NZ763357B2

NZ763357B2 - Methods of administering chimeric antigen receptor immunotherapy

Info

Publication number: NZ763357B2
Application number: NZ763357A
Authority: NZ
Inventors: Jeffrey S Wiezorek
Original assignee: Kite Pharma Inc
Filing date: 2018-10-18
Publication date: 2024-01-30

Abstract

The disclosure provides cells comprising CD19-directed chimeric antigen receptor (CAR) genetically modified autologous T cell immunotherapy for the treatment of, e.g., relapsed or refractory large B-cell lymphoma after two or more lines of systemic therapy, including diffuse large B-cell lymphoma (DLBCL) not otherwise specified, primary mediastinal large B-cell lymphoma, high grade B-cell lymphoma, and DLBCL arising from follicular lymphoma. Some aspects of the disclosure relate to methods of treatment and monitoring following infusion of T cell therapy provided herein. The monitoring is typically for CRS (cytokine release syndrome) and the monitoring only needs to occur for 7-10 days and wherein 8mg/kg tocilizumab is given daily when CRS grade 2 reaction is observed.

Description

METHODS OF ADMINISTERING IC ANTIGEN RECEPTOR IMMUNOTHERAPY CROSS-REFERENCE TO RELATED ATIONS This application claims priority to US Provisional ation No. 62/574,159, ?led October 18, 2017, which is incorporated by reference herein in its entirety.

TECHNICAL FIELD The present disclosure relates generally to T cell ies and more speci?cally to CDl9-directed genetically modi?ed autologous T cell immunotherapies comprising chimeric antigen receptors (CARs).

BACKGROUND Human cancers are by their nature comprised of normal cells that have undergone a genetic or epigenetic conversion to become abnormal cancer cells. In doing so, cancer cells begin to express proteins and other ns that are distinct from those expressed by normal cells. These aberrant tumor antigens may be used by the body’s innate immune system to speci?cally target and kill cancer cells. However, cancer cells employ various mechanisms to prevent immune cells, such as T and B lymphocytes, from successfully targeting cancer cells.

Chimeric antigen receptors (CARs), which comprise g domains capable of interacting with a particular tumor antigen, allow T cells to target and kill cancer cells that express the particular tumor antigen.

SUMMARY As described in detail below, the present disclosure is based, in part, on the surprising discovery that the stration methods disclosed herein identify and manage adverse side effects of CAR T-cell immunotherapy.

Any aspect or embodiment described herein may be ed with any other aspect or embodiment as disclosed herein. While the t invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the present invention, which is de?ned by the scope of the appended claims. Other aspects, advantages, and modi?cations are within the scope of the following claims.

In one aspect, the ion provides a method of treating relapsed or refractory diffuse large B-cell lymphoma (DLBCL) not ise speci?ed, primary mediastinal large B-cell lymphoma, high grade B-cell lymphoma, or DLBCL arising from follicular lymphoma after two or more lines of systemic therapy in a patient comprising: administering to the patient in need thereof aXicabtagene ciloleucel suspension by intravenous infusion at a dose between about 1 X 106 and about 2 X 106 sitive viable T cells per kg body weight up to a maXimum dose of about 1 X 108 CAR-positive viable T cells, wherein aXicabtagene ciloleucel is a CD19-directed genetically modi?ed autologous T cell immunotherapy, comprising the t’s own T cells harvested and genetically modi?ed eX vivo by retroviral transduction to express a chimeric antigen receptor (CAR) comprising an D19 single chain variable fragment (scFv) linked to CD28 and CD3-zeta co- stimulatory s.

In another aspect, the invention provides a method of treating relapsed or tory diffuse large B-cell lymphoma (DLBCL) and primary mediastinal large B-cell ma (PMBCL), after two or more lines of systemic therapy in a patient comprising: administering to the patient in need thereof aXicabtagene ciloleucel suspension by intravenous infusion at a dose between about 0.4 X 108 and about 2 X 108 CAR-positive viable T cells, wherein aXicabtagene ciloleucel is a CD19-directed genetically modi?ed autologous T cell immunotherapy, comprising the t’s own T cells harvested and genetically modi?ed eX vivo by retroviral uction to express a chimeric antigen receptor (CAR) comprising an anti-CD19 single chain variable fragment (scFv) linked to CD28 and ta mulatory domains.

In some ments, the intravenous infusion time is between 15 and 120 minutes. In some embodiments, the intravenous infusion time is up to 30 minutes.

In some embodiments, the infusion volume is between 50 and 100 mL. In some embodiments, the infusion volume is about 68 mL.

In some embodiments, the therapy is infused from an infusion bag. In some embodiments, the infusion bag is agitated during the infusion.

In some embodiments, the immunotherapy is stered within 3 hours after thawing.

In some embodiments, the suspension further comprises albumin. In some embodiments, albumin is present in an amount of about 2-3% (v/v). In some embodiments, albumin is present in an amount of about 2.5% (v/v). In some embodiments, albumin is human n.

In some embodiments, the suspension further comprises DMSO. In some embodiments, DMSO is t in an amount of about 4-6% (v/v). In some ments, DMSO is present in an amount of about 5% (v/v).

In one aspect, the invention provides a method of treating relapsed or refractory large B-cell lymphoma after two or more lines of systemic therapy in a t comprising: (a) administering to the patient in need thereof CDl9-directed genetically modi?ed autologous T cell immunotherapy, and (b) monitoring the patient following infusion for signs and symptoms of an adverse reaction.

In some ments, the relapsed or refractory large B-cell lymphoma is diffuse large B-cell lymphoma (DLBCL) not otherwise ied, y mediastinal large B-cell lymphoma, high grade B-cell lymphoma, or DLBCL arising from follicular lymphoma.

In some embodiments, the adverse reaction is selected from the group consisting of ne release syndrome (CR8), a neurologic toxicity, a hypersensitivity reaction, a serious infection, a cytopenia and hypogammaglobulinemia.

In some embodiments, the signs and symptoms of adverse ons are ed from the group ting of fever, hypotension, tachycardia, hypoxia, and chills, include cardiac arrhythmias (including atrial ?brillation and ventricular tachycardia), cardiac arrest, cardiac failure, renal insufficiency, capillary leak syndrome, hypotension, hypoxia, organ ty, hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS), seizure, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia.

In some embodiments, the method further comprises administering an IL-6 receptor inhibitor.

In some embodiments, the method r comprises administering an effective amount of tocilizumab to treat a symptom of an e reaction.

In some embodiments, tocilizumab is administered at a dose of about 8 mg/kg intravenously. In some embodiments, tocilizumab is administered intravenously over about 1 hour. In some embodiments, tocilizumab is administered about every 8 hours. In some embodiments, tocilizumab is administered for no more than about 24 hours.

In some embodiments, the method further comprises administering a osteroid to treat a symptom of an adverse on.

In some ments, the corticosteroid is at least one of methylprednisone or dexamethasone.

In some embodiments, methylprednisone is administered at a dose of about 1 mg/kg intravenously. In some embodiments, methylprednisone is administered twice daily. In some embodiments, methylprednisone is administered at a dose of about 1,000 mg per day intravenously. In some embodiments, methylprednisone is administered intravenously for about 3 days.

In some embodiments, dexamethasone is administered at a dose of about 10 mg. In some embodiments, dexamethasone is administered intravenously about every 6 hours.

In some embodiments, the adverse reaction is cytokine release syndrome (CRS). In some embodiments, the monitoring for signs and symptoms of cytokine release syndrome (CRS) is at least daily for about 7 days following infusion. In some embodiments, the monitoring for signs and symptoms of cytokine e syndrome (CRS) is at least daily for about 8 days, about 9 days, or about 10 days ing infusion. In some embodiments, the monitoring for signs and ms of cytokine release syndrome (CRS) is at least daily for about 10 days following infusion. In some embodiments, the ring for signs and symptoms of cytokine release syndrome (CRS) is for about 4 weeks following infusion.

In some embodiments, the adverse on is neurologic toxicity.

In some embodiments, the monitoring for signs and symptoms of neurologic toxicity up to about 8 weeks following infusion.

In some embodiments, the method further comprises administering a non- sedating, eizure medicine for seizure prophylaxis.

In some embodiments, the non-sedating, anti-seizure medicine is levetiracetam.

In some embodiments, the e reaction is a cytopenia. In some embodiments, the cytopenia is thrombocytopenia, neutropenia, and/or anemia.

In some embodiments, the method further comprises stering at least one of erythropoietin, darbepoetin alfa, platelet transfusion, colony-stimulating factor (CSF), granulocyte colony-stimulating factor, ?lgrastim, peg?lgrastim, or granulocyte-macrophage colony-stimulating factor.

In some embodiments, the method further comprises measuring cytokine and ine levels. In some embodiments, the level of at least one of IL-6, IL-8, IL-10, IL-l5, TNF-d, IFN—y, and SILZROL is measured.

In one , the invention provides a container comprising a suspension of irected genetically modi?ed autologous T cells, about 5% ylsulfoxide (DMSO) and about 2.5% human albumin (v/v). In another aspect, the container comprises a suspension of between about 0.4 X 108 — 2 X 108 CDl9-directed genetically modi?ed autologous T cells (CAR-positive viable T .

In some embodiments, the container is a sterile infusion bag. In some ments, the infusion bag volume is about 100 mL, 250 mL, 500 mL, 750 mL, 1000 mL, 1500 mL, 2000 mL or 3000 mL.

In one aspect, the invention provides a method of treating relapsed or refractory large B-cell lymphoma after two or more lines of systemic therapy in a human comprising administering to the human in need thereof CDl9-directed genetically modi?ed autologous T cell immunotherapy comprising: (a) administering to the t a composition comprising irected chimeric antigen receptor (CAR) positive viable T cells, (b) monitoring the patient following administration for signs and symptoms of an adverse on, and (c) if ne release syndrome (CRS) greater than Grade 2 is observed in (b), stering tocilizumab at a dose of about 8 mg/kg IV over 1 hour, repeating tocilizumab every 8 hours as needed if not responsive to IV ?uids or increasing supplemental oxygen, (d) if CRS symptoms observed in (b) do not improve after 24 hours of (c), administering methylprednisolone about 1 mg/kg IV twice daily or administering equivalent dexamethasone dose and continuing corticosteroids use until the event is Grade 1 or less, then tapering over 3 days, (e) if CRS Grade 3 is observed in (b), administering tocilizumab at a dose of 8 mg/kg IV over 1 hour, repeating tocilizumab every 8 hours as needed if not responsive to IV ?uids or increasing supplemental oxygen and administering methylprednisolone 1 mg/kg IV twice daily or administering equivalent dexamethasone dose and uing corticosteroids use until the event is Grade 1 or less, then tapering over 3 days, and (f) if CRS Grade 4 is observed in (b), administering tocilizumab at a dose of about 8 mg/kg IV over 1 hour, ing tocilizumab every 8 hours as needed if not responsive to IV ?uids or increasing supplemental oxygen and administering about 1,000 mg IV prednisolone per day for 3 days.

In one aspect, the invention provide a method of treating relapsed or refractory large B-cell lymphoma after two or more lines of systemic therapy in a patient comprising administering to the patient in need thereof CD19-directed genetically d autologous T cell immunotherapy comprising: (a) administering to the patient a composition comprising CD19-directed chimeric antigen receptor (CAR) positive viable T cells, (b) monitoring the t following administration for signs and symptoms of an adverse reaction, and (c) if cytokine release syndrome (CRS) and/or neurologic toxicity is observed, managing cytokine release syndrome (CRS) and/or neurologic ty according to Table 1 and/or Table 2.

