CD19-targeted chimeric antigen receptor T cell composition, method and use thereof

Abstract

The present invention provides cell compositions for treating subjects with diseases and conditions such as non-Hodgkin lymphoma (NHL), as well as related methods, compositions, uses, and manufactured articles. [Solution] A subject having or suspected of having B-cell NHL is administered a composition containing engineered T cells expressing a CD19-targeting chimeric antigen receptor (CAR).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under U.S. Provisional Patent Application No. 62 / 975,724, filed on 12 February 2020, titled "CD19-DIRECTED CHIMERIC ANTIGEN RECEPTOR T CELL COMPOSITIONS AND METHODS AND USES THEREOF," the contents of which are incorporated in their entirety by reference.

[0002] Inclusion by referencing sequence listings This application is filed together with an electronic sequence listing. The sequence listing is provided as a file named 735042022740SEQLIST.txt, created on February 6, 2021, and is 56.9 kilobytes in size. The information in the electronic sequence listing is incorporated entirely by reference.

[0003] field This disclosure relates, in several aspects, to adoptive cell therapy involving the administration of cell compositions for treating subjects with diseases and conditions such as non-Hodgkin lymphoma (NHL), as well as related methods, compositions, uses, and manufactured articles. [Background technology]

[0004] background Various immunotherapies and / or cell therapies are available to treat diseases and conditions. For example, adoptive cell therapies (e.g., those involving the administration of cells expressing chimeric receptors specific to the disease or disorder of interest, such as chimeric antigen receptors (CARs) and / or other recombinant antigen receptors, as well as other adoptive immunotherapy and adoptive T-cell therapies) may be beneficial in treating cancer or other diseases or disorders. Improved approaches are needed. This document provides methods, uses, and articles for manufacture that meet such needs. [Overview of the project]

[0005] overview In one aspect, provided herein is a method of treating B-cell non-Hodgkin lymphoma (B-cell NHL), the method comprising administering to a subject having or suspected of having B-cell NHL a composition comprising engineered T cells expressing a chimeric antigen receptor (CAR) that targets CD19, the composition comprising CD4 + T cells expressing the CAR and CD8 + T cells expressing the CAR, and the composition comprising 5×10 6 or about 5×10 6 CAR-expressing T cells to 25×10 6 or about 25×10 6 CAR-expressing T cells (including both end values), and at least 80% or at least about 80% of the cells in the composition being CD3 + cells. A method is provided.

[0006] In one aspect, provided herein is a method of treating B-cell non-Hodgkin lymphoma (B-cell NHL), the method comprising administering to a subject having or suspected of having B-cell NHL a composition comprising engineered T cells expressing a chimeric antigen receptor (CAR) that targets CD19, the composition comprising CD4 + T cells expressing the CAR and CD8 + T cells expressing the CAR in a ratio of about 1:2.5 to about 2.5:1, and the composition comprising 5×10 6 or about 5×10 6 CAR-expressing T cells to 100×10 6 or about 100×10 6 CAR-expressing T cells (including both end values), and at least 90% or at least about 90% of the cells in the composition being CD3 + cells. A method is provided.

[0007] In one aspect, this specification provides a method for treating B-cell non-Hodgkin lymphoma (B-cell NHL), the method comprising the step of administering to a subject having or suspected of having B-cell NHL a composition comprising engineered T cells expressing a CD19-targeting chimeric antigen receptor (CAR), the composition comprising CD4+ T cells expressing the CAR and CD8+ T cells expressing the CAR in a ratio of approximately 1:1 to approximately 2.5:1, and the composition is 5 × 10 6 Or approximately 5 x 10 6 From a single CAR-expressing T cell, 50 × 10 6 Or approximately 50 x 10 6 The composition contains CAR-expressing T cells (including values ​​at both ends), and at least 80% or at least about 80% of the cells in the composition are CD3 + It is a cell, and it provides a method.

[0008] In one aspect, this specification provides a method for treating B-cell non-Hodgkin lymphoma (r / r B-cell NHL), the method comprising the step of administering to a subject having or suspected of having B-cell NHL a composition comprising engineered T cells expressing a CD19-targeting chimeric antigen receptor (CAR), wherein the composition comprises CD4+ T cells expressing the CAR and CD8+ T cells expressing the CAR, and the composition is 5 × 10 6 Or approximately 5 x 10 6 From a single CAR-expressing T cell, 100 × 10⁶ 6 Or approximately 100 x 10 6 The composition contains CAR-expressing T cells (including values ​​at both ends), and at least 80% or at least about 80% of the cells in the composition are CD3 + Cells and CARs in the composition + This method provides a way in which at least 80% or at least about 80% of T cells exhibit a naive-like phenotype or a central memory phenotype.

[0009] In one aspect, this specification provides a method for treating B-cell non-Hodgkin lymphoma (r / r B-cell NHL), the method comprising the step of administering to a subject having or suspected of having B-cell NHL a composition comprising engineered T cells expressing a CD19-targeting chimeric antigen receptor (CAR), wherein the composition comprises CD4+ T cells expressing the CAR and CD8+ T cells expressing the CAR, and the composition is 5 × 10 6 Or approximately 5 x 10 6 From a single CAR-expressing T cell, 100 × 10⁶ 6 Or approximately 100 x 10 6 The composition contains CAR-expressing T cells (including values ​​at both ends), and at least 80% or at least about 80% of the cells in the composition are CD3 + A cell, and the CD4 in the composition + CAR + At least 50% or at least about 50% of T cells are CD27 + CCR7 + and / or the composition contains CD8 + CAR + At least 50% or at least about 50% of T cells are CD27 + CCR7 + It provides a method to achieve this.

[0010] In one aspect, this specification provides a method for treating B-cell non-Hodgkin lymphoma (B-cell NHL), the method comprising the step of administering to a subject having or suspected of having B-cell NHL a composition comprising engineered T cells expressing a CD19-targeting chimeric antigen receptor (CAR), wherein the composition comprises CD4+ T cells expressing the CAR and CD8+ T cells expressing the CAR, and the composition is 5 × 10 6 Or approximately 5 x 10 6 From a single CAR-expressing T cell, 50 × 10 6 Or approximately 50 x 10 6 The composition contains CAR-expressing T cells (including values ​​at both ends), and at least 96% or at least about 96% of the cells in the composition are CD3 + Cells and CARs in the composition+ This method provides a way in which at least 80% or at least about 80% of T cells exhibit a naive-like phenotype or a central memory phenotype.

[0011] In one aspect, this specification provides a method for treating B-cell non-Hodgkin lymphoma (r / r B-cell NHL), the method comprising the step of administering to a subject having or suspected of having B-cell NHL a composition comprising engineered T cells expressing a CD19-targeting chimeric antigen receptor (CAR), wherein the composition comprises CD4+ T cells expressing the CAR and CD8+ T cells expressing the CAR, and the composition is 5 × 10 6 Or approximately 5 x 10 6 From a single CAR-expressing T cell, 100 × 10⁶ 6 Or approximately 100 x 10 6 The composition contains CAR-expressing T cells (including values ​​at both ends), and at least 80% or at least about 80% of the cells in the composition are CD3 + Cells and CARs in the composition + This method provides a method in which the ratio of integrated vector copy number (iVCN) to total VCN in T cells is less than 0.9 on average or approximately less than 0.9.

[0012] In one aspect, this specification provides a method for treating B-cell non-Hodgkin lymphoma (r / r B-cell NHL), the method comprising the step of administering to a subject having or suspected of having B-cell NHL a composition comprising engineered T cells expressing a CD19-targeting chimeric antigen receptor (CAR), wherein the composition comprises CD4+ T cells expressing the CAR and CD8+ T cells expressing the CAR, and the composition is 5 × 10 6 Or approximately 5 x 10 6 From a single CAR-expressing T cell, 100 × 10⁶ 6 Or approximately 100 x 10 6 The composition contains CAR-expressing T cells (including values ​​at both ends), and at least 80% or at least about 80% of the cells in the composition are CD3 + Cells and CARs in the composition +This method provides that the number of integrated vector copies (iVCNs) of T cells is, on average, 0.4 copies / diploid genome to 3.0 copies / diploid genome or approximately 0.4 copies / diploid genome to 3.0 copies / diploid genome (including both end values).

[0013] In any embodiment of this specification, the composition is a CD4 that exhibits CAR + T cells and CD8 expressing CAR + It contains T cells in a ratio of approximately 1:2.5 to 2.5:1.

[0014] In any embodiment of this specification, the composition is 5 × 10 6 Or approximately 5 x 10 6 From a single CAR-expressing T cell, 50 × 10 6 Or approximately 50 x 10 6 The composition contains 5 × 10 CAR-expressing T cells (including values ​​at both ends). In any embodiment of this specification, the composition contains 5 × 10 6 Or approximately 5 x 10 6 From individual CAR-expressing T cells, 25 × 10⁶ 6 Or approximately 25 x 10 6 Includes 1 CAR-expressing T cell (including values ​​at both ends).

[0015] In any embodiment of this specification, the composition is a CD4 that exhibits CAR + T cells and CD8 expressing CAR + The composition may contain T cells in a ratio of approximately 1:2 to approximately 2:1, approximately 1:1.5 to approximately 1.5:1, or 1:1 or approximately 1:1. In any embodiment of this specification, the composition may contain CD4 expressing CAR. + T cells and CD8 expressing CAR + The composition may contain T cells in a ratio of approximately 1:1 to approximately 2.5:1, approximately 1.5:1 to approximately 2:1, 1.5:1 or approximately 1.5:1, or 2:1 or approximately 2:1. In any embodiment of this specification, the composition may contain 5 × 10 6 Or approximately 5 x 10 6 From a single CAR-expressing T cell, 10 × 10 6 Or approximately 10 x 106 may include individual CAR-expressing T cells (including the values at both ends). In any of the embodiments herein, the composition is 10×10 6 or about 10×10 6 individual CAR-expressing T cells, to 25×10 6 or about 25×10 6 individual CAR-expressing T cells (including the values at both ends). In any of the embodiments herein, the composition is 5×10 6 or about 5×10 6 individual CAR-expressing T cells. In any of the embodiments herein, the composition is 10×10 6 or about 10×10 6 individual CAR-expressing T cells. In any of the embodiments herein, the composition is 25×10 6 or about 25×10 6 individual CAR-expressing T cells. In any of the embodiments herein, the composition is 50×10 6 or about 50×10 6 individual CAR-expressing T cells. In any of the embodiments herein, the composition is 100×10 6 or about 100×10 6 individual CAR-expressing T cells.

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

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

[0018] In any of the aspects herein, at least 91% or at least about 91%, at least 92% or at least about 92%, at least 93% or at least about 93%, at least 94% or at least about 94%, at least 95% or at least about 95%, or at least 96% or at least about 96% of the cells in the composition are CD3 + cells. In any of the aspects herein, 5% or about 5% to 30% or about 30%, optionally 5% or about 5% to 30% 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 aspects herein, 10% or about 10% to 15% 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 aspects herein, 15% or about 15% to 20% 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 aspects herein, 20% or about 20% to 25% 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 aspects herein, 25% or about 25% to 30% 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 aspects herein, 5% or about 5%, 10% or about 10%, 15% or about 15%, 20% or about 20%, 25% or about 25%, or 30% 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 aspects herein, the CAR +80% or about 80% to 85% or about 85% of T cells exhibit a naive-like phenotype or a central memory phenotype. In any embodiment of this specification, CAR in the composition + 85% or about 85% to 90% or about 90% of T cells exhibit a naive-like phenotype or a central memory phenotype. In any embodiment of this specification, CAR in the composition + 90% or about 90% to 95% or about 95% of T cells exhibit a naive-like phenotype or a central memory phenotype. In any embodiment of this specification, CAR in the composition + 95% or about 95% to 99% or about 99% of T cells exhibit a naive-like phenotype or a central memory phenotype. In any embodiment of this specification, CAR in the composition + 85% or approximately 85%, 90% or approximately 90%, 95% or approximately 95%, or 99% or approximately 99% of T cells exhibit a naive-like phenotype or a central memory phenotype.

[0019] In some embodiments of this specification, the marker of apoptosis is annexin V. In some embodiments of this specification, the marker of apoptosis is activated caspase 3.

[0020] In any embodiment of this specification, at least 80% or at least about 80% of the composition is naive-like or central memory phenotype + T cells are surface-positive for markers expressed on naive-like T cells or central memory T cells. In some embodiments, the markers expressed on naive-like T cells or central memory T cells are selected from the group consisting of CD45RA, CD27, CD28, and CCR7.

[0021] In some of the embodiments, at least 80% or at least about 80% of the composition is naive-like phenotype or central memory phenotype.+ T cells are CCR7 + CD45RA + CD27 + CCR7 + , or CD62L - CCR7 + It has a phenotype selected from the following. In some of the embodiments, the composition contains CAR + 80% or about 80% of T cells, 85% or about 85%, 85% or about 85%, 90% or about 90%, 90% or about 90%, 95% or about 95%, 95% or about 95%, 99% or about 99% are CCR7 + CD45RA + CD27 + CCR7 + , or CD62L - CCR7 + The naive-like phenotype or central memory phenotype is selected from the following. In some of the embodiments, the CAR in the composition + 80% or approximately 80%, 85% or approximately 85%, 90% or approximately 90%, 95% or approximately 95%, or 99% or approximately 99% of T cells are CCR7 + CD45RA + CD27 + CCR7 + , or CD62L - CCR7 + The naive-like phenotype or central memory phenotype is selected from the following. In some of the embodiments, the CAR in the composition + 80% or approximately 80%, 85% or approximately 85%, 90% or approximately 90%, 95% or approximately 95%, or 99% or approximately 99% of T cells are CD27 + CCR7 + It is a naive-like phenotype or a central memory phenotype.

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

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

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

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

[0026] In any embodiment of this specification, CAR in the composition + At least 80% or at least about 80% of T cells are surface positive for markers expressed on naive-like T cells or central memory T cells. In any embodiment of this specification, the markers expressed on naive-like T cells or central memory T cells are selected from the group consisting of CD45RA, CD27, CD28, and CCR7. In any embodiment of this specification, CAR in the composition + At least 80% or at least about 80% of T cells are CCR7 + CD45RA + CD27 + CCR7 + and / or CD62L - CCR7 + In any embodiment of this specification, the CAR in the composition + 80% or about 80% of T cells, 85% or about 85%, 85% or about 85%, 90% or about 90%, 90% or about 90%, 95% or about 95%, 95% or about 95%, 99% or about 99% are CCR7 + CD45RA + CD27+ CCR7 + and / or CD62L - CCR7 + In any embodiment of this specification, the CAR in the composition + 80% or approximately 80%, 85% or approximately 85%, 90% or approximately 90%, 95% or approximately 95%, or 99% or approximately 99% of T cells are CCR7 + CD45RA + CD27 + CCR7 + and / or CD62L - CCR7 + In any embodiment of this specification, the CAR in the composition + 80% or approximately 80%, 85% or approximately 85%, 90% or approximately 90%, 95% or approximately 95%, or 99% or approximately 99% of T cells are CD27 + CCR7 + In any embodiment of this specification, CD4 in the composition + CAR + At least 50% or at least about 50% of T cells are CCR7 + CD45RA + or CCR7 + CD45RA - In any embodiment of this specification, the composition contains CD4 + CAR + At least 60% or at least about 60% of T cells are CCR7 + CD45RA + or CCR7 + CD45RA - In any embodiment of this specification, the composition contains CD4 + CAR + At least 70% or at least about 70% of T cells are CCR7 + CD45RA + or CCR7 + CD45RA - In any embodiment of this specification, the composition contains CD4 + CAR +At least 80% or at least about 80% of T cells are CCR7 + CD45RA + or CCR7 + CD45RA - In any embodiment of this specification, the composition contains CD4 + CAR + At least 85% or at least about 85% of T cells are CCR7 + CD45RA + or CCR7 + CD45RA - In any embodiment of this specification, CD4 in the composition + CAR + At least 50% or at least about 50% of T cells are CD27 + CCR7 + In any embodiment of this specification, the composition contains CD4 + CAR + At least 60% or at least about 60% of T cells are CD27 + CCR7 + In any embodiment of this specification, the composition contains CD4 + CAR + At least 70% or at least about 70% of T cells are CD27 + CCR7 + In any embodiment of this specification, the composition contains CD4 + CAR + At least 80% or at least about 80% of T cells are CD27 + CCR7 + In any embodiment of this specification, the composition contains CD4 + CAR + At least 85% or at least about 85% of T cells are CD27 + CCR7 + That is the case.

[0027] In any embodiment of this specification, CD8 in the composition + CAR + At least 50% or at least about 50% of T cells are CCR7 + CD45RA+ or CCR7 + CD45RA - In any embodiment of this specification, the composition contains CD8 + CAR + At least 60% or at least about 60% of T cells are CCR7 + CD45RA + or CCR7 + CD45RA - In any embodiment of this specification, the composition contains CD8 + CAR + At least 70% or at least about 70% of T cells are CCR7 + CD45RA + or CCR7 + CD45RA - In any embodiment of this specification, the composition contains CD8 + CAR + At least 80% or at least about 80% of T cells are CCR7 + CD45RA + or CCR7 + CD45RA - In any embodiment of this specification, the composition contains CD8 + CAR + At least 85% or at least about 85% of T cells are CCR7 + CD45RA + or CCR7 + CD45RA - In any embodiment of this specification, CD8 in the composition + CAR + At least 50% or at least about 50% of T cells are CD27 + CCR7 + In any embodiment of this specification, the composition contains CD8 + CAR + At least 60% or at least about 60% of T cells are CD27 + CCR7 + In any embodiment of this specification, the composition contains CD8 + CAR +At least 70% or at least about 70% of T cells are CD27 + CCR7 + In any embodiment of this specification, the composition contains CD8 + CAR + At least 80% or at least about 80% of T cells are CD27 + CCR7 + In any embodiment of this specification, the composition contains CD8 + CAR + At least 85% or at least about 85% of T cells are CD27 + CCR7 + That is the case.

[0028] In any embodiment of this specification, CAR in the composition + In T cells, the ratio of integrated vector copy numbers (iVCNs) to total VCNs is, on average, less than 0.9 or approximately less than 0.9.

[0029] In any embodiment of this specification, CAR in the composition + In T cells, the ratio of the integrated vector copy number (iVCN) to the total VCN is, on average, 0.9 or about 0.9–0.8 or about 0.8. In any embodiment of this specification, the CAR in the composition + In T cells, the ratio of integrated vector copy number (iVCN) to total VCN is, on average, less than 0.8 or about 0.8. In any embodiment of this specification, the CAR in the composition + In T cells, the ratio of the integrated vector copy number (iVCN) to the total VCN is, on average, 0.8 or about 0.8 to 0.7 or about 0.7. In any embodiment of this specification, the CAR in the composition + In T cells, the ratio of the integrated vector copy number (iVCN) to the total VCN is, on average, 0.7 or about 0.7 to 0.6 or about 0.6. In any embodiment of this specification, the CAR in the composition +In T cells, the ratio of the integrated vector copy number (iVCN) to the total VCN is, on average, 0.6 or about 0.6 to 0.5 or about 0.5. In any embodiment of this specification, the CAR in the composition + In T cells, the ratio of integrated vector copy numbers (iVCNs) to total VCNs is, on average, 0.5 or approximately 0.5 to 0.4 or approximately 0.4.

