NZ789091A - Methods for the treatment of b cell malignancies using adoptive cell therapy - Google Patents

Abstract

Provided are adoptive cell therapy methods involving the administration of doses of cells for treating B cell malignancies. The cells generally express recombinant receptors such as chimeric antigen receptors (CARs). In some embodiments, the methods are for treating subjects with chronic lymphocytic leukemia (CLL). In some embodiments, the methods are for treating subjects with non-Hodgkin lymphoma (NHL). In some embodiments, the methods involve prior administration of a lymphodepleting therapy, such as prior administration of fluradibine and/or another lymphodepleting chemotherapeutic agent, for example cyclophosphamide. In some embodiments, features of the methods include an increase in complete remission, overall survival and/or progression free survival of subjects treated in accord with the provided methods. leukemia (CLL). In some embodiments, the methods are for treating subjects with non-Hodgkin lymphoma (NHL). In some embodiments, the methods involve prior administration of a lymphodepleting therapy, such as prior administration of fluradibine and/or another lymphodepleting chemotherapeutic agent, for example cyclophosphamide. In some embodiments, features of the methods include an increase in complete remission, overall survival and/or progression free survival of subjects treated in accord with the provided methods.

Description

METHODS FOR THE ENT OF B CELL MALIGNANCIES USING ADOPTIVE CELL THERAPY Statement of Government Interest This invention was made with government support under CA136551 awarded by the National Institutes of Health. The government has certain rights in the ion.

Cross-Reference to Related Applications This application claims priority from U.S. provisional application No. 62/346,547, filed June 6, 2016, U.S. provisional application No. 62/417,292, filed er 3, 2016, and U.S. provisional application No. 62/429,737, filed December 3, 2016, the contents of which are incorporated by reference in their entirety.

Incorporation By Reference of Sequence Listing The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042006740seqlist.txt, created June 6, 2017, which is 36,277 kilobytes in size. The information in the electronic format of the Sequence Listing is orated by nce in its entirety.

Field The present disclosure relates to adoptive cell therapy involving the administration of doses of cells for treating B cell malignancies. The cells generally express recombinant ors such as chimeric antigen receptors (CARS). In some embodiments, the methods are for treating subjects with chronic lymphocytic ia (CLL). In some embodiments, the methods are for treating subjects with non-Hodgkin lymphoma (NHL). In some embodiments, the methods involve prior administration of a lymphodepleting y, such as prior administration of bine and/or another lymphodepleting herapeutic agent, for example hosphamide. In some embodiments, features of the methods include an increase in complete remission, overall survival and/or progression free survival of subjects treated in accord with the provided methods.

Background Various immunotherapy and/or cell therapy methods are ble for ng diseases and conditions. For example, adoptive cell therapies (including those involving the administration of cells expressing chimeric receptors specific for a disease or disorder of interest, such as chimeric antigen receptors (CARS) and/or other recombinant n receptors, as well as other adoptive immune cell and adoptive T cell therapies) can be effective in the treatment of cancer and other diseases and disorders. Improved methods are , for e, to increase efficacy of such methods. Provided are methods and uses that meet such needs.

Summary ed are methods and uses of ered cells (e.g., T cells) and compositions f, for the treatment of ts having a disease or condition, which generally is or includes a leukemia or lymphoma, most particularly a chronic cytic leukemia (CLL) and/or a non-Hodgkin lymphoma (NHL). In some aspects, the methods and uses provide for or achieve improved or more durable responses or efficacy and/or a reduced risk of toxicity or other side effects, as compared to certain alternative s, such as in particular groups of ts treated. In some embodiments, the methods are advantageous by virtue of the administration of specified numbers or relative numbers of the engineered cells, the administration of d ratios of particular types of the cells, the preconditioning of subjects with particular lymphodepleting therapies, treatment of particular patient populations, such as those having a particular risk profile, staging, and/or prior treatment history, and/or combinations thereof.

In some embodiments, the methods and uses include administering to the subject cells expressing genetically engineered (recombinant) cell surface receptors in adoptive cell therapy, which generally are chimeric receptors such as chimeric antigen receptors (CARS), recognizing an antigen sed by, associated with and/or specific to the leukemia or lymphoma and/or cell type from which it is derived. The cells are generally administered in a composition formulated for administration; the methods generally involve administering one or more doses of the cells to the subject, which dose(s) may include a particular number or relative number of cells or of the engineered cells, and/or a defined ratio of two or more sub—types within the composition, such as CD4 vs CD8 T cells.

The subject generally has been preconditioned with a lymphodepleting y, which in some aspects increases the persistence and/or efficacy of the cells following administration, as compared to s in which the preconditioning is not carried out or is carried out using a different lymphodepleting therapy. The lymphodepleting therapy generally es the administration of fludarabine, typically in combination with another chemotherapy or other agent, such as cyclophosphamide, which may be administered sequentially or aneously in either order.

In some embodiments, the methods involve ng a subject having or suspected of having a chronic lymphocytic leukemia (CLL). In some aspects, the methods include administering to the subject a dose of cells sing a ic antigen receptor (CAR) that specifically binds to a target antigen expressed by the CLL. In some aspects, the dose contains (a) at or about 2 x 105 of the cells per kilogram body weight of the subject (cells/kg); (b) at or about 2 x 106 of the cells/kg, (c) no more than at or about 2 x 106 of the cells/kg, (d) no more than at or about 2 x 105 of the cells/kg and/or (e) between at or about 2 x 105 of the cells/kg and at or about 2 x 106 of the cells/kg. In some aspects, prior to the administration, the t has been preconditioned with a depleting therapy that includes the administration of fludarabine.

Also provided is a method of treating a subject having or suspected of having a chronic lymphocytic ia (CLL), the method including administering to the subject a dose of cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen expressed by the CLL, said dose contianing (a) at or about 1 x 107 total cells or total CAR- expressing cells; (b) at or about 1.5 x 108 total cells or total CAR—expressing cells, (c) no more than at or about 1 x 107 total cells or total CAR—expressing cells, (d) no more than at or about 1.5 x 108 total cells or total CAR-expressing cells and/or (e) between at or about 1 x 107 total cells or total CAR—expressing cells and at or about 1.5 x 108 total cells or total CAR—expressing cells, wherein, prior to the administration, the subject has been preconditioned with a lymphodepleting y comprising the administration of fludarabine.

In some embodiments, the methods e treating a subject having a non-Hodgkin lymphoma (NHL), such as an aggressive NHL and/or an NHL of a particular sub—type such as a e large B cell lymphoma (DLBCL), a primary mediastinal large B cell lymphoma (PMBCL), a T cell/histocyte—rich large B cell lymphoma (TCHRBCL), a Burkitt’s lymphoma, and/or other aggressive NHL, a mantle cell lymphoma (MCL), and/or ular lymphoma (FL).

In some aspects, the methods include administering to the subject a dose of cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen expressed by the NHL. In some aspects, the dose contains (a) at or about 2 x 105 of the cells per kilogram body weight of the subject (cells/kg); (b) at or about 2 x 106 of the cells/kg, (c) no more than at or about 2 x 106 of the cells/kg, (d) no more than at or about 2 x 105 of the cells/kg and/or (e) between at or about 2 x 105 of the cells/kg and at or about 2 x 106 of the cells/kg. The dose lly r contains a defined ratio of particular subtypes of cells, such as CD4+ cells expressing the CAR to CD8+ cells expressing the CAR and/or of CD4+ cells to CD8+ cells.

In some aspects, prior to the administration, the t has been preconditioned with a lymphodepleting y that includes the administration of fludarabine.

In some aspects of any of the embodiments, the dose of cells administered includes a defined or termined or ered ratio of particular sub-types of cells. The ratio may include a ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR and/or of CD4+ cells to CD8+ cells. In some aspects, the CD4+ or CD8+ cells are enriched for a particular subtype, such as central-memory cells or are derived from such an enriched population, such as cells derived from CD8+ central memory cells, and/or those exhibiting sed expression of CD62L and/or CD45RO and/or CCR7 as compared to bulk T cells or bulk CD8+ T cells, or cells derived from bulk T cells or bulk CD8+ T cells. In some aspects, the ratio is or is approximately 1:1. In some s, it is between at or approximately 1:3 and at or approximately 3:1.

In some embodiments, at or prior to the administration of the dose of cells: the subject is or has been identified as having one or more cytogenetic abnormalities, ally ated with high—risk NHL; the subject is or has been identified as having high—risk NHL; and/or the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), T cell/histocyte— rich large B cell lymphoma (TCHRBCL), Burkitt’s lymphoma, mantle cell lymphoma (MCL), and/or follicular lymphoma (FL).

In some aspects of any of the embodiments, the subject is an adult and/or is over at or about 30, 40, 50, 60, or 70 years of age.

In some aspects, the lymphodepleting therapy further includes stering another chemotherapeutic agent other than the fludarabine, such as cyclophosphamide. In some aspects, the ditioning, e.g., via the lymphodepleting therapy such as fludarabine and/or cyclohphosphamide is initiated at a time that is at least at or about 24 or at least at or about 48 hours prior to, or is between at or about 48 and at or about 96 hours prior to, the administration of the cells. In some aspects, the lymphodepleting therapy includes the administration of cyclophosphamide at at or about 30-60 mg/kg of the subject (such as once daily for one, two or three days), and/or the fludai'abine, such as at 25 mg/m2 (e.g., daily for 2, 3, 4, or 5 or more days, such as for 3-5 days).

In some aspects of any of the embodiments, the stration of the cell dose and/or the depleting therapy is d out via outpatient delivery.

In some embodiments, at or prior to the administration of the dose of cells, the subject is or has been identified as having one or more cytogenetic abnormalities and/or other risk factors. In some aspects, the abnormalities or (s) are associated with high-risk or very- high risk disease, such as high—risk or very high risk CLL and/or NHL.

Such factors may be those detected or detectable by FISH and/or those not detectable by FISH. The abnormalities may include complex karyotype (CK), translocations, deletion of the long arm of chromosome 13 (del l3q), del 11, y 12, del 17p, del 6q, and del , optionally as detected by FISH. In some aspects, the abnormalities e CK and/or dell7p.

In some aspects, the subject treated by the methods and uses is or has been identified as having high-risk CLL or very high-risk CLL and optionally is selected for the treatment based on such classification and/or a particular ality. In some aspects of any of the embodiments, the subject is or has been identified as, and/or is selected for, having metastatic, aggressive, advanced, and/or extramedullary form of the disease or condition; and/or the subject is an adult and/or is over at or about 30, 40, 50, 60, or 70 years of age.

In some embodiments, prior to the administration of the dose of cells, the subject has been treated with two or more, optionally 3, 4, 5, 6, 7, 8, or 9 more, therapies for the leukemia or lymphoma, such as the NHL and/or the CLL and/or with two or more, optionally 3, 4, 5, 6, 7, 8, or 9 more, therapies other than the lymphodepleting therapy and/or other than another dose of cells expressing the CAR. In some embodiments, prior to the administration of the dose of cells, the t has been treated for the CLL with a kinase inhibitor, optionally an inhibitor of Btk, optionally ibrutinib, and/or a biologic and/or immunotherapy, such as onal antibody. In some embodiments, prior to the administration of the dose of cells, the subject has been d with two or more, optionally 2, 3, or 4 more, therapies for the leukemia or lymphoma, which generally are therapies other than the lymphodepleting therapy and/or the cells or another dose of cells expressing the CAR, optionally other than cells expressing a ent CAR.

In some embodiments, at or immediately prior to the time of the administration of the dose of cells, the subject has relapsed following ion after treatment with, or become refractory to, such one or more prior therapies.

In some embodiments, prior to the administration of the dose of cells, the subject has been treated for the leukemia or lymohoma (such as the CLL or NHL) with a monoclonal antibody such as one that specifically binds to an antigen expressed by, or previously expressed by, cells of the CLL or the NHL. In some embodiments, at or ately prior to the time of the administration of the dose of cells, the subject has relapsed following remission after treatment with, or become tory to, one or more prior therapies for the CLL.

In some embodiments, the method further includes, prior to the stration of the cell dose, administering the lymphodepleting therapy to the subject.

In some embodiments, the dose of cells ns a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR and/or of CD4+ cells to CD8+ cells, which optionally is approximately 1:1 or is between approximately 1:3 and approximately 3: 1.

In some embodiments, the dose of cells is administered parenterally, optionally intravenously.

In some ments of the s provided , at least 50% of subjects treated according to the method achieve complete remission (CR) and/or objective se (OR); and/or the subject ts CR, OR, lymph nodes of less than at or about 20 mm in size, within 1 month of the administration of the dose of cells; and/or a malignant immunoglobulin heavy chain (IGH) locus and/or an index clone of the disease or condition such as the CLL or NHL is not detected in the bone marrow of the subject (or in the bone marrow of greater than 50% of subjects treated according to the methods), optionally as assessed by IGH deep sequencing, optionally at a time that is at or about or at least at or about 1, 2, 3, 4, 5, 6, 12, 18, or 24 months following the administration of the cell dose.

In some embodiments of the methods provided herein, at least 50% of subjects that are treated according to the method, and that achieve complete ion (CR), exhibit progression-free survival (PFS) and/or overall survival (OS) of greater than 12 months; on average, subjects treated according to the method exhibit a median PFS or OS of greater than at or about 6 months, 12 months, or 18 months; and/or the subject exhibits PFS or OS following y for at least at or about 6, 12, 18 or more months.

In some embodiments, the antigen is a B cell antigen. In some aspects, the antigen is CD19. In some s, the antigen is or includes CD20, CD22, CD30, CD33, CD38, RORl, or other marker associated with B cells or B cell cancer.

In some embodiments, the CAR contains a binding domain, which typically is or comprises an scFv specific for the antigen, a transmembrane domain, and one or more cytoplasmic signaling domains or regions, which may be derived from natural or nous signaling molecules or functional variants thereof. In some aspects, the signaling region includes domains capable of delivering primary and ary signals to a T cell or other immune cell. The domains may include those derived from a costimulatory molecule, such as from a 4—1BB, e.g., a human 4—1BB, and/or a CD28 molecule, such as a human CD28. The domains may further include a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, such as a CD3zeta, e.g., human CD3zeta. In some embodiments, the CAR contains a spacer and/or hinge region, which may in some aspects be derived from a human IgG.

In some embodiments, at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, or at least at or about 90%, of subjects treated according to the method achieve complete ion (CR), such as measured by RECIST criteria and/or Lugano criteria, and/or any of a number of known criteria for assessing response. In some embodiments, at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, or at least at or about 90%, of subjects treated according to the method achieve objective response (OR), such as ed by RECIST criteria and/or Lugano ia, and/or any of a number of known criteria for ing response. In some embodiments, the subject, or at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, or at least at or about 90%, of subjects that are treated according to the method, and that achieve complete remission (CR), t progression—free survival (PFS) and/or overall survival (OS) of greater than 12, 13, 14, 15, l6, 17, 18, 19, 20, 21, 22, 23, 24, or more months, e.g., on average, and/or r than 6, 12, or 18 months. In some embodiments, the t, or at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, or at least at or about 90%, of subjects treated according to the method, exhibit(s) a median PFS or OS of r than at or about 6 , 12 months, or 18 months; and/or exhibits PFS or OS following therapy for at least at or about 6, 12, 18, or 24, or more months.

In some embodiments, the subject does not exhibit grade 3 or higher neurotoxicity and/or does not exhibit severe CR5, or does not do so within a certain period of time ing the treatment, such as within a week, two weeks, or one month of the administration of the cells.

Provided is method of treating a subject having a non-Hodgkin lymphoma (NHL), the method including administering to the subject a dose of cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen expressed by the NHL, wherein said dose (i) contains (a) at or about 2 x 105 of the cells per kilogram body weight of the subject (cells/kg); (b) at or about 2 x 106 of the kg, (c) no more than at or about 2 x 106 of the cells/kg, (d) no more than at or about 2 x 105 of the cells/kg and/or (6) between at or about 2 x 105 of the cells/kg and at or about 2 x 106 of the cells/kg, and (ii) contains a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR and/or of CD4+ cells to CD8+ cells, which ratio ally is imately 1:1 or is between approximately 1:3 and approximately 3:1 wherein, prior to the administration, the subject has been ditioned with a lymphodepleting therapy sing the administration of fludarabine.

Also provided is a method of treating a subject having a non-Hodgkin lymphoma (NHL), the method including administering to the t a dose of cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target n expressed by the NHL, wherein said dose (i) contains (a) at or about 1 x 107 total cells or total CAR-expressing cells; (b) at or about 1.5 x 108 total cells or total CAR-expressing cells, (c) no more than at or about 1 x 107 total cells or total pressing cells, (d) no more than at or about 1.5 x 108 total cells or total CAR—expressing cells and/or (e) between at or about 1 x 107 total cells or total CAR- expressing cells and at or about 1.5 x 108 total cells or total CAR—expressing cells, and (ii) contains a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR and/or of CD4+ cells to CD8+ cells, which ratio optionally is approximately 1:1 or is between approximately 1:3 and approximately 3: 1, wherein, prior to the administration, the subject has been preconditioned with a lymphodepleting therapy comprising the administration of fludarabine.

In some embodiments, at or prior to the administration of the dose of cells, the subject is or has been identified as having one or more cytogenetic abnormalities, optionally associated with high-risk NHL; the subject is or has been identified as having high-risk NHL; and/or the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), T cell/histocyte— rich large B cell ma (TCHRBCL), Burkitt’s lymphoma, mantle cell ma (MCL), and/or follicular lymphoma (FL); and/or the subject is an adult and/or is over at or about 30, 40, 50, 60, or 70 years of age.

In some of any such embodiments, wherein, prior to the administration of the dose of cells, the subject has been treated with two or more, optionally 2, 3, or 4 or more, therapies for the NHL other than the lymphodepleting therapy and/or other than another dose of cells expressing the CAR. In some of any such embodiments, at or immediately prior to the time of the administration of the dose of cells, the t has relapsed following remission after treatment with, or become refractory to, one or more prior therapies for the NHL.

In some of any such embodiments, the method further includes prior to the administration of the cell dose, administering the lymphodepleting y to the subject. In some embodiments, the lymphodepleting therapy (i) further includes administering another chemotherapeutic agent other than the fludarabine, which optionally is hosphamide; (ii) is initiated at a time that is at least at or about 48 hours prior to or is between at or about 48 and at or about 96 hours prior to the administration of the cells; and (iii) includes the administration of cyclophosphamide at about 30-60 mg/kg, optionally once daily for one or two days, and/or the fludarabine at about 25 mg/m2, daily for 3-5 days. In some of any such embodiments, the administration of the cell dose and/or the lymphodepleting y is carried out via ient delivery.

In some of any such embodiments, the defined ratio is a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR of at or about 1:1 and/or is a defined ratio of CD4+ cells to CD8+ cells, which is at or about 1:1. In some embodiments, the dose of cells is administered parenterally, ally enously.

In some of any such embodiments, at least 50% of subjects treated according to the method achieve complete remission (CR) and/or objective response (OR). In some embodiments of the method, at least 50% of subjects that are treated according to the method, and that e complete remission (CR), exhibit progression-free survival (PFS) and/or overall survival (OS) of r than 12 months; on average, subjects treated according to the method exhibit a median PFS or OS of greater than at or about 6 months, 12 , or 18 months; and/or the subject exhibits PFS or OS following therapy for at least at or about 6, 12, 18 or more months.

In some of any such embodiments, the antigen is a B cell antigen, which optionally is CD19. In some embodiments, the CAR contains an scFv specific for the antigen, a transmembrane , a cytoplasmic signaling domain d from a costimulatory molecule, which optionally is a 4-1BB, and a cytoplasmic signaling domain derived from a primary signaling ontaining molecule, which optionally is a CD3zeta. In some cases, the CAR contains a spacer and/or hinge region, each optionally derived from a human IgG.

In some of any such embodiments, the CAR contains, in order, an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a ulatory molecule, which optionally is or includes a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM—containing molecule, which optionally is a a signaling domain; or the CAR ns, in order, an scFv specific for the antigen, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is a 4—1BB signaling , and a cytoplasmic signaling domain d from a primary signaling ITAM—containing molecule, which optionally is or includes a CD3zeta signaling domain; and wherein the spacer is optionally a polypeptide spacer that (a) contains or consists of all or a portion of an immunoglobulin hinge or a modified version thereof or contains about 15 amino acids or less, and does not contain a CD28 extracellular region or a CD8 extracellular region, (b) contains or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof and/or ns about 15 amino acids or less, and does not contain a CD28 extracellular region or a CD8 extracellular region, or (c) is at or about 12 amino acids in length and/or contains or consists of all or a portion of an globulin hinge, optionally an IgG4, or a modified version f; or (d) has or consists of the sequence of SEQ ID NO: 1, a ce encoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID ON 34, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity o, or (e) includes or consists of the formula P, where X1 is glycine, cysteine or arginine and X2 is cysteine or threonine; and/or the costimulatory domain contains SEQ ID NO: 12 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; and/or the primary signaling domain contains SEQ ID NO: 13 or 14 or 15 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; and/or the scFv contains a CDRLl sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and/or a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37) and/or a CDRHl sequence of DYGVS (SEQ ID NO: 38), a CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), and/or a CDRH3 sequence of YAMDYWG (SEQ ID NO: 40) or n the scFv includes a variable heavy chain region of FMC63 and a variable light chain region of FMC63 and/or a CDRLl sequence of FMC63, a CDRL2 sequence of FMC63, a CDRL3 sequence of FMC63, a CDRHl ce of FMC63, a CDRH2 sequence of FMC63, and a CDRH3 sequence of FMC63 or binds to the same epitope as or competes for binding with any of the foregoing, and optionally n the scFv contains, in order, a VH, a linker, optionally comprising SEQ ID NO: 24, and a VL, and/or the scFv ns a flexible linker and/or es the amino acid sequence set forth as SEQ ID NO: 24.

Provided is a method of prognosis or staging, the method including detecting the presence or absence of a malignant immunoglobulin heavy chain locus (IGH) sequence in a sample from a subject having a B cell malignancy, said subject having previously received administration of a cell therapy comprising a dose or composition of genetically engineered cells expressing a recombinant receptor for treating the B cell malignancy, n ing the presence or absence of the malignant IGH sequence determines the prognosis of the subject in response to the cell therapy. In some aspects, the detecting the presence or absence of the ant IGH sequence is carried out within or within about or about 3 to 6 weeks after initiation of the cell therapy, optionally within or within about 4 weeks of initiation of administration of the cell therapy.

In some of any such embodiments, if the malignant IGH sequence is detected, the subject is identified as not responding or not exhibiting a complete se (CR) or an overall response (OR) to the cell therapy or as likely to relapse to the cell therapy. In some embodiments, if the malignant IGH ce is detected fying the t as a candidate for further treatment and/or for receiving an altered or alternative treatment. In some embodiments, if the malignant IGH sequence is detected tinuing administration of the cell therapy, administering to the subject a further dose of the cell therapy, administering to the subject a higher dose of the cell therapy, administering o the subject a different cell therapy, optionally a cell therapy expressing a different recombinant receptor, and/or administering to the subject an alternative therapeutic agent for treating the B cell malignancy.

In some of any such embodiments, if the malignant IGH sequence is not detected, the subject is identified as responding to the cell therapy and/or as exhibiting a complete se (CR) or overall response (OR) to the cell therapy or as likely not to relapse to the cell therapy.

In some embodiments, if the malignant IGH sequence is not ed, the subject is identified as a candidate for no further treatment and/or is not further treated, optionally is not further d with the cell therapy and/or is not further treated with an alternative therapy for the B cell malignancy.

Provided is a method of predicting durability of response to a cell therapy, the method ing detecting the ce or e of a malignant immunoglobulin heavy chain locus (IGH) sequence in a sample from a subject having a B cell malignancy, said subject having previously ed administration of a cell therapy including a dose or composition of cally engineered cells expressing a recombinant receptor for ng the B cell malignancy, wherein the presence or absence of the malignant IGH sequence predicts the durability of response to the cell therapy. In some aspects, the detecting the presence or absence of the malignant IGH sequence is carried out within or within about or about 4 weeks, 6 weeks, 8 weeks, 12 weeks or 16 weeks after initiation of the cell therapy.

In some of any such embodiments, if the malignant IGH sequence is not ed, the subject is predicted to exhibit or likely to exhibit a durable response to the cell therapy and/or to be at a low or relatively low risk of relapse within a certain period of time and/or to have a high likelihood of exhibiting progression free survival for at least a certain period of time. In some ments, if the malignant IGH sequence is not detected, the subject is predicted to exhibit survival without progression for greater than or about 3 months, greater than about 6 , greater than about 9 months or greater than about 12 months after initiation of the cell therapy; and/or to remain surviving for greater than or r than about 3 months, greater than or greater than about 6 months, greater than or greater than about about 9 months or greater than about 12 months after initiation of the cell therapy; and/or to exhibit e CR or OR for greater than or greater than about 3 months, greater than or greater than about 6 months or greater than or greater than about 9 months after initiation of the cell therapy; and/or not likely to relapse ing tion of administration of the cell therapy, optionally not likely to relapse within 3 months, 6 months or 9 months after initiation of administration of the cell therapy.

In some of any such embodiments, if the malignant IGH sequence is detected, the subject is predicted to exhibit or likely to exhibit a response to the cell therapy that is not durable and/or to be at a high or relatively high risk of relapse within a certain period of time and/or to have a low likelihood of exhibiting ssion free survival for at least a certain period of time.

In some embodiments, if the malignant IGH sequence is not detected, the subject is s predicted not to exhibit survival without progression for greater than or about 3 months, greater than about 6 months, greater than about 9 months or greater than about 12 months after initiation of the cell therapy; and/or not to remain ing for greater than or greater than about 3 months, greater than or greater than about 6 months, greater than or greater than about about 9 months or r than about 12 months after initiation of the cell therapy; and/or not to t durable CR or OR for greater than or greater than about 3 months, greater than or greater than about 6 months or greater than or greater than about 9 months after initiation of the cell therapy.

In some of any such ments, if the malignant IGH sequence is detected administering to the subject a further dose of the cell therapy, stering to the subject a higher dose of the cell therapy, administering o the subject a different cell therapy, optionally a cell therapy expressing a different inant receptor, and/or administering to the subject an alternative therapeutic agent for treating the B cell ancy. In some embodiments, the presence or absence of the malignant IGH sequence is determined by IGH sequencing, optionally comprising PCR amplification of IGH target DNA.

In some of any such embodiments, the sample contians B cells. In some embodiments, the sample contains a blood or bone marrow sample. In some aspects, the sample has been obtained from the subject. In some of any such embodiments, the method is carried out ex vivo.

In some of any such ments, the B cell malignancy is a . In some cases, the B cell malignancy is or es a leukemia. In some examples, the B cell malignancy is an antigen or is associated with an antigen selected from CD19, CD20, CD22, CD30, CD33 or CD38, RORl. In some embodiments, the B cell malignancy is selected from and/or is acute lymphoblastic leukemia (ALL), adult ALL, chronic blastic leukemia (CLL), non- Hodgkin lymphoma (NHL), and Diffuse Large B-Cell ma (DLBCL). In some examples, the B cell malignancy is or includes chronic lymphoblastic leukemia (CLL) or high- W0 14207 risk CLL. In some instances, the B cell malignancy is or includes non—Hodgkin lymphoma (NHL). In some aspects, the the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS (de novo and transformed from indolent), primary mediastinal large B cell lymphoma (PMBCL), T cell/histocyte—rich large B cell lymphoma (TCHRBCL), Burkitt’s lymphoma, mantle cell lymphoma (MCL), and/or follicular lymphoma (FL), optionally, follicular lymphoma Grade 3B (FL3B).

In some of any such embodiments, the recombinant receptor ically binds to an n associated with the disease or condition or expressed in cells of the environment of a lesion associated with the B cell malignancy. In some embodiments, the recombinant receptor is a T cell receptor or a functional non—T cell receptor. In some cases, the recombinant receptor is a ic antigen receptor (CAR). In some instances, the CAR contains an extracellular antigen—recognition domain that specifically binds to the n and an intracellular signaling domain comprising an ITAM, wherein optionally, the intracellular ing domain includes an ellular domain of a CD3-zeta (CD383 chain; and/or wherein the CAR further includes a costimulatory signaling region, which optionally includes a signaling domain of CD28 or 4— lBB. In some embodiments, the CAR ns an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain d from a costimulatory le, which optionally is a 4-1BB, and a cytoplasmic signaling domain derived from a primary ing ITAM—containing molecule, which optionally is a CD3zeta. In some embodiments, the CAR contains a spacer and/or hinge region, each optionally derived from a human IgG.

In some of any such embodiments, the CAR contains, in order, an scFv specific for the antigen, a transmembrane domain, a asmic signaling domain derived from a ulatory molecule, which optionally is or includes a 4-lBB signaling domain, and a cytoplasmic signaling domain d from a primary signaling ITAM—containing molecule, which ally is a CD3zeta signaling domain; or the CAR contains, in order, an scFv specific for the antigen, a spacer, a transmembrane domain, a cytoplasmic ing domain derived from a costimulatory molecule, which optionally is a 4— lBB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM—containing molecule, which optionally is or includes a CD3zeta signaling domain; and wherein the spacer is optionally a polypeptide spacer that (a) contains or consists of all or a portion of an immunoglobulin hinge or a ed version thereof or contains about 15 amino acids or less, and does not include a CD28 extracellular region or a CD8 extracellular region, (b) contains or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof and/or includes about 15 amino acids or less, and does not include a CD28 extracellular region or a CD8 extracellular region, or (c) is at or about 12 amino acids in length and/or includes or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4, or a modified version thereof; or (d) has or consists of the sequence of SEQ ID NO: 1, a sequence encoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID ON 34, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or (e) contains or consists of the formula X1PPX2P, where X1 is glycine, cysteine or arginine and X2 is cysteine or threonine; and/or the ulatory domain includes SEQ ID NO: 12 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence ty thereto; and/or the primary signaling domain includes SEQ ID NO: 13 or 14 or 15 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; and/or the scFv contains a CDRLl ce of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and/or a CDRL3 sequence of TFG (SEQ ID NO: 37) and/or a CDRHl sequence of DYGVS (SEQ ID NO: 38), a CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), and/or a CDRH3 sequence of YAMDYWG (SEQ ID NO: 40) or n the scFv includes a variable heavy chain region of FMC63 and a variable light chain region of FMC63 and/or a CDRLI sequence of FMC63, a CDRL2 sequence of FMC63, a CDRL3 sequence of FMC63, a CDRHl sequence of FMC63, a CDRH2 sequence of FMC63, and a CDRH3 sequence of FMC63 or binds to the same epitope as or competes for binding with any of the foregoing, and optionally wherein the scFv includes, in order, a VH, a linker, optionally comprising SEQ ID NO: 24, and a VL, and/or the scFv includes a flexible linker and/or includes the amino acid ce set forth as SEQ ID NO: 24.

In some of any such embodiments, the engineered cells contains T cells, optionally CD4+ and/or CD8+. In some cases, the T cells are primary T cells obtained from a subject. In some embodiments, the engineered cells are autologous to the subject. In some cases, the engineered cells are allogeneic to the t.

Provided is an article of cture ning one or more dose of a cell therapy, each dose comprising cells expressing a chimeric antigen receptor (CAR), and ctions for administering the cell therapy, wherein the instructions specify the dose of cells is to be administered to a t having a chronic lymphocytic leukemia (CLL); and the instructions specify administration of a number of CAR-expressing or a number of cells, or specify administration of an amount or volume of one or more ations corresponding to or containing said specified number of cells, wherein the specified number of cells to be administered contains a number to administer a dose of cells sing (a) at or about 2 x 105 of the cells per kilogram body weight of the subject (cells/kg); (b) at or about 2 x 106 of the cells/kg, (c) no more than at or about 2 x 106 of the cells/kg, ((1) no more than at or about 2 x 105 of the cells/kg and/or (e) between at or about 2 x 105 of the cells/kg and at or about 2 x 106 of the cells/kg.

Provided is an e of manufacture comprising one or more dose of a cell therapy, each dose containing cells expressing a chimeric antigen receptor (CAR), and instructions for administering the cell therapy, wherein the instructions specify the dose of cells is to be administered to a subject having a chronic lymphocytic leukemia (CLL); and the instructions specify administration of a number of CAR-expressing or a number of cells, or y administration of an amount or volume of one or more formulations corresponding to or containing said specified number of cells, wherein the specified number of cells to be administered contains a number to ster a dose of cells sing (a) at or about 1 x 107 total cells or total pressing cells; (b) at or about 1.5 x 108 total cells or total CAR- sing cells, (c) no more than at or about 1 x 107 total cells or total CAR—expressing cells, (d) no more than at or about 1.5 x 108 total cells or total CAR-expressing cells and/or (e) between at or about 1 x 107 total cells or total CAR-expressing cells and at or about 1.5 x 108 total cells or total CAR—expressing cells.