Other features and advantages of the disclosure will be apparent from the following Detailed Description, including the Examples, and the claims.

ED DESCRIPTION The present sure relates to engineered cells (e.g., T cells) comprising a CD19 CAR genetically modi?ed gous T cell immunotherapy indicated for the treatment of adult patients with relapsed or refractory large B-cell lymphoma after two or more lines of systemic therapy, ing diffuse large B-cell lymphoma (DLBCL) not otherwise specified, y mediastinal large B-cell lymphoma, high grade B-cell lymphoma, and DLBCL arising from follicular lymphoma. In some embodiments, the present disclosure provides methods of treatment using the engineered T cells for the treatment of a patient suffering from a cancer.

To prepare CD19-directed genetically modified autologous T cell immunotherapy, a t’s own T cells may be harvested and genetically modified ex vivo by iral transduction to express a chimeric antigen receptor (CAR) comprising a murine anti-CD19 single chain le fragment (scFv) linked to CD28 and CD3-zeta co- stimulatory domains. In some embodiments, the CAR comprises a murine anti-CD19 single chain variable fragment (scFv) linked to 4-1BB and CD3-zeta co-stimulatory domain. The D19 CAR T cells may be expanded and infused back into the patient, where they may recognize and eliminate CD19-expressing target cells. YESCARTA® (Axi-celTM, axicabtagene ciloleucel) is an example of such CD19-directed genetically d autologous T cell therapy. See Kochenderfer, el al., (J Immunother 2009;32:689 702). Additional CD19 directed CAR therapies include 7, 5, JCAR014, Kymriah (tisagenlecleucel). See in et al. Nature Rev. Cancer Vol. 3 (2003), Ruella et al., Curr Hematol Malig Rep., Springer, NY (2016) and Sadelain et al. Cancer ery (Apr 2013).

CD19-directed genetically modi?ed autologous T cell immunotherapy may be prepared from the patient’s peripheral blood mononuclear cells, which are typically obtained via a standard leukapheresis procedure. The mononuclear cells may be enriched for T cells and activated with anti-CD3 antibody in the presence of IL-2, then transduced with the replication incompetent retroviral vector containing the anti-CD19 CAR transgene. The transduced T cells may be expanded in cell culture, washed, formulated into a suspension, and/or cryopreserved. Typically, the product comprising genetically modi?ed autologous T cells must pass a sterility test before release for shipping as a frozen suspension in a patient- speci?c infusion container such as an infusion bag. Typically, the product is thawed prior to infusion.

In addition to T cells, CD19-directed genetically modi?ed autologous T cell immunotherapy may n NK and NK-T cells. In some embodiments, the CD19-directed genetically modi?ed autologous T cell immunotherapy formulation contains about 5% dimethylsulfoxide (DMSO) and about 2.5% albumin ) (v/v). irected genetically modi?ed autologous T cells bind to CD19-expressing cancer cells and normal B cells. Studies have demonstrated that, following anti-CD19 CAR T cell engagement with CD19-expressing target cells, the CD28 and CD3- zeta co-stimulatory domains activate downstream signaling cascades that lead to T-cell activation, proliferation, acquisition of effector functions and secretion of in?ammatory cytokines and chemokines. This sequence of events leads to killing of CD19-expressing cells.

In one aspect, the invention provides a method of treating relapsed or refractory diffuse large B-cell ma (DLBCL) not otherwise speci?ed, primary mediastinal large B-cell lymphoma, high grade B-cell lymphoma, or DLBCL arising from follicular ma after two or more lines of systemic therapy in a patient comprising: administering to the patient in need thereof a irected genetically modi?ed autologous T cell suspension by intravenous infusion at a dose between about 1 X 106 and about 2 X 106 CAR-positive viable T cells per kg body weight up to a maximum dose of about 1 X 108 CAR-positive viable T cells.

DEFINITIONS In order for the present invention to be more y understood, certain terms are ?rst de?ned below. Additional de?nitions for the following terms and other terms are set forth throughout the Speci?cation.

As used in this Speci?cation and the appended claims, the singular forms (4 77 "an" and "the" include plural referents unless the context clearly dictates ise.

Unless speci?cally stated or obvious from context, as used herein, the term "or" is understood to be inclusive and covers both "or" and "and".

The term "and/or" where used herein is to be taken as speci?c disclosure of each of the two speci?ed features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include A and B, A or B, A (alone), and B (alone). se, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to ass each of the following s: 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 ), and C (alone).

The terms "e.g.," and "i.e." as used herein, are used merely by way of example, without limitation intended, and should not be construed as referring only those items explicitly ated in the speci?cation.

The terms "or more", "at least", "more than", and the like, e.g., "at least one" are understood to include but not be limited to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15,16,17,18,1920,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,3& 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, 128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145, 146,147,148,149(n'150,200,300,400,500,600,700,800,900,1000,2000,3000,4000, 5000 or more than the stated value. Also included is any r number or fraction in Conversely, the term "no more than" includes each value less than the stated value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92,91,90,89,88,87,86,85,84,83,82,81,80,79,78,77,76,75,74,73,72,71,70,69,68, 67,66,65,64,63,62,61,60,59,58,57,56,55,54,53,52,51,50,49,48,47,46,45,44,43, 40,39,38,37,36,35,34,33,32,31,30,29,28,27,26,25,24,23,22,21,20,19,18, l7,l6,15,l4,l3,12,ll,10,9,8,7,6,5,4,3,2,l,and()nudeo?des Alm3nu?udedisany lesser number or fraction in between.

The terms lity", "at least two", "two or more", "at least second", and the like, are understood to include but not limited to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, 12, 13, 16,17,18,1920,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,3& 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, 128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145, 146,147,148,149(n'150,200,300,400,500,600,700,800,900,1000,2000,3000,4000, 5000 or more. Also included is any r number or on in between.

Throughout the speci?cation the word "comprising," or variations such as "comprises" or "comprising," will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of" and/or "consisting essentially of" are also provided.

Unless speci?cally stated or evident from context, as used herein, the term "about" refers to a value or ition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "approximately" may mean within one or more than one standard deviation per the practice in the art. "About" or "approximately" may mean a range of up to 10% (i.e., ::10%). Thus, "about" may be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg may include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological s or processes, the terms may mean up to an order of ude or up to 5-fold of a value. When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of "about" or ximately" should be assumed to be within an acceptable error range for that particular value or composition.

As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to be inclusive of the value of any integer within the d range and, when appropriate, fractions f (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

Units, pre?xes, and symbols used herein are provided using their Systeme International de Unites (SI) ed form. Numeric ranges are inclusive of the numbers de?ning the range.

Unless de?ned ise, all technical and scienti?c terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this sure is d. For example, Juo, "The Concise Dictionary of Biomedicine and Molecular Biology", 2nd ed., , CRC Press, "The Dictionary of Cell & Molecular Biology", 5th ed., , Academic Press, and "The Oxford Dictionary Of Biochemistry And Molecular Biology", Cammack et al. eds., 2nd ed, (2006), Oxford University Press, provide those of skill in the art with a general dictionary for many of the terms used in this disclosure.

"Administering" refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art.

Exemplary routes of administration for the formulations disclosed herein e intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, esional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, e.g., orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering may also be performed, for example, once, a plurality of times, and/or over one or more extended periods.

The term "antibody" (Ab) includes, without limitation, a glycoprotein immunoglobulin which binds cally to an antigen. In general, and antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disul?de 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 ses three constant domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region viated herein as VL) and a light chain constant . The light chain constant region is comprises one nt domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the Abs may mediate the g of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

Antibodies may include, for example, onal antibodies, recombinantly produced antibodies, monospecif1c antibodies, multispecif1c antibodies (including bispecif1c antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic dies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an dy light chain monomer, an dy heavy chain monomer, an dy light chain dimer, an antibody heavy chain dimer, an antibody light chain- antibody heavy chain pair, intrabodies, antibody fusions (sometimes referred to herein as ody conjugates"), heteroconjugate antibodies, single domain dies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab’)2 fragments, disulf1de-linked Fvs (dev), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above. In some embodiments, antibodies described herein refer to polyclonal antibody populations.

An "antigen binding molecule, 77 (L antigen binding portion," or "antibody fragment" refers to any le that comprises the antigen binding parts (e.g., CDRs) of the antibody from which the molecule is derived. An antigen binding molecule may include the antigenic complementarity determining regions (CDRs). Examples of antibody nts include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAb, linear antibodies, scFv dies, and multispeci?c antibodies formed from antigen binding molecules.

Peptibodies (i.e., Fc fusion molecules comprising peptide binding domains) are another e of suitable antigen binding molecules. In some embodiments, the n 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 a hyperproliferative disease or to a viral or bacterial antigen. In some ments, the antigen binding molecule binds to CD19. In further embodiments, the antigen binding molecule is an dy fragment that speci?cally binds to the antigen, ing one or more of the complementarity determining regions (CDRs) thereof. In further embodiments, the antigen binding molecule is a single chain variable fragment (scFv). In some embodiments, the antigen g molecule comprises or consists of avimers.

An "antigen" refers to any molecule that provokes an immune response or is capable of being bound by an dy or an antigen binding molecule. The immune response may involve either antibody tion, or the activation of speci?c immunologically- competent cells, or both. A person of skill in the art would y understand that any macromolecule, ing virtually all proteins or peptides, may serve as an antigen. An antigen may be endogenously expressed, i.e. expressed by genomic DNA, or may be recombinantly expressed. An antigen may be speci?c to a certain tissue, such as a cancer cell, or it may be broadly expressed. In addition, fragments of larger molecules may act as antigens. In some embodiments, antigens are tumor antigens. directed cally modi?ed autologous T cell therapy" refers to a suspension of chimeric antigen receptor (CAR)-positive T cells. An example of such immunotherapy is axicabtagene ciloleucel (also known as Axi-celTM, YESCARTA®), developed by Kite Pharmaceuticals, Inc.

The term "neutralizing" refers to an antigen binding molecule, scFv, antibody, or a nt thereof, that binds to a ligand and prevents or reduces the ical effect of that ligand. In some embodiments, the antigen binding molecule, scFv, antibody, or a fragment thereof, directly blocking a binding site on the ligand or otherwise alters the ligands y to bind through indirect means (such as structural or energetic alterations in the ligand). In some embodiments, the antigen g molecule, scFv, antibody, or a nt thereof prevents the protein to which it is bound from performing a biological function.

The term "autologous" refers to any al derived from the same individual to which it is later to be re-introduced. For example, the engineered autologous cell y (eACTTM) method described herein involves collection of lymphocytes from a patient, which are then engineered to express, e.g., a CAR construct, and then stered back to the same patient.

The term "allogeneic" refers to any material derived from one individual which is then introduced to another individual of the same species, e.g., allogeneic T cell transplantation.

The terms "transduction" and "transduced" refer to the process whereby foreign DNA is introduced into a cell via viral vector (see Jones et al., "Genetics: principles and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, a RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an irus associated vector, a lentiviral vector, or any combination thereof.