[0030] In any embodiment of this specification, CAR in the composition + The number of integrated vector copies (iVCNs) in T cells is, on average, 0.4 copies / diploid genome to 3.0 copies / diploid genome or approximately 0.4 copies / diploid genome to 3.0 copies / diploid genome (including both end values).

[0031] In any embodiment of this specification, CAR in the composition + The integrated vector copy number (iVCN) of T cells is, on average, 0.8 copies / diploid genome to 2.0 copies / diploid genome or approximately 0.8 copies / diploid genome to 2.0 copies / diploid genome (including both end values). In any embodiment of this specification, the CAR in the composition + The incorporated vector copy number (iVCN) of T cells is, on average, 0.8 copies / diploid genome to 1.0 copies / diploid genome or approximately 0.8 copies / diploid genome to 1.0 copies / diploid genome (including both end values). In any embodiment of this specification, the CAR in the composition + The number of integrated vector copies (iVCNs) of T cells is, on average, 1.0 copies / diploid genome to 1.5 copies / diploid genome or approximately 1.0 copies / diploid genome to 1.5 copies / diploid genome (including both end values). In any embodiment of this specification, the CAR in the composition + The number of integrated vector copies (iVCNs) in T cells is, on average, 1.5 copies / diploid genome to 2.0 copies / diploid genome or approximately 1.5 copies / diploid genome to 2.0 copies / diploid genome (including values ​​at both ends).

[0032] In any aspect of this specification, r / r B-cell NHL is selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), optionally unspecified DLBCL (DLBCL NOS; e.g., de novo DLBCL, or transformed DLBCL from follicular lymphoma or marginal zone lymphoma); high-grade B-cell lymphoma (HGBCL), optionally HGBCL with DLBCL histological features and rearrangement of MYC and BCL2 and / or BCL6; primary mediastinal large B-cell lymphoma (PMBCL); and follicular lymphoma (FL), optionally follicular lymphoma grade 3b (FL3B).

[0033] In any aspect of this specification, B-cell NHL is selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), optionally unspecified DLBCL; transformed DLBCL, optionally transformed DLBCL from follicular lymphoma or marginal zone lymphoma; high-grade B-cell lymphoma (HGBCL), optionally HGBCL with a DLBCL histological features and rearrangement of MYC and BCL2 and / or BCL6; primary mediastinal large B-cell lymphoma (PMBCL); and follicular lymphoma (FL), optionally follicular lymphoma grade 3b (FL3B).

[0034] In any aspect of this specification, B-cell NHL is diffuse large B-cell lymphoma. In any aspect of this specification, B-cell NHL is unspecified diffuse large B-cell lymphoma. In any aspect of this specification, B-cell NHL is newly diagnosed diffuse large B-cell lymphoma. In any aspect of this specification, B-cell NHL is diffuse large B-cell lymphoma transformed from follicular lymphoma. In any aspect of this specification, B-cell NHL is diffuse large B-cell lymphoma transformed from marginal zone lymphoma. In any aspect of this specification, B-cell NHL is high-grade B-cell lymphoma (HGBCL). In any aspect of this specification, B-cell NHL is high-grade B-cell lymphoma with rearrangement of MYC and BCL2 and / or BCL6. In any aspect of this specification, B-cell NHL is a high-grade B-cell lymphoma with DLBCL histological features. In any aspect of this specification, B-cell NHL is a double-hit or triple-hit lymphoma. In any aspect of this specification, B-cell NHL is a primary mediastinal large B-cell lymphoma. In any aspect of this specification, B-cell NHL is a follicular lymphoma. In any aspect of this specification, B-cell NHL is a grade 3b follicular lymphoma. In any aspect of this specification, B-cell NHL is histologically confirmed.

[0035] In any embodiment of this specification, at the time of administration of the composition comprising the manipulated T cells or immediately before such administration, the subject is either relapsed after remission following treatment with (i) two or more prior therapies for B-cell NHL and / or (ii) autologous stem cell transplantation (ASCT) therapy, or is resistant to such therapy.

[0036] In any aspect of this specification, at the time of administration of the composition containing the engineered T cells or immediately before such administration, the subject has relapsed after remission following treatment with two or more prior therapies for B-cell NHL, or is resistant to such therapy. In any aspect of this specification, at the time of administration of the composition containing the engineered T cells or immediately before such administration, the subject has relapsed after remission following treatment with three or more prior therapies for B-cell NHL, or is resistant to such therapy. In any aspect of this specification, the two or more prior therapies for r / r B-cell NHL do not include further doses of CAR-expressing cells.

[0037] In any aspect of this specification, two or more prior therapies for B-cell NHL include an anthracycline and a CD20-targeted agent. In any aspect of this specification, two or more prior therapies for B-cell NHL do not include those administered for prior indolent lymphoma. In any aspect of this specification, two or more prior therapies for B-cell NHL do not include anthracyclines administered for indolent DLBCL. In any aspect of this specification, two or more prior therapies for B-cell NHL include a CD20-targeted agent, and anthracyclines are excluded from two or more prior therapies for B-cell NHL if they have been administered for prior indolent lymphoma. In any aspect of this specification, the CD20-targeted agent includes an anti-CD20 monoclonal antibody. In any aspect of this specification, the CD20-targeted agent includes rituximab.

[0038] In any embodiment of this specification, at the time of administration of the composition containing the manipulated T cells or immediately before such administration, the subject is either relapsed after remission following treatment with autologous stem cell transplantation (ASCT) therapy, or is resistant to such therapy. In some embodiments, the subject has relapsed and / or resistant DLBCL. In some embodiments, an objective response (partial response (PR) or better) is not achieved with ASCT. In any embodiment of this specification, the subject's disease progresses after ASCT.

[0039] In any aspect of this specification, at the time of or prior to the administration of a composition containing manipulated T cells, the subject is identified as having an aggressive or high-risk disease, or as having a poor prognosis. In any aspect of this specification, at the time of or prior to the administration of a composition containing manipulated T cells, the subject is identified as having a chemotherapy-resistant disease, or as having a disease that persists after chemotherapy or has relapsed after chemotherapy. In any aspect of this specification, the subject has pathologically confirmed secondary central nervous system (CNS) infiltration due to a malignant tumor. In any aspect of this specification, the subject does not have infiltration of the central nervous system (CNS) alone due to a malignant tumor.

[0040] In any aspect of this specification, the subject has not received prior CAR T-cell therapy or genetically modified T-cell therapy. In any aspect of this specification, the subject has not received prior CD19-targeted therapy, such as anti-CD19 monoclonal antibodies or bispecific antibodies.

[0041] In any aspect of this specification, the method further includes the step of obtaining a leukocyte apheresis sample from a subject to produce a composition containing engineered T cells. In some aspects, the subject has optionally not received a therapeutic dose of corticosteroids within 14 days or about 14 days prior to the time of leukocyte apheresis. In some aspects, the subject has not received lymphotoxic chemotherapeutic agents or cytotoxic chemotherapeutic agents other than intrathecal ones within 7 days or about 7 days prior to the time of leukocyte apheresis. In any aspect of this specification, the subject has not received lymphotoxic chemotherapeutic agents within 4 weeks or about 4 weeks prior to the time of leukocyte apheresis. In any aspect of this specification, the subject has not received immunosuppressive therapy within 4 weeks or about 4 weeks prior to the time of leukocyte apheresis. In any aspect of this specification, the subject has not received radiation within 6 weeks or about 6 weeks prior to the time of leukocyte apheresis. In any aspect of this specification, the subject has not undergone autologous stem cell transplantation within three months prior to or approximately three months prior to the time of leukocyte apheresis.

[0042] In any aspect of this specification, the subject does not achieve complete remission (CR) in response to prior therapy. In any aspect of this specification, the subject does not achieve an objective response (partial response (PR) or better) in response to prior therapy.

[0043] In any embodiment of this specification, at the time of or prior to the administration of a composition containing manipulated T cells, the subject is evaluated for lymphoma or secondary CNS lymphoma with or associated with central nervous system (CNS) infiltration. In any aspect of this specification, at the time of or prior to the administration of a composition comprising manipulated T cells, the subject is identified as having adequate cardiac function, optionally having a left ventricular ejection fraction (LVEF) of 40% or about 40%, greater than 40%, or greater than about 40%; and / or adequate renal function, optionally having a calculated creatinine clearance of 45 mL / min or about 45 mL / min, greater than 45 mL / min, or greater than about 45 mL / min; and / or adequate hepatic function, optionally having aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at 2.5 times the upper limit of normal (ULN) or less than 2.5 times the ULN, and total bilirubin at less than 1.5 times the ULN; and / or adequate pulmonary function, optionally having dyspnea and oxygen saturation of ≤CTCAE grade 1 in air (e.g., SaO2 ≥ 92%). In any embodiment of this specification, at the time of or before administration of a composition containing manipulated T cells, the subject has a growth rate of 1.0 × 10 without the assistance of growth factors. 9 Or approximately 1.0 × 10 9 individual cells / L, 1.0 × 10⁻⁶ 9 More than 10 cells / L or approximately 1.0 × 10⁻⁶ 9 The cells are identified or have been identified as having a neutrophil absolute number (ANC) of more than 10 cells / L. In any embodiment herein, at the time of or before administration of a composition containing the engineered T cells, the subject is 50 × 10 without the assistance of blood transfusion. 9 Or approximately 50 x 10 9 individual cells / L, 50 × 10 9 More than 10 cells / L or approximately 50 × 10 9 It is identified or determined to have more than 1000 cells / L of platelets.

[0044] In any embodiment of this specification, at the time of or prior to the administration of a composition containing engineered T cells, the subject receives bridging chemotherapy between the time of leukocyte apheresis to prepare the composition containing engineered T cells and the administration of the composition containing engineered T cells. In any embodiment of this specification, at the time of or prior to the administration of a composition containing engineered T cells, the subject receives bridging chemotherapy for disease control after prior therapy.

[0045] In any aspect of this specification, a subject is identified or is identified as having an East Coast Cancer Clinical Trials Group Performance Status (ECOG PS) of 0 or 1. In any aspect of this specification, prior to administration of a composition containing engineered T cells, a subject is identified or is identified as having a high baseline tumor volume, measured, for example, by bidirectional sum of products (SPD) of orthogonal diameters, or a high serum lactate dehydrogenase (LDH), for example, LDH ≥ 500 U / L. In any aspect of this specification, at the time of or prior to administration of a composition containing engineered T cells, a subject has a positron emission tomography (PET) positive disease.

[0046] In any embodiment of this specification, the method further includes a step of identifying or selecting subjects for administration of the composition containing the manipulated T cells, prior to the administration of the composition containing the manipulated T cells.

[0047] In any embodiment of this specification, the subject is pre-treated with lymphocyte depletion therapy prior to administration. In any embodiment of this specification, the method further includes administering lymphocyte depletion therapy, including the administration of fludarabine and / or cyclophosphamide, to the subject immediately before administration of the composition containing the manipulated T cells.

[0048] In any embodiment of this specification, the administration of the composition comprising the manipulated T cells and / or lymphocyte depletion therapy is carried out on an outpatient basis. In some of the embodiments, the administration of the composition is carried out on an outpatient basis, optionally, until the subject exhibits a fever that has not fallen by more than 1°C after treatment with an antipyretic, or has not fallen In some of the embodiments, the composition may be administered without hospitalization and / or an overnight stay in a hospital, unless the subject exhibits a fever that has not fallen by more than 1°C after treatment with an antipyretic, or has not fallen or has not fallen,

[0049] In any embodiment of this specification, lymphocyte depletion therapy is 30 mg / m² daily. 2 Fludarabine for the target body surface area and 300 mg / m² daily 2This includes administering cyclophosphamide to the target body surface area for 3 days each. In any embodiment of this specification, the composition containing the manipulated T cells is administered for 9 days or about 9 days (including the values ​​at both ends) starting from 48 hours or about 48 hours after the completion of lymphocyte depletion therapy. In any embodiment of this specification, after receiving lymphocyte depletion therapy, the subject has a creatinine clearance of at least 30 mL / min or at least about 30 mL / min.

[0050] In any aspect of this specification, prior to the commencement of administration of the composition containing the manipulated T cells, the subject has not received any agents or treatments for the treatment, prevention, reduction, or attenuation of neurotoxicity and / or cytokine release syndrome or the risk thereof. In any aspect of this specification, the method further includes a step of administering to the subject an agent or treatment for the treatment, prevention, reduction, or attenuation of neurotoxicity and / or cytokine release syndrome or the risk thereof. In any aspect of this specification, the agent is or comprises an anti-IL-6 antibody, an anti-IL-6 receptor antibody, or a steroid. In any aspect of this specification, the agent is or comprises tocilizumab or methylprednisolone.

[0051] In any aspect of this specification, the T cells are primary T cells obtained from the subject.

[0052] In any aspect of this specification, the T cell is self to the subject.

[0053] In any aspect of this specification, at least 35%, at least 40%, or at least 50% of subjects treated according to this method achieve complete response (CR); at least 60%, 70%, 80%, 90%, or 95% of subjects that achieve CR exhibit a sustained CR for 3 months or more than 3 months or 6 months or more than 6 months; and / or at least 60%, 70%, 80%, 90%, or 95% of subjects that achieve CR within 1 month and / or 3 months remain in a state of response for 3 months or more than 3 months and / or 6 months or more than 6 months and / or 9 months or more than 9 months after achieving CR. The subjects are in a complete response (CR) state and / or are alive or alive without progression; and / or at least 50%, at least 60%, or at least 70% of subjects treated according to this method achieve an objective response (OR); at least 60%, 70%, 80%, 90%, or 95% of subjects who achieve an OR show a persistent OR for 3 months or more than 3 months or 6 months or more; and / or at least 35%, at least 40%, or at least 50% of subjects who achieve an OR remain in a response state or are alive for 3 months or more than 3 months and / or 6 months or more after achieving an OR.

[0054] In any aspect of this specification, at least 50% of subjects treated according to the Method achieve complete response (CR); at least 60% of subjects that achieve CR exhibit a persistent CR for 6 months or more; and / or at least 60% of subjects that achieve CR within 1 month and / or 3 months remain in response, remain in CR, and / or survive or survive without progression for 6 months or more after achieving CR; and / or at least 70% of subjects treated according to the Method achieve objective response (OR); at least 60% of subjects that achieve OR exhibit a persistent OR for 6 months or more; and / or at least 50% of subjects that achieve OR remain in response or survive for 6 months or more after achieving OR.

[0055] In any aspect of this specification, CR or OR is persistent for more than 3 months or more 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 this method reach a persistent CR for more than 3 months or more than 6 months; and / or at least 60%, 70%, 80%, 90%, or 95% of subjects treated with this method and who reach CR remain in a CR state, remain in a response state, or remain alive for 3 months or more than 3 months, or 6 months or more than 6 months, or 9 months or more than 9 months; and / or at least 60%, 70%, 80%, 90%, or 95% of subjects treated with this method who reach CR by 1 month and / or 3 months remain in a CR state for 3 months For longer than one month, or longer than three months, and / or six months or longer than six months, and / or nine months or longer than nine months, the patient remains in a state of response, remains in a state of complete response (CR), and / or is alive or alive without progression; and / or at least 50%, at least 60%, or at least 70% of subjects treated according to this method achieve an objective response (OR); at least 60%, 70%, 80%, 90%, or 95% of subjects achieve a sustained OR for three months or longer than three months, or six months or longer than six months; and / or at least 35%, at least 40%, or at least 50% of subjects treated with this method and who achieve an OR remain in a state of response or are alive for three months or longer than three months and / or six months or longer.

[0056] In any aspect of this specification, the cells are autologous to the subject, and the minimum absolute number of lymphocytes (ALC) of apheresis is not required and / or specified for the preparation of the therapy; and / or the cells are prepared by a process that can produce a cell product for administration by this method in at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the subject having B-cell NHL.

[0057] In any aspect of this specification, at least 35%, at least 40%, or at least 50% of subjects treated according to this method achieve complete response (CR) or remission of CNS disease; at least 60%, 70%, 80%, 90%, or 95% of subjects who achieve CR remain in CR state for 3 months or more than 3 months or 6 months or more than 6 months; and / or at least 60%, 70%, 80%, 90%, or 95% of subjects who achieve CR or remission of CNS disease within 1 month and / or 3 months remain in response, remain in CR state, and / or survive or survive without progression for longer than 3 months or more than 3 months and / or 6 months or more than 6 months and / or 9 months or more than 9 months; and / or at least 50% of subjects treated according to this method %, at least 60%, or at least 70% achieve an objective response (OR) or remission of the CNS disease; at least 60%, 70%, 80%, 90%, or 95% of subjects who achieve an OR remain in response for 3 months or more than 3 months or 6 months or more than 6 months; and / or at least 60%, 70%, 80%, 90%, or 95% of subjects who achieve an OR or remission of the CNS disease remain in response or are alive for 3 months or more than 3 months and / or 6 months or more; and / or the size or volume of brain lesions is reduced by 25%, 50%, 75%, or more or by about 25%, 50%, 75%, or more; and / or reduction, remission, or disappearance of the CNS disease is obtained in at least 35%, at least 40%, or at least 50% of subjects treated according to this method.

[0058] In any aspect of this specification, more than 50% or about 50%, about 60%, about 70%, or more than 80% of subjects treated according to this method will not exhibit grade 3 or higher cytokine release syndrome (CRS) and / or grade 3 or higher neurotoxicity, and / or more than 40%, 50%, or 55% of subjects treated according to this method will not exhibit any neurotoxicity or CRS. In any aspect of this specification, more than 80% or about 80% of subjects treated according to this method will not exhibit grade 3 or higher cytokine release syndrome (CRS) and / or grade 3 or higher neurotoxicity. In any aspect of this specification, more than 95% of subjects treated according to this method will not exhibit grade 3 or higher CRS. In any aspect of this specification, more than 85% of subjects treated according to this method will not exhibit grade 3 or higher neurotoxicity.

[0059] In any aspect of this specification, more than 30%, 35%, 40%, or 50% of subjects treated according to this method, or about 30%, 35%, 40%, or 50% of subjects, will not exhibit cytokine release syndrome (CRS) or neurotoxicity of any grade; and / or at least 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of subjects treated according to this method, or at least about 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% will not exhibit any neurotoxicity for more than 3 days after the start of administration. The subjects do not exhibit an early onset of CRS and / or an onset of neurotoxicity earlier than 5 days after the start of administration; and / or the median onset of neurotoxicity in subjects treated according to this method is the median peak of CRS in subjects treated according to this method, or after the median peak, or the median time to the disappearance of CRS in subjects treated according to this method; and / or the median onset of neurotoxicity in subjects treated according to this method is 8, 9, 10 or 11 days or about 8, 9, 10 or 11 days or more.

[0060] In any aspect of this specification, more than 50% or about 50% of subjects treated according to this method do not exhibit cytokine release syndrome (CRS) or neurotoxicity of any grade; and / or at least 45% or at least about 45% of subjects treated according to this method do not exhibit the onset of CRS earlier than 3 days after the start of administration and / or the onset of neurotoxicity earlier than 5 days after the start of administration; and / or the median onset of neurotoxicity in subjects treated according to this method is the median peak of CRS in subjects treated according to this method, or after the median peak, or the median time to the disappearance of CRS in subjects treated according to this method; and / or the median onset of neurotoxicity in subjects treated according to this method is more than 8 days or about 8 days.

