Method of administering chimeric antigen receptor immunotherapy drugs

Abstract

To provide a method for treating relapsed or refractory unspecified diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, or follicular lymphoma-related DLBCL in patients after two or more lines of systemic therapy. [Solution] The method involves administering an axicapbutagen siloleucel suspension to the patient by intravenous infusion in doses ranging from approximately 1 × 10⁶ to approximately 2 × 10⁶ CAR-positive viable T cells per kg of body weight up to a maximum dose of approximately 1 × 10⁸ CAR-positive viable T cells, wherein axicapbutagen siloleucel is a CD19-targeted genetically modified autologous T cell immunotherapy agent, comprising the patient's own T cells ex vivo genetically modified by retroviral transduction to express a chimeric antigen receptor containing an anti-CD19 single-strand variable fragment (scFv) linked to the costimulatory domains of CD28 and CD3 zeta.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims priority to U.S. Provisional Patent Application No. 62 / 574,159, filed October 18, 2017, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates generally to T - cell therapeutics, and more specifically to CD19 - directed genetically modified autologous T - cell immunotherapeutics comprising chimeric antigen receptors (CARs).

Background Art

[0003] Human cancers are essentially composed of normal cells that undergo genetic or epigenetic transformation into abnormal cancer cells. By doing so, cancer cells begin to express proteins and other antigens that are distinct from those expressed by normal cells. These abnormal tumor antigens can be used by the body's natural immune system to specifically target and kill cancer cells. However, cancer cells utilize various mechanisms to prevent immune cells, such as T - lymphocytes and B - lymphocytes, from successfully targeting cancer cells.

[0004] A chimeric antigen receptor (CAR) that includes a binding domain capable of interacting with a specific tumor antigen enables T - cells to target and kill cancer cells that express the specific tumor antigen.

Summary of the Invention

[0005] As described in detail below, the present disclosure is in part based on the surprising discovery that harmful side effects of CAR T - cell immunotherapeutics are identified and managed by the administration methods disclosed herein.

[0006] Any aspect or embodiment described herein can be combined with any other aspect or embodiment disclosed herein. While the invention has been described in detail, the foregoing description is intended to illustrate, not limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

[0007] In one embodiment, the present invention relates to a method for treating unspecified relapsed or refractory diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, or follicular lymphoma resulting in DLBCL after two or more lines of systemic therapy in a patient, In patients requiring treatment, axicapbutagen siloleucel suspension is administered intravenously by approximately 1 × 10⁶ units per kg of body weight. 6 pieces and approximately 2 x 10 6 Approximately 1 × 10⁶ CAR-positive surviving T cells were obtained between individuals. 8 This includes administering up to the maximum dose of individual CAR-positive surviving T cells, Here, we provide a method for a CD19-targeted genetically modified autologous T cell immunotherapy, comprising patient-owned T cells that have been ex vivo genetically modified to express a chimeric antigen receptor (CAR) containing an anti-CD19 single-strand variable fragment (scFv) linked to the CD28 and CD3 zeta costimulatory domains, which has been collected and transduced by retroviral mutation.

[0008] In another aspect, the present invention relates to relapsed or refractory diffuse large B-cell lymphoma (DLBCL) and primary mediastinal large B-cell lymphoma in patients after two or more lines of systemic therapy. A method for treating Parkinson's disease (PMBCL), comprising intravenous injection of approximately 0.4 × 10⁻¹⁰ axicapbutogen siloleucel suspension into patients requiring treatment. 8 pieces and approximately 2 x 10 8The present invention provides a CD19-targeted genetically modified autologous T cell immunotherapy agent comprising administering a dose of CAR-positive viable T cells between individuals, wherein axicapbutogen siloleucel is a CD19-targeted genetically modified autologous T cell immunotherapy agent comprising the patient's own T cells that have been ex vivo genetically modified to express a chimeric antigen receptor (CAR) containing an anti-CD19 single-strand variable fragment (scFv) linked to the costimulatory domains of CD28 and CD3 zeta by retroviral transduction.

[0009] In some embodiments, the duration of intravenous infusion is between 15 and 120 minutes. In some embodiments, the duration of intravenous infusion is up to 30 minutes.

[0010] In some embodiments, the injection volume is between 50 mL and 100 mL. In some embodiments, the injection volume is approximately 68 mL.

[0011] In some embodiments, the immunotherapy drug is injected from an infusion bag. In some embodiments, the infusion bag is agitated during infusion.

[0012] In some embodiments, the immunotherapy drug is administered within 3 hours of thawing.

[0013] In some embodiments, the suspension further comprises albumin. In some embodiments, albumin is present in an amount of approximately 2% (volume / volume) to 3% (volume / volume). In some embodiments, albumin is present in an amount of approximately 2.5% (volume / volume). In some embodiments, the albumin is human albumin.

[0014] In some embodiments, the suspension further contains DMSO. In some embodiments, DMSO is present in an amount of about 4% (volume / volume) to 6% (volume / volume). In some embodiments, DMSO is present in an amount of about 5% (volume / volume).

[0015] In one embodiment, the present invention relates to a method for treating relapsed or refractory large B-cell lymphoma in a patient after two or more lines of systemic therapy, (a) Administering CD19-targeted gene-modified autologous T-cell immunotherapy drugs to patients in need of treatment, (b) Monitoring the patient for signs and symptoms of adverse reactions after the injection, This provides a method that includes [something].

[0016] In some embodiments, the relapsed or refractory large B-cell lymphoma is DLBCL resulting from diffuse large B-cell lymphoma (DLBCL) not otherwise specified, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, or follicular lymphoma.

[0017] In some embodiments, the adverse reactions are selected from the group consisting of cytokine release syndrome (CRS), neurotoxicity, hypersensitivity reactions, severe infections, cytopenia, and hypogammaglobulinemia.

[0018] In some embodiments, the signs and symptoms of the adverse reactions are selected from the group consisting of fever, hypotension, tachycardia, hypoxia and chills, and include cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal failure, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), seizures, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia and anemia.

[0019] In some embodiments, the above method further includes administering an IL-6 receptor inhibitor.

[0020] In some embodiments, the above method further includes administering an effective amount of tocilizumab to treat the symptoms of adverse reactions.

[0021] In some embodiments, tocilizumab is administered intravenously at a dose of approximately 8 mg / kg. In some embodiments, tocilizumab is administered intravenously over approximately 1 hour. In some embodiments, tocilizumab is administered approximately every 8 hours. In some embodiments, tocilizumab is administered over a period of approximately 24 hours or less.

[0022] In some embodiments, the above method further includes administering corticosteroids to treat symptoms of adverse reactions.

[0023] In some embodiments, the corticosteroid is at least one of methylprednisone or dexamethasone.

[0024] In some embodiments, methylprednisone is administered intravenously at a dose of approximately 1 mg / kg. In some embodiments, methylprednisone is administered twice daily. In some embodiments, methylprednisone is administered intravenously at a dose of approximately 1000 mg per day. In some embodiments, methylprednisone is administered intravenously over a period of approximately 3 days.

[0025] In some embodiments, dexamethasone is administered at a dose of approximately 10 mg. In some embodiments, dexamethasone is administered intravenously approximately every 6 hours.

[0026] In some embodiments, the adverse reaction is cytokine release syndrome (CRS). In some embodiments, signs and symptoms of cytokine release syndrome (CRS) are monitored at least daily for approximately 7 days after injection. In some embodiments, signs and symptoms of cytokine release syndrome (CRS) are monitored at least daily for approximately 8, 9, or 10 days after injection. In some embodiments, signs and symptoms of cytokine release syndrome (CRS) are monitored at least daily for approximately 10 days after injection. In some embodiments, signs and symptoms of cytokine release syndrome (CRS) are monitored for approximately 4 weeks after injection.

[0027] In some embodiments, the adverse reaction is neurotoxicity.

[0028] In some embodiments, signs and symptoms of neurotoxicity are monitored for approximately 8 weeks after injection.

[0029] In some embodiments, the above method further includes administering a non-sedating antiepileptic drug for seizure prevention.

[0030] In some embodiments, the non-sedating antiepileptic drug is levetiracetam.

[0031] In some embodiments, the adverse reaction is cytopenia. In some embodiments, cytopenia is thrombocytopenia, neutropenia, and / or anemia.

[0032] In some embodiments, the above method involves erythropoietin, darbepoetin alfa, platelet transfusion, colony-stimulating factor (CSF), granulocyte colony-stimulating factor, filgrastim, The treatment further includes administering at least one of pegfilgrastim or granulocyte-macrophage colony-stimulating factor.

[0033] In some embodiments, the above method further includes measuring cytokine and chemokine levels. In some embodiments, levels of at least one of IL-6, IL-8, IL-10, IL-15, TNF-α, IFN-γ, and sIL2Rα are measured.

[0034] In one embodiment, the present invention provides a container comprising a suspension of CD19-directional gene-modified autologous T cells, about 5% dimethyl sulfoxide (DMSO), and about 2.5% human albumin (vol / vol). In another embodiment, the container comprises about 0.4 × 10 8 pieces~2×10 8 Contains a suspension of individual CD19-directed gene-modified autologous T cells (CAR-positive surviving T cells).

[0035] In some embodiments, the container is a sterile infusion bag. In some embodiments, the capacity of the infusion bag is approximately 100 mL, 250 mL, 500 mL, 750 mL, 1000 mL, 1500 mL, 2000 mL, or 3000 mL.

[0036] In one embodiment, the present invention relates to a method for treating relapsed or refractory large B-cell lymphoma in humans after two or more lines of systemic therapy, comprising administering a CD19-targeted gene-modified autologous T-cell immunotherapy agent to a person in need of treatment, (a) Administering the patient a composition containing CD19-directed chimeric antigen receptor (CAR)-positive viable T cells, (b) Monitoring the patient for signs and symptoms of adverse reactions after administration, If cytokine release syndrome (CRS) of grade 2 or higher is observed in (c)(b), administer tocilizumab at a dose of approximately 8 mg / kg (intravenously) over 1 hour, and if there is no response to increased intravenous fluid or oxygen supplementation, repeat tocilizumab every 8 hours as needed. If the CRS symptoms observed in (d)(b) do not improve 24 hours after (c), administer approximately 1 mg / kg of methylprednisolone (intravenously) twice daily or an equivalent dose of dexamethasone, continuing the use of corticosteroids until the event is grade 1 or lower, and then gradually reducing the dose over the following 3 days. If a Grade 3 CRS is observed in (e)(b), administer tocilizumab at a dose of 8 mg / kg (intravenously) over 1 hour, and if there is no response to increased intravenous fluid or oxygen supplementation, repeat tocilizumab every 8 hours as needed, administer methylprednisolone 1 mg / kg (intravenously) twice daily or an equivalent dose of dexamethasone, and continue the use of corticosteroids until the event is Grade 1 or lower, and then gradually reduce them over the following 3 days. If Grade 4 CRS is observed in (f)(b), administer tocilizumab at a dose of approximately 8 mg / kg (intravenously) over 1 hour, and if there is no response to increased intravenous fluid or oxygen supplementation, repeat tocilizumab every 8 hours as needed, and administer approximately 1000 mg (intravenously) of methylprednisolone per day for 3 days. This provides a method that includes [something].