A r" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also asize to distant parts of the body through the lymphatic system or bloodstream. A "cancer" or "cancer tissue" may include a tumor. Examples of cancers that may be treated by the methods disclosed herein include, but are not limited to, cancers of the immune system including lymphoma, leukemia, myeloma, and other leukocyte malignancies. In some embodiments, the methods disclosed herein may be used to reduce the tumor size of a tumor derived from, for example, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or cular malignant melanoma, e cancer, ovarian cancer, rectal , cancer of the anal region, stomach cancer, testicular cancer, uterine , carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, multiple myeloma, n's Disease, non-Hodgkin's lymphoma (NHL), y mediastinal large B cell lymphoma (PlV?3C), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the a, cancer of the penis, 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 of childhood, lymphocytic ma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T cell ma, environmentally induced cancers ing those induced by asbestos, other B cell malignancies, and combinations of said cancers. In some embodiments, the cancer is multiple myeloma. The particular cancer may be responsive to chemo- or radiation therapy or the cancer may be refractory. A refractor cancer refers to a cancer that is not amendable to surgical intervention and the cancer is either initially unresponsive to chemo- or radiation therapy or the cancer becomes unresponsive over time.

An "anti-tumor effect" as used herein, refers to a biological effect that may present as a decrease in tumor volume, a decrease in the number of tumor cells, a se in tumor cell proliferation, a decrease in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or amelioration of various physiological symptoms associated with the tumor. An anti-tumor effect may also refer to the prevention of the occurrence of a tumor, e.g., a vaccine.

A ine," as used herein, refers to a non-antibody protein that is ed by one cell in response to t with a specific n, wherein the cytokine interacts with a second cell to mediate a response in the second cell. ine" as used herein is meant to refer to proteins ed by one cell tion that act on another cell as intercellular mediators. A cytokine may be nously expressed by a cell or stered to a subject.

Cytokines may be released by immune cells, including macrophages, B cells, T cells, and mast cells to propagate an immune response. Cytokines may induce various responses in the recipient cell. Cytokines may include homeostatic cytokines, chemokines, pro-in?ammatory cytokines, ors, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (1L) 7 and IL-15, promote immune cell survival and proliferation, and pro- in?ammatory nes may promote an atory response. Examples of homeostatic cytokines include, but are not limited to, 1L-2, IL-4, lL-S, IL-7, IL-10, IL-12p40, 1L-12p70, IL-15, and interferon (IFN) gamma. es of pro-in?ammatory cytokines include, but are not limited to, lL-la, IL-lb, 1L-6, IL-l3, IL-l7a, tumor necrosis factor (TNF)-alpha, TNF- beta, ?broblast growth factor (FGF) 2, granulocyte macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion le 1 (sICAM-l), soluble vascular adhesion molecule 1 (sVCAM-l), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and tal growth factor (PLGF). Examples of effectors e, but are not limited to, granzyme A, granzyme B, soluble Fas ligand ), and perforin. Examples of acute phase- proteins e, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

"Chemokines" are a type of cytokine that es cell chemotaxis, or directional movement. Examples of ines include, but are not limited to, 1L-8, lL-l6, eotaxin, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemotactic protein 1 (MCP-l or CCL2), MCP-4, macrophage in?ammatory protein lOt (MIP-ld, MIP- la), MlP-lB (MlP-lb), gamma-induced protein 10 (lP-lO), and thymus and activation regulated chemokine (TARC or CCLl7).

A "therapeutically effective amount, 77 (L effective dose, 77 (L effective amount," or peutically ive dosage" of a therapeutic agent, e.g., engineered CAR T cells, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom- free periods, or a prevention of impairment or disability due to the disease af?iction. The y of a therapeutic agent to promote disease regression may be evaluated using a y of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of ef?cacy in humans, or by assaying the activity of the agent in in vitro assays.

The term "lymphocyte" as used herein includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell toxic) lymphocyte that represent a major component of the inherent immune system. NK cells reject tumors and cells infected by viruses. It works through the process of apoptosis or programmed cell death. They were termed "natural killers" because they do not require tion in order to kill cells. T cells play a major role in cell-mediated-immunity (no antibody involvement). Its T cell receptors (TCR) differentiate themselves from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the T cell’s maturation. There are six types of T cells, namely: Helper T cells (e.g., CD4+ cells), Cytotoxic T cells (also known as TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T cells or killer T cell), Memory T cells ((i) stem memory TSCM cells, like naive cells, are CD45RO—, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and L‘l', but they also express large s of CD95, IL-ZRB, CXCR3, and LFA-l, and show numerous functional attributes distinctive of memory cells), (ii) central memory TCM cells express L-selectin and the CCR7, they secrete IL-2, but not IFNv or IL-4, and (iii) effector memory TEM cells, however, do not express L-selectin or CCR7 but produce effector cytokines like IFNv and IL- 4), Regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), l Killer T cells (NKT) and Gamma Delta T cells. B-cells, on the other hand, play a principal role in humoral ty (with antibody ement). It makes dies and antigens and performs the role of antigen-presenting cells (APCs) and turns into memory B-cells after activation by antigen interaction. In mammals, immature B-cells are formed in the bone marrow, where its name is derived from.

The term "genetically engineered" or "engineered" refers to a method of ing the genome of a cell, ing, but not limited to, ng a coding or non-coding region or a portion thereof or inserting a coding region or a portion thereof. In some embodiments, the cell that is modi?ed is a lymphocyte, e.g., a T cell, which may either be obtained from a patient or a donor. The cell may be modi?ed to s an exogenous construct, such as, e.g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR), which is incorporated into the cell's genome.

An "immune response" refers to the action of a cell of the immune system (for example, T lymphocytes, B lymphocytes, l killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Abs, cytokines, and complement) that results in ive targeting, binding to, damage to, destruction of, and/or elimination from a vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological in?ammation, normal human cells or tissues.

The term "immunotherapy" refers to the treatment of a subject ed with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising ng, enhancing, suppressing or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T cell therapies. T cell therapy may include adoptive T cell therapy, tumor-in?ltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACTTM), and allogeneic T cell transplantation. r, one of skill in the art would recognize that the conditioning methods disclosed herein would enhance the effectiveness of any transplanted T cell therapy. Examples of T cell therapies are described in US. Patent Publication Nos. 2014/0154228 and 2002/0006409, US. Patent No. 7,741,465, US. Patent No. 6,319,494, US. Patent No. 5,728,388, and International Publication No.

The T cells of the immunotherapy may come from any source known in the art. For example, T cells may be differentiated in vitro from a hematopoietic stem cell population, or T cells may be obtained from a subject. T cells may be obtained from, e.g., peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node , cord blood, thymus tissue, tissue from a site of ion, ascites, pleural effusion, spleen tissue, and tumors. In addition, the T cells may be d from one or more T cell lines available in the art. T cells may also be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLLTM separation and/or apheresis. Additional methods of isolating T cells for a T cell therapy are disclosed in US.

Patent ation No. 2013/0287748, which is herein incorporated by references in its entirety.

The term eered Autologous Cell Therapy," which may be abbreviated as M," also known as adoptive cell transfer, is a process by which a patient's own T cells are collected and subsequently genetically altered to ize and target one or more antigens expressed on the cell surface of one or more speci?c tumor cells or malignancies. T cells may be engineered to express, for example, chimeric antigen receptors (CAR). CAR positive (+) T cells are engineered to express an extracellular single chain variable fragment (scFv) with speci?city for a ular tumor antigen linked to an intracellular signaling part comprising at least one costimulatory domain and at least one ting domain. The CAR scFv may be designed to target, for example, CD19, which is a transmembrane protein expressed by cells in the B cell lineage, including all normal B cells and B cell malignances, ing but not limited to diffuse large B-cell lymphoma (DLBCL) not otherwise specified, WO 79564 2018/056467 primary mediastinal large B-cell lymphoma, high grade B-cell lymphoma, and DLBCL arising from follicular lymphoma, NHL, CLL, and non-T cell ALL. Example CAR T cell therapies and constructs are described in US. Patent Publication Nos. 2013/0287748, 2014/0227237, 2014/0099309, and 2014/0050708, and these nces are incorporated by reference in their ty.

A "patient" as used herein includes any human who is af?icted with a cancer (e.g., a lymphoma or a leukemia). The terms "subject" and "patient" are used interchangeably herein.

As used herein, the term "in vitro cell" refers to any cell which is cultured ex vivo. In particular, an in vitro cell may include a T cell.

The terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids, and no limitation is placed on the maximum number of amino acids that may comprise a protein's or peptide's sequence. ptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are ed to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modi?ed polypeptides, derivatives, s, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic es, or a combination thereof.

"Stimulation," as used herein, refers to a primary se induced by binding of a stimulatory molecule with its cognate ligand, wherein the binding mediates a signal transduction event. A "stimulatory molecule" is a molecule on a T cell, e.g., the T cell receptor (TCR)/CD3 complex, that speci?cally binds with a cognate stimulatory ligand present on an n present cell. A "stimulatory ligand" is a ligand that when t on an n presenting cell (e.g., an APC, a dendritic cell, a B-cell, and the like) may speci?cally bind with a stimulatory le on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands include, but are not limited to, an D3 antibody, an MHC Class Imolecule loaded with a peptide, a superagonist anti-CD2 antibody, and a superagonist anti-CD28 antibody.

A "costimulatory signal," as used herein, refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to a T cell response, such as, but not limited to, proliferation and/or upregulation or down tion of key A "costimulatory ligand," as used herein, includes a molecule on an antigen presenting cell that speci?cally binds a cognate co-stimulatory molecule on a T cell. Binding of the costimulatory ligand provides a signal that mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A costimulatory ligand induces a signal that is in addition to the primary signal provided by a stimulatory molecule, for instance, by binding of a T cell receptor (TCR)/CD3 compleX with a major ompatibility compleX (MHC) molecule loaded with peptide. A co-stimulatory ligand may include, but is not limited to, 3/TR6, 4-lBB , agonist or dy that binds Toll ligand receptor B7-l , B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), 1LT4, immunoglobulin-like transcript (ILT) 3, ble costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), ligand that speci?cally binds with B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class 1 chain- related protein B (MICB), 0X40 , PD-L2, or programmed death (PD) Ll. A co- stimulatory ligand includes, without limitation, an antibody that speci?cally binds with a ulatory molecule t on a T cell, such as, but not limited to, 4-lBB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, ligand that speci?cally binds with CD83, lymphocyte function-associated antigen-l (LFA-l), natural killer cell receptor C (NKG2C), 0X40, PD-l, or tumor necrosis factor superfamily member 14 (TNF SFl4 or LIGHT).

A "costimulatory molecule" is a cognate binding partner on a T cell that speci?cally binds with a costimulatory , thereby mediating a ulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, A "costimulatory molecule" is a cognate binding partner on a T cell that speci?cally binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, 4-1BB/CD137, B7-H3, BAFFR, BLAME 8), BTLA, CD 33, CD 45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CDl9a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 , beta, delta, epsilon, gamma, zeta), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8alpha, CD8beta, CD9, CD96 (Tactile), CDl-la, , CDl-lc, CDl-ld, CDS, l, CRT AM, DAP-lO, DNAMl (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-l, ICAM-l, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, in, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-l, LFA-l, LIGHT, LIGHT (tumor necrosis factor superfamily member 14, TNFSFl4), LTBR, Ly9 (CD229), lymphocyte function-associated antigen-l (LFA-l (CDl la/CDl8), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRFl), 0X40, PAG/Cbp, PD-l, PSGLl, SELPLG (CD162), signaling lymphocytic activation molecule, SLAM (SLAMFl, CD150, IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, INF, TNFr, TNFR2, Toll ligand receptor, TRANCE/RANKL, VLAl, or VLA-6, or fragments, truncations, or combinations thereof.