[0061] In any aspect of this specification, at least 50% of subjects treated according to this method achieve complete response (CR); at least 70% of subjects treated according to this method achieve objective response (OR); and more than 50% or about 50% of subjects treated according to this method do not exhibit cytokine release syndrome (CRS) or neurotoxicity of any grade; and more than 80% or about 80% of subjects treated according to this method do not exhibit cytokine release syndrome (CRS) of grade 3 or higher, and / or neurotoxicity of grade 3 or higher.

[0062] In any aspect of this specification, CAR may comprise an antigen-specific extracellular antigen-binding domain, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule that is optionally 4-1BB, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule that is optionally CD3 zeta; in any several aspect, CAR may comprise, in order, an antigen-specific extracellular antigen-binding domain, 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 CAR may comprise an extracellular antigen-recognition domain that specifically binds to an antigen, and an intracellular signaling domain comprising a costimulatory signaling region that is a CD3-zeta (CD3ζ) chain and a 4-1BB signaling domain. In any aspect of this specification, CAR may comprise a CD19-specific extracellular antigen-binding domain, a transmembrane domain, a cytoplasmic signaling domain derived from 4-1BB, and a cytoplasmic signaling domain derived from CD3 zeta.

[0063] In any embodiment of this specification, CAR comprises a CD19-specific extracellular antigen-binding domain, a transmembrane domain, a cytoplasmic signaling domain derived from an optionally 4-1BB costimulatory molecule, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which is optionally CD3 zeta; CAR comprises, in turn, a CD19-specific extracellular antigen-binding domain, a transmembrane domain, a cytoplasmic signaling domain derived from an optional 4-1BB costimulatory molecule, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule; or CAR comprises an extracellular antigen-recognition domain that specifically binds to CD19, and an intracellular signaling domain comprising a costimulatory signaling region which is a CD3-zeta (CD3ζ) chain and a 4-1BB signaling domain.

[0064] In any embodiment of this specification, the CAR comprises a CD19-specific extracellular antigen-binding domain, a transmembrane domain, a cytoplasmic signaling domain derived from a 4-1BB costimulatory molecule, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which is a CD3 zeta.

[0065] In any aspect of this specification, the extracellular antigen-binding domain is scFv. In any aspect of this specification, scFv is The amino acid sequence of TIFF2026113543000001.tif4128, The amino acid sequence and / or of TIFF2026113543000002.tif4128 The amino acid sequence and / or of TIFF2026113543000003.tif4128 The amino acid sequence of TIFF2026113543000004.tif4128, The amino acid sequence and / or of TIFF2026113543000005.tif4128 The amino acid sequence of TIFF2026113543000006.tif4128 may be included. In any embodiment of this specification, the six CDR sequences of scFv (CDRL1-L3 and CDRH1-H3) are the amino acid sequence TIFF2026113543000007.tif17144 may be included. In some embodiments, the scFv includes the heavy chain variable region and the light chain variable region of FMC63. In some embodiments, the six CDR sequences (CDRL1-L3 and CDRH1-H3) of the scFv may include the CDRL1 sequence, CDRL2 sequence, CDRL3 sequence, CDRH1 sequence, CDRH2 sequence, and CDRH3 sequence of FMC63. In some embodiments, the scFv may bind to the same epitope as any one of the aforementioned, or compete with any one of the aforementioned for binding. In some embodiments, the scFv includes, in order, VH, a linker, optionally a linker shown for example in SEQ ID NO:24, and VL. In any embodiment of this specification, scFv comprises, in order, VL, linker, optionally a linker indicated as, for example, SEQ ID NO:24, and VH. In some embodiments, scFv comprises a flexible linker. In some embodiments, scFv comprises an amino acid sequence indicated as SEQ ID NO:43. In any embodiment of this specification, scFv is indicated as SEQ ID NO:43.

[0066] In any aspect of this specification, scFv is The amino acid sequence of TIFF2026113543000008.tif4128, The amino acid sequence of TIFF2026113543000009.tif4128 and / or The amino acid sequence of TIFF2026113543000010.tif4128 and / or The amino acid sequence of TIFF2026113543000011.tif4128, The amino acid sequence of TIFF2026113543000012.tif4128 and / or The scFv may include the amino acid sequence TIFF2026113543000013.tif4128. In some embodiments, the scFv includes the heavy chain variable region and the light chain variable region of FMC63. In some embodiments, the scFv includes the CDRL1 sequence, CDRL2 sequence, CDRL3 sequence, CDRH1 sequence, CDRH2 sequence, and CDRH3 sequence of FMC63. In some embodiments, the scFv includes VH, linker SEQ ID NO:24, and VL, respectively. In some embodiments, the scFv includes a flexible linker. In some embodiments, the scFv includes the amino acid sequence indicated as SEQ ID NO:43. In some embodiments, the CD19-directed scFv either binds to the same epitope as any one of the aforementioned or competes with any one of the aforementioned for binding.

[0067] In any aspect of this specification, scFv may include a heavy chain variable region and a light chain variable region of FMC63.

[0068] In some embodiments, the transmembrane domain is a transmembrane domain containing an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with respect to SEQ ID NO:8, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity. In some embodiments, the transmembrane domain is a transmembrane domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with respect to the amino acid sequence shown in SEQ ID NO:9, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity.

[0069] In any aspect of this specification, the co-stimulatory signaling region is the 4-1BB signaling domain. In any aspect of this specification, the co-stimulatory 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 higher sequence identity thereto. In any aspect of this specification, the primary signaling domain is the CD3 zeta signaling domain. In any aspect of this specification, 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 higher sequence identity thereto.

[0070] In any aspect of this specification, the cytoplasmic signaling domain derived from the co-stimulatory molecule is the 4-1BB signaling domain. In any aspect of this specification, the cytoplasmic signaling domain derived from the co-stimulatory molecule includes SEQ ID NO:12 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity.

[0071] In any aspect of this specification, the cytoplasmic signaling domain derived from the primary signaling ITAM-containing molecule is the signaling domain of CD3 zeta. In any aspect of this specification, the cytoplasmic signaling domain derived from the primary signaling ITAM-containing molecule includes 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 higher sequence identity to these.

[0072] In any aspect of this specification, the CAR further includes a spacer between the transmembrane domain and the extracellular antigen-binding domain. In some aspects, the spacer includes the sequence of SEQ ID NO:1, the 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 any one of the aforementioned variants having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to these, or the sequence of SEQ ID NO:1, the 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, SEQ ID The spacer consists of the sequence of NO:34, or any one of the aforementioned variants having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity thereto. In some embodiments, the spacer comprises or consists of the formula X1PPX2P, where X1 is glycine, cysteine, or arginine, and X2 is cysteine ​​or threonine.

[0073] In any embodiment of this specification, the CAR may further include a spacer between the transmembrane domain and the scFv. In any embodiment of this specification, the spacer is a polypeptide spacer comprising all or part of an immunoglobulin hinge or a modified form thereof, or comprising all or part of an immunoglobulin hinge or a modified form thereof. In some embodiments, the spacer is an IgG4 hinge or a modified form thereof. In any embodiment of this specification, the spacer is 12 or about 12 amino acids long. In any aspect of this specification, the spacer has any of the aforementioned variants having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID The spacer consists of a sequence encoded by NO:34, or any one of the aforementioned variants having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity thereto. In some embodiments, the spacer may comprise or consist of the formula X1PPX2P, where X1 is glycine, cysteine, or arginine, and X2 is cysteine ​​or threonine. In any embodiment of this specification, the spacer may comprise or consist of all or part of an immunoglobulin hinge or a modified form thereof, or is a polypeptide spacer comprising about 15 or fewer amino acids.In some embodiments, the spacer does not include the extracellular region of CD28 or CD8 and may include all or part of an immunoglobulin hinge, optionally an IgG4 hinge or a modified form thereof, or may consist of all or part of an immunoglobulin hinge, optionally an IgG4 hinge or a modified form thereof, and / or includes about 15 or fewer amino acids and does not include the extracellular region of CD28 or CD8.

[0074] In some embodiments, the spacer is 12 or approximately 12 amino acids long and / or comprises an immunoglobulin hinge, optionally containing all or part of IgG4 or a modified form thereof, or consisting of an immunoglobulin hinge, optionally containing all or part of IgG4 or a modified form thereof. In some embodiments, the spacer has any one variant of the aforementioned sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID The spacer may consist of a sequence encoded by NO:34, or any one of the aforementioned variants having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity thereto. In some embodiments, the spacer may contain or consist of the formula X1PPX2P, where X1 is glycine, cysteine, or arginine, and X2 is cysteine ​​or threonine. In some embodiments, the co-stimulatory domain may contain 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 higher sequence identity thereto.In any of the embodiments provided, 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 higher sequence identity to them. In any of the embodiments, scFv is... The amino acid sequence of TIFF2026113543000014.tif4128, The amino acid sequence of TIFF2026113543000015.tif4128, The amino acid sequence of TIFF2026113543000016.tif4128, The amino acid sequence of TIFF2026113543000017.tif4128, The amino acid sequence of TIFF2026113543000018.tif4128 and The amino acid sequence may include TIFF2026113543000019.tif4128. In some embodiments, the scFv includes the heavy chain variable region and the light chain variable region of FMC63. In some embodiments, the scFv includes six CDRs, including the CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3 sequences of FMC63. In some embodiments, the scFv is an scFv that binds to the same epitope as any one of the aforementioned, or competes with any one of the aforementioned for binding. In some embodiments, the scFv includes, in order, VH, a linker, a linker optionally containing SEQ ID NO:24, and VL. In some embodiments, the scFv includes, in order, V:, a linker, a linker optionally containing SEQ ID NO:24, and VH. In some embodiments, the scFv includes a flexible linker. In some embodiments, the amino acid sequence of the scFv is shown as SEQ ID NO:43.

[0075] In any aspect of this specification, (i)scFv is The amino acid sequence of TIFF2026113543000020.tif4128, The amino acid sequence of TIFF2026113543000021.tif4128, The amino acid sequence of TIFF2026113543000022.tif4128, The amino acid sequence of TIFF2026113543000023.tif4128, The amino acid sequence of TIFF2026113543000024.tif4128 and (ii) The spacer may include the amino acid sequence of TIFF2026113543000025.tif4128, for example scFv may include the amino acid sequence shown as SEQ ID NO:43; (ii) The spacer may be a polypeptide spacer consisting of (a) all or part of an immunoglobulin hinge or a modified form thereof, or all or part of an immunoglobulin hinge or a modified form thereof, comprising about 15 or fewer amino acids, and optionally including all or part of an immunoglobulin hinge, optionally an IgG4 hinge or a modified form thereof, or consisting of all or part of an immunoglobulin hinge, optionally an IgG4 hinge or a modified form thereof, optionally, for example the spacer may include the sequence of SEQ ID NO:1, or the sequence encoded by SEQ ID NO:2, or may consist of such sequence; (iii) The co-stimulatory domain is SEQ ID NO:12 or its variants having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity; and (iv) 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 higher sequence identity.

[0076] In any aspect of this specification, the CAR is (i) scFv comprises an amino acid sequence indicated as SEQ ID NO:43; (ii) the spacer is a polypeptide spacer comprising or consisting of the sequence encoded by SEQ ID NO:1 or SEQ ID NO:2; (iii) the cytoplasmic signaling domain derived from the co-stimulatory molecule is a co-stimulatory domain comprising 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 higher sequence identity; and (iv) the cytoplasmic signaling domain derived from the primary signaling ITAM-containing molecule is SEQ ID NO:13, 14, or 15, or a cytoplasmic signaling domain containing a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity.

[0077] In any embodiment of this specification, the CAR (i) comprises an extracellular antigen-binding domain comprising an scFv containing the heavy chain variable region and the light chain variable region of FMC63; a spacer being a polypeptide spacer comprising the sequence of SEQ ID NO:1; a cytoplasmic signaling domain derived from a co-stimulatory molecule comprising SEQ ID NO:12; and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule comprising SEQ ID NO:13, 14, or 15.

[0078] In any aspect of this specification, the spacer is a polypeptide spacer that may contain the sequence of SEQ ID NO:1; the co-stimulatory domain may contain SEQ ID NO:12; the primary signaling domain may contain SEQ ID NO:13, 14, or 15; and the antigen-binding domain may contain an scFv that includes the heavy chain variable region and the light chain variable region of FMC63. In any aspect of this specification, the cell composition is administered parenterally, optionally intravenously. In any aspect of this specification, the subject is a human subject.

[0079] In any particular embodiment of the method provided, the CAR comprises, in order from the N-terminus to the C-terminus, an extracellular antigen-binding domain which is an scFv shown as SEQ ID NO:43, a spacer shown as SEQ ID NO:1, a transmembrane domain shown as SEQ ID NO:8, a 4-1BB costimulatory signaling domain shown as SEQ ID NO:12, and a CD3-zeta (CD3ζ) chain signaling domain shown as SEQ ID NO:13.

[0080] In some of the embodiments, a composition comprising engineered T cells is prepared by a manufacturing process comprising: (i) an exposure step of exposing an input composition comprising a plurality of streptavidin mutein molecules to a stimulating reagent comprising an oligomeric particle reagent comprising a plurality of streptavidin mutein molecules under conditions that stimulate T cells, thereby generating a stimulated population, wherein the oligomeric particle reagent comprises a first agent comprising an anti-CD3 antibody or its antigen-binding fragment and a second agent comprising an anti-CD28 antibody or its antigen-binding fragment; (ii) an introduction step of introducing heterologous polynucleotides encoding CD19-targeting CARs into the T cells of the stimulated population, thereby generating a population of transformed cells; (iii) an incubation step of the population of transformed cells for up to 96 hours; and (iv) a collection step of collecting T cells from the population of transformed cells, thereby preparing a composition of engineered cells, wherein the collection is performed at a time between 24 and 120 hours (including both values) after the start of exposure to the stimulating reagent. In some embodiments, the input composition comprises autologous T cells enriched by immunoaffinity-based selection for CD3 T cells or CD4 and CD8 T cells from a subject, for example, a blood sample or apheresis (e.g., leukocyte apheresis) sample derived from the subject.

[0081] In any embodiment of this specification, a composition comprising engineered T cells is prepared by a manufacturing process comprising an exposure step, in which an input composition comprising primary T cells is exposed to a stimulating reagent comprising a plurality of avidin, streptavidin, avidin mutein, or streptavidin mutein molecules under conditions that stimulate T cells, thereby generating a stimulated population, the stimulating reagent being 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 co-stimulatory molecules. In any embodiment of this specification, the manufacturing process may further comprise an introduction step, in which heterologous polynucleotides encoding CD19-targeting CARs are introduced into the T cells of the stimulated population, thereby generating a population of transformed cells.

[0082] In some of the embodiments, the oligomer particle reagent comprises a first agent comprising an anti-CD3 antibody or an antigen-binding fragment thereof and a second agent comprising an anti-CD28 antibody or an antigen-binding fragment thereof. In some of the embodiments, the anti-CD3 antibody or antigen-binding fragment is Fab, and the anti-CD28 antibody or antigen-binding fragment is Fab. In some of the 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 oligomer particle reagent, and optionally the streptavidin-binding peptide comprises an amino acid sequence shown in any of SEQ ID NO: 78-82. In some of the embodiments, the streptavidin mutein molecule is a tetramer of streptavidin mutein containing the amino acid residues Val44-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47, and optionally the streptavidin mutein contains the sequence shown in SEQ ID NO: 69, 84, 87, 88, 90, 85, or 59. In some of the embodiments, the oligomeric particle reagent contains 1,000 to 5,000 streptavidin mutein tetramers (including the values ​​at both ends). In some of the embodiments, the method further comprises adding biotin or a biotin analog before cell collection, after incubation, or during incubation.

[0083] In any aspect of this specification, the manufacturing process may further include a step of incubating a population of transformed cells for up to 96 hours. In any aspect of this specification, the incubation is carried out in a basal medium lacking one or more recombinant cytokines. In any aspect of this specification, the manufacturing process may further include a collection step of collecting T cells from the population of transformed cells to thereby prepare a composition of engineered cells. In any aspect of this specification, the collection is carried out at 24 to 120 hours (inclusive) after the start of exposure to the stimulating agent. In any aspect of this specification, the collection is carried out at 48 to 120 hours (inclusive) after the start of exposure to the stimulating agent. In any aspect of this specification, the collection is carried out at a point when the integrated vector is detectable in the genome but before it reaches a stable, integrated vector copy number (iVCN) per diploid genome. In any aspect of this specification, the collection is carried out before the total number of viable cells at collection is more than three times or approximately three times the total number of viable cells in the stimulated population. In any aspect of this specification, collection is performed when the total number of viable cells at the time of collection is three times or approximately three times, two times or approximately two times, or equal to or approximately equal to the total number of viable cells in the stimulated population. In any aspect of this specification, collection is performed on CD27 + CCR7 + The proportion of cells is total T cells in the transformed cell population, and total CD3 cells in the population. + T cells, total CD4 in a population of transformed cells + All CD8 in a population of T cells or transformed cells + The collection is performed when the T cells, or the CAR-expressing cells in a population of transformed cells, represent more than 50% or approximately more than 50%. In any aspect of this specification, collection is performed at the CD45RA + CCR7 + and CD45RA - CCR7 + The proportion of cells is total T cells in the transformed cell population, total CD3 cells in the transformed cell population.+ T cells, total CD4 in a population of transformed cells + All CD8 in a population of T cells or transformed cells + This is performed when there are more than 60% or approximately more than 60% of T cells or these CAR-expressing cells.

[0084] In any aspect of this specification, the cells in the administered composition are produced by a manufacturing process for producing an output composition that (i) comprises engineered CD4+ T cells and engineered CD8+ T cells and (ii) exhibits predetermined characteristics, wherein the iteration of the manufacturing process is performed in multiple different individual subjects, optionally producing multiple output compositions from a human biological sample, the predetermined characteristics of the output composition among the multiple output compositions being: the average percentage of memory phenotype cells in the multiple output compositions is about 40% to about 65%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, or about 60% to about 65%; the average percentage of central memory phenotype cells in the multiple output compositions is about 40% to about 65%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, or about 55% %~approximately 60%, or approximately 60%~approximately 65%; the average percentage of cells in multiple output compositions that are CD27+, CD28+, CCR7+, CD45RA-, CD45RO+, CD62L+, CD3+, CD95+, granzyme B-, and / or CD127+ is approximately 40%~approximately 65%, approximately 40%~approximately 45%, approximately 45%~approximately 50%, approximately 50%~approximately 55%, and approximately 55%. %~approximately 60%, or approximately 60%~approximately 65%; the average percentage of cells that are CCR7+ / CD45RA- or CCR7+ / CD45RO+ in multiple output compositions is approximately 40%~approximately 65%, approximately 40%~approximately 45%, approximately 45%~approximately 50%, approximately 50%~approximately 55%, approximately 55%~approximately 60%, or approximately 60%~approximately 65%; manipulated CD4+ in multiple output compositions For T cells, the average percentage of central memory CD4+ T cells in any CAR+CD4+ T cells is approximately 40% to 65%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, or 60% to 65%; for manipulated CD8+ T cells of multiple output compositions, the average percentage of central memory CD8+ T cells in any CAR+CD8+ T cells is approximately 40% to 65%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, or 60% to 65%;Furthermore / or multiple output compositions are selected from those in which the mean proportions of central memory T cells, optionally CAR+ T cells, optionally CD4+ central memory T cells, and CD8+ central memory T cells are approximately 40%–65%, 40%–45%, 45%–50%, 50%–55%, 55%–60%, or 60%–65%. In some embodiments, multiple output compositions are prepared from human biological samples.