In some embodiments of the article of manufacture, further included are instructions for use with, after or in connection with a lymphodepleting therapy, the lympodepleting therapy comprising fludarabine. In some cases, the instructions specify that the cell therapy is to be administer to a subject that is or has been identified as having one or more cytogenetic alities, optionally associated with high—risk CLL, optionally selected from among: complex karyotype, deletion of the long arm of chromosome 13 (del l3q), del 11, trisomy 12, del 17p, del 6q, and del l3q.l4, optionally as detected by FISH; is or has been identified as having high-risk CLL; and/or is or has been fied as having extramedullary disease; and/or is or has been identified as having central nervous system (CNS) disease; and/or is an adult and/or is over at or about 30, 40, 50, 60, or 70 years of age.

In some ments, the instructions specify that the cell therapy is to be stered to a subject that has been treated with two or more, optionally 3, 4, 5, 6, 7, 8, or 9 or more, therapies for the CLL, other than the lymphodepleting therapy and/or other than another dose of cells expressing the CAR; and/or has been treated for the CLL with a kinase inhibitor, optionally an inhibitor of Btk, ally ibrutinib; and/or has been d for the CLL with a monoclonal antibody that specifically binds to an antigen expressed by, or previously expressed by, cells of the CLL; and/or has been treated for the CLL with venetoclax, a combination y comprising fludarabine and rituximab, radiation therapy and/or hematopoietic stem cell transplantation (HSCT).

In some of any such embodiments,the instructions specify that the cell therapy is to be administered to a subject that has ed following remission after treatment with, or become refractory to, one or more prior therapies for the CLL.

Provided is an e of manufacture containing one or more dose of a cell therapy, each dose containing cells expressing a chimeric antigen receptor (CAR), and ctions for administering the cell therapy, wherein the instructions specify the dose of cells is to be administered to a subject having a a non-Hodgkin lymphoma (NHL); and the ctions specify administration of a number of CAR-expressing or a number of cells, or specify administration of an amount or volume of one or more formulations corresponding to or containing said specified number of cells, wherein the specified number of cells to be administered contains a number to administer a dose of cells containing (a) at or about 2 x 105 of the cells per kilogram body weight of the subject (cells/kg); (b) at or about 2 x 106 of the cells/kg, (c) no more than at or about 2 x 106 of the cells/kg, (d) no more than at or about 2 x 105 of the kg and/or (e) between at or about 2 x 105 of the cells/kg and at or about 2 x 106 of the cells/kg.

Provided is an article of manufacture containing one or more dose of a cell therapy, each dose comprising cells expressing a chimeric antigen receptor (CAR), and instructions for administering the cell therapy, wherein the instructions specify the dose of cells is to be administered to a t having a a non-Hodgkin lymphoma (NHL); and the instructions specify administration of a number of CAR-expressing or a number of cells, or specify administration of an amount or volume of one or more formulations ponding to or containing said specified number of cells, wherein the specified number of cells to be administered contains a number to administer a dose of cells containing (a) at or about 1 x 107 total cells or total pressing cells; (b) at or about 1.5 x 108 total cells or total CAR— expressing cells, (c) no more than at or about 1 x 107 total cells or total CAR-expressing cells, (d) no more than at or about 1.5 x 108 total cells or total CAR—expressing cells and/or (e) between at or about 1 x 107 total cells or total CAR-expressing cells and at or about 1.5 x 108 total cells or total CAR-expressing cells. In some embodiments, the article of manufacture further contains instructions for use with, after or in connection with a lymphodepleting therapy, the lympodepleting y comprising fludarabine.

In some of any such embodiments, the instructions specify that the cell therapy is to be administer to a t that is or has been identified as having one or more cytogenetic abnormalities, optionally ated with high—risk NHL; is or has been fied as having high-risk NHL; and/or is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), T cell/histocyte—rich large B cell ma (TCHRBCL), Burkitt’s lymphoma, mantle cell lymphoma (MCL), and/or follicular lymphoma (FL); and/or is an adult and/or is over at or about , 40, 50, 60, or 70 years of age.

In some of any such embodiments, the instructions specify that the cell therapy is to be administered to a subject that has been treated with two or more, optionally 2, 3 or 4 or more, therapies for the NHL, other than the depleting therapy and/or other than r dose of cells expressing the CAR. In some embodiments, the instructions specify that the cell therapy is to be stered to a subject that has relapsed following remission after treatment with, or become refractory to, one or more prior therapies for the NHL.

In some of any such embodiments, the lymphodepleting therapy (i) r includes administering another chemotherapeutic agent other than the fludarabine, which optionally is cyclophosphamide; and/or (ii) includes the administration of cyclophosphamide at about 30—60 mg/kg, optionally once daily for one or two days, and/or the fludarabine at about 25 mg/m2, daily for 3-5 days. In some embodiments, the instructions specify that the lympodepleting therapy is initiated at a time that is at least at or about 48 hours prior to or is between at or about 48 and at or about 96 hours prior to the administration of the cell therapy.

In some of any such embodiments, the instructions specify administering the cell therapy at a defined ratio of CD4+ cells expressing the CAR to CD8+ cells, or specify administering amounts of volumes of the formulation(s) corresponding to such defined ratio, or includes a ation having the cells at such ratio or contains the cells at such ratio expressing the CAR and/or of CD4+ cells to CD8+ cells, which ratio optionally is approximately 1:1 or is between approximately 1:3 and approximately 3:1. In some of any such ments, the ctions further specify the cell therapy is for parenteral administration, optionally intravenous administration. In some of any such embodiments, the ctions further specify the administration of the cell therapy is to be or may be administered to the subject on an ient g and/or t admission of the subject to the hospital overnight or for one or more consecutive days and/or is without ion of the subject to the hospital for one or more days.

In some of any such embodiments,the cell therapy contains primary T cells obtained from a subject. In some cases, the T cells are autologous to the subject. In some cases, the T cells are allogeneic to the subject.

In some of any such embodiments, the CAR contains an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is a 4-1BB, and a cytoplasmic signaling domain derived from a primary signaling ITAM—containing molecule, which optionally is a CD3zeta. In some ments, the CAR contains a spacer and/or hinge , each optionally derived from a human IgG. In some embodiments, the antigen is a B cell antigen, which optionally is CD19.

In some of any such embodiments, the CAR contains, in order, an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is or contains a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is a CD3zeta signaling domain; or the CAR contains, in order, an scFv specific for the antigen, a spacer, a embrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is a 4—1BB ing , and a cytoplasmic signaling domain derived from a primary signaling ITAM—containing molecule, which optionally is or includes a CD3zeta signaling ; and wherein the spacer is optionally a ptide spacer that (a) includes or consists of all or a portion of an immunoglobulin hinge or a modified version thereof or includes about 15 amino acids or less, and does not include a CD28 extracellular region or a CD8 extracellular region, (b) contains or ts of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof and/or contains about 15 amino acids or less, and does not include a CD28 extracellular region or a CD8 extracellular region, or (c) is at or about 12 amino acids in length and/or contains or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4, or a modified version f; or (d) has or consists of the sequence of SEQ ID NO: 1, a sequence encoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID ON 34, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or (e) includes or consists of the formula XlPPX2P, where X1 is glycine, cysteine or arginine and X2 is cysteine or threonine; and/or the costimulatory domain contains SEQ ID NO: 12 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more ce identity thereto; and/or the primary signaling domain contains SEQ ID NO: 13 or 14 or 15 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; and/or the scFv contains a CDRLl sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and/or a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37) and/or a CDRHl sequence of DYGVS (SEQ ID NO: 38), a CDRH2 sequence of TTYYNSALKS (SEQ ID NO: 39), and/or a CDRH3 sequence of YAMDYWG (SEQ ID NO: 40) or n the scFv includes a variable heavy chain region of FMC63 and a variable light chain region of FMC63 and/or a CDRLl sequence of FMC63, a CDRL2 sequence of FMC63, a CDRL3 sequence of FMC63, a CDRHl sequence of FMC63, a CDRH2 sequence of FMC63, and a CDRH3 sequence of FMC63 or binds to the same epitope as or competes for binding with any of the ing, and ally wherein the scFv includes, in order, a VH, a linker, optionally comprising SEQ ID NO: 24, and a VL, and/or the scFv includes a e linker and/or includes the amino acid sequence set forth as SEQ ID NO: 24.

Provided is a composition containing cells expressing a chimeric antigen or (CAR) that specifically binds to a target antigen of a chronic lymphocytic leukemia (CLL) for use in treating a subject having or suspected of having CLL, wherein the treating includes administering to the subject a dose of cells expressing the CAR, said dose containing (a) at or about 2 x 105 of the cells per kilogram body weight of the subject (cells/kg); (b) at or about 2 x 106 of the cells/kg, (c) no more than at or about 2 x 106 of the kg, ((1) no more than at or about 2 x 105 of the cells/kg and/or (e) between at or about 2 x 105 of the kg and at or about 2 x 106 of the cells/kg,wherein, prior to the administration, the subject has been preconditioned with a lymphodepleting therapy sing the administration of fludarabine.

Provided is a composition containing cells expressing a chimeric n receptor (CAR) that specifically binds to a target n of a chronic lymphocytic leukemia (CLL) for use in treating a subject having or suspected of having CLL, wherein the treating includes stering to the t a dose of cells expressing the CAR, said dose comprising (a) at or about 1 x 107 total cells or total CAR-expressing cells; (b) at or about 1.5 x 108 total cells or total CAR-expressing cells, (c) no more than at or about 1 x 107 total cells or total CAR-expressing cells, (d) no more than at or about 1.5 x 108 total cells or total CAR-expressing cells and/or (6) between at or about 1 x 107 total cells or total CAR-expressing cells and at or about 1.5 x 108 total cells or total CAR-expressing cells, n, prior to the administration, the subject has been preconditioned with a lymphodepleting therapy comprising the stration of fludarabine.

In some embodiments of the use of the compositions described, the ition is for use in treating a subject in which, at or prior to the administration of the dose of cells: the subject is or has been identified as having one or more netic abnormalities, optionally associated with high—risk CLL, optionally selected from among: complex karyotype, deletion of the long arm of chromosome 13 (del l3q), del 11, trisomy 12, del 17p, del 6q, and del l3q.l4, optionally as detected by FISH; the subject is or has been identified as having high-risk CLL; and/or the subject is or has been identified as having extramedullary disease; and/or the subject is or has been identified as having central nervous system (CNS) disease; and/or the subject is an adult and/or is over at or about 30, 40, 50, 60, or 70 years of age.

In some of any such ments,the composition is for use in treating a subject in which, prior to the administration of the dose of cells, the subject has been treated with two or more, optionally 3, 4, 5, 6, 7, 8, or 9 or more, therapies for the CLL, other than the depleting therapy and/or other than another dose of cells expressing the CAR. In some of any such embodiments, the composition is for use in treating a subject in which, prior to the administration of the dose of cells, the subject has been treated with two or more, optionally 3, 4, , 6, 7, 8, or 9 or more, therapies for the CLL, other than another dose of cells expressing the CAR or other than r dose of cells expressing the CAR and the preconditioning therapy.

In some of any such embodiments,the composition is for use in treating a subject in which, prior to the administration of the dose of cells, the subject has been treated for the CLL with a kinase inhibitor, optionally an inhibitor of Btk, optionally ibrutinib. In some ments, the composition is for use in treating a subject in which, prior to the W0 14207 administration of the dose of cells, the subject has been treated for the CLL with a monoclonal antibody that specifically binds to an antigen expressed by, or previously expressed by, cells of the CLL.

In some of any such embodiments, the composition is for use in treating a subject in which, prior to the administration of the dose of cells, the subject has been treated for the CLL with clax, a combination y comprising fludarabine and rituximab, radiation therapy and/or hematopoietic stem cell lantation (HSCT). In some aspects, the composition is for use in treating a subject in which, at or immediately prior to the time of the administration of the dose of cells, the t has relapsed following remission after treatment with, or become refractory to, one or more prior therapies for the CLL.

Provided is a composition ning cells expressing a chimeric antigen receptor (CAR) that ically binds to a target antigen of a non—Hodgkin lymphoma (NHL) for use in treating a subject having or suspected of having NHL, wherein the treating es administering to the subject a dose of cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen expressed by the NHL, wherein the treating includes administering to the subject a dose of cells sing the CAR, said dose (i) contains (a) at or about 2 x 105 of the cells per kilogram body weight of the subject (cells/kg); (b) at or about 2 x 106 of the cells/kg, (c) no more than at or about 2 x 106 of the cells/kg, ((1) no more than at or about 2 x 105 of the cells/kg and/or (e) between at or about 2 x 105 of the cells/kg and at or about 2 x 106 of the cells/kg, and (ii) contains a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR and/or of CD4+ cells to CD8+ cells, which ratio optionally is imately 1:1 or is n approximately 1:3 and approximately 3:1,wherein, prior to the administration, the subject has been preconditioned with a lymphodepleting therapy comprising the administration of fludarabine.

Provided is a composition containing cells sing a chimeric antigen receptor (CAR) that specifically binds to a target antigen of a non-Hodgkin lymphoma (NHL) for use in treating a subject having or suspected of having NHL, wherein the treating includes administering to the subject a dose of cells expressing the CAR, said dose (i) contains (a) at or about 1 x 107 total cells or total CAR-expressing cells; (b) at or about 1.5 x 108 total cells or total CAR-expressing cells, (c) no more than at or about 1 x 107 total cells or total CAR-expressing cells, (d) no more than at or about 1.5 x 108 total cells or total CAR-expressing cells and/or (e) n at or about 1 x 107 total cells or total CAR-expressing cells and at or about 1.5 x 108 total cells or total pressing cells, and (ii) contains a defined ratio of CD4+ cells expressing the CAR to CD8+ cells sing the CAR and/or of CD4+ cells to CD8+ cells, which ratio optionally is imately 1:1 or is between approximately 1:3 and approximately 3:1, n, prior to the administration, the subject has been preconditioned with a lymphodepleting therapy comprising the administration of fludarabine.

In some embodiments, the composition is for use in treating a subject in which, at or prior to the administration of the dose of cells, the subject is or has been identified as having one or more cytogenetic abnormalities, optionally ated with high-risk NHL; the subject is or has been identified as having high-risk NHL; and/or the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), T cell/histocyte-rich large B cell lymphoma (TCHRBCL), Burkitt’ s lymphoma, mantle cell lymphoma (MCL), and/or ular lymphoma (FL); and/or the subject is an adult and/or is over at or about 30, 40, 50, 60, or 70 years of age.

In some of any such embodiments, the composition is for use in treating a subject in which, prior to the administration of the dose of cells, the subject has been treated with two or more, optionally 2, 3, or 4 or more, therapies for the NHL other than the lymphodepleting y and/or other than another dose of cells expressing the CAR. In some cases, the composition is for use in treating a t in which, at or immediately prior to the time of the administration of the dose of cells, the subject has relapsed following remission after treatment with, or become refractory to, one or more prior therapies for the NHL.

In some of any such embodiments, the lymphodepleting therapy (i) r includes administration of another chemotherapeutic agent other than the fludarabine, which optionally is cyclophosphamide; (ii) is initiated at a time that is at least at or about 48 hours prior to or is between at or about 48 and at or about 96 hours prior to the administration of the cells; and (iii) includes the administration of cyclophosphamide at about 30—60 mg/kg, optionally once daily for one or two days, and/or the fludarabine at about 25 mg/m2, daily for 3-5 days. In some embodiments, the treating includes stration of the cell dose and/or the lymphodepleting therapy via outpatient delivery.

In some of any such embodiments,the composition and/or the dose of cells contains a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR and/or of CD4+ cells to CD8+ cells, which optionally is approximately 1:1 or is between approximately 1:3 and approximately 3:1. In some of any such embodiments, the composition and/or dose of cells is formulated for parenteral administration, optionally intravenous stration.

In some embodiments, the antigen is a B cell antigen, which optionally is CD19. In some of any such ments, the CAR contains an scFv specific for the n, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is a 4-1BB, and a cytoplasmic signaling domain derived from a primary signaling ITAM—containing molecule, which optionally is a CD3zeta. In some cases, the CAR contains a spacer and/or hinge region, each optionally derived from a human IgG.

In some of any such embodiments, the CAR contains, in order, an scFv specific for the antigen, a transmembrane domain, a asmic signaling domain derived from a costimulatory molecule, which ally is or includes a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM—containing molecule, which optionally is a CD3zeta signaling domain; or the CAR ns, in order, an scFv specific for the antigen, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is a 4—1BB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM—containing molecule, which optionally is or es a CD3zeta signaling domain; and wherein the spacer is optionally a polypeptide spacer that (a) contains or consists of all or a portion of an globulin hinge or a modified version thereof or contains about 15 amino acids or less, and does not include a CD28 extracellular region or a CD8 extracellular region, (b) contains or ts of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof and/or includes about 15 amino acids or less, and does not include a CD28 extracellular region or a CD8 extracellular region, or (c) is at or about 12 amino acids in length and/or includes or consists of all or a portion of an immunoglobulin hinge, ally an IgG4, or a ed version thereof; or (d) has or consists of the sequence of SEQ ID NO: 1, a sequence encoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID ON 34, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or (e) includes or consists of the formula P, where X1 is glycine, cysteine or arginine and X2 is cysteine or ine; and/or the costimulatory domain includes SEQ ID NO: 12 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; and/or the primary signaling domain includes SEQ ID NO: 13 or 14 or 15 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence ty thereto; and/or the scFv includes a CDRLl sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and/or a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37) and/or a CDRHl sequence of DYGVS (SEQ ID NO: 38), a CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), and/or a CDRH3 sequence of G (SEQ ID NO: 40) or wherein the scFv contains a variable heavy chain region of FMC63 and a variable light chain region of FMC63 and/or a CDRLl sequence of FMC63, a CDRL2 sequence of FMC63, a CDRL3 sequence of FMC63, a CDRHl sequence of FMC63, a CDRH2 sequence of FMC63, and a CDRH3 sequence of FMC63 or binds to the same epitope as or competes for binding with any of the foregoing, and optionally wherein the scFv ns, in order, a VH, a linker, optionally sing SEQ ID NO: 24, and a VL, and/or the scFv contains a flexible linker and/or contains the amino acid sequence set forth as SEQ ID NO: 24.

Brief ption of the Drawings Figure 1A shows percent progression-free survival (PFS) curves for relapsed or refractory (R/R) CD19+ chronic lymphocytic leukemia (CLL) subjects treated with a single infusion of 2 x 105, 2 x 106 or 2 x 107 CAR—expressing T cells per am (kg) weight of the t. Separate curves are shown for subjects who achieved complete remission (CR) and for subjects that did not (non CR). Prior to the infusion, subjects were pre—conditioned with 60 mg/kg cyclophosphamide and 25 mg/m2 fludarabine (Flu) daily for 3-5 days. shows percent overall al (OS) curves for relapsed or refractory (R/R) CD19+ chronic lymphocytic leukemia (CLL) subjects treated with a single infusion of 2 X 105, 2 x 106 or 2 x 107 CAR-expressing T cells per kilogram (kg) weight of the subject. Separate curves are shown for subjects who achieved complete remission (CR) and for subjects that did not (non CR). Prior to the infusion, subjects were pre—conditioned with 60 mg/kg cyclophosphamide and 25 mg/m2 fludarabine (Flu) daily for 3—5 days. shows percent progression-free survival (PFS) curves for non-Hodgkin ma (NHL) subjects treated with a single infusion of 2 X 106 CAR—expressing T cells per kilogram (kg) weight of the subject. Separate curves are shown for subjects who ed complete remission (CR) and for subjects that did not (non CR). Prior to the infusion, subjects were pre—conditioned with 60 mg/kg cyclophosphamide and 25 mg/m2 fludarabine (Flu) daily for 3-5 days. shows percent overall survival (OS) curves for non—Hodgkin lymphoma (NHL) subjects treated with a single infusion of 2 x 106 CAR—expressing T cells per kilogram (kg) weight of the subject. Separate curves are shown for subjects who achieved te remission (CR) and for subjects that did not (non CR). Prior to the infusion, subjects were pre- conditioned with 60 mg/kg cyclophosphamide and 25 mg/m2 fludarabine (Flu) daily for 3-5 days. shows percent progression-free survival (PFS) curves for relapsed or refractory (R/R) CD19+ chronic lymphocytic ia (CLL) ts treated with a single on of 2 x 105, 2 x 106 or 2 x 107 CAR-expressing T cells per kilogram (kg) weight of the subject and pre—conditioned with 30—60 mg/kg cyclophosphamide and 25 mg/m2 fludarabine (Flu) daily for 3—5 days (n=l3).Separate curves are shown for subjects who achieved complete remission (CR) and for subjects that did not (non CR).

-E shows correlation of CAR—T cell ion with the percentage of abnormal B cells present in bone marrow (A), the tumor cross—sectional area (B), the absolute abnormal B cell count in blood (C), the maximum SUV on PET imaging (D) and the immune oint biomarker CD200 (E) for patients treated with Cy/Fu and 2x106 CAR-T kg. shows the overall survival (OS) and progression-free survival (PFS) for all CLL patients in the study. The median follow-up is 12.4 . and show progression free survival (PFS) and overall survival (OS) in patients as measured by IWCLL 2008 nodal response criteria, after Cy/Flu lymphodepletion and CAR-T cell infusion at 2x105 or 2x106 CAR-T cells/kg. shows PFS and OS in patients with CR, PR or that were non—responders (n:20). shows PFS and OS in patients with CR, PR or no response (SD/PD), which excluded one patient in that died prior to ing. For both and 6B, separate curves are shown for the group of subjects who achieved CR, subjects who achieved PR, and non—responders. The median PFS and OS follow—up for ts in CR/PR is 12.3 and 12.4 months, respectively. mPFS, median PFS; mOS, median OS; NR, not reached. shows percent progression-free survival (PFS) and overall-survival (OS) curves for fourteen subjects that achieved complete remission (CR) from bone marrow 4 weeks after CAR-T cell infusion by flow cytometry and had no detectable malignant IGH copies W0 14207 (IGHseq—negative) compared to those who had detectable malignant IGH copies (IGHseq— positive). shows CD4+/EGFRt+ and CD8+/EGFRt+ CAR—T cell counts for fourteen subjects that achieved complete remission (CR) and had IGH deep sequencing of bone marrow performed. shows the peak GFRt+ (left) and CD8+/EGFRt+ (right) CAR-T cell counts in blood from patients who did (Yes) or did not (No) clear disease from the bone marrow by esolution flow cytometry. shows the peak CD4+/EGFRt+ (left) and CD8+/EGFRt+ (right) CAR-T cell counts in blood from patients who cleared disease from the bone marrow by high—resolution flow cytometry and did or did not have detectable malignant IGH sequences in marrow.

A shows an estimated probability curve of response and an ted probability curve of ping Grade 3—5 neurotoxicity constructed based on the number of CD4+/EGFRt+ or CD8+/EGFRt+ CAR-T cells in the blood.

B shows estimated probability curves of bone marrow response, complete or partial remission (CR/PR) by IWCLL criteria, developing Grade 2-5 cytokine release me (CR8), and developing Grade 2-5 neurotoxicity (NT) constlucted based on the number of CD4+/EGFRt+ or CD8+/EGFRt+ CAR-T cells in the blood. shows a waterfall plot of the change in cross-sectional area of the 6 largest lymph nodes on CT scan by IWCLL (2008) g ia at best response in high-risk CLL patients after CAR-T cell immunotherapy. Four patients (2 CR, 1 SD, 1 died) without high- resolution imaging to enable tumor measurement are not shown.

Detailed Description I. METHODS AND USES OF CELL THERAPY WITH GENETICALLY ENGINEERED CELLS Provided are methods and compositions for use in cell y, for the treatment of diseases or conditions, including various cancers and tumors. The methods involve administering engineered cells expressing recombinant receptors designed to recognize and/or specifically bind to molecules associated with the disease or condition and result in a response, such as an immune response against such molecules upon g to such molecules. The receptors may include chimeric receptors, e.g., chimeric antigen receptors , and other transgenic antigen receptors including transgenic T cell ors (TCRs).

In some embodiments, the cells, populations, and itions are administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the methods involve treating a subject having a chronic lymphocytic leukemia (CLL) or a non-Hodgkin lymphoma (NHL) with a dose of antigen receptor-expressing cells (e.g. CAR—expressing .

In some embodiments, the subject has been preconditioned with an immunodepleting (e. g. lymphodepleting) therapy. Preconditioning subjects with immunodepleting (e. g., lymphodepleting) therapies can improve the effects of adoptive cell therapy (ACT).

Preconditioning with lymphodepleting agents, including combinations of cyclosporine and fludarabine, have been effective in improving the efficacy of transferred tumor infiltrating lymphocyte (TIL) cells in cell therapy, including to improve response and/or persistence of the erred cells. See, e.g., Dudley et al., 2002 Science, 298, ; Rosenberg et al., Clin Cancer Res 2011, l7(l3):45504557. Such preconditioning can be carried outwith the goal of reducing the risk of one or more of various es that could dampen efficacy of the therapy.

These e the phenomenon known as “cytokine sink,” by which T cells, B cells, NK cells e with TILs for homeostatic and activating cytokines, such as IL-2, IL-7, and/or IL-lS; suppression of TILs by regulatory T cells, NK cells, or other cells of the immune system; impact of negative regulators in the tumor microenvironment. ki et al., Nat Clin Pract Oncol. 2006 December; 3(12): 668—681.

Thus, in some embodiments, the methods comprise administration of a chemotherapeutic agent, e.g., a conditioning chemotherapeutic agent, for example, to reduce tumor burden prior to administering the dose of cells. In some embodiments, the methods include administering a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof. In some ments, the methods include administration of bine and, optionally, another chemotherapeutic other than fludarabine. In some embodiments, the other chemotherapeutic agent is cyclophosphamide. In some embodiments, the subject may be administered a lymphodepleting therapy at least 2 days prior, such as at least 3, 4, 5, 6, or 7 days prior, to the administration of the dose of cells. In some embodiments, the lymphodepleting y is administered or is initiated at least or at least about or at or about 48 hours or at least or at least about 96 hours prior to the administration of the dose of cells. In some embodiments, the lymphodepleting therapy is administered or is initiated between at or about 48 hours and at or about 96 hours prior to administration of the dose of cells.

Thus, in some embodiments, the methods include administering a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, to a subject prior to the first or subsequent dose. For example, the subject may be administered a preconditioning agent at least 2 days prior, such as at least 3, 4, 5, 6, or 7 days prior, to the first or subsequent dose. In some embodiments, the subject is administered a preconditioning agent no more than 7 days prior, such as no more than 6, 5, 4, 3, or 2 days prior, to the first or subsequent dose.

In some embodiments, the subject is preconditioned with cyclophosphamide at a dose n or between about 20 mg/kg and 100 mg/kg, such as between or between about 40 mg/kg and 80 mg/kg or n or n about 30 mg/kg and 60 mg/kg. In some aspects, the subject is preconditioned with or with about 60 mg/kg of cyclophosphamide. In some embodiments, the cyclophosphamide can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some embodiments, the cyclophosphamide is administered once daily for one or two days. In some embodiments, where the depleting agent comprises cyclophosphamide, the subject is administered hosphamide at a dose between or between about 100 mg/m2 and 500 mg/mz, inclusive, such as between or between about 200 mg/m2 and 400 mg/mz, or 250 mg/m2 and 350 mg/mz, inclusive. In some instances, the t is administered about 300 mg/m2 of cyclophosphamide. In some embodiments, the cyclophosphamide can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some embodiments, cyclophosphamide is administered daily, such as for l— days, for example, for 3 to 5 days. In some ces, the subject is administered about 300 mg/m2 of cyclophosphamide, daily for 3 days, prior to initiation of the cell therapy.

In some embodiments, where the lymphodepleting agent comprises fludarabine, the subject is administered bine at a dose between or between about 1 mg/m2 and 100 mg/mz, such as between or between about 10 mg/m2 and 75 mg/m2, 15 mg/m2 and 50 mg/m2, 20 mg/m2 and 40 mg/m2, 20 mg/m2 and 30 mg/m2, 24 mg/m2 and 35 mg/m2 or 24 mg/m2 and 26 mg/m2. In some instances, the t is administered 25 mg/m2 of fludarabine. In some instances, the subject is stered about 30 mg/m2 of fludarabine. In some embodiments, the fludarabine can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some embodiments, fludarabine is administered daily, such as for 1—5 days, for example, for 3 to 5 days. In some instances, the subject is administered about 30 mg/m2 of fludarabine, daily for 3 days, prior to initiation of the cell therapy.

In some ments, the lymphodepleting agent comprises a ation of agents, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of agents may include hosphamide at any dose or administration le, such as those described above, and fludarabine at any dose or administration schedule, such as those described above. For example, in some aspects, the subject is administered 30—60 mg/kg (~1—2 g/mz) of cyclophosphamide and 3 to 5 doses of 25 mg/m2 fludarabine prior to the dose of cells. In some aspects, the subject is administered 60 mg/kg (~2 g/mz) of cyclophosphamide and 3 to 5 doses of mg/m2 fludarabine prior to the first or subsequent dose. In some ments, lymphodepletion chemotherapy can be modified by reducing or omitting the dose of cyclophosphamide or administering a regimen of a lower total dose of cyclophosphamide administered concurrently with fludarabine, to ze toxicity in subjects, such as subjects who have received multiple previous cycles of herapy, have previously undergone allogeneic transplantation, have poor marrow reserve, and/or have other serious comorbidities.

In some embodiments, the antigen receptor (e.g. CAR) specifically binds to a target antigen associated with the e or condition, such as associated with CLL or NHL. In some embodiments, the n associated with the disease or disorder is selected from CD20, CD19, CD22, RORl, CD45, CD21, CD5, CD33, ngappa, Iglambda, CD79a, CD79b or CD30.

In some embodiments, the methods include administration of the cells or a composition containing the cells to a subject, tissue, or cell, such as one having, at risk for, or suspected of having the disease, condition or disorder. In some embodiments, the subject is the subject is an adult. In some embodiments, the subject is over at or about 30, 40, 50, 60, or 70 years of age.

In some embodiments, the provided methods involve ve cell therapy methods, e.g. CAR+ T cells, for treating chronic lymphocytic leukemia (CLL). CLL is the most common adult leukemia. In some cases, patients with high-risk disease, including those that manifest by del(17)(p13.1), p53 mutation, x kayotype or umutated immunoglobulin variable regions, e earlier therapy and/or have shorter survival (Dohner et a1. (2000) N. Engl. J. Med., 343:1910—1916; nbauer et a1. (2014) Blood, 123:3247—3254; Thompson et al. (2015) , 121 :3612—3621). Among treatments for high-risk CLL include herapy (Hallek et a1. (2010) Lancet 376: 1 164— 1 174), although recently the BTK inhibitor, nib, has recently been approved initially for relapsed and refractory disease and uently for first-line therapy (Burger et al. (2015) N. Engl J. Med., 373:2425-2437; Byrd et a1. (2013) N. Engl. J. Med., -42). While the overall response rate (ORR) to ibrutinib is high, the complete response rate (CR; or complete remission) can, in some cases, be low, and survival of patients who progress on ibrutinib may be short. Another ent for high-risk CLL is the BCL2-inhibitor, venetoclax, which as shown activity in some patients who failed ibrutinib therapy, but CR is rare and durability has not been reported (Stilgenbauer et a1. (2016) Lancet , 17:768—778).

T cell-based therapies, such as adoptive T cell therapies (including those involving the stration of cells expressing chimeric receptors specific for a disease or disorder of interest, such as ic antigen receptors (CARs) and/or other recombinant antigen receptors, as well as other adoptive immune cell and adoptive T cell therapies) can be effective in the treatment of cancer and other diseases and disorders. The engineered expression of recombinant receptors, such as chimeric antigen receptors (CARs), on the surface of T cells enables the redirection of T-cell specificity. In clinical studies, CAR-T cells, for e anti-CD19 CAR- T cells, have produced durable, complete responses in both leukemia and lymphoma patients (Porter et a1. (2015) Sci Transl Med., 7:303ra139; Kochenderfer (2015) J. Clin. Oncol., 33: 540— 9; Lee et a1. (2015) Lancet, 385:517-28; Maude et a1. (2014) N Engl J Med, 371:1507-17).