The terms "reducing" and "decreasing" are used interchangeably herein and indicate any change that is less than the original. "Reducing" and "decreasing" are ve terms, requiring a comparison between pre- and post- measurements. "Reducing" and "decreasing" include complete depletions.

"Treatment" or "treating" of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, ting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication or ion, or biochemical indicia associated with a disease. In some embodiments, "treatment" or "treating" includes a partial remission. In another ment, "treatment" or "treating" includes a te remission.

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

Chimeric n ors Chimeric antigen receptors (CARs or CAR-Ts) are genetically engineered receptors. These engineered receptors may be readily inserted into and sed by immune cells, including T cells in accordance with techniques known in the art. With a CAR, a single receptor may be programmed to both recognize a speci?c antigen and, when bound to that antigen, activate the immune cell to attack and destroy the cell bearing that antigen. When these antigens exist on tumor cells, an immune cell that ses the CAR may target and kill the tumor cell.

Engineered T cells and Use A CDl9-directed genetically modi?ed autologous T cell immunotherapy indicated for the treatment of patients with relapsed or refractory large B-cell lymphoma after two or more lines of systemic therapy, ing diffuse large B-cell lymphoma (DLBCL) not otherwise ed, primary mediastinal large B-cell ma, high grade B-cell lymphoma, and DLBCL arising from follicular lymphoma. In some embodiments, the irected genetically modi?ed autologous T cell immunotherapy is axicabtagene ciloleucel (Axi-celTM, YESCARTA®).

The cell of the present disclosure may be obtained through T cells obtained from a subject. T cells may be obtained from, e.g., eral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In on, the T cells may be derived from one or more T cell lines available in the art. T cells may also be obtained from a unit of blood collected from a subject using any number of ques known to the skilled artisan, such as FICOLLTM separation and/or apheresis. In some embodiments, the cells collected by apheresis are washed to remove the plasma fraction, and placed in an appropriate buffer or media for subsequent processing. In some ments, the cells are washed with PBS. As will be appreciated, a washing step may be used, such as by using a semiautomated ?ow through centrifuge, e.g., the CobeTM 2991 cell processor, the Baxter CytoMateTM, or the like. In some embodiments, the washed cells are resuspended in one or more biocompatible buffers, or other saline solution with or without buffer. In some embodiments, the undesired components of the apheresis sample are removed. Additional methods of isolating T cells for a T cell therapy are disclosed in US. Patent Pub. No. 2013/0287748, which is herein orated by references in its entirety.

In some embodiments, T cells are isolated from PBMCs by lysing the red blood cells and depleting the monocytes, e.g., by using centrifugation through a PERCOLLTM gradient. In some embodiments, a c subpopulation of T cells, such as CD4+, CD8+, CD28+, CD45RA+, and CD45RO+ T cells is further isolated by positive or negative selection techniques known in the art. For example, enrichment of a T cell population by negative selection may be lished with a combination of antibodies directed to surface markers unique to the negatively ed cells. In some ments, cell sorting and/or selection via negative magnetic immunoadherence or ?ow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively ed may be used. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically es antibodies to CD8, CD1 lb, CD14, CD16, CD20, and HLA-DR. In some embodiments, ?ow cytometry and cell sorting are used to isolate cell tions of interest for use in the present disclosure.

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

In some embodiments, CD8+ cells are further sorted into naive, central memory, and effector cells by fying cell surface antigens that are associated with each of these types of CD8+ cells. In some embodiments, the expression of phenotypic markers of l memory T cells includes CCR7, CD3, CD28, CD45RO, CD62L, and CD127 and are negative for granzyme B. In some embodiments, l memory T cells are CD8+, CD45RO+, and CD62L+ 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+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells may be sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens.

In some embodiments, the immune cells, e.g., T cells, are genetically modi?ed following ion using known methods, or the immune cells are activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modi?ed. In another embodiment, the immune cells, e.g., T cells, are genetically modi?ed with the chimeric antigen receptors described herein (e.g., uced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then are activated and/or expanded in vitro. Methods for activating and ing T cells are known in the art and are described, e.g., in US. Patent Nos. 6,905,874, 6,867,041, and 6,797,514, and PCT Publication No. WO 2012/079000, the contents of which are hereby incorporated by reference in their entirety.

Generally, such methods include contacting PBMC or isolated T cells with a stimulatory agent and costimulatory agent, such as anti-CD3 and anti-CD28 antibodies, lly attached to a bead or other e, in a culture medium with appropriate cytokines, such as IL-2. D3 and anti-CD28 antibodies attached to the same bead serve as a gate" antigen presenting cell (APC). One example is The Dynabeads® , a CD3/CD28 activator/stimulator system for logical activation of human T cells. In other embodiments, the T cells are activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in US. Patent Nos. 6,040,177 and 5,827,642 and PCT Publication No. which are hereby incorporated by reference in their entirety.

In some embodiments, the T cells are ed from a donor subject. In some embodiments, the donor subject is human patient af?icted with a cancer or a tumor. In some embodiments, the donor subject is a human patient not af?icted with a cancer or a tumor.

In some ments, the composition comprises a pharmaceutically acceptable carrier, diluent, solubilizer, f1er, preservative and/or adjuvant. In some embodiments, the composition comprises an excipient.

In some embodiments, the composition is selected for eral delivery, for inhalation, or for delivery through the ive tract, such as orally. The ation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art. In some embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. In some embodiments, when parenteral administration is contemplated, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising a 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 composition described herein, with or without at least one additional therapeutic agent, is formulated as a sterile, isotonic solution, properly preserved. In some embodiments, the preparation involves the formulation of the desired molecule with polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that provide for the controlled or sustained release of the product, which are then be red via a depot injection. In some embodiments, implantable drug delivery devices are used to introduce the desired molecule.

In some embodiments, the methods of ng a cancer in a subject in need thereof comprise a T cell y. In some embodiments, the T cell therapy sed herein is engineered Autologous Cell Therapy (eACTTM). According to this embodiment, the method may include collecting blood cells from the patient. The isolated blood cells (e.g., T cells) may then be engineered to eXpress a CAR or a TCR disclosed herein. In a particular ment, the CAR T cells or the TCR T cells are administered to the patient. In some embodiments, the CAR T cells or the TCR T cells treat a tumor or a cancer in the patient. In some ments the CAR T cells or the TCR T cells reduce the size of a tumor or a cancer.

In some embodiments, the donor T cells for use in the T cell therapy are obtained from the patient (e.g., for an autologous T cell therapy). In other embodiments, the donor T cells for use in the T cell therapy are obtained from a subject that is not the patient.

The T cells may be administered at a therapeutically effective amount. For example, a therapeutically effective amount of the T cells may be at least about 104 cells, at least about 105 cells, at least about 106 cells, at least about 107 cells, at least about 108 cells, at least about 109, or at least about 1010. In r embodiment, the therapeutically effective amount of the T cells is about 104 cells, about 105 cells, about 106 cells, about 107 cells, or about 108 cells. In some embodiments, the therapeutically effective amount of the CAR T cells is about 2 X 106 cells/kg, about 3 X 106 cells/kg, about 4 X 106 cells/kg, about 5 X 106 cells/kg, about 6 X 106 cells/kg, about 7 X 106 cells/kg, about 8 X 106 cells/kg, about 9 X 106 cells/kg, about 1 X 107 cells/kg, about 2 X 107 cells/kg, about 3 X 107 cells/kg, about 4 X 107 cells/kg, about X 107 cells/kg, about 6 X 107 cells/kg, about 7 X 107 cells/kg, about 8 X 107 cells/kg, or about 9 X 107 cells/kg. In some embodiments, the therapeutically effective amount of the CAR-positive viable T cells is between about 1 X 106 and about 2 X 106 CAR-positive viable T cells per kg body weight up to a maximum dose of about 1 X 108 CAR-positive viable T cells.

In some embodiments, the eutically effective amount of the CAR- ve viable T cells is between about 0.4 X 108 and about 2 X 108 CAR-positive viable T cells. In some ments, the therapeutically effective amount of the CAR-positive viable T cells is about 0.4 X 108, about 0.5 X 108, about 0.6 X 108, about 0.7 X 108, about 0.8 X 108, about 0.9 X 108, about 1.0 X 108, about 1.1 X 108, about 1.2 X 108, about 1.3 X 108, about 1.4 X 108, about 1.5 X 108, about 1.6 X 108, about 1.7 X 108, about 1.8 X 108, about 1.9 X 108, or about 2.0 X 108 CAR-positive viable T cells.

Methods of Treatment The s disclosed herein may be used to treat a cancer in a t, reduce the size of a tumor, kill tumor cells, prevent tumor cell proliferation, prevent growth of a tumor, eliminate a tumor from a patient, prevent relapse of a tumor, prevent tumor asis, induce remission in a patient, or any combination thereof. In some embodiments, the methods induce a complete se. In other embodiments, the methods induce a partial response.

Cancers that may be treated include tumors that are not vascularized, not yet substantially vascularized, or vascularized. The cancer may also include solid or non-solid tumors. In some embodiments, the cancer is a hematologic cancer. In some embodiments, the cancer is of the white blood cells. In other embodiments, the cancer is of the plasma cells. In some embodiments, the cancer is leukemia, lymphoma, or a. In some embodiments, the cancer is acute lymphoblastic leukemia (ALL) (including non T cell ALL), acute lymphoid leukemia (ALL), and hemophagocytic lymphohistocytosis , B cell prolymphocytic leukemia, B-cell acute id leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, chronic lymphocytic ia (CLL), chronic myelogenous leukemia (CML), chronic d leukemia (CML), chronic or acute granulomatous disease, 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 ting Syndrome (MAS), Hodgkin's Disease, large cell granuloma, leukocyte adhesion def1ciency, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell ma, Marginal zone lymphoma, monoclonal gammapathy of undetermined signif1cance (MGUS), multiple myeloma, myelodysplasia and ysplastic syndrome (MDS), myeloid diseases including but not limited to acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), plasma cell proliferative disorders (e.g., asymptomatic myeloma ering multiple myeloma or indolent myeloma), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, plasmacytomas (e.g., plasma cell sia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), POEMS syndrome (Crow-Fukase syndrome, Takatsuki disease, PEP me), primary mediastinal large B cell lymphoma (PMBC), small cell- or a large cell-follicular lymphoma, c marginal zone lymphoma (SMZL), systemic amyloid light chain dosis, T cell acute lymphoid leukemia ("TALL"), T cell lymphoma, transformed follicular lymphoma, Waldenstrom macroglobulinemia, or a combination thereof.

In some embodiments, the cancer is a myeloma. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is a ia. In some embodiments, the cancer is acute myeloid leukemia.

In some embodiments, the methods further comprise administering a chemotherapeutic. In some embodiments, the chemotherapeutic selected is a lymphodepleting (preconditioning) chemotherapeutic. Bene?cial preconditioning treatment regimens, along with correlative bene?cial biomarkers are described in US. Provisional Patent Applications 62/262, 143 and 62/167,750 which are hereby incorporated by reference in their entirety herein. These describe, e.g., methods of conditioning a patient in need of a T cell therapy comprising stering to the patient speci?ed bene?cial doses of cyclophosphamide (between 200 mg/mZ/day and 2000 mg/mZ/day) and speci?ed doses of ?udarabine (between 20 mg/mZ/day and 900 day). One such dose regimen involves treating a patient comprising administering daily to the patient about 500 mg/mZ/day of cyclophosphamide and about 60 mg/mZ/day of ?udarabine for three days prior to administration of a therapeutically effective amount of engineered T cells to the patient.