[0085] In any aspect of this specification, the administered composition is produced by a manufacturing process for producing an output composition exhibiting a predetermined characteristic, optionally, if it is performed on multiple different individual subjects, by a manufacturing process for producing an output composition exhibiting a certain threshold number of CAR-expressing cells in the output composition at least about 80%, about 90%, about 95%, about 97%, about 99%, about 100%, or 100% of the human biological sample. In any aspect of this specification, the composition containing genetically modified cells does not contain residual beads from the manufacturing process. In any aspect of this specification, B-cell NHL is relapsed and / or anti-treatment B-cell non-Hodgkin lymphoma (B-cell NHL). In any aspect of this specification, the predetermined characteristic is a certain threshold number of CAR-expressing cells in the output composition.

[0086] Furthermore, this specification provides a manufactured article comprising a composition containing genetically modified cells expressing a CD19-targeting chimeric antigen receptor (CAR), and instructions for administering the cell composition according to any of the methods provided herein. [Brief explanation of the drawing]

[0087] [Figure 1]This shows an exemplary quantification of cellular purity of T cell compositions prepared by a non-extended, manipulation process using different donor types (reference, patient), measured by flow cytometry. Cells were manipulated to express anti-CD19 CARs (CD19) or mock-transduced (mock). The percentage of CD3+ cells among viable CD45+ cells (left panel), the percentage of NK cells among viable CD45+ cells (middle panel), and the percentage of CD19+ cells among viable CD45+ cells (right panel) were examined. [Figure 2] This shows exemplary quantification of cellular phenotypes in manipulated processes with and without expanded proliferation using different donor types (reference, patient), as measured by flow cytometry. Cells were manipulated to express anti-CD19 CAR (CD19) or mock-transduced (mock). Figure 2 shows the percentage of CD3+CD8+ and CD3+CD4+ cells among viable CD45+ cells (left panel) and the percentage of CD8+CAR+ and CD4+CAR+ cells among viable CD45+ cells (right panel). [Figure 3] This shows exemplary quantification of cellular phenotypes in manipulated processes with and without expanded proliferation using different donor types (reference, patient), as measured by flow cytometry. Cells were manipulated to express anti-CD19 CAR (CD19) or mock-transduced (mock). Figure 3 shows the ratio of CD4+ cells to CD8+ cells and the ratio of CD4+CAR+ cells to CD8+CAR+ cells. [Figure 4] This shows an exemplary quantification of cell viability in T cell compositions prepared by a non-extended proliferation manipulation process using different donor types (reference, patient), as measured by flow cytometry. Cells were manipulated to express anti-CD19 CAR (CD19) or mock-transduced (mock). The proportion of aCas3+ cells among CD3+ cells was examined. [Figure 5-1]Figure 5A shows an exemplary relationship between the total copy number / cell (CNY) as assessed by standard VCN (without pulsed-field gel electrophoresis or PFGE) and the total copy number / cell (CNY) as assessed by iVCN (with PFGE) in cell compositions prepared from primary T cells derived from different human donors, which were engineered to express CAR using either a process with or without expansion (○) expansion (●). Figures 5B-5C show the relationship between the copy number / cell (CNY) in cell compositions assessed by standard VCN (Figure 5B) or iVCN (Figure 5C) and the surface expression of CAR, indicated by the percentage of CAR-expressing CD3+ cells among viable CD45+ cells (%CD3+CAR+) at the time of flow cytometry assessment. [Figure 5-2] See the explanation in Figure 5-1. [Figure 6A] Figures 6A–6B show exemplary proportions of cellular phenotypes resulting from manipulation processes with and without expanded proliferation using different donor types (reference, patient). Cells were manipulated to express anti-CD19 CAR (CD19) or mock-transduced (mock). Figure 6A shows exemplary proportions of CD45RA+CCR7+ cells among aCas-CD8+CAR+ and aCas-CD4+CAR+ cells (upper left panel), CD45RA-CCR7+ cells among aCas-CD8+CAR+ and aCas-CD4+CAR+ cells (upper right panel), CD45RA-CCR7- cells among aCas-CD8+CAR+ and aCas-CD4+CAR+ cells (lower left panel), and CD45RA+CCR7- cells among aCas-CD8+CAR+ and aCas-CD4+CAR+ cells (lower right panel). Figure 6B shows an exemplary proportion of CD27+CCR7+ cells among aCas-CD8+CAR+ and aCas-CD4+CAR+ cells. [Figure 6B] See the explanation in Figure 6A. [Figure 7-1]Figures 7A–7D show exemplary quantitative analysis of the displayed cellular phenotype before (primary) and after (secondary) long-term CAR-dependent stimulation (after 9–14 days of long-term stimulation with an anti-idiotype agonist antibody) cells were manipulated by flow cytometry to express anti-CD19 CARs in both donor-matched expansion and expansion processes. DP1 = diffuse large B-cell lymphoma (DLBCL) patient 1; DP2 = DLBCL patient 2; HD1 = healthy donor 1; MP1 = mantle cell lymphoma patient 1. [Figure 7-2] See the explanation in Figure 7-1. [Figure 8-1] Figures 8A–8F show exemplary in vitro proliferative capacity of cells produced by processes without and with expansion of growth after long-term anti-CD19 CAR-dependent stimulation with anti-ID antibodies. Viability (Figure 8A) and cell size (Figure 8B) were measured. The change in expansion multiplier was calculated by dividing the daily count by the starting cell number (Figure 8C), and then converted to the area under the curve (AUC) for either comparison between individual groups (Figure 8D) or comparison between manufacturing processes (Figure 8E, statistical significance by Mann-Whitney test; *p<0.05). The expansion multiplier in individual donors between manufacturing platforms was also measured directly by obtaining the daily expansion multiplier for compositions produced by the process without expansion of growth and dividing it by the donor-matched control value for compositions produced by the process with expansion of growth (Figure 8F). [Figure 8-2] See the explanation in Figure 8-1. [Figure 9A] Figures 9A–9B show exemplary cytolytic potential of anti-CD19 CAR T cells engineered by processes with or without expansion and expansion, at different effector-to-target ratios before (Figure 9A) and after (Figure 9B) long-term stimulation. Area under the curve (AUC) values ​​were compared either for individual groups (left panel of Figures 9A and 9B) or for the manufacturing process (right panel of Figures 9A and 9B, statistical significance by Mann-Whitney test; *p<0.05). [Figure 9B]See the explanation in Figure 9A. [Figure 10] This shows exemplary cytokine production by cells manipulated by processes without or with proliferative expansion, at 0 and 10 days after long-term antigen stimulation, as measured by a multifunctionality score using flow cytometry. Statistical significance was assessed by the Mann-Whitney U scale (*p<0.05). [Figure 11] Figures 11A–11B show exemplary tumor volume and circulating CAR-T cells over time in a Nalm6 leukemia model after treatment with anti-CD19 CAR-T cell compositions generated by matched donor manipulation processes with and without growth expansion. Figure 11A shows tumor growth for each group from day -1 (pre-treatment) to approximately day 25 post-treatment, calculated from the area under the bioluminescence (BLI) curve (AUC). Figure 11B shows the circulating anti-CD19 CAR-T cell count per μl of blood for each group from approximately day 5 to approximately day 20 post-treatment. [Figure 12A] Figures 12A-12B show exemplary tumor volume and circulating CAR-T cells over time in a Raji lymphoma model after treatment with anti-CD19 CAR-T cell compositions generated by matched donor manipulation processes with and without growth expansion. Figure 12A shows tumor growth for each group from day 1 (pre-treatment), calculated from the area under the BLI curve (AUC), up to approximately day 80 after treatment with high-dose (left panel) and low-dose (right panel) anti-CD19 CAR-T cell compositions generated by matched donor manipulation processes with and without growth expansion. Changes in BLI radiance (photons / second; y-axis) are shown for all groups (tumor volume). Figure 12B shows the circulating anti-CD19 CAR-T cell counts per μl of blood at the indicated post-treatment time points for each group (without growth expansion, NE; with growth expansion, E). The absolute counts at each time point are shown as group mean values. The differences were compared using the Mann-Whitney U test; *p<0.05. [Figure 12B] See the explanation in Figure 12A. [Modes for carrying out the invention]

[0088] Detailed explanation This specification provides methods and uses for the treatment of subjects having a disease or condition, and for the use of engineered cells (e.g., T cells) and / or compositions thereof, wherein the disease or condition is generally cancer or tumor, e.g., lymphoma, most particularly, B-cell malignancies that are non-Hodgkin lymphoma (NHL), e.g., aggressive subtype NHL, or B-cell malignancies that are non-Hodgkin lymphoma (NHL), e.g., aggressive subtype NHL. In certain aspects, the subject has aggressive NHL or high-risk NHL. In some embodiments of the methods provided, a therapeutic T-cell composition comprising engineered cells is administered to a subject having NHL, e.g., by adoptive cell therapy, e.g., adoptive T-cell therapy. In certain embodiments of any of the methods provided, the T cells are engineered with a chimeric antigen receptor (CAR) directed to differentiation antigen group 19 (CD19). In some aspects, the disease or condition is B-cell lymphoma. In some aspects, the disease or condition is large B-cell lymphoma. In some aspects, the disease or condition is diffuse large B-cell lymphoma (DLBCL) or a subtype thereof. In some aspects, the method and its use result in, or achieve, improved response and / or more sustained response or efficacy and / or a reduced risk of toxicity or other adverse effects compared to, for example, certain treatment groups, with respect to, certain alternative methods. In some aspects, the method is advantageous due to the administration of a specified number or relative number of manipulated cells, the administration of a specified ratio of a particular type of cell, the administration of a specific high percentage of poorly differentiated cells (e.g., naive-like cells or central memory cells or cells in an early differentiated state, e.g., CCR7+CD27+ cells), and the treatment of, and / or combinations thereof, a particular patient population, e.g., a specific risk profile, disease stage, and / or patient population with a history of prior treatment.

[0089] In some embodiments, the method and use involve administering to a subject T cells expressing genetically modified (recombinant) cell surface receptors in adoptive cell therapy, the cell surface receptors being generally chimeric receptors, such as chimeric antigen receptors (CARs), that recognize CD19 expressed by, associated with, and / or NHL and / or cell types derived therefrom, and specific to NHL and / or cell types derived therefrom. The cells are generally administered in a composition formulated for administration; the method generally involves administering single or multiple doses of cells to a subject, the single or multiple doses may contain a specific number or relative number of cells or modified cells. In some cases, the CD19-targeted CAR+ modified cells in the composition include two or more subtypes in a specified ratio or composition, such as CD4 T cells and CD8 T cells. In a particular embodiment, a cell composition for use or administration by the method provided comprises primary T cells engineered to express CD19-directed CARs, wherein (i) a small proportion (e.g., less than 40%, less than 30%, less than 20%, or less than 10%) of cells containing exhausted cells and / or cells exhibiting markers or phenotypes associated with exhaustion; and / or (ii) a relatively high proportion (e.g., greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%) of memory-like T cells, e.g., naive-like T cells, central memory T cells, or long-lived memory T cells. In the embodiments provided, the characteristics of the composition and the method provided result in improved or enhanced survival, growth, persistence, and / or antitumor activity compared to other CD19-targeted CAR T-cell therapies that involve the administration of a large number of cells exhibiting a high proportion of exhausted cells and / or exhaustion-associated phenotypes, and / or containing a small proportion of specific T cells, such as naive-like T cells, central memory T cells, or long-lived memory T cells.In the embodiments provided, the characteristics of the composition and the method of provision result in improved therapeutic efficacy, such as an increased percentage of patients achieving complete response (CR), compared to methods involving the administration of other CD19-directed CAR T-cell therapies that include a large number of cells exhibiting a high proportion of exhausted cells and / or phenotypes associated with exhaustion, and / or contain a small proportion of specific T cells, such as naive-like T cells, central memory T cells, or long-lived memory T cells. In the methods provided, the characteristics of the composition and the method of provision result in improved clinical durability of the therapeutic response, such as CR, for example, a response that persists for a certain period after the start of treatment, compared to methods involving the administration of other CD19-directed CAR T-cell therapies that include a large number of cells exhibiting a high proportion of exhausted cells and / or phenotypes associated with exhaustion, and / or contain a small proportion of memory-like T cells, such as naive-like T cells, central memory T cells, or long-lived memory T cells. The use or administration of a CD19-directed CAR T cell composition provided in a particular embodiment by the method provided may be carried out using a dose of cells that is more than twice as small, for example, five or ten times smaller, than the dose of a reference CD19-directed CAR T cell composition (e.g., manipulated by the same or similar CARs, for example, by the same antigen-binding domain, but the reference CD19-directed CAR T cell composition contains a large number of cells exhibiting a high proportion of exhausted cells and / or exhaustion-associated phenotypes, and / or contains a small proportion of memory-like T cells, e.g., 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 produced ex vivo by a process that involves culturing cells under conditions for expansion growth, for example, during the process for cell generation, resulting in cell proliferation or cell population doubling (e.g., doubling of the population of cells 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times compared to the start of the process).

[0090] In some embodiments, the CD19-directed CAR T cell compositions for use provided are produced by a relatively short process that does not involve the step of culturing cells under conditions designed for expansion or growth to expand the cells. Various processes are available for creating compositions containing genetically engineered T cell populations, for example, engineered T cells expressing a CAR, which typically involve culturing cells to expand or increase their growth, or steps designed for the purpose of culturing cells to expand or increase their growth. However, in certain contexts, some of these processes may require long or relatively long periods of time to produce the engineered cells. Also, in various contexts, some existing processes may have varying amounts of time required to successfully produce engineered T cells suitable for cell therapy, making it difficult to coordinate the administration of such cell therapy. In certain contexts, some of these processes may produce cell populations containing a relatively high proportion or relatively large number of exhausted cells, differentiated cells, or poorly differentiated cells. The CD19-targeted CAR T cell composition provided for use in the provided method addresses one or more of these problems.

[0091] In certain embodiments, the methods provided are used in connection with processes for efficiently generating or creating engineered cells suitable for use in cell therapy. In some embodiments, the compositions provided, including CD19-directed CAR engineered T cells, are produced by processes that do not require any additional steps for cell expansion and proliferation, e.g., without any expansion and proliferation unit operations and / or steps intended to cause cell expansion and proliferation. In various aspects of the process for producing a CD19-directed CAR T cell composition, the process includes one or more steps for stimulating and genetically engineering (e.g., transforming, transfecting, or transfecting) T cells to generate a population of engineered T cells that can be collected or formulated for use as a composition for cell therapy. In certain embodiments, the process includes the step of transfecting cells using a viral vector (e.g., a lentiviral vector) containing nucleic acid encoding a CD19-directed CAR. In some embodiments, the processes provided result in the stable incorporation of heterologous nucleic acid (expressed by the viral vector) into the cell genome. In several aspects, the provided process produces engineered CD19-targeted CAR T cells with improved differentiation potential compared to engineered T cell compositions produced by alternative processes, such as processes involving cell proliferation.

[0092] In certain contexts, the duration of the process for producing the composition to be provided may be measured from the time when the cells of the input cell population or input composition, such as T cells, first come into contact with or are exposed to the stimulating conditions (e.g., as described herein, e.g., in Section II-C), which is referred herein as the commencement of stimulation or stimulation, and also referred herein as exposure to the stimulating reagent, for example, when exposure to the stimulating reagent is initiated. In some embodiments, the duration required to collect or harvest the output population containing the manipulated cells (also referred herein as the output composition or the manipulated cells, e.g., the composition of manipulated T cells) is measured from the commencement of stimulation. In certain embodiments, the duration of the process is 120 hours, 108 hours, 96 hours, 84 hours, 72 hours, 60 hours, 48 ​​hours, 36 hours, or 30 hours, or approximately 120 hours, 108 hours, 96 hours, 84 hours, 72 hours, 60 hours, 48 ​​hours, 36 hours, or 30 hours, or less than 120 hours, 108 hours, 96 hours, 84 hours, 72 hours, 60 hours, 48 ​​hours, 36 hours, or 30 hours. In certain embodiments, the duration of the process is 5 days, 4 days, 3 days, 2 days, or 1 day, or approximately 5 days, 4 days, 3 days, 2 days, or 1 day, or less than 5 days, 4 days, 3 days, 2 days, or 1 day. In certain embodiments, the manipulated cells, e.g., cells in an output composition or output population, are more potent, persistent, or naive-like than cells manipulated using a process requiring a longer duration. In some aspects, the duration of the process provided, for example, the amount of time required to create or generate the manipulated T cell population, is 2, 3, 4, 5, 6, 7, or more than 7 days shorter than that of some existing processes, by approximately 2, 3, 4, 5, 6, 7, or more than 7 days, or by at least 2, 3, 4, 5, 6, 7, or more than 7 days.In some embodiments, the duration of the process provided is 75%, 60%, 50%, 40%, 30%, 25%, 15%, or 10% of the alternative or existing process, or approximately 75%, 60%, 50%, 40%, 30%, 25%, 15%, or 10%, or less than 75%, 60%, 50%, 40%, 30%, 25%, 15%, or 10%.

[0093] In certain embodiments, the process provided is carried out on a population of cells isolated, concentrated, or selected from a biological sample, e.g., CD3+, CD4+, and / or CD8+ T cells. In some aspects, by the method provided, the engineered T cell composition can be prepared or generated in a reduced amount of time from the time the biological sample was collected from the subject, compared to other methods or processes. In some embodiments, manipulated T cells can be produced or generated by the provided method, for example, when a biological sample or enriched, isolated, or selected cells are cryopreserved and stored, or in all cases, within 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, or 2 days, or within approximately 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, or 2 days, or within 120 hours, 96 hours, 72 hours, or 48 hours, or within approximately 120 hours, 96 hours, 72 hours, or 48 hours, from the time the biological sample is collected from the subject until the manipulated T cells are collected, gathered, or formulated (for example, for cryopreservation or administration).

