Cd19-directed chimeric antigen receptor t cell compositions and methods and uses thereof

Abstract

Provided in some aspects are compositions of cells for treating subjects with disease and conditions such as non-Hodgkin's lymphoma (NHL), and related methods, compositions, uses and articles of manufacture. In some embodiments, the disease or condition is a B-cell non-Hodgkin lymphoma (B-cell NHL). The cells generally express recombinant receptors such as chimeric antigen receptors (CARs) for targeting an antigen, such as CD 19, on cells of the lymphoma.

Description

CD19-DIRECTED CHIMERIC ANTIGEN RECEPTOR T CELL COMPOSITIONS ANDMETHODS AND USES THEREOFCross-Reference to Related Applications

[0001] This application claims priority from U.S. provisional application No. 62 / 975,724 filed February 12, 2020, entitled “CD19-DIRECTED CHIMERIC ANTIGEN RECEPTOR T CELL COMPOSITIONS AND METHODS AND USES THEREOF,” the contents of which are incorporated by reference in their entirety.Incorporation by Reference of Sequence Listing

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042022740SEQLIST.txt, created February 6, 2021, which is 56.9 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.Field

[0003] The present disclosure relates in some aspects to adoptive cell therapy involving the administration of compositions of cells for treating subjects with disease and conditions such as non- Hodgkin’s lymphoma (NHL), and related methods, compositions, uses and articles of manufacture.Background

[0004] Various immunotherapy and / or cell therapy methods are available for treating 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 antigen receptors, as well as other adoptive immune cell and adoptive T cell therapies) can be beneficial in the treatment of cancer or other diseases or disorders. Improved approaches are needed. Provided are methods, uses and articles of manufacture that meet such needs.Summary

[0005] In one aspect, provided herein is a method of treating a B-cell non-Hodgkin lymphoma (B- cell NHL), wherein the method includes administering to a subject having or suspected of having a B-cell NHL a composition including engineered T cells expressing a chimeric antigen receptor (CAR) thattargets CD19, wherein the composition includes CD4+T cells expressing the CAR and CD8+T cells expressing the CAR, and the composition includes between at or about 5 x 106CAR-expressing T cells and at or about 25 x 106CAR-expressing T cells, inclusive; and at least or at least about 80% of the cells in the composition are CD3+cells.

[0006] In one aspect, provided herein is a method of treating a B-cell non-Hodgkin lymphoma (B- cell NHL), wherein the method includes administering to a subject having or suspected of having a B-cell NHL a composition including engineered T cells expressing a chimeric antigen receptor (CAR) that targets CD19, wherein the composition includes CD4+T cells expressing the CAR and CD8+T cells expressing the CAR at a ratio between about 1:2.5 and about 2.5:1, and the composition includes between at or about 5 x 106CAR-expressing T cells and at or about 100 x 106CAR-expressing T cells, inclusive, and at least or at least about 90% of the cells in the composition are CD3+cells.

[0007] In one aspect, provided herein is a method of treating a B-cell non-Hodgkin lymphoma (B- cell NHL), the method including administering to a subject having or suspected of having a B-cell NHL, a composition including engineered T cells expressing a chimeric antigen receptor (CAR) that targets CD19, wherein the composition includes CD4+ T cells expressing the CAR and CD8+ T cells expressing the CAR at a ratio between about 1:1 and about 2.5:1, and the composition includes between at or about 5 x 106CAR-expressing T cells and at or about 50 x 106CAR-expressing T cells, inclusive, and at least or at least about 80% of the cells in the composition are CD3+cells.

[0008] In one aspect, provided herein is a method of treating a B-cell non-Hodgkin lymphoma (r / r B-cell NHL), the method including administering to a subject having or suspected of having a B-cell NHL a composition including engineered T cells expressing a chimeric antigen receptor (CAR) that targets CD19, wherein the composition includes CD4+ T cells expressing the CAR and CD8+ T cells expressing the CAR, and the composition includes between at or about 5 x 106CAR-expressing T cells and at or about 100 x 106CAR-expressing T cells, inclusive, and at least or at least about 80% of the cells in the composition are CD3+cells, and at least or at least about 80% of the CAR+T cells in the composition are of a naive-like or central memory phenotype.

[0009] In one aspect, provided herein is a method of treating a B-cell non-Hodgkin lymphoma (r / r B-cell NHL), the method including administering to a subject having or suspected of having a B-cell NHL a composition including engineered T cells expressing a chimeric antigen receptor (CAR) that targets CD19, wherein the composition includes CD4+ T cells expressing the CAR and CD8+ T cells expressing the CAR, and the composition includes between at or about 5 x 106CAR-expressing T cells and at or about 100 x 106CAR-expressing T cells, inclusive, and at least or at least about 80% of the cells in the composition are CD3+cells; and at least or at least about 50% of the CD4+CAR+T cells in the composition are CD27+CCR7+and / or at least or at least about 50% of the CD8+CAR+T cells in the composition are CD27+CCR7+.

[0010] In one aspect, provided herein is a method of treating a B-cell non-Hodgkin lymphoma (B- cell NHL), the method including administering to a subject having or suspected of having a B-cell NHL a composition including engineered T cells expressing a chimeric antigen receptor (CAR) that targets CD19, wherein the composition includes CD4+ T cells expressing the CAR and CD8+ T cells expressing the CAR, and the composition includes between at or about 5 x 106CAR-expressing T cells and at or about 50 x 106CAR-expressing T cells, inclusive, and at least or at least about 96% of the cells in the composition are CD3+cells, and at least or at least about 80% of the CAR+T cells in the composition are of a naive -like or central memory phenotype.

[0011] In one aspect, provided herein is a method of treating a B-cell non-Hodgkin lymphoma (r / r B-cell NHL), the method including administering to a subject having or suspected of having a B-cell NHL a composition including engineered T cells expressing a chimeric antigen receptor (CAR) that targets CD19, wherein the composition includes CD4+ T cells expressing the CAR and CD8+ T cells expressing the CAR, and the composition includes between at or about 5 x 106CAR-expressing T cells and at or about 100 x 106CAR-expressing T cells, inclusive, and at least or at least about 80% of the cells in the composition are CD3+cells; and the fraction of integrated vector copy number (iVCN) to total VCN in the CAR+T cells in the composition, on average, is less than or less than about 0.9.

[0012] In one aspect, provided herein is a method of treating a B-cell non-Hodgkin lymphoma (r / r B-cell NHL), the method including administering to a subject having or suspected of having a B-cell NHL a composition including engineered T cells expressing a chimeric antigen receptor (CAR) that targets CD19, wherein the composition includes CD4+ T cells expressing the CAR and CD8+ T cells expressing the CAR, and the composition includes between at or about 5 x 106CAR-expressing T cells and at or about 100 x 106CAR-expressing T cells, inclusive, and at least or at least about 80% of the cells in the composition are CD3+cells; and the integrated vector copy number (iVCN) of the CAR+T cells in the composition, on average, is between or between about 0.4 copies per diploid genome and 3.0 copies per diploid genome, inclusive.

[0013] In any of the embodiments herein, the composition comprises CD4+T cells expressing the CAR and CD8+T cells expressing the CAR at a ratio between about 1:2.5 and about 2.5:1.

[0014] In any of the embodiments herein, the composition comprises between at or about 5 x 106CAR-expressing T cells and at or about 50 x 106CAR-expressing T cells, inclusive. In any of the embodiments herein, the composition comprises between at or about 5 x 106CAR-expressing T cells and at or about 25 x 106CAR-expressing T cells, inclusive.

[0015] In any of the embodiments herein, the composition may include CD4+T cells expressing the CAR and CD8+T cells expressing the CAR at a ratio between about 1:2 and about 2:1, between about 1:1.5 and about 1.5:1, or at or at about 1:1. In any of the embodiments herein, the composition may include CD4+T cells expressing the CAR and CD8+T cells expressing the CAR at a ratio between about 1:1 and about 2.5:1, between about 1.5:1 and about 2:1, at or at about 1.5:1, or at or at about 2:1. In anyof the embodiments herein, the composition may include between at or about 5 x 106CAR-expressing T cells and at or about 10 x 106CAR-expressing T cells, inclusive. In any of the embodiments herein, the composition may include between at or about 10 x 106CAR-expressing T cells and at or about 25 x 106CAR-expressing T cells, inclusive. In any of the embodiments herein, the composition may include at or about 5 x 106CAR-expressing T cells. In any of the embodiments herein, the composition may include at or about 10 x 106CAR-expressing T cells. In any of the embodiments herein, the composition may include at or about 25 x 106CAR-expressing T cells. In any of the embodiments herein, the composition may include at or about 50 x 106CAR-expressing T cells. In any of the embodiments herein, the composition may include at or about 100 x 106CAR-expressing T cells.

[0016] In any of the embodiments herein, at least or at least about 90% of the cells in the composition are CD3+cells.

[0017] In any of the embodiments herein, at least or at least about 80% of the CAR+T cells in the composition are of a naive-like or central memory phenotype.

[0018] In any of the embodiments herein, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, or at least or at least about 96% of the cells in the composition are CD3+cells. In any of the embodiments herein, between at or about 5% and at or about 30%, optionally between at or about 5% and at or about 30%, of the CAR+T cells in the composition express a marker of apoptosis, optionally Annexin V or active Caspase 3. In any of the embodiments herein, between at or about 10% and at or about 15% of the CAR+T cells in the composition express a marker of apoptosis, optionally Annexin V or active Caspase 3. In any of the embodiments herein, between at or about 15% and at or about 20% of the CAR+T cells in the composition express a marker of apoptosis, optionally Annexin V or active Caspase 3. In any of the embodiments herein, between at or about 20% and at or about 25% of the CAR+T cells in the composition express a marker of apoptosis, optionally Annexin V or active Caspase 3. In any of the embodiments herein, between at or about 25% and at or about 30% of the CAR+T cells in the composition express a marker of apoptosis, optionally Annexin V or active Caspase 3. In any of the embodiments herein, at or about 5%, at or about 10%, at or about 15%, at or about 20%, at or about 25%, or at or about 30% of the CAR+T cells in the composition express a marker of apoptosis, optionally Annexin V or active Caspase 3. In any of the embodiments herein, between at or about 80% and at or about 85% of the CAR+T cells in the composition are of a naive -like or central memory phenotype. In any of the embodiments herein, between at or about 85% and at or about 90% of the CAR+T cells in the composition are of a naive-like or central memory phenotype. In any of the embodiments herein, between at or about 90% and at or about 95% of the CAR+T cells in the composition are of a naive -like or central memory phenotype. In any of the embodiments herein, between at or about 95% and at or about 99% of the CAR+T cells in the composition are of a naive-like or central memory phenotype. In any of theembodiments herein, at or about 85%, at or about 90%, at or about 95%, or at or about 99% of the CAR+T cells in the composition are of a naive -like or central memory phenotype.

[0019] In some of any of the embodiments herein, the marker of apoptosis is Annexin V. In some of any of the embodiments herein, the marker of apoptosis is active Caspase 3.

[0020] In any of the embodiments herein, the at least or at least about 80% of the CAR+T cells in the composition that are of a naive-like or central memory phenotype are surface positive for a marker expressed on naive -like or central memory T cells. In some embodiments, the marker expressed on naive-like or central memory T cell is selected from the group consisting of CD45RA, CD27, CD28, and CCR7.

[0021] In some of any embodiments, the at least or at least about 80% of the CAR+T cells in the composition that are of a naive -like or central memory phenotype have a phenotype selected from CCR7+CD45RA+, CD27+CCR7+, or CD62L CCR7+. In some of any embodiments, between at or about 80% and at or about 85%, between at or about 85% and at or about 90%, between at or about 90% and at or about 95%, between at or about 95% and at or about 99% of the CAR+T cells in the composition are of a naive -like or central memory phenotype selected from CCR7+CD45RA+, CD27+CCR7+, or CD62L CCR7+. In some of any embodiments, at or about 80%, at or about 85%, at or about 90%, at or about 95%, or at or about 99% of the CAR+T cells in the composition are of a naive-like or central memory phenotype selected from CCR7+CD45RA+, CD27+CCR7+, or CD62L CCR7+. In some of any embodiments, at or about 80%, at or about 85%, at or about 90%, at or about 95%, or at or about 99% of the CAR+T cells in the composition are of a naive-like or central memory phenotype that is CD27+CCR7+.

[0022] In some of any embodiments, at least or at least about 50% of the CD4+CAR+T cells in the composition are of a naive-like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA . In some of any embodiments, at least or at least about 60% of the CD4+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA . In some of any embodiments, at least or at least about 70% of the CD4+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA . In some of any embodiments, at least or at least about 80% of the CD4+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA . In some of any embodiments, at least or at least about 85% of the CD4+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA .

[0023] In some of any embodiments, at least or at least about 50% of the CD4+CAR+T cells in the composition are of a naive-like or central memory phenotype that is CD27+CCR7+. In some of any embodiments, at least or at least about 60% of the CD4+CAR+T cells in the composition are of a naive- like or central memory phenotype that is CD27+CCR7+. In some of any embodiments, at least or at leastabout 70% of the CD4+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CD27+CCR7+. In some of any embodiments, at least or at least about 80% of the CD4+CAR+T cells in the composition are of a naive-like or central memory phenotype that is CD27+CCR7+. In some of any embodiments, at least or at least about 85% of the CD4+CAR+T cells in the composition are of a naive-like or central memory phenotype that is CD27+CCR7+.

[0024] In some of any embodiments, at least or at least about 50% of the CD8+CAR+T cells in the composition are of a naive-like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA . In some of any embodiments, at least or at least about 60% of the CD8+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA . In some of any embodiments, at least or at least about 70% of the CD8+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA . In some of any embodiments, at least or at least about 80% of the CD8+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA . In some of any embodiments, at least or at least about 85% of the CD8+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA .

[0025] In some of any embodiments, at least or at least about 50% of the CD8+CAR+T cells in the composition are of a naive-like or central memory phenotype that is CD27+CCR7+. In some of any embodiments, at least or at least about 60% of the CD8+CAR+T cells in the composition are of a naive- like or central memory phenotype that is CD27+CCR7+. In some of any embodiments, at least or at least about 70% of the CD8+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CD27+CCR7+. In some of any embodiments, at least or at least about 80% of the CD8+CAR+T cells in the composition are of a naive-like or central memory phenotype that is CD27+CCR7+. In some of any embodiments, at least or at least about 85% of the CD8+CAR+T cells in the composition are of a naive-like or central memory phenotype that is CD27+CCR7+.

[0026] In any of the embodiments herein, at least or at least about 80% of the CAR+T cells in the composition are surface positive for a marker expressed on naive-like or central memory T cells. In any of the embodiments herein, the marker expressed on naive-like or central memory T cell is selected from the group consisting of CD45RA, CD27, CD28, and CCR7. In any of the embodiments herein, at least or at least about 80% of the CAR+T cells in the composition are CCR7+CD45RA+, CD27+CCR7+, and / or CD62L CCR7+. In any of the embodiments herein, between at or about 80% and at or about 85%, between at or about 85% and at or about 90%, between at or about 90% and at or about 95%, between at or about 95% and at or about 99% of the CAR+T cells in the composition are CCR7+CD45RA+, CD27+CCR7+, and / or CD62L CCR7+. In any of the embodiments herein, at or about 80%, at or about 85%, at or about 90%, at or about 95%, or at or about 99% of the CAR+T cells in the composition are CCR7+CD45RA+, CD27+CCR7+, and / or CD62L CCR7+. In any of the embodiments herein, at or about80%, at or about 85%, at or about 90%, at or about 95%, or at or about 99% of the CAR+T cells in the composition are CD27+CCR7+. In any of the embodiments herein, at least or at least about 50% of the CD4+CAR+T cells in the composition are CCR7+CD45RA+or CCR7+CD45RA . In any of the embodiments herein, at least or at least about 60% of the CD4+CAR+T cells in the composition are CCR7+CD45RA+or CCR7+CD45RA . In any of the embodiments herein, at least or at least about 70% of the CD4+CAR+T cells in the composition are CCR7+CD45RA+or CCR7+CD45RA . In any of the embodiments herein, at least or at least about 80% of the CD4+CAR+T cells in the composition are CCR7+CD45RA+or CCR7+CD45RA . In any of the embodiments herein, at least or at least about 85% of the CD4+CAR+T cells in the composition are CCR7+CD45RA+or CCR7+CD45RA . In any of the embodiments herein, at least or at least about 50% of the CD4+CAR+T cells in the composition are CD27+CCR7+. In any of the embodiments herein, at least or at least about 60% of the CD4+CAR+T cells in the composition are CD27+CCR7+. In any of the embodiments herein, at least or at least about 70% of the CD4+CAR+T cells in the composition are CD27+CCR7+. In any of the embodiments herein, at least or at least about 80% of the CD4+CAR+T cells in the composition are CD27+CCR7+. In any of the embodiments herein, at least or at least about 85% of the CD4+CAR+T cells in the composition are CD27+CCR7+.

[0027] In any of the embodiments herein, at least or at least about 50% of the CD8+CAR+T cells in the composition are CCR7+CD45RA+or CCR7+CD45RA . In any of the embodiments herein, at least or at least about 60% of the CD8+CAR+T cells in the composition are CCR7+CD45RA+or CCR7+CD45RA . In any of the embodiments herein, at least or at least about 70% of the CD8+CAR+T cells in the composition are CCR7+CD45RA+or CCR7+CD45RA . In any of the embodiments herein, at least or at least about 80% of the CD8+CAR+T cells in the composition are CCR7+CD45RA+or CCR7+CD45RA . In any of the embodiments herein, at least or at least about 85% of the CD8+CAR+T cells in the composition are CCR7+CD45RA+or CCR7+CD45RA . In any of the embodiments herein, at least or at least about 50% of the CD8+CAR+T cells in the composition are CD27+CCR7+. In any of the embodiments herein, at least or at least about 60% of the CD8+CAR+T cells in the composition are CD27+CCR7+. In any of the embodiments herein, at least or at least about 70% of the CD8+CAR+T cells in the composition are CD27+CCR7+. In any of the embodiments herein, at least or at least about 80% of the CD8+CAR+T cells in the composition are CD27+CCR7+. In any of the embodiments herein, at least or at least about 85% of the CD8+CAR+T cells in the composition are CD27+CCR7+.

[0028] In any of the embodiments herein, the fraction of integrated vector copy number (iVCN) to total VCN in the CAR+T cells in the composition, on average, is less than or less than about 0.9.

[0029] In any of the embodiments herein, the fraction of integrated vector copy number (iVCN) to total VCN in the CAR+T cells in the composition, on average, is between at or about 0.9 and at or about 0.8. In any of the embodiments herein, the fraction of integrated vector copy number (iVCN) to total VCN in the CAR+T cells in the composition, on average, is less than or less than about 0.8. In any of theembodiments herein, the fraction of integrated vector copy number (iVCN) to total VCN in the CAR+T cells in the composition, on average, is between at or about 0.8 and at or about 0.7. In any of the embodiments herein, the fraction of integrated vector copy number (iVCN) to total VCN in the CAR+T cells in the composition, on average, is between at or about 0.7 and at or about 0.6. In any of the embodiments herein, the fraction of integrated vector copy number (iVCN) to total VCN in the CAR+T cells in the composition, on average, is between at or about 0.6 and at or about 0.5. In any of the embodiments herein, the fraction of integrated vector copy number (iVCN) to total VCN in the CAR+T cells in the composition, on average, is between at or about 0.5 and at or about 0.4.

[0030] In any of the embodiments herein, the integrated vector copy number (iVCN) of the CAR+T cells in the composition, on average, is between or between about 0.4 copies per diploid genome and 3.0 copies per diploid genome, inclusive.

[0031] In any of the embodiments herein, the integrated vector copy number (iVCN) of the CAR+T cells in the composition, on average, is between or between about 0.8 copies per diploid genome and 2.0 copies per diploid genome, inclusive. In any of the embodiments herein, the integrated vector copy number (iVCN) of the CAR+T cells in the composition, on average, is between or between about 0.8 copies per diploid genome and 1.0 copies per diploid genome, inclusive. In any of the embodiments herein, the integrated vector copy number (iVCN) of the CAR+T cells in the composition, on average, is between or between about 1.0 copies per diploid genome and 1.5 copies per diploid genome, inclusive. In any of the embodiments herein, the integrated vector copy number (iVCN) of the CAR+T cells in the composition, on average, is between or between about 1.5 copies per diploid genome and 2.0 copies per diploid genome, inclusive.

[0032] In any of the embodiments herein, the r / r B-cell NHL is selected from the group consisting of a diffuse large B-cell lymphoma (DLBCL), optionally DLBCL not otherwise specified (DLBCL NOS; including de novo or transformed DLBCL, e.g., from follicular lymphoma or marginal zone lymphoma); high grade B-cell lymphoma (HGBCL), optionally HGBCL with MYC and BCL2 and / or BCL6 rearrangements with DLBCL histology; primary mediastinal large B cell lymphoma (PMBCL); and follicular lymphoma (FL), optionally follicular lymphoma grade 3b (FL3B).

[0033] In any of the embodiments herein, the B-cell NHL is selected from the group consisting of: diffuse large B-cell lymphoma (DLBCL), optionally DLBCL not otherwise specified; transformed DLBCL, optionally transformed DLBCL from follicular lymphoma or marginal zone lymphoma; high grade B-cell lymphoma (HGBCL), optionally HGBCL with MYC and BCL2 and / or BCL6 rearrangements with DLBCL histology; primary mediastinal large B cell lymphoma (PMBCL); and follicular lymphoma (FL), optionally follicular lymphoma grade 3b (FL3B).

[0034] In any of the embodiments herein, the B-cell NHL is a diffuse large B-cell lymphoma. In any of the embodiments herein, the B-cell NHL is a diffuse large B-cell lymphoma not otherwise specified.In any of the embodiments herein, the B-cell NHL is a de novo diffuse large B-cell lymphoma. In any ofthe embodiments herein, the B-cell NHL is a diffuse large B-cell lymphoma transformed from a follicular lymphoma. In any of the embodiments herein, the B-cell NHL is a diffuse large B-cell lymphoma transformed from a marginal zone lymphoma. In any of the embodiments herein, the B-cell NHL is a high grade B-cell lymphoma (HGBCL). In any of the embodiments herein, the B-cell NHL is a high grade B-cell lymphoma with MYC and BCL2 and / or BCL6 rearrangements. In any of the embodiments herein, the B-cell NHL is a high grade B-cell lymphoma with DLBCL histology. In any of the embodiments herein, the B-cell NHL is a double-hit lymphoma or triple -hit lymphoma. In any of the embodiments herein, the B-cell NHL is a primary mediastinal large B cell lymphoma. In any of the embodiments herein, the B-cell NHL is a follicular lymphoma. In any of the embodiments herein, the B- cell NHL is a follicular lymphoma grade 3b. In any of the embodiments herein, the B-cell NHL is histologically confirmed.

[0035] In any of the embodiments herein, at or immediately prior to the time of the administration of the composition comprising engineered T cells, the subject has relapsed following remission after treatment with, or become refractory to: (i) two or more prior therapies for the B-cell NHL and / or (ii) an autologous stem cell transplant (ASCT) therapy.

[0036] In any of the embodiments herein, at or immediately prior to the time of the administration of the composition comprising engineered T cells, the subject has relapsed following remission after treatment with, or become refractory to, two or more prior therapies for the B-cell NHL. In any of the embodiments herein, at or immediately prior to the time of the administration of the composition comprising engineered T cells, the subject has relapsed following remission after treatment with, or become refractory to, three or more prior therapies for the B-cell NHL. In any of the embodiments herein, the two or more prior therapies for the r / r B-cell NHL does not comprise another dose of cells expressing the CAR.

[0037] In any of the embodiments herein, the two or more prior therapies for the B-cell NHL comprise an anthracycline and a CD20-targeted agent. In any of the embodiments herein, the two or more prior therapies for the B-cell NHL do not include those given for prior indolent lymphoma. In any of the embodiments herein, the two or more prior therapies for the B-cell NHL do not include anthracycline given for indolent DLBCL. In any of the embodiments herein, the two or more prior therapies for the B- cell NHL comprise a CD20-targeted agent, and the two or more prior therapies for the B-cell NHL excludes an anthracycline if it is given for prior indolent lymphoma. In any of the embodiments herein, the CD20-targeted agent comprises an anti-CD20 monoclonal antibody. In any of the embodiments herein, the CD20-targeted agent comprises rituximab.

[0038] In any of the embodiments herein, at or immediately prior to the time of the administration of the composition comprising engineered T cells, the subject has relapsed following remission after treatment with, or become refractory to, an autologous stem cell transplant (ASCT) therapy. In some embodiments, the subject has relapsed and / or refractory DLBCL. In some embodiments, the ASCT failsto achieve an objective response (partial response (PR) or better). In any of the embodiments herein, after the ASCT, the disease of the subject has progressed.

[0039] In any of the embodiments herein, at or prior to the administration of the composition comprising engineered T cells, the subject has been identified as having an aggressive disease or a high- risk disease, or as having poor prognosis. In any of the embodiments herein, at or prior to the administration of the composition comprising engineered T cells, the subject has been identified as having a chemorefractory disease or as having a persistent or relapsed disease following chemotherapy.In any of the embodiments herein, the subject has a pathologically-confirmed secondary central nervous system (CNS) involvement by malignancy. In any of the embodiments herein, the subject does not have a central nervous system (CNS)-only involvement by malignancy.

[0040] In any of the embodiments herein, the subject has not received a prior CAR T cell or genetically-modified T cell therapy. In any of the embodiments herein, the subject has not received prior CD19-targeted therapy such as an anti-CD19 monoclonal antibody or bispecific antibody.

[0041] In any of the embodiments herein, the method further comprises obtaining a leukapheresis sample from the subject for manufacturing the composition comprising engineered T cells. In some embodiments, the subject has not received a therapeutic dose of a corticosteroid, optionally within at or about 14 days prior to the time of leukapheresis. In some embodiments, the subject has not received a cytotoxic chemotherapeutic agent, which is not a lymphotoxic chemotherapeutic agent or intrathecal therapy, within at or about 7 days prior to the time of leukapheresis. In any of the embodiments herein, the subject has not received a lymphotoxic chemotherapeutic agent within at or about 4 weeks prior to the time of leukapheresis. In any of the embodiments herein, the subject has not received an immunosuppressive therapy within at or about 4 weeks prior to the time of leukapheresis. In any of the embodiments herein, the subject has not received radiation within at or about 6 weeks prior to the time of leukapheresis. In any of the embodiments herein, the subject has not received an autologous stem-cell transplant within at or about 3 months prior to the time of leukapheresis.

[0042] In any of the embodiments herein, the subject has not achieved complete remission (CR) in response to a prior therapy. In any of the embodiments herein, the subject has not achieved an objective response (partial response (PR) or better) in response to a prior therapy.

[0043] In any of the embodiments herein, at or prior to the administration of the composition comprising engineered T cells, the subject is evaluated for a lymphoma associated with or involving central nervous system (CNS) involvement or a secondary CNS lymphoma. In any of the embodiments herein, at or prior to the administration of the composition comprising engineered T cells, the subject is or has been identified as having: an adequate cardiac function, optionally with a left ventricular ejection fraction (LVEF) at or about 40%, greater than 40%, or great than about 40%; and / or an adequate renal function, optionally with a calculated creatinine clearance of at or about 45 mL / min, greater than 45 mL / min, or greater than about 45 mL / min; and / or an adequate hepatic function, optionally with anaspartate aminotransferase (AST) and alanine aminotransferase (ALT) of or less than 2.5 times the upper limit of normal (ULN) and totol bilirubin less than 1.5 times the ULN; and / or an adequate pulmonary function, optionally with < CTCAE Grade 1 dyspnea and saturated oxygen (e.g., SaCL > 92%) on room air. In any of the embodiments herein, at or prior to the administration of the composition comprising engineered T cells, the subject is or has been identified as having an absolute neutrophil count (ANC) of at or about 1.0 x 109cells / L, greater than 1.0 x 109cells / L, or greater than about 1.0 x 109cells / L without growth factor support. In any of the embodiments herein, at or prior to the administration of the composition comprising engineered T cells, the subject is or has been identified as having platelets of at or about 50 x 109cells / L, greater than 50 x 109cells / L, or greater than about 50 x 109cells / L without transfusion support.