In some embodiments, the antigen binding molecule, transduced (or otherwise engineered) cells (such as CARs), and the herapeutic agent are administered each in an amount effective to treat the disease or condition in the subject.

In some embodiments, compositions comprising CAR-expressing immune effector cells disclosed herein may be administered in conjunction with any number of chemotherapeutic agents. Examples of chemotherapeutic agents e alkylating agents such as thiotepa and cyclophosphamide (CYTOXANTM), alkyl sulfonates such as busulfan, ulfan and piposulfan, ines such as benzodopa, uone, meturedopa, and uredopa, ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine , nitrogen mustards such as chlorambucil, chlomaphazine, hosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, nitrosureas such as carmustine, chlorozotocin, fotemustine, ine, nimustine, ranimustine, antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, omycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, bicin, ooxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin; pot?romycin; cin; quelamycin; bicin; streptonigrin; streptozocin; tubercidin; ubenimeX; zinostatin; cin; etabolites such as methotrexate and 5- ?uorouracil ; folic acid ues such as denopterin; methotrexate; pteropterin; trimetrexate; purine analogs such as ?udarabine; 6-mercaptopurine; prine; thioguanine; pyrimidine analogs such as ancitabine; azacitidine; 6-azauridine; carmofur; cytarabine; dideoxyuridine; doxi?uridine; enocitabine; ?oxuridine; 5-FU; ens such as calusterone; dromostanolone propionate; epitiostanol; ostane; testolactone; anti-adrenals such as aminoglutethimide; mitotane; trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; et; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizo?ran; spirogermanium; tenuazonic acid; triaziquone; 2; 2';2"-trichlorotriethylamine; urethan; ine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); hosphamide; thiotepa; taxoids; e. g. axel (TAXOLTM; Bristol-Myers Squibb) and doxetaxel (TAXOTERE®; Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-l6); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-l l; topoisomerase inhibitor RFSZOOO; di?uoromethylomithine (DMFO); ic acid derivatives such as TargretinTM (bexarotene); PanretinTM; (alitretinoin); M eukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts; acids or derivatives of any of the above. In some ments; compositions sing CAR- and/or TCR-expressing immune effector cells disclosed herein may be administered in conjunction with an anti-hormonal agent that acts to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen; raloxifene; aromatase inhibiting 4(5)-imidazoles; 4-hydroxytamoxifen; trioxifene; keoxifene; LYl 17018; onapristone; and toremifene (Fareston); and anti-androgens such as ?utamide; nilutamide; tamide; leuprolide; and goserelin; and pharmaceutically acceptable salts; acids or derivatives of any of the above. Combinations of chemotherapeutic agents are also administered where appropriate; including; but not limited to CHOP; i.e.; Cyclophosphamide (Cytoxan®); Doxorubicin (hydroxydoxorubicin); Vincristine (Oncovin®); and Prednisone.

In some embodiments, the chemotherapeutic agent is administered at the same time or within one week after the administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered from 1 to 4 weeks or from 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after the administration of the engineered cell or nucleic acid. In some ments, the chemotherapeutic agent is administered at least 1 month before administering the cell or nucleic acid. In some embodiments, the methods further comprise administering two or more chemotherapeutic agents.

A variety of additional eutic agents may be used in conjunction with the compositions described herein. For example, ially useful additional therapeutic agents include PD-l inhibitors such as nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), pembrolizumab, pidilizumab (CureTech), and atezolizumab (Roche).

Additional therapeutic agents suitable for use in combination with the compositions and methods disclosed herein include, but are not limited to, ibrutinib (l1V?3RUVICA®), ofatumumab (ARZERRA®), mab (RITUXAN®), zumab (AVASTIN®), trastuzumab PTIN®), trastuzumab emtansine (KADCYLA®), imatinib (GLEEVEC®), cetuXimab (ERBITUX®), panitumumab (VECTIBIX®), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuXimab, alemtuzumab, gemtuzumab, erlotinib, gef1tinib, vandetanib, ib, lapatinib, neratinib, aXitinib, masitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, aXitinib, cediranib, inib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, nib, crizotinib, cept,adipotide, denileukin diftitox, mTOR inhibitors such as Everolimus and Temsirolimus, hedgehog inhibitors such as sonidegib and Vismodegib, CDK tors such as CDK inhibitor (palbociclib).

In some embodiments, the composition comprising CAR immune cells are administered with an anti-in?ammatory agent. Anti-in?ammatory agents or drugs may include, but are not d to, steroids and glucocorticoids (including betamethasone, nide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, solone, prednisone, triamcinolone), nonsteroidal anti- in?ammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, le?unomide, NF medications, cyclophosphamide and mycophenolate.

Exemplary NSAle include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates. Exemplary sics include acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, ortisone, prednisolone, prednisolone, or prednisone. ary biological response modi?ers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine tors, such as the TNF antagonists, (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and in?iximab (REMICADE®), chemokine inhibitors and on molecule inhibitors. The biological response modi?ers include monoclonal antibodies as well as inant forms of les. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, mide, sulfasalazine, hydroxychloroquine, Gold (oral (aurano?n) and intramuscular), and minocycline.

In some embodiments, the compositions described herein are administered in conjunction with a cytokine. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N—methionyl human growth hormone, and bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, axin, glycoprotein hormones such as follicle stimulating hormone (F SH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH), hepatic growth factor (HGF), ?broblast growth factor (FGF), prolactin, placental lactogen, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular elial growth factor, integrin, thrombopoietin (TPO), nerve growth factors (NGFs) such as NGF-beta, platelet- growth factor, orming growth factors (TGFs) such as TGF-alpha and TGF-beta, insulin- like growth factor-1 and -II, erythropoietin (EPO, Epogen®, Procrit®), osteoinductive factors, interferons such as interferon-alpha, beta, and , colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF F), and granulocyte-CSF (G—CSF), interleukins (ILs) such as lL-l, IL-lalpha, 1L-2, 1L-3, IL-4, IL-5, IL-6, 1L-7, 1L-8, IL-9, IL-10, IL-1 1, lL-12, IL-15, a tumor necrosis factor such as INF-alpha or TNF-beta, and other ptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines.

Administration -directed genetically modi?ed autologous T cell therapy Indications and Usage In some embodiments, CDl9-directed genetically modi?ed autologous T cell immunotherapy indicated for the treatment of adult patients with relapsed or refractory large B-cell lymphoma after two or more lines of systemic therapy, ing diffuse large B-cell lymphoma (DLBCL) not otherwise speci?ed, primary mediastinal large B-cell lymphoma, high grade B-cell lymphoma, and DLBCL arising from ular lymphoma. In some embodiments, CDl9-directed genetically modi?ed autologous T cell immunotherapy is not indicated for the treatment of patients with primary l nervous system lymphoma.

Dosage andAdministration In some embodiments, an infusion bag of CDl9-directed genetically d autologous T cell immunotherapy comprises a suspension of chimeric antigen receptor (CAR)-positive T cells in approximately 68 mL. The target dose may be between about l X 106 and about 2 X 106 CAR-positive Viable T cells per kg body weight, with a maximum of 2 X 108 CAR-positive Viable T cells. In some ments the CDl9-directed genetically modi?ed autologous T cell immunotherapy is Axi-celTM RTA®, aXicabtagene ciloleucel).

CDl9-directed genetically modi?ed autologous T cell immunotherapy is for autologous use. The patient’s identity must match the patient identi?ers on the CD19- directed genetically modi?ed autologous T cell immunotherapy cassette and on bag. If the information on the patient-speci?c label does not match the ed patient, the CD19- directed genetically modi?ed autologous T cell immunotherapy cannot be administered.

In some embodiments, the availability of irected genetically modi?ed autologous T cell immunotherapy must be con?rmed prior to starting the lymphodepleting regimen.

In some embodiments, the patient is pre-treated prior to CDl9-directed genetically modi?ed autologous T cell therapy infusion with administration of lymphodepleting chemotherapy. In some embodiments, a lymphodepleting chemotherapy regimen of hosphamide 500 mg/m2 IV and ?udarabine 30 mg/m2 IV on the ?fth, fourth, and third day before infusion of CDl9-directed genetically modi?ed autologous T cell immunotherapy is administered.

In some embodiments, the patient is icated prior to CDl9-directed cally modi?ed autologous T cell immunotherapy on by oral administration of acetaminophen at a dose between about 500-1000 mg, about 600-1000 mg, about 700-1000 mg, about 800-1000 mg, about 900-1000 mg, about 500-900 mg, about 500-800 mg, about 500-700 mg, about 500-600 mg, about 600-900 mg, about 600-800 mg, about 600-700 mg, about 700-900 mg, about 700-800 mg, or about 800-900 mg. In some embodiments, the patient is premedicated prior to CDl9-directed genetically modi?ed autologous T cell immunotherapy infusion by oral administration of acetaminophen at a dose of about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg or about 1000 mg.

In some embodiments, the patient is premedicated prior to CDl9-directed genetically modi?ed autologous T cell immunotherapy infusion by administration of acetaminophen 650 mg by mouth and diphenhydramine 12.5 mg intravenously or by mouth approximately 1 hour before CDl9-directed genetically modi?ed autologous T cell immunotherapy infusion.

In some embodiments, the prophylactic use of systemic ds is avoided as it may interfere with the activity of CDl9-directed genetically modi?ed autologous T cell immunotherapy.

Preparation ofCD19—direcledgenetically modi?ed autologous T cell therapy for on The timing of CD19-directed genetically modi?ed autologous T cell immunotherapy thaw and infusion is coordinated. In some embodiments, the on time is con?rmed in advance, and the start time of CDl9-directed genetically modi?ed autologous T cell immunotherapy thaw is ed such that it will be available for infusion when the patient is ready.

In some embodiments, the patient identity is ed prior to CDl9-directed genetically modi?ed gous T cell immunotherapy thaw. Prior to CDl9-directed genetically modi?ed autologous T cell immunotherapy preparation, patient’s identity is matched with the patient ?ers on the CDl9-directed genetically modi?ed gous T cell immunotherapy cassette. In some embodiments, the CDl9-directed genetically modi?ed autologous T cell immunotherapy product bag is not removed from the cassette if the information on the patient-speci?c label does not match the intended patient.

In some embodiments, once patient identi?cation is ed, CDl9-directed genetically modi?ed gous T cell immunotherapy product bag is removed from the cassette and the patient information on the cassette label is con?rmed to match the bag label.

In some ments, the method comprises inspecting the product bag for any breaches of container integrity such as breaks or cracks before g. In some embodiments, the infusion bag is placed inside a second sterile bag per local guidelines.

In some embodiments, the method comprises thawing the CDl9-directed genetically modi?ed autologous T cell immunotherapy at approximately 37°C using either a water bath or dry thaw method until there is no e ice in the infusion bag. In some embodiments, the method comprises mixing or agitating the contents of the bag to disperse clumps of cellular material. In some embodiments, the contents of the bag are gently mixed or agitated. In some embodiments, the method comprises inspecting the bag for the presence of visible cell clumps remaining and mixing or agitation is continued. Small clumps of cellular material should disperse with gentle manual mixing. In some embodiments, the method does not comprise a wash, spin down, and/or re-suspension of CDl9-directed genetically modi?ed autologous T cell immunotherapy in new media prior to infusion.