[0094] In certain embodiments, a process for producing or manipulating a T cell population includes a step of stimulating cells, for example, before transduction using a viral vector. In various aspects of the process provided, stimulation is carried out using an irritant conjugate, such as an oligomeric stimulating reagent immobilized or conjugated with anti-CD3 / anti-CD28, such as a streptavidin mutein oligomer. Existing reagents for use in in vitro stimulation of T cells in the absence of exogenous growth factors or in small amounts of exogenous growth factors are known (see, for example, U.S. Patent 6,352,694 B1 and European Patent EP 0 700 430 B1). Generally, such reagents may be beads with a diameter greater than 1 μm, such as magnetic beads, immobilized with various conjugates (e.g., anti-CD3 antibodies and / or anti-CD28 antibodies). However, in some cases, such magnetic beads must be ensured to be completely removed, for example, before administering expanded T cells to the target, making it difficult to incorporate such magnetic beads into methods for stimulating cells under the conditions required for clinical trials or therapeutic purposes. In some situations, such removal, for example by exposing cells to a magnetic field, may reduce the yield of viable cells available for cell therapy. In certain cases, such reagents, such as stimulating reagents containing magnetic beads, must be incubated with cells for the minimum amount of time necessary to allow for the detachment of a sufficient amount of T cells from the stimulating reagent.

[0095] In the processes provided that utilize oligomer stimulating reagents, such as streptavidin mutein polymer, such potential limitations are overcome. For example, in some embodiments, the processes provided avoid or reduce the risk of stimulating reagents, such as those containing magnetic beads, remaining in the output cells produced or created by the process. In some embodiments, this also means that a process compliant with GMP standards can be established more easily than other methods, such as those requiring additional measures to ensure that the final operated T cell population is bead-free. In some embodiments, this can be easily done by adding a substance that dissociates the oligomer stimulating reagent from the cells, such as a competing reagent, for example, by simply rinsing or washing the cells, for example, by centrifugation. Therefore, in some aspects, the removal or separation of the oligomer stimulating reagent from the cells, for example by adding a substance or competing reagent, results in little to no cell loss compared to the removal or separation of bead-based stimulating reagents. In some aspects, the timing of the removal or separation of the oligomer stimulating reagent is unrestricted or less restrictive than that of the removal or separation of bead-based stimulating reagents. Therefore, in some cases, the oligomer-stimulating reagent can be removed or separated from the cells at any point or stage during the process being provided.

[0096] In some aspects, the use of oligomeric stimulating agents (e.g., anti-CD3 / anti-CD28 streptavidin mutein oligomers) may result in a generally reduced stimulating signal compared to alternative stimulating agents, such as anti-CD3 / anti-CD28 paramagnetic beads. Processes that may involve weaker or reduced stimulation may produce engineered CAR+ T cells that are as potent, persistent, or effective as, or even more potent, persistent, or effective than, CAR+ T cells produced by processes involving stronger stimulation conditions or larger amounts or higher concentrations of the stimulating agent, such as those produced after stimulation with anti-CD3 / anti-CD28 paramagnetic beads. Furthermore, in some embodiments, stimulating cells with small or relatively small amounts of the oligomeric stimulating agent may enhance the differentiation potential, efficacy, or persistence of the resulting engineered cell population compared to processes using larger amounts of the oligomeric stimulating agent. In such embodiments, it is assumed that such effects may persist even at doses low enough to sufficiently reduce the expression of the activation marker or the proportion of cells positive for the activation marker during and after the process.

[0097] In certain embodiments, the output composition or output population of engineered T cells produced or generated by the provided process, e.g., T cells expressing recombinant receptors, e.g., chimeric antigen receptors, is particularly effective or potent when used as cells for cell therapy. For example, in some aspects, the output composition containing engineered T cells produced by the provided process, e.g., CAR+ T cells, has much higher differentiation and / or proliferation potential than engineered T cells produced or created by existing alternative processes. In some aspects, the output composition containing engineered T cells produced by the provided process, e.g., CAR+ T cells, has improved antitumor activity or anticancer cell activity than engineered T cells produced by alternative or existing methods, e.g., CAR+ T cells.

[0098] In certain embodiments, a process for producing a CD19-directed T cell composition that does not involve the step of expanding and growing cells to a threshold amount or concentration has further advantages. In some aspects, a protocol that does not rely on cell expansion and growth to increase the number or concentration of cells from a starting cell population, e.g., an input population, does not require incubation or culture work that may vary between cell populations. For example, in some embodiments, cell populations obtained from different subjects, e.g., subjects with different diseases or disease subtypes, may be expected to divide or expand at different rates, particularly in the case of patients with NHL, e.g., high-risk, aggressive, and / or R / R NHL. In certain embodiments, eliminating a potentially variable step that requires cell expansion and growth allows for strict control over the duration of the entire process. In certain embodiments, variability in process duration is reduced or eliminated, which in some aspects may allow for improved coordination of appointments and procedures among physicians, patients, and technicians, and facilitate autologous cell therapy.

[0099] In some embodiments, the methods provided involve treating a specific group or subset of subjects, e.g., subjects identified as having a high-risk disease, e.g., high-risk NHL or high-risk large B-cell lymphoma. In some aspects, the methods treat subjects with a form of aggressive and / or poor-prognosis B-cell non-Hodgkin lymphoma (NHL), e.g., relapsed or resistant to standard treatment (R / R), and / or NHL with a poor prognosis. In some aspects, the methods treat subjects with relapsed or resistant to standard treatment (R / R) large B-cell lymphoma. In certain aspects, the manipulated cells are autologous to the subject and are administered after being generated by an ex vivo process that allows for the production of CAR-manipulated T cell compositions that are shortened compared to existing methods, do not include or involve a culture step for cell expansion and proliferation during the method of producing the manipulated cells, and / or are in a poorly differentiated state that allows for administration at low doses. As a result, the method provided is advantageous compared to existing methods because it can shorten the time until the manipulated T-cell therapy is available to patients, particularly in patients requiring treatment, for example, in subjects who have relapsed or are resistant to treatment after one or more other prior therapies to treat a disease or condition. In some aspects, the method, composition, use, and manufactured article provided yield improved or outstanding responses to available treatments. In some embodiments, the improved or outstanding response is relative to the current standard of care (SOC).

[0100] Non-Hodgkin lymphoma (NHL) is a group of lymphoid malignancies with diverse biological and clinical behaviors. In 2019, there were approximately 74,200 new diagnoses in the United States (US), and an estimated 19,970 deaths due to NHL (Siegel et al., CA Cancer J Clin. 2019;69(1):7-34). NHL can be divided into two prognostic groups: indolent lymphoma (slow growth, accompanied by regressive lymphadenopathy over several years) and aggressive lymphoma (rapid growth, leading to death within weeks if left untreated). The most common aggressive lymphoma is diffuse large B-cell lymphoma (DLBCL), which accounts for 30-40% of all NHL cases (Li et al., Pathology 2018;50(1):74-87). Other aggressive lymphomas include, but are not limited to, so-called double-hit (DHL) or triple-hit (THL) lymphomas such as high-grade B-cell lymphoma (HGBCL), mantle cell lymphoma (MCL), primary mediastinal large B-cell lymphoma (PMBCL), and follicular lymphoma grade 3b (FL3B). The progression and treatment of other aggressive lymphoma subtypes are similar to those of DLBCL. Approximately 50% of newly diagnosed DLBCL patients can be cured with first-line R-CHOP immunochemotherapy (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). However, approximately half of patients treated with R-CHOP relapse, mostly within the first two years after treatment (Coiffier et al., Blood 2010;116(12):2040-5; Vitolo et al., J Clin Oncol. 2017;35(31):3529-37). Autologous stem cell transplantation (ASCT) after salvage high-dose immunochemotherapy is the standard second-line treatment for relapsed or relapsed / reactive (R / R) DLBCL. Approximately half of R / R patients are unresponsive to salvage therapy or are ineligible for transplantation due to their disease stage, and the majority of patients relapse even after ASCT.In the multi-cohort retrospective NHL study of antitherapy-treated DLBCL (SCHOLAR-1; n=636), a very poor outcome was observed in this patient population, with an overall response rate (ORR) of 26% to the next line of treatment 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).

[0101] CD19 is a member of the immunoglobulin superfamily and a component of the B cell surface signaling complex, which positively regulates B cell receptor-mediated signaling. It is expressed by most B cell malignancies from early development to plasma cell differentiation (Stamenkovic et al., J Exp Med. 1988; 168(3): 1205-10). Because CD19 is expressed by most B cell malignancies, including B cell NHL, it is an attractive therapeutic target (Davila et al., Oncoimmunology. 2012; (9): 1577-83). Importantly, the CD19 antigen is not expressed on any normal tissue other than hematopoietic stem cells or B cell lineages. Furthermore, CD19 is not shedged into the circulatory system, which limits off-target adverse effects (Shank et al., Pharmacotherapy. 2017; 37(3): 334-45).

[0102] In certain embodiments, the methods provided herein are based on the administration of CD19-directed CAR T-cell therapy in which the CAR contains a CD19-directed scFv antigen-binding domain (e.g., derived from FMC63). The CAR further contains an intracellular signaling domain including a CD3 zeta-derived signaling domain, and also incorporates a 4-1BB costimulatory domain, which is associated with a lower incidence of cytokine release syndrome (CRS) and neurotoxicity (NE) compared to CD28-containing constructs (Lu et al. J Clin Oncol. 2018;36:3041).

[0103] The challenge in developing CAR T cells is to produce formulations that continuously expand and proliferate after infusion, remain present, and mediate a sustained antitumor response. Certain CD19-targeted CAR-T cell therapies, such as Kymriah® (tisagenlecleucel) (Kymriah PI) and Yescarta® (axicabutagen silolucel) (Yescarta PI), are available for the treatment of B-cell lymphoma. Both CAR T-cell therapies initially have a complete response rate (CRR) of 50-60% in patient populations for whom this treatment is difficult; however, the response rate decreases to 40-50% at 6 months, but the response group tends to have a sustained response (Locke et al., Mol Ther. 2017; 25(1): 285-95; Schuster et al., N Engl J Med. 2017; 377(26): 2545-54). Consequently, more than half of patients who receive these therapies do not have a long-lasting sustained response, and the overall survival (OS) prognosis is very poor.

[0104] The methods provided are based on the finding that the poor differentiation state of adopted T cells can affect the ability of such cells to persist and promote sustained antitumor immunity. In some embodiments, the CD19-directed CAR+ manipulated T cell compositions provided are prepared by methods that do not involve cell culture under expansion conditions, thereby limiting or reducing the number of population doublings of the final manipulated output composition and resulting in a poorly differentiated formulation. Furthermore, the compositions provided are also prepared by processes that result in a stably incorporated vector copy number (iVCN) to ensure consistent and reliable CAR expression, thereby resulting in a consistent cell product for administration to the target and reducing variability in CAR-expressing cells in the administered dose. In contrast, most protocols for T cell manipulation conventionally involve ex vivo expansion of T cells for 9–14 days or longer. The provided data illustrated herein support a model in which CAR T cell products with a high proportion of poorly differentiated memory T cells may exhibit improved sustained antitumor activity. These findings suggest that strategies aimed at minimizing effector differentiation in CAR T cell products may lead to improved clinical efficacy. This specification provides embodiments in which such an objective may be achieved.

[0105] The observations described herein support treating subjects with high-risk diseases according to the method provided with CD19-directed CAR T-cell therapy. For example, subjects with NHL, e.g., aggressive NHL, or patients with certain high-risk traits, e.g., patients with relapsed / anti-treatment (R / R) NHL, may be treated according to the method provided. In some embodiments, the method provided may be used to treat subjects who have undergone numerous pretreatments (e.g., with one, two, three, four or more prior therapies to treat the disease).

[0106] All publications referenced in this application, including patent documents, scientific articles, and databases, are incorporated by reference in whole for the same degree as each individual publication is incorporated by reference individually. If any definition provided herein conflicts with or otherwise contradicts any definition provided in a patent, patent application, published patent application, or other publication incorporated herein by reference, the definition provided herein shall prevail over the definition incorporated herein by reference.

[0107] Section headings used in this specification are for structural purposes only and should not be construed as limiting the subject matter described herein.

[0108] I. Methods and Use of CD19-Targeted Cell Therapy in B-Cell Non-Hodgkin Lymphoma This specification provides methods for treatment involving the administration of engineered cells or compositions comprising engineered cells, such as engineered T cells. It also provides methods and uses of the provided CD19-targeted CAR-engineered cells (e.g., T cells) and / or compositions, for example, for the treatment of subjects having B-cell non-Hodgkin lymphoma (NHL), such as aggressive NHL or high-risk NHL, such as R / R NHL, involving the administration of engineered cells and / or compositions. In some embodiments, the provided CD19-targeted CAR-engineered cells (e.g., T cells) and / or compositions, for example, for the treatment of subjects having R / R NHL that have failed at least two or more prior therapies. In certain embodiments, the method comprises administering to a subject a certain dose of T cells comprising CD4+ and CD8+ T cells, wherein the T cells contain a chimeric antigen receptor (CAR) that specifically binds to CD19.

[0109] In some embodiments, the method and use involve administering to a subject cells expressing genetically modified (recombinant) cell surface receptors in adoptive cell therapy, the cell surface receptors being generally chimeric receptors, such as chimeric antigen receptors (CARs), that recognize CD19 expressed by, associated with, and / or derived from lymphoma and / or cell types, and that is specific to lymphoma and / or derived from lymphoma and / or cell types. The cells are generally administered in a composition formulated for administration. In some embodiments, the cells are collected from the subject before treatment for the purpose of manipulating them with CD19-directed recombinant receptors (e.g., CARs). In some embodiments, the cells are collected by leukocyte apheresis. In some aspects, the cells are manipulated by an ex vivo method (hereinafter also referred to herein as a non-expansion process) that does not involve culturing the cells for expansion and growth. An exemplary non-expansion process for manipulating the CAR-expressing therapeutic composition provided is described in Section II-C.

[0110] In some aspects, the disease or condition is aggressive NHL. In some aspects, the disease or condition is aggressive NHL that was previously indolent. In some aspects, the NHL is a non-indolent type, such as aggressive lymphoma, which is rapidly progressive. Among these, the most common subtype of aggressive NHL is diffuse large B-cell lymphoma (DLBCL).

[0111] 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 according to the Lugano classification (see, e.g., Cheson et al., (2014) JCO 32(27):3059-3067; Cheson, BD (2015) Chin Clin Oncol 4(1):5). In some embodiments, the disease or condition is confirmed histologically. In any embodiment disclosed herein, the disease or condition or subtype or state may be determined prior to leukocyte apheresis in connection with the acquisition of T cells for autologous T-cell therapy.

[0112] In some embodiments, the disease or condition is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the disease or condition is unspecified DLBCL (NOS). In some embodiments, the disease or condition is high-grade B-cell lymphoma (HGBCL) with DLBCL histological features and rearrangement of MYC and BCL2 and / or BCL6. In some embodiments, the disease or condition is DLBCL transformed from follicular lymphoma (tFL). In some embodiments, the disease or condition is DLBCL transformed from marginal zone lymphoma (tMZL). In some embodiments, the disease or condition is primary mediastinal large B-cell lymphoma (PMBCL). In some embodiments, the disease or condition is follicular lymphoma (FL). In some embodiments, the disease or condition is follicular lymphoma grade 3B (FL3B).

[0113] In some embodiments, subjects prior to leukocyte apheresis have an 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 ECOG performance status index may be used to evaluate subjects, e.g., those with poor outcomes in prior therapies, or to select subjects, e.g., those with poor outcomes in prior therapies, for treatment (see, e.g., Oken et al., (1982) Am J Clin Oncol. 5:649-655). The ECOG Scale of performance status indicates the level of functionality in terms of a patient's ability to care for themselves, daily activities, and physical abilities (e.g., walking, working). In some embodiments, an ECOG performance status of 0 indicates that the subject is able to perform normal activities. In some cases, a subject with an ECOG performance status of 1 is able to walk well but shows some limitations in physical activity. In some cases, more than 50% of patients with an ECOG performance status of 2 are able to walk. Furthermore, in some cases, individuals with an ECOG performance status of 2 may be able to perform basic self-care activities; see, for example, Sorensen et al., (1993) Br J Cancer 67(4)773-775. The criteria reflecting ECOG performance status are listed in Table 1 below.

[0114] (Table 1) ECOG Performance Status Criteria TIFF2026113543000026.tif47160

[0115] In some embodiments, prior to the administration of the CD19-directed CAR T cell composition provided, for example at the time of administration, the subject is either relapsed after remission following treatment with one or more prior lines of treatment for NHL, or is antiretroviral to one or more prior lines of treatment for NHL. In any embodiment, prior to leukocyte apheresis in connection with the manipulation of the CD19-directed CAR T cell composition, the subject is either relapsed after remission following treatment with one or more prior lines of treatment for NHL, or is antiretroviral to one or more prior lines of treatment for NHL. Thus, in certain embodiments, prior to the time of treatment, for example prior to leukocyte apheresis, the subject has R / R NHL. In some embodiments, prior to the administration of cells expressing recombinant receptors, the subject has been previously treated with a disease or condition, e.g., NHL, or a therapy or therapeutic agent targeting NHL. In some embodiments, the subject has been previously treated with hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT or autologous HSCT. In some embodiments, the subjects had a poor prognosis after treatment with standard therapy and / or one or more previous lines of treatment were unresponsive. In some embodiments, the subjects had been treated with or had previously received at least one, two, three, four or more other therapies for treating NHL, e.g., aggressive NHL or high-risk NHL, e.g., DLBCL or its subtypes, or at least about one, two, three, four or more other therapies, or about one, two, three, four or more other therapies. In some embodiments, the subjects had been treated with or had previously received therapies including CD20-targeting agents (e.g., anti-CD20 antibodies) and alkylating agents. In some aspects, the subjects relapsed after an initial response of complete response (CR) or partial response (PR) to prior therapy. In some embodiments, the subjects are resistant to at least one or more prior therapies, and in the resistant treatment, the patient is either disease-stable (SD) or disease-progressing (PD) with the best response after the prior therapy.

[0116] In some embodiments, the subject is either relapsed after remission following treatment with at least two prior systemic treatment lines for the disease or condition, or is antiretroviral to at least two prior systemic treatment lines for the disease or condition. In some embodiments, at least one of the aforementioned prior systemic treatment lines includes treatment with an anthracycline and an anti-CD20 monoclonal antibody. Exemplary anti-CD20 antibodies include, but are not limited to, rituximab, ofatumumab, ocrelizumab (also known as GA101 or RO5072759), bertuzumab, obinutuzumab, TRU-015 (Trubion Pharmaceuticals), okalatuzumab (also known as AME-133v or okalatuzumab), and Pro131921 (Genentech). See, for example, Lim et al. Haematologica. (2010) 95(1):135-43. Rituximab is a chimeric mouse / human monoclonal antibody IgG1 kappa that binds to CD20 and induces cytolysis of CD20-expressing cells. In some embodiments, at least one of the aforementioned prior systemic treatment lines includes treatment with anthracycline and rituximab.

[0117] In some embodiments, subjects have relapsed after remission following treatment with autologous stem cell transplantation (ASCT) or have become resistant to ASCT. In some embodiments, relapse after remission following treatment with ASCT or resistant to ASCT prevents objective response from being achieved after ASCT. In some embodiments, relapse after remission following treatment with ASCT or resistant to ASCT prevents partial response (PR) or better from being achieved after ASCT. In some embodiments, relapse after remission following treatment with ASCT or resistant to ASCT includes disease progression after ASCT.