In some ments, the provided s and uses provide for or achieve improved or more durable responses or efficacy as compared to certain alternative methods, such as in particular groups of ts treated, such as in patients with a leukemia, such as CLL or NHL, including those with high—risk disease. In some embodiments, the methods are advantageous by virtue of administering T cell therapy, such as a composition including cells for adoptive cell therapy, e.g., such as a T cell therapy (6.g. CAR—expressing T cells) and a lymphodepleting therapy, e.g. such as cyclophosphamide, fludarabine, or combinations thereof.

In some embodiments, the provided methods are based on observations that a high rate of elimination of marrow disease and molecular CR can be achieved in patients with high-risk ibrutinib refractory CLL after lympodepletion and CLL-targeted CAR-T cell therapy, such as anti—CD19 CAR+ T cell therapy. This result was ed with relatively low incidence of serious toxicity that was lly manageable.

In some embodiments, the s include administration of cells to a subject selected or fied as having a certain prognosis or risk of CLL. Chronic lymphocytic leukemia (CLL) is a generally a variable disease. Some subjects with CLL may survive without treatment while others may require immediate ention. In some cases, subjects with CLL may be classified into groups that may inform disease prognosis and/or recommended treatment strategy. In some cases, these groups may be “low risk,” “intermediate risk,” “high risk,” and/or “very high risk” and patients may be classified as such depending on a number of factors ing, but not limited to, genetic abnormalities and/or morphological or physical characteristics. In some embodiments, subjects treated in accord with the method are classified or identified based on the risk of CLL. In some embodiments, the subject is one that has high risk CLL.

In some cases, one method of classifying subjects is the Rai system. In some aspects, the Rai system comprises 5 stages: Rai stage 0: lymphocytosis and no enlargement of the lymph nodes, spleen, or liver, and with near normal red blood cell and et counts; lymphocytes in blood > lSOOO/mcL, and > 40% cytes in the bone marrow. Rai stage I: lymphocytosis plus enlarged lymph nodes. The spleen and liver are not enlarged and the red blood cell and platelet counts are near normal. Rai stage II: cytosis plus an enlarged spleen (and possibly an enlarged liver), with or without enlarged lymph nodes. The red blood cell and platelet counts are near normal. Rai stage III: lymphocytosis plus anemia (too few red blood cells), with or without enlarged lymph nodes, , or liver. Platelet counts are near normal.

Rai stage IV: lymphocytosis plus thrombocytopenia (too few blood platelets), with or without anemia, enlarged lymph nodes, spleen, or liver. Rai stages may be further grouped into risk groups as follows: Stage 0 is considered low risk; stages I and II are considered intermediate risk; stages III and IV are considered high risk and in some grading systems also e disease—related anemia (hemoglobin level < 11.0 g/dL or hematocrit < 33%) or platelets < 100,000/mcL. In some cases, subjects may also be, or alternatively be, d into classes according to the Binet system, which in some respects involves assessing the number of affected lymphoid tissue groups (neck lymph nodes, groin lymph nodes, underarm lymph nodes, spleen, and liver) and by whether or not the patient has anemia (too few red blood cells) or thrombocytopenia (too few blood ets). In some s, the Binet system comprises three stages: Binet stage A: Hemoglobin Z 10 g/dL, platelets Z 100,000/mm3, and < 3 enlarged areas; Binet stage B: Hemoglobin Z 10 g/dL, platelets 2 100,000/mm3, and Z 3 enlarged areas; and Binet stage C: Hemoglobin < 10 g/dL, platelets < 100,000/mm3, and any number of enlarged areas and anemia and/or thrombocytopenia are present. (Rai KR, Keating HJ. Chronic lymphocytic leukemia. In: Cancer Medicine. 4th ed. Baltimore, Md: Williams and s; 1997. Vol II: 28; Hallek M. et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood 2008. 111:5446-56.).

Additional methods of classifying subjects with CLL involve genetic analysis and determination of the presence or absence of genetic abnormalities. In some cases, prognostic genetic abnormalities include netic abnormalities, which may include deletion of the long arm of some 13 (del 13q), del llq, y 12, del 17p and del 6q. ons of llq, 13q, 17p, and trisomy 12 can have prognostic value and may bute to CLL pathogenesis and evolution and inform outcome and eutic strategies. Abnormalities also include some that are not detectable by more traditional methods such as FISH is. In some aspects, chromosomal translocations, ing nced translocations, and complex karyotype, can be associated with poor outcomes and/or poor sis. Complex karyotypes (CK), generally defined as the presence of three or more chromosomal abnormalities, are detected in nearly 16% of CLL subjects and have been associated with unmutated IgHV status and CD38 expression.

CK can be predictive of shortened time to first therapy (TTFT) and overall survival (OS) in CLL subjects treated with salvage therapies, including chlorodeoxyadenosine (ch). The number of karyotypic abnormalities can be associated with shorter progression-free survival (PFS) and OS following hematopoietic stem cell transplantation (HSCT) following conditioning. Other genetic modifications that may indicate that a subject is high or very high risk include mutations of IgVH, ZAP70, and/or CD38. (Gribben, ASH Education Book; January 1, 2008, vol. 2008 no. 1, 444—449; Puiggros et al., BioMed Research International, Volume 2014 (2014), Article ID 435983). In some cases, high or very high risk patients may exhibit one or more genetic abnormalities.

In some embodiments, the subject exhibits one or more netic abnormalities, such as one or more of complex karyotype, deletion of the long arm of chromosome 13 (del 13q), del 11, trisomy 12, del 17p, del 6q, and del 13q.14. In some embodiments, any one or more of the netic abnormalities can be ed by fluorescence in situ hybridization (FISH).

In some embodiments, the subjects with CLL exhibit Richter’s syndrome (RS). RS is defined as the transformation of CLL into an aggressive lymphoma, most commonly diffuse large B—cell lymphoma (DLBCL) (see, e.g., Parikh et al. Blood 2014 123: 1647—1657).

In some embodiments, the subjects with CLL and/or RS exhibit neurological symptoms or neurological complications, such as symptoms or cations in the central nervous system (CNS). In some embodiments, magnetic resonance imaging (MRI) of the CNS (e.g. brain, spine) and/or a lumbar re with cerebral spinal fluid (CSF) analysis can be used to evaluate CNS-related symptoms, e.g., presence of monoclonal population of lymphocytes in the CSF (see, e.g., Mozzam et al., J. Neurooncol. 2012 106:185-200; Strati et 31., Haematologica 1z458—465).

In some embodiments, the methods include administration of cells to a subject selected or identified as having high—risk NHL. In some embodiments, the subject exhibits one or more cytogenetic abnormalities, such as associated with high—risk NHL. In some embodiments, the subject is selected or identified based on having a disease or condition characterized or determined to be aggressive NHL, diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma ), T cell/histocyte—rich large B cell lymphoma (TCHRBCL), Burkitt lymphoma, mantle cell lymphoma (MCL), and/or follicular lymphoma (FL).

In some embodiments, the t has been previously d with a therapy or a therapeutic agent targeting the e or condition, e.g. CLL or NHL, prior to stration of the cells expressing the recombinant antigen receptor. In some embodiments, the therapeutic agent is a kinase inhibitor, such as an inhibitor of Bruton’s tyrosine kinase (Btk), for example, ibrutinib. In some ments, the therapeutic agent is an inhibitor of B-cell lymphoma-2 (Bel—2), for example, venetoclax. In some embodiments, the therapeutic agent is an antibody (e.g. monoclonal dy) that specifically binds to an antigen expressed by the cells of the CLL or NHL, e.g. an antigen from any one or more of CD20, CD19, CD22, RORl, CD45, CD21, CD5, CD33, ngappa, da, CD79a, CD79b or CD30. In some embodiments, the therapeutic agent is an anti—CD20 antibody, e.g., rituximab. In some embodiments, the therapeutic agent is a ing chemotherapy that is a combination therapy that includes rituximab, e.g., a combination therapy of bine and rituximab or a combination therapy of anthracycline and rituximab. In some embodiments, the subject has been previously treated with hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT or autogenic HSCT. In some embodiments, the subject has been treated or has previously received at least or about at least or about 1, 2, 3, or 4 other therapies for treating the NHL or CLL other than the lymphodepleting therapy and/or the dose of cells expressing the antigen receptor. In some embodiments, the subject has been previously treated with chemotherapy or radiation therapy.

In some aspects, the subject is refractory or non-responsive to the other therapy or therapeutic agent. In some embodiments, the t has persistent or relapsed disease, e.g., following treatment with another therapy or therapeutic intervention, including chemotherapy or radiation.

In some cases, treatments or therapies (or those of particular categories) may not be recommended for CLL subjects in the low and intermediate risk ries. In some cases, treatment strategies for high risk and very high risk subjects may include fludarabine, cyclophosphamide, and mab (FCR), BTK inhibitors (e.g. ibrutinib), and/or allogeneic stem cell transplantation. (Puiggros et al., BioMed Research International, Volume 2014 (2014), e ID 435983). In some aspects, subjects treated for CLL exhibit poor long-term outcomes.

For example, in some cases, refractory (R/R) high—risk CLL subjects exhibit poor survival after Ibrutinib discontinuation (Jain et al. (2015) Blood 125(13):2062—2067). There is a need for improved methods of treating CLL, and in some aspects, for those appropriate for treating high and/or very high-risk CLL and/or subjects having relapsed or become refractory to multiple prior therapies.

In some embodiments, the provided methods are for use in subjects having a cancer in which the subject and/or the cancer is ant to inhibition by irbrutinib or comprises a population of cells that are resistant to inhibition by the inhibitor. In some aspects, the patient is one that is selected that is or is likely to become refractory to ibrutinib based on high—risk cytogenetics and/or based on the presence of, such as by early ion of, mutations prior to relapse that confer ibrutinib resistance. In some embodiments, provided methods are for use in a subject having a cancer in which the subject and/or the cancer comprises a mutation or disruption in a nucleic acid encoding BTK, in which such mutation is e of reducing or preventing inhibition of the BTK by the inhibitor, e.g. ibrutinib. In some aspects of any of the s provided herein, the mutation in the c acid ng BTK contains a substitution at on C481, optionally C4813 or C481R, and/or a tution at position T474, optionally T4741 or T474M. In some ments, the provided s are for use in a subject having a cancer in which at the time of administration of the adoptive cell therapy, e.g., such as a T cell therapy (e.g. CAR—expressing T cells) and/or at the time of administering the lymphodepleting therapy, the subject has relapsed following remission after treatment with, or been deemed refractory to treatment with ibrutinib.

Methods for stration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; US Patent No. 915 to erg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) m Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e6 1 3 3 8.

In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous er, in which the cells are ed and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a t, e.g., patient, in need of a treatment and the cells, and following isolation and processing are administered to the same subject.

In some embodiments, the cell therapy, e.g., ve cell y, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are stered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

The cells can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular ion, periocular injection, subretinal injection, intravitreal injection, trans—septal injection, eral ion, intrachoroidal injection, intracameral injection, subconjunctival injection, subconjunctival injection, sub-Tenon's injection, retrobulbar injection, peribulbar ion, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.

Parenteral infusions include intramuscular, enous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration of the cells. In some embodiments, it is administered by multiple bolus strations of the cells, for example, over a period of no more than 3 days, or by continuous infusion stration of the cells.

For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, r the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the ing ian. The compositions and cells are in some ments suitably administered to the subject at one time or over a series of treatments.

Once the cells are administered to the subject (e.g., human), the biological activity of the engineered cell populations in some aspects is measured by any of a number of known methods. Parameters to assess include specific binding of an engineered or l T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to y target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, derfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological s, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells also can be measured by ng expression and/or secretion of certain cytokines, such as CD107a, IFNy, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load. In some aspects, toxic outcomes, persistence and/or expansion of the cells, and/or presence or absence of a host immune response, are assessed.

In certain embodiments, engineered cells are modified in any number of ways, such that their therapeutic or prophylactic efficacy is increased. For e, the engineered CAR or TCR expressed by the population can be conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating nds, e.g., the CAR or TCR, to targeting moieties is known in the art. See, for instance, Wadwa et al., J. Drug Targeting 3: l 1 1 (1995), and U.S. Patent 5,087,616.

In some embodiments, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an dy or engineered cell or receptor or agent, such as a cytotoxic or therapeutic W0 14207 2017/036231 agent. The cells in some embodiments are co—administered with one or more onal therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co—administered with another therapy iently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agent includes a ne, such as IL—2, for example, to enhance persistence.

Also provided herein are s of prognosis or staging of subjects after treatment with cell therapy, e.g., such as a T cell therapy (e.g. CAR—expressing T cells), methods of monitoring response in subjects having received such a cell therapy and/or methods of predicting durability of response to such a cell therapy, in which such s involve sequencing of the immunoglobulin heavy chain (IGH) locus in samples containing or potentially containing tumor cells, obtained from of ts that have received the cell therapy. In some embodiments, such methods involve sequencing the IGH locus of harvested samples, such as those potentially ia cells, e.g. B cells, from marrow or blood, and/or samples prepared therefrom, to detect the presence or absence of residual tumor.

In some embodiments, typical s for prognosis, staging and/or monitoring response rates in subjects having a leukemia or lymphoma, e.g. CLL, to a therapy involve measuring lymph node size. For example, a common criteria for assessing response rates is the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) response criteria (Hallek, et al., Blood 2008, Jun 15', 111(12): 5446—5456), which, in some aspects, requires that all lymph nodes be 5 15 mm. In some cases, however, it is found that responses to cell therapy is rapid and may be achieved prior to measurable change in lymph node size. Thus, in some aspects, early restaging by tumor criteria alone, such as within or about 4 weeks after cell therapy, may not be an optimal determinant of prognosis. Based on ations provided herein, it is found that, even as early as 4 weeks after stration of a cell therapy, e.g. T cell therapy, such as CAR+ T cells, high rates of elimination of CLL from marrow is observed by IGH sequencing.

These results indicate that early restaging of subjects by IGH sequencing may provide for an efficient and earlier prognosis or predictor of response and durability of response compared to s that rely on tumor size criteria. In some embodiments of the ed methods, IGH sequencing is performed no more than 3 months, such as no more than 2 months or no more than 1 month after initiation of administration of a cell y. In some embodiments, IGH sequencing is carried out on at or about or within 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks after initiation of the cell therapy.

In some embodiments, the IGH sequence methods are used to provide prognostic information for stage a subject after treatment with a cell therapy. In some embodiments, if lack of able malignant IGH copies are detected or observed, the subject is identified as responding to the cell therapy, likely to be responding, and/or likely to t or develop a response such as a CR and/or a durable response. In some embodiments, lack of detection of malignant IGH copies by the ed methods is used to identify, such as identify early, e.g. within or about within 3—6 weeks of initiation of administration of the cell therapy, such as within or about 4 weeks of the initiation, r the subject is exhibiting or is likely to t a response to the cell therapy such as exhibiting a complete response (CR) or overall response (OR) to the cell therapy. In some aspects, if malignant IGH copies are detected, the subject is identified as not responding to the cell therapy and/or not exhibiting a complete response and/or to have poorer prognosis or to need additional treatment. In some embodiments, detection of malignant IGH copies by the provided methods is used to identify, such as fy early, e.g. within or about within 3-6 weeks of initiation of stration of the cell therapy, such as or about 4 weeks of the initiation, if the subject is not ting a response to the cell therapy or is not exhibiting a te response (CR) or overall response (OR) to the cell therapy. In some aspects, the patient may be identified for possible administration of alternative or additional therapeutic strategies to improve response or likelihood of response. In some embodiments, the methods are carried out on subjects receiving a cell therapy, such as containing ered T cells, e.g. CAR+ T cells, for treating a B cell malignancy, e.g. CLL or NHL. In some embodiments, the methods are carried out on subjects having received a cell therapy for treating In some embodiments, the IGH sequencing methods are used to assess or determine the durability of response to a cell therapy, e.g. CAR+ T cell therapy. In some ments, if lack of detectable malignant IGH copies are detected the t is predicted to exhibit or likely to exhibit a durable response to the cell therapy and/or to be at a low or relatively low risk of relapse or to have a high hood of exhibiting progression free survival for at least a certain period of time. In some embodiments, lack of ion of malignant IGH copies by the provided methods is used to identify, such as identify early, e.g. within or about 90 days or earlier of initiation of administration of the cell therapy, subjects ted to be at lower risk of e and/or to have increased likelihood of developing progression free survival (PFS) or a durable response, such as for greater than 3 months, greater than 6 months, greater than 9 months or more. In some s, if malignant IGH copies are detected, the subject is predicted not to exhibit or not likely to exhibit a durable response to the cell therapy and/or to be at a high or relatively high risk of e or to have a low likelihood of exhibiting progression free survival for at least a certain period of time. In some embodiments, detection of malignant IGH copies by the provided methods is used to identify, such as to identify early, e.g. within or about 90 days or earlier of initiation of administration of the cell therapy, subjects predicted to be at a high risk of relapse and/or to have decreased likelihood of developing progression free survival (PFS) or a durable response, such as for less than 3 months, less than 6 months, less than 9 months or less. In some aspects, the patient may be identified for le administration of alternative or additional therapeutic strategies to improve se efficacy and/or durability. In some embodiments, the methods are d out on ts receiving a cell therapy, such as containing ered T cells, e.g. CAR+ T cells, for treating a B cell malignancy, e.g. CLL or A. Dosing In some embodiments, a dose of cells is administered to subjects in accord with the provided methods. In some embodiments, the size or timing of the doses is determined as a function of the particular disease or condition in the subject. It is within the level of a skilled artisan to empirically determine the size or timing of the doses for a particular disease in View of the provided description.

In certain embodiments, the cells, or dual populations of sub—types of cells, are administered to the subject at a range of about 0.1 million to about 100 billion cells and/or that amount of cells per kilogram of body weight of the subject, such as, e.g., 0.1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 n cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing ), 1 million to about 50 n cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range d by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges and/or per kilogram of body weight of the subject. s may vary depending on attributes particular to the disease or disorder and/or patient and/or other treatments. In some embodiments, such values refer to numbers of recombinant receptor—expressing cells; in other embodiments, they refer to number of T cells or PBMCs or total cells administered.

In some embodiments, the cell therapy comprises administration of a dose sing a number of cells that is at least or at least about or is or is about 0.1 x 106 cells/kg body weight of the subject, 0.2 x 106 cells/kg, 0.3 x 106 cells/kg, 0.4 x 106 cells/kg, 0.5 x 106 cells/kg, 1 x 106 cell/kg, 2.0 x 106 cells/kg, 3 x 106 cells/kg or 5 x 106 cells/kg.

In some embodiments, the cell therapy comprises administration of a dose comprising a number of cells is between or between about 0.1 x 106 cells/kg body weight of the subject and 1.0 x 107 cells/kg, n or between about 0.5 x 106 cells/kg and 5 x 106 kg, between or between about 0.5 x 106 cells/kg and 3 x 106 cells/kg, between or between about 0.5 x 106 cells/kg and 2 x 106 kg, n or between about 0.5 x 106 cells/kg and 1 x 106 cell/kg, between or between about 1.0 x 106 cells/kg body weight of the t and 5 x 106 cells/kg, between or between about 1.0 x 106 cells/kg and 3 x 106 cells/kg, between or between about 1.0 x 106 cells/kg and 2 x 106 cells/kg, between or between about 2.0 x 106 cells/kg body weight of the subject and 5 x 106 cells/kg, between or between about 2.0 X 106 kg and 3 X 106 cells/kg, or between or between about 3.0 x 106 cells/kg body weight of the t and 5 x 106 kg, each inclusive.

In some embodiments, the dose of cells comprises between at or about 2 x 105 of the cells/kg and at or about 2 x 106 of the kg, such as n at or about 4 x 105 of the cells/kg and at or about 1 x 106 of the cells/kg or between at or about 6 x 105 of the cells/kg and at or about 8 x 105 of the cells/kg. In some embodiments, the dose of cells comprises no more than 2 x 105 of the cells (e.g. antigen-expressing, such as CAR-expressing cells) per kilogram body weight of the t (cells/kg), such as no more than at or about 3 x 105 cells/kg, no more than at or about 4 x 105 cells/kg, no more than at or about 5 x 105 cells/kg, no more than at or about 6 x 105 kg, no more than at or about 7 x 105 cells/kg, no more than at or about 8 x 105 cells/kg, no more than at or about 9 x 105 cells/kg, no more than at or about I x 106 cells/kg, or no more than at or about 2 x 106 cells/kg. In some embodiments, the dose of cells ses at least or at least about or at or about 2 x 105 of the cells (e.g. antigen-expressing, such as CAR- expressing cells) per kilogram body weight of the subject (cells/kg), such as at least or at least about or at or about 3 x 105 cells/kg, at least or at least about or at or about 4 x 105 cells/kg, at least or at least about or at or about 5 x 105 cells/kg, at least or at least about or at or about 6 x 105 cells/kg, at least or at least about or at or about 7 x 105 cells/kg, at least or at least about or at or about 8 x 105 cells/kg, at least or at least about or at or about 9 x 105 cells/kg, at least or at least about or at or about 1 x 106 cells/kg, or at least or at least about or at or about 2 x 106 cells/kg.

In some embodiments, for example, where the subject is a human, the dose includes fewer than about 1 x 108 total recombinant receptor (e.g., CAR)-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), e.g., in the range of about 1 x 106 to l x 108 such cells, such as 2 x 106, 5 x 106, l x 107, 5 x 107, or 1 x 108 or total such cells, or the range between any two of the foregoing values. In some embodiments, where the subject is a human, the dose includes between about 1 x 106 and 3 x 108 total recombinant receptor (e.g., CAR)- expressing cells, e.g., in the range of about 1 x 107 to 2 x 108 such cells, such as l x 107, 5 x 107, l x 108 or 1.5 x 108 total such cells, or the range n any two of the foregoing values. In some embodiments, the patient is administered multiple doses, and each of the doses or the total dose can be within any of the foregoing values. In some ments, the dose of cells comprises the administration of from or from about I x 105 to 5 x 108 total recombinant receptor—expressing T cells or total T cells, 1 x 105 to l x 108 total recombinant receptor— expressing T cells or total T cells, from or from about 5 X 105 to l x 107 total recombinant receptor-expressing T cells or total T cells, or from or from about I x 106 to l x 107 total inant receptor—expressing T cells or total T cells, each inclusive.

In the context of adoptive cell therapy, administration of a given “dose” encompasses stration of the given amount or number of cells as a single composition and/or single uninterrupted administration, e.g., as a single ion or continuous infusion, and also asses administration of the given amount or number of cells as a split dose, provided in multiple individual compositions or infusions, over a specified period of time, which is no more than 3 days. Thus, in some contexts, the dose is a single or uous administration of the specified number of cells, given or initiated at a single point in time. In some contexts, r, the dose is administered in multiple injections or infusions over a period of no more than three days, such as once a day for three days or for two days or by multiple infusions over a single day period.

Thus, in some aspects, the cells of the dose are administered in a single pharmaceutical composition. In some embodiments, the cells of the dose are stered in a plurality of compositions, collectively containing the cells of the dose.

The term “split dose” refers to a dose that is split so that it is administered over more than one day. This type of dosing is encompassed by the present s and is considered to be a single dose.

Thus, the dose of cells may be administered as a split dose. For example, in some embodiments, the dose may be stered to the subject over 2 days or over 3 days.

Exemplary methods for split dosing e administering 25% of the dose on the first day and administering the remaining 75% of the dose on the second day. In other embodiments, 33% of the dose may be administered on the first day and the remaining 67% administered on the second day. In some aspects, 10% of the dose is administered on the first day, 30% of the dose is administered on the second day, and 60% of the dose is administered on the third day. In some embodiments, the split dose is not spread over more than 3 days.

In some embodiments, the dose of cells is lly large enough to be effective in ng disease burden.

In some embodiments, the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type(s) and/or a desired ratio of cell types. Thus, the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub—types, such as the CD4+ to CD8+ ratio. In some embodiments, the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types. In some embodiments, the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.

In some embodiments, the populations or sub—types of cells, such as CD8+ and CD4+ T cells, are administered at or within a tolerated difference of a desired dose of total cells, such as a desired dose of T cells. In some aspects, the desired dose is a d number of cells or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg. In some aspects, the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body weight. In some aspects, among the total cells, administered at the desired dose, the individual populations or sub—types are present at or near a desired output ratio (such as CD4+ to CD8+ ratio), e.g., within a certain tolerated difference or error of such a ratio.

In some embodiments, the cells are administered at or within a tolerated difference of a desired dose of one or more of the individual populations or pes of cells, such as a d dose of CD4+ cells and/or a desired dose of CD8+ cells. In some s, the desired dose is a desired number of cells of the sub—type or population, or a desired number of such cells per unit of body weight of the t to whom the cells are administered, e.g., cells/kg. In some s, the desired dose is at or above a m number of cells of the population or sub— type, or minimum number of cells of the population or sub-type per unit of body weight.

Thus, in some embodiments, the dosage is based on a desired fixed dose of total cells and a desired ratio, and/or based on a desired fixed dose of one or more, e.g., each, of the individual sub-types or sub—populations. Thus, in some embodiments, the dosage is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4+ to CD8+ cells, and/or is based on a d fixed or minimum dose of CD4+ and/or CD8+ cells.

In some embodiments, the cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as CD4+ and CD8+ cells or sub—types. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios. for example, in some ments, the desired ratio (e.g., ratio of CD4+ to CD8+ cells) is between at or about 5:1 and at or about 5:1 (or greater than about 1:5 and less than about 5:1), or between at or about 1:3 and at or about 3:1 (or greater than about 1:3 and less than about 3: 1), such as between at or about 2:1 and at or about 1:5 (or greater than about 1:5 and less than about 2:1, such as at or about 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1,1.9:1,1.8:1,1.7:1,1.6:1,1.5:1, l.4:1,1.3:1,1.2:1,1.1:1,1:l,1:1.1,1:1.2,1:1.3,1:1.4,1:1.5,1:1.6,1:1.7,1:1.8,1:1.9:1:2,1:2.5, 1:3, 123.5, 1:4, 114.5, or 1:5. In some aspects, the tolerated difference is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in between these ranges.

In particular embodiments, the numbers and/or trations of cells refer to the number of inant receptor (e.g., CAR)—expressing cells. In other embodiments, the numbers and/or concentrations of cells refer to the number or concentration of all cells, T cells, or peripheral blood mononuclear cells (PBMCs) administered.

In some aspects, the size of the dose is determined based on one or more criteria such as response of the subject to prior treatment, e.g. chemotherapy, e burden in the subject, such as tumor load, bulk, size, or degree, extent, or type of metastasis, stage, and/or likelihood or incidence of the subject developing toxic outcomes, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, oxicity, and/or a host immune response against the cells and/or recombinant ors being administered.

In some embodiments, the methods also include administering one or more additional doses of cells expressing a chimeric antigen receptor (CAR) and/or depleting therapy, and/or one or more steps of the methods are repeated. In some embodiments, the one or more additional dose is the same as the initial dose. In some embodiments, the one or more onal dose is different from the initial dose, e.g., higher, such as , 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold or d or more higher than the initial dose, or lower, such as e.g., higher, such as 2-fold, , 4—fold, 5-fold, 6-fold, 7-fold, 8—fold, 9—fold or d or more lower than the initial dose. In some embodiments, administration of one or more onal doses is determined based on response of the subject to the initial treatment or any prior treatment, disease burden in the subject, such as tumor load, bulk, size, or degree, extent, or type of metastasis, stage, and/or likelihood or incidence of the subject developing toxic outcomes, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or a host immune response against the cells and/or recombinant receptors being administered.

B. Response, Efficacy and Survival In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy. In some embodiments, at least or about at least 50% of ts, at least or about at least 60% of the subjects, at least or about at least 70% of the subjects, at least or about at least 80% of the subjects or at least or about at least 90% of the subjects treated according to the method achieve complete remission (CR) and/or achieve an objective response (OR).

In some aspects, the administration in accord with the ed methods generally reduces or prevents the expansion or burden of the disease or condition in the subject. For example, where the disease or condition is a tumor, the s generally reduce tumor size, bulk, metastasis, percentage of blasts in the bone marrow or molecularly detectable cancer, and/or improve prognosis or survival or other m associated with tumor .

In some respects, progression-free survival (PFS) is described as the length of time during and after the treatment of a disease, such as cancer, that a subject lives with the disease but it does not get worse. In some aspects, objective response (OR) is described as a measurable se. In some aspects, ive response rate (ORR) is described as the proportion of patients who achieved CR or PR. In some aspects, overall survival (OS) is described as the length of time from either the date of diagnosis or the start of treatment for a disease, such as cancer, that subjects diagnosed with the disease are still alive. In some aspects, event-free survival (EFS) is described as the length of time after treatment for a cancer ends that the subject remains free of certain complications or events that the treatment was intended to prevent or delay. These events may include the return of the cancer or the onset of certain ms, such as bone pain from cancer that has spread to the bone, or death.

In some ments, the method reduces the burden of the disease or condition, e.g., number of tumor cells, size of tumor, duration of patient survival or event-free al, to a greater degree and/or for a greater period of time as compared to the reduction that would be observed with a comparable method using an ative dosing regimen, such as one in which the subject receives one or more alternative eutic agents and/or one in which the subject does not receive a dose of cells and/or a lymphodepleting agent in accord with the provided methods. In some embodiments, the burden of a disease or condition in the subject is detected, ed, or measured. Disease burden may be detected in some aspects by detecting the total number of disease or e—associated cells, e.g., tumor cells, in the subject, or in an organ, , or bodily fluid of the subject, such as blood or serum. In some aspects, survival of the subject, survival within a certain time period, extent of survival, presence or duration of event- free or symptom-free survival, or relapse-free survival, is assessed. In some embodiments, any symptom of the disease or condition is ed. In some embodiments, the measure of disease or condition burden is specified.

In some embodiments, the event—free survival rate or overall survival rate of the subject is improved by the methods, as compared with other methods, for example, methods in which the subject es one or more alternative therapeutic agents and/or one in which the subject does not receive a dose of cells and/or a lymphodepleting agent in accord with the provided methods. For example, in some embodiments, event-free survival rate or probability for subjects treated by the s at 6 months following the dose is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some aspects, overall survival rate is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some embodiments, the t treated with the methods exhibits event-free survival, relapse-free al, or survival to at least 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some embodiments, the time to progression is ed, such as a time to progression of greater than at or about 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

In some embodiments, following treatment by the method, the ility of relapse is reduced as ed to other methods, for example, methods in which the subject receives one or more alternative therapeutic agents and/or one in which the subject does not receive a dose of cells and/or a lymphodepleting agent in accord with the provided methods. For example, in some embodiments, the ility of relapse at 6 months following the first dose is less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10%.

Disease burden can encompass a total number of cells of the disease in the subject or in an organ, tissue, or bodily fluid of the subject, such as the organ or tissue of the tumor or another location, e.g., which would indicate metastasis. For example, tumor cells may be detected and/or quantified in the blood or bone marrow in the context of certain hematological malignancies. Disease burden can e, in some ments, the mass of a tumor, the number or extent of metastases and/or the percentage of blast cells present in the bone marrow.

In some aspects, response assessment utilizes any of clinical, hematologic, and/or lar methods.

W0 14207 1. lW6Z1 re Crllerl’d In some s, response rates in subjects, such as subjects with CLL, are based on the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) response criteria (Hallek, et al., Blood 2008, Jun 15; 111(12): 5446—5456; also called IWCLL 2008). In some aspects, these criteria are described as follows: complete remission (CR), which in some aspects requires the absence of peripheral blood clonal lymphocytes by immunophenotyping, e of lymphadenopathy, absence of hepatomegaly or splenomegaly, e of constitutional ms and actory blood counts; complete remission with incomplete marrow recovery (CRi), which in some aspects is described as CR above, but without normal blood counts; partial remission (PR), which in some s is described as 2 50% fall in lymphocyte count, 2 50% reduction in lymphadenopathy or Z 50% reduction in liver or spleen, together with improvement in peripheral blood counts; progressive disease (PD), which in some aspects is described as Z 50% rise in lymphocyte count to > 5 x109/L, 2 50% increase in lymphadenopathy, 2 50% increase in liver or spleen size, Richter’s transformation, or new cytopenias due to CLL; and stable disease, which in some aspects is described as not meeting criteria for CR, CRi, PR or PD.

In some embodiments, the subjects exhibits a CR or OR if, within 1 month of the administration of the dose of cells, lymph nodes in the subject are less than at or about 20 mm in size, less than at or about 10 mm in size or less than at or about 10 mm in size. 2. 013mm? 1'11 501M Mar/70W 0r 19/004 In some embodiments, a t has leukemia. The extent of disease burden can be determined by assessment of residual leukemia in blood or bone marrow.