[0037] In one embodiment, the present invention relates to a method for treating relapsed or refractory large B-cell lymphoma in a patient after two or more lines of systemic therapy, comprising administering a CD19-targeted gene-modified autologous T-cell immunotherapy agent to a patient in need of treatment, (a) Administering the patient a composition containing CD19-directed chimeric antigen receptor (CAR)-positive viable T cells, (b) Monitoring the patient for signs and symptoms of adverse reactions after administration, (c) If cytokine release syndrome (CRS) and / or neurotoxicity is observed, Tokine release syndrome (CRS) and / or neurotoxicity should be managed according to Table 1 and / or Table 2, This provides a method that includes [something].

[0038] Other features and advantages of this disclosure will become apparent from the following detailed description, including the examples, and from the claims. [Modes for carrying out the invention]

[0039] This disclosure relates to engineered cells (e.g., T cells) comprising a CD19 CAR gene-modified autologous T cell immunotherapy agent indicated for the treatment of adult patients with relapsed or refractory large B cell lymphoma after two or more lines of systemic therapy, including DLBCL resulting from unspecified diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma, high-grade B cell lymphoma, and follicular lymphoma. In some embodiments, this disclosure provides a therapeutic method using engineered T cells for the treatment of patients with cancer.

[0040] To prepare CD19-directed genetically modified autologous T-cell immunotherapy agents, the patient's own T cells can be harvested and ex vivo genetically modified by retroviral transduction to express a chimeric antigen receptor (CAR) containing a mouse anti-CD19 single-strand variable fragment (scFv) linked to the costimulatory domains of CD28 and CD3 zeta. In some embodiments, the CAR contains a mouse anti-CD19 single-strand variable fragment (scFv) linked to the costimulatory domains of 4-1BB and CD3 zeta. Anti-CD19 CAR T cells can be proliferated and reinjected into the patient, where they can recognize and eliminate target cells expressing CD19. YESCARTA® (Axi-cel®, axicapbutogen siloleucel) is one example of such a CD19-directed genetically modified autologous T-cell immunotherapy agent. See Kochenderfer, et al., (J Immunother 2009;32:689 702). Additional CD19-targeted CAR therapies include JCAR017, JCAR015, JCAR014, and Kymriah (tisagenlecleucel). See Sadelain et al. Nature Rev. Cancer Vol. 3 (2003), Ruella et al., Curr Hematol Malig Rep., Springer, NY (2016), and Sadelain et al. Cancer Discovery (Apr 2013).

[0041] CD19-targeted genetically modified autologous T-cell immunotherapy drugs are typically prepared from the patient's peripheral blood mononuclear cells obtained through standard leukocyte apheresis procedures. It can be obtained. T cells are concentrated from monocytes, activated with anti-CD3 antibody in the presence of IL-2, and then transduced with a replication-defective retroviral vector containing the anti-CD19 CAR transgene. The transduced T cells can be expanded in cell culture, washed, formulated into a suspension, and / or cryopreserved. Typically, a product containing genetically modified autologous T cells must pass a sterility test before being released for shipment as a frozen suspension in a patient-specific infusion container such as an infusion bag. Typically, the product is thawed before infusion.

[0042] The CD19-directed genetically modified autologous T cell immunotherapy agent may include NK cells and NK-T cells in addition to T cells. In some embodiments, the CD19-directed genetically modified autologous T cell immunotherapy agent formulation contains about 5% dimethyl sulfoxide (DMSO) and about 2.5% albumin (human) (volume / volume).

[0043] CD19-directed genetically modified autologous T cells bind to cancer cells and normal B cells that express CD19. Studies have demonstrated that after anti-CD19 CAR T cells bind to target cells expressing CD19, the co-stimulatory domains of CD28 and CD3 zeta activate downstream signaling cascades, thereby resulting in T cell activation, proliferation, acquisition of effector functions, and secretion of inflammatory cytokines and chemokines. This series of events causes the death of cells expressing CD19.

[0044] In one aspect, the present invention provides a method for treating refractory or treatment-resistant diffuse large B cell lymphoma (DLBCL) of unknown origin, primary mediastinal large B cell lymphoma, high-grade B cell lymphoma, or DLBCL resulting from follicular lymphoma in a patient after two or more lines of systemic therapy, the method comprising intravenously infusing a CD19-directed genetically modified autologous T cell suspension into a patient at a dose of about 1×10 6 cells to about 2×10 6 CAR-positive viable T cells per kg of body weight, and about 1×10 8The present invention provides a method that includes administering a dose up to the maximum dose of individual CAR-positive viable T cells.

[0045] definition To facilitate understanding of the present invention, certain terms are first defined below. Further definitions of the following terms and other terms are provided throughout this specification.

[0046] As used herein and in the appended claims, the singular forms "a," "an," and "the" include multiple subjects unless the context makes otherwise clear.

[0047] Unless otherwise specified or made clear from the context, the term “or” as used herein is understood to include and encompass both “or” and “and.”

[0048] As used herein, the term "and / or" is understood to mean a specific disclosure that includes, or excludes, each of two designated features or components. Thus, as used herein in phrases such as "A and / or B," the term "and / or" is intended to include A and B; A or B; A (alone); and B (alone). Similarly, as used in phrases such as "A, B, and / or C," the term "and / or" is intended to include each of the following embodiments: 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).

[0049] The terms “for example” and “that is” as used herein are used merely as examples and are not intended to be limiting, and should not be construed as referring only to those items explicitly listed herein.

[0050] Terms such as "at least," "at least," and "more than," for example, "at least one," are not limited to, but are 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, 3 5, 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, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 It is understood to include values ​​greater than or equal to 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. It also includes any larger number or fractions in between.

[0051] Conversely, the term "less than or equal to" includes each value smaller than the value indicated. For example, "less than or equal to 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, This includes 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 sets of nucleotides. It also includes any smaller number or fractions in between.

[0052] Terms such as "multiple," "at least two," "two or more," and "at least the second" are not limited to, but include 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 ,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,8 9, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, It is understood to include 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 and above. It also includes any larger number or fractions in between.

[0053] Throughout the specification, the words "includes," "comprising," or variations such as "comprises" or "comprising" refer to the elements, integers, or other specified elements. This implies that a process, or element, integer, or group of processes, is included, but is understood not to exclude any other element, integer, or process, or element, integer, or group of processes. Whenever an aspect is described in this specification with the words "including" or "contains," other similar aspects described in the terms "consisting of" and / or "essentially consisting of" are also provided.

[0054] Unless otherwise specifically stated or made clear from the context, the term “about” as used herein refers to a value or composition that falls within an acceptable range of error for a particular value or composition as determined by those skilled in the art, and which depends in part on how that value or composition is measured or determined, i.e., on the limitations of the measuring system. For example, “about” or “approximately” may mean within a range of one or more standard deviations per single implementation in the art. “About” or “approximately” may mean a range of up to 10% (i.e., ±10%). Thus, “about” may be understood to fall within a range of 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 indicated value. For example, about 5 mg may include any amount between 4.5 mg and 5.5 mg. Furthermore, the above usage may apply particularly with respect to biological systems or processes. The term may mean up to 10 times or up to 5 times a certain value. Where a particular value or composition is provided in this disclosure, unless otherwise indicated, the meaning of “about” or “approximately” is to include within an acceptable margin of error for that particular value or composition.

[0055] As described herein, any range of concentration, percentage, ratio, or integer is understood to include any integer, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0056] The units, prefixes, and symbols used herein are presented in their accepted forms within the International System of Units (SI). Numerical ranges are defined as follows: Includes a fixed number.

[0057] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which this disclosure relates. For example, Juo, "The Concise Dictionary of Biomedicine and Molecular Biology", 2 nd ed., (2001), CRC Press, "The Dictionary of Cell & Molecular Biology", 5 th ed., (2013), Academic Press, and "The Oxford Dictionary Of Biochemistry And Molecular Biology", Cammack et al. eds., 2 nd The ed. (2006), Oxford University Press, provides a general dictionary of many of the terms used in this disclosure for those skilled in the art.

[0058] "Administering" refers to various methods known to those skilled in the art. This refers to the physical delivery of a drug to a subject using either an or delivery system. Exemplary routes of administration for the formulations disclosed herein include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, vertebral, or other parenteral (extraintestinal) routes of administration by injection or infusion. As used herein, the term “parenteral administration” means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In some embodiments, the formulations are administered by a non-parenteral route, for example, orally. Other non-parenteral routes of administration include topical, epidermal, or mucosal routes of administration, for example, intranasal, transvaginal, rectal, sublingual, or topical. Furthermore, administration may be carried out, for example, once, multiple times, and / or over one or more extended periods.

[0059] The term “antibody” (Ab) is not limited to and includes glycoprotein immunoglobulins that specifically bind to an antigen. Generally, an antibody may comprise at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds, or an antigen-binding molecule thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, namely CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, namely CL. The VH and VL regions may be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interposed by more conserved regions called framework regions (FRs). VH and VL each contain three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of Ab can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).

[0060] Antibodies include, for example, monoclonal antibodies, recombinant antibodies, single-specific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, and antibody fusions (referred to herein as "antibodies"). Examples include heteroconjugate antibodies (sometimes referred to as "antibody conjugates"), single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv(scFv), camelized antibodies, affybodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv(sdFv), anti-idiotype (anti-Id) antibodies (e.g., including anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimes"), and any of the antigen-binding fragments described above. In some embodiments, the antibodies described herein refer to a population of polyclonal antibodies.

[0061] An "antigen-binding molecule," "antigen-binding region," or "antibody fragment" refers to any molecule containing the antigen-binding region (e.g., CDR) of an antibody, which is derived from the antibody. An antigen-binding molecule may contain an antigen complementarity-determining region (CDR). Examples of antibody fragments, but not limited to, include Fab, Fab', F(ab')2, and Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules containing peptide-binding domains) are defined as follows: Here are some other examples of suitable antigen-binding molecules. In some embodiments, the antigen-binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen-binding molecule binds to an antigen on a cell involved in hyperproliferative disease, or to a viral or bacterial antigen. In some embodiments, the antigen-binding molecule binds to CD19. In further embodiments, the antigen-binding molecule is an antibody fragment that specifically binds to an antigen, comprising one or more complementarity-determining regions (CDRs). In further embodiments, the antigen-binding molecule is a single-chain variable fragment (scFv). In some embodiments, the antigen-binding molecule is an avimer. It includes or consists of.

[0062] An "antigen" refers to any molecule that can trigger an immune response or be bound by an antibody or antigen-binding molecule. An immune response may include antibody production, activation of specific immunologically competent cells, or both. Those skilled in the art will readily understand that virtually any macromolecule, including proteins or peptides, can act as an antigen. Antigens can be endogenously expressed, i.e., expressed by genomic DNA, or expressed by recombination. Antigens can be specific to certain tissues, such as cancer cells, or they can be broadly expressed. Furthermore, larger molecular fragments can act as antigens. In some embodiments, the antigen is a tumor antigen.

[0063] "CD19-targeted gene-modified autologous T cell immunotherapy drugs" refer to suspensions of chimeric antigen receptor (CAR)-positive T cells. One example of such an immunotherapy drug is axicapbutagensiloleucel (also known as Axi-cel® or YESCARTA®), developed by Kite Pharmaceuticals, Inc.

[0064] The terms "neutralize" refer to antigen-binding molecules, scFvs, antibodies, or fragments thereof that bind to a ligand and interfere with or reduce the biological effects of that ligand. In some embodiments, antigen-binding molecules, scFvs, antibodies, or fragments thereof directly block the binding site on the ligand or modify the ligand's binding ability by indirect means (such as altering the ligand's structure or energy). In some embodiments, antigen-binding molecules, scFvs, antibodies, or fragments thereof prevent the bound protein from performing its biological function.