[0044] In any of the embodiments herein, at or prior to the administration of the composition comprising engineered T cells, the subject has received a bridging chemotherapy between the time of leukapheresis to produce the composition comprising engineered T cells and the administration of the composition comprising engineered T cells. In any of the embodiments herein, at or prior to the administration of the composition comprising engineered T cells, the subject has received a bridging chemotherapy for disease control after a prior therapy.

[0045] In any of the embodiments herein, the subject is or has been identified as having an Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 0 or 1. In any of the embodiments herein, prior to the administration of the composition comprising engineered T cells, the subject is or has been identified as having a high baseline tumor burden, such as measured by the sum of product of the perpendicular diameters (SPD), or high serum lactate dehydrogenase (LDH), such as LDH > 500 U / L. In any of the embodiments herein, at or prior to the administration of the composition comprising engineered T cells, the subject has a positron emission tomography (PET) -positive disease.

[0046] In any of the embodiments herein, the method further comprises, prior to administration of the composition comprising engineered T cells, identifying or selecting the subject for administration of the composition comprising engineered T cells.

[0047] In any of the embodiments herein, prior to the administration, the subject has been preconditioned with a lymphodepleting therapy. In any of the embodiments herein, the method further comprises, immediately prior to the administration of the composition comprising engineered T cells, administering a lymphodepleting therapy to the subject, wherein the lymphodepleting therapy comprises the administration of fludarabine and / or cyclophosphamide.

[0048] In any of the embodiments herein, the administration of the composition comprising engineered T cells and / or the lymphodepleting therapy is carried out via outpatient delivery. In some of any embodiments, the administration of the composition is carried out on an outpatient basis, optionally unless or until the subject exhibits a sustained fever or a fever that is or has not been reduced or not reduced by more than 1°C after treatment with an antipyretic. In some of any embodimetns, theadministration of the composition is without admitting the subject to a hospital and / or without an overnight stay at a hospital, optionally unless or until the subject exhibits a sustained fever or a fever that is or has not been reduced or not reduced by more than 1°C after treatment with an antipyretic. In some of any embodiments, the administration of the composition is without requiring admission to or an overnight stay at a hospital, optionally unless or until the subject exhibits a sustained fever or a fever that is or has not been reduced or not reduced by more than 1°C after treatment with an antipyretic.

[0049] In any of the embodiments herein, the lymphodepleting therapy comprises the administration of fludarabine at 30 mg / m2body surface area of the subject, daily, and cyclophosphamide at 300 mg / m2body surface area of the subject, daily, each for 3 days. In any of the embodiments herein, the composition comprising engineered T cells is administered between at or about 48 hours and at or about 9 days, inclusive, after completion of the lymphodepleting therapy. In any of the embodiments herein, the subject has a creatinine clearance of at least or at least about 30 mL / min when receiving the lymphodepleting therapy.

[0050] In any of the embodiments herein, prior to initiation of administration of the composition comprising engineered T cells, the subject has not been administered an agent or treatment for the treatment, prevention, reduction, or attenuation of a neurotoxicity and / or a cytokine release syndrome or risk thereof. In any of the embodiments herein, the method further comprises administering to the subject an agent or treatment for the treatment, prevention, reduction, or attenuation of a neurotoxicity and / or a cytokine release syndrome or risk thereof. In any of the embodiments herein, the agent is or comprises an anti-IL-6 antibody, an anti-IL-6 receptor antibody, or a steroid. In any of the embodiments herein, the agent is or comprises tocilizumab or methylprednisolone.

[0051] In any of the embodiments herein, the T cells are primary T cells obtained from a subject.

[0052] In any of the embodiments herein, the T cells are autologous to the subject.

[0053] In any of the embodiments herein, at least 35%, at least 40% or at least 50% of subjects treated according to the method achieve a complete response (CR); at least 60%, 70%, 80%, 90%, or 95% of subjects achieving a CR exhibit a CR that is durable for at or greater than 3 months or at or greater than 6 months; and / or at least 60%, 70%, 80%, 90%, or 95% of subjects achieving a CR by one month and / or by 3 months remain in response, remain in CR, and / or survive or survive without progression, for at or greater than 3 months and / or at or greater than 6 months and / or at greater than 9 months after achieving the CR; and / or at least 50%, at least 60% or at least 70% of the subjects treated according to the method achieve objective response (OR); at least 60%, 70%, 80%, 90%, or 95% of subjects achieving an OR exhibit an OR that is durable for at or greater than 3 months or at or greater than 6 months; and / or at least 35%, at least 40%, or at least 50% of subjects achieving an OR remain in response or survive for at or greater than 3 months and / or at or greater than 6 months after achieving the OR.

[0054] In any of the embodiments herein, at least 50% of subjects treated according to the method achieve a complete response (CR); at least 60% of subjects achieving a CR exhibit a CR that is durable for at or greater than 6 months; and / or at least 60% of subjects achieving a CR by 1 month and / or by 3 months remain in response, remain in CR, and / or survive or survive without progression, for at or greater than 6 months after achieving the CR; and / or at least 70% of the subjects treated according to the method achieve objective response (OR); at least 60% of subjects achieving an OR exhibit an OR that is durable for at or greater than 6 months; and / or at least 50% of subjects achieving an OR remain in response or survive for at or greater than 6 months after achieving the OR.

[0055] In any of the embodiments herein, the CR or the OR is durable for greater than 3 months or greater than 6 months; and / or at least 20%, at least 25%, at least 35%, at least 40% or at least 50% of subjects treated according to the method achieve a CR that is durable for greater than 3 months or greater than 6 months; and / or at least 60%, 70%, 80%, 90%, or 95% of subjects treated with the method and who achieve a CR, remain in CR or remain in response or remain surviving for at or greater than 3 months or at or greater than 6 months or at or greater than 9 months; and / or at least 60%, 70%, 80%, 90%, or 95% of subjects treated with the method who achieve a CR by one month and / or by 3 months remain in response, remain in CR, and / or survive or survive without progression, for greater at or greater than 3 months and / or at or greater than 6 months and / or at greater than 9 months; and / or at least 50%, at least 60% or at least 70% of the subjects treated according to the method achieve objective response (OR); at least 60%, 70%, 80%, 90%, or 95% of subjects achieve an OR that is durable for at or greater than 3 months or at or greater than 6 months; and / or at least at least 35%, at least 40%, or at least 50% of subjects treated with the method and achieving an OR remain in response or survive for at or greater than 3 months and / or at or greater than 6 months.

[0056] In any of the embodiments herein, the cells are autologous to the subject, and no minimum absolute lymphocyte count (ALC) for apheresis is required and / or specified for production of the therapy; and / or the cells are produced by a process which, for at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of subjects having the B-cell NHL, is capable of generating a cell product for administration according to the method.

[0057] In any of the embodiments herein, at least 35%, at least 40% or at least 50% of subjects treated according to the method achieve a complete response (CR) or remission of CNS disease; at least 60%, 70%, 80%, 90%, or 95% of subjects who achieve a CR remain in CR for at or greater than 3 months or at or greater than 6 months; and / or at least 60%, 70%, 80%, 90%, or 95% of subjects achieving a CR or remission of CNS disease by one month and / or by 3 months remain in response, remain in CR, and / or survive or survive without progression, for greater at or greater than 3 months and / or at or greater than 6 months and / or at greater than 9 months; and / or at least 50%, at least 60% or at least 70% of the subjects treated according to the method achieve objective response (OR) or remission of CNS disease; at least 60%, 70%, 80%, 90%, or 95% of subjects achieving the OR, for at or greaterthan 3 months or at or greater than 6 months; and / or at least 60%, 70%, 80%, 90%, or 95% of subjects achieving OR or remission of CNS disease remain in response or survive for at or greater than 3 months and / or at or greater than 6 months; and / or the brain lesion is reduced in size or volume by greater than or greater than about 25%, 50%, 75% or more; and / or reduction or remission or clearance of CNS disease is achieved in at least 35%, at least 40% or at least 50% of subjects treated according to the method.

[0058] In any of the embodiments herein, greater than or greater than about 50%, about 60%, about 70%, or about 80% of the subjects treated according to the method do not exhibit a grade 3 or greater cytokine release syndrome (CRS) and / or do not exhibit a grade 3 or greater neurotoxicity and / or greater than 40% or 50% or 55% of the subjects treated according to the method do not exhibit any neurotoxicity or CRS. In any of the embodiments herein, greater than or greater than about 80% of the subjects treated according to the method do not exhibit a grade 3 or greater cytokine release syndrome (CRS) and / or do not exhibit a grade 3 or greater neurotoxicity. In any of the embodiments herein, greater than 95% of the subjects treated according to the method do not exhibit grade 3 or greater CRS. In any of the embodiments herein, greater than 85% of the subjects treated according to the method do not exhibit grade 3 or greater neurotoxicity.

[0059] In any of the embodiments herein, greater than or greater than about 30%, 35%, 40%, or 50% of the subjects treated according to the method do not exhibit any grade of cytokine release syndrome (CRS) or neurotoxicity; and / or at least at or about 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of subjects treated according to the method do not exhibit onset of CRS earlier than 3 days following initiation of the administration and / or do not exhibit onset of neurotoxicity earlier than 5 days following initiation of the administration; and / or the median onset of neurotoxicity among subjects treated according to the method is at or after the median peak of, or median time to resolution of, CRS in subjects treated according to the method and / or the median onset of neurotoxicity among subjects treated according to the method is greater than at or about 8, 9, 10, or 11 days.

[0060] In any of the embodiments herein, greater than or greater than about 50% of the subjects treated according to the method do not exhibit any grade of cytokine release syndrome (CRS) or neurotoxicity; and / or at least at or about 45% of subjects treated according to the method do not exhibit onset of CRS earlier than 3 days following initiation of the administration and / or do not exhibit onset of neurotoxicity earlier than 5 days following initiation of the administration; and / or the median onset of neurotoxicity among subjects treated according to the method is at or after the median peak of, or median time to resolution of, CRS in subjects treated according to the method and / or the median onset of neurotoxicity among subjects treated according to the method is greater than at or about 8 days.

[0061] In any of the embodiments herein, at least 50% of subjects treated according to the method achieve a complete response (CR); at least 70% of the subjects treated according to the method achieve objective response (OR); and greater than or greater than about 50% of the subjects treated according to the method do not exhibit any grade of cytokine release syndrome (CRS) or neurotoxicity; and greaterthan or greater than about 80% of the subjects treated according to the method do not exhibit a grade 3 or greater cytokine release syndrome (CRS) and / or do not exhibit a grade 3 or greater neurotoxicity.

[0062] In any of the embodiments herein, the CAR may include an extracellular antigen-binding domain 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 any embodiments, the CAR may include, in order, an extracellular antigen-binding domain specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule; or the CAR may include an extracellular antigen-recognition domain that specifically binds to the antigen and an intracellular signaling domain including a CD3-zeta ( ϋ3z) chain and a costimulatory signaling region that is a signaling domain of 4-1BB. In any of the embodiments herein, the CAR may include an extracellular antigen-binding domain specific for CD19, a transmembrane domain, a cytoplasmic signaling domain derived from a 4- IBB, and a cytoplasmic signaling domain derived from a CD3zeta.

[0063] In any of the embodiments herein, the CAR comprises an extracellular antigen-binding domain specific for CD19, 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; the CAR comprises, in order, an extracellular antigen-binding domain specific for CD19, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule; or the CAR comprises an extracellular antigen-recognition domain that specifically binds to CD 19 and an intracellular signaling domain comprising a CD3-zeta (OΏ3z) chain and a costimulatory signaling region that is a signaling domain of 4- IBB.

[0064] In any of the embodiments herein, the CAR comprises an extracellular antigen-binding domain specific for CD19, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule that is 4- IBB, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule that is CD3zeta.

[0065] In any of the embodiments herein, the extracellular antigen-binding domain is an scFv. In any of the embodiments herein, the scFv may include an amino acid sequence of RASQDISKYLN (SEQ ID NO: 35), an amino acid sequence of SRLHSGV (SEQ ID NO: 36), and / or an amino acid sequence of GNTLPYTFG (SEQ ID NO: 37) and / or an amino acid sequence of DYGVS (SEQ ID NO: 38), an amino acid sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), and / or an amino acid sequence of YAMDYWG (SEQ ID NO: 40). In any of the embodiments herein, the six CDR sequences (CDRL1-L3 and CDRH1-H3) of the scFv may include an amino acid sequences RASQDISKYLN (SEQ ID NO: 35),SRLHSGV (SEQ ID NO: 36), GNTLPYTFG (SEQ ID NO: 37), DYGVS (SEQ ID NO: 38), VIWGSETTYYNSALKS (SEQ ID NO: 39), and YAMDYWG (SEQ ID NO: 40). In some embodimetnts, the scFv includes a variable heavy chain region of FMC63 and a variable light chain region of FMC63. In some embodiments, the six CDR sequences (CDRL1-L3 and CDRH1-H3) of the scFv may include 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. In some embodiments, the scFv may be an scFv that binds to the same epitope as or competes for binding with any one of the foregoing. In some embodiments, the scFv includes, in order, a VH, a linker, optionally including a linker set forth in SEQ ID NO: 24, and a VL. In any of the embodiments herein, the scFv includes, in order, a VL, a linker, optionally including a linker set forth in SEQ ID NO: 24, and a VH. In some embodiments, the scFv includes a flexible linker. In some embodiments, the scFv includes the amino acid sequence set forth as SEQ ID NO: 43. In any of the embodiments herein, the scFv is set forth as SEQ ID NO: 43.

[0066] In any of the embodiments herein, the scFv may comprise an amino acid sequence of RASQDISKYLN (SEQ ID NO: 35), an amino acid sequence of SRLHSGV (SEQ ID NO: 36), and / or an amino acid sequence of GNTLPYTFG (SEQ ID NO: 37) and / or an amino acid sequence of DYGVS (SEQ ID NO: 38), an amino acid sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), and / or an amino acid sequence of YAMDYWG (SEQ ID NO: 40). In some embodiments, the scFv comprises a variable heavy chain region of FMC63 and a variable light chain region of FMC63. In some embodiments, the scFv comprises 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. In some embodiments, the scFv comprises, in order, a VH, a linker SEQ ID NO: 24, and a VL. In some embodiments, the scFv comprises a flexible linker. In some embodiments, the scFv comprises the amino acid sequence set forth as SEQ ID NO: 43. In some embodiments, the CD19-directed scFv binds to the same epitope as or competes for binding with any one of the foregoing.

[0067] In any of the embodiments herein, the scFv may include a variable heavy chain region of FMC63 and a variable light chain region of FMC63.

[0068] In some embodiments, the transmembrane domain is a transmembrane 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 or at least about85%, 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 transmembrane- domain is a transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 9 or a sequence of amino acids having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0069] In any of the embodiments herein, the costimulatory signaling region is a signaling domain of 4-1BB. In any of the embodiments herein, the costimulatory domain may include 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. In any of the embodiments herein, the primary signaling domain is a CD3zeta signaling domain. In any of the embodiments herein, the primary signaling domain may include SEQ ID NO: 13, 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.

[0070] In any of the embodiments herein, the cytoplasmic signaling domain derived from a costimulatory molecule is a signaling domain of 4- IBB. In any of the embodiments herein, the cytoplasmic signaling domain derived from a costimulatory molecule 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.

[0071] In any of the embodiments herein, the cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule is a CD3zeta signaling domain. In any of the embodiments herein, the cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule comprises SEQ ID NO: 13, 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.

[0072] In any of the embodiments herein, the CAR further comprises a spacer between the transmembrane domain and the extracellular antigen-binding domain. In some embodiments, the spacer comprises or consists of the sequence of SEQ ID NO: 1, a sequence encoded by SEQ ID NO: 2, the sequence of SEQ ID NO: 30, the sequence of SEQ ID NO: 31, the sequence of SEQ ID NO: 32, the sequence of SEQ ID NO: 33, the sequence of SEQ ID NO: 34, or a variant of any one 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. In some embodiments, the spacer comprises or consists of the formula X1PPX2P, where Xi is glycine, cysteine or arginine and X2 is cysteine or threonine

[0073] In any of the embodiments herein, the CAR may further include a spacer between the transmembrane domain and the scFv. In any of the embodiments herein, the spacer is a polypeptide spacer that includes or consists of all or a portion of an immunoglobulin hinge or a modified version thereof. In some embodiments, the spacer is an IgG4 hinge, or a modified version thereof. In any of the embodiments herein, the spacer is at or about 12 amino acids in length. In any of the embodiments herein, the spacer 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 NO: 34, or a variant of any one 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. In some embodiments, the spacer may include or consist of the formula X1PPX2P, where Xi is glycine, cysteine or arginine and X2 is cysteine or threonine. In any of the embodiments herein, the spacer is a polypeptide spacer that may include orconsist of all or a portion of an immunoglobulin hinge or a modified version thereof or includes about 15 amino acids or less. In some embodiments, the spacer does not include a CD28 extracellular region or a CD8 extracellular region, and may include or consist 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.

[0074] In some embodiments, the spacer 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. In some embodiments, the spacer has or may consist 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 NO: 34, or a variant of any one 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. In some embodiments, the spacer may include or consist of the formula X1PPX2P, where Xi is glycine, cysteine or arginine and X2 is cysteine or threonine. In some of any of the embodiments, the costimulatory domain may include 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. In some of any of the provided embodiments, the primary signaling domain may include SEQ ID NO: 13, 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. In some of any embodiments, the scFv may include an amino acid sequence of RASQDISKYLN (SEQ ID NO: 35), an amino acid sequence of SRLHSGV (SEQ ID NO: 36), an amino acid sequence of GNTLPYTFG (SEQ ID NO: 37), an amino acid sequence of DYGVS (SEQ ID NO: 38), an amino acid sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), and an amino acid sequence of YAMDYWG (SEQ ID NO: 40). In some embodiments, the scFv includes a variable heavy chain region of FMC63 and a variable light chain region of FMC63. In some embodiments, the scFv includes six CDRs that includes 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. In some embodiments, the scFv is an scFv that binds to the same epitope as or competes for binding with any one of the foregoing. In some embodiemnts, the scFv includes, in order, a VH, a linker, optionally a linker including SEQ ID NO: 24, and a VL. In some embodiments, the scFv includes, in order, a V:, a linker, optionally a linker including SEQ ID NO: 24, and a VH. In some embodiments, the scFv includes a flexible linker. In some embodiments, the scFv amino acid sequence is set forth as SEQ ID NO: 43.

[0075] In any of the embodiments herein, (i) the scFv may comprise an amino acid sequence of RASQDISKYLN (SEQ ID NO: 35), an amino acid sequence of SRLHSGV (SEQ ID NO: 36), an amino acid sequence of GNTLPYTFG (SEQ ID NO: 37), an amino acid sequence of DYGVS (SEQ ID NO:38), an amino acid sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), and an amino acid sequence of YAMDYWG (SEQ ID NO: 40), such as the scFv may comprise the amino acid sequence set forth asSEQ ID NO: 43; (ii) the spacer may be a polypeptide spacer that (a) comprises or consists of all or a portion of an immunoglobulin hinge or a modified version thereof is about 15 amino acids or less, and comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof, optionally, such as wherein the spacer may comprise or consist of the sequence of SEQ ID NO: 1, or a sequence encoded by SEQ ID NO: 2; (iii) the costimulatory domain may comprise 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 (iv) the primary signaling domain may comprise SEQ ID NO: 13, 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.

[0076] In any of the embodiments herein, the CAR is one in which (i) the scFv comprises the amino acid sequence set forth as SEQ ID NO: 43; (ii) the spacer is a polypeptide spacer that comprises or consists of the sequence of SEQ ID NO: 1, or a sequence encoded by SEQ ID NO: 2; (iii) the cytoplasmic signaling domain derived from a costimulatory molecule is a costimulatory domain that 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 (iv) the cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule is a cytoplasmic signaling domain that comprises SEQ ID NO: 13, 14 or 15 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.

[0077] In any of the embodiments herein, the CAR is one in which (i) the extracellular antigen binding domain comprises an scFv that comprises a variable heavy chain region of FMC63 and a variable light chain region of FMC63; the spacer is a polypeptide spacer that comprises the sequence of SEQ ID NO: 1; the cytoplasmic signaling domain derived from a costimulatory molecule comprises SEQ ID NO: 12; and the cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule comprises SEQ ID NO: 13, 14 or 15.

[0078] In any of the embodiments herein, the spacer is a polypeptide spacer that may include the sequence of SEQ ID NO: 1; the costimulatory domain may include SEQ ID NO: 12; the primary signaling domain may include SEQ ID NO: 13, 14 or 15; the antigen binding domain may include an scFv that includes a variable heavy chain region of FMC63 and a variable light chain region of FMC63.In any of the embodiments herein, the composition of cells is administered parenterally, optionally intravenously. In any of the embodiments herein, the subject is a human subject.

[0079] In particular embodiments of any of the provided methods, the CAR contains in order from N-terminus to C-terminus: an extracellular antigen-binding domain that is the scFv set forth in SEQ ID NO: 43, the spacer set forth in SEQ ID NO:l, the transmembrane domain set forth in SEQ ID NO:8, the 4-1BB costimulatory signaling domain set forth in SEQ ID NO: 12, and the signaling domain of a CD3- zeta (OΏ3z) chain set forth in SEQ ID NO: 13.

[0080] In some of any embodiments, the composition comprising engineered T cells is produced by a manufacturing process comprising: (i) exposing an input composition comprising primary T cells with a stimulatory reagent comprising an oligomeric particle reagent comprising a plurality of streptavidin mutein molecules under conditions to stimulate T cells, thereby generating a stimulated population, wherein: the oligomeric particle reagent comprises a first agent comprising an anti-CD3 antibody or antigen binding fragment thereof and a second agent comprising an anti-CD28 antibody or antigen binding fragment thereof; (ii) introducing into T cells of the stimulated population, a heterologous polynucleotide encoding the CAR that targets CD 19, thereby generating a population of transformed cells; (iii) incubating the population of transformed cells for up to 96 hours; and (iv) harvesting T cells of the population of transformed cells, thereby producing a composition of engineered cells, wherein the harvesting is carried out at a time between 24 and 120 hours, inclusive, after the exposing to the stimulatory reagent is initiated. In some embodiments, the input composition comprises autologous T cells selected from the subject, such as enriched by immunoaffinity-based selection for CD3 T cells or CD4 and CD8 T cells from a blood or apheresis (e.g. leukarephesis) sample from the subject.

[0081] In any of the embodiments herein, the composition including engineered T cells is produced by a manufacturing process including exposing an input composition including primary T cells with a stimulatory reagent including an oligomeric particle reagent including a plurality of avidin, streptavidin, avidin mutein, or streptavidin mutein molecules under conditions to stimulate T cells, thereby generating a stimulated population, wherein the stimulatory reagent is capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules. In any of the embodiments herein, the manufacturing process further may include introducing into T cells of the stimulated population, a heterologous polynucleotide encoding the CAR that targets CD 19, thereby generating a population of transformed cells.

[0082] In some of any embodiments, the oligomeric particle reagent comprises a first agent comprising an anti-CD3 antibody or antigen binding fragment thereof and a second agent comprising an anti-CD28 antibody or antigen binding fragment thereof. In some of any embodiments, the anti-CD3 antibody or antigen binding fragment is a Fab and the anti-CD28 antibody or antigen binding fragment is a Fab. In some of any embodiments, the first agent and the second agent each comprise a streptavidin- binding peptide that reversibly binds the first agent and the second agent to the oligomeric particle reagent, optionally wherein the streptavidin-binding peptide comprises the sequence of amino acids set forth in any of SEQ ID NOS:78-82. In some of any embodiments, the streptavidin mutein molecule is a tetramer of a streptavidin mutein comprising amino acid residues Val44-Thr45-Ala46-Arg47 or Ile44- Gly45-Ala46-Arg47, optionally wherein the streptavidin mutein comprises the sequence set forth in any of SEQ ID NOS: 69, 84, 87, 88, 90, 85 or 59. In some of any embodiments, the oligomeric particle reagent comprises between 1,000 and 5,000 streptavidin mutein tetramers, inclusive. In some of anyembodiments, the method further comprises, prior to harvesting the cells, adding biotin or a biotin analog after or during the incubation.

[0083] In any of the embodiments herein, the manufacturing process may further include incubating the population of transformed cells for up to 96 hours. In any of the embodiments herein, the incubating is carried out in basal media lacking one or more recombinant cytokines. In any of the embodiments herein, the manufacturing process may further include harvesting T cells of the population of transformed cells, thereby producing a composition of engineered cells. In any of the embodiments herein, the harvesting is carried out at a time between 24 and 120 hours, inclusive, after the exposing to the stimulatory reagent is initiated. In any of the embodiments herein, the harvesting is carried out at a time between 48 and 120 hours, inclusive, after the exposing to the stimulatory reagent is initiated. In any of the embodiments herein, the harvesting is carried out at a time when integrated vector is detected in the genome but prior to achieving a stable integrated vector copy number (iVCN) per diploid genome. In any of the embodiments herein, the harvesting is carried out at a time before the total number of viable cells at the harvesting is more than or more than about three times as the number of total viable cells of the stimulated population. In any of the embodiments herein, the harvesting is carried out at a time when the total number of viable cells at the harvesting is at or about three times, at or about two times, or the same or about the same as the number of total viable cells of the stimulated population. In any of the embodiments herein, the harvesting is carried out at a time when the percentage of CD27+CCR7+cells is greater than or greater than about 50% among total T cells in the population of transformed cells, total CD3+T cells in the population, total CD4+T cells in the population of transformed cells, or total CD8+T cells in the population of transformed cells, or of CAR-expressing cells thereof, in the population of transformed cells. In any of the embodiments herein, the harvesting is carried out at a time when the percentage of CD45RA+CCR7+and CD45RA CCR7+cells is greater than or greater than about 60% among total T cells in the population of transformed cells, total CD3+T cells in the population of transformed cells, total CD4+T cells in the population of transformed cells, or total CD8+T cells, or of CAR-expressing cells thereof, in the population of transformed cells.

[0084] In any of the embodiments herein, the cells in the administered composition are produced by a manufacturing process to produce an output composition (i) comprising engineered CD4+ T cells and engineered CD8+ T cells and (ii) exhibiting a predetermined feature, wherein iterations of the manufacturing process produce a plurality of the output compositions, optionally from human biological samples, when carried out among a plurality of different individual subjects, in which the predetermined feature of the output composition among the plurality of output compositions is selected from: the mean percentage of cells of a memory phenotype in the plurality of the output compositions is between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%; the mean percentage of cells of a central memory phenotype in the plurality of the output compositions isbetween about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%; the mean percentage of cells that are CD27+, CD28+, CCR7+, CD45RA-, CD45RO+, CD62L+, CD3+, CD95+, granzyme B-, and / or CD127+ in the plurality of the output compositions is between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%; the mean percentage of cells that are CCR7+ / CD45RA- or CCR7+ / CD45RO+ in the plurality of the output compositions is between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%; the mean percentage of central memory CD4+ T cells in the engineered CD4+ T cells, optionally CAR+CD4+ T cells, of the plurality of the output compositions is between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%; the mean percentage of central memory CD8+ T cells in the engineered CD8+ T cells, optionally CAR+CD8+ T cells, of the plurality of the output compositions is between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%; and / or the mean percentage of central memory T cells, optionally CD4+ central memory T cells and CD8+ central memory T cells, in the engineered T cells, optionally CAR+ T cells, of the plurality of the output compositions is between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%. In some embodiments, the plurality of output compositions are produced from human biological samples.