In some ments, once thawed, CDl9-directed genetically modi?ed autologous T cell immunotherapy may be stored at room temperature (20°C to 25°C) for up to 3 hours.

Administration In some embodiments, the presently disclosed s of administration of irected genetically modi?ed autologous T cell immunotherapy comprise on or more of the following as steps or as considerations: 0 Ensure that tocilizumab and emergency equipment are available prior to infusion and during the ry period. 0 Do NOT use a leukodepleting ?lter. 0 Central venous access is recommended for the infusion of CDl9-directed cally modi?ed autologous T cell immunotherapy. 0 Con?rm the patient’s identity matches the patient ?ers on the irected genetically modi?ed autologous T cell immunotherapy product bag. 0 Prime the tubing with normal saline prior to infusion. o Infuse the entire contents of the CDl9-directed genetically modi?ed autologous T cell therapy bag within 30 minutes by either y or a peristaltic pump. CD19- directed genetically d autologous T cell immunotherapy is stable at room ature for up to 3 hours after thaw. o Gently agitate the product bag during CDl9-directed cally modi?ed autologous T cell immunotherapy infusion to prevent cell clumping. 0 After the entire content of the product bag is infused, rinse the tubing with normal saline at the same infusion rate to ensure all product is delivered. 0 CDl9-directed genetically modi?ed autologous T cell immunotherapy contains human blood cells that are genetically modi?ed with replication incompetent retroviral vector. Follow universal precautions and local biosafety guidelines for handling and disposal to avoid potential transmission of infectious diseases.

Monitoring In some embodiments, administration of CDl9-directed genetically modi?ed autologous T cell immunotherapy occurs at a certi?ed healthcare facility.

In some embodiments, the methods disclosed herein comprise monitoring patients at least daily for 7 days at the certi?ed healthcare facility following infusion for signs and symptoms of CR8 and neurologic toxicities. In some embodiments, the methods sed herein comprise monitoring patients at least daily for 10 days at the certi?ed healthcare facility following infusion for signs and symptoms of CR8 and neurologic toxicities.

In some embodiments, patients are instructed to remain within proximity of the certi?ed healthcare facility for at least 4 weeks following infusion. ment ofSevere Adverse Reactions In some embodiments, the method comprises management of adverse reactions. In some embodiments, the adverse reaction is ed from the group consisting of cytokine release syndrome (CRS), a neurologic ty, a hypersensitivity reaction, a serious infection, a cytopenia and mmaglobulinemia.

In some embodiments, the signs and symptoms of adverse reactions are selected from the group consisting of fever, hypotension, ardia, a, and chills, include cardiac arrhythmias (including atrial ation and ventricular tachycardia), c arrest, cardiac failure, renal insufficiency, capillary leak syndrome, hypotension, hypoxia, organ toxicity, agocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS), seizure, encephalopathy, he, tremor, dizziness, aphasia, delirium, insomnia anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia.

Cytokine Release Syndrome In some embodiments, the method comprises identifying CRS based on clinical presentation. In some embodiments, the method comprises evaluating for and ng other causes of fever, hypoxia, and hypotension. If CRS is observed or suspected, manage according to the recommendations in Table 1. Patients who ence 2 Grade 2 CRS (e.g., hypotension, not responsive to ?uids, or hypoxia requiring supplemental oxygenation) should be monitored with continuous cardiac telemetry and pulse oximetry. In some embodiments, for patients experiencing severe CRS, consider performing an echocardiogram 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 methods disclosed herein.

Table 1. CRS Grading and ment Guidance CRS Grade (a) Tocilizumab Corticosteroids Grade 1 N/A N/A Symptoms require symptomatic treatment only (e.g., fever, nausea, fatigue, headache, myalgia, malaise).

Grade 2 Administer tocilizumab (c) 8 Manage per Grade 3 if no mg/kg IV over 1 hour (not to improvement within 24 hours Symptoms require and exceed 800 mg). after starting tocilizumab. respond to moderate intervention. Repeat tocilizumab every 8 hours as needed if not Oxygen requirement less than 40% FiOz or responsive to IV ?uids or increasing supplemental hypotension responsive to ?uids or low-dose of one oxygen. vasopressor or Grade 2 Limit to a maximum of 3 organ toxicity (b). doses in a 24-hour ; maximum total of 4 doses if no clinical improvement in the signs and symptoms of Grade 3 Per Grade 2 ster prednisolone 1 mg/kg Symptoms require and IV twice daily or equivalent respond to aggressive dexamethasone (e.g., 10 mg intervention.

IV every 6 hours).

Oxygen requirement greater Continue osteroids use than or equal to 40% FlOz or until the event is Grade 1 or hypotension requiring high- dose or multiple less, then taper over 3 days. vasopressors or Grade 3 If not improving, manage as organ toxicity or Grade 4 Grade 4. transaminitis.

Grade 4 Per Grade 2 Administer methylprednisolone 1000 mg hreatening symptoms.

IV per day for 3 days, if Requirements for ventilator _ 1mproves, then manage as support, continuous veno- above. venous hemodialysis Consider alternate (CVVHD) or immunosuppressants if no Grade 4 or an toxicitg y . . . . 1mprovement or if cond1tlon (excluding transaminitis). worsens. (a) Lee DWet al., (2014). Current concepts in the diagnosis and management ofcytokine release me. Blood. 2014 Jul 10; 124(2): [887195. (b) Refer to Table 2for management ofneurologic toxicity. (c) Refer to ACEA/ITRA® (tocilizumab) Prescribing ationfor details, https.'//www.gene. wnload/pdf/actemraJarescribing.pdf (last accessed Oct. 18, 2017).

Initial US. al is indicated to be in 2010.

Neurologic Toxicity In some embodiments, the method comprises monitoring patients for signs and symptoms of neurologic toxicities (Table 2). In some embodiments, the method comprises ruling out other causes of neurologic ms. Patients who experience 2 Grade 2 neurologic toxicities should be monitored with continuous cardiac try and pulse oximetry. Provide intensive care supportive therapy for severe or life ening neurologic toxicities. Consider non-sedating, anti-seizure medicines (e.g., levetiracetam) for seizure prophylaxis for any 2 Grade 2 neurologic toxicities.

Table 2. ogic Toxicity Grading and Management Guidance Grading Concurrent CRS No concurrent CRS Administer tocilizumab per Table l for Administer dexamethasone 10 mg IV management of Grade 2 CRS. every 6 hours.

If no improvement within 24 hours after Continue dexamethasone use until the starting tocilizumab, administer event is Grade 1 or less, then taper over 3 g Concurrent CRS N0 concurrent CRS Assessment dexamethasone 10 mg IV every 6 hours if days. not already taking other steroids.

Continue dexamethasone use until the event is Grade 1 or less, then taper over 3 days.

Consider non-sedating, anti-seizure medicines (e.g., levetiracetam) for seizure prophylaxis.

Administer tocilizumab per Table l for Administer dexamethasone 10 mg IV management of Grade 2 CRS. every 6 hours.

In addition, administer dexamethasone 10 Continue dexamethasone use until the mg IV with the ?rst dose of tocilizumab event is Grade 1 or less, then taper over 3 and repeat dose every 6 hours. Continue days. thasone use until the event is Grade 1 or less, then taper over 3 days.

Administer tocilizumab per Table l for Administer prednisolone 1000 mg management of Grade 2 CRS. IV per day for 3 days, if improves, then Administer methylprednisolone 1000 mg manage as above.

IV per day with ?rst dose of tocilizumab and continue methylprednisolone 1000 mg IV per day for 2 more days, if es, then manage as above.

Dosage Forms And Strengths In some embodiments, CDl9-directed genetically modi?ed gous T cell immunotherapy is available as a cell suspension for infusion.

In some embodiments, a single dose of CDl9-directed genetically modi?ed autologous T cell immunotherapy comprises a target dose between about l X 106 and about 2 X 106 CAR-positive viable T cells per kg of body weight (or m of 2 X 108 CAR- positive viable T cells for patients 100 kg and above) in approximately 68 mL sion in an infusion bag. In some embodiments, the CDl9-directed genetically modi?ed autologous T cell immunotherapy is axicabtagene ciloleucel (YESCARTA®).

In some embodiments, a single dose of CDl9-directed genetically modi?ed autologous T cell immunotherapy is present in a container. Such container may be sterile. In some embodiments, the container is an infusion bag. In some embodiments, the infusion bag volume is about 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 500 mL, 750 mL, 1,000 mL, 1,500 mL, 2,000 mL or 3,000 mL.

Risk Evaluation andA/[itigation Strategy (REA/IS) Because of the risk of CR8 and ogic toxicities, in some embodiments, CDl9-directed genetically modi?ed autologous T cell immunotherapy is available through a restricted m under a Risk Evaluation and Mitigation Strategy (REMS). l components of the REMS are: 0 Healthcare facilities that dispense and administer CDl9-directed genetically modi?ed autologous T cell immunotherapy must be enrolled and comply with the REMS requirements. 0 Certi?ed healthcare facilities must have on-site, immediate access to tocilizumab, and ensure that a m of two doses of tocilizumab are available for each t for infusion within 2 hours after CDl9-directed genetically modi?ed autologous T cell immunotherapy infusion, if needed for treatment of CR8. 0 Certi?ed care facilities must ensure that healthcare providers who prescribe, se or ster CDl9-directed cally modi?ed autologous T cell immunotherapy are trained about the management of CR8 and neurologic toxicities.

WO 79564 2018/056467 Cytokine Release Syndrome [CR81 In some embodiments, the health care facility ensures that two doses of zumab are ble prior to infusion of CDl9-directed genetically modi?ed autologous T cell immunotherapy. In some embodiments, the health care facility s that four doses of tocilizumab are available prior to infusion of CDl9-directed cally d autologous T cell immunotherapy. In some embodiments, the method comprises monitoring patients at least daily for 7 days at the certi?ed care facility following infusion for signs and symptoms of CR8. In some embodiments, the method comprises monitoring patients at least daily for 7-10 days at the certi?ed healthcare facility following infusion for signs and symptoms of CR8. In some embodiments, the method comprises monitoring patients at least daily for 8 days at the certi?ed healthcare facility following infusion for signs and symptoms of CR8. In some embodiments, the method comprises monitoring patients at least daily for 9 days at the certi?ed healthcare facility following infusion for signs and symptoms of CR8. In some embodiments, the method comprises monitoring ts at least daily for 10 days at the certi?ed healthcare facility following infusion for signs and symptoms of CR8. In some embodiments, the method comprises monitoring patients for signs or symptoms of CR8 for 4 weeks after infusion. In some embodiments, the method comprises counseling patients to seek immediate medical attention should signs or symptoms of CR8 occur at any time. In some embodiments, the method comprises instituting treatment with supportive care, tocilizumab or zumab and osteroids as indicated at the ?rst sign of CR8.

Neurologic Toxicities In some embodiments, the method ses monitoring patients at least daily for 7 days at the ed healthcare facility following infusion for signs and symptoms of neurologic toxicities. In some embodiments, the method comprises monitoring patients at least daily for 7-10 days at the certi?ed healthcare facility following infusion for signs and symptoms of CR8. In some ments, the method comprises monitoring patients at least daily for 10 days at the certi?ed healthcare facility following infusion for signs and symptoms of CR8. In some embodiments, the method comprises monitoring patients for signs or symptoms of neurologic toxicities for 4 weeks after infusion and treat promptly.