[0065] The term “autologous” refers to any material originating from the same individual that is to be later reintroduced. For example, the engineered autologous cell therapy (eACT®) described herein involves collecting lymphocytes from a patient, which are then engineered to express, for example, a CAR construct and then returned to the same patient (administered back).

[0066] The term "allogeneic" refers to any material originating from one individual, which is then introduced into another individual of the same species (for example, allogeneic T cell transplantation).

[0067] The terms “transduction” and “transduced” refer to the process by which foreign DNA is introduced into a cell by a viral vector (see Jones et al., “Genetics: principles and analysis,” Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, DNA vector, RNA vector, adenovirus vector, baculovirus vector, Epstein-Barr virus vector, papovavirus vector, vaccinia virus vector, herpes simplex virus vector, adenovirus-associated vector, lentivirus vector, or any combination thereof.

[0068] "Cancer" refers to a broad group of diseases characterized by the uncontrolled proliferation of abnormal cells in the body. Uncontrolled cell division and proliferation can lead to the formation of malignant tumors, which can invade adjacent tissues and then metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" may include tumors. Examples of cancers that can be treated by the methods disclosed herein include, but are not limited to, lymphoma, leukemia, myeloma, and cancers of the immune system, including other leukocyte malignancies. In some embodiments, the methods disclosed herein are used, for example, to treat bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, multiple myeloma, Hodgkin's disease, non-Hodgkin lymphoma (NHL), primary mediastinal large B cell lymphoma (PMBC), and diffuse large B cell lymphoma (DLBCL). Large B-cell lymphoma, follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL) Lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft gland cancer Tissue sarcomas, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T cell ALL), chronic lymphocytic leukemia (CLL), solid tumors in childhood, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, It may be used to reduce the tumor size of tumors resulting from positivity sarcomas, epidermal carcinomas, squamous cell carcinomas, T-cell lymphomas, environment-induced cancers including those induced by asbestos, other B-cell malignancies, and combinations of the above cancers. In some embodiments, the cancer is multiple myeloma. Certain cancers may respond to chemotherapy or radiotherapy, but otherwise they may be refractory. A refractory cancer is one that is not suitable for surgical intervention, and is either initially unresponsive to chemotherapy or radiotherapy, or becomes unresponsive over time.

[0069] As used herein, “antitumor effect” refers to a biological effect that may be presented as a reduction in tumor volume, a reduction in tumor cell count, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall survival or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with the tumor. The term “antitumor effect” may also refer to the prevention of tumor development, such as through vaccination.

[0070] As used herein, "cytokines" are substances that react to contact with a specific antigen and are produced in a single cell. This refers to non-antibody proteins released, where cytokines interact with a second cell to mediate a response in that second cell. As used herein, “cytokines” means proteins released by one cell population that act on another cell as intercellular mediators. Cytokines can be endogenously expressed by cells or administered to a subject. Cytokines can be released by immune cells, including macrophages, B cells, T cells, and mast cells, to propagate an immune response. Cytokines can induce a variety of responses in recipient cells. Cytokines include homeostatic cytokines, chemokines, pro-inflammatory cytokines, These may include effectors and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, can promote the survival and proliferation of immune cells, while pro-inflammatory cytokines can promote inflammatory responses. Examples of homeostatic cytokines, but not limited to, include IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of pro-inflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF)2, granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular 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).

[0071] "Chemokines" are a type of cytokine that mediates chemotaxis or directional movement of cells. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokines (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1α (MIP-1α, MIP-1a), MIP-1β (MIP-1b), gamma-inducible protein 10 (IP-10), and thymic and activation-regulating chemokines (TARC or CCL17).

[0072] A "therapeutic effective dose," "effective dose," "effective amount," or "therapeutic effective dosage" of a therapeutic agent, such as engineered CAR T cells, is any amount that, when used alone or in combination with another therapeutic agent, protects the subject from the onset of the disease, or promotes disease regression, manifested by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of functional or physical impairment resulting from the onset of the disease. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to experienced physicians, for example, by assaying the activity of the drug in human subjects during clinical trials, in animal model systems to predict efficacy in humans, or in in vitro assays.

[0073] As used herein, the term “lymphocyte” includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic lymphocyte that constitutes a major component of the innate immune system. NK cells reject cells infected with tumors and viruses. NK cells act through the process of apoptosis or programmed cell death. NK cells are named “natural killers” because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (without antibody involvement). Their T cell receptors (TCRs) differentiate them from other types of lymphocytes. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells. There are six types of T cells: helper T cells (e.g., CD4-positive cells), cytotoxic T cells, and B cells. Cells (TCs, also known as cytotoxic T lymphocytes, CTLs, T-killer cells, cytolytic T cells, CD8-positive T cells, or killer T cells), memory T cells ((i) Stem memory TSCM cells, like naive cells, are CD45RO-negative, CCR7-positive, CD45RA-positive, CD62L-positive (L-selectin), CD27-positive, CD28-positive, and IL-7Rα-positive, but they express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1 and exhibit numerous functional attributes specific to memory cells; (ii) Central memory T CMCells express L-selectin and CCR7 and secrete IL-2 rather than IFNγ or IL-4; and (iii) effector memory TEM cells do not express L-selectin or CCR7 but produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Treg, suppressor T cells, or CD4-positive CD25-positive regulatory T cells), natural killer T cells (NKT), and gamma delta T cells. B cells, on the other hand, play the most important role in humoral immunity (involving antibodies). B cells produce antibodies and antigens, act as antigen-presenting cells (APCs), and become memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow, from which their name is derived.

[0074] The terms “genetically modified” or “manipulated” refer to methods of modifying the genome of a cell, including, but not limited to, deletions of coding regions or non-coding regions, or parts thereof, or insertions of coding regions or parts thereof. In some embodiments, the cells being modified are lymphocytes, for example, T cells which may be obtained from either a patient or a donor. T cells may be modified to express exogenous constructs, such as chimeric antigen receptors (CARs) or T cell receptors (TCRs), which are incorporated into the cell’s genome.

[0075] "Immune response" refers to the action of immune system cells (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including alpha, cytokines, and complement) produced by any of these cells or the liver, resulting in the selective targeting, binding to, damage to, destruction of, and / or elimination of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or normal human cells or tissues in the case of autoimmune or pathological inflammation.

[0076] The term "immunotherapy" refers to the treatment of subjects affected by disease, or at risk of developing or relapsing from disease, by methods including inducing, enhancing, suppressing, or modifying the immune response. Examples of immunotherapy include, but are not limited to, T-cell therapy. T-cell therapy includes adoptive T-cell therapy and tumor-infiltrating lymphocyte therapy. This may include globular (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT®), and allogeneic T cell transplantation. However, those skilled in the art will recognize that the conditioning methods disclosed herein may enhance the efficacy of any transplanted T cell therapy. Examples of T cell therapies are described in U.S. Patent Applications Publications 2014 / 0154228 and 2002 / 0006409, U.S. Patents 7,741,465, 6,319,494, 5,728,388 and International Publication 2008 / 081035.

[0077] T cells for immunotherapy may originate from any source known in the art. For example, T cells may be differentiated from a population of hematopoietic stem cells in vitro, or T cells may be obtained from a subject. T cells may be obtained from, for example, peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from the site of infection, ascites, pleural fluid, splenic tissue, and tumors. Furthermore, T cells may originate from one or more T cell lineages available in the art. Also, T cells may be isolated using FICOLL® and / Alternatively, they can be obtained from blood units collected from a subject using any of the many techniques known to those skilled in the art, such as apheresis. Further methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which in whole forms part of this Spec.

[0078] The term "engineered autologous cell therapy," which can be abbreviated as "eACT(trademark)" and is also known as adoptive cell transplantation, is a process in which a patient's own T cells are collected and then genetically modified to recognize and target one or more antigens expressed on the surface of one or more specific tumor cells or malignant tumor cells. T cells can be engineered, for example, to express chimeric antigen receptors (CARs). CAR-positive (+) T cells are engineered to express extracellular single-chain variable fragments (scFv) that have specificity to specific tumor antigens and are linked to an intracellular signaling region containing at least one costimulatory domain and at least one activating domain. CAR scFv can be designed to target CD19, a transmembrane protein expressed by cells in B cell lineages, including, but not limited to, all normal B cells, diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, and B-cell malignancies including DLBCL, NHL, CLL, and non-T-cell ALL, unless otherwise specified. Examples of CAR T-cell therapies and constructs are described in U.S. Patent Publications 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are incorporated herein by reference in whole.

[0079] As used herein, “patient” includes any human being affected by cancer (e.g., lymphoma or leukemia). The terms “subject” and “patient” are used interchangeably herein.

[0080] As used herein, the term “in vitro cells” refers to any cells cultured ex vivo. In particular, in vitro cells may include T cells.

[0081] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to one another by peptide bonds. As used herein, the above terms also refer to both short chains, commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as proteins, of which many types exist. Examples of “polypeptides” include, in particular, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0082] As used herein, “stimulus” refers to a primary response induced by the binding of a stimulating molecule to a homologous ligand, where the binding mediates a signaling event. “Stimulating molecule” refers to a molecule on a T cell, such as the T cell receptor (TCR) / CD3 complex that specifically binds to a homologous stimulating ligand present on an antigen-presenting cell. “Stimulating ligand” is a ligand that, when present on an antigen-presenting cell (e.g., APC, dendritic cell, B cell, etc.), can specifically bind to a stimulating molecule on a T cell, thereby mediating a primary response by T cells, including, but not limited to, T cell activation, initiation of an immune response, and proliferation. Examples of stimulating ligands include, but are not limited to, anti-CD3 antibodies, peptide-filled MHC class I molecules, superagonist anti-CD2 antibodies, and superagonist anti-CD28 antibodies. It can be done.

[0083] As used herein, "co-stimulatory signal" refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, elicits a T cell response, including but not limited to T cell proliferation and / or upregulation or downregulation of major molecules.

[0084] As used herein, “costimulatory ligands” include molecules on antigen-presenting cells that specifically bind to a congeneral costimulatory molecule on a T cell. The binding of a costimulatory ligand provides a signal that mediates T cell responses, including, but is not limited to, T cell proliferation, activation, and differentiation. In addition to primary signals, costimulatory ligands induce signals provided by stimulatory molecules, for example, by the binding of a T cell receptor (TCR) / CD3 complex to a peptide-loaded major histocompatibility complex (MHC) molecule. Examples of costimulatory ligands include, but are not limited to, 3 / TR6, 4-1BB ligands, agonists or antibodies that bind to Toll ligand receptors, B7-1 (CD80), B7-2 (CD86), CD30 ligands, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, and immunoglobulin-like molecules. Examples of costimulatory ligands include immunoglobulin-like transcript (ILT) 3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), ligands that specifically bind 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 cell death (PD) L1. Examples of costimulatory ligands include, but are not limited to, ligands that specifically bind to 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, CD83, and antibodies that specifically bind to costimulatory molecules present on T cells, such as 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).

[0085] "Co-stimulatory molecules" are congeneral binding partners on T cells that mediate co-stimulatory responses by T cells, such as proliferation, by specifically binding to co-stimulatory ligands, although these are not limited to those mentioned above. Examples of co-stimulatory molecules include, but are not limited to, 4-1BB / CD137, B7-H3, BAFFR, BLAME(SLAMF8), BTLA, CD33, CD45, CD100(SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160(BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276(B7-H3), CD28, CD29, C D3 (alpha; beta; delta; epsilon; gamma; zeta), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8 alpha, CD8 beta, CD9, CD96 (tactile), CD11a, CD11b, CD11c, CD11d, CDDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7 ITGB1, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18)), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activating molecules, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6, or their fragments, truncations, or The following combinations can be listed.