[0085] In any of the embodiments herein, the administered composition is produced by a manufacturing process to produce an output composition exhibiting a predetermined feature, optionally a threshold number of cells expressing the CAR in the output composition, in at least about 80%, about 90%, about 95%, about 97%, about 99%, about 100%, or is 100% of the human biological samples in which it is carried out among a plurality of different individual subjects. In any of the embodiments herein, the composition including genetically engineered cells does not contain residual beads from the manufacturing process. In any of the embodiments herein, the B-cell NHL is a relapsed and / or refractory B-cell non-Hodgkin lymphoma (B-cell NHL). In any of the embodiments herein, the predetermined feature is a threshold number of cells expressing the CAR in the output composition.

[0086] Also provided herein is an article of manufacture including a composition including genetically engineered cells expressing a chimeric antigen receptor (CAR) that targets CD19, and instructions for administering the composition of the cells in accordance with any of the methods provided herein.Brief Description of the Drawings

[0087] FIG. 1 shows exemplary quantifications determined by flow cytometry of cell purity of T cell compositions produced from non-expanded engineering processes using different donor types (Reference, Patient). Cells were engineered to express an anti-CD 19 CAR (CD 19) or were mock transduced (mock). Percentages of CD3+ cells of live CD45+ cells (left panel), percentages of NK cells of live CD45+ cells (middle panel), and percentages of CD19+ cells of live CD45+ cells (right panel) were determined.

[0088] FIGS. 2-3 shows exemplary quantifications of cell phenotypes determined by flow cytometry for expanded and non-expanded engineering processes using different donor types (Reference, Patient). Cells were engineered to express an anti-CD 19 CAR (CD 19) or were mock transduced (mock). FIG. 2 shows percentages of CD3+CD8+ and CD3+CD4+ cells of live CD45+ cells (left panel) and percentages of CD8+CAR+ and CD4+CAR+ cells of live CD45+ cells (right panel). FIG. 3 shows ratios of CD4+ cells to CD8+ cells, and CD4+CAR+ cells to CD8+CAR+ cells.

[0089] FIG. 4 shows exemplary quantifications determined by flow cytometry of cell viability of T cell compositions produced from non-expanded engineering processes using different donor types (Reference, Patient). Cells were engineered to express an anti-CD 19 CAR (CD 19) or were mock transduced (mock). Percentages of aCas3+ cells of CD3+ cells were determined.

[0090] FIG. 5A shows exemplary relationship between copy number per cell among total cells as assessed by standard VCN (without pulse field gel electrophoresis, or PFGE) and iVCN (with PFGE), in cell compositions produced from primary T cells from different human donors that had been engineered to express a CAR using an expanded process (o) or a non-expanded process (·). FIGS. 5B-5C show the relationship between the copy number per cell in the cell compositions as assessed by standard VCN (FIG. 5B) or iVCN (FIG. 5C) and the surface expression of the CAR, as indicated by the percentage of CAR-expressing CD3+ cells (%CD3+CAR+) among viable CD45+ cells assessed by flow cytometry.

[0091] FIGS. 6A-6B show exemplary percentages of cell phenotypes resulting from expanded and non-expanded engineering processes using different donor types (Reference, Patient). Cells were engineered to express an anti-CD19 CAR (CD19) or were mock transduced (mock). FIG. 6A shows exemplary percentages of CD45RA+CCR7+ cells of aCas-CD8+CAR+ and aCas-CD4+CAR+ cells (left top panel), CD45RA-CCR7+ cells of aCas-CD8+CAR+ and aCas-CD4+CAR+ cells (right top panel), CD45RA-CCR7- cells of aCas-CD8+CAR+ and aCas-CD4+CAR+ cells (left bottom panel), and CD45RA+CCR7- cells of aCas-CD8+CAR+ and aCas-CD4+CAR+ cells (right bottom panel). FIG. 6B shows exemplary percentages of CD27+CCR7+ cells of aCas-CD8+CAR+ and aCas-CD4+CAR+ cells.

[0092] FIGS. 7A-7D show exemplary quantifications of cell phenotypes as indicated determined by flow cytometry for donor-matched expanded and non-expanded engineering processes where cells were engineered to express an anti-CD 19 CAR, before the long-term CAR-dependent stimulation (primary)and after the long-term CAR-dependent stimulation (after 9 to 14 days of chronic stimulation with an agonistic anti-idiotype antibody) (secondary). DPI = diffuse large B-cell lymphoma (DLBCL) patient 1; DP2 = DLBCL patient 2; HD1 =healthy donor 1; MP1 = mantle cell lymphoma patient 1.

[0093] FIGS. 8A-8F show exemplary in vitro proliferative capacity of cells generated from non- expanded and expended processes following long-term anti-CD 19 CAR-dependent stimulation with an anti-ID antibody. Viability (FIG. 8A) and cell size (FIG. 8B) were measured. Fold change in expansion was calculated as the daily counts divided by the starting number of cells (FIG. 8C), and then transformed into an area under the curve (AUC) for comparison either for individual arms (FIG. 8D) or by manufacturing process (FIG. 8E, statistical significance with a Mann-Whitney test; *p < 0.05). Individual donor fold expansion between the manufacturing platforms was also directly measured by taking the daily fold expansion of compositions generated from the non-expanded process and dividing them by the donor-matched control values for compositions generated from the expanded process (FIG. 8F).

[0094] FIGS. 9A-9B show exemplary cytolytic potential of anti-CD 19 CAR T cells engineered by non-expanded or expanded processes before (FIG. 9A) and after chronic stimulation (FIG. 9B) at different effector to target ratios. Area under the curve (AUC) values were compared either for individual arms (left panels of FIG. 9A and FIG. 9B) or by manufacturing process (right panels of FIG. 9A and FIG. 9B, statistical significance with a Mann-Whitney test; *p < 0.05).

[0095] FIG. 10 shows exemplary cytokine productions by cells engineered by non-expanded or expanded processes, measured by a polyfunctional score using flow cytometry, at Day 0 and Day 10 of chronic antigen stimulation. Statistical significance was assessed by Mann-Whitney, * p<0.05.

[0096] FIGS. 11A-11B show exemplary tumor burden and circulating CAR-T cells in the Nalm6 leukemia model over time following treatment with anti-CD 19 CAR-T cell compositions generated from non-expanded and expanded matched-donor engineering processes. FIG. 11A shows tumor growth from Day -1 (before treatment) to about Day 25 post-treatment calculated from area under the curve (AUC) of bioluminescence (BLI) for each group. FIG. 11B shows circulating anti-CD 19 CAR-T cell counts per 1 pi of blood between about Day 5 and about Day 20 post-treatment for each group.

[0097] FIGS. 12A-12B show exemplary tumor burden and circulating CAR-T cells in Raji lymphoma model over time following treatment with anti-CD 19 CAR-T cell compositions generated from non-expanded and expanded matched-donor engineering processes. FIG. 12A shows tumor growth from Day -1 (before treatment) to about Day 80 post-treatment calculated from area under the curve (AUC) of BLI for each group, with higher (left panel) and lower (right panel) doses of anti-CD 19 CAR-T cell compositions generated from non-expanded and expanded matched-donor engineering processes. Changes in BLI radiance (photons / second; y-axis) are shown for all groups (tumor burden). FIG. 12B shows circulating anti-CD 19 CAR-T cell counts per 1 mΐ of blood at the indicated time points post-treatment for each group (non-expanded, NE; expanded, E). Absolute counts at each time point are shown as group means. Differences were compared using Mann-Whitney U test; *p < 0.05.Detailed Description

[0098] Provided herein are methods and uses of engineered cells (e.g., T cells) and / or compositions thereof, for the treatment of subjects having a disease or condition, which generally is or includes a cancer or a tumor, such as a lymphoma, most particularly a B cell malignancy that is a non-Hodgkin lymphoma (NHL), including aggressive subtypes of NHL. In particular aspects, the subject has an aggressive or high-risk NHL. In embodiments of the provided methods, the therapeutic T cell compositions containing the engineered cells are administered to a subject having NHL, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In particular embodiments of any of the provided methods, the T cells are engineered with a chimeric antigen receptor (CAR) that is directed against cluster of differentiation 19 (CD19). In some aspects, the disease or condition is a B cell lymphoma. In some aspects, the disease or condition is a large B cell lymphoma. In some aspects, the disease or condition is a diffuse large B-cell lymphoma (DLBCL) or a subtype thereof. In some aspects, the methods and uses provide for or achieve improved response and / or more durable responses or efficacy and / or a reduced risk of toxicity or other side effects, e.g., in particular groups of subjects treated, as compared to certain alternative methods. 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 defined ratios of particular types of the cells, the administration of cells of a particular high percentage of less differentiated cells (e.g. naive -like or central memory cells or cells of an early differentiation state, such as CCR7+CD27+ cells), treatment of particular patient populations, such as those having a particular risk profile, staging, and / or prior treatment history, and / or combinations thereof.

[0099] In some embodiments, the methods and uses include administering to the subject T cells expressing genetically engineered (recombinant) cell surface receptors in adoptive cell therapy, which generally are chimeric receptors such as chimeric antigen receptors (CARs), recognizing CD19 expressed by, associated with and / or specific to the NHL 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. In some cases, the CD 19 -directed CAR+ engineered cells in the composition include a defined ratio or compositions of two or more sub-types within the composition, such as CD4 vs. CD8 T cells. In particular embodiments, the compositions of cells for use or administration in the provided methods include primary T cells engineered to express a CD19-directed CAR that (i) contain a low percentage (e.g. less than 40%, less than 30%, less than 20%, or less than 10%) of exhausted cells and / or cells that display markers or phenotypes associated withexhaustion; and / or (ii) contain a relatively high percentage (e.g. greater than 50%, greater than 60%, greater than 70%, greater than 80% or greater than 90%) of memory-like T cells, such as naive-like T cells, central memory T cells or long-lived memory T cells. In provided embodiments, the features of the compositions and provided methods result in improved or enhanced survival, expansion, persistence, and / or anti-tumor activity compared to methods involving administration other CD19-directed CAR T cell therapies that contain a higher percentage of exhausted cells and / or a higher number of cells that display phenotypes associated with exhaustion and / or that contain a lower percentage of certain T cells, such as naive-like T cells, central memory T cells or long-lived memory T cells. In provided embodiments, the features of the compositions and provided methods result in improved therapeutic efficacy, e.g. increased percentage of patients achieving a complete response (CR), compared to methods involving administration of other CD19-directed CAR T cell therapies that contain a higher percentage of exhausted cells and / or a higher number of cells that display phenotypes associated with exhaustion and / or that containa lower percentage of certain T cells, such as naive -like T cells, central memory T cells or long-lived memory T cells. In provided methods, the features of the compositions and provided methods result in improved clinical durability of therapeutic response, such as CR, e.g., response that persists after a period of time from initiation of therapy, compared to methods involving administration of other CD 19- directed CAR T cell therapies that contain a higher percentage of exhausted cells and / or a higher number of cells that display phenotypes associated with exhaustion and / or that contain a lower percentage of memory-like T cells, such as naive-like T cells, central memory T cells or long-lived memory T cells. In particular embodiments, the use or administration of the provided CD19-directed CAR T cell compositions in the provided methods can be achieved with doses of cells that are more than 2-fold lower, such as 5-fold or 10-fold, lower than doses of reference CD19-directed CAR T cell compositions (e.g. engineered with the same or similar CAR, such as with the same antigen-binding domain) but in which the reference CD19-directed CAR T cell composition contain a higher percentage of exhausted cells and / or a higher number of cells that display phenotypes associated with exhaustion and / or that contains a lower percentage of memory-like T cells, such as naive -like T cells, central memory T cells or long-lived memory T cells. In some embodiments, the reference CD19-directed CAR T cell composition is a composition that is produced ex vivo by processes that involve steps of cultivating the cells under conditions for expansion, such as resulting in proliferation of cells or population doubling of cells (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doublings of cells in the population compared to the start of the process) during the process for producing the cells.

[0100] In some embodiments, the CD19-directed CAR T cell compositions for use in the provided methods and uses are produced by a relatively short process that do not include a step for cultivating the cells under conditions for expansion designed for expanding or proliferating the cells. Different processes are available for generating compositions containing genetically engineered T cell populations, including for generating engineered T cells that express a CAR, which typically include a step designed for or forthe purpose of cultivating the cells to expand or increase proliferation of the cells. However, in particular aspects, some of these processes may require a long or a relatively long amount of time to generate the engineered cells. In addition, in various aspects, some existing processes may vary in the amount of time required to successfully produce engineered T cells suitable for cell therapy, making it difficult to coordinate that administration of the cell therapy. In certain aspects, some of these processes may produce populations of cells that include a relatively high percentage or amount of exhausted cells, differentiated cells, or cells with a low potency. The provided CD19-directed CAR T cell compositions for use in the provided methods address one or more of these problems.

[0101] In particular embodiments, the provided methods are used in connection with a process for efficiently producing or generating engineered cells that are suitable for use in a cell therapy. In some embodiments, provided compositions containing CD19-directed CAR engineered T cells are produced by a process without the need for any additional steps for expanding the cells, e.g. without an expansion unit operation and / or without steps intended to cause expansion of cells. In aspects of processes for producing CD19-directed CAR T cell composition, the processes include one or more steps for stimulating and genetically engineering (e.g., transforming, transducing or transfecting) T cells to produce a population of engineered T cells that may be collected or formulated for use as a composition for cell therapy. In particular embodiments, the processes include a step of transducing cells with a viral vector (e.g. lentiviral vector) that contains a nucleic acid encoding the CD19-directed CAR. In some aspects, the provided processes result in the stable integration of the heterologous nucleic acid (expressed from the viral vector) into the genome of the cells. In some aspects, the provided processes generate engineered CD19-directed CAR T cells with enhanced potency as compared to engineered T cell compositions produced from alternative processes, such as those that involve expanding the cells.

[0102] In particular aspects, the durations of the processes for producing the provided compositions can be measured from when cells, e.g., T cells of an input cell population or input composition, are first contacted or exposed to stimulating conditions (e.g., as described herein such as in Section II-C), referred to herein as the initiation of the stimulation or stimulating and also referred to herein as the exposing to the stimulatory reagent, e.g., as in when the exposing to the stimulatory reagent is initiated. In some embodiments, the duration of time required to harvest or collect an output population (also referred to herein as an output composition or as a composition of engineered cells, e.g., engineered T cells) containing engineered cells is measured from initiation of the stimulation. In particular embodiments, the duration of the process is, is about, or is less than 120 hours, 108 hours, 96 hours, 84 hours, 72 hours, 60 hours, 48 hours, 36 hours, or 30 hours. In particular embodiments, the duration of the process is, is about, or is less than 5 days, 4 days, 3 days, 2 days, or one day. In particular embodiments, the engineered cells, e.g., the cells of the output composition or population, are more potent, persistent or naive-like than cells that are engineered with processes that require longer amounts of time. In some aspects, the duration, e.g., the amount of time required to generate or produce an engineered population of T cells, of theprovided processes are shorter than those of some existing processes by, by about, or by at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days. In some embodiments, the duration of the provided process is, is about, or is less than 75%, 60%, 50%, 40%, 30%, 25%, 15%, or 10% of alternative or existing processes.

[0103] In certain embodiments, the provided processes are performed on a population of cells, e.g., CD3+, CD4+, and / or CD8+ T cells, that are isolated, enriched, or selected from a biological sample. In some aspects, the provided methods can produce or generate a composition of engineered T cells from when a biological sample is collected from a subject within a shortened amount of time as compared to other methods or processes. In some embodiments, the provided methods can produce or generate engineered T cells, including any or all times where biological samples, or enriched, isolated, or selected cells are cryopreserved and stored, within or within about 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, or 2 days, or within or within about 120 hours, 96 hours, 72 hours, or 48 hours, from when a biological sample is collected from a subject to when the engineered T cells are collected, harvested, or formulated (e.g., for cryopreservation or administration).

[0104] In particular embodiments, the processes for producing or engineering T cell populations include a step of stimulating the cells, such as prior to transduction with a viral vector. In aspects of the provided processes, stimulation is carried out with an oligomeric stimulatory reagent, such as a streptavidin mutein oligomer, to which is immobilized or attached a stimulatory binding agent(s), e.g. anti-CD3 / anti-CD28. Existing reagents for use in stimulating T cells in vitro, such as in the absence of exogenous growth factors or low amounts of exogenous growth factors, are known (see e.g. US Patent 6,352,694 B1 and European Patent EP 0700430 Bl). In general, such reagents may employ beads, e.g., magnetic beads, of greater than 1 pm in diameter to which various binding agents (e.g. anti-CD3 antibody and / or anti-CD28 antibody) are immobilized. However, in some cases, such magnetic beads are, for example, difficult to integrate into methods for stimulating cells under conditions required for clinical trials or therapeutic purposes since it has to be made sure that these magnetic beads are completely removed before administering the expanded T cells to a subject. In some aspects, such removal, such as by exposing the cells to a magnetic field, may decrease the yield of viable cells available for the cell therapy. In certain cases, such reagents, e.g., stimulatory reagents containing magnetic beads, must be incubated with the cells for a minimal amount of time to allow a sufficient amount of detachment of the T cells from the stimulatory reagent.

[0105] The provided processes utilizing oligomeric stimulatory reagents, e.g. streptavidin mutein polymer, overcome such potential limitations. For example, in some embodiments, the provided processes avoid or reduce risk of residual stimulatory reagent, e.g., reagents containing magnetic beads, in the output cells generated or produced by the processes. In some embodiments, this also means that a process that is compliant with GMP standards can be more easily established compared to other methods, such as those where additional measures have to be taken to ensure that the final engineered T cellpopulation is free of beads. In some embodiments, this may be readily accomplished in the present embodiments by the addition of a substance, e.g., a competition reagent, that dissociates the oligomeric stimulatory reagents from the cells, e.g., by simply rinsing or washing the cells e.g., by centrifugation. Thus, in some aspects, removal or separation of oligomeric stimulatory reagent from cells, such as by the addition of a substance or competition reagent, results in little or no cell loss as compared to removal or separation of bead based stimulatory reagents. In some aspects, the timing of the oligomeric stimulatory reagent removal or separation is not limited or is less limited than the removal or separation of bead based stimulatory reagents. Thus, in some aspects, the oligomeric stimulatory reagent may be removed or separated from the cells at any time or stage during the provided processes.

[0106] In some aspects, the use of oligomeric stimulatory reagents (e.g. anti-CD3 / anti-CD28 streptavidin mutein oligomers) can result in an overall reduced stimulatory signal compared to alternative stimulatory reagents, such as anti-CD3 / anti-CD28 paramagnetic beads. The provided process, which can involve a weaker or reduced stimulation, can generate engineered CAR+ T cells that are as, or even more, potent, persistent, or efficacious as CAR+ T cells generated by processes that involve stronger stimulatory conditions or higher amounts or concentrations of stimulatory reagent, such as may occur following stimulation with anti-CD3 / anti-CD28 paramagnetic beads. In addition, in some embodiments, stimulating cells with a lower amount or relatively low amount of oligomeric stimulatory reagents may increase the potency, efficacy, or persistency of the resulting engineered cell population, as compared to processes using higher amounts of oligomeric stimulatory reagent. Such embodiments contemplate that such effects may persist even at doses sufficiently low enough to reduce the expression of activation markers or the portion of cells positive for the activation markers during and after the process.

[0107] In certain embodiments, the engineered T cells, e.g., output composition or populations of T cells containing T cells expressing a recombinant receptor, such as a chimeric antigen receptor, produced or generated by the provided processes are particularly effective or potent when utilized as cells for a cell therapy. For example, in some aspects, an output composition containing engineered T cells, e.g., CAR+ T cells, that are generated from the provided processes have a much higher degree of potency and / or proliferative capacity than engineered T cells generated or produced by alternative existing processes. In some aspects, an output composition containing engineered T cells, e.g., CAR+ T cells, produced by the provided processes have enhanced anti-tumor or anti-cancer cell activity than engineered T cells, e.g., CAR+ T cells, produced by alternative or existing methods.

[0108] In particular embodiments, the processes for producing the provided CD19-directed T cell compositions that do not contain steps where the cells are expanded to a threshold amount or concentration have further advantages. In some aspects, protocols that do not rely on expanding the cells to increase the number or concentration of cells from a starting cell population, e.g., an input population, do not require incubations or cultivations that may vary between cell populations. For example, some embodiments contemplate that cell populations obtained from different subjects, such as subjects havingdifferent diseases or disease subtypes, particularly as is the case for patients with NHL, including high- risk, aggressive and / or R / R NHL, may divide or expand at different rates. In certain aspects, eliminating potentially variable steps requiring cell expansion allows for the duration of the whole process to be tightly controlled. In certain embodiments, the variability of the process duration is reduced or eliminated which may, in some aspects, allow for improved coordination for appointments and treatment between doctors, patients, and technicians to facilitate autologous cell therapies.

[0109] In some embodiments, the provided methods involve treating a specific group or subset of subjects, e.g., subjects identified as having high-risk disease, e.g., high-risk NHL or a high-risk large B cell lymphoma. In some aspects, the methods treat subjects having a form of aggressive and / or poor prognosis B-cell non-Hodgkin lymphoma (NHL), such as NHL that has relapsed or is refractory (R / R) to standard therapy and / or has a poor prognosis. In some aspects, the methods treat subjects having a large B cell lymphoma that has relapsed or is refractory (R / R) to standard therapy. In particular aspects, the engineered cells are autologous to the subject and are administered following generation by ex vivo processes that are shortened compared to existing methods, that do not include or involve a cultivation step for expanding the cells during the methods of producing the engineered cells, and / or that are able to produce a CAR-engineered T cell composition that is less differentiated permitting administration of lower doses. As a result, the provided methods are advantageous compared to existing methods because they can shorten the time until the engineered T cell therapy is available to the patient, particularly among patients who are in need of treatment, such as subjects that have relapsed to or are refractory to treatment following one or more other prior therapies for treating the disease or condition. In some aspects, the provided methods, compositions, uses and articles of manufacture achieve improved and superior responses to available therapies. In some embodiments, the improved or superior responses are to current standard of care (SOC).

[0110] Non-Hodgkin lymphoma (NHL) is a group of lymphoid malignant neoplasms with diverse biological and clinical behavior. It is estimated there will be approximately 74,200 new cases diagnosed and 19,970 deaths from NHL in the United States (US) in 2019 (Siegel et al., CA Cancer J Clin. 2019; 69(l):7-34). Non-Hodgkin lymphoma can be divided into 2 prognostic groups: the indolent lymphoma (slowly growing with waxing and waning lymphadenopathy for years) and the aggressive lymphoma (rapidly growing and resulting death within a few weeks if not treated). The most common aggressive lymphoma is diffuse large B-cell lymphoma (DLBCL) accounting for 30 to 40% of all NHL (Li et al., Pathology 2018; 50(l):74-87). Other aggressive lymphomas include but are not limited to high grade B- cell lymphoma (HGBCL) so-called as double hit (DHL) or triple hit lymphoma (THL), mantle cell lymphoma (MCL), primary mediastinal large B-cell lymphoma (PMBCL), and follicular lymphoma grade 3b (FL3B). Prognosis and therapy of other aggressive lymphoma subtypes are similar to DLBCL. About 50% of newly diagnosed patients with DLBCL can be cured with first-line R-CHOP immunochemotherapy (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone).However, approximately half of the patients treated with R-CHOP will relapse, mostly within the first 2 years after therapy (Coiffier et al., Blood 2010; 116(12):2040-5; Vitolo et al., J Clin Oncol. 2017;35(31):3529-37). Salvage high-dose immunochemotherapy followed by autologous stem-cell transplantation (ASCT) is the standard secondline therapy for relapsed or refractory (R / R) DLBCL. Approximately half of the R / R patients will not be transplant eligible due to no response to salvage therapy or medical condition, and significant portion of patients will relapse even after ASCT. A multicohort retrospective NHL research study in refractory DLBCL (SCHOLAR-1; n = 636), demonstrated very poor outcomes in this patient population with an overall response rate (ORR) of 26% to the next line of therapy and a median overall survival (OS) of 6.3 months (Crump et al., Blood 2017; 130(16): 1800-8; Gisselbrecht et al., Br J Haematol. 2018; 182(5):633-43).

[0111] CD19 is a member of the immunoglobulin superfamily and a component of the B-cell surface signal transduction complex that positively regulates signal transduction through the B-cell receptor. It is expressed by most B-cell malignancies from early development until differentiation into plasma cells (Stamenkovic et al., J Exp Med. 1988; 168(3): 1205-10). CD19 is an attractive therapeutic target because it is expressed by most B-cell malignancies, including B-cell NHL (Davila et al., Oncoimmunology. 2012;(9): 1577-83). Importantly, the CD19 antigen is not expressed on hematopoietic stem cells or on any normal tissue apart from those of the B-cell lineage. Additionally, CD 19 is not shed in the circulation, which limits off-target adverse effects (Shank et al., Pharmacotherapy. 2017;37(3):334- 45).

[0112] In particular embodiments, the methods provided herein are based on administration of a CD19-directed CAR T cell therapy in which the CAR contains a CD19-directed scFv antigen binding domain (e.g. from FMC63). The CAR further contains an intracellular signaling domain containing a signaling domain from CD3zeta, and also incorporates a 4-1BB costimulatory domain, which has been associated with lower incidence of cytokine release syndrome (CRS) and neurotoxicity (NE) compared with CD28 -containing constructs (Lu et al. J Clin Oncol. 2018;36:3041).

[0113] A challenge in CAR T cell development is to generate a product that consistently expands, persists, and mediates durable antitumor responses after infusion. Certain CD19-directed CAR-T cell therapies are available for the treatment of B cell lymphoma, including Kymriah™ (tisagenlecleucel) (Kymriah PI) and Yescarta® (axicabtagene ciloleucel) (Yescarta PI). Both CAR T cell therapies have initial 50 to 60% of complete response rates (CRR) in this difficult to treat patient population; however, at 6 months the response rate drops to the 40 to 50% range with responders more likely to have ongoing durable responses (Locke et al., Mol Ther. 2017; 25(l):285-95; Schuster et al., N Engl J Med. 2017; 377(26):2545-54). Thus, more than half of the patients who receive these therapies will not have a long lasting, durable response, and the prognosis for overall survival (OS) is very poor.

[0114] The provided methods are based on findings that a lower differentiation state of adoptively transferred T cells can influence the ability of these cells to persist and promote durable antitumorimmunity. In some embodiments, the provided CD19-directed CAR+ engineered T cell compositions are produced by a method in which the cells are not cultivated under conditions of expansion, thereby limiting or reducing the number of population doublings of the final engineered output composition and resulting in a less differentiated product. Yet, the provided compositions also are produced via processes that result in stably integrated vector copy number (iVCN) to ensure consistent and reliable expression of the CAR, thereby resulting in a consistent cell product for administration to subjects and low variability among CAR-expressing cells in administered doses. In contrast, most protocols for T cell engineering routinely expand T cells ex vivo for 9 to 14 days or more. Provided data exemplified herein support a model in which CAR T cell products with an increased composition of less differentiated memory T cells may exhibit enhanced durable antitumor activity. These findings reveal that strategies aimed at minimizing effector differentiation in CAR T cell products could result in improved clinical efficacy. Provided herein are embodiments that can meet such aims.

[0115] The observations herein support treating subjects with high-risk disease with a CD19- directed CAR T cell therapy in accord with the provided methods. For example, subjects with NHL, including patients with aggressive NHL or certain high-risk features, such as those with relapsed / refractory (R / R) NHL, can be treated in accord with the provided methods. In some embodiments, the provided methods can be used to treat subjects that have been heavily pretreated (e.g. with one, two, three, four, or more prior therapies for treating the disease).