Hypersensitivity Reactions Allergic reactions may occur with the infusion of CDl9-directed genetically modi?ed autologous T cell immunotherapy. In some embodiments, serious hypersensitivity reactions including anaphylaXis, may be due to dimethyl sulfoxide (DMSO) or residual gentamicin in CDl9-directed genetically modi?ed autologous T cell immunotherapy.

Viral Reactivation In some embodiments, Hepatitis B virus (HBV) reactivation, in some cases resulting in fulminant hepatitis, hepatic failure and death, may occur in patients treated with drugs directed against B cells. In some embodiments, the method comprises performing screening for HBV, HCV, and HIV in accordance with clinical guidelines before tion of cells for manufacturing.

Prolonged Cytopenias In some embodiments, patients may eXhibit cytopenias for several weeks following lymphodepleting chemotherapy and irected genetically modi?ed autologous T cell immunotherapy infusion. In some embodiments, the method comprises monitoring blood counts after CDl9-directed genetically modi?ed autologous T cell therapy infusion. mmaglobulinemia In some embodiments, B-cell aplasia and hypogammaglobulinemia may occur in ts receiving treatment with CDl9-directed genetically modi?ed autologous T cell immunotherapy. In some embodiments, the method comprises monitoring immunoglobulin levels after treatment with irected genetically modi?ed autologous T cell immunotherapy and managing using infection precautions, antibiotic prophylaxis and immunoglobulin replacement.

In some embodiments, vaccination with live virus vaccines is not recommended for at least 6 weeks prior to the start of lymphodepleting chemotherapy, during CDl9-directed cally d autologous T cell therapy treatment, and until immune recovery following treatment with CDl9-directed genetically modi?ed gous T cell immunotherapy.

Secondary Malignancies In some embodiments, patients treated with CDl9-directed genetically modi?ed gous T cell immunotherapy may develop secondary malignancies. In some ments, the method comprises monitoring life-long for secondary malignancies.

Tumour lysis syndrome [TLS] Patients treated with CDl9-directed genetically modi?ed autologous T cell therapy may develop TLS, which may be severe. To minimise risk of TLS, in some embodiments, the method comprises evaluating patients for elevated uric acid or high tumour burden and administering allopurinol, or an alternative prophylaxis, prior to axicabtagene ciloleucel infusion. Signs and ms of TLS should be red and events managed according to standard guidelines.

Effects on y to Drive and Use Machines Due to the potential for ogic events, including altered mental status or seizures, patients receiving CDl9-directed genetically modi?ed gous T cell therapy are at risk for altered or decreased consciousness or coordination in the 8 weeks following CDl9-directed genetically modi?ed autologous T cell immunotherapy infusion. In some embodiments, the method comprises advising patients to refrain from driving and engaging in hazardous occupations or activities, such as operating heavy or potentially dangerous machinery, during this initial period.

Sforage andHandling In some embodiments, CDl9-directed genetically modi?ed autologous T cell immunotherapy is supplied in an infusion bag containing approximately 68 mL of frozen suspension of genetically modi?ed autologous T cells in 5% DMSO and 2.5% albumin (human). In some embodiments, CDl9-directed genetically d gous T cell immunotherapy is ed in an infusion bag containing imately 68 mL of frozen suspension of genetically modi?ed autologous T cells in 5% DMSO and 2.5% albumin (human) (NDC 7128701). In some embodiments, CDl9-directed genetically modi?ed autologous T cell immunotherapy comprises Cryostor CSlO. In some embodiments, CD19- directed genetically modi?ed autologous T cell immunotherapy comprises 300 mg sodium per infusion. In some embodiments, CDl9-directed genetically modi?ed autologous T cell immunotherapy is supplied in an infusion bag containing approximately 50-100 mL, 50-90 mL, 50-80 mL, 50-70 mL, 60-70 mL, 60-75 mL, or 65-75 mL, of suspension of genetically modi?ed autologous T cells in 5% DMSO and 2.5% albumin (human). In some ments, irected cally d autologous T cell immunotherapy 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 modi?ed gous T cells in 5% DMSO and 2.5% albumin (human). In some embodiments, CDl9-directed genetically modi?ed autologous T cell immunotherapy is supplied in an infusion bag containing greater than 50 mL, greater than 60 mL, greater than 65 mL, greater than 66 mL, greater than 67 mL, or greater than 68 mL, of sion of genetically modi?ed autologous T cells in 5% DMSO and 2.5% albumin (human). In some embodiments, the suspension is frozen.

In some embodiments, the irected genetically modi?ed gous T cell immunotherapy on bag is supplied in ethylene-vinyl e cryostorage bag with sealed addition tube and two available spike ports, containing approximately 68 mL of cell sion.

In some embodiments, the CDl9-directed genetically modi?ed autologous T cell immunotherapy infusion bag is individually packed in a metal cassette. In some embodiments, the CDl9-directed genetically modi?ed gous T cell immunotherapy infusion bag is individually packed in a metal cassette (NDC 7128702). In some embodiments, the CDl9-directed genetically modi?ed autologous T cell immunotherapy infusion bag is stored in the vapor phase of liquid nitrogen. In some embodiments, the CDl9-directed genetically modi?ed gous T cell immunotherapy infusion bag is supplied in a liquid nitrogen dry shipper.

In some embodiments, the method comprises ng the identity of the patient with the patient identi?ers on the cassette and infusion bag upon receipt. In some embodiments, irected genetically modi?ed autologous T cell immunotherapy is stored frozen in the vapor phase of liquid nitrogen (less than or equal to minus 150°C). In some embodiments, the CDl9-directed genetically modi?ed autologous T cell immunotherapy is thaw before using.

EXAMPLES Example 1: Clinical Studies of Relapsed 0r Refractory Large B-Cell Lymphoma A single-arm, open-label, multicenter trial evaluated the ef?cacy of a single infusion of Axi-celTM RTA®) in adult patients with relapsed or refractory aggressive B-cell non-Hodgkin ma. Eligible patients had refractory disease to the most recent therapy or relapse within 1 year after autologous hematopoietic stem cell lantation (HSCT). The study excluded patients with prior allogeneic HSCT, any history of central nervous system lymphoma, ECOG performance status of 2 or greater, absolute lymphocyte count less than 100/uL, creatinine nce less than 60 mL/min, hepatic transaminases more than 2.5 times the upper limit of normal, cardiac ejection fraction less than 50%, or active serious infection.

Following lymphodepleting herapy, AXi-celTM was administered as a single IV infusion at a target dose of 2 X 106 CAR-positive viable T cells/kg (maximum permitted dose: 2 X 108 cells). The lymphodepleting regimen consisted of cyclophosphamide 500 mg/m2 IV and ?udarabine 30 mg/m2 IV, both given on the fifth, fourth, and third day before AXi-celTM. Bridging chemotherapy between leukapheresis and lymphodepleting chemotherapy was not permitted. All ts were alized for Axi-celTM infusion and for a m of 7 days afterward.

Of 1 11 patients who underwent leukapheresis, 101 received lTM. Of the patients treated, the median age was 58 years (range: 23 to 76), 67% were male, and 89% were white. Most (76%) had DLBCL, 16% had transformed follicular lymphoma, and 8% had primary mediastinal large B-cell lymphoma. The median number of prior ies was 3 (range: 1 to 10), 77% of the patients had refractory disease to a second or greater line of therapy, and 21% had relapsed within 1 year of autologous HSCT.

One out of 1 11 patients did not receive the product due to cturing failure. Nine other patients were not treated, primarily due to progressive disease or serious e reactions following leukapheresis. The median time from leukapheresis to product delivery was 17 days (range: 14 to 51 days), and the median time from leukapheresis to infusion was 24 days (range: 16 to 73 days). The median dose was 2.0 X 106 CAR-positive viable T cells/kg (range: 1.1 to 2.2 X 106 kg).

Ef?cacy was established on the basis of complete remission (CR) rate and on of response (DOR), as determined by an independent review committee (Table 3 and Table 4). The median time to response was 0.9 months (range: 0.8 to 6.2 months). se durations were longer in patients who achieved CR, as compared to patients with a best response of partial remission (PR) (Table 4). Of the 52 patients who achieved CR, 14 initially had stable disease (7 patients) or PR (7 patients), with a median time to improvement of 2.1 months (range: 1.6 to 5.3 months).

Table 3. Response Rate Recipients of Axi-celTM (N = 101) Objective Response Rate" 73 (72%) (95% C1) (62, 81) Complete ion Rate 52 (51%) (95% CI) (41, 62) Partial Remission Rate 21 (21%) (95% C1) (13, 30) CI, con?dence interval.

"Per 2007 revisedInternational Working Group criteria, as assessed by the independent review committee.

Table 4. Duration of Response From N of 101 Number of Responders 73 DOR (Months)" Medianl7 9.2 (95% CI) (5.4, NE) Rangec 003+, 144+ DOR if Best se is CR s) Medianl7 NE (95% CI) (8.1, NE) Range 0.4, 144+ DOR if Best Response is PR (Months) Medianl7 2.1 (95% CI) (1.3, 5.3) Range 003+, 8.4+ Median Follow-up for DOR (Months)"’ b 7.9 CR; complete remission; DOR; duration of response; NE; not ble; PR; partial remission.

"Among all responders. DOR is measuredfrom the date offirst objective response to the date ofprogression or deathfrom relapse or toxicity. bKaplan-Meier estimate.

CA + sign tes a censored value.

Example 2: Pharmacodynamics and cokinetics After Axi-celTM infusion After lTM infusion; pharmacodynamic responses were evaluated over a 4-week al by measuring transient elevation of cytokines; chemokines and other molecules in blood. Levels of cytokines and chemokines such as 1L-6; 1L-8; lL-lO; IL-15; TNF-d; lFN—y; and SILZROL were analyzed. Peak elevation was ed within the ?rst 14 days after infusion; and levels generally returned to baseline within 28 days. Due to the on- target effect of AXi-celTM; a period of B-cell aplasia is expected.

Following infusion of AXi-celTM; anti-CD19 CAR T cells exhibited an initial rapid ion followed by a decline to near baseline levels by 3 months. Peak levels of anti-CD19 CAR T cells ed within the ?rst 7-14 days after Axi-celTM infusion. Age (range: 23 — 76 years) and gender had no signi?cant impact on 2sd) and CmaX of Axi- celTM.

The number of anti-CD19 CAR T cells in blood was positively associated with objective response ete remission (CR) or partial remission (PR)). The median anti- CD19 CAR T-cell CmaX levels in ders (n=73) were 205% higher compared to the corresponding level in nonresponders (n=23) (43.6 uL vs 21.2 cells/uL). Median AUC Day 0-28 in responding patients (n=73) was 251% of the corresponding level in nonresponders (n=23) (557.1 days*cells/uL vs. 222.0 days*cells/uL).

Some patients required tocilizumab and corticosteroids for management of CR8 and neurologic toxicities. Patients treated with tocilizumab (n=44) had 262% and 232% higher anti-CD19 CAR T cells as measured by AUC(o-2sd) and CmaX respectively; as compared to patients who did not receive tocilizumab (n=57). Similarly; patients that received corticosteroids (n=26) had 217% and 155% higher AUC(o.2sd) and CmaX compared to patients who did not receive corticosteroids (n=75).

Example 3: Management of e ons after CD19-directed genetically modified autologous T cell immunotherapy Because clinical trials are conducted under widely varying conditions, adverse reaction rates observed in the clinical trials of a drug cannot be directly compared to rates in the clinical trials of another drug and may not re?ect the rates observed in practice.