[0086] The terms "reducing" and "decreasing" are used interchangeably herein and refer to any change that is less than what it originally was. "Reducing" and "decreasing" are relative terms that require a comparison between the measured and measured states. "Reducing" and "decreasing" include complete deficiency.

[0087] The "treatment" of the subject or the "treating" of the subject. "Treatment" refers to any type of treatment or process performed on a subject, or administration of an active ingredient to a subject, for the purpose of altering, alleviating, improving, inhibiting, delaying, or preventing the onset, progression, manifestation, severity, or recurrence of symptoms, complications, or disease-related biochemical indicators. In some embodiments, "treatment" or "to treat" includes partial remission. In other embodiments, "treatment" or "to treat" includes complete remission.

[0088] Various aspects of the present invention are described in further detail in the following subsections.

[0089] Chimeric antigen receptor Chimeric antigen receptors (CARs or CAR-Ts) are genetically modified receptors. These modified receptors can be readily inserted into immune cells, including T cells, by techniques known in the art, and expressed by those cells. CARs can be programmed so that a single receptor recognizes a specific antigen and, upon binding to that antigen, activates immune cells to attack and destroy cells possessing that antigen. When these antigens are present on tumor cells, immune cells expressing CARs can target and kill the tumor cells.

[0090] Manipulated T cells and use CD19-directed gene-modified autologous T-cell immunotherapy agents are indicated for the treatment of patients with relapsed or treatment-resistant large B-cell lymphoma after two or more lines of systemic therapy, including DLBCL resulting from unspecified diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, and follicular lymphoma. In some embodiments, the CD19-directed gene-modified autologous T-cell immunotherapy agent is axicaptagen siloleucel (Axi-cel®, YESCARTA®).

[0091] The cells of this disclosure can be obtained through T cells obtained from a subject. T cells may be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from an infection site, ascites, pleural fluid, splenic tissue, and tumors. Furthermore, T cells may be derived from one or more T cell lines available in the art. Also, T cells may be obtained from blood units collected from a subject using numerous techniques known to those skilled in the art, such as FICOLL® isolation and / or apheresis. In some embodiments, cells collected by apheresis are washed to remove the plasma fraction and placed in a buffer or medium suitable for subsequent processing. In some embodiments, cells are washed with PBS. As understood, the washing step may be performed by using, for example, a Cobe® 2991 cell processing device, a semi-automatic flow-through centrifuge such as a Baxter CytoMate®, etc. In some embodiments, the washed cells are resuspended in one or more biocompatible buffers, or other physiological saline solutions with or without buffers. Several embodiments Then, undesirable components are removed from the apheresis sample. An additional method for isolating T cells for T cell therapy is disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is incorporated herein by reference in whole.

[0092] In some embodiments, T cells are isolated from PBMCs by monocyte depletion, for example, by erythrocyte lysis and centrifugation using a PERCOLL® gradient. In some embodiments, specific subpopulations of T cells, such as CD4-positive, CD8-positive, CD28-positive, CD45RA-positive, and CD45RO-positive T cells, are further isolated by positive or negative selection techniques known in the art. For example, enrichment of T cell populations by negative selection can be carried out by a combination of antibodies against surface markers specific to negatively selected cells. In some embodiments, cell sorting and / or selection by negative magnetic immunoadherence or flow cytometry is used, using a cocktail of monoclonal antibodies against cell surface markers present on negatively selected cells. For example, to enrich CD4-positive cells by negative selection, a monoclonal antibody cocktail typically includes antibodies against CD8, CD11b, CD14, CD16, CD20, and HLA-DR. In some embodiments, flow cytometry and cell sorting are used to isolate the cell population of interest for use in this disclosure.

[0093] In some embodiments, PBMCs are directly used for genetic modification by immune cells (such as CARs) using the methods described herein. In some embodiments, after isolating the PBMCs, T lymphocytes are further isolated, and both cytotoxic T lymphocytes and helper T lymphocytes are sorted into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion.

[0094] In some embodiments, CD8-positive cells are further sorted into naive cells, central memory cells, and effector cells by identifying cell surface antigens associated with these various CD8-positive cells. In some embodiments, the expression of phenotypic markers in central memory T cells includes CCR7, CD3, CD28, CD45RO, CD62L, and CD127, and is negative for granzyme B. In some embodiments, central memory T cells are CD8-positive, CD45RO-positive, and CD62L-positive T cells. In some embodiments, effector T cells are negative for CCR7, CD28, CD62L, and CD127, and positive for granzyme B and perforin. In some embodiments, CD4-positive T cells are further sorted into subpopulations. For example, CD4-positive helper T cells can be sorted into naive cells, central memory cells, and effector cells by identifying cell populations possessing cell surface antigens.

[0095] In some embodiments, immune cells, such as T cells, are genetically modified using known methods following isolation, or are activated and expanded (or, in the case of progenitor cells, differentiated) in vitro prior to genetic modification. In other embodiments, immune cells, such as T cells, are genetically modified with chimeric antigen receptors described herein (e.g., transduced by a viral vector containing one or more nucleotide sequences encoding a CAR), and then activated and / or expanded in vitro. 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 International Publication No. 2012 / 079000, the contents of which are incorporated herein by reference in whole. Generally, such methods involve contacting PBMCs or isolated T cells with stimulants and co-stimulants, such as anti-CD3 antibodies and anti-CD28 antibodies, typically attached to beads or other surfaces, in a culture medium containing appropriate cytokines such as IL-2. The anti-CD3 and anti-CD28 antibodies attached to the same beads act as "surrogate" antigen-presenting cells (APCs). One example is human T cells. The Dynabeads® system is a CD3 / CD28 activator / stimulator system for physiological activation. In other embodiments, T cells are activated, stimulated, and proliferated by feeder cells, as well as appropriate antibodies and cytokines, using methods such as those described in U.S. Patent No. 6,040,177 and No. 5,827,642, and PCT International Publication No. 2012 / 129514 (the contents of which are incorporated herein by reference in whole).

[0096] In some embodiments, T cells are obtained from a donor subject. In some embodiments, the donor subject is a human patient with cancer or a tumor. In some embodiments, the donor subject is a human patient who does not have cancer or a tumor.

[0097] In some embodiments, the composition comprises a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, and / or adjuvant. In some embodiments, the composition comprises an excipient.

[0098] In some embodiments, the composition is selected for parenteral delivery, inhalation, or delivery via the gastrointestinal tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the scope of those skilled in the art. In some embodiments, a buffer is used to maintain the composition at a physiological pH or slightly lower, typically in the range of about 5 to about 8. In some embodiments, when parenteral administration is considered, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the composition described herein, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle. In some embodiments, the vehicle for parenteral injection is sterile distilled water, in which the composition described herein is formulated as a sterile isotonic solution in which it is adequately stored, with or without at least one additional therapeutic agent. In some embodiments, the preparation involves formulation of the desired molecule with a polymer (such as polylactic acid or polyglycolic acid), beads, or liposomes that provide controlled or sustained release of the product, which is then delivered by depot injection. In some embodiments, an implantable drug delivery device is used to introduce a desired molecule.

[0099] In some embodiments, methods for treating cancer in a subject requiring treatment include T-cell therapy. In some embodiments, the T-cell therapy disclosed herein is engineered autologous cell therapy (eACT™). According to this embodiment, the method may include collecting blood cells from a patient. The isolated blood cells (e.g., T cells) may then be engineered to express CAR or TCR as disclosed herein. In certain embodiments, CAR T cells or TCR T cells are administered to the patient. In some embodiments, CAR T cells or TCR T cells treat a tumor or cancer in the patient. In some embodiments, CAR T cells or TCR T cells reduce the size of the tumor or cancer.

[0100] In some embodiments, donor T cells for use in T cell therapy are obtained from a patient (for example, for autologous T cell therapy). In other embodiments, donor T cells for use in T cell therapy are obtained from a non-patient subject. T cells may be administered in a therapeutically effective dose. For example, a therapeutically effective dose of T cells may be at least about 10 4 individual cells, at least about 10 5 individual cells, at least about 10 6 individual cells, at least about 10 7 individual cells, at least about 10 8 individual cells, at least about 10 9 A number of cells, or at least about 10 10 They can become individual cells. In another embodiment, the therapeutically effective dose of T cells is about 10 4 individual cells, approximately 10 5 individual cells, approximately 10 6 individual cells, approximately 10 7 A single cell, or about 10 8 These are individual cells. In some embodiments, the therapeutically effective dose of CAR T cells is approximately 2 × 10⁶ 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×106 cells / kg, approximately 9×10 6 cells / k g, about 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 Cells / kg, or approximately 9 x 10⁻⁶ 7 The value is cells / kg. In some embodiments, a therapeutically effective dose of CAR-positive viable T cells is approximately 1 × 10⁶ 8 Approximately 1 × 10¹¹ CAR-positive surviving T cells per kg of body weight 6 From each piece, approximately 2 x 10 6 These are CAR-positive surviving T cells between individuals.

[0101] In some embodiments, a therapeutically effective amount of CAR-positive viable T cells is approximately 0.4 × 10⁶ 8 pieces and approximately 2 x 10 8 These are CAR-positive surviving T cells between individuals. In some embodiments, a therapeutically effective amount of CAR-positive surviving T cells is approximately 0.4 × 10⁶ 8 pieces, approximately 0.5×10 8 pieces, approximately 0.6×10 8 pieces, approximately 0.7×10 8 pieces, approximately 0.8×10 8 pieces, approximately 0.9×10 8 pieces, approximately 1.0×10 8 pieces, approximately 1.1×10 8 pieces, approximately 1.2×10 8 pieces, approximately 1.3×10 8 pieces, approximately 1.4×10 8 pieces, approximately 1.5×10 8 pieces, approximately 1.6×10 8 pieces, approximately 1.7×10 8 pieces, approximately 1.8×10 8 pieces, approximately 1.9×10 8 1 or approximately 2.0 × 10 8 These are individual CAR-positive surviving T cells.

[0102] Treatment method The methods disclosed herein may be used to treat cancer in a subject, reduce tumor size, kill tumor cells, prevent tumor cell proliferation, prevent tumor growth, eliminate tumor from a patient, prevent tumor recurrence, prevent tumor metastasis, induce remission in a patient, or any combination thereof. In some embodiments, the methods induce complete response. In other embodiments, the methods induce partial response.

[0103] Cancers that can be treated include non-angiogenic tumors, tumors that are not yet essentially angiogenic, or angiogenic tumors. Cancers may also include solid tumors or non-solid tumors. In some embodiments, cancer is a hematological cancer. In some embodiments, cancer is a leukocyte cancer. In other embodiments, cancer is a plasma cell cancer. In some embodiments, cancer is leukemia, lymphoma, or myeloma. In some embodiments, cancer is acute lymphoblastic leukemia (ALL) (including non-T cell ALL), acute lymphoblastic leukemia (ALL) and hemophagocytic lymphohistiocytosis (HLH), B-cell prelymphoblastic leukemia, B-cell acute lymphoblastic leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasms, Burkitt lymphoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic or acute granulomatous diseases, Chronic or acute leukemia, diffuse large B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, follicular lymphoma (FL), hairy cell leukemia, hemophagocytic syndrome (macrophage activation syndrome (MAS)), Hodgkin's disease, large cell granuloma, leukocyte adhesion disorder, malignant lymphoproliferative disorder, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, monoclonal gammaglobulinemia of unknown significance (MGUS) ), multiple myeloma, myelodysplasia and myelodysplastic syndromes (MDS), bone marrow diseases including but not limited to acute myeloid leukemia (AML), non-Hodgkin lymphoma (NHL), plasma cell proliferation disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or painless myeloma), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, plasmacytomas (e.g., plasma cell hyperplasia; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple myeloma) These include stoichioma, POEMS syndrome (Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome), mediastinal large B-cell lymphoma (PMBC), small cell or large cell follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoblastic leukemia ("TALL"), T-cell lymphoma, post-transformed follicular lymphoma, Waldenström macroglobulinemia, or combinations thereof.