[0116] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually 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 definition that is incorporated herein by reference.

[0117] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. METHODS AND USES OF CD19-TARGETED CELL THERAPY IN B CELL NON- HODGKIN’S LYMPHOMA

[0118] Provided herein are methods of treatment that involve administering engineered cells or compositions containing engineered cells, such as engineered T cells. Also provided are methods and uses of provided CD19-directed CAR engineered cells (e.g., T cells) and / or compositions thereof, including methods for the treatment of subjects having a B cell non-Hodgkin’s lymphoma (NHL), including aggressive or high-risk NHL, such as R / R NHL, that involves administration of the engineered cells and / or compositions thereof. In some embodiments, the methods and use of provided CD19- directed CAR engineered cells (e.g., T cells) and / or compositions thereof, including methods for thetreatment of subjects with R / R NHL that have failed at least two or more prior therapies. In particular embodiments, the method includes administering to the subject a dose of T cells that includes CD4+ and CD8+ T cells, wherein the T cells comprises a chimeric antigen receptor (CAR) that specifically binds to CD19.

[0119] 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 CD19 expressed by, associated with and / or specific to the lymphoma and / or cell type from which it is derived. The cells are generally administered in a composition formulated for administration. In some embodiments, cells are collected from the subject prior to treatment for the purpose of engineering the cells with the CD 19- directed recombinant receptor (e.g. CAR). In some embodiments, the cells are collected by leukapheresis. In some aspects, the cells are engineered by ex vivo methods that do not involve cultivating the cells for expansion (hereinafter also called non-expanded process). Exemplary non-expanded processes for engineering the provided CAR-expressing therapeutic compositions are described in Section II-C.

[0120] In some embodiments, the disease or condition is an aggressive NHL. In some embodiments, the disease or condition is an aggressive NHL that was previously indolent. In some embodiments, the NHL is non-indolent, such is an aggressive lymphoma that is fast moving. Among the most common aggressive NHL subtype is Diffuse large B-cell lymphoma (DLBCL).

[0121] In some embodiments, the disease or condition is confirmed using positron emission tomography (PET). In some embodiments, the disease or condition is confirmed using PET and staged based on the Lugano Classification (see, e.g., Cheson et al., (2014) JCO 32(27):3059-3067; Cheson, B.D. (2015) Chin Clin Oncol 4(1):5). In some embodiments, the disease or condition is histologically confirmed. In any embodiments disclosed herein, the disease or conditions, or subtype or state, can be determined prior to leukapheresis in connection with obtaining T cells for autologous T cell therapy.

[0122] In some embodiments, the disease or condition is a diffuse large B cell lymphoma (DLBCL). In some embodiments, the disease or condition is a DLBCL not otherwise specified (NOS). In some embodiments, the disease or condition is a high-grade B cell lymphoma with MYC and BCL2 and / or BCL6 rearrangements with DLBCL histology (HGBCL). In some embodiments, the disease or condition is a DLBCL transformed from follicular lymphoma (tFL). In some embodiments, the disease or condition is a DLBCL transformed from marginal zone lymphoma (tMZL). In some embodiments, the disease or condition is a primary mediastinal B cell lymphoma (PMBCL). In some embodiments, the disease or condition is a follicular lymphoma (FL). In some embodiments, the disease or condition is a follicular lymphoma grade 3B (FL3B).

[0123] In some embodiments, the subject prior to leukapheresis has an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 (see, e.g., Oken et al., (1982) Am J Clin Oncol. 5:649-655). In some embodiments, the Eastern Cooperative Oncology Group (ECOG) performancestatus indicator can be used to assess or select subjects for treatment, e.g., subjects who have had poor performance from prior therapies (see, e.g., Oken et al., (1982) Am J Clin Oncol. 5:649-655). The ECOG Scale of Performance Status describes a patient’s level of functioning in terms of their ability to care for themselves, daily activity, and physical ability (e.g., walking, working, etc.). In some embodiments, an ECOG performance status of 0 indicates that a subject can perform normal activity. In some aspects, subjects with an ECOG performance status of 1 exhibit some restriction in physical activity but the subject is fully ambulatory. In some aspects, patients with an ECOG performance status of 2 is more than 50% ambulatory. In some cases, the subject with an ECOG performance status of 2 may also be capable of self-care; see e.g., Sprensen et al., (1993) Br J Cancer 67(4) 773-775. The criteria reflective of the ECOG performance status are described in Table 1 below:

[0124] In some embodiments, prior to, such as at the time of, administration of the provided CD19- directed CAR T cell compositions, the subject has relapsed following remission after treatment with, or become refractory to, one or more lines of prior therapy for the NHL. In any embodiments, at a time prior to leukapheresis in connection with engineering the CD19-directed CAR T cell composition, the subject has relapsed following remission after treatment with, or become refractory to, one or more lines of prior therapy for treating the NHL. Thus, in particular embodiments, prior to the time of treatment, such as prior to leukapheresis, the subject has a R / R NHL. In some embodiments, the subject has been previously treated with a therapy or a therapeutic agent targeting the disease or condition, e.g., a NHL or a, prior to administration of the cells expressing the recombinant receptor. In some embodiments, the subject has been previously treated with a hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT or autologous HSCT. In some embodiments, the subject has had poor prognosis after treatment with standard therapy and / or has failed one or more lines of previous therapy. In some embodiments, the subject has been treated or has previously received at least or at least about or about 1, 2, 3, 4, or more other therapies for treating the NHL, such as an aggressive or high-risk NHL, such as DLBCL or a subtype thereof. In some embodiments, the subject has been treated or has previously received a therapy that includes a CD20 targeted agent (e.g. anti-CD20 antibody) and an alkylating agent. In some aspects, the subject has relapsed after an initial response of complete response (CR) or partialresponse (PR) to the prior therapy. In some embodiments, the subject is refractory to treatment with the at least one or more prior therapy, and the refractory treatment is a best response of stable disease (SD) or progressive disease (PD) after the prior therapy.

[0125] In some embodiments, the subject has relapsed following remission after treatment with, or become refractory to, at least two lines of prior systemic therapy for the disease or condition. In some embodiments, at least one said line of prior systemic therapy comprises anthracycline and anti-CD20 monoclonal antibody treatment. Exemplary anti-CD20 antibodies include but are not limited to rituximab, ofatumumab, ocrelizumab (also known as GA101 or RO5072759), veltuzumab, obinutuzumab, TRU-015 (Trubion Pharmaceuticals), ocaratuzumab (also known as AME-133v or ocaratuzumab), and Prol31921 (Genentech). See, e.g., Lim et al. Haematologica. (2010) 95(1): 135-43. Rituximab is a chimeric mouse / human monoclonal antibody IgGl kappa that binds to CD20 and causes cytolysis of a CD20 expressing cell. In some embodiments, at least one said line of prior systemic therapy comprises anthracycline and rituximab treatment.

[0126] In some embodiments, the subject has relapsed following remission after treatment with, or become refractory to, autologous stem-cell transplantation (ASCT). In some embodiments, relapse following remission after treatment with or becoming refractory to ASCT is failure to achieve an objective response after ASCT. In some embodiments, relapse following remission after treatment with or becoming refractory to ASCT is failure to achieve a partial response (PR) or better after ASCT. In some embodiments, relapse following remission after treatment with or becoming refractory to ASCT includes disease progression after ASCT.

[0127] In some embodiments, the subject has relapsed following remission after treatment with, or become refractory to, at least two lines of prior systemic therapy and ASCT for the disease or condition. In some embodiments, at least one said line of prior systemic therapy comprises anthracycline and anti- CD20 monoclonal antibody treatment. In some embodiments, at least one said line of prior systemic therapy comprises anthracycline and rituximab treatment.

[0128] In some embodiments, prior to leukapheresis, the subject does not show central nervous system (CNS)-only involvement by the disease or condition. In some embodiments, the subject has pathologically-confirmed secondary CNS involvement by the disease or condition.

[0129] In some embodiments, the subject has not previously received CAR T cell therapy prior to administration of the CD19-directed engineered CAR T cells in accord with the provided methods. In some embodiments, the subject has not received genetically-modified T cell therapy. In some embodiments, the subject has not received CD19-targeted therapy. Exemplary CD19-targeted therapies include, but are not limited to, anti-CD19 monoclonal antibodies or anti-CD19 bispecific antibodies. In some embodiments, the subject does not have hypersensitivity to fludarabine and / or cyclophosphamide. In some embodiments, the subject does not have an active autoimmune disease requiring immunosuppressive therapy.

[0130] In some embodiments, the subject has not received therapeutic doses of corticosteroids less than 14 days prior to leukapheresis. In some embodiments, a therapeutic dose of corticosteroids is defined as greater than 20 mg / day of prednisone or equivalent. In some embodiments, the subject has not received cytotoxic chemotherapeutic agents that are not considered lymphotoxic less than 7 days prior to leukapheresis. In some embodiments, the subject has not received intrathecal therapy (IT) less than 7 days prior to leukapheresis. Exemplary cytotoxic chemotherapeutic agents include, but are not limited to, doxorubicin, vincristine, gemcitabine, oxaliplatin, carboplatin, and etoposide. In some embodiments, the subject has not received an oral chemotherapeutic agent less than 5 half-lives prior to leukapheresis. Exemplary oral chemotherapeutic agents include, but are not limited to, lenalidomide and ibrutinib. In some embodiments, the subject has not received lymphotoxic chemotherapeutic agents less than 4 weeks prior to leukapheresis. Exemplary lymphotoxic chemotherapeutic agents include, but are not limited to, cyclophosphamide, ifosfamide, and bendamustine. In some embodiments, the subject has not received any experimental therapy less than 8 weeks (for biologies) or 5 half-lives (for small molecules) prior to leukapheresis treatment. In some embodiments, the subject has not received immunosuppressive therapies less than 4 weeks prior to leukapheresis. Exemplary immunosuppressive therapies include, but are not limited to, calcineurin inhibitors, methotrexate or other chemotherapeutics, mycophenolate, rapamycin, immunosuppressive antibodies such as anti-TNF, anti-IL6, or anti-IL6R. In some embodiments, the subject has not received donor lymphocyte infusions less than 6 weeks prior to leukapheresis. In some embodiments, the subject has not received radiation therapy for multiple lesions less than 6 weeks of leukapheresis. In some embodiments, the subject has not received radiation therapy for a single lesion, if additional non-irradiated PET-positive lesions are present, less than 14 days prior to leukapheresis. In some embodiments, the subject has not received an autologous stem-cell transplant (SCT) less than 3 months prior to leukapheresis. In some embodiments, the subject has not received an allogenic SCT less than 6 months prior to leukapheresis.

[0131] In some embodiments, the eligibility of subjects for treatment involving administering engineered cells is determined prior to leukapheresis. In some embodiments, the subject prior to leukapheresis has adequate vascular access for leukapheresis. In some embodiments, the subject prior to leukapheresis has adequate organ function. In some embodiments, adequate organ function is indicated by, among other factors, an absolute neutrophil count (ANC) greater than 1.0 x 109cells / L without growth factor support within 7 days of determination of eligibility; a platelet count greater than 50 x 109cells / L without transfusion support within 7 days of determination of eligibility; a calculated creatinine clearance rate (CrCl, Cockcroft- Gault formula) greater than 45 mL / min; an aspartate aminotransferase (AST) level less than or equal to 2.5 times the upper limit of normal (ULN); an alanine aminotransferase (ALT) level less than or equal to 2.5 times the ULN; a total bilirubin level less than 1.5 times the ULN; a direct bilirubin level less than 1.5 times the ULN, in the case of Gilbert’s syndrome or lymphomatous infiltration of the liver; adequate pulmonary function, for instance less than or equal to CTCAE Grade 1dyspnea and saturated oxygen (Sa02 greater than 92%) on room air; adequate cardiac function, for instance left ventricular ejection fraction (LVEF) greater than or equal to 40% as assessed by echocardiogram (ECHO) or a multiple uptake gated acquisition (MUGA) scan performed within 30 days of determination of eligibility; adequate organ function to receive lymphodepleting (LD) chemotherapy; or a combination of any of the foregoing.

[0132] In particular embodiments, prior to administration of the dose of CD19-directed engineered CAR T cells, the subject is administered or has received a lymohodepleting chemotherapy. Lymphodepletion may improve the engraftment and activity of CAR T cells through homeostatic cytokines, reduction of CD4+CD25+ regulatory T cells, increase of SDF-1 within bone marrow microenvironment, and stimulatory effects on antigen presenting cells (Grossman et al., Nat Rev Immunol. 2004; 4(5):387-395; Stachel et al., Pediatr Blood Cancer 2004; 43(6):644-50; Pinthus et al., J Clin Invest 2004; 114(12): 1774-81 ; Turk et al., J Exp Med 2004; 200(6):771-82). In addition, LD chemotherapy may further reduce the subject’s tumor burden and potentially lower the risk and severity of cytokine release syndrome (CRS).

[0133] 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 administration of engineered cells. For example, the subject may be administered a preconditioning agent at least 2 days prior, such as at least 3, 4, 5, 6, 7, 8, or 9 days prior, to the administration of engineered cells. In some embodiments, the subject is administered a preconditioning agent no more than 9 days prior, such as no more than 8, 7, 6, 5, 4, 3, or 2 days prior, to the administration of engineered cells.

[0134] In some embodiments, the subject is preconditioned with cyclophosphamide at a dose between or between about 20 mg / kg and 100 mg / kg body weight of the subject, such as between or between about 40 mg / kg and 80 mg / kg. In some aspects, the subject is preconditioned or administered 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 lymphodepleting agent comprises cyclophosphamide, the subject is administered cyclophosphamide at a dose between or between about 100 mg / m2and 500 mg / m2body surface area of the subject, such as between or between about 200 mg / m2and 400 mg / m2, or 250 mg / m2and 350 mg / m2, inclusive. In some instances, the subject is administered about 100 mg / m2of cyclophosphamide. In some instances, the subject is administered about 150 mg / m2of cyclophosphamide. In some instances, the subject is administered about 200 mg / m2of cyclophosphamide. In some instances, the subject is administered about 250 mg / m2of cyclophosphamide. In some instances, the subject is administered about 300 mg / m2of cyclophosphamide. In some embodiments, the cyclophosphamide can be administered in a single dose orcan 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 1-5 days, for example, for 3 to 5 days. In some instances, the subject is administered about 300 mg / m2body surface area of the subject, of cyclophosphamide, daily for 3 days, prior to initiation of the cell therapy. In some embodiments, the subject is administered a total of at or about 300 mg / m2, 400 mg / m2, 500 mg / m2, 600 mg / m2, 700 mg / m2, 800 mg / m2, 900 mg / m2, 1000 mg / m2, 1200 mg / m2, 1500 mg / m2, 1800 mg / m2, 2000 mg / m2, 2500 mg / m2, 2700 mg / m2, 3000 mg / m2, 3300 mg / m2, 3600 mg / m2, 4000 mg / m2or 5000 mg / m2cyclophosphamide, or a range defined by any of the foregoing, prior to initiation of the cell therapy.

[0135] In some embodiments, where the lymphodepleting agent comprises fludarabine, the subject is administered fludarabine at a dose between at or about 1 mg / m2and at or 100 mg / m2, such as between at or about 10 mg / m2and at or about 75 mg / m2, at or about 15 mg / m2and at or about 50 mg / m2, at or about 20 mg / m2and at or about 40 mg / m2, at or about or 24 mg / m2and at or about 35 mg / m2, inclusive.In some instances, the subject is administered at or at or about 10 mg / m2of fludarabine. In some instances, the subject is administered at or about 15 mg / m2of fludarabine. In some instances, the subject is administered at or about 20 mg / m2of fludarabine. In some instances, the subject is administered at or about 25 mg / m2of fludarabine. In some instances, the subject is administered at or about 30 mg / m2of 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 at or about 30 mg / m2body surface area of the subject, of fludarabine, daily for 3 days, prior to initiation of the cell therapy. In some embodiments, the subject is administered a total of at or about 10 mg / m2, 20 mg / m2, 25 mg / m2, 30 mg / m2, 40 mg / m2, 50 mg / m2, 60 mg / m2, 70 mg / m2, 80 mg / m2, 90 mg / m2, 100 mg / m2, 120 mg / m2, 150 mg / m2, 180 mg / m2, 200 mg / m2,250 mg / m2, 270 mg / m2, 300 mg / m2, 330 mg / m2, 360 mg / m2, 400 mg / m2or 500 mg / m2cyclophosphamide, or a range defined by any of the foregoing, prior to initiation of the cell therapy.

[0136] In some embodiments, the lymphodepleting agent comprises a single agent, such as cyclophosphamide or fludarabine. In some embodiments, the subject is administered cyclophosphamide only, without fludarabine or other lymphodepleting agents. In some embodiments, prior to the administration, the subject has received a lymphodepleting therapy comprising the administration of cyclophosphamide at or about 200-400 mg / m2body surface area of the subject, optionally at or about 300 mg / m2, daily, for 2-4 days. In some embodiments, the subject is administered fludarabine only, for example, without cyclophosphamide or other lymphodepleting agents. In some embodiments, prior to the administration, the subject has received a lymphodepleting therapy comprising the administration of fludarabine at or about 20-40 mg / m2body surface area of the subject, optionally at or about 30 mg / m2, daily, for 2-4 days.

[0137] In some embodiments, the lymphodepleting agent comprises a combination of agents, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of agents may include cyclophosphamide at any dose or administration schedule, 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 at or about 60 mg / kg (~2 g / m2) of cyclophosphamide and 3 to 5 doses of 25 mg / m2fludarabine prior to the first or subsequent dose. In some the subject is administered fludarabine (30 mg / m2 / day for 3 days) and cyclophosphamide (300 mg / m2 / day for 3 days) (flu / cy) concurrently, intravenously, prior to administration of the cells. In some embodiments, the subject is administered a reduced, delayed or eliminated dose of one or more doses of the lymphodepleting agent(s).

[0138] In some embodiments, after collecting the cells from the subject and prior to administering lymphodepleting (LD) chemotherapy, the subject can receive bridging therapy for disease control. Any of a variety of therapies can be administered as part of a bridging therapy based on the judgment of a skilled practitioner for treating the particular disease or condition, including based on factors such as the age of the patient, severity or extent of the disease, potential for side effects, timing of the administration prior to the LD chemotherapy, previous therapies and other factors. Exemplary therapies that can be given as a bridge prior to the lymphodepleting therapy include, but are not limited to, corticosteroids, vincristine, cyclophosphamide, rituximab, dexamethasone, prednisone, lenalidomide, gemcitabine, oxaliplatin, Brentuximab vedotin, ibrutininb, methotrexate, cytosine arabinoside, cytarabine, bendamustine, or any combination of any of the foregoing.

[0139] In some embodiments, the subjects are premedicated, e.g., to minimize the risk of infusion reaction. In some aspects, the premedication includes administering pain reliever and / or an antihistamine. In some embodiments, the premedication includes administering an acetaminophen and / or a diphenhydramine, or another HI -antihistamine. In some embodiments, the patient with acetaminophen (e.g., 650 mg orally) and diphenhydramine (e.g., 25-50 mg, IV or orally), or another HI -antihistamine, at or about 30 to 60 minutes prior to treatment with the cell therapy.

[0140] In some embodiments, the subject is at least 18 years of age. In embodiments of any of the provided methods, the subject is a human subject.A. Dosing

[0141] In some embodiments, a dose of engineered cells is administered to subjects in accordance with the provided methods, and / or with the provided articles of manufacture or compositions. In some embodiments, the size or timing of the doses is determined as a function of the particular disease or condition in the subject. In some cases, the size or timing of the doses for a particular disease in view of the provided description may be empirically determined.

[0142] In some of any of the provided embodiments, the dose of T cells, such as engineered T cells expressing a recombinant receptor, includes is enriched for, or comprises a cell composition or a cell population that is enriched for, CD3+ T cells, CD4+ T cells, CD8+ T cells or CD4+ T cells and CD8+ T cells. In some of any such embodiments, greater than at or about 70%, 75%, 80%, 85%, 90%, 95% or 98% of the cells in the dose of T cells are CD3+ T cells, CD4+ T cells, CD8+ T cells or CD4+ T cells and CD8+ T cells. In some of any such embodiments, greater than at or about 70%, 75%, 80%, 85%, 90%, 95% or 98% of the cells in the dose of T cells are CD3+ T cells. In some of any of the provided embodiments, the dose of T cells comprises both CD4+ cells and CD8+ cells. In some of any such embodiments, greater than at or about 70%, 75%, 80%, 85%, 90%, 95% or 98% of the cells in the dose of T cells are CD4+ T cells and CD8+ T cells.

[0143] In some embodiments, the dose of cells comprises between at or about 0.1 x 105of the CD 19 -directed CAR engineered cells per kilogram body weight of the subject (cells / kg) and at or about 2 x 106cells / kg, such as between at or about 0.1 x 105cells / kg and at or about 0.5 x 105cells / kg, between at or about 0.5 x 105cells / kg and at or about 1 x 105cells / kg, between at or about 1 x 105cells / kg and at or about 1.5 x 105cells / kg, between at or about 1.5 x 105cells / kg and at or about 2 x 105cells / kg, between at or about 2 x 105cells / kg and at or about 2.5 x 105cells / kg, between at or about 2.5 x 105cells / kg and at or about 3 x 105cells / kg, between at or about 3 x 105cells / kg and at or about 3.5 x 105cells / kg, between at or about 3.5 x 105cells / kg and at or about 4 x 105cells / kg, between at or about 4 x 105cells / kg and at or about 4.5 x 105cells / kg, between at or about 4.5 x 105cells / kg and at or about 5 x 105cells / kg, between at or about 5 x 105cells / kg and at or about 5.5 x 105cells / kg, between at or about 5.5 x 105cells / kg and at or about 6 x 105cells / kg, between at or about 6 x 105cells / kg and at or about 6.5 x 105cells / kg, between at or about 6.5 x 105cells / kg and at or about 7 x 105cells / kg, between at or about 7 x 105cells / kg and at or about 7.5 x 105cells / kg, between at or about 7.5 x 105cells / kg and at or about 8 x 105cells / kg, or between at or about 8 x 105of the cells / kg and at or about 10 x 105of the cells / kg. In some embodiments, the dose of cells comprises no more than 2 x 105of the CD19-directed CAR engineered cells per kilogram body weight of the subject (cells / kg), such as no more than at or about 3 x 105cells / kg, no more than at or about 4 x 105cells / kg, no more than at or about 5 x 105cells / kg, no more than at or about 6 x 105cells / kg, no more than at or about 7 x 105cells / kg, no more than at or about 8 x 105cells / kg, no more than at or about 9 x 105cells / kg, no more than at or about 1 x 106cells / kg, or no more than at or about 2 x 106cells / kg. In some embodiments, the dose of cells comprises at least or at least about or at or about 0.1 x 105of the CD19-directed CAR engineered cells per kilogram body weight of the subject (cells / kg), such as at least or at least about or at or about 0.2 x 105cells / kg, at least or at least about or at or about 0.3 x 105cells / kg, at least or at least about or at or about 0.4 x 105cells / kg, at least or at least about or at or about 0.5 x 105cells / kg, at least or at least about or at or about 0.6 x 105cells / kg, at least or at least about or at or about 0.7 x 105cells / kg, at least or at least about or at or about 0.8 x 105cells / kg, at least or at least about or at or about 0.9 x 105cells / kg, at least or at least about or ator about 0.1 x 106cells / kg, or at least or at least about or at or about 0.2 x 106cells / kg. In some embodiments, the number of cells is the number of such cells that are viable cells, e.g., viable T cells such as viable CD3+ cells expressing the CD19-directed CAR.

[0144] In certain embodiments, the cells, or individual populations of sub-types of cells, are administered to the subject at a range of at or about 0.1 million to at or about 100 billion cells and / or that amount of cells per kilogram of body weight of the subject, such as, e.g. , at or about 0.1 million to at or about 50 billion cells (e.g., at or about 5 million cells, at or about 25 million cells, at or about 500 million cells, at or about 1 billion cells, at or about 5 billion cells, at or about 20 billion cells, at or about 30 billion cells, at or about 40 billion cells, or a range defined by any two of the foregoing values), at or about 1 million to at or about 50 billion cells (e.g., at or about 5 million cells, at or about 25 million cells, at or about 500 million cells, at or about 1 billion cells, at or about 5 billion cells, at or about 20 billion cells, at or about 30 billion cells, at or about 40 billion cells, or a range defined by any two of the foregoing values), such as at or about 10 million to at or about 100 billion cells (e.g., at or about 20 million cells, at or about 30 million cells, at or about 40 million cells, at or about 60 million cells, at or about 70 million cells, at or about 80 million cells, at or about 90 million cells, at or about 10 billion cells, at or about 25 billion cells, at or about 50 billion cells, at or about 75 billion cells, at or about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases at or about 100 million cells to at or about 50 billion cells (e.g., at or about 120 million cells, at or about 250 million cells, at or about 350 million cells, at or about 650 million cells, at or about 800 million cells, at or about 900 million cells, at or about 3 billion cells, at or about 30 billion cells, at or about 45 billion cells) or any value in between these ranges and / or per kilogram of body weight of the subject. Dosages 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 total cells in the composition administered. In some embodiments, the number of cells is the number of such cells that are viable cells.

[0145] In some embodiments, the dose of cells is a flat dose of cells or fixed dose of cells such that the dose of cells is not tied to or based on the body surface area or weight of a subject.