In some embodiments, a subject exhibits morphologic disease if there are greater than or equal to 5% blasts in the bone marrow, for example, as detected by light microscopy, such as greater than or equal to 10% blasts in the bone marrow, greater than or equal to 20% blasts in the bone marrow, greater than or equal to 30% blasts in the bone marrow, greater than or equal to 40% blasts in the bone marrow or greater than or equal to 50% blasts in the bone marrow. In some embodiments, a subject exhibits complete or clinical remission if there are less than 5% blasts in the bone marrow.

In some embodiments, a subject may exhibit complete remission, but a small proportion of morphologically undetectable (by light microscopy techniques) residual ic cells are t. A subject is said to exhibit minimum residual disease (MRD) if the subject exhibits less than 5% blasts in the bone marrow and ts larly detectable cancer. In some embodiments, molecularly detectable cancer can be ed using any of a variety of molecular techniques that permit sensitive detection of a small number of cells. In some aspects, such techniques include PCR assays, which can determine unique ell receptor gene rearrangements or fusion ripts produced by chromosome translocations. In some embodiments, flow cytometry can be used to fy cancer cell based on leukemia-specific immunophenotypes. In some embodiments, molecular detection of cancer can detect as few as l leukemia cell in 10,000 normal cells or 1 leukemia cell in 100,000 normal cells. In some embodiments, a t exhibits MRD that is molecularly detectable if at least or greater than 1 leukemia cell in 10,000 cells detected or 1 leukemia cell in 100,000 cells is detected, such as by PCR or flow cytometry. In some ments, the e burden of a subject is molecularly undetectable or MRD‘, such that, in some cases, no leukemia cells are able to be detected in the subject using PCR or flow cytometry ques.

In some embodiments, an index clone of the leukemia, e.g. CLL, is not detected in the bone marrow of the subject (or in the bone marrow of greater than 50%, 60%, 70%, 80%, 90% or more of the subjects treated according to the methods. In some embodiments, an index clone of the leukemia, e.g. CLL, is assessed by IGH deep sequencing. In some embodiments, the index clone is not detected at a time that is at or about or at least at or about 1, 2, 3, 4, 5, 6, 12, 18 or 24 months following the administration of the cells. a. Determination of MRD by flow cytometry In some aspects MRD is ed by flow cytometry. Flow cytometry can be used to monitor bone marrow and peripheral blood samples for cancer cells. In particular aspects, flow cytometry is used to detect or monitor the presence of cancer cells in bone marrow. In some aspects, multiparameter immunological detection by flow cytometry is used to detect cancer cells (see for example, Coustan—Smith et al., (1998) Lancet 35 11550—554). In some aspects, multiparameter immunological detection by mass cytometry is used to detect cancer cells. In some examples, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13,14, 15, 20, 25, 30, 35, 40, 45 or 50 parameters can be used to detect cancer cells. The antigens used for detection are selected based on the cancer being detected (Foon and Todd (1986) Blood 68: 1-31).

W0 14207 In some examples, bone marrow is ted by bone marrow aspirates or bone marrow biopsies, and lymphocytes are isolated for analysis. onal and/or polyclonal antibodies conjugated to a fluorochrome (e.g., fluorescein isothiocyanate (FITC), phycoerythrin, peridinin phyll protein, or biotin) can be used to detect epitopes, such as terminal deoxynucleotidyl transferase (TdT), CD3, CD10, CDl lc, CD13, CD14, CD33, CD19, CD20, CD21, CD22, CD23, CD34, CD45, CD56, CD79b, IgM, and/or KORSA3544, on isolated lymphocytes. Labeled cells can then be detected using flow cytometry, such as arameter flow cytometry, or mass cytometry, to detect multiple epitopes.

Lymphoid cells can be identified and gated based on a light-scatter dot plot and then secondarily gated to identify cell populations expressing the immunophenotypic features of interest. Exemplary es are set forth in Table 1 below. Other immunologic fication of leukemias and lymphomas are ed by Foon and Todd (Blood (1986) 68(1): 1—31). In some aspects, flow cytometric assessment of MRD can be achieved by quantifying live lymphocytes bearing one or more CLL immunophenotypes (e. g., low forward/side scatter; CD3neg; CD5+; CD 14mg; CD19+; CD23+; CD455 CD56neg).

Table 1. Exem lar Immnuno henot e and C t0_entics Characteristics Disease Immunophenotype Cytogenetics Chronic Pan—B+; CD5+; Trisomy12 Lymphocytic CD23+; CD79b/CD22 del( 13)(q14_3) Leukemia (CLL) weak; FMC7-; sIg del llq22—q23 del 17p13 (p53) t(l l;l4)(ql3;q32) BCLl/IgH rearrangement t(l4; l9)(q32;ql3) IgH deletion (l4q32) del(6q) +8q24 del 6q2l Small lymphocytic Pan—B+; CD5+; (q21—23) lymphoma (SLL) CD23+; CD 10—; sIgM+ faint Table 1. Exem lar Immnuno henot e and C t0_entics Characteristics Disease n henoty; e Cyt01enetics Lymphoplasmacytic Pan—B+; CD5—; CD10— t(9;l4)(p13;q32) PAX5/IgH lymphoma ; cngM+ Follicle centre cell Pan-B+; CD10+/—; t(14;18)(q32;q21) / BCL2 Rearr lymphoma CD5—; sIg+ Diffuse large cell CD19+; CD22+; t(14;18) and p53 mutations lymphoma CD10-/+; SIg+ t(3;V)(q27;V)/ BCL6 Rearr variants c-MYC Rearr Burkitt's ma Pan-B+; TdT-; t(8;14)(q24;q32) or variants / c-MYC R earr CD10+; CD5-; sIgM+ Burkitt—like Pan-B+; TdT-; CD10- t(8;14) or variants lymphoma /+ CD5-; sIg+ t(8;14)+ t(14;18) Mantle cell Pan—B +; CD5+; t(11;14)(q13;q32)/BCL1Rearr lymphoma CD23—; CD 10—/+; sIgM+ bright Marginal zone B-cell pan-B+; CD5—/+; t(11;18)(q21;q21) / P12 / MLT fusion: Extralymphoma CD10-; CD23-; nodal low—grade MALT lymphoma; (MZBCL) CD11c+/—; cng + indolent e (40% of the cells), t(1;14)(p21;q32): Extra-nodal MALT sIgM+ bright; sIgD- ma del(7)(q22—3 1): Splenic MZBCL /+3q: Nodal, extra—nodal and Splenic MZBCL +: positive in >90% of the cases +/—: ve in more than 50% of the cases —/+: positive in less than 50% of cases —: ve in <10% of the cases Pan—B markers: e.g., CD19, CD20, CD79a SIG: surface immunoglobulins cng: cytoplasmic immunoglobulins b. IGH Deep Sequencing In some aspects, deep sequencing of the immunoglobulin heavy chain (IGH) locus of ted B cells can be used to detect minimal residual disease (MRD). Clonal presence of a particular IgG rearrangement can provide a marker to detect the presence of B cell ancies, such as CLL or NHL and/or residual ce of malignant cells thereof. In some aspects cells such as a population containing or suspected of containing B cells are harvested and isolated from blood. In some aspects, cells are harvested and isolated from bone , e.g., from bone marrow aspirates or bone marrow biopsies and/or from other biological samples. In some aspects, polymerase chain reaction (PCR) amplification of the mentarity ining region 3 (CDR3) is achieved using primers to highly conserved sequences within the V and J regions of the gene locus, which may be used to identify clonal populations of cells for purposes of assessing minimal residual disease. Other methods for detecting clonal populations, such as single cell sequencing approaches, including those ing information regarding number of cells of a particular lineage and/or expressing a particular variable chain such as variable heavy chain or binding site thereof, such as a clonal tion, may be used. In some aspects, the IGH DNA is amplified using a degenerate primers or primers izing regions of variable chains shared among ent cell clones, such as those recognizing consensus V and degenerate consensus J region of the IGH sequence. An exemplary sequence of the V region is ACACGGCCTCGTGTATTACTGT (SEQ ID NO: 17). An exemplary degenerate consensus sequence of the J region is ACCTGAGGAGACGGTGACC (SEQ ID NO: 18).

The PCR product or sequencing result in some aspects is specific to the rearranged allele and serves as a clonal marker for MRD detection. Following PCR amplification of the CDR3 region, PCR products can be sequenced to yield patient-specific oligonucleotides constructed as probes for allele-specific PCR for sensitive detection of MRD ing treatment of B—cell malignancies with CAR—T cell therapy, e.g. CD19 CAR— T cell therapy. In examples where a PCR product is not generated using the consensus primers, V region family- specific primers for the framework region 1 can be used instead.

In some aspects, persistence of PCR—detectable tumor cells such as cells of the B cell malignancy such as the NHL or CLL, such as detectable IGH sequences corresponding to the malignant or clonal IGH sequences, after ent is associated with increased risk of relapse.

In some aspects, patients who are negative for malignant IGH ces following treatment (in some aspects, even in the context of other criteria indicating progressive disease or only a partial response, such as persistence of enlarged lymph nodes or other criteria that may in some contexts be associated with disease or lack of complete response) may be deemed to have sed hood of PFS or to enter into CR or e CR or prolonged survival, compared to ts with persistent malignant IGH ces. In some ments, such prognostic and g determinations are particularly relevant for treatments in which clearance of malignant cells is observed within a short period of time following administration of the therapy, e.g., in comparison to resolution of other al symptoms such as lymph node size or other staging criteria. For example, in some such aspects, absence of detectable IGH or minimal residual disease in a sample such as the bone marrow may be a preferred readout for response or hood of response or durability f, as compared to other available staging or prognostic approaches. In some aspects, results from MRD, e.g., IGH deep sequencing information, may inform further intervention or lack thereof. For example, the methods and other provided embodiments in some contexts provide that a subject deemed negative for malignant IGH may in some aspects be not further treated or not be r stered a dose of the therapy provided, or that the subject be administered a lower or reduced dose. sely, it may be provided or specified that a subject exhibiting MRD via IGH deep cing be further treated, e.g., with the therapy initially administered at a similar or higher dose or with a further treatment. 3. [11574110 crib/1'4 . In some respects, response is assessed using the Lugano criteria (Cheson et al., JCO September 20, 2014 vol. 32 no. 27 3059—3067; Johnson et al., (2015) Imaging for staging and response assessment in lymphoma. Radiology 2:323—338). Lugano criteria include evaluation by imaging, tumor bulk measurements, and assessments of spleen, liver, and bone marrow involvement.

In some s, response assessed using the Lugano criteria involves the use of positron emission tomography (PET)—computed tomography (CT) and/or CT as appropriate for imaging evaluation. PET—CT evaluations may further comprise the use of deoxyglucose (FDG), to assess FDG uptake, in FDG-avid lymphomas. FDG—avid lymphomas include Hodgkin lymphoma (HL) and certain non-Hodgkin lymphomas (NHL), including diffuse large B cellular lymphoma (DLBCL), marginal zone NHL with an aggressive transformation, and FDG-avid nodal mas (essentially all histologic types except: chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, lymphoplasmacytic lymphoma / Waldenstrom macroglobulinaemia, and mycosis fungoides). In some cases, for non—FDG—avid histologies, CT is the preferred imaging . In some aspects, the post—treatment scans are taken as long as le after administration of treatment. In some aspects the post-treatment scans are taken a minimum of 3 weeks after therapy, such as 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12, weeks or more after administration of treatment.

In some aspects, where PET-CT will be used to assess response in FDG-avid histologies, a 5-point scale, such as the Deauville five-point scale (Deauville 5ps), may be used for evaluation or stagingThe Deauville score is based on visual interpretation of fluorodeoxyglucose (FDG) uptake, visualized by PET/CT scans, of each lesion, compared to two reference organs, the mediastinum (i.e., blood pool) and the liver. One assessment al g) is made prior to treatment and a second round of FDG PET/CT scans is used to evaluate residual masses (in comparison to the FDG update in the nce organs) during and/or after treatment. The scale ranges from 1 to 5, where 1 is best and 5 is the worst. Each FDG—avid (or usly id) lesion is rated independently. In some respects, the 5—point scale comprises the following criteria: 1, no uptake above background; 2, uptake 5 mediastinum; 3, uptake > mediastinum but S liver; 4, uptake tely > liver; 5, uptake markedly higher than liver (e. g., maximum standard uptake value (SUVMAX >2x liver; 5a) and/or new lesion (on response evaluation) that is possibly related to lymphoma (5b); X, new areas of uptake unlikely to be related to lymphoma.

A Deauville score of 1 or 2 is considered to represent complete metabolic response (CMR) at interim and end of treatment. A Deauville score of 3 also ents CMR, but interpretation of score 3 depends on the timing of the assessment, the clinical context and the treatment. A Deauville score of 4 or 5 at interim is considered to represent partial lic response. However, a Deauville score of 4 or 5 at the end of treatment represents residual metabolic disease, if the uptake has reduced from baseline; no metabolic response (NMR) if there is no change in uptake from baseline; and progressive metabolic disease (PMD) if there in an increase in uptake from baseline and/or there are new lesions. At interim and end of treatment, NMR or PMD tes treatment failure.

W0 14207 In some aspects, a complete response at the end of treatment, as bed using the Lugano criteria, involves a complete metabolic se and a complete radiologic response at various measureable sites. In some aspects, these sites include lymph nodes and ymphatic sites, wherein a CR is described as a score of l, 2, or 3 with or without a residual mass on the 5- point scale, when PET-CT is used. In some aspects, in Waldeyer's ring or extranodal sites with high physiologic uptake or with activation within spleen or marrow (e.g., with herapy or myeloid colony-stimulating s), uptake may be greater than normal mediastinum and/or liver. In this stance, complete metabolic response may be inferred if uptake at sites of initial involvement is no greater than surrounding normal tissue even if the tissue has high physiologic uptake. In some aspects, response is assessed in the lymph nodes using CT, n a CR is described as no extralymphatic sites of disease and target nodes/nodal masses must regress to S 1.5 cm in longest transverse diameter of a lesion (LDi). Further sites of assessment include the bone marrow n PET—CT—based assessment should indicate a lack of evidence of FDG-avid disease in marrow and a CT-based assessment should indicate a normal morphology, which if indeterminate should be IHC negative. Further sites may include assessment of organ enlargement, which should regress to normal. In some aspects, non— measured lesions and new lesions are assessed, which in the case of CR should be .

(Cheson et al., JCO September 20, 2014 vol. 32 no. 27 3059-3067; Johnson et al., (2015) Imaging for staging and se assessment in lymphoma. Radiology 2:323—338). 4. [Peary/Isa[Pd/”alibi! Criteria i/z Solid/7111710” ME(757) critaria In some s, Response Evaluation Criteria in Solid Tumors (RECIST) criteria are used to determine objective tumor response; in some aspects, in solid tumors. (Eisenhauer et al., European Journal of Cancer 45 (2009) 228-247.) In some aspects, the RECIST criteria are used to determine objective tumor se for target s. In some respects, a complete response as ined using RECIST criteria is described as the disappearance of all target lesions and any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm. In other aspects, a partial response as determined using RECIST criteria is described as at least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters. In other aspects, progressive disease (PD) is described as at least a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on study (this W0 14207 includes the baseline sum if that is the smallest on study). In addition to the relative se of %, the sum must also demonstrate an absolute increase of at least 5 mm (in some aspects the appearance of one or more new lesions is also considered progression). In other aspects, stable disease (SD) is described as neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study.

C. Toxicity In some embodiments, the method does not cause or reduces the likelihood of toxicity or toxic outcomes, such as cytokine release syndrome (CRS), severe CRS , macrophage activation syndrome, tumor lysis syndrome, fever of at least at or about 38 degrees s for three or more days and a plasma level of CRP of at least at or about 20 mg/dL, neurotoxicity and/or neurotoxicity. In some embodiments, at least 50% of subjects treated according to the method (e.g. at least 60%, at least 70%, at least 80%, at least 90% or more of the subjects d) do not exhibit a toxic outcome (e.g. CRS or neurotoxicity) or do not t a severe toxic outcome (e.g. severe CRS or severe neurotoxicity). In some embodiments, the subject does not exhibit grade 3 or higher neurotoxicity and/or does not exhibit severe CRS, or does not do so within a certain period of time following the ent, such as within a week, two weeks, or one month of the administration of the cells.

Administration of adoptive T cell therapy, such as treatment with T cells expressing chimeric antigen receptors, can induce toxic effects or outcomes such as cytokine release syndrome and neurotoxicity. In some examples, such effects or outcomes parallel high levels of circulating cytokines, which may underlie the observed toxicity.

In some aspects, the toxic outcome is or is associated with or indicative of cytokine release me (CRS) or severe CRS (sCRS). CRS, e.g., sCRS, can occur in some cases following adoptive T cell therapy and administration to subjects of other ical products.

See DaVila et al., Sci Transl Med 6, 224ra25 (2014); Brentjens et al., Sci. Transl. Med. 5, l77ra38 (2013); Grupp et al., N. Engl. J. Med. 368, 1509—1518 (2013); and Kochenderfer et al., Blood 119, 2709—2720 (2012); Xu et al., Cancer Letters 343 (2014) 172-78.

Typically, CRS is caused by an exaggerated systemic immune response ed by, for example, T cells, B cells, NK cells, monocytes, and/or macrophages. Such cells may release a large amount of atory mediators such as cytokines and chemokines. Cytokines may trigger an acute inflammatory response and/or induce endothelial organ damage, which may result in microvascular leakage, heart failure, or death. Severe, life—threatening CRS can lead to ary infiltration and lung injury, renal failure, or inated intravascular coagulation.

Other severe, life—threatening toxicities can include cardiac toxicity, atory distress, neurologic ty and/or hepatic failure.

CRS may be treated using anti-inflammatory therapy such as an anti-IL-6 therapy, e.g., anti-IL-6 antibody, e.g., tocilizumab, or antibiotics. Outcomes, signs and symptoms of CRS are known and include those described herein. In some embodiments, where a particular dosage regimen or administration effects or does not effect a given CRS-associated outcome, sign, or symptom, particular outcomes, signs, and symptoms and/or quantities or s f may be specified.

In the context of administering pressing cells, CRS typically occurs 6-20 days after infusion of cells that express a CAR. See Xu et al., Cancer Letters 343 (2014) 172— 78. In some cases, CRS occurs less than 6 days or more than 20 days after CAR T cell infusion.

The incidence and timing of CR8 may be related to baseline cytokine levels or tumor burden at the time of infusion. ly, CRS involves elevated serum levels of interferon (IFN)—y, tumor necrosis factor (TNF)-0t, and/or interleukin (IL)-2. Other cytokines that may be rapidly induced in CRS are IL-lB, IL-6, IL-8, and IL-10.

Exemplary outcomes associated with CRS include fever, rigors, chills, hypotension, dyspnea, acute respiratory distress syndrome (ARDS), encephalopathy, ALT/AST elevation, renal failure, cardiac disorders, a, neurologic disturbances, and death. Neurological complications include delirium, seizure-like ty, confusion, word-finding difficulty, aphasia, and/or becoming obtunded. Other lated outcomes include fatigue, nausea, headache, seizure, tachycardia, myalgias, rash, acute vascular leak me, liver function impairment, and renal failure. In some aspects, CRS is associated with an increase in one or more factors such as serum—ferritin, d—dimer, aminotransferases, lactate dehydrogenase and triglycerides, or with hypofibrinogenemia or hepatosplenomegaly.

In some embodiments, outcomes associated with CRS include one or more of: persistent fever, e.g., fever of a ied ature, e.g., greater than at or about 38 degrees Celsius, for two or more, e.g., three or more, e.g., four or more days or for at least three consecutive days; fever greater than at or about 38 degrees Celsius; elevation of cytokines, such as a max fold change, e.g., of at least at or about 75, compared to pre—treatment levels of at least two cytokines (e.g., at least two of the group consisting of interferon gamma (IFNy), GM-CSF, IL—6, IL—10, Flt—3 L, fracktalkine, and IL—5, and/or tumor necrosis factor alpha (TNFOL)), or a max fold change, e.g., of at least at or about 250 of at least one of such cytokines; and/or at least one al sign of toxicity, such as hypotension (e.g., as ed by at least one intravenous vasoactive pressor); hypoxia (e.g., plasma oxygen (P02) levels of less than at or about 90%); and/or one or more neurologic disorders (including mental status changes, obtundation, and seizures).

Exemplary CRS-related outcomes include increased or high serum levels of one or more factors, including cytokines and chemokines and other factors associated with CRS.

Exemplary outcomes further include ses in synthesis or secretion of one or more of such factors. Such synthesis or secretion can be by the T cell or a cell that interacts with the T cell, such as an innate immune cell or B cell.

In some embodiments, the CRS—associated serum factors or CRS—related outcomes include inflammatory cytokines and/or chemokines, including interferon gamma (IFN—y), TNF— a, IL-lB, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, sIL-2Ra, granulocyte macrophage colony stimulating factor (GM—CSF), macrophage inflammatory protein (MIP)—l, tumor necrosis factor alpha (TNFOL), IL—6, and IL- 10, IL—lB, IL-8, IL—2, MIP-l, Flt—3L, fracktalkine, and/or IL—S. In some embodiments, the factor or outcome includes C ve protein (CRP). In addition to being an early and easily measurable risk factor for CRS, CRP also is a marker for cell expansion. In some embodiments, subjects that are ed to have high levels of CRP, such as 2 15 mg/dL, have CRS. In some embodiments, subjects that are measured to have high levels of CRP do not have CRS. In some embodiments, a measure of CR8 includes a measure of CRP and another factor tive of CRS.

In some embodiments, one or more atory cytokines or chemokines are monitored before, during, or after CAR treatment. In some aspects, the one or more cytokines or chemokines e IFN—y, TNF—a, IL—2, IL—IB, IL—6, IL—7, IL—8, IL—10, IL—12, sIL—ZRd, granulocyte macrophage colony stimulating factor (GM-CSF), or hage inflammatory protein (MIP). In some embodiments, IFN—y, TNF—oc, and IL—6 are monitored.

CRS criteria that appear to correlate with the onset of CR8 to predict which patients are more likely to be at risk for developing sCRS have been developed (see Davilla et al.

Science translational medicine. 2014;6(224):224ra25). Factors include fevers, hypoxia, hypotension, neurologic changes, elevated serum levels of inflammatory cytokines, such as a set of seven cytokines (IFNy, IL-5, IL-6, IL-10, , lkine, and GM-CSF) whose treatment- W0 14207 induced elevation can correlate well with both atment tumor burden and sCRS symptoms. Other guidelines on the diagnosis and management of CRS are known (see e.g., Lee et al, Blood. 2014; 124(2): 188—95). In some embodiments, the criteria reflective of CRS grade are those detailed in Table 2 below.

Table 2: Exemplary Grading Criteria for CRS Grade Description of Symptoms Not life-threatening, require only symptomatic treatment such as antipyretics Mild and anti-emetics (e. g., fever, nausea, fatigue, headache, myalgias, malaise) Require and respond to moderate intervention: Moderate O Oxygen requirement < 40%, or O Hypotension responsive to fluids or low dose of a single vasopressor, or O Grade 2 organ toxicity (by CTCAE v4.0) Require and respond to aggressive intervention: Severe O Oxygen requirement 2 40%, or O Hypotension requiring high dose of a single vasopressor (e. g., norepinephrine 2 20 ug/kg/min, dopamine 2 10 pg/kg/min, phenylephrine 2 200 pg/kg/min, or epinephrine 2 10 pg/kg/min), or Hypotension requiring multiple essors (e.g., vasopressin + one of me above agents, or combination vasopressors equivalent to 2 20 pg/kg/min norepinephrine), or O Grade 3 organ ty or Grade 4 transaminitis (by CTCAE v4.0) 4 Life-threatening: ireatening 0 Requirement for ventilator support, or O Grade 4 organ toxicity (excluding transaminitis) Death As used herein, a subject is deemed to develop “severe CRS” (“sCRS”) in response to or secondary to administration of a cell therapy or dose of cells thereof, if, ing administration, the t displays: (1) fever of at least 38 degrees Celsius for at least three days; (2) cytokine elevation that includes either (a) a max fold change of at least 75 for at least two of the following group of seven cytokines compared to the level immediately following the administration: interferon gamma (IFNy), GM—CSF, IL—6, IL—10, Flt—3L, fracktalkine, and IL—5 and/or (b) a max fold change of at least 250 for at least one of the following group of seven cytokines compared to the level immediately following the administration: interferon gamma (IFNy), GM—CSF, IL—6, IL—10, Flt—3L, fracktalkine, and IL—5; and (c) at least one clinical sign of toxicity such as hypotension (requiring at least one intravenous vasoactive r) or a (P02 < 90%) or one or more neurologic er(s) (including mental status changes, obtundation, and/or seizures). In some embodiments, severe CRS includes CRS with a grade of 3 or greater, such as set forth in Table 2.

In some embodiments, the CRS encompasses a combination of (l) persistent fever (fever of at least 38 degrees Celsius for at least three days) and (2) a serum level of CRP of at least at or about 20 mg/dL.

In some embodiments, the CRS encompasses hypotension requiring the use of two or more vasopressors or respiratory failure requiring ical ventilation.

The method of measuring or detecting the various outcomes may be specified.

In some aspects, the toxic outcome is or is associated with neurotoxicity. In some embodiments, symptoms ated with a clinical risk of neurotoxicity include confusion, delirium, expressive aphasia, obtundation, myoclonus, lethargy, altered mental status, convulsions, seizure—like activity, seizures (optionally as confirmed by electroencephalogram [EEG]), elevated levels of beta amyloid (AB), elevated levels of glutamate, and elevated levels of oxygen radicals. In some ments, neurotoxicity is graded based on severity (e.g., using a Grade 1—5 scale (see, e.g., Guido Cavaletti & Paola Marmiroli Nature Reviews Neurology 6, 657—666 (December 2010); National Cancer Institute—Common Toxicity Criteria version 4.03 (NCI-CTCAE V4.03).

In some instances, ogic symptoms may be the st symptoms of sCRS. In some embodiments, neurologic ms are seen to begin 5 to 7 days after cell therapy infusion. In some embodiments, duration of neurologic changes may range from 3 to 19 days.

In some cases, recovery of neurologic changes occurs after other symptoms of sCRS have ed. In some embodiments, time or degree of resolution of neurologic s is not hastened by treatment with anti-IL—6 and/or steroid(s).

As used herein, a subject is deemed to develop “severe neurotoxicity” in response to or secondary to administration of a cell therapy or dose of cells thereof, if, following administration, the subject ys symptoms that limit self-care (e.g. bathing, dressing and undressing, feeding, using the toilet, taking medications) from among: 1) symptoms of eral motor neuropathy, including inflammation or degeneration of the peripheral motor nerves; 2) symptoms of peripheral sensory athy, including inflammation or degeneration of the peripheral sensory , dysesthesia, such as distortion of sensory tion, resulting in an abnormal and unpleasant sensation, neuralgia, such as intense l sensation along a nerve or a group of nerves, and/or paresthesia, such as functional disturbances of sensory neurons resulting in abnormal cutaneous sensations of tingling, numbness, pressure, cold and warmth in the absence of stimulus. In some embodiments, severe neurotoxicity includes neurotoxicity with a grade of 3 or greater, such as set forth in Table 3.

Table 3: Exemplary Grading Criteria for neurotoxicity ption of Symptoms Mild or asymptomatic symptoms omatic 01 Mild Modzerate Presence of symptoms that limit instrumental activities of daily living (ADL), such as preparing meals, shopping f01 groceries or s, using the telephone, managing money 3 Presence of symptoms that limit self-care ADL, such as bathing, dressing and Severe undressing, g self, using the toilet, taking medications 4 Symptoms that are hreatening, requiring urgent intervention Life-threatening Death Fatal In some embodiments, the methods reduce ms ated with neurotoxicity compared to other methods. For example, subjects treated according to the present methods may have reduced ms of neurotoxicity, such as limb weakness or numbness, loss of , vision, and/or intellect, uncontrollable obsessive and/or compulsive behaviors, ons, headache, cognitive and behavioral problems including loss of motor control, cognitive deterioration, and autonomic nervous system dysfunction, and sexual dysfunction, compared to subjects treated by other methods. In some embodiments, subjects treated according to the present methods may have reduced symptoms associated with peripheral motor neuropathy, peripheral sensory athy, dysethesia, neuralgia or paresthesia.

In some embodiments, the methods reduce outcomes associated with neurotoxicity including damages to the nervous system and/or brain, such as the death of neurons. In some aspects, the methods reduce the level of factors associated with neurotoxicity such as beta amyloid (AB), glutamate, and oxygen radicals. 11. RECOMBINANT ANTIGEN RECEPTORS EXPRESSED BY THE CELLS In some embodiments, the cells for use in or administered in connection with the provided methods contain or are engineered to contain an engineered receptor, e.g., an engineered antigen receptor, such as a chimeric antigen receptor (CAR), or a T cell receptor (TCR). Also provided are populations of such cells, compositions containing such cells and/or enriched for such cells, such as in which cells of a certain type such as T cells or CD8+ or CD4+ cells are enriched or selected. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell y. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., ts, in accord with the provided methods.

In some embodiments, the cells include one or more nucleic acids introduced via genetic ering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, gene transfer is accomplished by first stimulating the cells, such as by combining it with a stimulus that induces a se such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to s sufficient for clinical applications.

The cells generally express recombinant receptors, such as antigen ors including functional non—TCR antigen receptors, e.g., ic antigen receptors (CARS), and other n-binding receptors such as transgenic T cell receptors (TCRs). Also among the ors are other chimeric receptors.

A. Chimeric Antigen ors (CARS) In some embodiments of the provided methods and uses, chimeric receptors, such as a chimeric antigen receptors, contain one or more s that combine a ligand-binding domain (e.g. antibody or dy fragment) that provides icity for a desired antigen (e.g., tumor antigen) with intracellular ing domains. In some embodiments, the intracellular signaling domain is an activating intracellular domain portion, such as a T cell activating domain, providing a primary tion signal. In some embodiments, the intracellular signaling domain contains or onally contains a costimulatory signaling domain to facilitate effector functions. In some embodiments, chimeric receptors when genetically engineered into immune cells can modulate T cell activity, and, in some cases, can modulate T cell differentiation or homeostasis, thereby resulting in genetically engineered cells with improved longevity, survival and/or tence in viva, such as for use in adoptive cell therapy methods.

Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in international patent application publication numbers WO2000l4257, 126726, WO20l2/129514, WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061 US. patent application publication numbers US2002131960, U82013287748, US20130149337, US. Patent Nos.: 6,451,995, 7,446,190, 8,252,592, , 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416,and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388—398; Davila et al. (2013) PLoS ONE 8(4): 661338; Turtle et al., Curr. Opin. Immunol, 2012 October; 24(5): 633—39; Wu et al., , 2012 March 18(2): 160—75. In some aspects, the antigen receptors include a CAR as bed in US. Patent No.: 7,446,190, and those described in International Patent ation Publication No.: WO/2014055668 A1. Examples of the CARs include CARs as disclosed in any of the entioned publications, such as 031687, US 8,339,645, US 7,446,179, US 2013/0149337, US. Patent No.: 7,446,190, US Patent No.: 8,389,282, derfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, U.S. Patent No.: 7,446,190, and US Patent No.: 282.

The chimeric ors, such as CARs, generally include an extracellular antigen binding domain, such as a portion of an antibody molecule, generally a le heavy (VH) chain region and/or variable light (VL) chain region of the dy, e.g., an scFv antibody fragment.

In some embodiments, the antigen targeted by the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or rgeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on the engineered cells.

Antigens targeted by the receptors in some embodiments e antigens ated with a B cell malignancy, such as any of a number of known B cell marker. In some embodiments, the antigen targeted by the receptor is CD20, CD19, CD22, RORl, CD45, CD21, CD5, CD33, ngappa, Iglambda, CD79a, CD79b or CD30.