[0118] In some embodiments, the subject has relapsed after remission following treatment with at least two previous systemic treatment lines and ASCT for the disease or condition, or has been anti-therapeutic to at least two previous systemic treatment lines and ASCT for the disease or condition. In some embodiments, at least one of the aforementioned previous systemic treatment lines includes treatment with an anthracycline and an anti-CD20 monoclonal antibody. In some embodiments, at least one of the aforementioned previous systemic treatment lines includes treatment with an anthracycline and rituximab.

[0119] In some embodiments, prior to leukocyte apheresis, the subjects do not show infiltration into the central nervous system (CNS) alone due to the disease or condition. In some embodiments, the subjects have pathologically confirmed secondary CNS infiltration due to the disease or condition.

[0120] In some embodiments, the subjects have not previously received CAR T-cell therapy prior to being administered CD19-targeted modified CAR T cells according to the method provided. In some embodiments, the subjects have not received genetically modified T-cell therapy. In some embodiments, the subjects have not received CD19-targeted therapy. Examples of CD19-targeted therapies include, but are not limited to, anti-CD19 monoclonal antibodies or anti-CD19 bispecific antibodies. In some embodiments, the subjects do not have hypersensitivity to fludarabine and / or cyclophosphamide. In some embodiments, the subjects do not have an active autoimmune disease requiring immunosuppressive therapy.

[0121] In some aspects, subjects have not received a therapeutic dose of corticosteroids within 14 days prior to leukocyte apheresis. In some aspects, a therapeutic dose of corticosteroids is defined as prednisone or equivalent at doses greater than 20 mg / day. In some aspects, subjects have not received cytotoxic chemotherapeutic agents that are not considered lymphocytotoxic within 7 days prior to leukocyte apheresis. In some aspects, subjects have not received intrathecal therapy (IT) within 7 days prior to leukocyte apheresis. Examples of cytotoxic chemotherapeutic agents include, but are not limited to, doxorubicin, vincristine, gemcitabine, oxaliplatin, carboplatin, and etoposide. In some aspects, subjects have not received oral chemotherapeutic agents within 5 half-lives prior to leukocyte apheresis. Examples of oral chemotherapeutic agents include, but are not limited to, lenalidomide and ibrutinib. In some aspects, subjects have not received lymphocytotoxic chemotherapeutic agents within 4 weeks prior to leukocyte apheresis. Examples of lymphotoxic chemotherapeutic agents include, but are not limited to, cyclophosphamide, ifosfamide, and bendamustine. In some embodiments, subjects have not received any experimental treatment within 8 weeks (for biologics) or within 5 half-lives (for small molecules) prior to leukocyte apheresis. In some embodiments, subjects have not received immunosuppressive therapy within 4 weeks prior to leukocyte apheresis. Examples of immunosuppressive therapies include, but are not limited to, calcineurin inhibitors, methotrexate or other chemotherapy, mycophenolates, rapamycin, and immunosuppressive antibodies, such as anti-TNF, anti-IL6, or anti-IL6R. In some embodiments, subjects have not received donor lymphocyte infusion within 6 weeks prior to leukocyte apheresis. In some embodiments, subjects have not received radiotherapy for multiple lesions within 6 weeks of leukocyte apheresis. In some embodiments, subjects have not received radiotherapy for a single lesion within 14 days prior to leukocyte apheresis, if additional non-irradiated PET-positive lesions are present.In some embodiments, subjects have not undergone autologous stem cell transplantation (SCT) within 3 months prior to leukocyte apheresis. In some embodiments, subjects have not undergone allogeneic SCT within 6 months prior to leukocyte apheresis.

[0122] In some embodiments, the eligibility of subjects for treatment involving the administration of manipulated cells is determined prior to leukocyte apheresis. In some embodiments, subjects prior to leukocyte apheresis have sufficient vascular access for leukocyte apheresis. In some embodiments, subjects prior to leukocyte apheresis have sufficient organ function. In some embodiments, sufficient organ function is, among several factors, 1.0 × 10⁶ without growth factor support within 7 days of eligibility determination. 9 Neutrophil absolute count (ANC) of more than 10 cells / L; 50 × 10⁶ cells without transfusion assistance within 7 days of eligibility determination. 9 Platelet count greater than 100 cells / L; calculated creatinine clearance rate greater than 45 mL / min (CrCl, Cockcroft-Gault formula); aspartate aminotransferase (AST) level 2.5 times the upper limit of normal (ULN) or less than 2.5 times the ULN; alanine aminotransferase (ALT) level 2.5 times the ULN or less than 2.5 times the ULN; total bilirubin level less than 1.5 times the ULN; direct bilirubin level less than 1.5 times the ULN in cases of Gilbert's syndrome or lymphomatous hepatic infiltration; adequate lung function, e.g., CTCAE grade 1 or less than CTCAE grade 1 dyspnea and oxygen saturation (SaO2 higher than 92%) in the atmosphere; adequate cardiac function, e.g., echocardiography (ECHO) or multiple uptake gated echocardiography performed within 30 days of eligibility assessment. This is indicated by a left ventricular ejection fraction (LVEF) greater than 40% or 40% at the time of evaluation by acquisition (MUGA) scan; sufficient organ function to receive lymphocyte depletion (LD) chemotherapy; or any combination of the above.

[0123] In certain embodiments, subjects are administered lymphocyte depletion chemotherapy prior to the administration of a dose of CD19-targeted manipulated CAR T cells, or are undergoing lymphocyte depletion chemotherapy. Lymphocyte depletion can improve CAR T cell engraftment and activity by reducing homeostatic cytokines, CD4+CD25+ regulatory T cells, increasing SDF-1 in the bone marrow microenvironment, and stimulating 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). Furthermore, LD chemotherapy can further reduce the target tumor burden, potentially lowering the risk and severity of cytokine release syndrome (CRS).

[0124] Accordingly, in some embodiments, the method includes administering a pre-treatment agent, such as a lymphocyte depletion agent or a chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, to the subject prior to administration of the manipulated cells. For example, the subject may be administered the pre-treatment agent at least two days before administration of the manipulated cells, for example, at least three, four, five, six, seven, eight, or nine days prior. In some embodiments, the subject is administered the pre-treatment agent within nine days prior to administration of the manipulated cells, for example, within eight, seven, six, five, four, three, or two days.

[0125] In some embodiments, subjects are pre-treated with cyclophosphamide at a dose of 20 mg / kg to 100 mg / kg of body weight or approximately 20 mg / kg to 100 mg / kg of body weight, for example, 40 mg / kg to 80 mg / kg or approximately 40 mg / kg to 80 mg / kg. In some aspects, subjects are pre-treated with 60 mg / kg or approximately 60 mg / kg of cyclophosphamide, or are administered 60 mg / kg or approximately 60 mg / kg of cyclophosphamide. In some embodiments, cyclophosphamide may be administered as a single dose or as multiple doses, for example, daily, every other day, or every three days. In some embodiments, cyclophosphamide is administered once daily for one or two days. In some embodiments, if the lymphocyte depletion agent contains cyclophosphamide, subjects receive 100 mg / m² of cyclophosphamide. 2 ~500 mg / m 2 Alternatively, approximately 100 mg / m² 2 ~500 mg / m 2 Target body surface area, e.g., 200 mg / m² 2 ~400 mg / m 2 Alternatively, approximately 200 mg / m² 2 ~400 mg / m 2 or 250 mg / m² 2 ~350 mg / m 2 The drug is administered at doses (including both extreme values). In some cases, the subject is given approximately 100 mg / m². 2 Cyclophosphamide is administered. In some cases, the subject receives approximately 150 mg / m². 2 Cyclophosphamide is administered. In some cases, the subject receives approximately 200 mg / m². 2 Cyclophosphamide is administered. In some cases, the subject receives approximately 250 mg / m². 2 Cyclophosphamide is administered. In some cases, the subject receives approximately 300 mg / m². 2Cyclophosphamide is administered. In some embodiments, cyclophosphamide may be administered as a single dose or as multiple doses, for example, daily, every other day, or every three days. In some embodiments, cyclophosphamide is administered daily, for example, for 1 to 5 days, or for example, for 3 to 5 days. In some cases, the subject receives approximately 300 mg / m² before the initiation of cell therapy. 2 The target body surface area is administered with cyclophosphamide daily for 3 days. In some embodiments, the subject receives a total dose of 300 mg / m² before the initiation of cell therapy. 2 , 400 mg / m² 2 500 mg / m² 2 , 600 mg / m² 2 700 mg / m² 2 , 800 mg / m² 2 900 mg / m² 2 , 1000 mg / m² 2 , 1200 mg / m² 2 , 1500 mg / m² 2 , 1800 mg / m² 2 , 2000 mg / m² 2 , 2500 mg / m² 2 , 2700 mg / m² 2 , 3000 mg / m² 2 , 3300 mg / m² 2 , 3600 mg / m² 2 , 4000 mg / m² 2 Alternatively, 5000 mg / m² 2 , or approximately 300 mg / m² 2 , 400 mg / m² 2 500 mg / m² 2 , 600 mg / m² 2 700 mg / m² 2 , 800 mg / m² 2 900 mg / m² 2 , 1000 mg / m² 2 , 1200 mg / m² 2 , 1500 mg / m² 2 , 1800 mg / m² 2 , 2000 mg / m² 2 , 2500 mg / m² 2 , 2700 mg / m² 2, 3000 mg / m² 2 , 3300 mg / m² 2 , 3600 mg / m² 2 , 4000 mg / m² 2 Alternatively, 5000 mg / m² 2 Alternatively, cyclophosphamide is administered within the range defined by any of the aforementioned methods.

[0126] In some embodiments, if the lymphocyte depletion agent contains fludarabine, the target is given fludarabine at a dose of 1 mg / m². 2 Alternatively, approximately 1 mg / m² 2 From 100 mg / m² 2 Alternatively, 100 mg / m² 2 For example, 10 mg / m² 2 Alternatively, approximately 10 mg / m² 2 From 75 mg / m² 2 Alternatively, approximately 75 mg / m² 2 , 15 mg / m² 2 Alternatively, approximately 15 mg / m² 2 From 50 mg / m² 2 Alternatively, approximately 50 mg / m² 2 , 20 mg / m² 2 Alternatively, approximately 20 mg / m² 2 From 40 mg / m² 2 Alternatively, approximately 40 mg / m² 2 , 24 mg / m² 2 Alternatively, approximately 24 mg / m² 2 Alternatively, 24 mg / m² 2 From 35 mg / m² 2 Alternatively, approximately 35 mg / m² 2 The drug is administered at doses (including both extreme values). In some cases, the subject is given 10 mg / m². 2 or 10 mg / m² 2 Or approximately 10 mg / m² 2 Fludarabine is administered. In some cases, the subject is given 15 mg / m². 2 Or approximately 15 mg / m² 2 Fludarabine is administered. In some cases, the subject is given 20 mg / m². 2 Or approximately 20 mg / m² 2Fludarabine is administered. In some cases, the subject is given 25 mg / m². 2 Or approximately 25 mg / m² 2 Fludarabine is administered. In some cases, the subject is given 30 mg / m². 2 Or approximately 30 mg / m² 2 Fludarabine is administered. In some embodiments, fludarabine may be administered as a single dose or as multiple doses, for example, daily, every other day, or every three days. In some embodiments, fludarabine is administered daily, for example, for 1 to 5 days, or for example, for 3 to 5 days. In some cases, the subject is given 30 mg / m² before the start of cell therapy. 2 Or approximately 30 mg / m² 2 Fludarabine is administered daily for 3 days, equal to the target body surface area. In some embodiments, the subject receives a total dose of 10 mg / m² before the initiation of cell therapy. 2 , 20 mg / m² 2 , 25 mg / m² 2 , 30 mg / m² 2 40 mg / m² 2 50 mg / m² 2 , 60 mg / m² 2 70 mg / m² 2 , 80 mg / m² 2 90 mg / m² 2 , 100 mg / m² 2 , 120 mg / m² 2 , 150 mg / m² 2 , 180 mg / m² 2 , 200 mg / m² 2 , 250 mg / m² 2 , 270 mg / m² 2 , 300 mg / m² 2 , 330 mg / m² 2 , 360 mg / m² 2 , 400 mg / m² 2 Alternatively, 500 mg / m² 2 , or approximately 10 mg / m² 2 , 20 mg / m² 2 , 25 mg / m² 2 , 30 mg / m² 2 40 mg / m² 2 50 mg / m² 2, 60 mg / m² 2 70 mg / m² 2 , 80 mg / m² 2 90 mg / m² 2 , 100 mg / m² 2 , 120 mg / m² 2 , 150 mg / m² 2 , 180 mg / m² 2 , 200 mg / m² 2 , 250 mg / m² 2 , 270 mg / m² 2 , 300 mg / m² 2 , 330 mg / m² 2 , 360 mg / m² 2 , 400 mg / m² 2 Alternatively, 500 mg / m² 2 Alternatively, cyclophosphamide is administered within the range defined by any of the aforementioned methods.

[0127] In some embodiments, the lymphocyte depletion agent comprises a single drug, such as cyclophosphamide or fludarabine. In some embodiments, the subject is administered cyclophosphamide alone without fludarabine or other lymphocyte depletion agents. In some embodiments, prior to administration, the subject receives 200-400 mg / m² 2 Target body surface area or approximately 200-400 mg / m² 2 Target body surface area, 300 mg / m² (optional). 2 Or approximately 300 mg / m² 2 The subjects receive lymphocyte depletion therapy, including daily administration of cyclophosphamide for 2-4 days. In some embodiments, the subjects are administered fludarabine alone, without, for example, cyclophosphamide or other lymphocyte depletion agents. In some embodiments, prior to administration, the subjects are given 20-40 mg / m² 2 Target body surface area or approximately 20-40 mg / m² 2 Target body surface area, 30 mg / m² (optional) 2 Or approximately 30 mg / m² 2 They are receiving lymphocyte depletion therapy, which includes daily administration of fludarabine for 2 to 4 days.

[0128] In some embodiments, lymphocyte depletion agents include combinations of drugs, for example, a combination of cyclophosphamide and fludarabine. Thus, the drug combination may include any dose or administration schedule, for example, cyclophosphamide in the above and any dose or administration schedule, for example, fludarabine in the above. For example, in some aspects, the subject may receive 60 mg / kg or about 60 mg / kg (about 2 g / m²) before the first dose or subsequent dose. 2 ) Cyclophosphamide and 25 mg / m² in 3-5 doses 2 Fludarabine is administered. In some cases, subjects are given fludarabine (30 mg / m²) before cell administration. 2 (3 days at a rate of / day) and cyclophosphamide (300 mg / m²) 2 (flu / cy) is administered intravenously concurrently for 3 days at a rate of / day. In some embodiments, subjects are administered a single or multiple dose of a reduced, delayed, or reduced dose of a lymphocyte depletion agent.

[0129] In some embodiments, after cell collection from the subject and before administration of lymphocyte depletion (LD) chemotherapy, the subject may receive bridging therapy for disease control. Various arbitrary therapies may be administered as part of bridging therapy based on the judgment of those skilled in the art to address the specific disease or condition, based on factors such as the patient's age, the severity or extent of the disease, the possibility of side effects, the timing of administration before LD chemotherapy, previous therapies, and other factors. Exemplary therapies that may be administered as bridging therapy before lymphocyte depletion therapy include, but are not limited to, corticosteroids, vincristine, cyclophosphamide, rituximab, dexamethasone, prednisone, lenalidomide, gemcitabine, oxaliplatin, brentuximab vedotin, ibrutinib, methotrexate, cytosine arabinoside, cytarabine, bendamustine, or any combination of any of the aforementioned.

[0130] In some embodiments, subjects are premedicated, for example, to minimize the risk of acute fluid reactions. In some aspects, premedication includes administering analgesics and / or antihistamines. In some embodiments, premedication includes administering acetaminophen and / or diphenhydramine or another H1-antihistamine. In some embodiments, patients are administered acetaminophen (e.g., 650 mg orally) and diphenhydramine (e.g., 25-50 mg, IV or orally) or another H1-antihistamine 30-60 minutes or approximately 30-60 minutes before the cell therapy procedure.

[0131] In some embodiments, the subjects are at least 18 years of age. In some embodiments of the method of provision, the subjects are human subjects.

[0132] A. Medication In some embodiments, a certain dose of the manipulated cells is administered to the subject according to the method provided and / or in the manufactured article or composition provided. In some embodiments, the size or timing of the dose is determined as a function of the specific disease or condition of the subject. In some cases, the size or timing of the dose for a particular disease in light of the description provided may be determined empirically.

[0133] In any of the embodiments provided, the dose of T cells, for example engineered T cells expressing recombinant receptors, comprises CD3+ T cells, CD4+ T cells, CD8+ T cells, or CD4+ T cells and CD8+ T cells, or is enriched with CD3+ T cells, CD4+ T cells, CD8+ T cells, or CD4+ T cells and CD8+ T cells, or comprises a cell composition or cell population enriched with CD3+ T cells, CD4+ T cells, CD8+ T cells, or CD4+ T cells and CD8+ T cells. In any of such embodiments, more than 70%, 75%, 80%, 85%, 90%, 95%, or 98% of the dose of T cells, or about 70%, 75%, 80%, 85%, 90%, 95%, or more than 98% of the cells, are CD3+ T cells, CD4+ T cells, CD8+ T cells, or CD4+ T cells and CD8+ T cells. In some of the available embodiments, more than 70%, 75%, 80%, 85%, 90%, 95%, or 98% of the T cell dose, or about 70%, 75%, 80%, 85%, 90%, 95%, or more than 98% of the cells, are CD3+ T cells. In some of the available embodiments, the T cell dose includes both CD4+ and CD8+ cells. In some of the available embodiments, more than 70%, 75%, 80%, 85%, 90%, 95%, or 98% of the T cell dose, or about 70%, 75%, 80%, 85%, 90%, 95%, or more than 98% of the cells, are CD4+ T cells and CD8+ T cells.