[0146] In some embodiments, the dose of genetically engineered cells comprises from at or about 1 x 105to at or about 1 x 10stotal T cells expressing the CD19-directed CAR, from at or about 1 x 105to at or about 0.8 x 10stotal T cells expressing the CD19-directed CAR, from at or about 1 x 105to at or about 0.6 x 108total T cells expressing the CD19-directed CAR, from at or about 1 x 105to at or about 0.4 x 108total T cells expressing the CD19-directed CAR, from at or about 1 x 105to at or about 0.2 x 108total T cells expressing the CD19-directed CAR, from at or about 1 x 105to at or about 1.0 x 107total T cells expressing the CD19-directed CAR, from at or about 1 x 105to at or about 0.8 x 107total T cells expressing the CD19-directed CAR, from at or about 1 x 105to at or about 0.6 x 107total T cells expressing the CD19-directed CAR, from at or about 1 x 105to at or about 0.4 x 107total T cellsexpressing the CD19-directed CAR, from at or about 1 x 105to at or about 0.2 x 107total T cells expressing the CD19-directed CAR, from at or about 1 x 105to at or about 1.0 x 106total T cells expressing the CD19-directed CAR, from at or about 1 x 106to at or about 1.0 x 10stotal T cells expressing the CD19-directed CAR, from at or about 1 x 106to at or about 0.8 x 10stotal T cells expressing the CD19-directed CAR, from at or about 1 x 106to at or about 0.6 x 108total T cells expressing the CD19-directed CAR, from at or about 1 x 106to at or about 0.4 x 108total T cells expressing the CD19-directed CAR, from at or about 1 x 106to at or about 0.2 x 108total T cells expressing the CD19-directed CAR, from at or about 1 x 106to at or about 1.0 x 107total T cells expressing the CD19-directed CAR, from at or about 1 x 106to at or about 0.8 x 107total T cells expressing the CD19-directed CAR, from at or about 1 x 106to at or about 0.6 x 107total T cells expressing the CD19-directed CAR, from at or about 1 x 106to at or about 0.4 x 107total T cells expressing the CD19-directed CAR, from at or about 1 x 106to at or about 0.2 x 107total T cells expressing the CD19-directed CAR, from at or about 5 x 106to at or about 1.0 x 108total T cells expressing the CD19-directed CAR, from at or about 5 x 106to at or about 0.8 x 108total T cells expressing the CD19-directed CAR, from at or about 5 x 106to at or about 0.6 x 108total T cells expressing the CD19-directed CAR, from at or about 5 x 106to at or about 0.4 x 108total T cells expressing the CD19-directed CAR, from at or about 5 x 106to at or about 0.2 x 108total T cells expressing the CD19-directed CAR, from at or about 5 x 106to at or about 1.0 x 107total T cells expressing the CD19-directed CAR, from at or about 5 x 106to at or about 0.8 x 107total T cells expressing the CD19-directed CAR, from at or about 5 x 106to at or about 0.6 x 107total T cells expressing the CD19-directed CAR, from at or about 10 x 106to at or about 1.0 x 108total T cells expressing the CD19-directed CAR, from at or about 10 x 106to at or about 0.9 x 108total T cells expressing the CD19-directed CAR, from at or about 10 x 106to at or about 0.8 x 108total T cells expressing the CD19-directed CAR, from at or about 10 x 106to at or about 0.7 x 108total T cells expressing the CD19-directed CAR, from at or about 10 x 106to at or about 0.6 x 108total T cells expressing the CD19-directed CAR, from at or about 10 x 106to at or about 0.5 x 108total T cells expressing the CD19-directed CAR, from at or about 10 x 106to at or about 0.4 x 108total T cells expressing the CD19-directed CAR, from at or about 10 x 106to at or about 0.3 x 108total T cells expressing the CD19-directed CAR, from at or about 10 x 106to at or about 0.2 x 108total T cells expressing the CD19-directed CAR, from at or about 10 x 106to at or about 15 x 106total T cells expressing the CD19-directed CAR, from at or about 15 x 106to at or about 1.0 x 108total T cells expressing the CD19-directed CAR, from at or about 15 x 106to at or about 0.9 x 108total T cells expressing the CD19-directed CAR, from at or about 15 x 106to at or about 0.8 x 108total T cells expressing the CD19-directed CAR, from at or about 15 x 106to at or about 0.7 x 108total T cells expressing the CD19-directed CAR, from at or about 15 x 106to at or about 0.6 x 108total T cells expressing the CD19-directed CAR, from at or about 15 x 106to at or about 0.5 x 108total T cellsexpressing the CD19-directed CAR, from at or about 15 x 106to at or about 0.4 x 10stotal T cells expressing the CD19-directed CAR, from at or about 15 x 106to at or about 0.3 x 10stotal T cells expressing the CD19-directed CAR, from at or about 15 x 106to at or about 0.2 x 108total T cells expressing the CD19-directed CAR, from at or about 20 x 106to at or about 1.0 x 108total T cells expressing the CD19-directed CAR, from at or about 20 x 106to at or about 0.9 x 108total T cells expressing the CD19-directed CAR, from at or about 20 x 106to at or about 0.8 x 108total T cells expressing the CD19-directed CAR, from at or about 20 x 106to at or about 0.7 x 108total T cells expressing the CD19-directed CAR, from at or about 20 x 106to at or about 0.6 x 108total T cells expressing the CD19-directed CAR, from at or about 20 x 106to at or about 0.5 x 108total T cells expressing the CD19-directed CAR, from at or about 20 x 106to at or about 0.4 x 108total T cells expressing the CD19-directed CAR, from at or about 20 x 106to at or about 0.3 x 108total T cells expressing the CD19-directed CAR, from at or about 20 x 106to at or about 25 x 106total T cells expressing the CD19-directed CAR, from at or about 25 x 106to at or about 1.0 x 108total T cells expressing the CD19-directed CAR, from at or about 25 x 106to at or about 0.9 x 108total T cells expressing the CD19-directed CAR, from at or about 25 x 106to at or about 0.8 x 108total T cells expressing the CD19-directed CAR, from at or about 25 x 106to at or about 0.7 x 108total T cells expressing the CD19-directed CAR, from at or about 25 x 106to at or about 0.6 x 108total T cells expressing the CD19-directed CAR, from at or about 25 x 106to at or about 0.5 x 108total T cells expressing the CD19-directed CAR, from at or about 25 x 106to at or about 0.4 x 108total T cells expressing the CD19-directed CAR, from at or about 25 x 106to at or about 0.3 x 108total T cells expressing the CD19-directed CAR, from at or about 30 x 106to at or about 1.0 x 108total T cells expressing the CD19-directed CAR, from at or about 30 x 106to at or about 0.9 x 108total T cells expressing the CD19-directed CAR, from at or about 30 x 106to at or about 0.8 x 108total T cells expressing the CD19-directed CAR, from at or about 30 x 106to at or about 0.7 x 108total T cells expressing the CD19-directed CAR, from at or about 30 x 106to at or about 0.6 x 108total T cells expressing the CD19-directed CAR, from at or about 30 x 106to at or about 0.5 x 108total T cells expressing the CD19-directed CAR, from at or about 30 x 106to at or about 0.4 x 108total T cells expressing the CD19-directed CAR, from at or about 30 x 106to at or about 35 x 106total T cells expressing the CD19-directed CAR, from at or about 35 x 106to at or about 1.0 x 108total T cells expressing the CD19-directed CAR, from at or about 35 x 106to at or about 0.9 x 108total T cells expressing the CD19-directed CAR, from at or about 35 x 106to at or about 0.8 x 108total T cells expressing the CD19-directed CAR, from at or about 35 x 106to at or about 0.7 x 108total T cells expressing the CD19-directed CAR, from at or about 35 x 106to at or about 0.6 x 108total T cells expressing the CD19-directed CAR, from at or about 35 x 106to at or about 0.5 x 108total T cells expressing the CD19-directed CAR, from at or about 35 x 106to at or about 0.4 x 108total T cells expressing the CD19-directed CAR, from at or about 40 x 106to at or about 1.0 x 108total T cellsexpressing the CD19-directed CAR, from at or about 40 x 106to at or about 0.9 x 10stotal T cells expressing the CD19-directed CAR, from at or about 40 x 106to at or about 0.8 x 10stotal T cells expressing the CD19-directed CAR, from at or about 40 x 106to at or about 0.7 x 108total T cells expressing the CD19-directed CAR, from at or about 40 x 106to at or about 0.6 x 108total T cells expressing the CD19-directed CAR, from at or about 40 x 106to at or about 0.5 x 108total T cells expressing the CD19-directed CAR, or from at or about 40 x 106to at or about 45 x 106total T cells expressing the CD19-directed CAR. In some embodiments, the number of cells is the number of such cells that are viable cells, such as viable T cells.

[0147] In some embodiments, the dose of genetically engineered cells comprises at least or at least about 1 x 105T cells expressing the CD19-directed CAR, at least or at least about 2.5 x 105T cells expressing the CD19-directed CAR, at least or at least about 5 x 105T cells expressing the CD 19- directed CAR, at least or at least about 1 x 106T cells expressing the CD19-directed CAR, at least or at least about 2.5 x 106T cells expressing the CD19-directed CAR, at least or at least about 5 x 106T cells expressing the CD19-directed CAR, at least or at least about 1 x 107T cells expressing the CD 19- directed CAR, at least or at least about 2.5 x 107T cells expressing the CD19-directed CAR, or at least or at least about 5 x 107T cells expressing the CD19-directed CAR. In some embodiments, the number of cells is the number of such cells that are viable cells, such as viable T cells.

[0148] In some embodiments, the dose of genetically engineered cells comprises less than or less than about 1 x 105T cells expressing the CD19-directed CAR, less than or less than about 2.5 x 105T cells expressing the CD19-directed CAR, less than or less than about 5 x 105T cells expressing the CD19-directed CAR, less than or less than about 1 x 106T cells expressing the CD19-directed CAR, less than or less than about 2.5 x 106T cells expressing the CD19-directed CAR, less than or less than about 5 x 106T cells expressing the CD19-directed CAR, less than or less than about 1 x 107T cells expressing the CD19-directed CAR, less than or less than about 1.5 x 107T cells expressing the CD19-directed CAR, less than or less than about 2 x 107T cells expressing the CD19-directed CAR, less than or less than about 2.5 x 107T cells expressing the CD19-directed CAR, less than or less than about 3 x 107T cells expressing the CD19-directed CAR, less than or less than about 3.5 x 107T cells expressing the CD19-directed CAR, less than or less than about 4 x 107T cells expressing the CD19-directed CAR, less than or less than about 4.5 x 107T cells expressing the CD19-directed CAR, or less than or less than about 5 x 107T cells expressing the CD19-directed CAR. In some embodiments, the number of cells is the number of such cells that are viable cells, such as viable T cells.

[0149] In some embodiments, the cell therapy comprises administration of a dose comprising a number of cell from or from about 1 x 105to or to about 5 x 108total recombinant receptor-expressing cells or total T cells, from or from about 5 x 105to or to about 1 x 107total recombinant receptor expressing cells or total T cells, or from or from about 1 x 106to or to about 1 x 107total recombinant receptor-expressing cells or total T cells,, each inclusive. In some embodiments, the cell therapycomprises administration of a dose comprising a number of cell from or from about 1 x 105to or to about 1 x 10stotal recombinant receptor-expressing cells, or total T cells, from or from about 5 x 105to or to about 1 x 10stotal recombinant receptor-expressing cells, or total T cells, from or from about 1 x 106to or to about 50 x 106total recombinant receptor-expressing cells, or total T cells, from or from about 5 x106to or to about 45 x 106total recombinant receptor-expressing cells or total T cells, or from or from about 10 x 106to or to about 25 x 106total recombinant receptor-expressing cells or total T cells, each inclusive. In some embodiments, the cell therapy comprises administration of a dose of cells comprising a number of cells at least or at least about 1 x 105total recombinant receptor-expressing cells or total T cells, such at least or at least 1 x 106, at least or at least about 1 x 107, at least or at least about 1 x 108of such cells. In some embodiments, the number of cells is the number of such cells that are viable cells, such as viable T cells.

[0150] In some embodiments, the number is with reference to the total number of CD3+, CD8+, or CD4+ and CD8+, in some cases also recombinant receptor-expressing (e.g. CAR+) cells. In some embodiments, the number of cells is the number of such cells that are viable cells.

[0151] In some embodiments, the cell therapy comprises administration of a dose comprising a number of cell from or from about 1 x 105to or to about 1 x 108CD3+, CD8+or CD4+and CD8+total T cells or CD3+, CD8+or CD4+and CD8+recombinant receptor (e.g. CAR)-expressing cells, from or from about 5 x 105to or to about 5 x 107CD3+, CD8+or CD4+and CD8+total T cells or CD3+, CD8+or CD4+and CD8+recombinant receptor (e.g. CAR)-expressing cells, or from or from about 1 x 106to or to about 2.5 x 107CD3+, CD8+or CD4+and CD8+total T cells or CD3+, CD8+or CD4+and CD8+recombinant receptor (e.g. CAR)-expressing cells, each inclusive. In some embodiments, the cell therapy comprises administration of a dose comprising a number of cell from or from about 1 x 105to or to about 1 x 108total CD3+ / CAR+, CD8+ / CAR+or CD4+ / CD8+ / CAR+cells, from or from about 5 x 105to or to about 5 x107total CD3+ / CAR+, CD8+ / CAR+or CD4+ / CD8+ / CAR+cells, or from or from about 1 x 106to or to about 2.5 x 107total CD3+ / CAR+, CD8+ / CAR+or CD4+ / CD8+ / CAR+cells, each inclusive. In some embodiments, the number of cells is the number of such cells that are viable cells.

[0152] In some embodiments, the dose of genetically engineered cells comprises at least or at least about 0.1 x 107CD3+ / CAR+, CD8+ / CAR+, or CD4+ / CD8+ / CAR+T cells, at least or at least about 0.5 x 107CD3+ / CAR+, CD87CAR+, or CD47CD87CAR+T cells, at least or at least about 1.0 x 107CD3+ / CAR+, CD87CAR+, or CD47CD87CAR+T cells, at least or at least about 2.5 x 107CD3+ / CAR+, CD87CAR+, or CD47CD87CAR+T cells, or at least or at least about 5 x 107CD3+ / CAR+, CD87CAR+, or CD4+ / CD8+ / CAR+T cells. In some embodiments, the dose of genetically engineered cells comprises less than or less than about 0.1 x 107CD3+ / CAR+, CD8+ / CAR+, or CD4+ / CD8+ / CAR+T cells, less than or less than about 0.5 x 107CD3+ / CAR+, CD8+ / CAR+, or CD4+ / CD8+ / CAR+T cells, less than or less than about 1.0 x 107CD3+ / CAR+, CD8+ / CAR+, or CD4+ / CD8+ / CAR+T cells, less than or less than about 2.5 x 107CD3+ / CAR+, CD87CAR+, or CD47CD87CAR+T cells, less than or less than about 5 x 107CD3+ / CAR+, CD8+ / CAR+, or CD4+ / CD8+ / CAR+T cells, less than or less than about 7.5 x 107CD3+ / CAR+, CD8+ / CAR+, or CD4+ / CD8+ / CAR+T cells, or less than or less than about less than or less than about 10 x 107CD3+ / CAR+, CD8+ / CAR+, or CD4+ / CD8+ / CAR+T cells. In some embodiments, the dose of genetically engineered cells comprises at or about 0.5 x 107CD3+ / CAR+, CD8+ / CAR+, or CD47CD87CAR+T cells, at or about 1.0 x 107CD3+ / CAR+, CD87CAR+, or CD47CD87CAR+T cells, at or about 1.5 x 107CD3+ / CAR+, CD87CAR+, or CD47CD87CAR+T cells, at or about 2.0 x 107CD3+ / CAR7 CD87CAR7 or CD47CD87CAR+T cells, at or about 2.5 x 107CD3+ / CAR+,CD87CAR7 or CD47CD87CAR+T cells, at or about 3.0 x 107CD3+ / CAR+, CD87CAR+, or CD47CD 87C AR+T cells, at or about 3.5 x 107CD3+ / CAR+, CD87CAR+, or CD47CD87CAR+T cells, at or about 4.0 x 107CD3+ / CAR+, CD87CAR+, or CD47CD87CAR+T cells, at or about 4.5 x 107CD3+ / CAR7 CD87CAR7 or CD47CD87CAR+T cells, at or about 5 x 107CD3+ / CAR+, CD87CAR+, or CD47CD87CAR+T cells. In some embodiments, the number of cells is the number of such cells that are viable cells.

[0153] In some embodiments, the dose of T cells comprises: at or about 0.5 x 107recombinant receptor (e.g. CAR)-expressing T cells, at or about 1.0 x 107recombinant receptor (e.g. CAR)-expressing T cells, at or about 1.5 x 107recombinant receptor (e.g. CAR)-expressing T cells, at or about 2.0 x 107recombinant receptor (e.g. CAR)-expressing T cells, at or about 2.5 x 107recombinant receptor (e.g. CAR)-expressing CD8+T cells, at or about 3.0 x 107recombinant receptor (e.g. CAR)-expressing T cells, at or about 3.5 x 107recombinant receptor (e.g. CAR)-expressing T cells, at or about 4.0 x 107recombinant receptor (e.g. CAR)-expressing T cells, or at or about 4.5 x 107recombinant receptor (e.g. CAR)-expressing T cells. In some embodiments, the dose of T cells comprises: at or about 1 x 10srecombinant receptor (e.g. CAR)-expressing T cells or at or about 5 x 107recombinant receptor (e.g. CAR)-expressing CD8+T cells. In some embodiments, the dose of T cells comprises: at or about 1.5 x 10srecombinant receptor (e.g. CAR)-expressing T cells or at or about 0.75 x 108recombinant receptor (e.g. CAR) -expressing CD8+T cells. In some embodiments, the number of cells is the number of such cells that are viable cells.

[0154] In some embodiments, the T cells of the dose include CD4+T cells, CD8+T cells or CD4+and CD8+T cells.

[0155] In some embodiments, for example, where the subject is human, the CD8+ T cells of the dose, including in a dose including CD4+ and CD8+ T cells, includes between at or about 1 x 106and at or about 1 x 108total recombinant receptor (e.g., CAR)-expressing CD8+ cells, e.g., in the range of from at or about 1 x 106to at or about 1 x 108such cells, such as 1 x 106, 2.5 x 106, 5 x 106, 7.5 x 106, 1 x 107, 2.5 x 107, 5 x 107, or 7.5 x 107total such cells, or the range between 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 embodiments, the dose of cells comprises the administration of from or from about 1 x 106to or to about 5 x 107total recombinant receptor-expressingCD8+ T cells, from or from about 5 x 106to or to about 2.5 x 107total recombinant receptor-expressing CD8+ T cells, from or from about 10 x 106to or to about 2.5 x 107total recombinant receptor-expressing CD8+ T cells, each inclusive. In some embodiments, the number of cells is the number of such cells that are viable cells.

[0156] In some embodiments, for example, where the subject is human, the CD4+ T cells of the dose, including in a dose including CD4+ and CD8+ T cells, includes between at or about 1 x 106and at or about 1 x 10stotal recombinant receptor (e.g., CAR)-expressing CD4+ cells, e.g., in the range of from at or about 1 x 106to at or about 1 x 10ssuch cells, such as 1 x 106, 2.5 x 106, 5 x 106, 7.5 x 106, 1 x 107, 2.5 x 107, 5 x 107, or 7.5 x 107total such cells, or the range between 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 embodiments, the dose of cells comprises the administration of from or from about 1 x 106to or to about 5 x 107total recombinant receptor-expressing CD4+ T cells, from or from about 5 x 106to or to about 2.5 x 107total recombinant receptor-expressing CD4+ T cells, from or from about 10 x 106to or to about 2.5 x 107total recombinant receptor-expressing CD4+ T cells, each inclusive. In some embodiments, the number of cells is the number of such cells that are viable cells.

[0157] In some embodiments, for example, where the subject is a human, the dose includes fewer than about 5 x 108total recombinant receptor (e.g., CAR)-expressing cells or T cells, e.g., in the range of at or about 1 x 106to at or about 1 x 108such cells, such as at or about 2 x 106, 5 x 106, 1 x 107, 5 x 107, or 1 x 108total such cells, or the range between any two of the foregoing values. In some embodiments, the number of cells is the number of such cells that are viable cells.

[0158] 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 embodiments, the dose of cells comprises the administration of from or from about 1 x 105to or to about 1 x 108total recombinant receptor (e.g. CAR)-expressing T cells or total T cells, from or from about 1 x 105to or to about 0.5 x 108total recombinant receptor (e.g. CAR)-expressing T cells or total T cells, from or from about 1 x 105to or to about 0.5 x 108total recombinant receptor (e.g. CAR)-expressing T cells or total T cells, from or from about 5 x 105to or to about 5 x 107total recombinant receptor (e.g. CAR)-expressing T cells or total T cells, or from or from about 1 x 106to or to about 1 x 107total recombinant receptor (e.g. CAR)- expressing T cells or total T cells, each inclusive.

[0159] In some embodiments, the T cells of the dose include CD4+T cells, CD8+T cells or CD4+and CD8+T cells.

[0160] In some embodiments, the dose of cells, e.g., recombinant receptor-expressing T cells, is administered to the subject as a single dose or is administered only one time within a period of two weeks, one month, 3 months, six months, 1 year or more.

[0161] In the context of adoptive cell therapy, administration of a given “dose” encompasses administration of the given amount or number of cells as a single composition and / or single uninterrupted administration, e.g., as a single injection or continuous infusion, and also encompasses administration of the given amount or number of cells as a split dose or as a plurality of compositions, provided in multiple individual compositions or infusions, over a specified period of time, such as over no more than 3 days. Thus, in some contexts, the dose is a single or continuous administration of the specified number of cells, given or initiated at a single point in time. In some contexts, however, 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.

[0162] In particular embodiments, the numbers and / or concentrations of cells refer to the number of recombinant receptor (e.g., CAR)-expressing cells. In other embodiments, the numbers and / or concentrations of cells refer to the number or concentration of T cells administered.

[0163] In some embodiments, the subject receives multiple doses, e.g., two or more doses or multiple consecutive doses, of the cells. In some embodiments, two doses are administered to a subject. In some embodiments, the subject receives the consecutive dose, e.g., second dose, is administered approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days after the first dose. In some embodiments, multiple consecutive doses are administered following the first dose, such that an additional dose or doses are administered following administration of the consecutive dose. In some aspects, the number of cells administered to the subject in the additional dose is the same as or similar to the first dose and / or consecutive dose. In some embodiments, the additional dose or doses are larger than prior doses.

[0164] 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, 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.

[0165] In some aspects, the time between the administration of the first dose and the administration of the consecutive dose is about 9 to about 35 days, about 14 to about 28 days, or 15 to 27 days. In some embodiments, the administration of the consecutive dose is at a time point more than about 14 days after and less than about 28 days after the administration of the first dose. In some aspects, the time between the first and consecutive dose is about 21 days. In some embodiments, an additional dose or doses, e.g. consecutive doses, are administered following administration of the consecutive dose. In some aspects, the additional consecutive dose or doses are administered at least about 14 and less than about 28 days following administration of a prior dose. In some embodiments, the additional dose is administered less than about 14 days following the prior dose, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days after theprior dose. In some embodiments, no dose is administered less than about 14 days following the prior dose and / or no dose is administered more than about 28 days after the prior dose.

[0166] In some embodiments, the dose of cells is generally large enough to be effective in reducing disease burden.

[0167] In particular embodiments, the numbers and / or concentrations of cells refer to the number of recombinant 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.

[0168] In some embodiments, the methods also include administering one or more additional doses of cells expressing a chimeric antigen receptor (CAR) and / or lymphodepleting 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 additional dose is different from the initial dose, e.g., higher, such as at or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold or more higher than the initial dose, or lower, such as e.g., higher, such as 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold or more lower than the initial dose. In some embodiments, administration of one or more additional 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

[0169] In some embodiments, the administration in accord with the provided methods effectively treats the subject despite the subject having become resistant to another therapy. In some embodiments, at least 30%, at least 35%, at least 40% at least 50%, at least 60%, at least 70%, or at least 80%, of subjects treated according to the method achieve complete remission (CR). In some embodiments, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least 80%, or at least 90% of the subjects treated according to the method achieve an objective response (OR). In some embodiments, at least or at least about 50% of subjects, at least or at least about 60% of the subjects, at least or at least about 70% of the subjects, at least or at least about 80% of the subjects or at least or at least about 90% of the subjects treated according to the method achieve CR and / or achieve an objective response (OR). In some embodiments, criteria assessed for effective treatment includes overall response rate (ORR; also known in some cases as objective response rate), complete response (CR; also known in some cases as complete remission), duration of response (DOR), progression-free survival (PFS), and / or overall survival (OS).

[0170] In some embodiments, at least 40% or at least 50% of subjects treated according to the methods provided herein achieve complete remission (CR; also known in some cases as complete response), exhibit progression-free survival (PFS) and / or overall survival (OS) of greater than at or about 3 months, 6 months or 12 months or greater than 13 months or approximately 14 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 therapy for at least at or about 6, 12, 18 or more months or longer.

[0171] In some embodiments, the subjects treated according to the provided methods exhibits a CRR of at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, the complete response rate (CRR) is calculated as the percentage of subjects with the best overall response (BOR) up to 12 months, up to 18 months, up to 24 months, up to 36 months or longer.

[0172] In some aspects, response rates in subjects, such as subjects with NHL, are based on the Lugano criteria. (Cheson et al., (2014) JCO 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323- 338; Cheson, B.D. (2015) Chin Clin Oncol 4(1):5). In some aspects, response assessment utilizes any of clinical, hematologic, and / or molecular methods. In some aspects, response assessed using the Lugano criteria involves the use of positron emission tomography (PET)-computed tomography (CT) and / or CT as appropriate. PET-CT evaluations may further comprise the use of fluorodeoxyglucose (FDG) for FDG-avid lymphomas. In some aspects, where PET-CT will be used to assess response in FDG-avid histologies, a 5-point scale may be used. In some respects, the 5-point scale comprises the following criteria: 1, no uptake above background; 2, uptake < mediastinum; 3, uptake > mediastinum but < liver;4, uptake moderately > liver; 5, uptake markedly higher than liver and / or new lesions; X, new areas of uptake unlikely to be related to lymphoma.

[0173] In some aspects, a complete response as described using the Lugano criteria involves a complete metabolic response and a complete radiologic response at various measureable sites. In some aspects, these sites include lymph nodes and extralymphatic sites, wherein a CR is described as a score of 1, 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 chemotherapy or myeloid colony-stimulating factors), uptake may be greater than normal mediastinum and / or liver. In this circumstance, 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, wherein a CR is described as no extralymphatic sites of disease and target nodes / nodal masses must regress to < 1.5 cm in longest transverse diameter of a lesion (LDi). Further sites of assessment include the bone marrow wherein 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. Insome aspects, non-measured lesions and new lesions are assessed, which in the case of CR should be absent (Cheson et al., (2014) JCO 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323-338; Cheson, B.D. (2015) Chin Clin Oncol 4(1):5).

[0174] In some aspects, a partial response (PR; also known in some cases as partial remission) as described using the Lugano criteria involves a partial metabolic and / or radiological response at various measureable sites. In some aspects, these sites include lymph nodes and extralymphatic sites, wherein a PR is described as a score of 4 or 5 with reduced uptake compared with baseline and residual mass(es) of any size, when PET-CT is used. At interim, such findings can indicate responding disease. At the end of treatment, such findings can indicate residual disease. In some aspects, response is assessed in the lymph nodes using CT, wherein a PR is described as >50% decrease in SPD of up to 6 target measureable nodes and extranodal sites. If a lesion is too small to measure on CT, 5 mm x 5 mm is assigned as the default value; if the lesion is no longer visible, the value is 0 mm x 0 mm; for a node >5 mm x 5 mm, but smaller than normal, actual measurements are used for calculation. Further sites of assessment include the bone marrow wherein PET-CT-based assessment should indicate residual uptake higher than uptake in normal marrow but reduced compared with baseline (diffuse uptake compatible with reactive changes from chemotherapy allowed). In some aspects, if there are persistent focal changes in the marrow in the context of a nodal response, consideration should be given to further evaluation with MRI or biopsy, or an interval scan. In some aspects, further sites may include assessment of organ enlargement, where the spleen must have regressed by >50% in length beyond normal. In some aspects, non-measured lesions and new lesions are assessed, which in the case of PR should be absent / normal, regressed, but no increase. No response / stable disease (SD) or progressive disease (PD) can also be measured using PET- CT and / or CT based assessments. (Cheson et al., (2014) JCO 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323-338; Cheson, B.D. (2015) Chin Clin Oncol 4(1):5).

[0175] 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 response. In some aspects, objective response rate (ORR; also known in some cases as overall response rate) 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 symptoms, such as bone pain from cancer that has spread to the bone, or death.

[0176] In some embodiments, the measure of duration of response (DOR) includes the time from documentation of tumor response to disease progression. In some embodiments, the parameter forassessing response can include durable response, e.g., response that persists after a period of time from initiation of therapy. In some embodiments, durable response is indicated by the response rate at approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18 or 24 months after initiation of therapy. In some embodiments, the response is durable for greater than 3 months or greater than 6 months.

[0177] In some aspects, the RECIST criteria is 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 is used to determine objective tumor response for target lesions. In some respects, a complete response as determined 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 includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, 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.

[0178] In some embodiments survival rates in subjects with follicular lymphoma (FL) are based on scoring systems developed by the Italian Lymphoma Intergroup (ILI) and / or the International Follicular Lymphoma Prognostic Factor Project (IFLPFP), generally as described above (Luminari et al., (2012) Rev. Brad. Hematol. Hemoter., 34:54-59). In some embodiments, the extent of disease such as a FL may be assessed by the Ann Arbor staging system, tumor burden, bulky disease, number of nodal or extranodal sites of disease, and / or bone marrow involvement, generally as described above.