In some embodiments the scFv and/or VH domains is derived from FMC63. FMC63 generally refers to a mouse monoclonal IgGl antibody raised against Nalm-l and -16 cells expressing CD19 of human origin (Ling, N. R., et al. . Leucocyte typing III. 302). The FMC63 dy comprises CDRHl and H2 set forth in SEQ ID NOS: 38, 39 respectively, and CDRH3 seq forth in SEQ ID NOS: 40 or 54 and CDRLl set forth in SEQ ID NOS: 35 and CDR L2 36 or 55 and CDR L3 sequences 37 or 56. The FMC63 dy comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 41 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the stv comprises a variable light chain containing the CDRLl sequence of 35, a CDRL2 sequence of 36, and a CDRL3 sequence of 37 and/or a le heavy chain containing a CDRHl sequence of 38, a CDRH2 sequence of 39, and a CDRH3 ce of 40. In some embodiments, the scFv comprises a variable heavy chain region of FMC63 set forth in SEQ ID NO:41 and a variable light chain region of FMC63 set forth in 42. In some embodiments, the variable heavy and variable light chain are connected by a . In some embodiments, the linker is set forth in SEQ ID NO:24. In some embodiments, the scFv comprises, in order, a VH, a linker, and a VL. In some embodiments, the scFv comprises, in order, a VL, a linker, and a VH. In some embodiments, the stc is encoded by a sequence of nucleotides set forth in SEQ ID NO:25 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:25. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO:43 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:43.

In some embodiments the scFv is d from SJ25C1. SJ25C1 is a mouse monoclonal IgGl antibody raised against Nalm—l and -16 cells sing CD19 of human origin (Ling, N. R., et al. (1987). Leucocyte typing III. 302). The SJ25C1 antibody comprises CDRHl, H2 and H3 set forth in SEQ ID NOS: 47-49, respectively, and CDRLl, L2 and L3 sequences set forth in SEQ ID NOS: 44-46, respectively. The SJ25C1 antibody comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 50 and the light chain le region (VL) sing the amino acid sequence of SEQ ID NO: 51. In some embodiments, the stv comprises a variable light chain containing hte CDRLl sequence of 44, a CDRL2 sequence of 45, and a CDRL3 sequence of 46 and/or a variable heavy chain containing a CDRHl sequence of 47, a CDRH2 sequence of 48, and a CDRH3 sequence of 49. In some embodiments, the scFv comprises a variable heavy chain region of SJ25C1 set forth in SEQ ID NO:50 and a variable light chain region of SJ25C1 set forth in 51. In some embodiments, the variable heavy and variable light chain are connected by a linker. In some embodiments, the linker is set forth in SEQ ID NO:52. In some embodiments, the scFv comprises, in order, a VH, a , and a VL. In some embodiments, the scFv comprises, in order, a VL, a linker, and a VH. In some ments, the scFv ses the sequence of amino acids set forth in SEQ ID NO:53 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:53.

In some embodiments, the chimeric antigen or includes an ellular portion containing an antibody or antibody fragment. In some s, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment includes an scFv.

In some embodiments, the antibody portion of the recombinant receptor, e.g., CAR, further includes at least a portion of an immunoglobulin constant , such as a hinge region, e.g., an IgG4 hinge region, and/or a CHI/CL and/or Fc region. In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgGl. In some aspects, the portion of the constant region serves as a spacer region between the antigen—recognition component, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for sed responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, international patent application publication number W02014031687, US. Patent No. 8,822,647 or hed app. No. US2014/0271635.

In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgGl. In some embodiments, the spacer has the ce ESKYGPPCPPCP (set forth in SEQ ID NO: 1), and is encoded by the ce set forth in SEQ ID NO: 2. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 3. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 4. In some embodiments, the constant region or portion is of IgD. In some ments, the spacer has the sequence set forth in SEQ ID NO: 5. In some embodiments, the spacer has a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 1, 3, 4 or 5. In some ments, the spacer has the sequence set forth in SEQ ID NOS: 26-34. In some embodiments, the spacer has a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence ty to any of SEQ ID NOS: 26-34.

In some embodiments, the antigen receptor comprises an ellular domain linked directly or indirectly to the extracellular domain. In some embodiments, the ic antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain ses an ITAM. For example, in some s, the n recognition domain (e.g. extracellular domain) generally is linked to one or more intracellular signaling components, such as signaling ents that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and/or signal via another cell surface receptor. In some embodiments, the chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g. scFv) and intracellular signaling domain . Thus, in some embodiments, the antigen-binding ent (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains.

In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane—bound or transmembrane n. Transmembrane regions include those derived from (i.e. comprise at least the embrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154. atively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane . In some ments, the linkage is by linkers, s, and/or transmembrane domain(s). In some aspects, the transmembrane domain contains a transmembrane portion of CD28.

In some embodiments, the extracellular domain and transmembrane domain can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein. In some embodiments, the receptor contains extracellular portion of the molecule from which the transmembrane domain is derived, such as a CD28 extracellular portion.

Among the intracellular signaling domains are those that mimic or approximate a signal through a natural n receptor, a signal h such a receptor in combination with a costimulatory receptor, and/or a signal h a costimulatory receptor alone. In some ments, a short oligo— or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e. g., glycine-serine doublet, is t and forms a linkage between the transmembrane domain and the asmic signaling domain of the CAR.

T cell activation is in some aspects described as being mediated by two s of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation h the TCR (primary cytoplasmic ing sequences), and those that act in an antigen- independent manner to provide a secondary or co-stimulatory signal dary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.

The receptor, e.g., the CAR, generally includes at least one intracellular signaling component or components. In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine—based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences e those derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.

W0 14207 In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the antigen—binding n is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling s, and/or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further es a portion of one or more additional molecules such as Fc receptor v, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR or other chimeric receptor includes a chimeric molecule between CD3—zeta (CD3—C) or Fc receptor 7 and CD8, CD4, CD25 or CD16.

In some embodiments, upon ligation of the CAR or other chimeric receptor, the cytoplasmic domain or intracellular ing domain of the receptor activates at least one of the normal or functions or responses of the immune cell, e.g., T cell ered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an ellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co—receptors that in the l context act in concert with such receptors to initiate signal transduction ing antigen receptor engagement.

In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to e full tion, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for ting the secondary or costimulatory signal.

In some embodiments, the ic antigen receptor contains an intracellular domain of a T cell costimulatory molecule. In some embodiments, the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-lBB, 0X40, DAPlO, and ICOS. In some aspects, the same CAR includes both the activating and costimulatory componentsln some embodiments, the chimeric antigen receptor contains an intracellular domain derived from a T cell costimulatory molecule or a functional variant W0 14207 thereof, such as between the transmembrane domain and intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 4lBB.

In some embodiments, the activating domain is included within one CAR, whereas the costimulatory component is provided by another CAR recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, costimulatory CARs, both sed on the same cell (see WO2014/055668). In some aspects, the cells include one or more stimulatory or activating CAR and/or a ulatory CAR. In some ments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than the one associated with and/or specific for the e or condition whereby an activating signal red through the disease-targeting CAR is shed or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off—target effects.

In certain embodiments, the ellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a ic CD28 and CD137 (4-lBB, TNFRSF9) co—stimulatory domains, linked to a CD3 zeta ellular domain.

In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation , e.g., y activation domain, in the asmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.

In some embodiments, the antigen receptor further includes a marker and/or cells expressing the CAR or other antigen receptor further includes a surrogate marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor. In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor, such as truncated version of such a cell surface receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A, P2A, E2A or F2A, e.g. set forth in any of SEQ ID NOS: 6 or 19—23. For example, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2OI4031687. For example, the marker can be a truncated EGFR (tEGFR) that is, ally, linked to a linker sequence, such as a T2A cleavable linker sequence.

An exemplary polypeptide for a truncated EGFR (e.g. tEGFR) comprises the sequence of amino acids set forth in SEQ ID NO: 7 or 16 or a sequence of amino acids that ts at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7 or 16. An exemplary T2A linker sequence comprises the sequence of amino acids set forth in SEQ ID NO: 6 or a sequence of amino acids that ts at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 6.

In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the e of T cells, or a portion thereof.

In some embodiments, the molecule is a non—self molecule, e.g., non—self n, i.e., one that is not recognized as “self ’ by the immune system of the host into which the cells will be adoptively transferred.

In some embodiments, the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic ering, e.g., for ing cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some d effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive er and encounter with ligand.

In some cases, CARs are referred to as first, second, and/or third generation CARs.

In some aspects, a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding; in some aspects, a second-generation CARS is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR is one that includes multiple costimulatory domains of different ulatory receptors.

For example, in some embodiments, the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular ing domain containing a signaling portion of CD28 or functional variant f and a signaling portion of CD3 zeta or functional t thereof. In some embodiments, the CAR contains an antibody, e.g., antibody fragment, a embrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a ing portion of a 4-1BB or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof.

In some such ments, the receptor further includes a spacer containing a portion of an Ig le, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4 hinge, such as a hinge- only spacer.

In some embodiments, the transmembrane domain of the recombinant or, e.g., the CAR, is or includes a transmembrane domain of human CD28 (e.g. Accession No.

P01747.1) or variant thereof, such as a embrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 8 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8; in some embodiments, the embrane-domain containing portion of the recombinant receptor comprises the sequence of amino acids set forth in SEQ ID NO: 9 or a sequence of amino acids having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

In some embodiments, the intracellular signaling component(s) of the inant receptor, e. g. the CAR, contains an intracellular costimulatory signaling domain of human CD28 or a functional t or portion thereof, such as a domain with an LL to GG substitution at ons 186-187 of a native CD28 protein. For example, the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 10 or 11 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10 or 11. In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of 4-1BB (e.g.

(Accession No. Q07011.1) or functional variant or portion thereof, such as the sequence of amino acids set forth in SEQ ID NO: 12 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12.

In some embodiments, the intracellular signaling domain of the inant or, e. g. the CAR, comprises a human CD3 zeta stimulatory signaling domain or functional variant thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human CD38; (Accession No.: P209632) or a CD3 zeta signaling domain as described in US. Patent No.: 7,446,190 or US. Patent No. 8,911,993. For example, in some embodiments, the intracellular signaling domain comprises the sequence of amino acids as set forth in SEQ ID NO: 13, 14 or 15 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13, 14 or 15.

In some aspects, the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgGl, such as the hinge only spacer set forth in SEQ ID NO: 1. In other embodiments, the spacer is or contains an Ig hinge, e.g., an IgG4-derived hinge, optionally linked to a CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains, such as set forth in SEQ ID NO: 4. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a CH3 domain only, such as set forth in SEQ ID NO: 3. In some embodiments, the spacer is or ses a glycine-serine rich sequence or other flexible linker such as known flexible s.

For example, in some embodiments, the CAR includes an antibody such as an antibody fragment, including scFvs, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more nt regions of a heavy chain molecule, such as an Ig-hinge containing , a transmembrane domain containing all or a n of a CD28—derived transmembrane domain, a CD28—derived intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR includes an antibody or fragment, such as scFv, a spacer such as any of the Ig-hinge containing spacers, a CD28-derived transmembrane domain, a 4-1BB-derived intracellular signaling , and a CD3 zeta—derived signaling domain.

In some ments, nucleic acid molecules encoding such CAR constructs further includes a sequence encoding a T2A ribosomal skip t and/or a tEGFR sequence, e.g., downstream of the sequence encoding the CAR. In some embodiments, the ce encodes a T2A mal skip element set forth in SEQ ID NO: 6, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 6. In some embodiments, T cells expressing an antigen receptor (e.g. CAR) can also be generated to express a truncated EGFR (EGFRt) as a non—immunogenic ion epitope (e.g. by introduction of a construct encoding the CAR and EGFRt separated by a T2A ribosome switch to express two proteins from the same construct), which then can be used as a marker to detect such cells (see e.g. US. Patent No. 8,802,374). In some ments, the sequence encodes a tEGFR sequence set forth in SEQ ID NO: 7 or 16, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7 or 16.

The recombinant receptors, such as CARs, expressed by the cells administered to the subject generally recognize or specifically bind to a molecule that is expressed in, associated with, and/or specific for the disease or condition or cells thereof being treated. Upon specific binding to the molecule, e.g., antigen, the or generally delivers an immunostimulatory signal, such as an ITAM—transduced signal, into the cell, thereby promoting an immune se targeted to the disease or condition. For example, in some embodiments, the cells express a CAR that specifically binds to an antigen expressed by a cell or tissue of the disease or condition or ated with the disease or condition.

B. TCRs In some embodiments, the genetically ered antigen receptors include recombinant T cell receptors (TCRs) and/or TCRs cloned from naturally occurring T cells. In some embodiments, a high—affinity T cell clone for a target antigen (e.g., a cancer antigen) is fied, ed from a patient, and introduced into the cells. In some embodiments, the TCR clone for a target antigen has been generated in transgenic mice engineered with human immune system genes (e.g., the human leukocyte antigen , or HLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al. (2009) Clin Cancer Res. 15: 169—180 and Cohen et al. (2005) J Immunol. 175:579975808. In some embodiments, phage display is used to isolate TCRs against a target antigen (see, e.g., Varela-Rohena et al. (2008) Nat Med. 14: 1390—1395 and Li (2005) Nat Biotechnol. 23:349—354.

In some embodiments, after the T—cell clone is obtained, the TCR alpha and beta chains are isolated and cloned into a gene expression vector. In some embodiments, the TCR alpha and beta genes are linked via a picornavirus 2A ribosomal skip peptide so that both chains are coexpression. In some embodiments, genetic er of the TCR is lished via retroviral or lentiviral vectors, or via transposons (see, e.g., Baum et al. (2006) Molecular y: The Journal of the American Society of Gene y. 13: 1050—1063; Frecha et al. (2010) Molecular Therapy: The Journal of the American Society of Gene Therapy. 18: 1748, 1757', an Hackett et al. (2010) Molecular Therapy: The Journal of the an Society of Gene Therapy. 18:674—683.

III. GENETICALLY ENGINEERED CELLS AND METHODS OF PRODUCING CELLS In some embodiments, the provided methods involve administering to a t having a disease or condition cells expressing a recombinant antigen receptor. s methods for the introduction of cally engineered components, e.g., recombinant receptors, e.g., CARs or TCRs, are well known and may be used with the provided methods and compositions.

Exemplary methods include those for transfer of nucleic acids encoding the ors, including via viral, e.g., retroviral or iral, transduction, transposons, and electroporation.

Among the cells expressing the receptors and administered by the provided methods are engineered cells. The genetic engineering lly involves introduction of a nucleic acid encoding the recombinant or engineered component into a composition containing the cells, such as by retroviral transduction, transfection, or ormation.

A. Vectors and Methods for Genetic Engineering In some embodiments, inant nucleic acids are transferred into cells using recombinant infectious Virus particles, such as, e.g., vectors derived from simian Virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso—Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550—557.

In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma Virus (MPSV), murine nic stem cell Virus , murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV).

Most retroviral s are derived from murine retroviruses. In some embodiments, the retroviruses include those d from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980—990; , A. D. (1990) Human Gene Therapy 1:5—14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 109.

Methods of lentiviral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637— 1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. : 497—505.

In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 143 1— 1437). In some embodiments, recombinant nucleic acids are erred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427—437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115—126). Other methods of introducing and expressing genetic material in immune cells include m phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten le—facilitated microparticle bombardment (Johnston, Nature, 346: 7 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

Other approaches and vectors for transfer of the nucleic acids encoding the recombinant ts are those described, e.g., in international patent application, Publication No.: WO2014055668, and US. Patent No. 7,446,190.

In some embodiments, the cells, e.g., T cells, may be transfected either during or after expansion e.g. with a T cell receptor (TCR) or a chimeric antigen receptor (CAR). This transfection for the introduction of the gene of the desired receptor can be carried out with any suitable retroviral vector, for example. The genetically modified cell population can then be liberated from the initial stimulus (the CD3/CD28 stimulus, for e) and subsequently be stimulated with a second type of stimulus e.g. via a de novo introduced receptor). This second type of stimulus may include an nic stimulus in form of a peptide/MHC molecule, the cognate -linking) ligand of the genetically introduced receptor (e. g. l ligand of a CAR) or any ligand (such as an antibody) that directly binds within the framework of the new receptor (e.g. by recognizing constant regions within the receptor). See, for example, Cheadle et al, ric antigen receptors for T-cell based therapy” Methods Mol Biol. 2012; 907:645—66 or Barrett et al., Chimeric Antigen or Therapy for Cancer Annual Review of Medicine Vol. 65: 333-347 (2014).

In some cases, a vector may be used that does not require that the cells, e.g., T cells, are ted. In some such instances, the cells may be selected and/or transduced prior to activation. Thus, the cells may be engineered prior to, or subsequent to culturing of the cells, and in some cases at the same time as or during at least a portion of the culturing.

Among additional nucleic acids, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., M01. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319—338 (1992); see also the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant ve selectable marker with a negative selectable marker. See, e.g., Riddell et al., US Patent No. 6,040,177, at columns 14—17.

B. Cells and Preparation of Cells for Genetic Engineering In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from r sm or cell, which for example, is not rily found in the cell being engineered and/or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in , including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.

The cells generally are otic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as otent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and . In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and ulations thereof, such as those defined by on, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or tment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Among the methods include off-the-shelf methods. In some s, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some ments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, and re— introducing them into the same subject, before or after cryopreservation.

Among the pes and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor—infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as THl cells, THZ cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, ular helper T cells, alpha/beta T cells, and delta/gamma T cells.

In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are tes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.

In some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another sm or cell, which for example, is not rily found in the cell being engineered and/or an organism from which such cell is d. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one sing chimeric combinations of c acids encoding s domains from multiple different cell types.

In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for introduction of the c acid encoding the transgenic receptor such as the CAR, may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, sed, and/or engineered.

Accordingly, the cells in some embodiments are primary cells, e.g., y human cells. The samples include , fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The ical sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed s derived rom.

In some s, the sample from which the cells are derived or isolated is blood or a derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, ia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa ated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, s, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic s.

In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non—human primate, and pig.

In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to ular reagents.

In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to ular components.

In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and ets.

In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate ed saline (PBS). In some embodiments, the wash on lacks calcium and/or magnesium and/or many or all nt cations. In some s, a washing step is accomplished a semi—automated “flow—through” fuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's ctions.

In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Calf/Mg++ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.

In some ments, the methods include density-based cell tion methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface s, e.g., surface proteins, ellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the tion is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that ically binds to such markers, followed generally by washing steps and tion of cells having bound the antibody or g partner, from those cells having not bound to the antibody or binding partner.

Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically fies a cell type in a heterogeneous population, such that separation is best carried out based on markers sed by cells other than the desired population.

The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a , refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

In some examples, multiple rounds of separation steps are carried out, where the positively or vely selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative ion. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of dies or binding partners expressed on the various cell types.

For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28“: CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or ve selection techniques.

For e, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 28 T Cell Expander).

In some ments, isolation is carried out by enrichment for a particular cell population by positive ion, or depletion of a particular cell population, by negative selection. In some embodiments, positive or ve selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (markerhigh) on the positively or negatively selected cells, respectively.

In some embodiments, T cells are ted from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, tes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative ion for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.

In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by ve or ve selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some s is particularly robust in such sub-populations. See Terakura et a1. (2012) Blood.1:72—82; Wang et a1. (2012) J Immunother. 35(9):689—701. In some embodiments, combining TCM'CanCth CD8+ T cells and CD4+ T cells r enhances efficacy.

In embodiments, memory T cells are present in both CD62L+ and CD62L' subsets of CD8+ eral blood lymphocytes. PBMC can be ed for or depleted of CD62L'CD8+ and/or CD62LJ’CD8+ fractions, such as using anti—CD8 and anti—CD62L antibodies.

In some embodiments, the enrichment for central memory T (TCM) cells is based on ve or high surface expression of , CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells ed based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 sion-based ion step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub— population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further ve or negative selection steps.

In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained.

The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on a marker teristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.

CD4Jr T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO', CD45RA+, , CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and +. In some embodiments, effector CD4+ cells are CD62L' and CD45RO'.

In one example, to enrich for CD4+ cells by negative selection, a onal antibody cocktail typically es antibodies to CD14, CD20, CD1 lb, CD16, HLA—DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative ion. For example, in some embodiments, the cells and cell tions are ted or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 5 8: asis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U.

Schumacher © Humana Press Inc., Totowa, NJ).

In some aspects, the sample or ition of cells to be separated is incubated with small, magnetizable or magnetically sive al, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.

In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific g , such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable ic particles include those described in Molday, US. Pat.

No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat.

No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.

The incubation generally is d out under conditions whereby the antibodies or g partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the ic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.

In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles ed thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further tion steps.

In ceitain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or avidin. In certain ments, the magnetic les are attached to cells via a coating of y antibodies ic for one or more markers. In n embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type ic secondary antibody- or other binding partner (e.g., streptavidin)—coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

In some embodiments, the magnetically responsive particles are left attached to the cells that are to be uently ted, cultured and/or engineered; in some aspects, the particles are left ed to the cells for administration to a t. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non—labeled antibodies, and magnetizable particles or antibodies ated to cleavable linkers.

In some embodiments, the magnetizable particles are biodegradable.

In some embodiments, the affinity-based selection is Via ic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are tially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are .

Then, after this first n step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and red. In certain embodiments, the rget cells are labelled and depleted from the heterogeneous population of cells.

In n embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the ion, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Number W02009/072003, or US 03380 A1.

In some embodiments, the system or tus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self—contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various s of the processing, isolation, engineering, and formulation steps.

In some aspects, the separation and/or other steps is carried out using CliniMACS system (Miltenyi Biotic), for example, for ted separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and s pinch valves. The ated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.

The CliniMACS system in some aspects uses dy-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic on. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set ts of pre—assembled sterile tubing, including a pre-column and a tion , and are for single use only. After tion of the tion program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the s described herein are eluted from the column after removal of the ic field, and are collected within the cell collection bag.

In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy system nyi Biotec). The CliniMACS Prodigy system in some aspects is equipped with a cell processing unity that permits automated washing and onation of cells by fugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by ning the macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an ated cell cultivation chamber which accomplishes cell culture ols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651—660, Terakura et al. (2012) Blood.l:72—82, and Wang et al. (2012) J Immunother. 35(9):689-701.

In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell e markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via ative scale (FACS)—sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., (2008) J Biophoton. 1(5):355—376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.

In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence- activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.

In some ments, the preparation methods include steps for ng, e.g., cryopreserving, the cells, either before or after isolation, tion, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, tes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are lly then frozen to —80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid en storage tank.

In some embodiments, the cells are incubated and/or ed prior to or in connection with c engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or ation. The tion and/or ering may be d out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, Vial, culture dish, bag, or other container for culture or cultivating cells. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic ering, such as for the introduction of a recombinant n receptor.

The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or atory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR, e.g. anti-CD3. In some embodiments, the stimulating conditions include one or more agent, e.g. ligand, which is capable of stimulating a costimulatory receptor, e.g., anti-CD28. In some embodiments, such agents and/or ligands may be, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agents include IL—2, IL—15 and/or IL—7. In some aspects, the IL-2 concentration is at least about 10 units/mL.

In some aspects, incubation is carried out in accordance with ques such as those described in US Patent No. 6,040,177 to l et al., Klebanoff et a1.(2012) J Immunother. 35(9): 651—660, ra et al. (2012) Blood.1:72—82, and/or Wang et al. (2012) J ther. 35(9):689—701.

In some embodiments, the T cells are expanded by adding to a culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the ing tion of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the l population to be expanded); and incubating the culture (e.g. for a time ient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are ated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.

In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. ally, the incubation may r comprise adding non—dividing EB V—transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads.

The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.

In embodiments, antigen—specific T cells, such as antigen—specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen.

For example, antigen—specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same n.

IV. COMPOSITIONS AND FORMULATIONS In some embodiments, the dose of cells comprising cells engineered with a recombinant antigen or, e.g. CAR or TCR, is provided as a composition or formulation, such as a pharmaceutical composition or ation. Such compositions can be used in accord with the provided methods, such as in the prevention or treatment of diseases, conditions, and ers, or in detection, diagnostic, and prognostic methods.

The term “pharmaceutical ation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or vative.

In some aspects, the choice of carrier is determined in part by the particular cell or agent and/or by the method of administration. Accordingly, there are a variety of suitable ations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for e, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's ceutical Sciences 16th edition, Osol, A. Ed. (1980). ceutically able carriers are generally nontoxic to recipients at the dosages and trations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and nine; preservatives (such as cyldimethylbenzyl ammonium chloride; hexamethonium chloride; konium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3—pentanol; and m—cresol); low molecular weight (less than about 10 es) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, gine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; ing agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non—ionic surfactants such as polyethylene glycol (PEG).

Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, ium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, ton: The Science and Practice of cy, Lippincott Williams & s; 21st ed. (May 1, 2005).

The formulation or composition may also contain more than one active ingredient useful for the particular tion, disease, or condition being prevented or treated with the cells or agents, where the respective ties do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., ginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, yurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the agents or cells are administered in the form of a salt, e.g., a pharmaceutically able salt. Suitable pharmaceutically acceptable acid addition salts include those d from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, , malic, , fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p-toluenesulphonic acid.

Active ingredients may be entrapped in microcapsules, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano—particles and nanocapsules) or in mulsions. In certain embodiments, the pharmaceutical composition is formulated as an inclusion complex, such as cyclodextrin inclusion complex, or as a liposome. mes can serve to target the agent or host cells (e.g., T—cells or NK cells) to a particular . Many methods are available for preparing mes, such as those described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 , and US. s 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

The pharmaceutical ition in some aspects can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be d. Many types of release delivery systems are available and known. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the t and the physician.

The pharmaceutical composition in some embodiments contains agents or cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, ing on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

The agents or cells can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal ion, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, jectval injection, subconjuntival injection, sub—Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are stered by parenteral, ulmonary, and intranasal, and, if desired for local treatment, intralesional stration.

Parenteral infusions e intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration of the cells or agent. In some embodiments, it is administered by multiple bolus administrations of the cells or agent, for e, over a period of no more than 3 days, or by continuous infusion administration of the cells or agent.

For the tion or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of agent or agents, the type of cells or inant receptors, the severity and course of the disease, whether the agent or cells are administered for preventive or eutic purposes, us therapy, the subject's al history and response to the agent or the cells, and the discretion of the attending physician. The itions are in some embodiments suitably administered to the subject at one time or over a series of treatments.

The cells or agents may be administered using standard administration techniques, formulations, and/or devices. ed are formulations and devices, such as syringes and Vials, for storage and administration of the compositions. With respect to cells, stration can be autologous or heterologous. For example, immunoresponsive cells or progenitors can be obtained from one subject, and stered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex Vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or eral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell or an agent that treats or ameliorates symptoms of neurotoxicity), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).

Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some ments, the agent or cell tions are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the agent or cell tions are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or s compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to e than gels, other Viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the riate Viscosity range to provide longer contact periods with specific s. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and le mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the agent or cells in a solvent, such as in admixture with a le carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized.

The compositions can contain auxiliary nces such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, ing upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.

Various additives which e the stability and sterility of the compositions, including crobial preservatives, antioxidants, ing agents, and buffers, can be added.

Prevention of the action of microorganisms can be d by various antibacterial and antifungal agents, for example, ns, chlorobutanol, phenol, sorbic acid, and the like.

Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sustained—release ations may be prepared. Suitable examples of sustained— release ations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generally sterile. Sterility may be readily lished, e.g., by filtration through sterile filtration membranes.

V. ARTICLES OF MANUFACTURE AND KITS Also provided are es of manufacture and kits containing engineered cells expressing a recombinant receptor or compositions f, and optionally ctions for use, for example, instructions for stering, according to the ed methods.

In some embodiments, provided are articles of manufacture and/or kits that include a composition comprising a therapeutically effective amount of any of the engineered cells described herein, and instructions for administering, to a subject for treating a disease or condition. In some embodiments, the instructions can specify some or all of the elements of the methods provided herein. In some embodiments, the instructions specify particular instructions for administration of the cells for cell therapy, e.g., doses, timing, selection and/or identification of subjects for administration and conditions for administration. In some embodiments, the articles of manufacture and/or kits further comprise an agent for depleting therapy, and optionally further includes instructions for administering the lymphodepleting therapy. In some embodiments, the instructions can be included as a label or package insert accompanying the compositions for administration.

In some embodiments, the instructions specify the criteria for ion or identification of subjects for therapy. In some embodiments, such ia include subjects having a high—risk CLL. In some embodiments, the instructions specify the subject is or has been identified as having one or more cytogenetic alities, optionally ated with high—risk CLL, optionally selected from among: complex karyotype, deletion of the long arm of chromosome 13 (del l3q), del 11, trisomy 12, del 17p, del 6q, and del l3q.l4, optionally as detected by FISH; the subject is or has been identified as having high-risk CLL; and/or the subject is or has been identified as having extramedullary e; and/or the subject is an adult and/or is over at or about 30, 40, 50, 60, or 70 years of age. In some embodiments, the ia for ion or identification include that the subjects have a high risk NHL. In some embodiments, the subject is or has been fied as having one or more cytogenetic abnormalities, optionally associated with high-risk NHL; the subject is or has been identified as having high-risk NHL; and/or the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma ), primary tinal large B cell lymphoma (PMBCL), T cell/histocyte—rich large B cell lymphoma (TCHRBCL), Burkitt’s lymphoma, mantle cell lymphoma (MCL), and/or follicular lymphoma (FL); and/or the subject is an adult and/or is over at or about 30, 40, 50, 60, or 70 years of age.

In some embodiments, the instructions specify that the subject is one that has received one or more previous or prior therapies for treating the leukemia, e.g. CLL or NHL, such as has received 2, 3, or 4 more prior therapies. In some embodiments, the ctions specify the subject is selected for ent with the cell therapy, e.g. CAR+ T cells, if the subject is identified to be or likely to be refractory to and/or has relapsed after receiving one or more of such prior therapies.

In some embodiments, the instructions specify the dose of cells to be administered.

For example, in some embodiments, the instructions specify the dose (i) comprises (a) at or about 2 x 105 of the cells per kilogram body weight of the subject (cells/kg); (b) at or about 2 x 106 of the cells/kg, (c) no more than at or about 2 x 106 of the kg, ((1) no more than at or about 2 x 105 of the cells/kg and/or (e) between at or about 2 x 105 of the cells/kg and at or about 2 x 106 of the cells/kg. In some embodiments, the instructions specify particulars of the cell therapy, ing the target of the cell therapy, e.g. that the cell y is a argeted cell therapy. In some ments, the instructions specify that the cell therapy includes administration of CAR+ engineered cells that comprise a defined ratio of CD4+ cells expressing the CAR to CD8+ cells sing the CAR and/or of CD4+ cells to CD8+ cells, which ratio optionally is imately 111 or is between approximately 1:3 and approximately 3:1.

In some embodiments, the articles of manufacture and/or kits further e one or more additional agents for therapy, e.g., lymphodepleting therapy and/or combination therapy, as described herein, and optionally instructions for administering the additional agents. In some embodiments, the articles of manufacture and/or kits further include one or more ts for assaying biological samples, e.g., ical samples from subjects who are candidates for administration or who have been administered the therapy, and optionally instructions for use of the reagents or . In some embodiments, the ts can be used prior to the administration of the cell therapy or after the administration of cell therapy, for diagnostic purposes, to identify subjects and/or to assess treatment outcomes and/or toxicities. For example, in some embodiments, the e of manufacture and/or kits further contain reagents for measuring the level of particular biomarkers, e.g., cytokines, that are associated with toxicity, and instructions for measuring. In some embodiments, the reagents include ents for performing an in vitro assay to measure the kers, such as an immunoassay, an aptamer—based assay, a histological or cytological assay, or an mRNA expression level assay. In some embodiments, the in vitro assay is selected from among an enzyme linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry assay, surface plasmon resonance (SPR), chemiluminescence assay, lateral flow immunoassay, inhibition assay and avidity assay.

In some aspects, the reagent is a binding reagent that specifically binds the biomarkers. In some cases, the binding reagent is an antibody or antigen-binding fragment thereof, an aptamer or a nucleic acid probe.

The articles of manufacture and/or kits may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container in some embodiments holds a ition which is by itself or combined with r composition effective for treating, preventing and/or diagnosing the condition. In some embodiments, the container has a sterile access port.

Exemplary containers e an intravenous solution bags, vials, including those with stoppers able by a needle for injection, or bottles or vials for orally administered agents. The label or package insert may te that the composition is used for treating a disease or condition, such as a CLL or NHL.

The article of manufacture may include a container with a composition contained therein, wherein the composition includes engineered cells expressing a recombinant or; and, in some cases, one or more further containers with a composition contained therein, n the composition includes one or more further agent or agents. The article of manufacture may further include a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further include r or the same ner comprising a pharmaceutically-acceptable buffer.

It may further include other materials such as other s, diluents, filters, needles, and/or syringes.