[0134] In some embodiments, the cell dose is 0.1 × 10⁻⁶ 5 Or approximately 0.1 × 10 5 From individual CD19-directed CAR-operated cells / kilogram target body weight (cells / kg), 2 × 10 6 Or approximately 2 × 10 6 cells / kg, e.g., 0.1 × 10⁻⁶ 5 Or approximately 0.1 × 10 5 From cells / kg, 0.5 or approximately 0.5 × 10 5 Cells / kg, 0.5 or approximately 0.5 × 10⁻⁶ 5 From cells / kg, 1 × 10 5 Or approximately 1 x 10 5cells / kg, 1×10 5 Or approximately 1 x 10 5 From cells / kg, 1.5 × 10 5 Or approximately 1.5 x 10 5 cells / kg, 1.5×10 5 Or approximately 1.5 x 10 5 From cells / kg, 2 × 10 5 Or approximately 2 x 10 5 cells / kg, 2×10 5 Or approximately 2 x 10 5 From cells / kg, 2.5 × 10 5 Or approximately 2.5 x 10 5 cells / kg, 2.5×10 5 Or approximately 2.5 x 10 5 From cells / kg, 3 × 10 5 Or approximately 3 x 10 5 cells / kg, 3×10 5 Or approximately 3 x 10 5 From cells / kg, 3.5 × 10 5 Or approximately 3.5 x 10 5 cells / kg, 3.5×10 5 Or approximately 3.5 x 10 5 From cells / kg, 4 × 10 5 Or approximately 4 x 10 5 cells / kg, 4×10 5 Or approximately 4 x 10 5 From cells / kg, 4.5 × 10 5 Or approximately 4.5 x 10 5 cells / kg, 4.5×10 5 Or approximately 4.5 x 10 5 From cells / kg, 5 × 10 5 Or approximately 5 x 10 5 cells / kg, 5×10 5 Or approximately 5 x 10 5 From cells / kg, 5.5 × 10 5 Or approximately 5.5 x 10 5 cells / kg, 5.5×10 5 Or approximately 5.5 x 10 5 From cells / kg, 6 × 10 5 Or approximately 6 x 10 5 cells / kg, 6×10 5 Or approximately 6 x 105 From cells / kg, 6.5 × 10 5 Or approximately 6.5 x 10 5 cells / kg, 6.5×10 5 Or approximately 6.5 x 10 5 From cells / kg, 7 × 10 5 Or approximately 7 x 10 5 cells / kg, 7×10 5 Or approximately 7 x 10 5 From cells / kg, 7.5 × 10 5 Or approximately 7.5 x 10 5 cells / kg, 7.5×10 5 Or approximately 7.5 x 10 5 From cells / kg, 8 × 10 5 Or approximately 8 x 10 5 cells / kg, or 8 × 10⁶ 5 Or approximately 8 x 10 5 From the number of such cells / kg, 10 × 10 5 Or approximately 10 x 10 5 It contains 10 cells / kg. In some embodiments, the dose of cells is 2 × 10 5 Less than 10⁴ CD19-directed CAR-operated cells / kilogram of target body weight (cells / kg), e.g., 3 × 10⁶ 5 Less than or approximately 3 x 10 5 Less than one cell / kg, 4 × 10 5 Less than or approximately 4 x 10 5 Less than one cell / kg, 5 × 10 5 Less than or approximately 5 x 10 5 Less than one cell / kg, 6 × 10 5 Less than or approximately 6 x 10 5 Less than one cell / kg, 7 × 10 5 Less than or approximately 7 x 10 5 Less than one cell / kg, 8 × 10 5 Less than or approximately 8 x 10 5 Less than one cell / kg, 9 × 10 5 Less than or approximately 9 x 10 5 Less than 1 cell / kg, 1 × 10⁻⁶ 6 Less than or equal to 1 x 10 6 Less than one cell / kg or 2 × 10⁶ cells 6 Less than or approximately 2 x 10 6Contains no more than 10 cells / kg. In some embodiments, the cell dose is at least 0.1 × 10⁶ 5 Or at least about 0.1 × 10⁻⁶ 5 Or 0.1 × 10 5 Or approximately 0.1 × 10 5 Individual CD19-directed CAR-operated cells / kilogram of target body weight (cells / kg), e.g., at least 0.2 × 10⁶ 5 Or at least about 0.2 × 10 5 Or 0.2 × 10 5 Or approximately 0.2 × 10 5 cells / kg, at least 0.3 × 10⁻⁶ 5 Or at least about 0.3 × 10 5 Or 0.3 × 10 5 Or approximately 0.3 × 10 5 cells / kg, at least 0.4 × 10⁻⁶ 5 Or at least about 0.4 × 10 5 Or 0.4 × 10 5 Or approximately 0.4 × 10 5 cells / kg, at least 0.5 × 10⁻⁶ 5 Or at least about 0.5 × 10 5 Or 0.5 × 10 5 Or approximately 0.5 × 10 5 cells / kg, at least 0.6 × 10⁻⁶ 5 Or at least about 0.6 × 10 5 Or 0.6 × 10 5 Or approximately 0.6 × 10 5 cells / kg, at least 0.7 × 10⁻⁶ 5 Or at least about 0.7 × 10 5 Or 0.7 × 10 5 Or approximately 0.7 × 10 5 cells / kg, at least 0.8 × 10⁻⁶ 5 Or at least about 0.8 × 10 5 Or 0.8 × 10 5 Or approximately 0.8 × 10 5 cells / kg, at least 0.9 × 10⁻⁶ 5 Or at least about 0.9 × 10 5 Or 0.9 × 10 5 Or approximately 0.9 × 105 cells / kg, at least 0.1 × 10⁻⁶ 6 Or at least about 0.1 × 10⁻⁶ 6 Or 0.1 × 10 6 Or approximately 0.1 × 10 6 cells / kg or at least 0.2 × 10⁻⁶ 6 Or at least about 0.2 × 10 6 Or 0.2 × 10 6 Or approximately 0.2 × 10 6 The number of cells per kg is the number of living cells expressing CD19-directed CARs, such as living T cells, such as living CD3+ cells. In some embodiments, the number of such cells is the number of living cells expressing CD19-directed CARs, such as living T cells, such as living CD3+ cells.

[0135] In certain embodiments, the cell population or individual subtype of cell populations in the subject ranges from 100,000 or about 100,000 to 100 billion or about 100 billion cells and / or the amount of such cells per kilogram of the subject's body weight, for example, from 100,000 or about 100,000 to 50 billion or about 50 billion cells (for example, 5 million or about 5 million cells, 25 million or about 25 million cells, 500 million or about 500 million cells, 1 billion or about 1 billion cells, 5 billion or about 5 billion cells, 20 billion or about 20 billion cells). , 30 billion or approximately 30 billion cells, 40 billion or approximately 40 billion cells, or a range defined by any two of the aforementioned values), 1 million or approximately 1 million to 50 billion or approximately 50 billion cells (for example, 5 million or approximately 5 million cells, 25 million or approximately 25 million cells, 500 million or approximately 500 million cells, 1 billion or approximately 1 billion cells, 5 billion or approximately 5 billion cells, 20 billion or approximately 20 billion cells, 30 billion or approximately 30 billion cells, 40 billion or approximately 40 billion Cells (or a range defined by any two of the aforementioned values), for example, from 10 million or approximately 10 million to 100 billion or approximately 100 billion cells (for example, 20 million or approximately 20 million cells, 30 million or approximately 30 million cells, 40 million or approximately 40 million cells, 60 million or approximately 60 million cells, 70 million or approximately 70 million cells, 80 million or approximately 80 million cells, 90 million or approximately 90 million cells, 10 billion or approximately 10 billion cells, and 25 billion cells). or approximately 25 billion cells, 50 billion or approximately 50 billion cells, 75 billion or approximately 75 billion cells, 90 billion or approximately 90 billion cells, or a range defined by any two of the aforementioned values), and in some cases, from 100 million or approximately 100 million to 50 billion or approximately 50 billion cells (for example, 120 million or approximately 120 million cells, 250 million or approximately 250 million cells, 350 million or approximately 350 million cells, 650 million or approximately 650 million cells,The drug is administered in a number of cells (800 million or approximately 800 million cells, 900 million or approximately 900 million cells, 3 billion or approximately 3 billion cells, 30 billion or approximately 30 billion cells, 45 billion or approximately 45 billion cells) or any value in between these ranges and / or any value per kilogram of the subject's body weight. The dosage may vary depending on the disease or disorder and / or attributes specific to the patient and / or other treatment. In some embodiments, such value indicates the number of recombinant receptor-expressing cells; in other embodiments, it indicates the number of T cells or total cells in the administered composition. In some embodiments, the number of cells is the number of such cells that are living cells.

[0136] In some embodiments, the cell dose is a constant or fixed dose of cells such that the cell dose is independent of or not based on the body surface area or body weight of the subject.

[0137] In some embodiments, the dose of genetically engineered cells is 1 × 10⁻⁶ 5 Or approximately 1 x 10 5 From, 1 x 10 8 Or approximately 1 x 10 8 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 5 Or approximately 1 x 10 5 From, 0.8 × 10 8 Or approximately 0.8 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 5 Or approximately 1 x 10 5 From, 0.6 × 10 8 Or approximately 0.6 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 5 Or approximately 1 x 10 5 From, 0.4 × 10 8 Or approximately 0.4 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 5 Or approximately 1 x 10 5 From, 0.2 × 10 8 Or approximately 0.2 × 10 8Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 5 Or approximately 1 x 10 5 From, 1.0 × 10 7 Or approximately 1.0 × 10 7 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 5 Or approximately 1 x 10 5 From, 0.8 × 10 7 Or approximately 0.8 × 10 7 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 5 Or approximately 1 x 10 5 From, 0.6 × 10 7 Or approximately 0.6 × 10 7 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 5 Or approximately 1 x 10 5 From, 0.4 × 10 7 Or approximately 0.4 × 10 7 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 5 Or approximately 1 x 10 5 From, 0.2 × 10 7 Or approximately 0.2 × 10 7 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 5 Or approximately 1 x 10 5 From, 1.0 × 10 6 Or approximately 1.0 × 10 6 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 6 Or approximately 1 x 10 6 From, 1.0 × 10 8 Or approximately 1.0 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 6 Or approximately 1 x 10 6 From, 0.8 × 10 8 Or approximately 0.8 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 6 Or approximately 1 x 10 6 From, 0.6 × 10 8 Or approximately 0.6 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 6 Or approximately 1 x 10 6From, 0.4 × 10 8 Or approximately 0.4 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 6 Or approximately 1 x 10 6 From, 0.2 × 10 8 Or approximately 0.2 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 6 Or approximately 1 x 10 6 From, 1.0 × 10 7 Or approximately 1.0 × 10 7 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 6 Or approximately 1 x 10 6 From, 0.8 × 10 7 Or approximately 0.8 × 10 7 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 6 Or approximately 1 x 10 6 From, 0.6 × 10 7 Or approximately 0.6 × 10 7 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 6 Or approximately 1 x 10 6 From, 0.4 × 10 7 Or approximately 0.4 × 10 7 Individual whole CD19-directed CAR-expressing T cells, 1 × 10⁶ 6 Or approximately 1 x 10 6 From, 0.2 × 10 7 Or approximately 0.2 × 10 7 5 × 10¹ whole CD19-directed CAR-expressing T cells 6 Or approximately 5 x 10 6 From, 1.0 × 10 8 Or approximately 1.0 × 10 8 5 × 10¹ whole CD19-directed CAR-expressing T cells 6 Or approximately 5 x 10 6 From, 0.8 × 10 8 Or approximately 0.8 × 10 8 5 × 10¹ whole CD19-directed CAR-expressing T cells 6 Or approximately 5 x 10 6 From, 0.6 × 10 8 Or approximately 0.6 × 10 85 × 10¹ whole CD19-directed CAR-expressing T cells 6 Or approximately 5 x 10 6 From, 0.4 × 10 8 Or approximately 0.4 × 10 8 5 × 10¹ whole CD19-directed CAR-expressing T cells 6 Or approximately 5 x 10 6 From, 0.2 × 10 8 Or approximately 0.2 × 10 8 5 × 10¹ whole CD19-directed CAR-expressing T cells 6 Or approximately 5 x 10 6 From, 1.0 × 10 7 Or approximately 1.0 × 10 7 5 × 10¹ whole CD19-directed CAR-expressing T cells 6 Or approximately 5 x 10 6 From, 0.8 × 10 7 Or approximately 0.8 × 10 7 5 × 10¹ whole CD19-directed CAR-expressing T cells 6 Or approximately 5 x 10 6 From, 0.6 × 10 7 Or approximately 0.6 × 10 7 Individual whole CD19-directed CAR-expressing T cells, 10 × 10 6 Or approximately 10 x 10 6 From, 1.0 × 10 8 Or approximately 1.0 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 10 × 10 6 Or approximately 10 x 10 6 From, 0.9 × 10 8 Or approximately 0.9 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 10 × 10 6 Or approximately 10 x 10 6 From, 0.8 × 10 8 Or approximately 0.8 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 10 × 10 6 Or approximately 10 x 10 6 From, 0.7 × 10 8 Or approximately 0.7 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 10 × 10 6 Or approximately 10 x 106 From, 0.6 × 10 8 Or approximately 0.6 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 10 × 10 6 Or approximately 10 x 10 6 From, 0.5 × 10 8 Or approximately 0.5 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 10 × 10 6 Or approximately 10 x 10 6 From, 0.4 × 10 8 Or approximately 0.4 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 10 × 10 6 Or approximately 10 x 10 6 From, 0.3 × 10 8 Or approximately 0.3 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 10 × 10 6 Or approximately 10 x 10 6 From, 0.2 × 10 8 Or approximately 0.2 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 10 × 10 6 Or approximately 10 x 10 6 From, 15×10 6 Or approximately 15 x 10 6 Individual whole CD19-directed CAR-expressing T cells, 15 × 10⁶ 6 Or approximately 15 x 10 6 From, 1.0 × 10 8 Or approximately 1.0 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 15 × 10⁶ 6 Or approximately 15 x 10 6 From, 0.9 × 10 8 Or approximately 0.9 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 15 × 10⁶ 6 Or approximately 15 x 10 6 From, 0.8 × 10 8 Or approximately 0.8 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 15 × 10⁶ 6 Or approximately 15 x 10 6 From, 0.7 × 10 8 Or approximately 0.7 × 108 Individual whole CD19-directed CAR-expressing T cells, 15 × 10⁶ 6 Or approximately 15 x 10 6 From, 0.6 × 10 8 Or approximately 0.6 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 15 × 10⁶ 6 Or approximately 15 x 10 6 From, 0.5 × 10 8 Or approximately 0.5 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 15 × 10⁶ 6 Or approximately 15 x 10 6 From, 0.4 × 10 8 Or approximately 0.4 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 15 × 10⁶ 6 Or approximately 15 x 10 6 From, 0.3 × 10 8 Or approximately 0.3 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 15 × 10⁶ 6 Or approximately 15 x 10 6 From, 0.2 × 10 8 Or approximately 0.2 × 10 8 20 × 10¹ whole CD19-directed CAR-expressing T cells 6 Or approximately 20 x 10 6 From, 1.0 × 10 8 Or approximately 1.0 × 10 8 20 × 10¹ whole CD19-directed CAR-expressing T cells 6 Or approximately 20 x 10 6 From, 0.9 × 10 8 Or approximately 0.9 × 10 8 20 × 10¹ whole CD19-directed CAR-expressing T cells 6 Or approximately 20 x 10 6 From, 0.8 × 10 8 Or approximately 0.8 × 10 8 20 × 10¹ whole CD19-directed CAR-expressing T cells 6 Or approximately 20 x 10 6 From, 0.7 × 10 8 Or approximately 0.7 × 10 8 20 × 10¹ whole CD19-directed CAR-expressing T cells 6or about 20×10 6 to 0.6×10 8 or about 0.6×10 8 total CD19-directed CAR-expressing T cells, 20×10 6 or about 20×10 6 to 0.5×10 8 or about 0.5×10 8 total CD19-directed CAR-expressing T cells, 20×10 6 or about 20×10 6 to 0.4×10 8 or about 0.4×10 8 total CD19-directed CAR-expressing T cells, 20×10 6 or about 20×10 6 to 0.3×10 8 or about 0.3×10 8 total CD19-directed CAR-expressing T cells, 20×10 6 or about 20×10 6 to 25×10 6 or about 25×10 6 total CD19-directed CAR-expressing T cells, 25×10 6 or about 25×10 6 to 1.0×10 8 or about 1.0×10 8 total CD19-directed CAR-expressing T cells, 25×10 6 or about 25×10 6 to 0.9×10 8 or about 0.9×10 8 total CD19-directed CAR-expressing T cells, 25×10 6 or about 25×10 6 to 0.8×10 8 or about 0.8×10 8 total CD19-directed CAR-expressing T cells, 25×10 6 or about 25×10 6 to 0.7×10 8 or about 0.7×10 8 total CD19-directed CAR-expressing T cells, 25×10 6 or about 25×10 6 to 0.6×10 8Or approximately 0.6 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 25 × 10⁶ 6 Or approximately 25 x 10 6 From, 0.5 × 10 8 Or approximately 0.5 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 25 × 10⁶ 6 Or approximately 25 x 10 6 From, 0.4 × 10 8 Or approximately 0.4 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 25 × 10⁶ 6 Or approximately 25 x 10 6 From, 0.3 × 10 8 Or approximately 0.3 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 30 × 10⁶ 6 Or approximately 30 x 10 6 From, 1.0 × 10 8 Or approximately 1.0 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 30 × 10⁶ 6 Or approximately 30 x 10 6 From, 0.9 × 10 8 Or approximately 0.9 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 30 × 10⁶ 6 Or approximately 30 x 10 6 From, 0.8 × 10 8 Or approximately 0.8 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 30 × 10⁶ 6 Or approximately 30 x 10 6 From, 0.7 × 10 8 Or approximately 0.7 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 30 × 10⁶ 6 Or approximately 30 x 10 6 From, 0.6 × 10 8 Or approximately 0.6 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 30 × 10⁶ 6 Or approximately 30 x 10 6 From, 0.5 × 10 8 Or approximately 0.5 × 10 8Individual total CD19-directed CAR-expressing T cells, 30×10 6 or approximately 30×10 6 to 0.4×10 8 or approximately 0.4×10 8 Individual total CD19-directed CAR-expressing T cells, 30×10 6 or approximately 30×10 6 to 35×10 6 or approximately 35×10 6 Individual total CD19-directed CAR-expressing T cells, 35×10 6 or approximately 35×10 6 to 1.0×10 8 or approximately 1.0×10 8 Individual total CD19-directed CAR-expressing T cells, 35×10 6 or approximately 35×10 6 to 0.9×10 8 or approximately 0.9×10 8 Individual total CD19-directed CAR-expressing T cells, 35×10 6 or approximately 35×10 6 to 0.8×10 8 or approximately 0.8×10 8 Individual total CD19-directed CAR-expressing T cells, approximately 35×10 6 or approximately 35×10 6 to 0.7×10 8 or approximately 0.7×10 8 Individual total CD19-directed CAR-expressing T cells, 35×10 6 or approximately 35×10 6 to 0.6×10 8 or approximately 0.6×10 8 Individual total CD19-directed CAR-expressing T cells, 35×10 6 or approximately 35×10 6 to 0.5×10 8 or approximately 0.5×10 8 Individual total CD19-directed CAR-expressing T cells, 35×10 6 or approximately 35×10 6 to 0.4×10 8 or approximately 0.4×10 8 Individual total CD19-directed CAR-expressing T cells, 40×10 6Or approximately 40 x 10 6 From, 1.0 × 10 8 Or approximately 1.0 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 40 × 10⁶ 6 Or approximately 40 x 10 6 From, 0.9 × 10 8 Or approximately 0.9 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 40 × 10⁶ 6 Or approximately 40 x 10 6 From, 0.8 × 10 8 Or approximately 0.8 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 40 × 10⁶ 6 Or approximately 40 x 10 6 From, 0.7 × 10 8 Or approximately 0.7 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 40 × 10⁶ 6 Or approximately 40 x 10 6 From, 0.6 × 10 8 Or approximately 0.6 × 10 8 Individual whole CD19-directed CAR-expressing T cells, 40 × 10⁶ 6 Or approximately 40 x 10 6 From, 0.5 × 10 8 Or approximately 0.5 × 10 8 Individual whole CD19-directed CAR-expressing T cells, or 40 × 10⁶ 6 Or approximately 40 x 10 6 From, 45×10 6 Or approximately 45 x 10 6 It contains all CD19-directed CAR-expressing T cells. In some embodiments, the number of cells is the number of such cells, which are living cells, for example, living T cells.