[0179] In some aspects, the administration in accord with the provided methods, and / or with the provided articles of manufacture or compositions, 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 methods 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 symptom associated with tumor burden.

[0180] 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 include, in some embodiments, the mass of a tumor, the number or extent of metastases and / or the percentage of blast cells present in the bone marrow.

[0181] In some aspects, minimal residual disease (MRD) is detected 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 351:550-554). 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).

[0182] In some examples, bone marrow is harvested by bone marrow aspirates or bone marrow biopsies, and lymphocytes are isolated for analysis. Monoclonal and / or polyclonal antibodies conjugated to a fluorochrome (e.g., fluorescein isothiocyanate (FITC), phycoerythrin, peridinin chlorophyll protein, or biotin) can be used to detect epitopes, such as terminal deoxynucleotidyl transferase (TdT), CD3,CD 10, CDllc, 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 multiparameter flow cytometry, or mass cytometry, to detect multiple epitopes.

[0183] 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 epitopes are set forth in Table 2 below. Other immunologic classification of leukemias and lymphomas are provided 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+; CD14neg; CD19+; CD23+; CD45+; CD56neg).

[0184] In some aspects, deep sequencing of the immunoglobulin heavy chain (IGH) locus of harvested 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 malignancies, such as CLL or NHL and / or residual presence 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 marrow, 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 complementarity determining 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 clonalpopulations of cells for purposes of assessing minimal residual disease. Other methods for detecting clonal populations, such as single cell sequencing approaches, including those providing 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 population, may be used. In some aspects, the IGH DNA is amplified using a degenerate primers or primers recognizing regions of variable chains shared among different cell clones, such as those recognizing consensus V and degenerate consensus J region of the IGH sequence. An exemplary sequence of the V region isACACGGCCTCGTGTATTACTGT (SEQ ID NO: 57). An exemplary degenerate consensus sequence of the J region is ACCT G AGG AG ACGGT G ACC (SEQ ID NO: 58).

[0185] 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 following 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.

[0186] 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 treatment is associated with increased risk of relapse. In some aspects, patients who are negative for malignant IGH sequences 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 increased likelihood of PFS or to enter into CR or durable CR or prolonged survival, compared to patients with persistent malignant IGH sequences. In some embodiments, such prognostic and staging 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 clinical 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 likelihood of response or durability thereof, 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 further administered a dose of the therapy provided, or that the subject be administered a lower or reduced dose. Conversely, it may be provided or specified that a subject exhibiting MRD via IGH deep sequencing be further treated, e.g., with the therapy initially administered at a similar or higher dose or with a furthertreatment. In some aspects, the disease or condition persists following administration of the first dose and / or administration of the first dose is not sufficient to eradicate the disease or condition in the subject.

[0187] In some embodiments, 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 survival, 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 alternative dosing regimen, such as one 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, and / or with the provided articles of manufacture or compositions. In some embodiments, the burden of a disease or condition in the subject is detected, assessed, or measured. Disease burden may be detected in some aspects by detecting the total number of disease or disease-associated cells, e.g., tumor cells, in the subject, or in an organ, tissue, 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 assessed. In some embodiments, the measure of disease or condition burden is specified.

[0188] 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 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, and / or with the provided articles of manufacture or compositions. For example, in some embodiments, event-free survival rate or probability for subjects treated by the methods 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 subject treated with the methods exhibits event-free survival, relapse-free survival, 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 improved, 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.

[0189] In some embodiments, following treatment by the method, the probability of relapse is reduced as compared 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, and / or with the provided articles of manufacture or compositions. For example, in some embodiments, the probability 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%.

[0190] In some cases, the pharmacokinetics of administered cells, e.g., adoptively transferred cells are determined to assess the availability, e.g., bioavailability of the administered cells. Methods for determining the pharmacokinetics of adoptively transferred cells may include drawing peripheral blood from subjects that have been administered engineered cells, and determining the number or ratio of the engineered cells in the peripheral blood. Approaches for selecting and / or isolating cells may include use of chimeric antigen receptor (CAR)-specific antibodies (e.g., Brentjens et ah, Sci. Transl. Med. 2013 Mar; 5(177): 177ra38) Protein L (Zheng et al., J. Transl. Med. 2012 Feb; 10:29), epitope tags, such as Strep-Tag sequences, introduced directly into specific sites in the CAR, whereby binding reagents for Strep-Tag are used to directly assess the CAR (Liu et al. (2016) Nature Biotechnology, 34:430; international patent application Pub. No. WO2015095895) and monoclonal antibodies that specifically bind to a CAR polypeptide (see international patent application Pub. No. WO2014190273). Extrinsic marker genes may in some cases be utilized in connection with engineered cell therapies to permit detection or selection of cells and, in some cases, also to promote cell suicide. A truncated epidermal growth factor receptor (EGFRt) in some cases can be co-expressed with a transgene of interest (a CAR) in transduced cells (see e.g. U.S. Patent No. 8,802,374). EGFRt may contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibody or binding molecule, which can be used to identify or select cells that have been engineered with the EGFRt construct and another recombinant receptor, such as a chimeric antigen receptor (CAR), and / or to eliminate or separate cells expressing the receptor. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434).

[0191] In some embodiments, the number of CAR+T cells in a biological sample obtained from the patient, e.g., blood, can be determined at a period of time after administration of the cell therapy, e.g., to determine the pharmacokinetics of the cells. In some embodiments, number of CAR+T cells, optionally CAR+CD8+T cells and / or CAR+CD4+T cells, detectable in the blood of the subject, or in a majority of subjects so treated by the method, is greater than 1 cells per pL, greater than 5 cells per pL or greater than per 10 cells per pL.C. Toxicity

[0192] In some embodiments, the provided methods are designed to or include features that result in a lower rate and / or lower degree of toxicity, toxic outcome or symptom, toxicity-promoting profile, factor, or property, such as a symptom or outcome associated with or indicative of cytokine release syndrome (CRS) or neurotoxicity (NT), for example, compared to administration of an alternative cell therapy, such as an alternative CAR+T cell composition and / or an alternative dosing of cells, e.g. a dosing of cells that is not administered at a defined ratio. Cytokine release syndrome (CRS) and neurotoxicity) can be graded according to the American Society for Transplantation and Cellular Therapy(ASTCT) Consensus Grading System (see e.g., Lee et al. Biol Blood Marrow Transplant. 2019 Apr;25(4):625-38)).

[0193] In some aspects, although the lower differentiation state of the engineered T cells administered as part of the methods provided herein (e.g., the higher proportion of engineered T cells having a naive-like or central memory phenotype, such as a phenotype selected from CCR7+CD45RA+, CD27+CCR7+, or CD62L CCR7+) are expected to be more active than cells that are more differentiated, findings indicate that safety of the cell therapy can be successfully managed. In some aspects, providing a lower dose of the composition, e.g. compared to a cell composition produced by a process in which the cells are more differentiated, such as a process that includes expansion of the cells, achieves robust efficacy and high safety. In some aspects, it is found that even higher doses of cells of the provided anti- BCMA CAR compositions can be administered while maintaining alower degree of toxicity, such as a severe cytokine release syndrome (CRS) or severe neurotoxicity. Thus, the provided methods in some embodiments include the administration of higher doses of engineered T cells (e.g., greater than 50 x 106CAR-expressing T cells, such as at or about 100 x 106CAR-expressing T cells), compared to methods that include the administration of an alternative cell therapy, such as an alternative CAR+T cell composition with engineered T cells that are more differentiated than those administered herein.

[0194] In some embodiments, the provided methods do not result in a high rate or likelihood of toxicity or toxic outcomes, or reduces the rate or likelihood of toxicity or toxic outcomes, such as neurotoxicity (NT), cytokine release syndrome (CRS), such as compared to certain other cell therapies.In some embodiments, the methods do not result in, or do not increase the risk of, severe NT (sNT), severe CRS (sCRS), macrophage activation syndrome, tumor lysis syndrome, fever of at least at or about 38 degrees Celsius for three or more days and a plasma level of CRP of at least at or about 20 mg / dL. In some embodiments, greater than or greater than about 30%, 35%, 40%, 50%, 55%, 60% or more of the subjects treated according to the provided methods do not exhibit any grade of CRS or any grade of neurotoxcity. In some embodiments, no more than 50% of subjects treated (e.g. at least 60%, at least 70%, at least 80%, at least 90% or more of the subjects treated) exhibit a cytokine release syndrome (CRS) higher than grade 2 and / or a neurotoxicity higher than grade 2. 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 treated) do not exhibit a severe toxic outcome (e.g. severe CRS or severe neurotoxicity), such as do 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 treatment, such as within a week, two weeks, or one month of the administration of the cells. In some embodiments, parameters assessed to determine certain toxicities include adverse events (AEs), dose-limiting toxicities (DLTs), CRS and NT.

[0195] 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 andneurotoxicity. In some examples, such effects or outcomes parallel high levels of circulating cytokines, which may underlie the observed toxicity.

[0196] In some aspects, the toxic outcome is or is associated with or indicative of cytokine release syndrome (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 biological products. See Davila et al., Sci Transl Med 6, 224ra25 (2014); Brentjens et al., Sci. Transl. Med. 5, 177ra38 (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.

[0197] Typically, CRS is caused by an exaggerated systemic immune response mediated by, for example, T cells, B cells, NK cells, monocytes, and / or macrophages. Such cells may release a large amount of inflammatory mediators such as cytokines and chemokines. Cytokines may trigger an acute inflammatory response and / or induce endothelial organ damage, which may result in micro vascular leakage, heart failure, or death. Severe, life-threatening CRS can lead to pulmonary infiltration and lung injury, renal failure, or disseminated intravascular coagulation. Other severe, life-threatening toxicities can include cardiac toxicity, respiratory distress, neurologic toxicity and / or hepatic failure. In some aspects, fever, especially high fever (> 38.5°C or > 101.3°F), is associated with CRS or risk thereof. In some cases, features or symptoms of CRS mimic infection. In some embodiments, infection is also considered in subjects presenting with CRS symptoms, and monitoring by cultures and empiric antibiotic therapy can be administered. Other symptoms associated with CRS can include cardiac dysfunction, adult respiratory distress syndrome, renal and / or hepatic failure, coagulopathies, disseminated intravascular coagulation, and capillary leak syndrome.

[0198] 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 or other agents as described. 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 degrees thereof may be specified.

[0199] In the context of administering CAR-expressing 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 CRS may be related to baseline cytokine levels or tumor burden at the time of infusion. Commonly, CRS involves elevated serum levels of interferon (IFN)-y, tumor necrosis factor (TNF)-a, and / or interleukin (IL)-2. Other cytokines that may be rapidly induced in CRS are IL-Ib, IL-6, IL-8, and IL-10.

[0200] Exemplary outcomes associated with CRS include fever, rigors, chills, hypotension, dyspnea, acute respiratory distress syndrome (ARDS), encephalopathy, ALT / AST elevation, renal failure, cardiac disorders, hypoxia, neurologic disturbances, and death. Neurological complications include delirium,seizure-like activity, confusion, word-finding difficulty, aphasia, and / or becoming obtunded. Other CRS- related outcomes include fatigue, nausea, headache, seizure, tachycardia, myalgias, rash, acute vascular leak syndrome, 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. Other exemplary signs or symptoms associated with CRS include hemodynamic instability, febrile neutropenia, increase in serum C-reactive protein (CRP), changes in coagulation parameters (for example, international normalized ratio (INR), prothrombin time (PTI) and / or fibrinogen), changes in cardiac and other organ function, and / or absolute neutrophil count (ANC).

[0201] In some embodiments, outcomes associated with CRS include one or more of: persistent fever, e.g., fever of a specified temperature, 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-3L, fracktalkine, and IL-5, and / or tumor necrosis factor alpha (TNFa)), 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 clinical sign of toxicity, such as hypotension (e.g., as measured by at least one intravenous vasoactive pressor); hypoxia (e.g., plasma oxygen (PO2) levels of less than at or about 90%); and / or one or more neurologic disorders (including mental status changes, obtundation, and seizures). In some embodiments, neurotoxicity (NT) can be observed concurrently with CRS.

[0202] 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 increases 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.

[0203] In some embodiments, the CRS-associated serum factors or CRS-related outcomes include inflammatory cytokines and / or chemokines, including interferon gamma (IFN-g), TNF-a, IL-Ib, 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 (TNFa), IL-6, and IL-10, IL-Ib, IL-8, IL-2, MIP-1, Flt-3L, fracktalkine, and / or IL-5. In some embodiments, the factor or outcome includes C reactive 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 measured to have high levels of CRP, such as > 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 CRS includes a measure of CRP and another factor indicative of CRS.

[0204] In some embodiments, one or more inflammatory cytokines or chemokines are monitored before, during, or after CAR treatment. In some aspects, the one or more cytokines or chemokines include IFN-g, TNF-a, IL-2, IL-Ib, IL-6, IL-7, IL-8, IL-10, IL-12, sIL-2Ra, granulocyte macrophage colony stimulating factor (GM-CSF), or macrophage inflammatory protein (MIP). In some embodiments, IFN-g, TNF-a, and IL-6 are monitored.

[0205] CRS criteria that appear to correlate with the onset of CRS 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 (IHNg, IL-5, IL-6, IL- 10, Flt-3L, fractalkine, and GM-CSF) whose treatment-induced elevation can correlate well with both pretreatment 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; Lee et al., Biol Blood Marrow Transplant 2019; 25(4):625-38). In some embodiments, the criteria reflective of CRS grade are those detailed in Table 3 below.

[0206] In some embodiments, a criteria reflective of CRS grade are those detailed in Table 4 below.

[0207] In some embodiments, high-dose vasopressor therapy include those described in Table 5 below.

[0208] In some embodiments, the toxic outcome is a severe CRS. In some embodiments, the toxic outcome is the absence of severe CRS (e.g. moderate or mild CRS). In some embodiments, 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, following administration, the subject 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 pressor) or hypoxia (PO2 < 90%) or one or more neurologic disorder(s) (including mental status changes, obtundation, and / or seizures). In someembodiments, severe CRS includes CRS with a grade of 3 or greater, such as set forth in Table 3 and Table 4.

[0209] In some embodiments, the level of the toxic outcome, e.g. the CRS-related outcome, e.g. the serum level of an indicator of CRS, is measured by ELISA. In some embodiments, fever and / or levels of C-reactive protein (CRP) can be measured. In some embodiments, subjects with a fever and a CRP > 15 mg / dL may be considered high-risk for developing severe CRS. In some embodiments, the CRS- associated serum factors or CRS-related outcomes include an increase in the level and / or concentration of inflammatory cytokines and / or chemokines, including Flt-3L, fracktalkine, granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-1 beta (IL-Ib), IL-2, IL-5, IL-6, IL-7, IL-8, IL-10, IL- 12, interferon gamma (IFN-g), macrophage inflammatory protein (MIP)-l, MIP-1, sIL-2Ra, or tumor necrosis factor alpha (TNFa). In some embodiments, the factor or outcome includes C reactive 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 measured to have high levels of CRP, such as > 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 CRS includes a measure of CRP and another factor indicative of CRS.

[0210] In some embodiments, outcomes associated with severe CRS or grade 3 CRS or greater, such as grade 4 or greater, include one or more of: persistent fever, e.g., fever of a specified temperature, 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-3L, fracktalkine, and IL-5, and / or tumor necrosis factor alpha (TNFa)), 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 clinical sign of toxicity, such as hypotension (e.g., as measured by at least one intravenous vasoactive pressor); hypoxia (e.g., plasma oxygen (PO2) levels of less than at or about 90%); and / or one or more neurologic disorders (including mental status changes, obtundation, and seizures). In some embodiments, severe CRS includes CRS that requires management or care in the intensive care unit (ICU).

[0211] In some embodiments, the CRS, such as severe CRS, encompasses a combination of (1) 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 mechanical ventilation. In some embodiments, the dosage of vasopressors is increased in a second or subsequent administration.

[0212] In some embodiments, severe CRS or grade 3 CRS encompasses an increase in alanine aminotransferase, an increase in aspartate aminotransferase, chills, febrile neutropenia, headache, left ventricular dysfunction, encephalopathy, hydrocephalus, and / or tremor.

[0213] The method of measuring or detecting the various outcomes may be specified.

[0214] In some aspects, the toxic outcome is or is associated with neurotoxicity. In some embodiments, symptoms associated with a clinical risk of neurotoxicity include confusion, delirium, aphasia, 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 embodiments, 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).

[0215] In some instances, neurologic symptoms may be the earliest symptoms of sCRS. In some embodiments, neurologic symptoms 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 resolved. In some embodiments, time or degree of resolution of neurologic changes is not hastened by treatment with anti-IL-6 and / or steroid(s).

[0216] In some embodiments, 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 displays symptoms that limit self-care (e.g. bathing, dressing and undressing, feeding, using the toilet, taking medications) from among: 1) symptoms of peripheral motor neuropathy, including inflammation or degeneration of the peripheral motor nerves; 2) symptoms of peripheral sensory neuropathy, including inflammation or degeneration of the peripheral sensory nerves, dysesthesia, such as distortion of sensory perception, resulting in an abnormal and unpleasant sensation, neuralgia, such as intense painful 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 6.

[0217] In some embodiments, the methods reduce symptoms associated with CRS or neurotoxicity compared to other methods. In some aspects, the provided methods reduce symptoms, outcomes or factors associated with CRS, including symptoms, outcomes or factors associated with severe CRS or grade 3 or higher CRS, compared to other methods. For example, subjects treated according to the present methods may lack detectable and / or have reduced symptoms, outcomes or factors of CRS, e.g. severe CRS or grade 3 or higher CRS, such as any described, e.g. set forth in Table 3 and Table 4. In some embodiments, subjects treated according to the present methods may have reduced symptoms of neurotoxicity, such as limb weakness or numbness, loss of memory, vision, and / or intellect, uncontrollable obsessive and / or compulsive behaviors, delusions, 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 neuropathy, dysethesia, neuralgia or paresthesia.

[0218] 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.

[0219] In some embodiments, the toxicity outcome is a dose-limiting toxicity (DLT). In some embodiments, the toxic outcome is a dose-limiting toxicity. In some embodiments, the toxic outcome is the absence of a dose-limiting toxicity. In some embodiments, a dose-limiting toxicity (DLT) is defined as any grade 3 or higher toxicity as assessed by any known or published guidelines for assessing the particular toxicity, such as any described above and including the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.0.

[0220] In some embodiments, the low rate, risk or likelihood of developing a toxicity, e.g. CRS or neurotoxicity or severe CRS or neurotoxicity, e.g. grade 3 or higher CRS or neurotoxicity, observed with administering a dose of T cells in accord with the provided methods, and / or with the provided articles of manufacture or compositions, permits administration of the cell therapy on an outpatient basis. In some embodiments, the administration of the cell therapy, e.g. dose of T cells (e.g. CAR+T cells) in accord with the provided methods, and / or with the provided articles of manufacture or compositions, is performed on an outpatient basis or does not require admission to the subject to the hospital, such as admission to the hospital requiring an overnight stay.

[0221] In some aspects, subjects administered the cell therapy, e.g. dose of T cells (e.g. CAR+T cells) in accord with the provided methods, and / or with the provided articles of manufacture orcompositions, including subjects treated on an outpatient basis, are not administered an intervention for treating any toxicity prior to or with administration of the cell dose, unless or until the subject exhibits a sign or symptom of a toxicity, such as of a neurotoxicity or CRS. Exemplary agents for treating, delaying, attenuating or ameliorating a toxicity are described in Section I-C.

[0222] In some embodiments, if a subject administered the cell therapy, e.g. dose of T cells (e.g. CAR+T cells), including subjects treated on an outpatient basis, exhibits a fever the subject is given or is instructed to receive or administer a treatment to reduce the fever. In some embodiments, the fever in the subject is characterized as a body temperature of the subject that is (or is measured at) at or above a certain threshold temperature or level. In some aspects, the threshold temperature is that associated with at least a low-grade fever, with at least a moderate fever, and / or with at least a high-grade fever. In some embodiments, the threshold temperature is a particular temperature or range. For example, the threshold temperature may be at or about or at least at or about 38, 39, 40, 41, or 42 degrees Celsius, and / or may be a range of at or about 38 degrees Celsius to at or about 39 degrees Celsius, a range of at or about 39 degrees Celsius to at or about 40 degrees Celsius, a range of at or about 40 degrees Celsius to at or about 41 degrees, or a range of at or about 41 degrees Celsius to at or about 42 degrees Celsius.

[0223] In some embodiments, the treatment designed to reduce fever includes treatment with an antipyretic. An antipyretic may include any agent, e.g., compound, composition, or ingredient, that reduces fever, such as one of any number of agents known to have antipyretic effects, such as NSAIDs (such as ibuprofen, naproxen, ketoprofen, and nimesulide), salicylates, such as aspirin, choline salicylate, magnesium salicylate, and sodium salicylate, paracetamol, acetaminophen, Metamizole, Nabumetone, Phenaxone, antipyrine, febrifuges. In some embodiments, the antipyretic is acetaminophen. In some embodiments, acetaminophen can be administered at a dose of 12.5 mg / kg orally or intravenously up to every four hours. In some embodiments, it is or comprises ibuprofen or aspirin.

[0224] In some embodiments, if the fever is a sustained fever, the subject is administered an alternative treatment for treating the toxicity, such as any described in Section I-C. For subjects treated on an outpatient basis, the subject is instructed to return to the hospital if the subject has and / or is determined to or to have a sustained fever. In some embodiments, the subject has, and / or is determined to or considered to have, a sustained fever if he or she exhibits a fever at or above the relevant threshold temperature, and where the fever or body temperature of the subject is not reduced, or is not reduced by or by more than a specified amount (e.g., by more than 1 °C, and generally does not fluctuate by about, or by more than about, 0.5 °C, 0.4 °C, 0.3 °C, or 0.2 °C), following a specified treatment, such as a treatment designed to reduce fever such as treatment with an antipyreticm, e.g. NSAID or salicylates, e.g. ibuprofen, acetaminophen or aspirin. For example, a subject is considered to have a sustained fever if he or she exhibits or is determined to exhibit a fever of at least at or about 38 or 39 degrees Celsius, which is not reduced by or is not reduced by more than at or about 0.5 °C, 0.4 °C, 0.3 °C, or 0.2 °C, or by at or about 1%, 2%, 3%, 4%, or 5%, over a period of 6 hours, over a period of 8 hours, or over a period of 12hours, or over a period of 24 hours, even following treatment with the antipyretic such as acetaminophen. In some embodiments, the dosage of the antipyretic is a dosage ordinarily effective in such as subject to reduce fever or fever of a particular type such as fever associated with a bacterial or viral infection, e.g., a localized or systemic infection.

[0225] In some embodiments, the subject has, and / or is determined to or considered to have, a sustained fever if he or she exhibits a fever at or above the relevant threshold temperature, and where the fever or body temperature of the subject does not fluctuate by about, or by more than about, 1 °C, and generally does not fluctuate by about, or by more than about, 0.5 °C, 0.4 °C, 0.3 °C, or 0.2 °C. Such absence of fluctuation above or at a certain amount generally is measured over a given period of time (such as over a 24-hour, 12-hour, 8-hour, 6-hour, 3-hour, or 1-hour period of time, which may be measured from the first sign of fever or the first temperature above the indicated threshold). For example, in some embodiments, a subject is considered to or is determined to exhibit sustained fever if he or she exhibits a fever of at least at or about or at least at or about 38 or 39 degrees Celsius, which does not fluctuate in temperature by more than at or about 0.5°C, 0.4 °C, 0.3 °C, or 0.2 °C, over a period of 6 hours, over a period of 8 hours, or over a period of 12 hours, or over a period of 24 hours.

[0226] In some embodiments, the fever is a sustained fever; in some aspects, the subject is treated at a time at which a subject has been determined to have a sustained fever, such as within one, two, three, four, five six, or fewer hours of such determination or of the first such determination following the initial therapy having the potential to induce the toxicity, such as the cell therapy, such as dose of T cells, e.g. CAR+T cells.

[0227] In some embodiments, one or more interventions or agents for treating the toxicity, such as a toxicity-targeting therapies, is administered at a time at which or immediately after which the subject is determined to or confirmed to (such as is first determined or confirmed to) exhibit sustained fever, for example, as measured according to any of the aforementioned embodiments. In some embodiments, the one or more toxicity-targeting therapies is administered within a certain period of time of such confirmation or determination, such as within 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, or 8 hours thereof.II. CELL THERAPY AND ENGINEERING CELLS

[0228] In some embodiments, the cell therapy (e.g., T cell therapy) methods disclosed herein includes administering engineered cells expressing recombinant receptors (e.g. CAR) designed to recognize and / or specifically bind to antigens associated with the disease or condition, such as r / r / B-cell NHL. In particular embodiments, the antigen that is bound or recognized by the recombinant receptor (e.g. CAR) is CD19. In some embodiments, binding to the antigen results in a response, such as an immune response against such antigens. In some embodiments, the cells contain or are engineered to contain the recombinant receptor, such as a chimeric antigen receptor (CAR). The recombinant receptor,such as a CAR, generally includes an extracellular antigen (or ligand) binding domain specific to the antigen that is linked to one or more intracellular signaling components, in some aspects via linkers and / or transmembrane domain(s). In some aspects, the engineered cells are provided as pharmaceutical compositions and formulations suitable for administration to a subjects, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.

[0229] 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, gene transfer is accomplished by first stimulating the cells, such as by combining it with a stimulus that induces a response 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 numbers sufficient for clinical applications.A. Chimeric Antigen Receptors

[0230] In some embodiments of the provided methods and uses, the engineered cells, such as T cells, express a chimeric receptors, such as a chimeric antigen receptors (CAR), that contains one or more domains that combine a ligand-binding domain (e.g. antibody or antibody fragment) that provides specificity for a desired antigen (e.g., tumor antigen) with intracellular signaling domains. In some embodiments, the intracellular signaling domain is an activating intracellular domain portion, such as a T cell activating domain, providing a primary activation signal. In some embodiments, the intracellular signaling domain contains or additionally contains a costimulatory signaling domain to facilitate effector functions. Upon specific binding to the molecule, e.g., antigen, the receptor generally delivers an immunostimulatory signal, such as an IT AM-transduced signal, into the cell, thereby promoting an immune response targeted to the disease or condition. 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 persistence in vivo, such as for use in adoptive cell therapy methods.

[0231] 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 W0200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, W02013 / 123061, U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. 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): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75.In some aspects, the antigen receptors include a CAR as described in U.S. Patent No.: 7,446,190, and those described in International Patent Application Publication No.: WO / 2014055668 Al. Examples of the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, U.S. Patent No.: 7,446,190, US Patent No.: 8,389,282, Kochenderfer 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.: 8,389,282.

[0232] In some embodiments, the engineered cells, such as T cells, express a recombinant receptor such as a chimeric antigen receptor (CAR) with specificity for a particular antigen (or marker or ligand), such as an antigen expressed on the surface of a particular cell type. 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 non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or is expressed on the engineered cells.

[0233] Antigens targeted by the receptors in some embodiments include antigens associated 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, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30. In particular aspects, the antigen is CD19. In some embodiments, any of such antigens are antigens expressed on human B cells.

[0234] The chimeric receptors, such as CARs, generally include an extracellular antigen binding domain that is an antigen-binding portion or portions of an antibody molecule. In some embodiments, the antigen-binding domain is a portion of an antibody molecule, generally a variable heavy (VH) chain region and / or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment. In some embodiments, the antigen-binding domain is a single domain antibody (sdAb), such as sdFv, nanobody, VHH and VNAR. In some embodiments, an antigen-binding fragment comprises antibody variable regions joined by a flexible linker.