VI. EXEMPLARY EMBODIMENTS Among the provided embodiments are: l. A method of treating a subject having or suspected of having a chronic lymphocytic leukemia (CLL), the method comprising stering to the subject a dose of cells expressing a chimeric n receptor (CAR) that specifically binds to a target antigen sed by the CLL, said dose comprising (a) at or about 2 x 105 of the cells per kilogram body weight of the subject (cells/kg); (b) at or about 2 x 106 of the cells/kg, (c) no more than at or about 2 x 106 of the cells/kg, (d) no more than at or about 2 x 105 of the cells/kg and/or (e) between at or about 2 x 105 of the kg and at or about 2 x 106 of the cells/kg, wherein, prior to the administration, the subject has been preconditioned with a lymphodepleting therapy comprising the administration of fludarabine. 2. A method of treating a subject having or suspected of having a chronic lymphocytic leukemia (CLL), the method comprising administering to the subject a dose of cells expressing a chimeric antigen receptor (CAR) that ically binds to a target antigen expressed by the CLL, said dose comprising (a) at or about 1 x 107 total cells or total CAR- expressing cells; (b) at or about 1.5 x 108 total cells or total CAR-expressing cells, (c) no more than at or about 1 x 107 total cells or total CAR-expressing cells, ((1) no more than at or about 1.5 x 108 total cells or total CAR—expressing cells and/or (e) between at or about 1 x 107 total cells or total CAR—expressing cells and at or about 1.5 x 108 total cells or total CAR-expressing cells, wherein, prior to the administration, the t has been preconditioned with a lymphodepleting therapy comprising the administration of fludarabine. 3. The method of embodiment 1, wherein, at or prior to the administration of the dose of cells: the subject is or has been identified as having one or more cytogenetic abnormalities, optionally associated with high-risk CLL, optionally ed from among: complex karyotype, deletion of the long arm of chromosome 13 (del 13q), del 11, trisomy 12, del 17p, del 6q, and del 13q. 14, optionally as detected by FISH; the subject is or has been identified as having high-risk CLL; and/or the subject is or has been identified as having extramedullary disease; and/or the subject is or has been fied as having central nervous system (CNS) disease; and/or the subject is an adult and/or is over at or about 30, 40, 50, 60, or 70 years of age. 4. The method of embodiment 1 or embodiment 3, wherein, prior to the stration of the dose of cells, the subject has been treated with two or more, optionally 3, 4, , 6, 7, 8, or 9 or more, therapies for the CLL, other than the lymphodepleting y and/or other than another dose of cells sing the CAR.

. The method of embodiment 1 or embodiment 3, wherein, prior to the administration of the dose of cells, the subject has been treated with two or more, optionally 3, 4, , 6, 7, 8, or 9 or more, therapies for the CLL, other than another dose of cells expressing the CAR or other than another dose of cells sing the CAR and the preconditioning y. 6. The method of any of embodiments 1-5, wherein, prior to the administration of the dose of cells, the t has been treated for the CLL with a kinase inhibitor, optionally an inhibitor of Btk, optionally ibrutinib. 7. The method of any of embodiments 1—6, wherein, prior to the administration of the dose of cells, the subject has been treated for the CLL with a monoclonal antibody that specifically binds to an antigen expressed by, or previously expressed by, cells of the CLL. 8. The method of any of embodiments 1—7, wherein, prior to the administration of the dose of cells, the subject has been treated for the CLL with venetoclax, a combination therapy comprising fludarabine and mab, radiation therapy and/or hematopoietic stem cell transplantation (HSCT). 9. The method of any of embodiments 1-8, wherein, at or immediately prior to the time of the administration of the dose of cells, the subject has relapsed following remission after treatment with, or become refractory to, one or more prior therapies for the CLL.

. The method of any of embodiments 1-9, further comprising, prior to the stration of the cell dose, administering the lymphodepleting therapy to the subject. 11. The method of any of embodiments 1—10, wherein the lymphodepleting therapy: (i) further comprises administering another chemotherapeutic agent other than the bine, which optionally is cyclophosphamide; (ii) is initiated at a time that is at least at or about 48 hours prior to or is n at or about 48 and at or about 96 hours prior to the administration of the cells; and (iii) comprises the administration of cyclophosphamide at about 30-60 mg/kg, optionally once daily for one or two days, and/or the bine at about 25 mg/m2, daily for 3-5 days. 12. The method of any of embodiments 1-11, wherein the administration of the cell dose and/or the lymphodepleting therapy is carried out via outpatient delivery. 13. The method of any of embodiments 1—12, wherein the dose of cells comprises a d ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR and/or of CD4+ cells to CD8+ cells, which optionally is approximately 1:1 or is between approximately 1:3 and imately 3:1. 14. The method of any of ments 1-13, wherein the dose of cells is administered parenterally, optionally intravenously.

. The method of any of embodiments 1—14, wherein: at least 50% of subjects treated according to the method achieve complete remission (CR) and/or objective response (OR); and/or the subject ts CR, OR, or lymph nodes of less than at or about 20 mm in size, within 1 month of the administration of the dose of cells; and/or wherein a malignant immunoglobulin heavy chain (IGH) locus and/or an index clone of the CLL is not detected in the bone marrow of the subject (or in the bone marrow of greater than 50% of subjects treated ing to the methods), optionally as assessed by IGH deep cing, optionally at a time that is at or about or at least at or about 1, 2, 3, 4, 5, 6, 12, 18, or 24 months following the administration of the cell dose. 16. The method of any of ments 1-15, n: at least 50% of subjects treated according to the method achieve complete remission (CR), exhibit progression-free survival (PFS) and/or overall survival (OS) of greater than 12 months; on average, ts treated according to the method exhibit a median PFS or OS of r than at or about 6 months, 12 months, or 18 months; and/or the subject exhibits PFS or OS following y for at least at or about 6, 12, 18 or more months. 17. The method of any of embodiments 1-16, wherein the antigen is a B cell antigen, which optionally is CD19. 18. The method of any of embodiments 1—17, wherein the CAR comprises an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is a 4-1BB, and a cytoplasmic signaling domain derived from a primary signaling ITAM—containing molecule, which optionally is a CD3zeta. 19. The method of any of embodiments 1-18, wherein the CAR comprises a spacer and/or hinge region, each optionally derived from a human IgG.

. A method of treating a subject having a non—Hodgkin lymphoma (NHL), the method comprising stering to the subject a dose of cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen expressed by the NHL, wherein: said dose (i) comprises (a) at or about 2 x 105 of the cells per kilogram body weight of the t /kg); (b) at or about 2 x 106 of the cells/kg, (c) no more than at or about 2 x 106 of the cells/kg, ((1) no more than at or about 2 x 105 of the cells/kg and/or (e) between at or about 2 x 105 of the cells/kg and at or about 2 x 106 of the cells/kg, and (ii) comprises a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR and/or of CD4+ cells to CD8+ cells, which ratio ally is approximately 1:1 or is between approximately 1:3 and approximately 3:1, wherein, prior to the administration, the subject has been ditioned with a lymphodepleting therapy sing the stration of fludarabine. 21. A method of treating a subject having a non—Hodgkin ma (NHL), the method comprising administering to the subject a dose of cells expressing a chimeric antigen receptor (CAR) that ically binds to a target antigen expressed by the NHL, wherein: said dose (i) comprises (a) at or about 1 x 107 total cells or total CAR-expressing cells; (b) at or about 1.5 x 108 total cells or total CAR-expressing cells, (c) no more than at or about 1 x 107 total cells or total CAR-expressing cells, (d) no more than at or about 1.5 x 108 total cells or total CAR-expressing cells and/or (e) between at or about 1 x 107 total cells or total CAR- expressing cells and at or about 1.5 x 108 total cells or total CAR—expressing cells, and (ii) comprises a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR and/or of CD4+ cells to CD8+ cells, which ratio optionally is approximately 1:1 or is between approximately 1:3 and approximately 3: 1, wherein, prior to the stration, the subject has been preconditioned with a lymphodepleting therapy comprising the administration of fludarabine. 22. The method of embodiment 20, wherein, at or prior to the administration of the dose of cells: the subject is or has been identified as having one or more cytogenetic abnormalities, optionally associated with high-risk NHL; the subject is or has been identified as having high-risk NHL; and/or the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), T cell/histocyte— rich large B cell lymphoma (TCHRBCL), Burkitt’s lymphoma, mantle cell lymphoma (MCL), and/or follicular lymphoma (FL); and/or the subject is an adult and/or is over at or about 30, 40, 50, 60, or 70 years of age. 23. The method of embodiment 20 or embodiment 22, wherein, prior to the administration of the dose of cells, the subject has been treated with two or more, ally 2, 3, or 4 or more, therapies for the NHL other than the lymphodepleting y and/or other than another dose of cells expressing the CAR. 24. The method of any of embodiments 20-23, wherein, at or immediately prior to the time of the administration of the dose of cells, the subject has relapsed ing remission after treatment with, or become refractory to, one or more prior therapies for the NHL.

. The method of any of embodiments 20—24, further comprising, prior to the administration of the cell dose, administering the lymphodepleting therapy to the subject. 26. The method of any of embodiments 20—25, wherein the depleting therapy: (i) further comprises administering another herapeutic agent other than the abine, which optionally is cyclophosphamide; (ii) is initiated at a time that is at least at or about 48 hours prior to or is between at or about 48 and at or about 96 hours prior to the administration of the cells; and (iii) comprises the administration of cyclophosphamide at about 30-60 mg/kg, optionally once daily for one or two days, and/or the fludarabine at about 25 mg/m2, daily for 3-5 days. 27. The method of any of embodiments 20—26, wherein the administration of the cell dose and/or the lymphodepleting therapy is carried out via ient delivery. 28. The method of any of embodiments 20—27, wherein the defined ratio is a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR of at or about 1:1 and/or is a defined ratio of CD4+ cells to CD8+ cells, which is at or about 1:1. 29. The method of any of embodiments 20—28, n the dose of cells is administered parenterally, optionally intravenously.

. The method of any of embodiments 20-29, wherein at least 50% of subjects treated according to the method achieve complete remission (CR) and/or objective response (OR). 31. The method of any of embodiments 20-30, wherein: at least 50% of ts that are treated according to the method, and that e complete remission (CR), exhibit progression—free survival (PFS) and/or overall survival (OS) of greater than 12 months; on average, subjects treated according to the method t a median PFS or OS of greater than at or about 6 months, 12 months, or 18 months; and/or the subject exhibits PFS or OS following therapy for at least at or about 6, 12, 18 or more months. 32. The method of any of embodiments 20—3 1, wherein the antigen is a B cell antigen, which optionally is CD19. 33. The method of any of embodiments 20-32, n the CAR comprises an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a ulatory molecule, which optionally is a 4—1BB, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is a CD3zeta. 34. The method of any of embodiments 20—33, wherein the CAR comprises a spacer and/or hinge , each optionally derived from a human IgG.

. The method of any of embodiments 1-34, wherein: the CAR comprises, in order, an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is or comprises a 4-1BB signaling , and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is a CD3zeta signaling domain; the CAR comprises, in order, an scFv specific for the antigen, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is a 4—1BB signaling , and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing le, which optionally is or comprises a CD3zeta signaling domain; and n: the spacer is optionally a polypeptide spacer that (a) comprises or consists of all or a portion of an immunoglobulin hinge or a modified version thereof or comprises about 15 amino acids or less, and does not se a CD28 extracellular region or a CD8 extracellular region, (b) comprises or consists of all or a n of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof and/or comprises about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 extracellular region, or (c) is at or about 12 amino acids in length and/or comprises or consists of all or a portion of an globulin hinge, optionally an IgG4, or a ed version thereof; or (d) has or ts of the sequence of SEQ ID NO: 1, a sequence encoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID ON 34, or a t of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or (6) comprises or consists of the formula X1PPX2P, where X1 is glycine, cysteine or arginine and X2 is cysteine or threonine; and/or the costimulatory domain comprises SEQ ID NO: 12 or a variant f having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; and/or the primary ing domain comprises SEQ ID NO: 13 or 14 or 15 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity o; and/or the scFv comprises a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and/or a CDRL3 sequence of TFG (SEQ ID NO: 37) and/or a CDRH1 ce of DYGVS (SEQ ID NO: 38), a CDRH2 sequence of TTYYNSALKS (SEQ ID NO: 39), and/or a CDRH3 sequence of YAMDYWG (SEQ ID NO: 40) or n the scFv comprises a variable heavy chain region of FMC63 and a variable light chain region of FMC63 and/or a CDRL1 sequence of FMC63, a CDRL2 sequence of FMC63, a CDRL3 sequence of FMC63, a CDRH1 sequence of FMC63, a CDRH2 sequence of FMC63, and a CDRH3 sequence of FMC63 or binds to the same epitope as or competes for binding with any of the foregoing, and optionally wherein the scFv comprises, in order, a VH, a linker, optionally comprising SEQ ID NO: 24, and a VL, and/or the scFv comprises a flexible linker and/or comprises the amino acid sequence set forth as SEQ ID NO: 24. 36. A method of prognosis, the method comprising detecting the presence or e of a malignant immunoglobulin heavy chain (IGH) locus sequence in a sample from a subject having a B cell malignancy, said subject having previously received stration of a cell therapy comprising a dose or composition of genetically engineered cells expressing a recombinant receptor for treating the B cell malignancy, wherein detecting the presence or absence of the malignant IGH sequence determines the prognosis of the subject in response to the cell therapy. 37. The method of embodiment 36, wherein the detecting the presence or e of the malignant IGH sequence is carried out within or within about or about 3 to 6 weeks after initiation of the cell therapy, optionally within or within about 4 weeks of initiation of administration of the cell y. 38. The method of embodiment 36 or embodiment 37, wherein if the malignant IGH sequence is detected, the subject is identified as not responding or not exhibiting a complete response (CR) or an overall response (OR) to the cell therapy or as likely to relapse to the cell therapy. 39. The method of any of ments 36-38, wherein if the malignant IGH sequence is detected identifying the subject as a candidate for further treatment and/or for receiving an altered or alternative treatment. 40. The method of any of embodiments 36—38, wherein if the malignant IGH sequence is detected discontinuing administration of the cell therapy, administering to the t a further dose of the cell y, administering to the subject a higher dose of the cell therapy, administering o the subject a different cell therapy, ally a cell therapy expressing a different recombinant receptor, and/or administering to the subject an alternative therapeutic agent for treating the B cell ancy. 41. The method of embodiment 36 or embodiment 37, wherein if the malignant IGH sequence is not detected, the subject is identified as responding to the cell therapy and/or as exhibiting a complete response (CR) or overall response (OR) to the cell y or as likely not to relapse to the cell y. 42. The method of any of embodiments 36, 37 and 41 wherein if the ant IGH sequence is not detected, the subject is identified as a candidate for no further treatment and/or is not further treated, optionally is not further treated with the cell therapy and/or is not further treated with an alternative therapy for the B cell malignancy. 43. A method of predicting durability of response to a cell therapy, the method comprising detecting the presence or absence of a malignant immunoglobulin heavy chain locus (IGH) sequence in a sample from a subject having a B cell malignancy, said subject having previously received administration of a cell therapy comprising a dose or composition of genetically engineered cells expressing a recombinant receptor for treating the B cell ancy, wherein the presence or absence of the malignant IGH sequence predicts the durability of response to the cell therapy. 44. The method of embodiment 43, wherein the detecting the presence or absence of the malignant IGH sequence is carried out within or within about or about 4 weeks, 6 weeks, 8 weeks, 12 weeks or 16 weeks after tion of the cell therapy. 45. The method of ment 43 or embodiment 44, wherein if the ant IGH ce is not ed, the subject is predicted to exhibit or likely to exhibit a durable response to the cell y and/or to be at a low or relatively low risk of relapse within a certain period of time and/or to have a high likelihood of exhibiting progression free survival for at least a certain period of time. 46. The method of any of embodiments 43-45, wherein if the malignant IGH sequence is not detected, the subject is predicted: to exhibit survival without progression for greater than or about 3 months, greater than about 6 months, greater than about 9 months or greater than about 12 months after initiation of the cell therapy; and/or to remain surviving for greater than or greater than about 3 months, greater than or greater than about 6 months, greater than or greater than about about 9 months or greater than about 12 months after initiation of the cell therapy; and/or to exhibit durable CR or OR for r than or greater than about 3 months, greater than or greater than about 6 months or greater than or greater than about 9 months after initiation of the cell therapy; and/or not likely to relapse following tion of administration of the cell therapy, ally not likely to e within 3 months, 6 months or 9 months after initiation of administration of the cell therapy. 47. The method of embodiment 43 or embodiment 44, wherein if the malignant IGH sequence is detected, the subject is predicted to exhibit or likely to exhibit a se to the cell therapy that is not durable and/or to be at a high or vely high risk of relapse within a certain period of time and/or to have a low likelihood of ting progression free survival for at least a certain period of time. 48. The method of embodiment 43 or embodiment 44, wherein if the malignant IGH sequence is not detected, the subject is s predicted: not to exhibit al without progression for greater than or about 3 months, r than about 6 months, greater than about 9 months or greater than about 12 months after initiation of the cell therapy; and/or not to remain surviving for greater than or greater than about 3 months, greater than or r than about 6 months, greater than or greater than about about 9 months or greater than about 12 months after initiation of the cell therapy; and/or not to exhibit durable CR or OR for greater than or r than about 3 months, r than or greater than about 6 months or greater than or greater than about 9 months after initiation of the cell therapy. 49. The method of embodiment 43, 44 and 48, wherein if the malignant IGH sequence is detected administering to the subject a further dose of the cell therapy, administering to the subject a higher dose of the cell therapy, administering o the subject a different cell therapy, optionally a cell therapy expressing a different recombinant or, and/or administering to the subject an alternative therapeutic agent for treating the B cell malignancy. 50. The method of any of embodiments 36—49, wherein the presence or e of the malignant IGH sequence is determined by IGH sequencing, optionally comprising PCR amplification of IGH target DNA. 51. The method of any of embodiments 36-38, wherein the sample comprises B cells. 52. The method of any of embodiments 36—5 1, wherein the sample comprises a blood or bone marrow sample. 53. The method of any of embodiments 36-52, wherein the sample has been obtained from the subject. 54. The method of any of ments 36-53, n the method is carried out ex Vivo. 55. The method of any of embodiments 36—54, wherein the B cell malignancy is a cancer. 56. The method of any of embodiments 36—55, wherein the B cell ancy is or comprises a leukemia. 57. The method of any of embodiment 37-56, wherein the B cell malignancy comprises an antigen or is associated with an n selected from CD19, CD20, CD22, CD30, CD33 or CD38, RORl. 58. The method of any of embodiments 36-57, wherein the B cell ancy is selected from and/or is acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non—Hodgkin lymphoma (NHL), and Diffuse Large B—Cell Lymphoma (DLBCL). 59. The method of any of embodiments 37—5 8, n the B cell malignancy is or comprises chronic lymphoblastic leukemia (CLL) or high—risk CLL. 60. The method of any of embodiments 37-58, wherein the B cell malignancy is or comprises non—Hodgkin lymphoma (NHL). 61. The method of ment 60, wherein the the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS (de novo and transformed from indolent), y mediastinal large B cell lymphoma (PMBCL), T cell/histocyte-rich large B cell lymphoma (TCHRBCL), Burkitt’s lymphoma, mantle cell lymphoma (MCL), and/or follicular lymphoma (FL), optionally, follicular lymphoma Grade 3B (FL3B). 62. The method of any of ments 37—61, wherein the recombinant receptor specifically binds to an antigen associated with the disease or condition or expressed in cells of the environment of a lesion ated with the B cell malignancy. 63. The method of any of embodiments 37-62, wherein the recombinant receptor is a T cell receptor or a functional non—T cell receptor. 64. The method of any of embodiments 37-63, wherein the recombinant receptor is a ic antigen receptor (CAR). 65. The method of embodiment 64, wherein the CAR comprises an extracellular antigen-recognition domain that specifically binds to the antigen and an intracellular signaling domain comprising an ITAM, wherein optionally, the intracellular signaling domain comprises an ellular domain of a CD3—zeta (CD3 8;) chain; and/or wherein the CAR r comprises a costimulatory signaling , which optionally comprises a signaling domain of CD28 or 4— 66. The method of any of embodiments 37—65, wherein the CAR comprises an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is a 4-1BB, and a cytoplasmic signaling domain derived from a primary signaling ITAM—containing molecule, which optionally is a CD3zeta. 67. The method of any of embodiments 37-66, wherein the CAR comprises a spacer and/or hinge region, each optionally derived from a human IgG. 68. The method of any of embodiments 37—67, n: the CAR comprises, in order, an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is or comprises a 4—lBB signaling domain, and a cytoplasmic ing domain derived from a primary signaling ITAM-containing le, which optionally is a CD3zeta signaling domain; the CAR comprises, in order, an scFv ic for the antigen, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which ally is a 4-lBB signaling domain, and a cytoplasmic signaling domain derived from a primary ing ITAM-containing molecule, which optionally is or comprises a a signaling domain; and wherein: the spacer is optionally a polypeptide spacer that (a) comprises or consists of all or a portion of an immunoglobulin hinge or a modified version thereof or comprises about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 extracellular region, (b) comprises or ts of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified n thereof and/or comprises about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 extracellular region, or (c) is at or about 12 amino acids in length and/or comprises or consists of all or a portion of an immunoglobulin hinge, ally an IgG4, or a modified version thereof; or (d) has or consists of the ce of SEQ ID NO: 1, a ce encoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID ON 34, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more ce identity thereto, or (e) comprises or consists of the formula X1PPX2P, where X1 is glycine, cysteine or arginine and X2 is cysteine or threonine; and/or the ulatory domain comprises SEQ ID NO: 12 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity o; and/or the primary signaling domain comprises SEQ ID NO: 13 or 14 or 15 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; and/or the scFv comprises a CDRLl sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and/or a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37) and/or a CDRHl sequence of DYGVS (SEQ ID NO: 38), a CDRH2 ce of VIWGSETTYYNSALKS (SEQ ID NO: 39), and/or a CDRH3 sequence of YAMDYWG (SEQ ID NO: 40) or wherein the scFv comprises a variable heavy chain region of FMC63 and a le light chain region of FMC63 and/or a CDRLl sequence of FMC63, a CDRL2 sequence of FMC63, a CDRL3 sequence of FMC63, a CDRHl sequence of FMC63, a CDRH2 sequence of FMC63, and a CDRH3 sequence of FMC63 or binds to the same epitope as or competes for binding with any of the foregoing, and optionally wherein the scFv comprises, in order, a VH, a linker, optionally comprising SEQ ID NO: 24, and a VL, and/or the scFv comprises a flexible linker and/or comprises the amino acid sequence set forth as SEQ ID NO: 24. 69. The method of any of embodiments 37—68, wherein the engineered cells comprise T cells, optionally CD4+ and/or CD8+. 70. The method of embodiment 69, wherein the T cells are primary T cells obtained from a subject. 71. The method of any of any of ments 37-70, wherein the engineered cells are autologous to the subject. 72. The method of any of ments 37—71, wherein the engineered cells are allogeneic to the subject. 73. An article of manufacture comprising one or more dose of a cell therapy, each dose comprising cells expressing a chimeric antigen receptor (CAR), and instructions for administering the cell therapy, wherein: the instructions specify the dose of cells is to be stered to a subject having a chronic lymphocytic leukemia (CLL); and the ctions specify administration of a number of CAR—expressing or a number of cells, or specify administration of an amount or volume of one or more formulations corresponding to or ning said specified number of cells, wherein the specified number of cells to be stered comprises a number to administer a dose of cells comprising (a) at or about 2 x 105 of the cells per kilogram body weight of the subject (cells/kg); (b) at or about 2 x 106 of the cells/kg, (c) no more than at or about 2 x 106 of the cells/kg, ((1) no more than at or about 2 x 105 of the cells/kg and/or (e) between at or about 2 x 105 of the cells/kg and at or about 2 x 106 of the cells/kg. 74. An article of manufacture comprising one or more dose of a cell therapy, each dose comprising cells expressing a chimeric antigen receptor (CAR), and instructions for administering the cell therapy, wherein: the instructions specify the dose of cells is to be administered to a subject having a chronic cytic leukemia (CLL); and the instructions specify administration of a number of pressing or a number of cells, or specify administration of an amount or volume of one or more ations corresponding to or containing said specified number of cells, wherein the specified number of cells to be administered comprises a number to administer a dose of cells comprising (a) at or about 1 x 107 total cells or total CAR—expressing cells; (b) at or about 1.5 x 108 total cells or total CAR-expressing cells, (c) no more than at or about 1 x 107 total cells or total CAR-expressing cells, ((1) no more than at or about 1.5 x 108 total cells or total CAR—expressing cells and/or (e) between at or about 1 x 107 total cells or total CAR-expressing cells and at or about 1.5 x 108 total cells or total CAR—expressing cells. 75. The e of manufacture of embodiment 73, further sing instructions for use with, after or in connection with a depleting therapy, the lympodepleting therapy comprising fludarabine. 76. The article of cture of embodiment 73 or embodiment 75, n the instructions specify that the cell therapy is to be administer to a subject that: is or has been identified as having one or more cytogenetic abnormalities, optionally associated with high—risk CLL, optionally selected from among: complex karyotype, deletion of the long arm of chromosome 13 (del 13q), del 11, y 12, del 17p, del 6q, and del 13q.14, optionally as detected by FISH; is or has been identified as having high—risk CLL; and/or is or has been identified as having extramedullary disease; and/or is or has been fied as having central nervous system (CNS) disease; and/or is an adult and/or is over at or about 30, 40, 50, 60, or 70 years of age. 77. The article of manufacture of any of embodiments 73-76, wherein the instructions y that the cell therapy is to be administered to a subject that: has been treated with two or more, optionally 3, 4, 5, 6, 7, 8, or 9 or more, therapies for the CLL, other than the lymphodepleting therapy and/or other than another dose of cells expressing the CAR; and/or has been treated for the CLL with a kinase tor, optionally an inhibitor of Btk, optionally ibrutinib; and/or has been treated for the CLL with a monoclonal antibody that specifically binds to an antigen expressed by, or previously expressed by, cells of the CLL; and/or has been treated for the CLL with venetoclax, a combination therapy comprising fludarabine and rituximab, radiation therapy and/or hematopoietic stem cell transplantation (HSCT). 78. The article of manufacture of any of embodiments 73-77, wherein the instructions specify that the cell therapy is to be administered to a subject that has relapsed W0 14207 following remission after treatment with, or become refractory to, one or more prior therapies for the CLL. 79. An article of manufacture comprising one or more dose of a cell therapy, each dose comprising cells expressing a chimeric antigen receptor (CAR), and instructions for administering the cell therapy, wherein: the instructions specify the dose of cells is to be administered to a subject having a a non—Hodgkin lymphoma (NHL); and the instructions specify administration of a number of CAR-expressing or a number of cells, or specify administration of an amount or volume of one or more formulations corresponding to or containing said specified number of cells, n the specified number of cells to be administered comprises a number to administer a dose of cells comprising (a) at or about 2 x 105 of the cells per kilogram body weight of the subject (cells/kg); (b) at or about 2 x 106 of the cells/kg, (c) no more than at or about 2 x 106 of the cells/kg, ((1) no more than at or about 2 x 105 of the cells/kg and/or (e) between at or about 2 x 105 of the cells/kg and at or about 2 x 106 of the cells/kg. 80. An article of manufacture comprising one or more dose of a cell therapy, each dose comprising cells expressing a chimeric antigen receptor (CAR), and ctions for administering the cell therapy, wherein: the instructions specify the dose of cells is to be administered to a subject having a a non-Hodgkin ma (NHL); and the instructions specify administration of a number of CAR-expressing or a number of cells, or specify administration of an amount or volume of one or more formulations corresponding to or containing said specified number of cells, wherein the ied number of cells to be administered ses a number to administer a dose of cells sing (a) at or about 1 x 107 total cells or total CAR—expressing cells; (b) at or about 1.5 x 108 total cells or total CAR-expressing cells, (c) no more than at or about 1 x 107 total cells or total CAR-expressing cells, (d) no more than at or about 1.5 x 108 total cells or total CAR—expressing cells and/or (e) between at or about 1 x 107 total cells or total pressing cells and at or about 1.5 x 108 total cells or total CAR-expressing cells. 81. The article of manufacture of embodiment 79, further comprising instructions for use with, after or in connection with a lymphodepleting therapy, the lympodepleting y comprising fludarabine. 82. The article of manufacture of embodiment 79 or embodiment 81, wherein the instructions specify that the cell therapy is to be ster to a t that: is or has been identified as having one or more cytogenetic abnormalities, optionally associated with high-risk NHL; is or has been fied as having high-risk NHL; and/or is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), T cell/histocyte—rich large B cell lymphoma (TCHRBCL), Burkitt’s lymphoma, mantle cell lymphoma (MCL), and/or follicular ma (FL); and/or is an adult and/or is over at or about 30, 40, 50, 60, or 70 years of age. 83. The article of manufacture of any of embodiments 79-82, wherein the instructions specify that the cell therapy is to be administered to a subject that has been treated with two or more, optionally 2, 3 or 4 or more, therapies for the NHL, other than the lymphodepleting therapy and/or other than another dose of cells expressing the CAR. 84. The article of manufacture of any of embodiments 79—83, wherein the instructions specify that the cell therapy is to be administered to a subject that has relapsed following remission after treatment with, or become refractory to, one or more prior therapies for the NHL. 85. The article of manufacture of any of embodiments 73—84, n the lymphodepleting y: (i) further comprises administering another chemotherapeutic agent other than the fludarabine, which optionally is cyclophosphamide; and/or (ii) comprises the administration of cyclophosphamide at about 30-60 mg/kg, optionally once daily for one or two days, and/or the fludarabine at about 25 mg/m2, daily for 3— days. 86. The article of manufacture of any of embodiments 73-84, wherein the instructions specify that the lympodepleting therapy is initiated at a time that is at least at or about 48 hours prior to or is between at or about 48 and at or about 96 hours prior to the administration of the cell therapy. 87. The e of manufacture of any of embodiments 73-86, wherein the instructions y administering the cell therapy at a defined ratio of CD4+ cells expressing the CAR to CD8+ cells, or specify stering amounts of volumes of the formulation(s) corresponding to such defined ratio, or comprises a formulation having the cells at such ratio or comprises the cells at such ratio expressing the CAR and/or of CD4+ cells to CD8+ cells, which ratio optionally is approximately 1:1 or is between approximately 1:3 and approximately 3:1. 88. The article of manufacture of any of embodiments 73—87, wherein the instructions further specify the cell therapy is for parenteral administration, optionally intravenous administration. 89. The article of manufacture of any of embodiments 73—88, wherein the instructions further specify the administration of the cell therapy is to be or may be administered to the subject on an ient setting and/or without ion of the subject to the hospital overnight or for one or more consecutive days and/or is without admission of the subject to the hospital for one or more days. 90. The article of manufacture of any of embodiments 73—89, wherein the cell therapy comprises primary T cells obtained from a subject. 91. The article of manufacture of embodiment 89, n the T cells are autologous to the subject. 92. The article of manufacture of embodiment 91, n the T cells are allogeneic to the subject. 93. The article of manufacture of any of embodiments 73-92, wherein the CAR comprises an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a ulatory molecule, which optionally is a 4-1BB, and a asmic signaling domain derived from a primary ing ITAM-containing molecule, which optionally is a CD3zeta. 94. The article of manufacture of any of embodiments 73-93, wherein the CAR comprises a spacer and/or hinge region, each optionally derived from a human IgG. 95. The article of manufacture of embodiment 93 or ment 94, wherein the antigen is a B cell antigen, which optionally is CD19. 96. The e of manufacture of any of ments 73—95, wherein: the CAR ses, in order, an scFv specific for the antigen, a transmembrane , a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is or comprises a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is a CD3zeta signaling domain; the CAR comprises, in order, an scFv specific for the n, a spacer, a transmembrane domain, a cytoplasmic signaling domain d from a costimulatory molecule, which optionally is a 4—1BB signaling domain, and a cytoplasmic ing domain derived from a primary signaling ITAM-containing molecule, which optionally is or comprises a CD3zeta signaling domain; and wherein: the spacer is optionally a polypeptide spacer that (a) ses or consists of all or a portion of an immunoglobulin hinge or a modified version thereof or comprises about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 ellular region, (b) comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified n thereof and/or comprises about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 extracellular region, or (c) is at or about 12 amino acids in length and/or comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4, or a modified version thereof; or (d) has or consists of the sequence of SEQ ID NO: 1, a sequence encoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID O:N 34, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or (e) comprises or consists of the formula X1PPX2P, where X1 is glycine, cysteine or arginine and X2 is cysteine or ine; and/or the costimulatory domain comprises SEQ ID NO: 12 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; and/or the primary signaling domain comprises SEQ ID NO: 13 or 14 or 15 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; and/or the scFv comprises a CDRLl sequence of SKYLN (SEQ ID NO: 35), a CDRL2 sequence of V (SEQ ID NO: 36), and/or a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37) and/or a CDRHl sequence of DYGVS (SEQ ID NO: 38), a CDRH2 ce of TTYYNSALKS (SEQ ID NO: 39), and/or a CDRH3 sequence of YAMDYWG (SEQ ID NO: 40) or wherein the scFv comprises a variable heavy chain region of FMC63 and a variable light chain region of FMC63 and/or a CDRLl sequence of FMC63, a CDRL2 sequence of FMC63, a CDRL3 sequence of FMC63, a CDRHl sequence of FMC63, a CDRH2 sequence of FMC63, and a CDRH3 sequence of FMC63 or binds to the same epitope as or competes for binding with any of the foregoing, and optionally wherein the scFv ses, in order, a VH, a linker, optionally comprising SEQ ID NO: 24, and a VL, and/or the scFv comprises a flexible linker and/or comprises the amino acid sequence set forth as SEQ ID NO: 24. 97. A composition comprising cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen of a chronic cytic leukemia (CLL) for use in treating a subject having or ted of having CLL, wherein the treating comprises administering to the subject a dose of cells expressing the CAR, said dose comprising (a) at or about 2 x 105 of the cells per kilogram body weight of the subject (cells/kg); (b) at or about 2 x 106 of the cells/kg, (c) no more than at or about 2 x 106 of the kg, (d) no more than at or about 2 x 105 of the cells/kg and/or (e) between at or about 2 x 105 of the cells/kg and at or about 2 x 106 of the cells/kg, wherein, prior to the administration, the subject has been preconditioned with a lymphodepleting therapy comprising the stration of fludarabine. 98. A composition comprising cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen of a chronic lymphocytic leukemia (CLL) for use in treating a subject having or suspected of having CLL, wherein the treating comprises administering to the subject a dose of cells expressing the CAR, said dose comprising (a) at or about 1 x 107 total cells or total CAR—expressing cells; (b) at or about 1.5 x 108 total cells or total pressing cells, (c) no more than at or about 1 x 107 total cells or total CAR-expressing cells, (d) no more than at or about 1.5 x 108 total cells or total CAR-expressing cells and/or (e) between at or about 1 x 107 total cells or total CAR—expressing cells and at or about 1.5 x 108 total cells or total CAR-expressing cells, n, prior to the administration, the subject has been preconditioned with a lymphodepleting therapy comprising the stration of fludarabine. 99. The use of embodiment 97 or embodiment 98, wherein the composition is for use in ng a subject in which, at or prior to the administration of the dose of cells: the subject is or has been fied as having one or more cytogenetic abnormalities, optionally ated with high-risk CLL, optionally selected from among: complex karyotype, deletion of the long arm of chromosome 13 (del 13q), del 11, trisomy 12, del 17p, del 6q, and del 13q. 14, optionally as detected by FISH; the subject is or has been identified as having high-risk CLL; and/or the subject is or has been identified as having extramedullary disease; and/or the t is or has been identified as having central nervous system (CNS) disease; and/or the t is an adult and/or is over at or about 30, 40, 50, 60, or 70 years of age. 100. The use of any of embodiments 97-99, wherein the composition is for use in treating a subject in which, prior to the administration of the dose of cells, the subject has been treated with two or more, optionally 3, 4, 5, 6, 7, 8, or 9 or more, therapies for the CLL, other than the lymphodepleting therapy and/or other than another dose of cells expressing the CAR. 101. The use of any of embodiments 97-100, wherein the composition is for use in treating a subject in which, prior to the administration of the dose of cells, the subject has been treated with two or more, optionally 3, 4, 5, 6, 7, 8, or 9 or more, therapies for the CLL, other than another dose of cells expressing the CAR or other than another dose of cells expressing the CAR and the preconditioning therapy. 102. The use of any of embodiments 97-101, wherein the composition is for use in treating a subject in which, prior to the administration of the dose of cells, the subject has been treated for the CLL with a kinase inhibitor, optionally an inhibitor of Btk, optionally nib. 103. The use of any of embodiments 97-102, wherein the composition is for use in treating a subject in which, prior to the administration of the dose of cells, the subject has been treated for the CLL with a monoclonal antibody that specifically binds to an antigen expressed by, or previously expressed by, cells of the CLL. 104. The use of any of embodiments 97-102, n the composition is for use in treating a subject in which, prior to the administration of the dose of cells, the subject has been treated for the CLL with venetoclax, a combination y comprising bine and rituximab, radiation therapy and/or hematopoietic stem cell transplantation (HSCT). 105. The use of any of embodiments 97—102, n the composition is for use in ng a subject in which, at or immediately prior to the time of the administration of the dose of cells, the t has relapsed following remission after treatment with, or become refractory to, one or more prior ies for the CLL. 106. A composition comprising cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen of a non-Hodgkin ma (NHL) for use in treating a subject having or ted of having NHL, wherein the treating comprises administering to the t a dose of cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen expressed by the NHL, wherein the ng comprises administering to the subject a dose of cells sing the CAR, said dose (i) comprises (a) at or about 2 x 105 of the cells per kilogram body weight of the subject /kg); (b) at or about 2 x 106 of the cells/kg, (c) no more than at or about 2 x 106 of the cells/kg, (d) no more than at or about 2 x 105 of the cells/kg and/or (e) between at or about 2 x 105 of the kg and at or about 2 x 106 of the cells/kg, and (ii) comprises a d ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR and/or of CD4+ cells to CD8Jr cells, which ratio optionally is approximately 1:1 or is between approximately 1:3 and approximately 3: 1, wherein, prior to the administration, the subject has been preconditioned with a lymphodepleting y comprising the stration of fludarabine. 107. A composition comprising cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen of a non—Hodgkin lymphoma (NHL) for use in treating a subject having or suspected of having NHL, wherein the treating comprises administering to the subject a dose of cells expressing the CAR, said dose (i) comprises (a) at or about 1 x 107 total cells or total CAR—expressing cells; (b) at or about 1.5 x 108 total cells or total CAR— expressing cells, (c) no more than at or about 1 x 107 total cells or total pressing cells, (d) no more than at or about 1.5 x 108 total cells or total CAR-expressing cells and/or (e) between at or about 1 x 107 total cells or total CAR-expressing cells and at or about 1.5 x 108 total cells or total CAR—expressing cells, and (ii) comprises a d ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR and/or of CD4+ cells to CD8+ cells, which ratio optionally is approximately 1:1 or is between approximately 1:3 and approximately wherein, prior to the administration, the subject has been ditioned with a lymphodepleting therapy comprising the administration of fludarabine. 108. The use of embodiment 106 or embodiment 107, wherein the composition is for use in treating a subject in which, at or prior to the administration of the dose of cells: the subject is or has been identified as having one or more cytogenetic abnormalities, optionally associated with high-risk NHL; the subject is or has been identified as having high-risk NHL; and/or the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell ma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), T cell/histocyte— W0 14207 rich large B cell lymphoma (TCHRBCL), Burkitt’s lymphoma, mantle cell lymphoma (MCL), and/or follicular lymphoma (FL); and/or the subject is an adult and/or is over at or about 30, 40, 50, 60, or 70 years of age. 109. The use of any of embodiments 106—108, wherein the composition is for use in treating a subject in which, prior to the administration of the dose of cells, the subject has been treated with two or more, optionally 2, 3, or 4 or more, therapies for the NHL other than the lymphodepleting therapy and/or other than another dose of cells expressing the CAR. 110. The use of any of embodiments 106-109, wherein the composition is for use in treating a subject in which, at or immediately prior to the time of the stration of the dose of cells, the subject has ed following remission after treatment with, or become refractory to, one or more prior therapies for the NHL. 111. The use of any of embodiments 97—110, wherein the lymphodepleting y: (i) further comprises administration of another chemotherapeutic agent other than the fludarabine, which optionally is cyclophosphamide; (ii) is initiated at a time that is at least at or about 48 hours prior to or is between at or about 48 and at or about 96 hours prior to the administration of the cells; and (iii) ses the administration of cyclophosphamide at about 30-60 mg/kg, optionally once daily for one or two days, and/or the fludarabine at about 25 mg/m2, daily for 3-5 days. 112. The use of any of embodiments 97—101, n the treating comprises administration of the cell dose and/or the lymphodepleting therapy via outpatient delivery. 113. The use of any of embodiments 97-112, n the composition and/or the dose of cells comprises a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR and/or of CD4+ cells to CD8+ cells, which optionally is approximately 1:1 or is between approximately 1:3 and approximately 3:1. 114. The use of any of embodiments 97—113, wherein the composition and/or dose of cells is formulated for parenteral administration, optionally intravenous stration. 115. The use of any of embodiments 97—114, wherein the antigen is a B cell antigen, which optionally is CD19. 116. The use of any of embodiments , wherein the CAR ses an scFv specific for the antigen, a transmembrane domain, a cytoplasmic ing domain derived from a costimulatory molecule, which optionally is a 4-1BB, and a cytoplasmic signaling domain derived from a y signaling ITAM-containing molecule, which optionally is a CD3zeta. 117. The use of any of embodiments 97—116, wherein the CAR comprises a spacer and/or hinge region, each optionally derived from a human IgG. 118. The method of any of embodiments 97—117, wherein: the CAR comprises, in order, an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a ulatory molecule, which optionally is or comprises a 4-1BB ing domain, and a cytoplasmic signaling domain derived from a primary ing ITAM-containing molecule, which ally is a CD3zeta signaling domain; the CAR comprises, in order, an scFv specific for the antigen, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is a 4-1BB signaling domain, and a cytoplasmic ing domain derived from a primary signaling ontaining molecule, which optionally is or ses a CD3zeta signaling domain; and wherein: the spacer is optionally a polypeptide spacer that (a) comprises or consists of all or a portion of an immunoglobulin hinge or a modified version thereof or ses about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 extracellular region, (b) comprises or consists of all or a portion of an globulin hinge, optionally an IgG4 hinge, or a modified version thereof and/or comprises about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 extracellular region, or (c) is at or about 12 amino acids in length and/or comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4, or a modified version thereof; or (d) has or consists of the sequence of SEQ ID NO: 1, a sequence encoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID O:N 34, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or (e) comprises or consists of the formula X1PPX2P, where X1 is glycine, ne or arginine and X2 is cysteine or threonine; and/or the costimulatory domain comprises SEQ ID NO: 12 or a t thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence ty thereto; and/or the primary signaling domain comprises SEQ ID NO: 13 or 14 or 15 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; and/or the scFv comprises a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and/or a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37) and/or a CDRH1 sequence of DYGVS (SEQ ID NO: 38), a CDRH2 sequence of TTYYNSALKS (SEQ ID NO: 39), and/or a CDRH3 sequence of YAMDYWG (SEQ ID NO: 40) or wherein the scFv comprises a variable heavy chain region of FMC63 and a variable light chain region of FMC63 and/or a CDRL1 sequence of FMC63, a CDRL2 sequence of FMC63, a CDRL3 sequence of FMC63, a CDRH1 sequence of FMC63, a CDRH2 sequence of FMC63, and a CDRH3 sequence of FMC63 or binds to the same e as or competes for binding with any of the foregoing, and optionally wherein the scFv comprises, in order, a VH, a linker, optionally comprising SEQ ID NO: 24, and a VL, and/or the scFv comprises a flexible linker and/or comprises the amino acid sequence set forth as SEQ ID NO: 24.