[0104] In some embodiments, the cancer is myeloma. In some embodiments, the cancer is multiple myeloma. In some embodiments, cancer is leukemia. In some embodiments, cancer is acute myeloid leukemia.

[0105] In some embodiments, the above method further includes administering a chemotherapeutic agent. In some embodiments, the selected chemotherapeutic agent is a lymphocyte-depleting (preconditioning) chemotherapeutic agent. Beneficial preconditioning treatment plans, along with corresponding beneficial biomarkers, are described in U.S. Provisional Patent Applications 62 / 262,143 and 62 / 167,750, which are incorporated herein by reference in their entirety. These include, for example, identified beneficial doses of cyclophosphamide (200 mg / m²). 2 2000 mg / m² per day 2 (for a period of / day) and a specified dose of fludarabine (20 mg / m²) 2 900 mg / m² per day 2 The text describes a method for conditioning patients requiring T-cell therapy, which involves administering the patient approximately 500 mg / m² over three days prior to administering a therapeutically effective dose of engineered T cells to the patient. One such dosing regimen involves administering the patient approximately 500 mg / m² over three days prior to administering the engineered T cells to the patient. 2 Cyclophosphamide and approximately 60 mg / m³ per day 2 The patient's treatment involves administering fludarabine daily.

[0106] In some embodiments, antigen-binding molecules, transduced (or manipulated) cells (e.g., CARs), and chemotherapeutic agents are administered in amounts effective to treat a disease or condition in the subject.

[0107] In some embodiments, compositions comprising immunoeffector cells expressing the CARs disclosed herein may be administered in combination with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®), alkyl sulfonic acid esters such as busulfan, improsulfan and biposulfan, aziridines such as benzodopa, carbocone, meturedopa and uredopa, ethyleneimines and methylmelamines including regimes of altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine, chlorambucil, and chloro Lunafadin, cyclophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, novoenicin, phenestrin, prednimustine, trophosphamide, nitrogen mustards such as uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine and other nitrosoureas, acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, kakutinomycin, calitiamicin, carabicin, carmino Mycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, Zinosta Antibiotics such as tin and zolubicin, antimetabolites such as methotrexate and 5-fluorouracil (5-FU), folic acid analogs such as denopterin, methotrexate, pteropterin, and trimethrexate, purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5 - Pyrimidine analogs such as FU, carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone and other androgens, anti-adrenal agents such as aminoglutethimide, mitotane, trilostane, folic acid and other folic acid solutions, acegraton, aldofsphamide glycoside, aminolevulinic acid, amsacrin, bestrabusil, bisanthren, edatraxate, defofamine, demecoltin, dia Dicon, elformithine, eriptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, ronidamin, mitoglucon, mitoxantrone, mopidamol, nitracrine, pentostatin, fenamet, pirarubicin, podophyllic acid, 2-ethylhydrazide, procarbazine, PSK (trademark), razoxane, schizophyllan, spirogermanium, tenuazonic acid, triadicone, 2,2',2''-trichlorotriethylamine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitractol, pipobromane, gacytosine, arabinoside ("Ara-C"), cyclophosphamide, thiotepa, taxoid, e.g., paclitaxel (TAXOL (trademark), Bristol-Myers Squibb Inc. and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer Inc.), chlorambutyl, gemcitabine, 6-Citrate Examples include platinum analogs such as ogwanine, mercaptopurine, methotrexate, cisplatin and carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, navelbine, novantrone, teniposide, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS2000, difluoromethylornithine (DMFO), retinoic acid derivatives such as Targretin (trademark) (bexarotene) and Panretin (trademark) (allitretinoin), ONTAK (trademark) (denileukin diffitox), esperamicin, capecitabine, and any of the above pharmaceutically acceptable salts, acids, or derivatives. In some embodiments, compositions comprising immune effector cells expressing CARs and / or TCRs disclosed herein are used with antihormone agents that act to modulate or inhibit hormonal effects on tumors, such as tamoxifen, raloxifen, aromatase inhibitory 4(5)-imidazoles, 4-hydroxytamoxifen, trioxyfen, keoxifen, and LY117018. It may be administered in combination with anti-estrogen agents including onapristone and toremifene (Fareston), as well as anti-androgen agents such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, and any pharmaceutically acceptable salt, acid, or derivative of any of the above. Depending on the circumstances, but not limited to, a combination of chemotherapeutic agents including CHOP, i.e., cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone, may also be administered.

[0108] In some embodiments, the chemotherapeutic agent is administered simultaneously with or within one week after the administration of the engineered cells or nucleic acids. In other embodiments, the chemotherapeutic agent is administered one to four weeks, one week to one month, one week to two months, one week to three months, one week to six months, one week to nine months, or one week to twelve months after the administration of the engineered cells or nucleic acids. In some embodiments, the chemotherapeutic agent is administered at least one month before the administration of the cells or nucleic acids. In some embodiments, the above method further includes the administration of two or more chemotherapeutic agents.

[0109] Various additional therapeutic agents may be used in conjunction with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), pembrolizumab, pidilizumab (CureTech), and atezolizumab (Roche).

[0110] Additional therapeutic agents suitable for use in combination with the compositions and methods disclosed herein include, but are not limited to, ibrutinib (IMBRUVICA®), ofatumumab (ARZERRA®), rituximab (RITUXAN®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), trastuzumab emtansine (KADCYLA®), imatinib (GLEEVEC®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), catumakisomab, ibritumomab, ofatumumab, and tositumomab. Brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, macitinib, pazopanib, sunitinib, sorafenib, toceranib, restaurtinib, axitinib, cejiranib, lenvatinib, nintedanib, pazopanib, regorafenib, semacsanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib Examples include mTOR inhibitors such as dasatinib, nilotinib, ponatinib, radotinib, bosutinib, restaurtinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin-diffitox, everolimus, and temsirolimus; hedgehog inhibitors such as sonidecib and bismodegib; and CDK inhibitors such as palbociclib.

[0111] In some embodiments, the composition containing CAR immune cells is administered together with an anti-inflammatory agent. Examples of anti-inflammatory agents include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone), aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF agents, cyclophosphamide, and non-steroidal anti-inflammatory drugs (NSAIDs) including mycophenolates. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialates. Exemplary analgesics include acetaminophen, oxycodone, tramadol, or propoxyfene hydrochloride. Examples of glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Examples of biological response modifiers include molecules targeting cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®)), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers also include monoclonal antibodies and recombinant forms of molecules. Examples of DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (Auranofin) and intramuscular), and minocycline.

[0112] In some embodiments, the compositions described herein are administered in combination with cytokines. Examples of cytokines include lymphokines, monokines, and conventional polypeptide hormones. Among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; glycoprotein hormones such as parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF), fibroblast growth factor (FGF), prolactin, placental lactogen, Müllerian duct inhibitor, mouse sex gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), nerve growth factor (NGF) such as NGF-beta, platelet growth factor, and T Transforming growth factors (TGF) such as GF-alpha and TGF-beta, insulin-like growth factors I and II, erythropoietin (EPO, Epogen®, Procrit®), bone induction factors, interferons such as interferon-alpha, beta and gamma, colony-stimulating factors (CSF) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF), interleukins (IL) such as IL-1, IL-1-alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, and IL-15, TNF Examples include tumor necrosis factors such as alpha or TNF-beta, as well as other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins of natural origin or derived from recombinant cell cultures, and bioactive equivalents of cytokines of natural sequences.

[0113] Administration of CD19-targeted gene-modified autologous T-cell immunotherapy drugs Adaptation and Use In some embodiments, CD19-directed gene-modified autologous T-cell immunotherapy agents are indicated for the treatment of adult patients with relapsed or refractory large B-cell lymphoma after two or more lines of systemic therapy, including DLBCL (unspecified diffuse large B-cell lymphoma), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, and DLBCL resulting from follicular lymphoma. In some embodiments, CD19-directed gene-modified autologous T-cell immunotherapy agents are not indicated for the treatment of patients with primary central nervous system lymphoma.

[0114] Medication and administration In some embodiments, the infusion bag of the CD19-targeted genetically modified autologous T-cell immunotherapy agent contains approximately 68 mL of a suspension of chimeric antigen receptor (CAR)-positive T cells. The target dose is approximately 1 × 10⁶ per kg of body weight. 6 pieces and approximately 2 x 10 6 These are CAR-positive surviving T cells between individuals, up to 2 × 10⁶ 8 This can be a number of CAR-positive viable T cells. In some embodiments, the CD19-targeted gene-modified autologous T cell immunotherapy agent is Axi-cel® (YESCARTA®, axicapbutagensiloleucel).

[0115] CD19-directed genetically modified autologous T-cell immunotherapy drugs are for self-administration. The patient's identity must match the patient identifier on the CD19-directed genetically modified autologous T-cell immunotherapy drug cassette and infusion bag. If the information on the patient-specific label does not match the patient, the CD19-directed genetically modified autologous T-cell immunotherapy drug cannot be administered.

[0116] In some embodiments, the drug efficacy of CD19-targeted gene-modified autologous T-cell immunotherapy agents must be confirmed before initiating a lymphocyte depletion regimen.

[0117] In some embodiments, patients are pre-treated with lymphocyte-depleting chemotherapy drugs prior to the infusion of CD19-targeted genetically modified autologous T-cell immunotherapy drugs. In some embodiments, cyclophosphamide 500 mg / m² is administered on days 5, 4, and 3 prior to the infusion of CD19-targeted genetically modified autologous T-cell immunotherapy drugs. 2 (intravenously) and fludarabine 30 mg / m² 2 A lymphocyte depletion chemotherapy regimen (intravenous) is administered.

[0118] In some embodiments, patients are premedicated by orally administering acetaminophen in doses between approximately 500 mg and 1000 mg, approximately 600 mg and 1000 mg, approximately 700 mg and 1000 mg, approximately 800 mg and 1000 mg, approximately 900 mg and 1000 mg, approximately 500 mg and 900 mg, approximately 500 mg and 800 mg, approximately 500 mg and 700 mg, approximately 500 mg and 600 mg, approximately 600 mg and 900 mg, approximately 600 mg and 800 mg, approximately 600 mg and 700 mg, approximately 700 mg and 900 mg, or approximately 700 mg and 800 mg before infusion of the CD19-targeted genetically modified autologous T-cell immunotherapy agent. In some embodiments, patients are premedicated by orally administering acetaminophen in doses of approximately 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, or 1000 mg prior to the infusion of the CD19-directed genetically modified autologous T-cell immunotherapy agent.

[0119] In some embodiments, patients are premedicated by receiving 650 mg of acetaminophen orally and 12.5 mg of diphenhydramine intravenously or orally approximately one hour before the infusion of the CD19-targeted genetically modified autologous T-cell immunotherapy agent.