[0235] In some embodiments, the antibody or an antigen-binding fragment (e.g. scFv or VHdomain) specifically recognizes an antigen, such as CD19. In some embodiments, the antibody or antigen-binding fragment is derived from, or is a variant of, antibodies or antigen-binding fragment that specifically binds to CD19. In some embodiments, the antigen is CD19. In some embodiments, the scFv contains a VHand a VLderived from an antibody or an antibody fragment specific to CD 19. In some embodiments, the antibody or antibody fragment that binds CD 19 is a mouse derived antibody such as FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. US 2016 / 0152723.

[0236] In some embodiments the antigen-binding domain includes a VHand / or VLderived from FMC63, which, in some aspects, can be an scFv. FMC63 generally refers to a mouse monoclonal IgGl antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, N. R., et al.(1987). Leucocyte typing III. 302). In some embodiments, the FMC63 antibody comprises CDR-H1 and CDR-H2 set forth in SEQ ID NO: 38 and 39, respectively, and CDR-H3 set forth in SEQ ID NO: 40 or 54 and CDR-L1 set forth in SEQ ID NO: 35 and CDR-L2 set forth in SEQ ID NO: 36 or 55 and CDR-L3 sequences set forth in SEQ ID NO: 37 or 56. In some embodiments, the FMC63 antibody 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.

[0237] In some embodiments, the scFv comprises a variable light chain containing the CDR— LI sequence of SEQ ID NO:35, a CDR-L2 sequence of SEQ ID NO:36, and a CDR-L3 sequence of SEQ ID NO:37 and / or a variable heavy chain containing a CDR-H1 sequence of SEQ ID NO:38, a CDR-H2 sequence of SEQ ID NO:39, and a CDR-H3 sequence of SEQ ID NO:40, 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. 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 SEQ ID NO:42, 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. In some embodiments, the variable heavy and variable light chains are connected by a linker. 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 scFv 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.

[0238] In some embodiments the antigen-binding domain includes a VHand / or VLderived from SJ25C1, which, in some aspects, can be an scFv. SJ25C1 is a mouse monoclonal IgGl antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, N. R., et al. (1987). Leucocyte typing III. 302). In some embodiments, the SJ25C1 antibody comprises CDR-H1, CDR-H2 and CDR-H3 set forth in SEQ ID NOS: 47-49, respectively, and CDR-L1, CDR-L2 and CDR-L3 sequences set forth in SEQ ID NOS: 44-46, respectively. In some embodiments, the SJ25C1 antibody comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 50 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 51. In some embodiments, the svFv comprises a variable light chain containing a CDR-L1 sequence of SEQ ID NO:44, a CDR-L2sequence of SEQ ID NO: 45, and a CDR-L3 sequence of SEQ ID NO:46 and / or a variable heavy chain containing a CDR-H1 sequence of SEQ ID NO:47, a CDR-H2 sequence of SEQ ID NO:48, and a CDR- H3 sequence of SEQ ID NO:49„ 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. 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 SEQ ID NO:51, 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. In some embodiments, the variable heavy and variable light chains 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 linker, and a VL- In some embodiments, the scFv comprises, in order, a VL, a linker, and a VH- In some embodiments, the scFv comprises 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.

[0239] The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab’)2fragments, Fab’ fragments, Fv fragments, recombinant IgG (rlgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody, VHH or VNAR) or fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di- scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD. In some aspects, the CAR is a bispecific CAR, e.g., containing two antigen-binding domains with different specificities.

[0240] In some embodiments, the antigen-binding proteins, antibodies and antigen binding fragments thereof specifically recognize an antigen of a full-length antibody. In some embodiments, the heavy and light chains of an antibody can be full-length or can be an antigen-binding portion (a Fab, F(ab’)2, Fv or a single chain Fv fragment (scFv)). In other embodiments, the antibody heavy chain constant region is chosen from, e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE, particularly chosen from, e.g., IgGl, IgG2, IgG3, and IgG4, more particularly, IgGl (e.g., human IgGl). In another embodiment, the antibody light chain constant region is chosen from, e.g., kappa or lambda, particularly kappa.

[0241] The terms “complementarity determining region,” and “CDR,” synonymous with “hypervariable region” or “HVR,” are known, in some cases, to refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and / or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). “Framework regions” and “FR” are known, in some cases, to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR- H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).

[0242] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Rabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Flealth Service, National Institutes of Health, Bethesda, MD (“Rabat” numbering scheme); Al-Lazikani et al, (1997) JMB 273,927-948 (“Chothia” numbering scheme); MacCallum et al. , J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732- 745.” (“Contact” numbering scheme); Lefranc MP et al, “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(l):55-77 (“IMGT” numbering scheme); Honegger A and Pliickthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70, (“Aho” numbering scheme); and Martin et al, “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272, (“AbM” numbering scheme).

[0243] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Rabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Rabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between Rabat and Chothia definitions based on that used by Oxford Molecular’s AbM antibody modeling software.

[0244] Table 7, below, lists exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Rabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Rabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FR-L1 located before CDR-L1, FR-L2 located between CDR- L1 and CDR-L2, FR-L3 located between CDR-L2 and CDR-L3 and so forth. It is noted that because theshown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.Table 7. Boundaries of CDRs according to various numbering schemes.1 - Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD2 - Al-Lazikani et al, (1997) JMB 273,927-948

[0245] Thus, unless otherwise specified, a “CDR” or “complementary determining region,” or individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the aforementioned schemes, or other known schemes. For example, where it is stated that a particular CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given VHor VLregion amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the aforementioned schemes, or other known schemes. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of provided antibodies are described using various numbering schemes, although it is understood that a provided antibody can include CDRs as described according to any of the other aforementioned numbering schemes or other numbering schemes known to a skilled artisan.

[0246] Likewise, unless otherwise specified, a FR or individual specified FR(s) (e.g., FR-H1, FR- H2, FR-H3, FR-H4), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) framework region as defined by any of the known schemes.In some instances, the scheme for identification of a particular CDR, FR, or FRs or CDRs is specified, such as the CDR as defined by the Kabat, Chothia, AbM or Contact method, or other known schemes. In other cases, the particular amino acid sequence of a CDR or FR is given.

[0247] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable regions of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al.Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VHor VLdomain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VHor VLdomain from an antibody that binds the antigen to screen a library of complementary VLor VHdomains, respectively. See, e.g., Portolano et al, J. Immunol. 150:880-887 (1993); Clarkson et ai, Nature 352:624-628 (1991).

[0248] Among the provided antibodies are antibody fragments. An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; variable heavy chain (VH) regions, single chain antibody molecules such as scFvs and single-domain VHsingle antibodies; and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single -chain antibody fragments comprising a variable heavy chain region and / or a variable light chain region, such as scFvs.

[0249] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al.Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VHor VLdomain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VHor VLdomain from an antibody that binds the antigen to screen a library of complementary VLor VHdomains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0250] Single-domain antibodies (sdAb) are antibody fragments comprising ah or a portion of the heavy chain variable domain or ah or a portion of the light chain variable domain of an antibody. In certain embodiments, a single -domain antibody is a human single -domain antibody. In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds the antigen, such as a cancer marker or cell surface antigen of a cell or disease to be targeted, such as a tumor cell or a cancer cell, such as any of the target antigens described herein or known. Exemplary single-domain antibodies include sdFv, nanobody, VHH or VNAR-

[0251] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and / or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some embodiments, the antibody fragments are scFvs.

[0252] A “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0253] In some aspects, the recombinant receptor, e.g., a chimeric antigen receptor, includes an extracellular portion containing one or more ligand- (e.g., antigen-) binding domains, such as an antibody or fragment thereof, and one or more intracellular signaling region or domain (also interchangeably called a cytoplasmic signaling domain or region). In some aspects, the recombinant receptor, e.g., CAR, further includes a spacer and / or a transmembrane domain or portion. In some aspects, the spacer and / or transmembrane domain can link the extracellular portion containing the ligand- (e.g., antigen-) binding domain and the intracellular signaling region(s) or domain(s)

[0254] In some embodiments, the recombinant receptor such as the CAR, further includes a spacer, which may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region, e.g., an IgG4 hinge region, and / or a CH1 / CLand / or Fc region. In some embodiments, the recombinant receptor further comprises a spacer and / or a hinge 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 increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. In some examples, the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include IgG4 hinge alone, IgG4 hinge linked to CF12 and CF13 domains, or IgG4 hinge linked to the CF13 domain. Exemplary spacers include, but are not limited to, those described in Fludeceket al. (2013) Clin. Cancer Res., 19:3153, Hudeceket al. (2015) Cancer Immunol Res. 3(2): 125-135 or international patent application publication number WO2014031687.

[0255] In some embodiments, 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, and encoded by the sequence set forth in SEQ ID NO: 2. In some embodiments, the spacer is an Ig hinge, e.g., and IgG4 hinge, 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 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 comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers. In some embodiments, the constant region or portion is of IgD. In some embodiments, 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 or at least about 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 and 5.

[0256] In some aspects, the spacer is 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 consists of all or a portion 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 immunoglobulin hinge, optionally an IgG4, or a modified version thereof; or (d) consists or comprises the sequence of amino acids set forth in SEQ ID NOS: 1, 3-5, 27-34 or 24, 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 Xi is glycine, cysteine or arginine and X2 is cysteine or threonine.

[0257] In some embodiments, the antigen receptor comprises an intracellular domain linked directly or indirectly to the extracellular domain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an IT AM. For example, in some aspects, the antigen recognition domain (e.g. extracellular domain) generally is linked to one or more intracellular signaling components, such as signaling components 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 component (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains.

[0258] 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 ormodified 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.

[0259] 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 protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 (4-1BB), or CD154. Alternatively 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 domain. In some embodiments, the linkage is by linkers, spacers, and / or transmembrane domain(s). In some aspects, the transmembrane domain contains a transmembrane portion of CD28 or a variant thereof. The extracellular domain and transmembrane can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein.

[0260] In some embodiments, the transmembrane domain of the receptor, e.g., the CAR is a transmembrane domain of human CD28 or variant thereof, e.g., a 27-amino acid transmembrane domain of a human CD28 (Accession No.: P10747.1), or is a transmembrane 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 or at least about85%, 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 transmembrane-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 or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0261] In some embodiments, the recombinant receptor, e.g., CAR, includes at least one intracellular signaling component or components, such as an intracellular signaling region or domain. T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components. Among the intracellular signaling region are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and / or a signal through a costimulatory receptor alone. In some embodiments, 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, ispresent and form a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0262] In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling region of the CAR activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered 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 intracellular signaling region 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 regions, e.g., comprising intracellular 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 natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and / or any derivative or variant of such molecules, and / or any synthetic sequence that has the same functional capability. In some embodiments, the intracellular signaling regions, e.g., comprising intracellular domain or domains, include the cytoplasmic sequences of a region or domain that is involved in providing costimulatory signal.

[0263] 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 IT AMs. Examples of IT AM containing primary cytoplasmic signaling sequences include 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.

[0264] 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 portion is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and / or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor g, CD8alpha,CD8beta, CD4, CD25, or CD16. For example, in some aspects, the CAR or other chimeric receptor includes a chimeric molecule between CD3-zeta (CD3^) or Fc receptor g and CD8alpha, CD8beta, CD4, CD25 or CD 16.

[0265] In some embodiments, the intracellular (or cytoplasmic) signaling region comprises a human CD3 chain, optionally a CD3 zeta stimulatory signaling domain or functional variant thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human CD3z (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Patent No.: 7,446,190 or U.S. Patent No. 8,911,993. In someembodiments, the intracellular signaling region comprises the sequence of amino acids set forth in SEQ ID NO: 13, 14 or 15 or a sequence of amino acids that exhibits at least or at least about 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.

[0266] 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 promote full activation, 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 generating the secondary or costimulatory signal.

[0267] In some embodiments, the chimeric 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, 4-1BB, 0X40 (CD134), CD27, DAP10, DAP12, ICOS and / or other costimulatory receptors. In some embodiments, the CAR includes a costimulatory region or domain of CD28 or 4-1BB, such as of human CD28 or human 4-1BB.

[0268] In some embodiments, the intracellular signaling region or domain comprises an intracellular costimulatory signaling domain of human CD28 or functional variant or portion thereof, such as a 41 amino acid domain thereof and / or such a domain with an LL to GG substitution at positions 186-187 of a native CD28 protein. In some embodiments, 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 or at least about 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 region comprises an intracellular costimulatory signaling domain of 4- IBB or functional variant or portion thereof, such as a 42-amino acid cytoplasmic domain of a human 4- IBB (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 or at least about 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12.

[0269] In some aspects, the same CAR includes both the primary (or activating) cytoplasmic signaling regions and costimulatory signaling components.

[0270] 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 expressed on the same cell (see WO2014 / 055668). In some aspects, the cells include one or more stimulatory or activating CAR and / or a costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CARrecognizing an antigen other than the one associated with and / or specific for the disease or condition whereby an activating signal delivered through the disease-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.

[0271] In some embodiments, the two receptors induce, respectively, an activating and an inhibitory signal to the cell, such that ligation of one of the receptor to its antigen activates the cell or induces a response, but ligation of the second inhibitory receptor to its antigen induces a signal that suppresses or dampens that response. Examples are combinations of activating CARs and inhibitory CARs (iCARs). Such a strategy may be used, for example, to reduce the likelihood of off-target effects in the context in which the activating CAR binds an antigen expressed in a disease or condition but which is also expressed on normal cells, and the inhibitory receptor binds to a separate antigen which is expressed on the normal cells but not cells of the disease or condition.

[0272] In some aspects, the chimeric receptor is or includes an inhibitory CAR (e.g. iCAR) and includes intracellular components that dampen or suppress an immune response, such as an ITAM- and / or co stimulatory-promoted response in the cell. Exemplary of such intracellular signaling components are those found on immune checkpoint molecules, including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptors, EP2 / 4 Adenosine receptors including A2AR. In some aspects, the engineered cell includes an inhibitory CAR including a signaling domain of or derived from such an inhibitory molecule, such that it serves to dampen the response of the cell, for example, that induced by an activating and / or costimulatory CAR.

[0273] 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 CD 137; in some aspects, a third generation CAR in some aspects is one that includes multiple costimulatory domains of different costimulatory receptors.

[0274] In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.

[0275] 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. For example, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2014031687. Forexample, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence.

[0276] 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 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. An exemplary T2A linker sequence comprises the sequence of amino acids set forth in SEQ ID NO: 6 or 17 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 or 17.

[0277] 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 surface of T cells, or a portion thereof. In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self’ by the immune system of the host into which the cells will be adoptively transferred.

[0278] In some embodiments, the marker serves no therapeutic function and / or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired 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 transfer and encounter with ligand.

[0279] In some embodiments, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment includes an scFv or a single -domain VHantibody and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain includes a signaling domain of a zeta chain of a CD3-zeta ^Ώ3z) chain. In some embodiments, the CD3-zeta chain is a human CD3-zeta chain. In some embodiments, the intracellular signaling region further comprises a CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain. In some embodiments, the CD28 is a human CD28. In some embodiments, the 4-1BB is a human 4-1BB. In some embodiments, the chimeric antigen receptor includes a transmembrane domain disposed between the extracellular domain and the intracellular signaling region. In some aspects, the transmembrane domain contains a transmembrane portion of CD28. The extracellular domain and transmembrane can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein.

[0280] 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 variantthereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. For example, in some embodiments, the CAR includes an antibody such as an antibody fragment, including scFvs, e.g. specific for CD 19 such as any described above, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and / or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3 zeta signaling domain.

[0281] In some embodiments, the CAR contains an antibody, e.g., antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4- IBB or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some such embodiments, the receptor further includes a spacer containing a portion of an Ig molecule, 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 CAR includes an antibody or fragment, such as scFv, e.g. specific for CD19 such as any described above, a spacer such as any of the Ig-hinge containing spacers, a CD28-derived transmembrane domain, a 4- IBB -derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.

[0282] In particular embodiments of any of the provided methods, the CAR contains in order from N-terminus to C-terminus: an extracellular antigen-binding domain that is the scFv set forth in SEQ ID NO: 43, the spacer set forth in SEQ ID NO:l, the transmembrane domain set forth in SEQ ID NO:8, the 4-1BB costimulatory signaling domain set forth in SEQ ID NO: 12, and the signaling domain of a CD3- zeta (0O3z) chain set forth in SEQ ID NO: 13.B. Nucleic Acids, Vectors and Methods for Genetic Engineering

[0283] In some embodiments, the cells, e.g., T cells, are genetically engineered to express a recombinant receptor. In some embodiments, the engineering is carried out by introducing polynucleotides that encode the recombinant receptor. Also provided are polynucleotides encoding a recombinant receptor, and vectors or constructs containing such nucleic acids and / or polynucleotides.

[0284] In some cases, the nucleic acid sequence encoding the recombinant receptor contains a signal sequence that encodes a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. In other aspects, the signal sequence may encode a heterologous or non-native signal peptide, such as the exemplary signal peptide of the GMCSFR alpha chain set forth in SEQ ID NO:65 and encoded by the nucleotide sequence set forth in SEQ ID NO:66. In some cases, the nucleic acid sequence encoding the recombinant receptor, e.g., chimeric antigen receptor (CAR) contains a signal sequence that encodes a signal peptide. Non-limiting exemplary examples ofsignal peptides include, for example, the GMCSFR alpha chain signal peptide set forth in SEQ ID NO:65 and encoded by the nucleotide sequence set forth in SEQ ID NO:66, or the CD8 alpha signal peptide set forth in SEQ ID NO:67.

[0285] In some embodiments, the polynucleotide encoding the recombinant receptor contains at least one promoter that is operatively linked to control expression of the recombinant receptor. In some examples, the polynucleotide contains two, three, or more promoters operatively linked to control expression of the recombinant receptor.

[0286] In certain cases where nucleic acid molecules encode two or more different polypeptide chains, e.g., a recombinant receptor and a marker, each of the polypeptide chains can be encoded by a separate nucleic acid molecule. For example, two separate nucleic acids are provided, and each can be individually transferred or introduced into the cell for expression in the cell. In some embodiments, the nucleic acid encoding the recombinant receptor and the nucleic acid encoding the marker are operably linked to the same promoter and are optionally separated by an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, which optionally is a T2A, a P2A, an E2A or an F2A. In some embodiments, the nucleic acids encoding the marker and the nucleic acid encoding the recombinant receptor are operably linked to two different promoters. In some embodiments, the nucleic acid encoding the marker and the nucleic acid encoding the recombinant receptor are present or inserted at different locations within the genome of the cell. In some embodiments, the polynucleotide encoding the recombinant receptor is introduced into a composition containing cultured cells, such as by retroviral transduction, transfection, or transformation.

[0287] In some embodiments, such as those where the polynucleotide contains a first and second nucleic acid sequence, the coding sequences encoding each of the different polypeptide chains can be operatively linked to a promoter, which can be the same or different. In some embodiments, the nucleic acid molecule can contain a promoter that drives the expression of two or more different polypeptide chains. In some embodiments, such nucleic acid molecules can be multicistronic (bicistronic or tricistronic, see e.g., U.S. Patent No. 6,060,273). In some embodiments, transcription units can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows coexpression of gene products ((e.g. encoding the marker and encoding the recombinant receptor) by a message from a single promoter. Alternatively, in some cases, a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF), two or three genes (e.g. encoding the marker and encoding the recombinant receptor) separated from one another by sequences encoding a self-cleavage peptide (e.g., 2A sequences) or a protease recognition site (e.g., furin). The ORF thus encodes a single polypeptide, which, either during (in the case of 2A) or after translation, is processed into the individual proteins. In some cases, the peptide, such as a T2A, can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2 A sequence and the next peptide downstream (see, for example, de Felipe,Genetic Vaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)). Various 2A elements are known. Examples of 2A sequences that can be used in the methods and system disclosed herein, without limitation, 2A sequences from the foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 21), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 20), Thosea asigna virus (T2A, e.g., SEQ ID NO: 6 or 17), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 18 or 19) as described in U.S. Patent Publication No. 20070116690.

[0288] Any of the recombinant receptors described herein can be encoded by polynucleotides containing one or more nucleic acid sequences encoding recombinant receptors, in any combinations or arrangements. For example, one, two, three or more polynucleotides can encode one, two, three or more different polypeptides, e.g., recombinant receptors. In some embodiments, one vector or construct contains a nucleic acid sequence encoding marker, and a separate vector or construct contains a nucleic acid sequence encoding a recombinant receptor, e.g., CAR. In some embodiments, the nucleic acid encoding the marker and the nucleic acid encoding the recombinant receptor are operably linked to two different promoters. In some embodiments, the nucleic acid encoding the recombinant receptor is present downstream of the nucleic acid encoding the marker.

[0289] In some embodiments, the vector backbone contains a nucleic acid sequence encoding one or more marker(s). In some embodiments, the one or more marker(s) is a transduction marker, surrogate marker and / or a selection marker.

[0290] In some embodiments, the marker is a transduction marker or a surrogate marker. A transduction marker or a surrogate marker can be used to detect cells that have been introduced with the polynucleotide, e.g., a polynucleotide encoding a recombinant receptor. In some embodiments, the transduction marker can indicate or confirm modification of a cell. In some embodiments, the surrogate marker is a protein that is made to be co-expressed on the cell surface with the recombinant receptor, e.g. CAR. In particular embodiments, such a surrogate marker is a surface protein that has been modified to have little or no activity. In certain embodiments, the surrogate marker is encoded on the same polynucleotide that encodes the recombinant receptor. In some embodiments, the nucleic acid sequence encoding the recombinant receptor is operably linked to a nucleic acid sequence encoding a marker, optionally separated by an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, such as a 2A sequence, such as a T2A, a P2A, an E2A or an F2A. Extrinsic marker genes may in some cases be utilized in connection with engineered cell to permit detection or selection of cells and, in some cases, also to promote cell suicide.

[0291] Exemplary surrogate markers can include truncated forms of cell surface polypeptides, such as truncated forms that are non-functional and to not transduce or are not capable of transducing a signal or a signal ordinarily transduced by the full-length form of the cell surface polypeptide, and / or do not or are not capable of internalizing. Exemplary truncated cell surface polypeptides including truncated forms of growth factors or other receptors such as a truncated human epidermal growth factor receptor 2(tHER2), a truncated epidermal growth factor receptor (tEGFR, exemplary tEGFR sequence set forth in SEQ ID NO:7 or 16) or a prostate-specific membrane antigen (PSMA) or modified form thereof. tEGFR may contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibody or binding molecule, which can be used to identify or select cells that have been engineered with the tEGFR construct and an encoded exogenous protein, and / or to eliminate or separate cells expressing the encoded exogenous protein. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434). In some aspects, the marker, e.g. surrogate marker, includes all or part (e.g., truncated form) of CD34, a NGFR, a CD19 or a truncated CD19, e.g., a truncated non-human CD 19, or epidermal growth factor receptor (e.g., tEGFR).

[0292] In some embodiments, the marker is or comprises a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as super-fold GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), and variants thereof, including species variants, monomeric variants, and codon-optimized and / or enhanced variants of the fluorescent proteins. In some embodiments, the marker is or comprises an enzyme, such as a luciferase, the lacZ gene from E. coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyl transferase (CAT). Exemplary light-emitting reporter genes include luciferase (luc), b-galactosidase, chloramphenicol acetyltransferase (CAT), b-glucuronidase (GUS) or variants thereof.

[0293] In some embodiments, the marker is a selection marker. In some embodiments, the selection marker is or comprises a polypeptide that confers resistance to exogenous agents or drugs. In some embodiments, the selection marker is an antibiotic resistance gene. In some embodiments, the selection marker is an antibiotic resistance gene confers antibiotic resistance to a mammalian cell. In some embodiments, the selection marker is or comprises a Puromycin resistance gene, a Hygromycin resistance gene, a Blasticidin resistance gene, a Neomycin resistance gene, a Geneticin resistance gene or a Zeocin resistance gene or a modified form thereof.

[0294] In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self’ by the immune system of the host into which the cells will be adoptively transferred.

[0295] In some embodiments, the marker serves no therapeutic function and / or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired 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 transfer and encounter with ligand.

[0296] 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., a T2A. For example, a marker, and optionally a linker sequence, can be any as disclosed in PCT Pub. No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, 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 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.

[0297] In some embodiments, the marker is or comprises a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as super-fold GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), and variants thereof, including species variants, monomeric variants, and codon-optimized and / or enhanced variants of the fluorescent proteins. In some embodiments, the marker is or comprises an enzyme, such as a luciferase, the lacZ gene from E. coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyl transferase (CAT). Exemplary light-emitting reporter genes include luciferase (luc), b-galactosidase, chloramphenicol acetyltransferase (CAT), b-glucuronidase (GUS) or variants thereof.

[0298] In some embodiments, the marker is a selection marker. In some embodiments, the selection marker is or comprises a polypeptide that confers resistance to exogenous agents or drugs. In some embodiments, the selection marker is an antibiotic resistance gene. In some embodiments, the selection marker is an antibiotic resistance gene confers antibiotic resistance to a mammalian cell. In some embodiments, the selection marker is or comprises a Puromycin resistance gene, a Hygromycin resistance gene, a Blasticidin resistance gene, a Neomycin resistance gene, a Geneticin resistance gene or a Zeocin resistance gene or a modified form thereof.

[0299] In some embodiments, recombinant 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 gamma-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.

[0300] In some embodiments, the viral vector is an adeno-associated virus (AAV).

[0301] 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 embryonic stem cell virus (MESV), murine stem cell virus (MSCV) orspleen focus forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived 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; Miller, A. D. (1990) Fluman 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. 3:102-109.

[0302] 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. 102(2): 497- 505.

[0303] 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): 1431-1437). In some embodiments, recombinant nucleic acids are transferred 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 calcium 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 particle -facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

[0304] Other approaches and vectors for transfer of the nucleic acids encoding the recombinant products are those described, e.g., in international patent application, Publication No.: WO2014055668, and U.S. Patent No. 7,446,190.

[0305] In some embodiments, the cells, e.g., T cells, may be transfected either during or after expansion e.g. with 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 anti-CD3 / anti-CD28 stimulus, for example) 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 antigenic stimulus in form of a peptide / MHC molecule, the cognate (cross-linking) ligand of the genetically introduced receptor (e.g. natural 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, “Chimeric antigen receptors for T-cell based therapy” Methods Mol Biol. 2012; 907:645-66 or Barrettet al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol. 65: 333-347 (2014).

[0306] In some cases, a vector may be used that does not require that the cells, e.g., T cells, are activated. 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.

[0307] 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., Mol. 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 positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., US Patent No. 6,040,177, at columns 14-17.

[0308] Cells and Preparation of Cells for Genetic Engineeringln 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 organism or cell, which for example, is not ordinarily 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 nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.

[0309] The cells generally are eukaryotic 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 multipotent 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 frozen. 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 subpopulations thereof, such as those defined by function, 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 compartment, 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 aspects, 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 embodiments, the methods include isolating cells from thesubject, preparing, processing, culturing, and / or engineering them, and re-introducing them into the same subject, before or after cryopreservation.

[0310] Among the sub-types and subpopulations of T cells and / or of CD4+ and / or of CD8+ T cells are naive T (TN) cells, effector T cells (TEEF), 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 TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells.

[0311] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0312] 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 organism or cell, which for example, is not ordinarily 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 nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.

[0313] In some embodiments, preparation of the engineered cells includes one or more culture and / or preparation steps. The cells for introduction of the nucleic 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, processed, and / or engineered.

[0314] Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells.The samples include tissue, 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 biological 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 samples derived therefrom.

[0315] In some aspects, the sample from which the cells are derived or isolated is blood or a blood- derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samplesinclude whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, 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 sources.

[0316] 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.

[0317] 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 particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[0318] 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 platelets.

[0319] 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 buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated “flow-through” centrifuge (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 instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++ / Mg++free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.

[0320] In some embodiments, the methods include density-based cell separation 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.