The safety data bed in this section re?ect exposure to Axi-celTM in the clinical trial (Study 1) in which 108 patients with relapsed/refractory B-cell NHL received CAR-positive T cells based on a ended dose which was weight-based. ts with a history of CNS disorders (such as seizures or cerebrovascular ischemia) or autoimmune disease requiring systemic immunosuppression were ineligible. The median duration of follow up was 8.7 months. The median age of the study population was 58 years (range: 23 to 76 years); 68% were men. The baseline ECOG mance status was 43% with ECOG 0, and 57% with ECOG 1.

The most common adverse reactions (incidence Z 20%) include CRS, fever, hypotension, encephalopathy, tachycardia, fatigue, headache, sed appetite, chills, diarrhea, febrile neutropenia, infections-pathogen unspeci?ed, nausea, hypoxia, , cough, vomiting, dizziness, constipation, and cardiac arrhythmias. Serious adverse reactions occurred in 52% of patients. The most common serious adverse ons (> 2%) include encephalopathy, fever, lung infection, febrile neutropenia, c arrhythmia, cardiac failure, y tract infection, renal insuf?ciency, aphasia, cardiac arrest, Clostridium dif?cile infection, delirium, hypotension, and a.

The most common (2 10%) Grade 3 or higher reactions include febrile neutropenia, fever, CRS, encephalopathy, infections-pathogen unspeci?ed, hypotension, hypoxia and lung infections.

Forty-?ve t (49/108) of patients received tocilizumab after infusion of Axi-celTM.

Table 5 summarizes the adverse reactions that occurred in at least 10% of patients treated with Axi-celTM and Table 6 describes the laboratory abnormalities of Grade 3 or 4 that occurred in at least 10% of patients.

Table 5. Summary of Adverse Reactions Observed in at Least 10% of the Patients Treated with Axi-celTM in Study 1 e Reaction Any Grade (%) Grade 3 or Higher (%) Cardiac Disorders Tachycardia" 57 2 Arrhythmial7 23 7 Gastrointestinal Disorders Diarrhea 3 8 4 Nausea 34 O Vomiting 26 1 pation 23 O Abdominal painC 14 1 Dry mouth 11 0 l Disorders AndAdministration Site Conditions Fever 86 1 6 Fatigue61 46 3 Chills 40 O Edemae 1 9 1 Immune System Disorders Cytokine release syndrome 94 13 Hypogammaglobulinemiaf 15 O Infections AndInfestations Infections-pathogen i?ed 26 16 Viral infections 16 4 Bacterial Infections 13 9 Investigations Decreased appetite 44 2 Weight decreased 16 O Dehydration 1 1 3 Musculoskelatal And Connective Tissue Disorders Motor dysfunctiong 19 1 Pain in extremityh 17 2 Back pain 15 1 Muscle pain 14 1 Arthralgia 1 O O Nervous System Disorders alopathyi 57 29 Headache’ 45 1 Tremor 3 1 2 Dizzinessk 21 1 Adverse Reaction Any Grade (%) Grade 3 or Higher (%) Aphasial l 8 6 Psychiatric Disorders Deliriumm l 7 6 Respiratory, Thoracic AndMediastinal a" 3 2 l l Cougho 30 O Dyspneap 1 9 3 Pleural effusion 13 2 Renal and Urinary Disorders Renal insuf?ciency 12 5 Vascular Disorders Hypotensionq 5 7 1 5 Hypertension 1 5 6 Thrombosi sr 10 l The following events were also counted in the nce of CR8: tachycardia, arrhythmia, fever, chills, hypoxia, renal insuf?ciency, and hypotension. cardia includes tachycardia, sinus tachycardia.

Z7Arrhythmia includes arrhythmia, atrialfibrillation, atrialflutter, atrioventricular block, bundle branch block right, electrocardiogram QTprolonged, extra-systoles, heart rate lar, supraventricular extra systoles, entricular tachycardia, ventricular arrhythmia, cular tachycardia.

CAbdominalpain includes nalpain, abdominalpain lower, abdominalpain upper. dFatigue includesfatigue, malaise. eEdema includesface edema, generalized edema, local swelling, localized edema, edema, edema genital, edemaperipheral, periorbital edema, peripheral swelling, scrotal edema. nypogammaglobulinemia includes hypogammaglobulinemia, blood immunoglobulin D decreased, blood immunoglobulin G decreased gMotor dysfunction includes muscle spasms, muscular weakness. hPain in extremity includes pain not otherwise specified, pain in extremity. lEncephalopathy includes ive disorder, confusional state, depressed level of consciousness, disturbance in attention, encephalopathy, hypersomnia, leukoencephalopathy, memory impairment, mental status s, paranoia, ence, stupor.

JHeadache includes headache, head discomfort, sinus headache, procedural headache. kDizziness includes dizziness, presyncope, syncope. lAphasia includes aphasia, dysphasia. mDelirium includes agitation, delirium, delusion, disorientation, hallucination, hyperactivity, irritability, restlessness.

"Hypoxia includes hypoxia, oxygen saturation decreased 0Cough includes cough, productive cough, upper-airway cough syndrome. prspnea es acute atoryfailure, dyspnea, orthopnea, respiratory distress. qHypotension es lic hypotension, hypotension, orthostatic hypotension. rThrombosis includes deep vein thrombosis, sm, embolism venous, pulmonary embolism, splenic infarction, splenic vein thrombosis, subclavian vein thrombosis, thrombosis, thrombosis in device.

WO 79564 2018/056467 Other clinically important adverse reactions that occurred in less than 10% of patients treated with AXi-celTM include the following: 0 Blood and lymphatic system disorders: Coagulopathy (2%) 0 Cardiac ers: c failure (6%) and cardiac arrest (4%) o Immune system disorders: Hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS) (1%), hypersensitivity (1%) o Infections and infestations disorders: Fungal infections (5%) o s system disorders: Ataxia (6%), seizure (4%), dyscalculia (2%), and myoclonus (2%) 0 Respiratory, thoracic and mediastinal disorders: Pulmonary edema (9%) 0 Skin and subcutaneous tissue disorders: Rash (9%) 0 Vascular ers: Capillary leak me (3%) Laboratory Abnormalities: Table 6. Grade 3 or 4 Laboratory Abnormalities Occurring in 2 10% of Patients in Study 1 Following Treatment with Axi-cel based on CTCAE (N=108) Grades 3 or 4 (%) Lymphopenia 100 Leukopenia 96 Neutropenia 93 Anemia 66 Thrombocytopenia 5 8 Hypophosphatemia 50 Hyponatremia 19 Uric acid increased 13 Direct Bilirubin increased 13 Hypokalemia 10 Alanine Aminotransferase increased 10 Cytokine Release Syndrome CRS, including fatal or life-threatening reactions, occurred following treatment with Axi-celTM. In Study 1, CRS occurred in 94% (101/108) of patients receiving Axi-celTM, including 2 Grade 3 (Lee grading systeml) CRS in 13% 8) of patients.

Among patients who died after receiving Axi-celTM, four had ongoing CRS events at the time of death. The median time to onset was 2 days (range: 1 to 12 days) and the median on of CRS was 7 days (range: 2 to 58 days). Key manifestations 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 ?brillation and ventricular tachycardia), cardiac arrest, cardiac failure, renal insuf?ciency, capillary leak syndrome, hypotension, hypoxia, and hemophagocytic lymphohistiocytosis/macrophage activation me AS).

Neurologic Toxicities Neurologic toxicities, that were fatal or life-threatening, occurred following treatment with Axi-celTM. Neurologic toxicities occurred in 87% of patients. -eight percent of all neurologic toxicities occurred within the ?rst 8 weeks of Axi-celTM infusion, with a median time to onset of 4 days (range: 1 to 43 days). The median duration of neurologic toxicities was 17 days. Grade 3 or higher neurologic ties occurred in 31% of patients.

The most common neurologic toxicities ed encephalopathy (57%), headache (44%), tremor (31%), dizziness (21%), aphasia (18%), delirium (17%), insomnia (9%) and anxiety (9%). Prolonged encephalopathy lasting up to 173 days was noted. Serious events including leukoencephalopathy and seizures occurred with Axi-celTM. Fatal and serious cases of cerebral edema have ed in patients treated with Axi-celTM.

Serious Infections Severe or life-threatening infections occurred in patients after Axi-celTM infusion. In Study 1, infections (all grades) occurred in 38% of patients. Grade 3 or higher infections occurred in 23% of patients. Grade 3 or higher infections with an unspeci?ed en occurred in 16% of patients, bacterial infections in 9%, and viral infections in 4%.

Axi-celTM should not be administered to ts with clinically signi?cant active ic infections. Monitor ts for signs and symptoms of infection before and after Axi-celTM on and treat appropriately. Administer prophylactic anti-microbials according to local guidelines.

Febrile neutropenia was observed in 36% of patients after Axi-celTM infusion and may be rent with CRS. In the event of febrile neutropenia, evaluate for infection and manage with broad spectrum antibiotics, ?uids and other supportive care as medically Immunogenicity Axi-celTM has the potential to induce anti-product antibodies. The genicity of Axi-celTM has been evaluated using an enzyme-linked immunosorbent assay (ELISA) for the detection of binding antibodies against FMC63, the ating antibody of the anti-CD19 CAR. Three patients tested ve for pre-dose anti-FMC63 antibodies at baseline and months 1, 3, or 6 in Study 1. There is no evidence that the kinetics of initial expansion and persistence of Axi-celTM, or the safety or effectiveness of Axi-celTM, was altered in these patients.

In Study 1, Grade 3 or higher cytopenias not resolved by Day 30 following Axi-celTM infusion occurred in (28%) of patients and included thrombocytopenia (18%), neutropenia (15%), and anemia (3%). Monitor blood counts after Axi-celTM infusion.

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

All publications, s, patent applications, and references, including prescribing information, that are mentioned in this ication are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present invention. To the extent that any of the definitions or terms provided in the nces orated by reference differ from the terms and discussion provided herein, the present terms and de?nitions control. 1004790493

Claims

1. Use of an axicabtagene ciloleucel suspension in the manufacture of a medicament for ng relapsed or refractory diffuse large B-cell lymphoma (DLBCL) not otherwise specified, primary tinal large B-cell lymphoma, high grade B-cell lymphoma, or DLBCL arising from follicular lymphoma after two or more lines of systemic therapy in a patient; wherein the medicament is to be stered by enous infusion at a dose between about 1 × 106 and about 2 × 106 CAR-positive viable T cells per kg body weight up to a maximum dose of about 1 x 108 sitive viable T cells; and the patient is to be monitored for signs and symptoms of cytokine release syndrome (CRS) at least daily, for a period of only 7-10 days following the administration of the medicament, if CRS Grade 2 is observed, the patient is to be administered zumab 8 mg/kg IV over 1 hour.

2. The use of claim 1, n following stering the medicament, if CRS Grade 3 is observed, tocilizumab is to be administered at a dose of 8 mg/kg IV over 1 hour, tocilizumab every 8 hours as needed, and methylprednisolone 1 mg/kg IV is to be administered at least two times or equivalent dexamethasone (10 mg IV every 6 hour) is to be administered.

3. The use of claim 1, wherein following administering the medicament, if CRS Grade 4 is observed, tocilizumab is to be administered at a dose of about 8 mg/kg IV over 1hour, ing tocilizumab every 8 hours as needed; and about 1,000 mg IV methylprednisolone is to be administered per day for 3 days.

4. The the use of any one of claims 1-3, wherein the intravenous infusion time for the axicabtagene ciloleucel suspension infusion is between 15 and