[0120] In some embodiments, prophylactic use of systemic steroids is avoided because systemic steroids may interfere with the activity of CD19-targeted genetically modified autologous T-cell immunotherapy agents.

[0121] Preparation of CD19-targeted gene-modified autologous T-cell immunotherapy drugs for injection The timing of thawing and infusion of the CD19-directed gene-modified autologous T-cell immunotherapy drug is adjusted. In some embodiments, the infusion time is confirmed in advance, and the start time of thawing the CD19-directed gene-modified autologous T-cell immunotherapy drug is adjusted so that it is available for infusion when the patient is ready.

[0122] In some embodiments, patient identity is verified before thawing the CD19-directed genetically modified autologous T-cell immunotherapy drug. Before preparing the CD19-directed genetically modified autologous T-cell immunotherapy drug, patient identity is matched against a patient identifier on the CD19-directed genetically modified autologous T-cell immunotherapy drug cassette. In some embodiments, if the information on the patient-specific label does not match the patient in question, the product bag of the CD19-directed genetically modified autologous T-cell immunotherapy drug is not removed from the cassette.

[0123] In some embodiments, once patient matching is confirmed, the product bag of the CD19-directed gene-modified autologous T-cell immunotherapy drug is removed from the cassette, and the patient information on the cassette label is checked and matched with the label on the bag.

[0124] In some embodiments, the method includes inspecting the product bag for any breach of container integrity, such as damage or cracks, before thawing. In some embodiments, the infusion bag is placed inside a second sterile bag in accordance with local guidelines.

[0125] In some embodiments, the method includes thawing the CD19-directed genetically modified autologous T-cell immunotherapy drug at approximately 37°C using either a water bath or dry thawing method until no visible ice remains in the infusion bag. In some embodiments, the method includes mixing or agitating the contents of the bag to disperse any clumps of cellular material. In some embodiments, the contents of the bag are gently mixed or agitated. In some embodiments, the method includes inspecting the bag for the presence of any visible residual cell clumps, and mixing or agitation is continued. Small clumps of cellular material should be dispersed by gentle manual mixing. In some embodiments, the method does not include washing, centrifuging, and / or resuspending the CD19-directed genetically modified autologous T-cell immunotherapy drug before infusion.

[0126] In some embodiments, once thawed, the CD19-targeted gene-modified autologous T-cell immunotherapy drug can be stored at room temperature (20°C to 25°C) for up to 3 hours.

[0127] Administration In some embodiments, the administration method of a CD19-targeted gene-modified autologous T-cell immunotherapy agent disclosed herein includes one or more of the following as steps or precautions: Ensure that tocilizumab and emergency equipment are available before infusion and during the recovery period. Do not use a leukocyte removal filter. Central venous access is recommended for the infusion of CD19-targeted gene-modified autologous T-cell immunotherapy drugs. Verify that the patient's identity matches the patient identifier on the product bag of the CD19-directed gene-modified autologous T-cell immunotherapy drug. Prime the tube with saline solution before infusion. The entire contents of the CD19-targeted genetically modified autologous T-cell immunotherapy drug bag are injected within 30 minutes using either gravity or a peristaltic pump. The CD19-targeted genetically modified autologous T-cell immunotherapy drug is stable at room temperature for up to 3 hours after thawing. To prevent cell aggregation, gently agitate the product bag during the infusion of the CD19-targeted gene-modified autologous T-cell immunotherapy drug. After injecting the entire contents of the product bag, rinse the tube with saline solution at the same injection rate to ensure that all product is dispensed. CD19-targeted genetically modified autologous T-cell immunotherapy drugs contain human blood cells genetically modified with replication-deficient retroviruses. Universal precautions and local biosafety guidelines should be followed for handling and disposal to avoid the potential transmission of infection.

[0128] monitoring In some embodiments, the administration of CD19-targeted gene-modified autologous T-cell immunotherapy drugs is performed in an authorized medical facility.

[0129] In some embodiments, the methods disclosed herein include monitoring the patient for signs and symptoms of CRS and neurotoxicity at an authorized medical facility for at least 7 days daily after infusion. In some embodiments, the methods disclosed herein include monitoring the patient for signs and symptoms of CRS and neurotoxicity at an authorized medical facility for at least 10 days daily after infusion.

[0130] In some embodiments, patients are instructed to remain within the vicinity of an authorized medical facility for at least four weeks after the injection.

[0131] Management of severe adverse reactions In some embodiments, the above method includes the management of adverse reactions. In some embodiments, adverse reactions are selected from the group consisting of cytokine release syndrome (CRS), neurotoxicity, hypersensitivity reactions, severe infection, cytopenia, and hypogammaglobulinemia.

[0132] In some embodiments, signs and symptoms of adverse reactions are selected from the group consisting of fever, hypotension, tachycardia, hypoxia, and chills, and include cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal failure, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), seizures, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia.

[0133] Cytokine release syndrome In some embodiments, the method includes identifying CRS based on clinical symptoms. In some embodiments, the method includes evaluating and treating fever, hypoxia, and other causes of hypotension. When CRS is observed or suspected, it should be managed according to the recommendations in Table 1. Patients experiencing Grade 2 or higher CRS (e.g., non-fluid-responsive hypotension or hypoxia requiring oxygen supplementation) should be monitored using continuous telecardiogram and pulse oximetry. In some embodiments, for patients experiencing severe CRS, echocardiography should be considered to assess cardiac function. For severe or life-threatening CRS, intensive care supportive therapy may be considered. In some embodiments, a biosimilar or equivalent of tocilizumab may be used instead of tocilizumab in the method disclosed herein. It is possible.

[0134] [Table 1]

[0135] Neurotoxicity In some embodiments, the method includes monitoring the patient for signs and symptoms of neurotoxicity (Table 2). In some embodiments, the method includes ruling out other causes of neurological symptoms. Patients who have experienced Grade 2 or higher neurotoxicity should be monitored using continuous telecardiogram and pulse oximetry. In cases of severe or life-threatening neurotoxicity, intensive supportive care should be provided. In all cases of Grade 2 or higher neurotoxicity, non-sedating antiepileptic drugs (e.g., levetiracetam) should be considered for seizure prevention.

[0136] [Table 2]

[0137] Dosage form and quantity In some embodiments, the CD19-targeted gene-modified autologous T-cell immunotherapy agent is available as a cell suspension for injection.

[0138] In some embodiments, a single dose of CD19-targeted gene-modified autologous T-cell immunotherapy is approximately 1 × 10⁶ units per kg of body weight in a suspension of approximately 68 mL in an infusion bag. 6 pieces and approximately 2 x 10 6 CAR-positive surviving T cells between individuals (or up to 2 × 10 for patients weighing 100 kg or more) 8 It includes a targeted dose of (number of CAR-positive surviving T cells). In some embodiments, the CD19-directed gene-modified autologous T cell immunotherapy agent is axicapbutogen siloleucel (YESCARTA®).

[0139] In some embodiments, a single dose of the CD19-targeted gene-modified autologous T-cell immunotherapy agent is contained in a container. Such containers may be sterile. In some embodiments, the container is an infusion bag. In some embodiments, the capacity of the infusion bag is approximately 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 500 mL, 750 mL, 1000 mL, 1500 mL, 2000 mL, or 3000 mL.

[0140] Risk Assessment and Mitigation Strategy (REMS) Due to the risks of CRS and neurotoxicity, in some embodiments, CD19-targeted genetically modified autologous T-cell immunotherapy agents are available through a restricted program under a risk assessment and mitigation strategy (REMS). Typical components of a REMS are as follows: Medical facilities that compound and administer CD19-targeted gene-modified autologous T-cell immunotherapy drugs must be registered and comply with REMS requirements. Authorized medical facilities must ensure that tocilizumab is readily available on-site and, if required for the treatment of CRS, that at least two doses of tocilizumab are available for infusion for each patient within two hours of infusion of the CD19-directed genetically modified autologous T-cell immunotherapy agent. Accredited healthcare facilities must ensure that healthcare providers prescribing, compounding, or administering CD19-targeted genetically modified autologous T-cell immunotherapy agents are proficient in managing CRS and neurotoxicity.

[0141] Cytokine release syndrome (CRS) In some embodiments, the medical facility ensures that two doses of tocilizumab are available before infusion of the CD19-directed genetically modified autologous T-cell immunotherapy agent. In some embodiments, the medical facility ensures that four doses of tocilizumab are available before infusion of the CD19-directed genetically modified autologous T-cell immunotherapy agent. In some embodiments, the method includes monitoring the patient for signs and symptoms of CRS at an authorized medical facility for at least seven days daily after infusion. In some embodiments, the method includes monitoring the patient for signs and symptoms of CRS at an authorized medical facility for at least seven to ten days daily after infusion. In some embodiments, the method includes monitoring the patient for signs and symptoms of CRS at an authorized medical facility for at least eight days daily after infusion. In some embodiments, the method includes monitoring the patient for signs and symptoms of CRS at an authorized medical facility for at least nine days daily after infusion. In some embodiments, the method includes monitoring the patient for signs and symptoms of CRS at an authorized medical facility for at least 10 days daily after infusion. In some embodiments, the method includes monitoring the patient for signs or symptoms of CRS for 4 weeks after infusion. In some embodiments, the method includes advising the patient to seek immediate medical attention whenever signs or symptoms of CRS occur. In some embodiments, the method includes providing supportive care, tocilizumab, or tocilizumab as indicated when the first signs of CRS are observed. This includes initiating treatment with mab and corticosteroids.

[0142] Neurotoxicity In some embodiments, the method includes monitoring the patient for signs and symptoms of neurotoxicity at an authorized medical facility for at least 7 days daily after the injection. In some embodiments, the method includes monitoring the patient for signs and symptoms of CRS at an authorized medical facility for at least 7 to 10 days daily after the injection. In some embodiments, the method includes monitoring the patient for signs and symptoms of CRS at an authorized medical facility for at least 10 days daily after the injection. In some embodiments, the method includes monitoring the patient for signs or symptoms of neurotoxicity for 4 weeks after the injection and providing prompt treatment.

[0143] Hypersensitivity Infusion of CD19-targeted genetically modified autologous T-cell immunotherapy agents may cause allergic reactions. In some embodiments, severe hypersensitivity reactions, including anaphylaxis, may be due to residual dimethyl sulfoxide (DMSO) or gentamicin in the CD19-targeted genetically modified autologous T-cell immunotherapy agent.

[0144] Virus reactivation In some embodiments, reactivation of hepatitis B virus (HBV), which may result in fulminant hepatitis, liver failure, and death, may occur in patients treated with drugs targeting B cells. In some embodiments, the above method includes screening for HBV, HCV, and HIV in accordance with clinical guidelines before collecting cells for production.

[0145] Long-term cytopenia In some embodiments, patients may exhibit cytopenia for several weeks following lymphocyte depletion chemotherapy and infusion of CD19-directed genetically modified autologous T-cell immunotherapy agents. In some embodiments, the method includes monitoring blood cell counts after infusion of CD19-directed genetically modified autologous T-cell immunotherapy agents.

[0146] Hypogammaglobulinemia In some embodiments, B-cell aplasia and hypogammaglobulinemia may occur in patients receiving treatment with CD19-directed genetically modified autologous T-cell immunotherapy agents. In some embodiments, the method includes monitoring immunoglobulin levels after treatment with CD19-directed genetically modified autologous T-cell immunotherapy agents and managing them using infection precautions, antibiotic prophylaxis, and immunoglobulin supplementation.