[0321] 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 markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation isaffinity- 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 specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[0322] 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 identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

[0323] 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 marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0324] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively 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 selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[0325] 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 negative selection techniques.

[0326] For example, CD3+, CD28+T cells can be positively selected using anti-CD3 / anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0327] In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative 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 orexpressed (marker+) at a relatively higher level (markerhlgh) on the positively or negatively selected cells, respectively.

[0328] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, 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 selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations.

[0329] 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 positive or negative 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 aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood, 1 :72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TcM-enriched CD8+T cells and CD4+T cells further enhances efficacy.

[0330] In embodiments, memory T cells are present in both CD62L+and CD62L subsets of CD8+peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L CD8+and / or CD62L+CD8+fractions, such as using anti-CD8 and anti-CD62L antibodies.

[0331] In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; 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, CD 14, 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 selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 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 expression-based selection 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 positive or negative selection steps.

[0332] 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, andpositive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.

[0333] CD4+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+, CD62L+, CD4+T cells. In some embodiments, central memory CD4+cells are CD62L+and CD45RO+. In some embodiments, effector CD4+cells are CD62L and CD45RO .

[0334] In one example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDllb, 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 selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Sch...

Claims

CLAIMS1. A method of treating a B-cell non-Hodgkin lymphoma (r / r B-cell NHL), the method comprising administering to a subject having or suspected of having a B-cell NHL a composition comprising engineered T cells expressing a chimeric antigen receptor (CAR) that targets CD19, wherein: the composition comprises CD4+ T cells expressing the CAR and CD8+ T cells expressing theCAR; the composition comprises between at or about 5 x 106CAR-expressing T cells and at or about 100 x 106CAR-expressing T cells, inclusive; at least or at least about 80% of the cells in the composition are CD3+cells; and at least or at least about 80% of the CAR+T cells in the composition are of a naive-like or central memory phenotype.

2. A method of treating a B-cell non-Hodgkin lymphoma (r / r B-cell NHL), the method comprising administering to a subject having or suspected of having a B-cell NHL a composition comprising engineered T cells expressing a chimeric antigen receptor (CAR) that targets CD19, wherein: the composition comprises CD4+ T cells expressing the CAR and CD8+ T cells expressing theCAR; the composition comprises between at or about 5 x 106CAR-expressing T cells and at or about 100 x 106CAR-expressing T cells, inclusive; at least or at least about 80% of the cells in the composition are CD3+cells; and at least or at least about 50% of the CD4+CAR+T cells in the composition are CD27+CCR7+and / or at least or at least about 50% of the CD8+CAR+T cells in the composition are CD27+CCR7+.

3. The method of claim 1 or claim 2, wherein the composition comprises CD4+T cells expressing the CAR and CD8+T cells expressing the CAR at a ratio between about 1:2.5 and about 2.5:1.

4. The method of any of claims 1-3, wherein the composition comprises CD4+T cells expressing the CAR and CD8+T cells expressing the CAR at a ratio between about 1:2 and about 2:1, between about 1:1.5 and about 1.5:1, or at or at about 1:1.

5. The method of any of claims 1-3, wherein the composition comprises CD4+T cells expressing the CAR and CD8+T cells expressing the CAR at a ratio between about 1:1 and about 2.5:1, between about 1.5:1 and about 2:1, at or at about 1.5:1, or at or at about 2:1.

6. The method of any of claims 1-5, wherein the composition comprises between at or about 5 x 106CAR-expressing T cells and at or about 50 x 106CAR-expressing T cells, inclusive.

7. The method of any of claims 1-6, wherein the composition comprises between at or about 5 x 106CAR-expressing T cells and at or about 25 x 106CAR-expressing T cells, inclusive.

8. The method of any of claims 1-7, wherein the composition comprises between at or about 5 x 106CAR-expressing T cells and at or about 10 x 106CAR-expressing T cells, inclusive.

9. The method of any of claims 1-7, wherein the composition comprises between at or about 10 x 106CAR-expressing T cells and at or about 25 x 106CAR-expressing T cells, inclusive.

10. The method of any of claims 1-8, wherein the composition comprises at or about 5 x 106CAR-expressing T cells.

11. The method of any of claims 1-9, wherein the composition comprises at or about 10 x 106CAR-expressing T cells.

12. The method of any of claims 1-7 and 9, wherein the composition comprises at or about 25 x 106CAR-expressing T cells.

13. The method of any of claims 1-12, wherein at least or at least about 90% of the cells in the composition are CD3+cells.

14. The method of any of claims 1-13, wherein at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, or at least or at least about 96% of the cells in the composition are CD3+cells.

15. The method of any of claims 1-14, wherein between at or about 5% and at or about 30% of the CAR+T cells in the composition express a marker of apoptosis, optionally Annexin V or active Caspase 3.

16. The method of any of claims 1-15, wherein between at or about 10% and at or about 15% of the CAR+T cells in the composition express a marker of apoptosis, optionally Annexin V or active Caspase 3.

17. The method of any of claims 1-15, wherein between at or about 15% and at or about 20% of the CAR+T cells in the composition express a marker of apoptosis, optionally Annexin V or active Caspase 3.

18. The method of any of claims 1-15, wherein between at or about 20% and at or about 25% of the CAR+T cells in the composition express a marker of apoptosis, optionally Annexin V or active Caspase 3.

19. The method of any of claims 1-15, wherein between at or about 25% and at or about 30% of the CAR+T cells in the composition express a marker of apoptosis, optionally Annexin V or active Caspase 3.

20. The method of any of claims 1-15, wherein at or about 5%, at or about 10%, at or about 15%, at or about 20%, at or about 25%, or at or about 30% of the CAR+T cells in the composition express a marker of apoptosis, optionally Annexin V or active Caspase 3.

21. The method of any of claims 2-20, wherein at least or at least about 80% of the CAR+T cells in the composition are of a naive-like or central memory phenotype.

22. The method of any of claims 1-21, wherein between at or about 80% and at or about 85% of the CAR+T cells in the composition are of a naive -like or central memory phenotype.

23. The method of any of claims 1-21, wherein between at or about 85% and at or about 90% of the CAR+T cells in the composition are of a naive -like or central memory phenotype.

24. The method of any of claims 1-21, wherein between at or about 90% and at or about 95% of the CAR+T cells in the composition are of a naive -like or central memory phenotype.

25. The method of any of claims 1-21, wherein between at or about 95% and at or about 99% of the CAR+T cells in the composition are of a naive -like or central memory phenotype.

26. The method of any of claims 1-21, wherein at or about 85%, at or about 90%, at or about 95%, or at or about 99% of the CAR+T cells in the composition are of a naive-like or central memory phenotype.

27. The method of any of claims 1 and 3-26, wherein the at least or at least about 80% of the CAR+T cells in the composition that are of a naive-like or central memory phenotype are surface positive for a marker expressed on naive-like or central memory T cells.

28. The method of claim 27, wherein the marker expressed on naive -like or central memory T cell is selected from the group consisting of CD45RA, CD27, CD28, and CCR7.

29. The method of any of claims 1 and 3-28, wherein the at least or at least about 80% of the CAR+T cells in the composition that are of a naive-like or central memory phenotype have a phenotype selected from CCR7+CD45RA+, CD27+CCR7+, or CD62L CCR7+.

30. The method of any of claims 1-29, wherein between at or about 80% and at or about 85%, between at or about 85% and at or about 90%, between at or about 90% and at or about 95%,between at or about 95% and at or about 99% of the CAR+T cells in the composition are of a nai ve -like or central memory phenotype selected from CCR7+CD45RA+, CD27+CCR7+, or CD62L CCR7+.

31. The method of any of claims 1-29, wherein at or about 80%, at or about 85%, at or about 90%, at or about 95%, or at or about 99% of the CAR+T cells in the composition are of a naive -like or central memory phenotype selected from CCR7+CD45RA+, CD27+CCR7+, or CD62L CCR7+.

32. The method of any of claims 1-31, wherein at or about 80%, at or about 85%, at or about 90%, at or about 95%, or at or about 99% of the CAR+T cells in the composition are of a naive -like or central memory phenotype that is CD27+CCR7+.

33. The method of any of claims 1-32, wherein at least or at least about 50% of the CD4+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA .

34. The method of any of claims 1-33, wherein at least or at least about 60% of the CD4+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA .

35. The method of any of claims 1-34, wherein at least or at least about 70% of the CD4+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA .

36. The method of any of claims 1-35, wherein at least or at least about 80% of the CD4+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA .

37. The method of any of claims 1-36, wherein at least or at least about 85% of the CD4+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA .

38. The method of any of claims 1-37, wherein at least or at least about 50% of the CD4+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CD27+CCR7+.

39. The method of any of claims 1-38, wherein at least or at least about 60% of the CD4+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CD27+CCR7+.

40. The method of any of claims 1-39, wherein at least or at least about 70% of the CD4+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CD27+CCR7+.

41. The method of any of claims 1-40, wherein at least or at least about 80% of the CD4+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CD27+CCR7+.

42. The method of any of claims 1-41, wherein at least or at least about 85% of the CD4+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CD27+CCR7+.

43. The method of any of claims 1-42, wherein at least or at least about 50% of the CD8+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA .

44. The method of any of claims 1-43, wherein at least or at least about 60% of the CD8+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA .

45. The method of any of claims 1-44, wherein at least or at least about 70% of the CD8+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA .

46. The method of any of claims 1-45, wherein at least or at least about 80% of the CD8+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA .

47. The method of any of claims 1-46, wherein at least or at least about 85% of the CD8+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CCR7+CD45RA+or CCR7+CD45RA .

48. The method of any of claims 1-47, wherein at least or at least about 50% of the CD8+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CD27+CCR7+.

49. The method of any of claims 1-48, wherein at least or at least about 60% of the CD8+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CD27+CCR7+.

50. The method of any of claims 1-49, wherein at least or at least about 70% of the CD8+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CD27+CCR7+.

51. The method of any of claims 1-50, wherein at least or at least about 80% of the CD8+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CD27+CCR7+.

52. The method of any of claims 1-51, wherein at least or at least about 85% of the CD8+CAR+T cells in the composition are of a naive -like or central memory phenotype that is CD27+CCR7+.

53. The method of any of claims 1-52, wherein the fraction of integrated vector copy number (iVCN) to total VCN in the CAR+T cells in the composition, on average, is less than or less than about 0.9.

54. The method of any of claims 1-53, wherein the fraction of integrated vector copy number (iVCN) to total VCN in the CAR+T cells in the composition, on average, is between at or about 0.9 and at or about 0.8.

55. The method of any of claims 1-53, wherein the fraction of integrated vector copy number (iVCN) to total VCN in the CAR+T cells in the composition, on average, is less than or less than about 0.8.

56. The method of any of claims 1-53 and 55, wherein the fraction of integrated vector copy number (iVCN) to total VCN in the CAR+T cells in the composition, on average, is between at or about 0.8 and at or about 0.7.

57. The method of any of claims 1-53 and 55, wherein the fraction of integrated vector copy number (iVCN) to total VCN in the CAR+T cells in the composition, on average, is between at or about 0.7 and at or about 0.6.

58. The method of any of claims 1-53 and 55, wherein the fraction of integrated vector copy number (iVCN) to total VCN in the CAR+T cells in the composition, on average, is between at or about 0.6 and at or about 0.5.

59. The method of any of claims 1-53 and 55, wherein the fraction of integrated vector copy number (iVCN) to total VCN in the CAR+T cells in the composition, on average, is between at or about 0.5 and at or about 0.4.

60. The method of any of claims 1-59, wherein the integrated vector copy number (iVCN) of the CAR+T cells in the composition, on average, is between or between about 0.4 copies per diploid genome and 3.0 copies per diploid genome, inclusive.

61. The method of any of claims 1-60, wherein the integrated vector copy number (iVCN) of the CAR+T cells in the composition, on average, is between or between about 0.8 copies per diploid genome and 2.0 copies per diploid genome, inclusive.

62. The method of any of claims 1-61, wherein the integrated vector copy number (iVCN) of the CAR+T cells in the composition, on average, is between or between about 0.8 copies per diploid genome and 1.0 copies per diploid genome, inclusive.

63. The method of any of claims 1-61, wherein the integrated vector copy number (iVCN) of the CAR+T cells in the composition, on average, is between or between about 1.0 copies per diploid genome and 1.5 copies per diploid genome, inclusive.

64. The method of any of claims 1-61, wherein the integrated vector copy number (iVCN) of the CAR+T cells in the composition, on average, is between or between about 1.5 copies per diploid genome and 2.0 copies per diploid genome, inclusive.

65. The method of any of claims 1-64, wherein the B-cell NHL is selected from the group consisting of: diffuse large B-cell lymphoma (DLBCL), optionally DLBCL not otherwise specified; transformed DLBCL, optionally transformed DLBCL from follicular lymphoma or marginal zone lymphoma; high grade B-cell lymphoma (HGBCL), optionally HGBCL with MYC and BCL2 and / or BCL6 rearrangements with DLBCL histology; primary mediastinal large B cell lymphoma (PMBCL); and follicular lymphoma (FL), optionally follicular lymphoma grade 3b (FL3B).

66. The method of any of claims 1-65, wherein at or immediately prior to the time of the administration of the composition comprising engineered T cells, the subject has relapsed following remission after treatment with, or become refractory to: (i) two or more prior therapies for the B-cell NHL and / or (ii) an autologous stem cell transplant (ASCT) therapy.

67. The method of claim 66, wherein the two or more prior therapies for the B-cell NHL comprise an anthracycline and a CD20-targeted agent, optionally wherein the CD20-targeted agent comprises rituximab.

68. The method of any of claims 1-67, wherein the subject has not received a prior CAR T cell or genetically-modified T cell therapy.

69. The method of any of claims 1-68, further comprising obtaining a leukapheresis sample from the subject for manufacturing the composition comprising engineered T cells.

70. The method of any of claims 1-69, wherein prior to the administration, the subject has been preconditioned with a lymphodepleting therapy.

71. The method of any of claims 1-69, wherein the method further comprises, immediately prior to the administration of the composition comprising engineered T cells, administering a lymphodepleting therapy to the subject, wherein the lymphodepleting therapy comprises the administration of fludarabine and / or cyclophosphamide.

72. The method of any of claims 1-71, wherein the administration of the composition comprising engineered T cells and / or the lymphodepleting therapy is carried out via outpatient delivery.

73. The method of any of claims 70-72, wherein the lymphodepleting therapy comprises the administration of fludarabine at 30 mg / m2body surface area of the subject, daily, and cyclophosphamide at 300 mg / m2body surface area of the subject, daily, each for 3 days.

74. The method of any of claims 70-73, wherein the composition comprising engineered T cells is administered between at or about 48 hours and at or about 9 days, inclusive, after completion of the lymphodepleting therapy.

75. The method of any of claims 1-74, further comprising administering to the subject an agent or treatment for the treatment, prevention, reduction, or attenuation of a neurotoxicity and / or a cytokine release syndrome or risk thereof.

76. The method of claim 75, wherein the agent is or comprises an anti-IL-6 antibody, an anti-IL-6 receptor antibody, or a steroid.

77. The method of 75 or claim 76, wherein the agent is or comprises tocilizumab or methylprednisolone.

78. The method of any of claims 1-77, wherein the T cells are autologous to the subject.

79. The method of any of claims 1-78, wherein: at least 35%, at least 40% or at least 50% of subjects treated according to the method achieve a complete response (CR); at least 60%, 70%, 80%, 90%, or 95% of subjects achieving a CR exhibit a CR that is durable for at or greater than 3 months or at or greater than 6 months; and / orat least 60%, 70%, 80%, 90%, or 95% of subjects achieving a CR by one month and / or by 3 months remain in response, remain in CR, and / or survive or survive without progression, for at or greater than 3 months and / or at or greater than 6 months and / or at greater than 9 months after achieving the CR; and / or at least 50%, at least 60% or at least 70% of the subjects treated according to the method achieve objective response (OR); at least 60%, 70%, 80%, 90%, or 95% of subjects achieving an OR exhibit an OR that is durable for at or greater than 3 months or at or greater than 6 months; and / or at least 35%, at least 40%, or at least 50% of subjects achieving an OR remain in response or survive for at or greater than 3 months and / or at or greater than 6 months after achieving the OR.

80. The method of any of claims 1-79, wherein the cells are autologous to the subject, and no minimum absolute lymphocyte count (ALC) for apheresis is required and / or specified for production of the therapy; and / or the cells are produced by a process which, for at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of subjects having the B-cell NHL, is capable of generating a cell product for administration according to the method.

81. The method of any of claim 1-80, wherein: greater than or greater than about 50%, about 60%, about 70%, or about 80% of the subjects treated according to the method do not exhibit a grade 3 or greater cytokine release syndrome (CRS) and / or do not exhibit a grade 3 or greater neurotoxicity and / or greater than 40% or 50% or 55% of the subjects treated according to the method do not exhibit any neurotoxicity or CRS.

82. The method of any of claim 1-81, wherein greater than or greater than about 80% of the subjects treated according to the method do not exhibit a grade 3 or greater cytokine release syndrome (CRS) and / or do not exhibit a grade 3 or greater neurotoxicity.

83. The method of any of claims 1-82, wherein greater than 95% of the subjects treated according to the method do not exhibit grade 3 or greater CRS.

84. The method of any of claims 1-83, wherein greater than 85% of the subjects treated according to the method do not exhibit grade 3 or greater neurotoxicity.

85. The method of any of claims 1-84, wherein: greater than or greater than about 30%, 35%, 40%, or 50% of the subjects treated according to the method do not exhibit any grade of cytokine release syndrome (CRS) or neurotoxicity; and / orat least at or about 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of subjects treated according to the method do not exhibit onset of CRS earlier than 3 days following initiation of the administration and / or do not exhibit onset of neurotoxicity earlier than 5 days following initiation of the administration; and / or the median onset of neurotoxicity among subjects treated according to the method is at or after the median peak of, or median time to resolution of, CRS in subjects treated according to the method and / or the median onset of neurotoxicity among subjects treated according to the method is greater than at or about 8, 9, 10, or 11 days.

86. The method of any of claims 1-85, wherein: at least 50% of subjects treated according to the method achieve a complete response (CR); at least 70% of the subjects treated according to the method achieve objective response (OR); and greater than or greater than about 50% of the subjects treated according to the method do not exhibit any grade of cytokine release syndrome (CRS) or neurotoxicity; and greater than or greater than about 80% of the subjects treated according to the method do not exhibit a grade 3 or greater cytokine release syndrome (CRS) and / or do not exhibit a grade 3 or greater neurotoxicity.

87. The method of any of claims 1-86, wherein: the CAR comprises an extracellular antigen-binding domain specific for CD 19, 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; the CAR comprises, in order, an extracellular antigen-binding domain specific for CD19, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule; or the CAR comprises an extracellular antigen-recognition domain that specifically binds to CD19, a transmembrne domain, and an intracellular signaling domain comprising a CD3-zeta ^ϋ3z) chain and a costimulatory signaling region that is a signaling domain of 4- IBB.

88. The method of any of claims 1-87, wherein the CAR comprises an extracellular antigen binding domain specific for CD19, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule that is 4- IBB, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule that is CD3zeta.

89. The method of claim 87 or claim 88, wherein the extracellular antigen-binding domain is an scFv.

90. The method of claim 89, wherein the scFv comprises an amino acid sequence of RASQDISKYLN (SEQ ID NO: 35), an amino acid sequence of SRLFiSGV (SEQ ID NO: 36), an amino acid sequence of GNTLPYTFG (SEQ ID NO: 37), an amino acid sequence of DYGVS (SEQ ID NO:38), an amino acid sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), an amino acid sequence of YAMDYWG (SEQ ID NO: 40).

91. The method of claim 89 or claim 90, wherein the scFv comprises a variable heavy chain region of FMC63 and a variable light chain region of FMC63.

92. The method of any of claims 89-91, wherein the scFv is set forth as SEQ ID NO: 4393. The method of any of claims 87-92, wherein the cytoplasmic signaling domain derived from a costimulatory molecule is a signaling domain of 4- IBB, optionally wherein the cytoplasmic signaling domain derived from a costimulatory molecule 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.

94. The method of any of claims 87-93, wherein the cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule is a CD3zeta signaling domain, optionally wherein the cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule comprises SEQ ID NO: 13, 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.

95. The method of any of claims 87-94, wherein the CAR further comprises a spacer between the transmembrane domain and the extracellular antigen-binding domain.

96. The method of claim 95, wherein the spacer is a polypeptide spacer that comprises or consists of all or a portion of an immunoglobulin hinge or a modified version thereof, optionally an IgG4 hinge, or a modified version thereof.

97. The method of claim 95 or claim 96, wherein the spacer is at or about 12 amino acids in length.

98. The method of any of claims 96-97, wherein: the spacer comprises or consists of the sequence of SEQ ID NO: 1, or a sequence encoded by SEQ ID NO: 2..

99. The method of any of claims 95-98, wherein: the spacer is a polypeptide spacer that comprises the sequence of SEQ ID NO: 1; the cytoplasmic signaling domain derived from a costimulatory molecule comprises SEQ ID NO:12; the cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule comprises SEQ ID NO: 13, 14 or 15; and the extracellular antigen binding domain comprises an scFv that comprises a variable heavy chain region of FMC63 and a variable light chain region of FMC63.

100. The method of any of claims 87-99, wherein the transmembrane domain is a transmembrane domain from CD28, optionally a transmembrane 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 or at least about85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8.

101. The method of any of claims 96-100, wherein the CAR contains in order from N- terminus to C-terminus: an extracellular antigen-binding domain that is the scFv set forth in SEQ ID NO: 43, the spacer set forth in SEQ ID NO:1, the transmembrane domain set forth in SEQ ID NO: 8, the 4- 1BB costimulatory signaling domain set forth in SEQ ID NO: 12, and the signaling domain of a CD3- zeta (0O3z) chain set forth in SEQ ID NO: 13.

102. The method of any of claims 1-101, wherein the composition comprising engineered T cells is produced by a manufacturing process comprising:(i) exposing an input composition comprising primary T cells, optionally an input composition comprising autologous T cells selected from the subject, with a stimulatory reagent comprising an oligomeric particle reagent comprising a plurality of streptavidin mutein molecules under conditions to stimulate T cells, thereby generating a stimulated population, wherein: the oligomeric particle reagent comprises a first agent comprising an anti-CD3 antibody or antigen binding fragment thereof and a second agent comprising an anti-CD28 antibody or antigen binding fragment thereof;(ii) introducing into T cells of the stimulated population, a heterologous polynucleotide encoding the CAR that targets CD 19, thereby generating a population of transformed cells;(iii) incubating the population of transformed cells for up to 96 hours; and(iv) harvesting T cells of the population of transformed cells, thereby producing a composition of engineered cells, wherein the harvesting is carried out at a time between 24 and 120 hours, inclusive, after the exposing to the stimulatory reagent is initiated.

103. The method of claim 102, wherein the anti-CD3 antibody or antigen binding fragment is a Fab and the anti-CD28 antibody or antigen binding fragment is a Fab.

104. The method of claim 102 or claim 103, wherein the first agent and the second agent each comprise a streptavidin-binding peptide that reversibly binds the first agent and the second agent to the oligomeric particle reagent, optionally wherein the streptavidin-binding peptide comprises the sequence of amino acids set forth in any of SEQ ID NOS:78-82.

105. The method of any of claims 102-104, wherein the streptavidin mutein molecule is a tetramer of a streptavidin mutein comprising amino acid residues Val44-Thr45-Ala46-Arg47 or Ile44- Gly45-Ala46-Arg47, optionally wherein the streptavidin mutein comprises the sequence set forth in any of SEQ ID NOS: 69, 84, 87, 88, 90, 85 or 59.

106. The method of any of claims 102-105, wherein the oligomeric particle reagent comprises between 1,000 and 5,000 streptavidin mutein tetramers, inclusive.

107. The method of any of claims 102-106, wherein the method further comprises, prior to harvesting the cells, adding biotin or a biotin analog after or during the incubation .

108. The method of any of claims 102-107, wherein the harvesting is carried out at a time between 48 and 120 hours, inclusive, after the exposing to the stimulatory reagent is initiated.

109. The method of any of claims 102-108, wherein the harvesting is carried out at a time when integrated vector is detected in the genome but prior to achieving a stable integrated vector copy number (iVCN) per diploid genome.

110. The method of any of claims 102-109, wherein the harvesting is carried out at a time before the total number of viable cells at the harvesting is more than or more than about three times the number of total viable cells of the stimulated population.

111. The method of any of claims 102-110, wherein the harvesting is carried out at a time when the total number of viable cells at the harvesting is at or about three times, at or about two times, or the same or about the same as the number of total viable cells of the stimulated population.

112. The method of any of claims 102-111, wherein the harvesting is carried out at a time when the percentage of CD27+CCR7+cells is greater than or greater than about 50% among total T cells in the population of transformed cells , total CD3+T cells in the population of transformed cells , total CD4+T cells in the population of transformed cells , or total CD8+T cells in the population of transformed cells , or of CAR-expressing cells thereof, in the population of transformed cells .

113. The method of any of claims 102-112, wherein the harvesting is carried out at a time when the percentage of CD45RA+CCR7+and CD45RA CCR7+cells is greater than or greater than about 60% among total T cells in the population of transformed cells , total CD3+T cells in the population of transformed cells , total CD4+T cells in the population of transformed cells , or total CD8+T cells, or of CAR-expressing cells thereof, in the population of transformed cells .

114. The method of any of claims 1-113, wherein the cells in the administered composition are produced by a manufacturing process to produce an output composition (i) comprising engineered CD4+ T cells and engineered CD8+ T cells and (ii) exhibiting a predetermined feature, wherein iterations of the manufacturing process produce a plurality of the output compositions, optionally from human biological samples, when carried out among a plurality of different individual subjects, in which the predetermined feature of the output composition among the plurality of output compositions is selected from: the mean percentage of cells of a memory phenotype in the plurality of the output compositions is between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%; the mean percentage of cells of a central memory phenotype in the plurality of the output compositions is between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%; the mean percentage of cells that are CD27+, CD28+, CCR7+, CD45RA-, CD45RO+, CD62L+, CD3+, CD95+, granzyme B-, and / or CD127+ in the plurality of the output compositions is between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%; the mean percentage of cells that are CCR7+ / CD45RA- or CCR7+ / CD45RO+ in the plurality of the output compositions is between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%; the mean percentage of central memory CD4+ T cells in the engineered CD4+ T cells, optionally CAR+CD4+ T cells, of the plurality of the output compositions is between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%; the mean percentage of central memory CD8+ T cells in the engineered CD8+ T cells, optionally CAR+CD8+ T cells, of the plurality of the output compositions is between about 40% and about 65%,between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%; and / or the mean percentage of central memory T cells, optionally CD4+ central memory T cells and CD8+ central memory T cells, in the engineered T cells, optionally CAR+ T cells, of the plurality of the output compositions is between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%.

115. The method of any of claims 1-114, wherein the administered composition is produced by a manufacturing process to produce an output composition exhibiting a predetermined feature, optionally a threshold number of cells expressing the CAR in the output composition, in at least about 80%, about 90%, about 95%, about 97%, about 99%, about 100%, or in 100% of the human biological samples in which it is carried out among a plurality of different individual subjects.

116. The method of any of claims 102-115, wherein the composition comprising genetically engineered cells does not contain residual beads from the manufacturing process.

117. The method of any of claims 1-116, wherein the B-cell NHL is a relapsed and / or refractory B-cell non-Hodgkin lymphoma (B-cell NHL).

118. An article of manufacture comprising a composition comprising genetically engineered cells expressing a chimeric antigen receptor (CAR) that targets CD19, and instructions for administering the composition of cells in accordance with the method of any of claims 1-117.

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