number/percentage of CDRA45+ CCR7+ (naive) T cells, and/or decreasing the number/percentage of differentiated cells in the T cell product, decreasing the level of IFN- γ production by the T cells during production, wherein the improvement is observed relative to the effectiveness of a T cell product prepared without any deliberate manipulation of the number/percentage of CDRA45+ CCR7+ (naive) T cells and/or the number/percentage of differentiated cells in the T cell product.