[0147] In some embodiments, vaccination with live viral vaccines is not recommended for at least six weeks prior to the initiation of lymphocyte apheresis chemotherapy, during CD19-targeted genetically modified autologous T-cell immunotherapy, and until immune recovery after treatment with CD19-targeted genetically modified autologous T-cell immunotherapy.

[0148] Secondary malignant tumors In some embodiments, patients treated with CD19-targeted gene-modified autologous T-cell immunotherapy agents may develop secondary malignancies. In some embodiments, the method includes monitoring the patient's lifetime for secondary malignancies.

[0149] Tumor lysis syndrome (TLS) Patients treated with CD19-targeted genetically modified autologous T-cell immunotherapy drugs may develop TLS, which can be severe. To minimize the risk of TLS, several measures are taken. In one embodiment, the method includes evaluating the patient for elevated uric acid levels or high tumor burden and administering allopurinol or an alternative prophylactic agent before infusion of axicapbutagen siloleucel. Signs and symptoms of TLS should be monitored, and the event should be managed according to standard guidelines.

[0150] Impact on the ability to operate and use the machine Due to the possibility of changes in mental state or neurological events, including seizures, patients receiving CD19-directed genetically modified autologous T-cell immunotherapy are at risk of changes or decreased consciousness or motor coordination within eight weeks after infusion of the CD19-directed genetically modified autologous T-cell immunotherapy drug. In some embodiments, the method includes advising patients to refrain from engaging in hazardous occupations or activities, such as driving a car or operating heavy machinery or potentially dangerous equipment, during this initial period.

[0151] Storage and handling In some embodiments, the CD19-directed genetically modified autologous T cell immunotherapy agent is supplied in an infusion bag containing approximately 68 mL of a frozen suspension of genetically modified autologous T cells in 5% DMSO and 2.5% albumin (human). In some embodiments, the CD19-directed genetically modified autologous T cell immunotherapy agent is supplied in an infusion bag containing approximately 68 mL of a frozen suspension of genetically modified autologous T cells in 5% DMSO and 2.5% albumin (human) (NDC 71287-119-01). In some embodiments, the CD19-directed genetically modified autologous T cell immunotherapy agent contains Cryostor CS10. In some embodiments, the CD19-directed genetically modified autologous T cell immunotherapy agent contains 300 mg of sodium per infusion. In some embodiments, the CD19-directed genetically modified autologous T cell immunotherapy agent is supplied in an infusion bag containing approximately 50 mL–100 mL, 50 mL–90 mL, 50 mL–80 mL, 50 mL–70 mL, 60 mL–70 mL, 60 mL–75 mL, or 65 mL–75 mL of suspension of genetically modified autologous T cells in 5% DMSO and 2.5% albumin (human). In some embodiments, the CD19-directed genetically modified autologous T cell immunotherapy agent is supplied in an infusion bag containing less than 100 mL, less than 90 mL, less than 80 mL, less than 70 mL, less than 70 mL, less than 72 mL, or less than 75 mL of suspension of genetically modified autologous T cells in 5% DMSO and 2.5% albumin (human). In some embodiments, the CD19-directed genetically modified autologous T-cell immunotherapy agent is supplied in an infusion bag containing more than 50 mL, more than 60 mL, more than 65 mL, more than 66 mL, more than 67 mL, or more than 68 mL of suspension of genetically modified autologous T-cells in 5% DMSO and 2.5% albumin (human). In some embodiments, the suspension is frozen.

[0152] In some embodiments, the infusion bag of the CD19-targeted genetically modified autologous T-cell immunotherapy agent is supplied in an ethylene-vinyl acetate cryopreservation bag containing approximately 68 mL of cell dispersion, equipped with a sealed addition tube and two available spike ports.

[0153] In some embodiments, the infusion bags of the CD19-directed genetically modified autologous T-cell immunotherapy drug are individually packaged in a metal cassette. In some embodiments, the infusion bags of the CD19-directed genetically modified autologous T-cell immunotherapy drug are individually packaged in a metal cassette (NDC 71287-119-02). In some embodiments, the infusion bags of the CD19-directed genetically modified autologous T-cell immunotherapy drug are stored in the gas phase of liquid nitrogen. In some embodiments, the infusion bags of the CD19-directed genetically modified autologous T-cell immunotherapy drug are supplied in a liquid nitrogen dry shipper.

[0154] In some embodiments, the above method includes matching the patient's identity with a patient identifier on the cassette and infusion bag at the time of receipt. In some embodiments, the CD19-directed genetically modified autologous T-cell immunotherapy drug is administered in the gas phase of liquid nitrogen (below -150°C). It is stored frozen. In some embodiments, the CD19-targeted gene-modified autologous T-cell immunotherapy drug is thawed before use. [Examples]

[0155] Example 1: Clinical study of relapsed or refractory large B-cell lymphoma A single-arm, open-label, multicenter trial evaluated the efficacy of a single infusion of Axi-cel® (YESCARTA®) in adult patients with relapsed or refractory invasive B-cell non-Hodgkin lymphoma. Eligible patients had disease refractory to most modern therapies or had relapsed within one year of autologous hematopoietic stem cell transplantation (HSCT). This study excluded patients with a prior allogeneic HSCT, a history of any central nervous system lymphoma, two or more ECOG performance statuses, an absolute lymphocyte count <100 / μL, creatinine clearance <60 mL / min, hepatic transaminases greater than 2.5 times the upper limit of normal, cardiac ejection fraction <50%, or active severe infection.

[0156] After lymphocyte apheresis chemotherapy, Axi-cel (trademark) is administered in 2 x 10 doses. 6Target dose of CAR-positive viable T cells / kg (maximum tolerable dose: 2 × 10⁶) 8 It was administered as a single intravenous infusion (individual cells). The lymphocyte depletion regimen was cyclophosphamide 500 mg / m². 2 (intravenously) and fludarabine 30 mg / m² 2 The treatment consisted of intravenous administration, both administered on days 5, 4, and 3 prior to Axi-cel® administration. No bridging chemotherapy between leukocyte apheresis and lymphocyte apheresis was observed. All patients were hospitalized for Axi-cel® infusion and remained hospitalized for a minimum of 7 days thereafter.

[0157] Of the 111 patients who underwent leukocytosis apheresis, 101 received Axi-cel™. The median age of the treated patients was 58 years (range: 23–76 years), 67% were male and 89% were Caucasian. The majority (76%) had DLBCL, 16% had transformed follicular lymphoma, and 8% had primary mediastinal large B-cell lymphoma. The median number of prior therapies was 3 (range: 1–10), 77% of patients had treatment-resistant disease to second-line or higher therapy, and 21% relapsed within one year of autologous HSCT.

[0158] Of the 111 patients, one did not receive the product due to a manufacturing failure. The other nine patients were not treated, mainly due to progressive disease or severe adverse reactions after leukocyte apheresis. The median time from leukocyte apheresis to product delivery was 17 days (range: 14 to 51 days), and the median time from leukocyte apheresis to infusion was 24 days (range: 16 to 73 days). The median dose was 2.0 × 10⁻⁶. 6 CAR-positive viable T cells / kg (range: 1.1 × 10⁻¹⁴) 6 Cells per kg ~ 2.2 × 10⁻⁶ 6 It was (number of cells / kg).

[0159] Efficacy was established based on the complete remission (CR) rate and duration of response (DOR) determined by an independent review committee (Tables 3 and 4). The median duration of response was 0.9 months (range: 0.8 months to 6.2 months). The duration of response was longer in patients who achieved CR compared to patients with the best partial remission (PR) (Table 4). Of the 52 patients who achieved CR, 14 initially had stable disease (7 patients) or PR (7 patients), and the median time to improvement was 2.1 months (range: 1.6 months to 5.3 months).

[0160] [Table 3]

[0161] [Table 4]

[0162] Example 2: Pharmacodynamics and pharmacokinetics after injection of Axi-cel® Pharmacodynamic responses were assessed over a 4-week interval by measuring transient increases in blood cytokines, chemokines, and other molecules after Axi-cel® infusion. Levels of cytokines and chemokines such as IL-6, IL-8, IL-10, IL-15, TNF-α, IFN-γ, and sIL2Rα were analyzed. Peak increases were observed within the first 14 days after infusion, and levels generally returned to baseline within 28 days. Due to the on-target effect of Axi-cel®, a period of B-cell aplasia is expected.

[0163] Following Axi-cel® injection, anti-CD19 CAR T cells showed initial rapid proliferation, followed by a decline to near baseline levels by 3 months. Peak levels of anti-CD19 CAR T cells occurred within the first 7–14 days after Axi-cel® injection. Age (range: 23–76 years) and sex were used in relation to the AUC of Axi-cel®. (0日目~28日目) It did not have a significant effect on Cmax.

[0164] The number of anti-CD19 CAR T cells in the blood was positively associated with objective response (complete remission (CR) or partial remission (PR)). The median Cmax level of anti-CD19 CAR T cells in responders (n=73) was 205% higher than the corresponding level in non-responders (n=23) (43.6 cells / μL vs. 21.2 cells / μL). The median AUC from day 0 to day 28 in responding patients (n=73) was 251% of the corresponding level in non-responders (n=23) (557.1 days × cells / μL vs. 222.0 days × cells / μL).

[0165] Some patients required tocilizumab and corticosteroids to manage CRS and neurotoxicity. Patients treated with tocilizumab (n=44) had a higher AUC compared to patients who did not receive tocilizumab (n=57). (0日目~28日目) They had 262% and 232% higher anti-CD19 CAR T cells, as measured by Cmax, respectively. Similarly, patients treated with corticosteroids (n=26) had 217% and 155% higher AUC compared to patients not treated with corticosteroids (n=75). (0日目~28日目) It also had Cmax.

[0166] Example 3: Management of adverse reactions after CD19-directed gene-modified autologous T-cell immunotherapy Because clinical trials are conducted under a wide variety of conditions, the adverse reaction rates observed in a clinical trial of one drug cannot be directly compared to rates in a clinical trial of another drug and may not reflect the actual observed rates.

[0167] The safety data described in this section reflect exposure to Axi-cel® in a clinical trial (Study 1), in which 108 patients with relapsed / refractory B-cell NHL were administered CAR-positive T cells based on a recommended dose according to body weight. Patients with a history of CNS disorders (e.g., seizures or cerebral ischemia) or autoimmune diseases requiring systemic immunosuppression were ineligible. The median follow-up period was 8.7 months. The median age of the study population was 58 years (range: 23–76 years), and 68% were male. Baseline ECOG performance status was ECOG0 for 43% and ECOG1 for 57%.

[0168] The most common adverse reactions (incidence ≥20%) include CRS, fever, hypotension, encephalopathy, tachycardia, malaise, headache, loss of appetite, chills, diarrhea, febrile neutropenia, infection by an unspecified pathogen, nausea, hypoxia, tremor, cough, vomiting, dizziness, constipation, and cardiac arrhythmias. Serious adverse reactions occurred in 52% of patients. The most common serious adverse reactions (≥2%) include encephalopathy, fever, pulmonary infection, febrile neutropenia, cardiac arrhythmias, heart failure, urinary tract infection, renal failure, aphasia, cardiac arrest, Clostridium difficile infection, delirium, hypotension, and hypoxia.

[0169] The most common (over 10%) grade 3 or higher reactions include febrile neutropenia, fever, CRS, encephalopathy, infection by an unspecified pathogen, hypotension, hypoxia, and pulmonary infection.

[0170] Tocilizumab was administered to 45% of patients (49 out of 108) after Axi-cel® injection.