VII. DEFINITIONS The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum . Polypeptides, including the provided receptors and other polypeptides, e.g., linkers or peptides, may e amino acid es including l and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, the ptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as h mutations of hosts which produce the ns or errors due to PCR amplification.

As used herein, a “subject” is a mammal, such as a human or other , and typically is human. In some embodiments, the t, e.g., patient, to whom the agent or agents, cells, cell populations, or compositions are administered, is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non—primate mammal, such as a rodent.

As used herein, “treatment” (and grammatical ions thereof such as “treat” or “treating”) refers to te or partial amelioration or reduction of a disease or condition or er, or a symptom, adverse effect or outcome, or phenotype associated ith. ble effects of treatment include, but are not d to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.

As used , “delaying development of a disease" means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.

“Preventing,” as used herein, includes providing laxis with respect to the occurrence or recurrence of a e in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. In some ments, the provided cells and compositions are used to delay development of a disease or to slow the progression of a disease.

As used herein, to “suppress” a on or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or ter of interest, or alternatively, as compared to another condition. For example, cells that suppress tumor growth reduce the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the cells.

An “effective amount” of an agent, e.g., a pharmaceutical formulation, cells, or composition, in the context of administration, refers to an amount ive, at dosages/amounts and for periods of time necessary, to e a desired result, such as a therapeutic or prophylactic result.

A “therapeutically effective amount” of an agent, e.g., a ceutical formulation or cells, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired eutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered. In some embodiments, the provided methods e administering the cells and/or compositions at effective amounts, e.g., therapeutically ive amounts.

A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to e the d prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. In the context of lower tumor burden, the prophylactically effective amount in some aspects will be higher than the eutically effective .

The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

As used , the singular forms 4; 39 “ a an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” Throughout this disclosure, various aspects of the claimed t matter are presented in a range format. It should be understood that the ption in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub—ranges as well as individual cal values within that range. For example, where a range of values is provided, it is understood that each ening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject . The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

As used , a composition refers to any mixture of two or more ts, nces, or compounds, including cells. It may be a solution, a suspension, , powder, a paste, aqueous, non—aqueous or any combination thereof.

As used herein, “enriching” when referring to one or more ular cell type or cell population, refers to increasing the number or percentage of the cell type or population, e.g., compared to the total number of cells in or volume of the composition, or relative to other cell types, such as by positive selection based on markers expressed by the population or cell, or by negative selection based on a marker not present on the cell population or cell to be depleted.

The term does not require complete removal of other cells, cell type, or populations from the composition and does not require that the cells so enriched be present at or even near 100% in the enriched composition.

As used herein, a statement that a cell or tion of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface , the term refers to the presence of e expression as detected by flow cytometry, for example, by staining with an dy that ically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level ntially above the staining detected carrying out the same procedure with an isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.

As used herein, a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level ntially above the staining detected carrying out the same procedure with an isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical ions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level ntially similar as compared to that for a cell known to be negative for the marker.

The term r,” as used herein, refers to a c acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of c acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

All publications, ing patent documents, scientific articles and databases, referred to in this application are orated by reference in their ty for all purposes to the same extent as if each individual publication were dually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the tion that is incorporated herein by reference.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

VIII. EXAMPLES The following es are included for illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLE 1 Treatment of Subjects with Relapsed 0r tory 1R/R! CD19+ c Lymphocytic Leukemia [CLLQ with Chimeric Antigen-Receptor [CAR] Specific for CD19 Autologous T cells expressing a chimeric n-receptor (CAR) specific for CD19 were administered to thirteen (13) adult human subjects with relapsed or refractory (R/R) CD19+ chronic lymphocytic leukemia (CLL). The ts ranged in age from forty (40) to seventy- three (73), with an average age of sixty—one (61). No subjects were excluded based on lymphopenia, circulating tumor, prior transplant, or test expansion. The group of subjects exhibited high-risk cytogenetics (10/13 (77%) dell7p', 8/13 (62%) complex karyotype); 12/13 (92%) exhibited extramedullary disease. All subjects had been usly treated with one or more other therapies for CLL (with a median number of five (5), and a range of three (3) to nine (9), prior lines of treatment), including, in each case, ibrutinib (with seven (7) (54%) having being refractory and two (2) (15%) having been intolerant). Just prior to treatment, the median tage of abnormal B cells in the bone marrow among all subjects was 66% (with a range of 0.4% to 90%).

The CAR included an scFv (in a ker-VH orientation) specific for CD19, with variable regions derived from FMC63, an IgG hinge region, a transmembrane , and intracellular signaling domains derived from human 41BB and CD3zeta. The construct further d a truncated EGFR (EGFRt), which served as a ate marker for CAR expression; the EGFRt—coding region was separated from the CAR ce by a T2A skip sequence. Prior to administration of the cells, patients underwent leukapheresis; CD4+ and CD8+ populations were selected by immunoaffinity—based enrichment methods, transduced with a viral vector with the CAR construct, and expanded in e over fifteen (15) days. For the manufacture of CAR+ T cells, CD8+ central memory T cell populations were engineered, except bulk CD8+ T cells were engineered in in patients with severe lymphopenia resulting in a low CD8+ central memory T cells. N0 difference in clinical outcome was observed between patients who received CAR+ T cells manufactured from CD4+ T cells and either bulk CD8+ T cells or CD8+ central memory T cells.

Beginning at least forty—eight (48) (and up to ninety—six (96)) hours prior to CAR+ T cell infusion, subjects received a lymphodepleting chemotherapy with either (a) hosphamide (Cy, 60 mg/kg) with or without etoposide (2/13 subjects), or (b) hosphamide (Cy, 60 mg/kg) in combination with bine (flu, 25 mg/m2 daily for 3—5 days (cy/flu, 11/13 subjects).

Cells for administration generally were formulated at a CAR+ CD4+ T cell to CAR+ CD8+ T cell ratio of approximately l:l. Therapeutic compositions were successfully produced for all subjects. For l/l3 subjects, fewer than the target dose (2x106/kg CAR+) of cells were produced.

Subjects were infused with a composition having approximately a 1:1 ratio of CD8+ CAR+ T cells to CD4+ CAR-T cells, at one of three different dose levels (2x105 (N24) 2x106 (N28) or 2x107 (N21) CAR+ T cells per kilogram (kg) weight of the subject). Lymphodepleting therapy and T cell infusions were administered on an ient basis. After one CLL patient developed grade 4 CRS and grade 3 neurotoxicity after ing 2x107 CAR+ T cells/kg, a maximal dose of 2x106 CAR+ T cells/kg was selected for subsequent CLL patients.

Patients underwent whole—body imaging with a stic quality CT scan before and 4 weeks after administration of the CAR+ T cell composition. Responses were determined by International Workshop on c Lymphocytic Leukemia (IWCLL) response criteria (Hallek, et al., Blood 2008, Jun 15; ): 5446—5456; IWCLL (2008)). Nodal tumor bulk was ed as the sum of the cross-sectional areas of the 6 largest index lymph nodes identified on a diagnostic quality CT scan. In some cases, whole body PEG imaging by Lugano criteria (Cheson et al., JCO September 20, 2014 vol. 32 no. 27 3059—3067) also was performed.

Marrow response was assessed by bone marrow aspirate and biopsy obtained 4 weeks after CAR—T cell infusion in those with bone marrow disease prior to depletion and 4 weeks after stration of the CAR+ T cell composition. Morphology analysis and high-resolution flow cytometry were performed on the marrow, with conventional karyotyping and FISH in patients with an identified cytogenetic ality. IGH deep sequencing (Adaptive Biotechnologies) was performed on marrow from patients who had no detectable marrow disease by flow cytometry 4 weeks after CAR-T cell infusion and had an identified malignant clonal sequence before lymphodepletion. esolution flow cytometry was performed on blood 2 weeks, and l, 2, 3, 6 and 12 months after CAR—T cell infusion.

Toxicity was graded using the National Cancer Institute—Common Toxicity Criteria version 4.03 (NCI—CTCAE v4.03), except cytokine release syndrome was graded as bed in Lee et al, Blood. 2014;124(2):188—95.

Among the eleven (1 1) subjects preconditioned with the combination of cyclophosphamide and fludarabine (cy/flu), the objective response rate (ORR) was 91% (10/11 subjects), and 10/11 subjects (91%) were observed to be negative for tumor cells in the bone marrow as measured by flow cytometry, and five (5) of the 11 (45%) were observed to e complete remission (CR) as measured by Lugano ia. Among those two subjects not having been preconditioned with cy/flu, the ORR was 50% (1/2 subjects), with 1 of the 2 subjects observed as negative for tumor cells in the bone marrow as measured by flow cytometry, but not W0 14207 ed to have achieved CR by Lugano criteria. In those two subjects assessed as achieving partial remission (PR), max 17-18 mm lymph nodes were observed. In four (4) of the subjects having achieved CR, IGH deep sequencing of bone marrow was carried out after treatment. In 4/4 (100%) of these subjects, the index clone was not detected in the bone marrow.

Progression-free survival (PFS) and l survival (OS) among the 11 subjects preconditioned with cyclophosphamide are shown in and 1B, with separate curves shown for the group of subjects who achieved CR, versus those who did not. Median PFS was not reached in the subjects who had achieved CR; PFS ranged from 3+ to 19+ months from first CAR+ T cell infusion.

In this study, twenty—three t (23%) of the 13 subjects exhibited severe cytokine release syndrome (CRS), ed according to Lee et al, Blood. 2014;124(2):188—95; 23% exhibited grade 3 or higher neurotoxicity.

The results demonstrated that administration of the CD19—specific CAR+—T cells at defined CD4+/CD8+ ratios ed in durable CR in a majority of the subjects with high-risk relapsed/refractory CLL.

EXAMPLE 2 Treatment of Subjects with Non-Hodgkin lymphoma [NHL] with ic Antigen- Receptor [CAR] Specific for CD19 Autologous T cells expressing a chimeric antigen-receptor (CAR) specific for CD19 were administered to forty-one (41) adult human subjects with CD19+ dgkin lymphoma (NHL). The group of subject ranged in age from twenty—eight (28) to seventy (70), with an average age of fifty-six (56). No ts were excluded based on lymphopenia, circulating tumor, prior transplant, or test expansion. The group of subjects exhibited a number of disease types including aggressive NHL (30/41 subjects, including e large B—cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), T cell/histocyte—rich large B cell lymphoma (TCHRBCL), and Burkitt lymphoma), mantle cell lymphoma (MCL; 5/41 ts), and ular lymphoma (FL; 6/41 subjects). All subjects had been previously treated, with a median number of four (4) prior treatments and a range of one (1) to eleven (11) prior treatments, among all subjects, and twenty-seven (27) subjects having been treated with greater than or equal to 4 prior therapies. Nineteen (19) of the 41 subjects had received prior auto- and/or allo- poietic stem cell transplantation (HSCT).

The CAR included an D19 scFv (in a VL—linker—VH ation) with variable regions derived from FMC63, an IgG hinge region, a transmembrane region derived from human CD28, and intracellular signaling s derived from human 41BB and CD3zeta. The construct further encoded a truncated EGFR (EGFRt), which served as a ate marker for CAR expression; the EGFRt-coding region was separated from the CAR ce by a T2A skip sequence. Prior to administration of the cells, patients underwent leukapheresis; CD4+ and CD8+ populations were ed by immunoaffinity-based enrichment methods, transduced with a viral vector with the CAR construct, and expanded in culture over 15 days. Cells for administration generally were formulated at a CD4+ T cell to CD8+ T cell ratio of approximately 1:1, prior to administration. Therapeutic compositions were successfully produced for all subjects.

Beginning at least forty-eight (48) (and up to ninety-six (96)) hours prior to CAR+ T cell infusion, 39 subjects received a lymphodepleting chemotherapy with either (a) cyclophosphamide (Cy, 60 mg/kg) with or without etoposide (12/39 subjects), or (b) cyclophosphamide (Cy, 60 mg/kg) in combination with fludarabine (flu, 25 mg/m2 daily for 3-5 days (cy/flu, 27/39 subjects).

Subjects were d with a ition having approximately a 1:1 ratio of CD8+ CAR+ T cells to CD4+ CAR-T cells, at one of three different dose levels (2x105 (N25) 2x 106 (N227) or 2x107 (N29) CAR+ T cells per kilogram (kg) weight of the subject).

Lymphodepleting therapy and T cell infusions were administered out on an outpatient basis. In this study, the maximum tolerated dose was dose level 2 (2x106 CAR+ T cells per kg).

Among the twenty—seven (27) subjects preconditioned with cyclophosphamide and fludarabine (cy/flu), the ive response rate (ORR) was 74% (20/27 subjects). Twelve (12) of the 27 (44%) were observed to e complete ion (CR).

Among the 27 subjects preconditioned with cy/flu, twenty (20) subjects, across all disease subtypes outlined above, were administered dose level 2 (2x106 CAR+ T cells per kg).

Of these subjects, the ORR was 80% (16/20) and 10 of the 20 subjects (50%) were observed to achieve CR. Sixteen (16) of the thirty (30) subjects with aggressive lymphoma were preconditioned with the cy/flu therapy and administered dose level 2. Among these 16 subjects, the ORR was 81% (13/16 subjects) and eight (8) subjects (50%) achieved CR. Two (2) of the 6 subjects with FL were preconditioned with cy/flu and administered dose level 2. Among these two subjects, the ORR was 50% (1/2) and 1 subject (50%) achieved CR. Two (2) of the five (5) subjects with MCL were preconditioned with cy/flu and administered dose level 2. Among these subjects, the ORR was 100% (2/2) and 1 subject (50%) achieved CR.

Progression-free survival (PFS) and overall survival (OS) among the 20 subjects preconditioned with cyclophosphamide and fludarabine (cy/flu) and administered 2x106 CAR+ T cells per kg are shown in and 2B, with separate curves for subjects who achieved CR, versus those who did not. Median PFS was not reached in the subjects who achieved CR; PFS ranged from 3 to 11+ months from first CAR+ T cell infusion. Median PFS was 4.1 months in subjects who did not achieve CR.

In this study, seventeen percent (17%) of ts at all dose levels of subjects exhibited severe cytokine release syndrome (CRS), assessed ing to Lee et al, Blood. 2014;124(2):188—95; five percent (5%, 2/41) exhibited grade 5 CR8, and 20% ted grade 3 or higher oxicity. Among those subjects who were administered 2x106 CAR+ T cells per kg following preconditioning with cy/flu (20 subjects), only ten percent (10%) exhibited severe cytokine release syndrome (CR8) and only 10% ted grade 3 or higher neurotoxicity.

EXAMPLE 3 Treatment of Subjects with Relapsed 0r Refractory gR/R! CD19+ CLL with Chimeric Antigen-Receptor [CAR] Specific for CD19: Additional Patients In an extension of the study described in Example 1, additional ts with relapsed or refractory (R/R) CD19+ chronic lymphocytic leukemia (CLL) were evaluated.

Eighteen (18) adult human subjects were administered the autologous T cells expressing a chimeric antigen-receptor (CAR) specific for CD19 and evaluated as described below.

The subjects ranged in age from forty (40) to seventy—three (73), with a median age of sixty (60). Twelve (12) subjects had complex karyotype and eleven (11) ts had 17p on. All subjects had extramedullary disease and two (2) had central s system (CNS) disease. All subjects had been previously treated with one or more other therapies for CLL (with a median number of five (5), and a range of three (3) to nine (9), prior lines of treatment), ing, in each case, ibrutinib (with eleven (11) (61%) having been refractory; three (3) (17%) having been intolerant). Three (3) (17%) subjects had failed prior allogeneic stem cell transplant and four (4) (22%) subjects were refractory to venetoclax. All subjects also were refractory to or had relapsed after receiving a depleting chemotherapy regimen containing fludarabine and rituximab. Just prior to ent, the median percentage of abnormal B cells in the bone marrow among all ts was 77% (with a range of 0.4% to 90%).

Autologous CAR- T cells were manufactured for all subjects and administered to subjects as described in Example 1, but additional subjects were d. 16/18 subjects received a cell composition with a CAR+ CD4+ T cell to CARJr CD8+ T cell ratio of approximately 1:1.

Subjects were infused with the cell composition at the different dose levels as follows: (2 x 105 (N24); 2 x 106 (N213); or 2 x 107 (N21) CAR+ T cells per kilogram (kg) weight of the subject).

Prior to CAR—T cell infusion, the treated subjects received a lymphodepleting chemotherapy with either (a) cyclophosphamide 30-60 mg/kg x 1 in combination with fludarabine 25 mg/mz/day x 3 days , 15/18 subjects), (b) fludarabine 25 mg/mz/day x 3 days (flu, 2/18 ts) or (c) cyclophosphamide 60 mg/kg (cy, 1/ 18 ts). Four (4) subjects with persistent disease received a second cycle of lymphodepletion chemotherapy and CAR+ T cells at a 10—fold higher dose than the first infusion.

Subjects were ed 4 weeks after the last CAR+ T cell infusion as described in Seventeen subjects completed response and toxicity assessment. Among the seventeen (17) subjects assessed, the ive se rate (ORR) was 76% (13/17 subjects; 8 with partial remission (PR) and 5 with complete remission (CR)). Two (2) subjects with PR based on the lymph node size criteria (IWCLL 2008) had negative PET scans after therapy.

Among thirteen (13) ibrutinib—refractory or intolerant subjects, the ORR was 77% (10/13 ts, 7 PR and 3 CR). Among four (4) venetoclax—refractory subjects, the ORR was 50% (2/4 subjects, 2 PR). Among those three (3) subjects not having been preconditioned with the combination of cy/flu, the ORR was 33% (1/3 subjects).

At day 28, among the thirteen (13) subjects who received cy/flu lymphodepletion and 2 x 105 or 2 x 106 CAR+ T cells/kg, eleven (11) (85%) exhibited complete ation of marrow disease by flow cytometry; 10/13 (77%) with nodal disease exhibited PR or CR; 1/ 13 (8%) had a mixed response; and 2/13 (15%) exhibited progressive disease (PD), In four (4) of the subjects having achieved CR, IGH deep sequencing of bone marrow was performed. No malignant sequences were detected in 4/4 (100%) of these subjects.

Progression—free survival (PFS) among the thirteen (13) subjects who received cy/flu lymphodepletion and 2 x 105 or 2 x 106 CAR+ T cells/kg, are shown in with separate curves shown for the group of subjects who achieved CR, versus those who did not. Overall survival (OS) was 100% in this group. No subjects who achieved CR had relapsed or died after a median follow-up of 8.4 months. The results also showed that subjects who had achieved CR exhibited a higher peak percentage of CD8+ (p:0.006), but not CD4+ CAR+ T cells in blood.

Robust CAR—T cell expansion was seen in some non—responders.

The result r demonstrated that administration of the CD19-specific CAR+-T cells at defined CD4+/CD8+ ratios resulted in a high response rate and e CR in a majority of the subjects with isk relapsed/refractory CLL, such as patients who have failed ibrutinib EXAMPLE 4 Treatment of Subjects with Relapsed 0r Refractory gR/R) CD19+ CLL with Chimeric Antigen-Receptor [CAR] Specific for CD19: r Patients A. Subjects and Treatment In an extension of the studies described in Examples 1 and 3, additional subjects with ed or refractory (R/R) CD19+ chronic lymphocytic leukemia (CLL) were evaluated. In total, twenty—four (24) adult human subjects, who had received previous therapies, were administered the autologous T cells expressing a ic antigen—receptor (CAR) specific for CD19, following lymphodepletion, and evaluated as described in Examples 1 and 3 and below.

As described in Table 4, the subjects ranged in age from 40 to 73, with a median age of 61. n (16) subjects had x karyotype and 14 subjects had 17p deletion. Eight (8) subjects had high-risk histology. —three (23) subjects had extramedullary disease. All subjects had been previously treated with one or more other therapies for CLL, with a median of (range 3—9) prior lines of treatment, which included, in each case, treatment with ibrutinib (with 19 (79%) having been refractory; 3 (13%) having been intolerant). Nine (9) of the 18 ibrutinib—refractory subjects had a BTK or PLCG2 mutation (50%; BTK, n27; PLCG2, n22).

Ibrutinib was discontinued in all subjects before lymphodepletion. Four (4) (17%) ts had failed prior allogeneic stem cell transplant and 6 (25%) subjects were refractory to clax.

Just prior to treatment, the median percentage of abnormal B cells in the bone marrow among all subjects was 61.6%% (range 0.0%—96%). Just prior to treatment, the median abnormal B cell count in the blood among all subjects was 1.1 x 103 ALL (range of 0.0—76.68 x 103 ALL).