[0171] Table 5 summarizes adverse reactions that occurred in at least 10% of patients treated with Axi-cel®, and Table 6 lists grade 3 or grade 4 abnormal clinical laboratory values ​​that occurred in at least 10% of patients.

[0172] [Table 5] TIFF2026048753000006.tif174170

[0173] Other clinically important adverse reactions that occurred in less than 10% of patients treated with Axi-cel (trademark) include the following: Blood and lymphatic system disorders: Coagulation disorders (2%) Cardiac disorders: Heart failure (6%) and cardiac arrest (4%) Immune system disorders: Hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS) (1%), allergy (1%) Infections and parasitic diseases: Fungal infections (5%) Nervous system disorders: Ataxia (6%), seizures (4%), dyscalculia (2%) and myoclonus (2%) Respiratory, thoracic and mediastinal disorders: Pulmonary edema (9%) Skin and subcutaneous tissue disorders: Rash (9%) Vascular disorders: Capillary leak syndrome (3%)

[0174] Abnormal clinical test values:

[0175] [Table 6]

[0176] Cytokine release syndrome After treatment with Axi-cel (trademark), CRS occurred, including fatal or life-threatening reactions. In Study 1, CRS occurred in 94% (101 / 108) of patients who received Axi-cel (trademark), including grade 3 or higher (Lee grading system 1 ) CRS in 13% (14 / 108) of patients. Axi-cel (trademark Of the patients who died after administration of the drug, four had advanced CRS events at the time of death. The median time to onset was 2 days (range: 1 to 12 days), and the median duration of CRS was 7 days (range: 2 to 58 days). The main symptoms of CRS include fever (78%), hypotension (41%), tachycardia (28%), hypoxia (22%), and chills (20%). Serious events that may be associated with CRS include cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal failure, capillary leak syndrome, hypotension, hypoxia, and hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS).

[0177] Neurotoxicity Fatal or life-threatening neurotoxicity occurred after treatment with Axi-cel®. Neurotoxicity occurred in 87% of patients. 98% of all neurotoxicities occurred within the first 8 weeks of Axi-cel® infusion, with a median time to onset of 4 days (range: 1 to 43 days). The median duration of neurotoxicity was 17 days. Grade 3 or higher neurotoxicity occurred in 31% of patients.

[0178] The most common neurotoxicities included encephalopathy (57%), headache (44%), tremor (31%), dizziness (21%), aphasia (18%), delirium (17%), insomnia (9%), and anxiety (9%). Long-term encephalopathy lasting up to 173 days was observed. Serious events, including leukoencephalopathy and seizures, occurred with Axi-cel®. Fatal and severe cases of cerebral edema occurred in patients treated with Axi-cel®.

[0179] Severe infection Severe or life-threatening infections occurred in patients after Axi-cel® administration. In Study 1, infections (of all grades) occurred in 38% of patients. Grade 3 or higher infections occurred in 23% of patients. Grade 3 or higher infections caused by non-specific pathogens occurred in 16% of patients, bacterial infections in 9%, and viral infections in 4%. Axi-cel® should not be administered to patients with clinically significant active systemic infections. Monitor the patient for signs and symptoms of infection before and after Axi-cel® injection and treat appropriately. Administer prophylactic antibiotics according to local guidelines.

[0180] Febrile neutropenia is observed in 36% of patients after Axi-cel® infusion, and may occur concurrently with CRS. In cases of febrile neutropenia, infection should be evaluated and managed with broad-spectrum antibiotics, fluid replacement, and other medically appropriate supportive therapies.

[0181] immunogenicity Axi-cel® may induce antibodies against its anti-products. The immunogenicity of Axi-cel® was evaluated using enzyme-linked immunosorbent assay (ELISA) to detect antibodies bound to FMC63, an antibody derived from anti-CD19 CARs. Pre-administration anti-FMC63 antibodies were positive in three patients at baseline and at 1, 3, or 6 months in Study 1. There is no evidence that these patients were affected by changes in the pharmacokinetics of Axi-cel®'s initial proliferation or long-term survival, or by the safety or efficacy of Axi-cel®.

[0182] In Study 1, grade 3 or higher cytopenia that did not resolve by 30 days after Axi-cel® infusion occurred in 28% of patients, including thrombocytopenia (18%), neutropenia (15%), and anemia (3%). Blood cell counts should be monitored after Axi-cel® infusion.

[0183] In Study 1, hypogammaglobulinemia occurred in 15% of patients.

[0184] All publications, patents, patent applications, and references cited herein constitute part of this specification to the same extent as each individual publication, patent, or patent application is specifically indicated as constituting part of this specification by reference. However, the references herein should not be construed as an acknowledgment that such references are prior art of the present invention. To the extent that any definition or term contained in a reference that constitutes part of this specification by reference differs from the terms and discussions contained herein, the terms and definitions herein shall prevail.

Claims

1. A method for treating relapsed or refractory unspecified diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, or follicular lymphoma-related DLBCL in patients after two or more lines of systemic therapy, In patients requiring treatment, axicapbutagen siloleucel suspension is administered intravenously by approximately 1 x 10⁶ units per kg of body weight. ６ Each and approximately 2 x 10 ６ Approximately 1 × 10⁶ CAR-positive surviving T cells were obtained between individuals. ８ This includes administering up to the maximum dose of individual CAR-positive viable T cells. Herein, axicapbutagen siloleucel is a CD19-targeted genetically modified autologous T cell immunotherapy agent comprising patient-derived T cells ex vivo genetically modified by retroviral transduction to express a chimeric antigen receptor (CAR) containing an anti-CD19 single-strand variable fragment (scFv) linked to the CD28 and CD3 zeta costimulatory domains, in a method.

2. The method according to claim 1, wherein the time of intravenous infusion is between 15 and 120 minutes.

3. The method according to claim 1, wherein the time of intravenous infusion is a maximum of 30 minutes.

4. The method according to any one of claims 1 to 3, wherein the injection volume is between 50 mL and 100 mL.

5. The method according to any one of claims 1 to 4, wherein the injection volume is approximately 68 mL.

6. The method according to any one of claims 1 to 5, wherein the immunotherapy drug is injected from an infusion bag.

7. The method according to claim 6, wherein the injection bag is agitated during injection.

8. The method according to any one of claims 1 to 7, wherein the immunotherapy drug is administered within 3 hours after thawing.

9. The method according to any one of claims 1 to 8, wherein the suspension further comprises albumin.

10. The method according to claim 9, wherein albumin is present in an amount of approximately 2% (volume / volume) to 3% (volume / volume).

11. The method according to claim 10, wherein albumin is present in an amount of approximately 2.5% (volume / volume).

12. The method according to any one of claims 9 to 11, wherein the albumin is human albumin.

13. The method according to any one of claims 1 to 12, wherein the suspension further comprises DMSO.

14. A method for treating relapsed or refractory large B-cell lymphoma in patients after two or more lines of systemic therapy, (a) Administering CD19-targeted gene-modified autologous T-cell immunotherapy drugs to patients in need of treatment, (b) Monitoring the patient for signs and symptoms of adverse reactions after the injection, Methods that include...

15. The aforementioned relapsed or refractory large B-cell lymphoma is diffuse large B-cell lymphoma that cannot be specified. The method according to claim 14, wherein the DLBCL is caused by cellular lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, or follicular lymphoma.

16. The method according to claim 14, wherein the adverse reaction is selected from the group consisting of cytokine release syndrome (CRS), neurotoxicity, hypersensitivity reaction, severe infection, cytopenia, and hypogammaglobulinemia.

17. The method according to claim 14, wherein the signs and symptoms of the adverse reaction are selected from the group consisting of fever, hypotension, tachycardia, hypoxia and chills, and include cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal failure, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), seizures, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia, anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia and anemia.

18. The method according to claim 14, further comprising administering an effective amount of tocilizumab to treat the symptoms of an adverse reaction.

19. The method according to claim 18, further comprising administering a corticosteroid to treat symptoms of an adverse reaction.

20. The method according to claim 14, wherein the adverse reaction is cytokine release syndrome (CRS).

21. The method according to claim 20, comprising monitoring for signs and symptoms of cytokine release syndrome (CRS) at least daily for about seven days after infusion.

22. The method according to claim 14, wherein the adverse reaction is neurotoxic.

23. The method according to claim 22, further comprising administering a non-sedating antiepileptic drug for seizure prevention.

24. The method according to claim 14, wherein the adverse reaction is cytopenia.

25. The method according to claim 24, further comprising administering erythropoietin, darbepoetin alfa, platelet transfusion, colony-stimulating factor (CSF), granulocyte colony-stimulating factor, filgrastim, pegfilgrastim, or granulocyte-macrophage colony-stimulating factor.

26. The method according to claim 14, further comprising measuring cytokine levels and chemokine levels.

27. The method according to claim 26, wherein at least one level of IL-6, IL-8, IL-10, IL-15, TNF-α, IFN-γ, and sIL2Rα is measured.

28. A container containing a suspension of CD19-directed gene-modified autologous T cells, approximately 5% dimethyl sulfoxide (DMSO), and approximately 2.5% human albumin (volume / volume).

29. A method for treating relapsed or refractory large B-cell lymphoma in humans after two or more lines of systemic therapy, comprising administering a CD19-targeted gene-modified autologous T-cell immunotherapy agent to a person in need of treatment, (a) A composition containing CD19-directed chimeric antigen receptor (CAR)-positive viable T cells is administered to the patient. Administering the drug, (b) Monitoring the patient for signs and symptoms of adverse reactions after administration, (c) If cytokine release syndrome (CRS) of grade 2 or higher is observed in (b), administer tocilizumab at a dose of approximately 8 mg / kg (intravenously) over 1 hour, and if there is no response to increased intravenous fluid resuscitation or oxygen supplementation, repeat tocilizumab every 8 hours as needed. If the CRS symptoms observed in (d) and (b) do not improve 24 hours after (c), administer approximately 1 mg / kg of methylprednisolone (intravenously) twice daily or an equivalent dose of dexamethasone, continue using corticosteroids until the event is grade 1 or lower, and then gradually reduce the dose over the following three days. If Grade 3 CRS is observed in (e)(b), administer tocilizumab at a dose of 8 mg / kg (intravenously) over 1 hour, and if there is no response to increased intravenous fluid or oxygen supplementation, repeat tocilizumab every 8 hours as needed, administer methylprednisolone 1 mg / kg (intravenously) twice daily or an equivalent dose of dexamethasone, and continue the use of corticosteroids until the event is Grade 1 or lower, and then gradually reduce the dose over the following 3 days. If grade 4 CRS is observed in (f)(b), administer tocilizumab at a dose of approximately 8 mg / kg (intravenously) over 1 hour, and if there is no response to increased intravenous fluid or oxygen supplementation, repeat tocilizumab every 8 hours as needed, and administer approximately 1000 mg (intravenously) of methylprednisolone per day for 3 days. Methods that include...

30. A method for treating relapsed or refractory large B-cell lymphoma in a patient after two or more lines of systemic therapy, comprising administering a CD19-targeted gene-modified autologous T-cell immunotherapy agent to a patient in need of treatment, (a) Administering the patient a composition containing CD19-directed chimeric antigen receptor (CAR)-positive viable T cells, (b) Monitoring the patient for signs and symptoms of adverse reactions after administration, (c) If cytokine release syndrome (CRS) and / or neurotoxicity are observed, cytokine release syndrome (CRS) and / or neurotoxicity shall be managed in accordance with Table 1 and / or Table 2, Methods that include...

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