Table 4. High-Risk CLL Population Characteristics Characteristic N224 Age at infusion, median [range], years 61 [40-73] Prior lines of therapy, median [range] 5 [3—9] Prior neic HCT 4 (17%) Prior Ibrutinib, median duration 13 mo. [range 0.75—39 mo.] 24 (100%) o Ibrutinib—refractory 19 (79%) o BTK or PLCG2 mutation 9/19 (47%) o Ibrutinib-intolerant 3 (13%) claX-refractory 6 (25%) Refractory or relapsed after fludarabine with rituximab 23 (96%) Failed bendamustine/rituximab 1 High-risk cytogenetics, N (%) 23 (96%) 0 Complex karyotype 16 (67%) o 17p del 14 (58%) High—risk histology (Richter’s/IPC/PLL), N (%) 8 (33%) Marrow abnormal B cells before lymphodepletion . 64.5 [0—96] chemotherapy, median ], % Extramedullary disease, N (%) 23 (96%) 2 3093 [546'20406] o Cross—sectional area, median ], m o FDG-avid e on PET, N (%) 14/15 (93%) 7'1 [3-4'27-5] 0 Maximum SUV, median [range] Active CNS disease 2 (8%) Measureable disease by CT and FDG-avid disease on PET, N (%) 14/15 (93%) 0 Maximum SUV, median [range] 7.1 [34—275] Autologous CAR—T cells were manufactured for all subjects and administered to ts as described in Examples 1 and 3, but with additional subjects treated. Twenty-two (22) of the 24 subjects received a cell composition with a CAR+ CD4+ T cell to CAR+ CD8+ T cell ratio of approximately 1:1, and 2 patients received less than the target CD8+ CAR-T cell dose (58.5% and . Subjects were infused with the cell composition the different dose levels as follows: 2 x 105 (N24); 2 x 106 (N219); or 2 x 107 (N21) CAR+ T cells per kilogram (kg) weight of the subject. Prior to CAR-T cell infusion, 21 subjects received lymphodepleting chemotherapy as outlined in Table 5. The treated subjects received a lymphodepleting chemotherapy received either (a) hosphamide 30-60 mg/kg (1—2 g/mz) x 1 in combination with fludarabine 25 mg/mZ/day x 3 days (cy/flu, 18/24 subjects), (b) cyclophosphamide 60 mg/kg (1—2 g/mz) x l in combination with fludarabine 25 mg/mZ/day x 5 days u, 1/24 subjects), (c) fludarabine 25 mg/mZ/day x 3 days (flu, 2/24 subjects), (d) cyclophosphamide 60 mg/kg (cy, 1/24 subjects) or (e) 500 mg/m2 x 3 with bine 25 mg/mZ/day x 3 days (cy/flu, 2/24 subjects). A total of 15 patients in the study received Cy/Flu lymphodepletion and 2 x 106 CAR+ T cells.

Table 5. Lymphodepletion and Immunotherapy in High-Risk CLL Patients depleting herapy o Cyclophosphamide/fludarabine (Cy/Flu) 21 (87%)* o Non—Cy/Flu 3 (13%) CAR—T cell manufacturing + + 0 CD4 and CD8 central memory 7(29%) + + 0 CD4 and CD8 bulk 17 (71%) CD19 CAR—T cell dose level + 0 BL] (2x10 EGFRt cells/kg) 3(13%) 6 + o DL2 (2X10 EGFRt cells/kg) 20 (83%) 7 + o DL3 (2x10 EGFRt cells/kg) 1(4%) Single cycle 18 (75%) Second c cle for residual disease or relase 6 (25%) *1 patient died prior to restaging Six (6) subjects with persistent e received a second cycle of lymphodepletion chemotherapy and CAR+ T cell infusion at the same (N=1) or at a 10-fold higher dose (N=5) than the first infusion.

During the 3 weeks between leukapheresis and lymphodepletion chemotherapy, 6 patients required high-dose corticosteroids to l progressive disease and 2 others required treatment for tumor—associated hypercalcemia.

B. Response to Treatment Subjects were ed for response 4 weeks after the last CAR+ T cell infusion as described in e 1. The assessment of twenty-one (21) subjects receiving Cy/Flu lymphodepletion demonstrated high response rates in high—risk CLL patients at four weeks post— infusion as shown in Table 8. ses were measured in subjects by: (a) bone marrow is; (b) PET-CT; and (c) International Workshop Group on CLL (IWCLL) criteria.

Table 8: Res onse Rates in Sub'ects at Four Weeks Post-infusion Lymphodepletion Non-Cymlu C mlul m hode letion (N=21)* (N=3 restaged) All patients Ibrutinib-refractory (N=19 resta_ed) (N=16 resta_ed) 0/37(Q%) 4/19g21%) 74/16(25%) 7 VCR(at4weeks)r , , 7 7 7 7 , , , , , 7 —VVV V FlwahégVthiveVV—VV V1V/3V(V33V%)V V V V V V V1V5/V1i(88%j V V V V V 12/14(8V6%)V V V V ORR (at 4 weeks) 0/1 (0%) 8/11 (73%) 8/11 (73%) CR (at 4 weeks) 0/1 (0%) 7/11 (64%)** 7/11 (64%)** **One additional patient ed as SD (IWCLL, PET) at 4 weeks ed PR (IWCLL, max 16mm) and CR (PET) 8 weeks after CAR—T cell infusion without additional therapy *1 patient died prior to restaging 1 CAIP-I'cellExpat/211w! /‘flk/e/Ice After CAR—T cell infusion, CAR-T cells were detected in blood by flow cytometry in all patients to assess expansion and persistence. Among subjects who received cyclophosphamide/fludarabine (Cy/Flu) lymphodepletion and were infused with 2 x 106 CAR—T cells, r CAR-T cell expansion was positively correlated with the, percentage of abnormal B cells present in bone marrow (r=0.67, p=0.006), tumor cross-sectional area (r=0.57, p=0.025) and absolute abnormal B cell count in blood as shown in —4C, respectively. In addition, there was an inverse ation between CAR-T cell expansion and the SUVmax in those subjects with FDG-avid e on pretreatment PEG scans (). CAR-T cells were detected by QPCR in blood at 2 6 months in all patients (n=1l) that were evaluated and did not o subsequent allogeneic HCT.

In the subjects who received Cy/Flu lymphodepletion and were infused with 2 x 106 CAR-T cells, CAR-T cell expansion was inversely correlated with the immune checkpoint biomarker CD200 (r=-O.25, p=0.4) as shown in . CAR-T cell expansion was also inversely correlated with immune checkpoint biomarkers PDLl and PDL2.

Z. lympfl Node[Papa/lye [Pd/e Twenty three (23) patents were evaluated for lymph node response 4 weeks after CAR-T cell infusion by IWCLL criteria. Among the 23 restaged patients, the overall response rate (ORR) at 4 weeks after CAR+ T cell infusion by IWCLL lymph node criteria was 70% (16/23). Among the 3 patients who did not receive Cy/Flu lymphodepletion, 1 cleared marrow disease, 1 had a partial se (PR), and all developed progressive e.

Among 19 subjects who completed IWCLL analysis and received Cy/Flu lymphodepletion and a single CD19 CAR-T cell infusion at 52x106 CAR-T cells/kg, a lymph node response by IWCLL criteria was obseryed in 74% of patients (14/19) [95% confidence interval (CI): 49—91%]: 21% (4/19) CR; 53% (10/19) PR. Similar response rates were observed when considering only the 16 patients that were ibrutinib-refractory: 69% (l 1/ 16) ORR [95% CI: 41-89%]; 25% (4/26) CR).

Where feasible, lymph node se was also assessed by PET imaging 4 weeks after CAR—T cell infusion. The CR rate in ibrutinib—refractory patients after PET-CT restaging was 64% (7/11) [95% CI: 31-89%], with a Deauville score of 1-2. More patients were staged as CR by PET—CT than by IWCLL (64% vs. 25%). Four (4) of 5 patients who achieved PR by IWCLL and underwent PET g had no FDG-acid disease after CAR—T cell infusion. One additional patient with stable disease according to PET-CT, 4 weeks after CAR-T cell infusion, subsequently achieved CR on a —up PET—CT, 8 weeks later. 3. Allaflflflell/ ofmalty/Mill[6'17Segue/Icesfl‘fllll Marrow Twenty-two of 24 patients had marrow disease before treatment and 21 patients had a bone marrow evaluation 4 weeks after CAR+ T cell administration. een of 21 patients (81%) had no marrow e detected by high tion flow cytometry. Of the subjects who received Cy/Flu depeletion, administration of 52x106 CAR-T cells/kg and had bone marrow involvement prior to therapy, fifteen out of seventeen (15/17) exhibited clearance of bone marrow disease by high—resolution flow cytometry (88% [95% CI: 64—99%]). The flow— negative marrow se rate in the subset of ibrutinib-refractory patients was similar (12/14; 86% [95% CI: ]). FISH and conventional karyotyping did not identify residual CLL in patients without detectable e by flow cytometry; however, 2 patients had abnormalities considered to be due to the effects of prior chemotherapy on the myeloid lineage and one had a persistent constitutional translocation.

Twelve patients who cleared marrow by flow cytometry, after Cy/Flu and 52x106 CAR-T kg on, also had an identified clonal malignant IGH sequence in CLL cells before treatment. Seven of the 12 (7/12; 58%) subjects, identified as having a clonal malignant IGH sequence, exhibited nce of bone marrow disease by IGH sequencing 4 weeks after CAR-T cell infusion.

Of the 6 patients that received a second cycle of lymphodepletion chemotherapy and CAR—T cell on at the same (n=1) or lO-fold higher dose (n=5), 2 (2/6; 33%) achieved CR (PET-CT) and eliminated bone marrow disease by flow cytometry and IGH sequencing. Four of the 6 patients (4/6; 67%) developed CRS (2 grade 23) and one developed reversible neurologic toxicity (grade 3) after the second CAR—T cell on.

A subset of patients who achieved PR by IWCLL at initial restaging at 4 weeks had no FDG—avid disease by PET—CT ia (4/5), and/or had no detectable malignant IGH sequence in marrow (4/6). This observation is consistent with a finding that the IWCLL criteria might underestimate the response achieved with CAR-T cells, which is further ted by the survival data described below, measured by IWCLL response, showing an equivalent PFS in patients who achieved PR or CR, and ongoing tumor regression after initial response in one 4. Frag/ambit Frye Sll/‘Vl'lld/fl/ldOverdIISl/er'm/l Progression free al (PFS) and overall survival (OS) for all CLL patients are shown in A lymph node response (CR/PR by IWCLL) was associated with longer PFS and OS compared to failure to respond (SD/PD). Progression free survival (PFS) and overall al (OS), as measured by IWCLL nodal response criteria, among the subjects who received Cy/Flu lymphodepletion and 2 x 105 or 2 x 106 CAR—T cells/kg, are shown in . also depicts PFS and OS as ed by IWCLL among the same group of subjects as , except excluding one patient that died prior to restaging. For both FIGS. 6A and 6B, separate curves are shown for the group of subjects who achieved CR, subjects who achieved PR, and non-responders, and indicate that patients who achieved PR by IWCLL criteria did not have inferior PFS and OS compared to those who achieved CR. OS was 100% in subjects who achieved CR or PR. N0 subjects who achieved CR or PR died within 24 months of the first CAR-T cell infusion.

The survival of patients who cleared marrow by flow try was analyzed for the presence (detected) or absence (none) of malignant IGH sequences in marrow 4 weeks after CAR—T cell infusion. Among 14 subjects, 7 subjects had malignant sequences detected by IGH deep sequencing. PFS and OS are shown in PFS and OS were reduced in the 7 subjects with malignant ces, compared to the 7 subjects without malignant sequences. Thus, independent of the IWCLL response, patients who were negative for malignant IGH sequences had better PFS compared to those with persistent malignant IGH ces. Median OS was not reached in either group. The positive effect of marrow nce by IGH sequencing on outcome was also observed when the analysis was restricted to PFS patients who responded (CR/PR) by IWCLL criteria 63, mPFS for IGHseq—positive = 8.5 months, mPFS for IGHseq-negative not reached).

The results further trated that administration of the CDl9—specific CAR+—T cells at defined CD4+/CD8+ ratios resulted in a high response rate and durable CR in a majority of the subjects with high-risk relapsed/refractory CLL, such as patients who have failed ibrutinib and/or venetoclax treatment. Additionally, the results indicated that the detection of malignant sequences by IGH deep cing of bone marrow after CAR T— cell therapy may provide early signs of durable ses.

C. Toxicity Twenty—four (24) subjects were assessed for symptoms of cytokine release syndrome (CRS) and neurotoxicity as shown in Table 6 and Table 7, respectively.

Table 6. CR8 in CLL Subjects after CAR T cell therapy CLL (n=24) 4 (17%) CRS grade 8 (33%) (Lee et al, Blood. 10 (42%) 2014;124(2):188-95) Table 7. Neurotoxicity in CLL Subjects after CAR T cell y Neurotoxicit CLL (n=24) Neurotoxicity grade 2 2 (8%) (CTCAE V4.03) 3 5 (21%) 4 0 (0%) 1 (4%) Tocilizumab (4-8 mg/kg I.V.) and dexamethasone (10 mg bid I.V.) were administered to patients who either required management in the intensive care unit (ICU) or were under evaluation for ICU care. ention was initiated in patients with grade 2-3 cytokine release me (Lee et al, Blood, 2014) that were not responding to intravenous fluids and/or low dose vasopressor support and grade 2—3 neurotoxicity. Of treated patients, 1 patient progressed to grade 3-4 CRS from day 4 that was refractory to zumab and dexamethasone, developed cerebral edema on day 9 that was refractory to siltuximab and mannitol and died 11 days after CAR—T cell infusion. No other patients developed greater than grade 2 CR8 and only 4 patients developed grade 3 neurotoxicity. Only 6/24 patients exhibited clinical symptoms sufficiently severe to e an intervention therapy, and CR3 and neurotoxicity resolved in all patients treated according to these criteria, with the exception of the t with fatal cerebral edema.

EXAMPLE 5 Factors Correlating with Response and/or Toxicity Based on Peak CAR T Cell ion in High-Risk CLL Patients Following the evaluation of expansion and response to CD19 CAR-T cell therapy for relapsed or refractory (R/R) CD19+ chronic lymphocytic ia (CLL), as described above in es 1, 3, and 4, subjects with relapsed or refractory (R/R) CD19+ chronic lymphocytic leukemia (CLL) were evaluated for expansion and response.

Elevated peak CD4+ or CD8+ CAR—T cell counts after infusion were associated with better bone marrow responses in isk CLL subjects as assessed by the presence (detected) or absence (none) of malignant IGH sequences in marrow 4 weeks after CAR—T cell infusion (. Peak CD4+/EGFRt+ and CDES‘L/EGFRt+ CAR-T cell counts were higher in patients who ultimately cleared bone marrow by flow try compared to those who failed to eliminate CLL from marrow (), and also higher in those who achieved CR by flow cytometry and had no malignant IGH sequences detected in marrow compared to those who had CR by flow cytometry with detectable malignant IGH sequences (FIG 9B). ility curves depicting the probability estimated by logistic regression of clinical outcomes associated with peak CD4+/EGFRt+ and CD8+/EGFRt+ CAR-T cell counts in blood were generated and are depicted in FIGA. 10A and 10B. An estimated probability curve of response and an estimated probability of developing Grade 3-5 neurotoxicity were ucted based on the number of CD4+/EGFRt+ or GFRt+ CAR-T cells in the blood (A). An estimated probability curve of response and an estimated probability of developing Grade 2—5 neurotoxicity or CRS were constructed based on the number of CD4+/EGFRt+ or CD8+/EGFRt+ CAR-T cells in the blood (3). Generally, as the number of CAR-T cells increased, the probability of bone marrow response increased then ued while the probability of developing a toxicity, Grade 3—5 oxicity (A) or Grade 2—5 oxicity or CRS (B) increased. These curves demonstrate a therapeutic window of peak CD4+/EGFRt+ and CD8+/EGFRt+ CAR—T cell counts in which marrow clearance might be achieved in most patients without a high risk of neurotoxicity or CRS.

Robust umor activity, as determined by s in cross-sectional area of 6 largest lymph nodes on CT scan by IWCLL imaging criteria, was seen in a subset of patients with large lymph node tumor burdens, including those with Richter’s transformation (), however patients with higher lymph node tumor bulk were overall less likely to respond to CAR-T cells (CR vs PR vs NR by IWCLL, p=0.098), as were those with fewer prior therapies (CR/PR vs NR by IWCLL; 5.5 vs 4; p=0.04). This suggests that bulky and aggressive nodal disease might be less amenable to CAR-T cell therapy. The relationship between the peak CAR- T cell count in blood and the probability of a response in lymph nodes was less robust than that noted for marrow, but higher peak CD3Jr/EGFRt+ CAR—T cell counts in blood were associated with reduced risk of disease progression and death in high-risk CLL patients (HR 0.56, 95% confidence interval 0.34—0.93, p=0.025).

The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are ed, for example, to illustrate various s of the invention.

Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the sure and are intended to fall within the scope of the present disclosure.

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APPS?QPATYTCVVS{TDSRTPKVASRSRTVSYVTDH 6 R4GGG*G?GSRILCGDV**WPGPQ TZA 7 MRR.V"S.LRC71P{?AFL.IPR<VCNGIGIG3FKDSLSINAT'IKHFK tEGFR NCTSISGDLHIJPVAFRGDSFTHTPPRD?Q*LDIR< VK‘IiGbLRIQA WPPV? D.HAb*NR*IIRGQiKQiGQbSLAVVSLNITSLGLRSJKEISD artificia; GDVIISGVKNLCYAN"INW<KLFGTSGQ<T<IISN¥GENSCKATGQVCH ALCS?*GCWGP*PRDCVSCRNVSRGRECVD<C LL<G*P?*EV*NS*CI QCH?ECL?QAMVI C GRG?DNCIQCAHYIDG?HCVKTC?AGV GEVNT LVW{YADAGHVCHJCHPNCTYGCTGPGLEGCPTNG?KIPSIATGMVGAL TIL.VVA.GIG.FM 8 FWVLVVVGGVLACYSLLVTVAFIIFWV CD28 (amino acids 153— 179 Of Accession No. ) Homo sapiens 9 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP CD28 (amino FWVLVVVGGVLACYSLLVTVAFIIFWV acids 114* 179 of Accession No. ) Homo sapiens RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (amino acids 180— 220 of PlO747) Homo sapiens 11 RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (LL to Homo sapiens 12 KRGRKKLLYIEKQPEMRPVQLLQ4*DGCSCREP“*‘GGC*1 4-lBB (amino acids 214— 255 of Q0701l.l) Homo sapiens 13 RVKFSRSADA9AYQQGQ QJYNEJNLGRRLLYDVJD(RRGRDPEMGGK? C33 zeta 4GLY-4LQK3<WA4AY84IGWKGLR?RG<G€DG.YQG.S A < D"Y3AL{MQA4?PR Homo sapiens 14 RVKFSRSAEP9AYQQGQ Q-YNT.NRGRR4*YDVTD<RRGRDPEMGGK? C33 zeta RRKVPQ4GRY 41QK3< AdAYS‘IG KG*RRRG<G-DGIYQGIS A < D"Y3AL{MQAL?PR Homo sapiens RVKFSRSA3A9AYKQGQ Q-YN7.NRGRR4*YDVK3<RRGRDPEMGGK? C33 zeta 461Y 41QK3< AdAYSdIG KGFRKRG<G-DG-YQG.S A < D”Y3AL{MQAL?P? Homo sapiens 16 RKVCNGIGIGTFKDS.SINATNI<HF<NCTSISG34-IL?VAF?GDSFT tEGFR H P?1D?Q*RDIT V<4IiGbl.IQAWP4VRiDTiAb4W14IIRGR < QHGQFSTAVVS.NI"SRGRRSTKTISDGDVIISGW<\LCYA "INWKKL artificial FGTSGQ<T{IISNRGEVSCKATGQVC4ATCS?*GCWGP49RDCVSCRNV SRGRECVD<C .1464??*EV*NS*CIQC{?ECL?QAMVITC"GRGPDV CIQCAHYIDG?&CVK"CPAGV VW<YADAG-VC4LCHPNCTYG CTGPGLEGCPTWGPKI?SIATGMVGALALLLVVALGIGLFM 17 ACACGGCCTCGTGTA"TACTGT exemplary degenerate sequence of the V region of IGH 18 ACCTGAGGAGACGGTGACC exemplary degenerate sequence of the J region of IGH 19 7GRGSR11CGDV4 PGP T2A GSGATNFSJJKQA DVEENPGP ?2A 21 ATNFSLLKQAGDV *N?GP 92A 22 QCTNYALL<LAGDVES PGP 32A 23 VKQTLNFDJJKLAGDVESNPGP FZA WO 14207 24 GSTSGSGKPGSGEGSTKG Linker gacatccaga tgacccagac cacctccagc ctgagcgcca Sequence encoding gcctgggcga ccgggtgacc atcagctgcc gggccagcca ggacatcagc aagtacctga actggtatca gcagaagccc gacggcaccg tcaagctgct gatctaccac accagccggc tgcacagcgg cgtgcccagc cggtttagcg gcagcggctc cggcaccgac tacagcctga ccatctccaa cctggaacag gaagatatcg ccacctactt ttgccagcag ggcaacacac tgccctacac ctttggcggc ggaacaaagc tggaaatcac cggcagcacc tccggcagcg gcaagcctgg cgag ggcagcacca agggcgaggt gaagctgcag ggcc ctggcctggt ggcccccagc cagagcctga gcgtgacctg caccgtgagc ggcgtgagcc tgcccgacta cggcgtgagc tggatccggc agccccccag gaagggcctg ctgg gcgtgatctg gggcagcgag accacctact acaacagcgc gagc acca tcatcaagga caacagcaag agccaggtgt agat gaacagcctg cagaccgacg acaccgccat ctactactgcgccaagcact actactacgg cggcagctac gccatggact actggggcca cagc gtgaccgtga gcagc 26 XlPPXZP Hinge X1 is glycine, cysteine or arginine X2 is cysteine or threonine 27 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Hinge Pro Cys Pro 28 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Hinge 29 LLK PLGDlHiCPRCPLPKSCDiPPPCPRCPEPKSCDTPPPCPRCPEPK Hinge SCD"PPPCPRCP Glu Ser Lys "yr Gly Pro Pro Cys Pro Ser Cys Pro Hinge 31 Glu Ser Lys "yr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 32 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 33 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 34 Glu Val Val Val Lys Tyr Gly Pro Pro Cys Pro Pro Hinge Cys Pro RASQDISKYLN FMC63 CDR 41 36 SRLHSGV FMC63 CDR 42 37 GNTLPY"FG FMC63 CDR i3 38 )YGVS FMC63 CDR 41 39 VIWGS; KS FMC63 CDR 42 40 YAMDYWG FMC63 CDR i3 41 EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLG FMC63 VH VIWGSL YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKH YYYGGSYAMDYWGQGTSVTVSS 42 DIQM Q SSLSASLGDRV"ISCRASQDISKYLNWYQQKPDG"V<LLIY FMC63 VL H"SRLHSGVPSRFSGSGSG DYS: ISVLdeDIA YECQQGN LPY r GGG KLLI 43 DIQM Q LGDRV"ISCRASQDISKYLNWYQQKPDG"V<LLIY FMC63 SCFV H"SRLHSGVPSRFSGSGSG DYSJ ISWLLQLDIA YhCQQGN LPY h GGG KLLI GSiSGSGKPGSGLGS KGLVKLQLSGPGLVAPSQSLSV"C TVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL"IIK DNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSV"VSS 44 SC25C" CDR KASQNVGTNVA L1 45 SC25C; CDR SATYRNS L2 46 SCZSC” CDR QQYNRYPYT L3 47 SC25C" CDR SYWMN H1 48 SC25C“ CDR QIYPGDGDT-YNGKFKG H2 49 SCZSC» CDR ("ISSVVDFYFDY H3 50 TVKLQQSGATLVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIG SCZSC; VH QIYPGDGDL GQA L ADKSSSTAYMQLSGLTSEDSAVYFCAR KNISSVVDFYFDYWGQGTTVTVSS 51 DIELTQSPKFMSTSVGDRVSV"CKASQNVGTNVAWYQQK?GQSPK?LIY SCZSCL VL SATYRNSGVPDRFTGSGSG"DFTLTITNVQSKDLADYFCQQYNRY?YTS 52 GGGGSGGGGSGGGGS Linker 53 EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIG SJ25C1 SCFV QIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGL SLDSAVYECAR KTISSVVDFYFDYWGQGTTVhVSSGGGGSGGGGSGGGGSDIELTQSPKF MSTSVGDRVSVTCKASQNVGhNVAWYQQKPGQSPKPLIYSATYRNSGVP DRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPY"SGGGTKLEIKR 54 HYYYGGSYAMDY FMC63 CDR i3 55 HTSRLHS FMC63 CDR 42 56 QQGNTLPYT FMC63 CDR 43

Claims (39)

1. Use of a dose of cells expressing a ic antigen receptor (CAR) that specifically binds to a target n expressed by a chronic lymphocytic leukemia (CLL) for the manufacture of a medicament for treating a subject having or suspected of having a CLL, wherein the dose ses between about 0.1 x 106 cells/kg body weight of the subject (cells/kg) and about 1.0 x 107 cells/kg.

2. The use of claim 1, wherein the dose comprises between about 1.0 x 106 cells/kg and about 5.0 x 106 cells/kg.

3. Use of a dose of cells expressing a ic antigen receptor (CAR) that specifically binds to a target antigen expressed by a chronic lymphocytic leukemia (CLL) for the manufacture of a medicament for treating a subject having or suspected of having a CLL, wherein the dose ses between about 1 x 105 total CAR-expressing cells and about 5 x 108 total CAR-expressing cells.

4. The use of claim 3, wherein the dose comprises between about 1 x 107 total CAR- expressing cells and about 1.5 x 108 total CAR-expressing cells.

5. The use of any of claims 1-4, wherein, at or prior to treatment with the medicament: the subject is or has been identified as having one or more netic abnormalities; the t is or has been identified as having high-risk CLL; the subject is or has been identified as having extramedullary disease; the subject is or has been identified as having central nervous system (CNS) disease; or the subject is an adult.

6. The use of any one of claims 1-5, wherein, at the time of treatment with the medicament, the subject has ed following remission after treatment with, or become refractory to, one or more prior ies for the CLL.

7. The use of any one of claims 1-6, wherein, at the time of ent with the medicament, the subject has relapsed following remission after treatment with, or become refractory to, two or more prior therapies for the CLL.

8. The use of claim 6 or claim 7, wherein the prior therapies for the CLL are other than the lymphodepleting therapy and/or another dose of cells expressing the CAR.

9. The use of any one of claims 6-8, wherein the prior therapies comprise an inhibitor of Bruton’s tyrosine kinase (BTK).

10. The use of any of claims 6-9, wherein the prior ies comprise ibrutinib.

11. The use of any one of claims 6-10, n the prior therapies comprise a monoclonal antibody that specifically binds to an antigen expressed by, or previously expressed by, cells of the CLL.

12. The use of any one of claims 6-11, wherein the prior therapies se venetoclax, a combination therapy comprising fludarabine and rituximab, radiation therapy and/or hematopoietic stem cell transplantation (HSCT).

13. The use of any one of claims 6-12, wherein the prior ies comprise venetoclax.

14. The use of any one of claims 1-13, wherein the dose of cells comprises a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR.

15. The use of any one of claims 1-14, n the dose of cells comprises a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR, which is approximately 1:1 or is between approximately 1:3 and approximately 3:1.

16. Use of a dose of cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen expressed by a non-Hodgkin lymphoma (NHL) for the manufacture of a medicament for treating a subject having a NHL, wherein the dose: (i) comprises between about 0.1 x 106 cells/kg body weight of the subject (cells/kg) and about 1.0 x 107 cells/kg; and (ii) comprises a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR.

17. The use of claim 16, wherein the dose comprises n about 1.0 x 106 cells/kg and about 5.0 x 106 cells/kg.

18. Use of a dose of cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen sed by a dgkin lymphoma (NHL) for the manufacture of a medicament for treating a subject having a NHL, wherein the dose: (i) comprises between about 1 x 106 total CAR-expressing cells and about 3 x 108 total CAR-expressing cells; and (ii) comprises a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR.

19. The use of claim 18, wherein the dose comprises between about 1 x 107 total CAR-expressing cells and about 1.5 x 108 total CAR-expressing cells.

20. The use of any of claims 16-19, wherein, at or prior to the treatment with the medicament: the subject is or has been identified as having one or more cytogenetic abnormalities the subject is or has been identified as having high-risk NHL; the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell ma (DLBCL), primary mediastinal large B cell ma (PMBCL), T istocyterich large B cell lymphoma (TCHRBCL), t’s lymphoma, mantle cell lymphoma (MCL), and follicular lymphoma (FL); or the subject is an adult.

21. The use of any one of claims 16-20, wherein the NHL is DLBCL.

22. The use of any one of claims 16-21, wherein, at the time of treatment with the medicament, the subject has ed following ion after treatment with, or become refractory to, one or more prior therapies for the NHL.

23. The use of any one of claims 16-22, wherein, prior to treatment with the medicament, the subject has ed following remission after treatment with, or become refractory to, two or more prior therapies for the NHL.

24. The use of claim 22 or claim 23, wherein the prior therapies for the NHL are other than the lymphodepleting therapy and/or another dose of cells expressing the CAR.

25. The use of any one of claims 1-24, wherein the lymphodepleting therapy: (i) further comprises cyclophosphamide; (ii) is initiated at a time that is at least about 48 hours prior to, or is between about 48 hours and about 96 hours prior to, the administration of the medicament; and (iii) comprises the administration of cyclophosphamide at about 30-60 mg/kg and/or the fludarabine at about 25 mg/m2, daily for 3-5 days.

26. The use of any one of claims 1-25, wherein treatment with the medicament is carried out via outpatient delivery.

27. The use of any one of claims 14-26, wherein the defined ratio is approximately

28. The use of any one of claims 1-27, wherein at least 50% of subjects treated with the medicament achieve complete remission (CR) or objective response (OR).

29. The use of any one of claims 1-28, wherein the antigen is a B cell antigen.

30. The use of any one of claims 1-29, wherein the antigen is CD19.

31. The use of any one of claims 1-30, wherein the CAR comprises an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, and a cytoplasmic signaling domain derived from a CD3zeta.

32. The use of any one of claims 1-31, wherein the CAR comprises a spacer and/or a hinge region, each derived from a human IgG.

33. The use of any one of claims 1-32, wherein the CAR comprises, in order, an scFv specific for the n, a , a transmembrane domain, a cytoplasmic signaling domain derived from a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a CD3zeta signaling domain.

34. The use of any one of claims 1-33, wherein: (a) the spacer ses the sequence of SEQ ID NO: 1; a ce encoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or a variant of any of the foregoing having at least 90% or more sequence identity thereto; (b) the costimulatory domain comprises SEQ ID NO: 12 or a variant thereof having at least 90% or more sequence ty thereto; (c) the primary signaling domain comprises SEQ ID NO: 13 or 14 or 15 having at least 90% or more sequence ty thereto; and/or (d) the scFv ses a CDR-L1 sequence of SEQ ID NO: 35, a CDR-L2 sequence of SEQ ID NO: 36, a CDR-L3 sequence of SEQ ID NO: 37, a CDR-H1 ce of SEQ ID NO: 38, a CDRH-2 sequence of SEQ ID NO: 39, and a CDR-H3 sequence of SEQ ID NO: 40.

35. A method of sis or staging, the method comprising detecting the presence or absence of a malignant immunoglobulin heavy chain locus (IGH) sequence in a sample from a subject having a B cell malignancy, said subject having previously received administration of a cell therapy comprising a dose or composition of cally engineered cells expressing a recombinant receptor for treating the B cell malignancy, wherein detecting the presence or absence of the malignant IGH sequence determines the prognosis of the t in response to the cell therapy.

36. The method of claim 35, wherein the detecting the presence or absence of the malignant IGH sequence is carried out within or within about or about 3 to 6 weeks after initiation of the cell therapy.

37. A method of predicting durability of response to a cell therapy, the method sing detecting the presence or absence of a malignant immunoglobulin heavy chain locus (IGH) sequence in a sample from a subject having a B cell malignancy, said subject having previously received administration of a cell therapy comprising a dose or ition of genetically engineered cells sing a recombinant receptor for treating the B cell malignancy, wherein the presence or absence of the malignant IGH sequence predicts the durability of response to the cell therapy.

38. The method of claim 37, wherein the ing the presence or e of the malignant IGH sequence is carried out within or within about 16 weeks after initiation of the cell therapy.

39. The use of any of claims 1-34, wherein, prior to ent with the medicament, the subject has been preconditioned with a lymphodepleting therapy comprising fludarabine. 11W??- HG. 1A auwivai 3% Magrwfiamfiw 3:} a $ $22 $3“ "$3336. sinw first C§i§$¥~§§§¥ §nfizsian (mami’zsfi R6. 18 {€52} 3;:er fii} fivgraii I 1 a 8 33 “38 Kim gi‘me first (smug: maxim {33mm} wo