[0138] In some embodiments, the dose of genetically engineered cells is at least 1 × 10⁶ 5 Or at least about 1 × 10 5 Individual CD19-directed CAR-expressing T cells, at least 2.5 × 10⁶ 5 Or at least about 2.5 × 10 5 Individual CD19-directed CAR-expressing T cells, at least 5 × 10⁶ 5Or at least about 5 x 10 5 Individual CD19-directed CAR-expressing T cells, at least 1 × 10⁶ 6 Or at least about 1 × 10 6 Individual CD19-directed CAR-expressing T cells, at least 2.5 × 10⁶ 6 Or at least about 2.5 × 10 6 Individual CD19-directed CAR-expressing T cells, at least 5 × 10⁶ 6 Or at least about 5 x 10 6 Individual CD19-directed CAR-expressing T cells, at least 1 × 10⁶ 7 Or at least about 1 × 10 7 Individual CD19-directed CAR-expressing T cells, at least 2.5 × 10⁶ 7 Or at least about 2.5 × 10 7 individual CD19-directed CAR-expressing T cells or at least 5 × 10⁶ 7 Or at least about 5 x 10 7 It contains CD19-directed CAR-expressing T cells. In some embodiments, the number of cells is the number of such cells, which are living cells, for example, living T cells.

[0139] In some embodiments, the dose of genetically engineered cells is 1 × 10⁻⁶ 5 Less than one or approximately 1 x 10 5 Less than 10 CD19-directed CAR-expressing T cells, 2.5 × 10⁶ 5 Less than one or approximately 2.5 × 10 5 Less than 5 CD19-directed CAR-expressing T cells, 5 × 10⁶ 5 Less than one or approximately 5 x 10 5 Less than 1 CD19-directed CAR-expressing T cell, 1 × 10⁶ 6 Less than one or approximately 1 x 10 6 Less than 10 CD19-directed CAR-expressing T cells, 2.5 × 10⁶ 6 Less than one or approximately 2.5 × 10 6 Less than 5 CD19-directed CAR-expressing T cells, 5 × 10⁶ 6 Less than one or approximately 5 x 10 6 Less than 1 CD19-directed CAR-expressing T cell, 1 × 10⁶ 7 Less than one or approximately 1 x 10 7Less than 10 CD19-directed CAR-expressing T cells, 1.5 × 10⁶ 7 Less than one or approximately 1.5 × 10 7 Less than 10 CD19-directed CAR-expressing T cells, 2 × 10 7 Less than 10 or approximately 2 x 10 7 Less than 10 CD19-directed CAR-expressing T cells, 2.5 × 10⁶ 7 Less than one or approximately 2.5 × 10 7 Less than 3 CD19-directed CAR-expressing T cells, 3 × 10⁶ 7 Less than one or approximately 3 x 10 7 Less than 10 CD19-directed CAR-expressing T cells, 3.5 × 10⁶ 7 Less than one or approximately 3.5 × 10 7 Less than 10 CD19-directed CAR-expressing T cells, 4 × 10 7 Less than one or approximately 4 x 10 7 Less than 10 CD19-directed CAR-expressing T cells, 4.5 × 10⁶ 7 Less than one or approximately 4.5 × 10 7 Less than 5 CD19-directed CAR-expressing T cells or 5 × 10⁶ 7 Less than one or approximately 5 x 10 7 It contains fewer than 1 CD19-directed CAR-expressing T cells. In some embodiments, the number of cells is the number of such cells, which are living cells, e.g., living T cells.

[0140] In some embodiments, cell therapy is 1 × 10 5 Or approximately 1 x 10 5 From, 5 x 10 8 Or approximately 5 x 10 8 5 × 10¹ fully recombinant receptor-expressing cells or fully T cells 5 Or approximately 5 x 10 5 From, 1 x 10 7 Or approximately 1 x 10 7 10⁴ fully recombinant receptor-expressing cells or all T cells or 1 × 10⁶ 6 Or approximately 1 x 10 6 From, 1 x 10 7 Or approximately 1 x 10 7 The administration of a dose comprising the number of total recombinant receptor-expressing cells or total T cells (each including the values ​​at both ends) comprises 1 × 10⁶ cells. In some embodiments, the cell therapy comprises 1 × 10⁶ cells.5 Or approximately 1 x 10 5 From, 1 x 10 8 Or approximately 1 x 10 8 5 × 10¹ fully recombinant receptor-expressing cells or fully T cells 5 Or approximately 5 x 10 5 From, 1 x 10 8 Or approximately 1 x 10 8 1 x 10¹ fully recombinant receptor-expressing cells or all T cells 6 Or approximately 1 x 10 6 From, 50 x 10 6 Or approximately 50 x 10 6 5 × 10¹ fully recombinant receptor-expressing cells or fully T cells 6 Or approximately 5 x 10 6 From, 45×10 6 Or approximately 45 x 10 6 10⁴ fully recombinant receptor-expressing cells or all T cells, or 10 × 10⁶ 6 Or approximately 10 x 10 6 From, 25×10 6 Or approximately 25 x 10 6 The administration of a dose comprising the number of total recombinant receptor-expressing cells or total T cells (each including the values ​​at both ends) is included. In some embodiments, the cell therapy comprises at least 1 × 10⁶ cells. 5 Or at least about 1 × 10 5 A total number of fully recombinant receptor-expressing cells or total T cells, for example (such) at least 1 × 10⁶ 6 Or at least 1 × 10 6 , at least 1 × 10 7 Or at least about 1 × 10 7 , at least 1 × 10 8 Or at least about 1 × 10 8 The method includes administering a cell dose containing the number of such cells. In some embodiments, the number of cells is the number of such cells, which are living cells, for example, living T cells.

[0141] In some embodiments, this number is CD3 + CD8 +Or it relates to the total number of CD4+ and CD8+, and in some cases, recombinant receptor expression (e.g., CAR + This relates to the total number of cells. In some embodiments, the number of cells is the number of such cells that are living cells.

[0142] In some embodiments, cell therapy is 1 × 10 5 Or approximately 1 x 10 5 From, 1 x 10 8 Or approximately 1 x 10 8 individual CD3 + CD8 + Or CD4 + and CD8 + All T cells or CD3 + CD8 + Or CD4 + and CD8 + Recombinant receptor (e.g., CAR) expressing cells, 5 × 10 5 Or approximately 5 x 10 5 From, 5 x 10 7 Or approximately 5 x 10 7 individual CD3 + CD8 + Or CD4 + and CD8 + All T cells or CD3 + CD8 + Or CD4 + and CD8 + Recombinant receptor (e.g., CAR) expressing cells or 1 × 10 6 Or approximately 1 x 10 6 From, 2.5 × 10 7 Or approximately 2.5 x 10 7 individual CD3 + CD8 + Or CD4 + and CD8 + All T cells or CD3 + CD8 + Or CD4 + and CD8 + The administration of a dose comprising the number of recombinant receptor (e.g., CAR) expressing cells (each including the values ​​at both ends) is included. In some embodiments, the cell therapy is 1 × 10⁶5 Or approximately 1 x 10 5 From, 1 x 10 8 Or approximately 1 x 10 8 All CD3 + / CAR + CD8 + / CAR + Or CD4 + / CD8 + / CAR + cells, 5 x 10 5 Or approximately 5 x 10 5 From, 5 x 10 7 Or approximately 5 x 10 7 All CD3 + / CAR + CD8 + / CAR + Or CD4 + / CD8 + / CAR + Cells or 1 × 10 6 Or approximately 1 x 10 6 From, 2.5 × 10 7 Or approximately 2.5 x 10 7 All CD3 + / CAR + CD8 + / CAR + Or CD4 + / CD8 + / CAR + The administration of a dose including the number of cells (each including the values ​​at both ends). In some embodiments, the number of cells is the number of such cells that are living cells.

[0143] In some embodiments, the dose of genetically engineered cells is at least 0.1 × 10⁶ 7 Or at least about 0.1 × 10⁻⁶ 7 CD3+ / CAR + CD8 + / CAR + Or CD4 + / CD8 + / CAR + T cells, at least 0.5 × 10 7 Or at least about 0.5 × 10 7 CD3+ / CAR + CD8+ / CAR + Or CD4 + / CD8 + / CAR + T cells, at least 1.0 × 10⁶ 7 Or at least about 1.0 × 10 7 CD3+ / CAR + CD8 + / CAR + Or CD4 + / CD8 + / CAR + T cells, at least 2.5 × 10 7 Or at least about 2.5 × 10 7 CD3+ / CAR + CD8 + / CAR + Or CD4 + / CD8 + / CAR + T cells or at least 5 × 10⁻⁶ 7 Or at least about 5 x 10 7 CD3+ / CAR + CD8 + / CAR + Or CD4 + / CD8 + / CAR + Includes T cells. In some embodiments, the dose of genetically engineered cells is 0.1 × 10⁻⁶ 7 Less than one or approximately 0.1 × 10 7 Less than one CD3+ / CAR + CD8 + / CAR + Or CD4 + / CD8 + / CAR + T cells, 0.5 x 10 7 Less than one or approximately 0.5 × 10 7 Less than one CD3+ / CAR + CD8 + / CAR + Or CD4 + / CD8 + / CAR + T cells, 1.0×10 7 Less than one or approximately 1.0 × 10 7Less than one CD3+ / CAR + CD8 + / CAR + Or CD4 + / CD8 + / CAR + T cells, 2.5 x 10 7 Less than one or approximately 2.5 × 10 7 Less than one CD3+ / CAR + CD8 + / CAR + Or CD4 + / CD8 + / CAR + T cells, 5 x 10 7 Less than one or approximately 5 x 10 7 Less than one CD3+ / CAR + CD8 + / CAR + Or CD4 + / CD8 + / CAR + T cells, 7.5 x 10 7 Less than one or approximately 7.5 x 10 7 Less than one CD3+ / CAR + CD8 + / CAR + Or CD4 + / CD8 + / CAR + T cells, or 10 × 10 7 Less than one or approximately 10 x 10 7 Less than 10 x 10 7 Less than one or approximately 10 x 10 7 Less than one CD3+ / CAR + CD8 + / CAR + Or CD4 + / CD8 + / CAR + Includes T cells. In some embodiments, the dose of genetically engineered cells is 0.5 × 10⁻⁶ 7 Or approximately 0.5 × 10 7 CD3+ / CAR + CD8 + / CAR + or CD4 + / CD8 + / CAR +T cells, 1.0×10 7 Or approximately 1.0 × 10 7 CD3+ / CAR + CD8 + / CAR + or CD4 + / CD8 + / CAR + T cells, 1.5 x 10 7 Or approximately 1.5 × 10 7 CD3+ / CAR + CD8 + / CAR + or CD4 + / CD8 + / CAR + T cells, 2.0×10 7 Or approximately 2.0 × 10 7 CD3+ / CAR + CD8 + / CAR + or CD4 + / CD8 + / CAR + T cells, 2.5 x 10 7 Or approximately 2.5 × 10 7 CD3+ / CAR + CD8 + / CAR + or CD4 + / CD8 + / CAR + T cells, 3.0×10 7 Or approximately 3.0 × 10 7 CD3+ / CAR + CD8 + / CAR + or CD4 + / CD8 + / CAR + T cells, 3.5 x 10 7 Or approximately 3.5 × 10 7 CD3+ / CAR + CD8 + / CAR + or CD4 + / CD8 + / CAR + T cells, 4.0×10 7 Or approximately 4.0 × 10 7 CD3+ / CAR+ CD8 + / CAR + or CD4 + / CD8 + / CAR + T cells, 4.5 x 10 7 Or approximately 4.5 × 10 7 CD3+ / CAR + CD8 + / CAR + or CD4 + / CD8 + / CAR + T cells, 5 x 10 7 Or approximately 5 x 10 7 CD3+ / CAR + CD8 + / CAR + or CD4 + / CD8 + / CAR + This includes T cells. In some embodiments, the number of cells is the number of such cells that are living cells.

[0144] In some aspects, the dose of T cells is 0.5 × 10⁻⁶ 7 Or approximately 0.5 × 10 7 1.0 × 10⁶ recombinant receptor (e.g., CAR) expressing T cells 7 Or approximately 1.0 × 10 7 1.5 × 10⁶ recombinant receptor (e.g., CAR) expressing T cells 7 Or approximately 1.5 x 10 7 2.0 × 10⁶ recombinant receptor (e.g., CAR) expressing T cells 7 Or approximately 2.0 × 10 7 2.5 × 10⁶ recombinant receptor (e.g., CAR) expressing T cells 7 Or approximately 2.5 x 10 7 Individual recombinant receptor (e.g., CAR) expression CD8 + T cells, 3.0×10 7 Or approximately 3.0 x 10 7 3.5 × 10⁶ recombinant receptor (e.g., CAR) expressing T cells 7 Or approximately 3.5 x 10 7 4.0 × 10⁶ recombinant receptor (e.g., CAR) expressing T cells7 Or approximately 4.0 x 10 7 1 recombinant receptor (e.g., CAR) expressing T cells or 4.5 × 10⁶ 7 Or approximately 4.5 x 10 7 It contains recombinant receptor (e.g., CAR) expressing T cells. In some embodiments, the dose of T cells is 1 × 10⁶ 8 Or approximately 1 x 10 8 1 recombinant receptor (e.g., CAR) expressing T cells or 5 × 10 7 Or approximately 5 x 10 7 Individual recombinant receptor (e.g., CAR) expression CD8 + It contains T cells. In some embodiments, the dose of T cells is 1.5 × 10⁻⁶ 8 Or approximately 1.5 x 10 8 1 recombinant receptor (e.g., CAR) expressing T cells or 0.75 × 10⁶ 8 Alternatively, approximately 0.75 × 10 8 Individual recombinant receptor (e.g., CAR) expression CD8 + This includes T cells. In some embodiments, the number of cells is the number of such cells that are living cells.

[0145] In some embodiments, the dose of T cells is CD4 + T cells, CD8 + T cells or CD4 + and CD8 + Includes T cells.

[0146] In some embodiments, for example, when the subject is human, the dose of CD8+ T cells is, for example, 1 × 10⁶ in a dose containing CD4+ and CD8+ T cells. 6 Or approximately 1 x 10 6 From, 1 x 10 8 Or approximately 1 x 10 8 A total recombinant receptor (e.g., CAR) expressing CD8+ cell, e.g., 1 × 10⁶ 6 Or approximately 1 x 10 6 From, 1 x 10 8 Or approximately 1 x 10 8 A cell within a range of 1 × 10⁶ 6 , 2.5×10 6 , 5×106 , 7.5×10 6 , 1 x 10 7 , 2.5×10 7 , 5×10 7 Or 7.5 x 10 7 or the total number of such cells in a range between any two of the aforementioned values. In some embodiments, the patient is administered repeated doses, and each dose or the total dose may be within the range of any of the aforementioned values. In some embodiments, the dose of cells is 1 × 10⁻⁶ 6 Or approximately 1 x 10 6 From, 5 x 10 7 Or approximately 5 x 10 7 5 × 10¹ fully recombinant receptor-expressing CD8+ T cells 6 Or approximately 5 x 10 6 From, 2.5 × 10 7 Or approximately 2.5 × 10 7 10 × 10¹⁶ CD8+ T cells expressing all recombinant receptors 6 Or approximately 10 x 10 6 From, 2.5 × 10 7 Or approximately 2.5 × 10 7 The administration consists of a total number of fully recombinant receptor-expressing CD8+ T cells (each including the values ​​at both ends). In some embodiments, the number of cells is the number of such cells that are living cells.

[0147] In some embodiments, for example, when the subject is human, the dose of CD4+ T cells is, for example, 1 × 10⁶ in a dose containing both CD4+ and CD8+ T cells. 6 Or approximately 1 x 10 6 From, 1 x 10 8 Or approximately 1 x 10 8 A total recombinant receptor (e.g., CAR) expressing CD4+ cell, e.g., 1 × 10⁶ 6 Or approximately 1 x 10 6 From, 1 x 10 8 Or approximately 1 x 10 8 A cell within a range of 1 × 10⁶ 6 , 2.5×10 6 , 5×10 6 , 7.5×10 6 , 1 x 10 7 , 2.5×10 7 , 5×107 Or 7.5 x 10 7 or the total number of such cells in a range between any two of the aforementioned values. In some embodiments, the patient is administered repeated doses, and each dose or the total dose may be within the range of any of the aforementioned values. In some embodiments, the dose of cells is 1 × 10⁻⁶ 6 Or approximately 1 x 10 6 From, 5 x 10 7 Or approximately 5 x 10 7 5 × 10¹ fully recombinant receptor-expressing CD4+ T cells 6 Or approximately 5 x 10 6 From, 2.5 × 10 7 Or approximately 2.5 × 10 7 10 × 10¹⁶ CD4+ T cells expressing all recombinant receptors 6 Or approximately 10 x 10 6 From, 2.5 × 10 7 Or approximately 2.5 × 10 7 The administration consists of a total number of fully recombinant receptor-expressing CD4+ T cells (each including the values ​​at both ends). In some embodiments, the number of cells is the number of such cells that are living cells.

[0148] In some embodiments, for example, when the subject is human, the dose is approximately 5 × 10 8 Fewer fully recombinant receptor (e.g., CAR) expressing cells or T cells, e.g., 1 × 10⁶ 6 Or approximately 1 x 10 6 From, 1 x 10 8 Or approximately 1 x 10 8 A cell that is affected by a range of individuals, for example, 2 × 10 6 , 5×10 6 , 1 x 10 7 , 5×10 7 Or 1 x 10 8 , or approximately 2 x 10 6 , 5×10 6 , 1 x 10 7 , 5×10 7 Or 1 x 10 8 Alternatively, it includes all such cells in the range between any two of the aforementioned values. In some embodiments, the number of cells is the number of such cells that are living cells.

[0149] In some embodiments, the patient is administered repeated doses, and each dose or the total dose may be within any of the aforementioned values. 5 Or approximately 1 x 10 5 From, 1 x 10 8 Or approximately 1 x 10 8 1 x 10¹ fully recombinant receptor (e.g., CAR) expressing T cells or whole T cells 5 Or approximately 1 x 10 5 From, 0.5 × 10 8 Or approximately 0.5 × 10 8 1 x 10¹ fully recombinant receptor (e.g., CAR) expressing T cells or whole T cells 5 Or approximately 1 x 10 5 From, 0.5 × 10 8 Or approximately 0.5 × 10 8 5 × 10¹ fully recombinant receptor (e.g., CAR) expressing T cells or whole T cells 5 Or approximately 5 x 10 5 From, 5 x 10 7 Or approximately 5 x 10 7 Individual total recombinant receptor (e.g., CAR) expressing T cells or total T cells or 1 × 10⁶ 6 Or approximately 1 x 10 6 From, 1 x 10 7 Or approximately 1 x 10 7 This constitutes the administration of a total recombinant receptor (e.g., CAR) expressing T cells or total T cells (each including values ​​at both ends).

[0150] In some embodiments, the dose of T cells is CD4 + T cells, CD8 + T cells or CD4 + and CD8 + Includes T cells.

[0151] In some embodiments, the dose of cells, such as recombinant receptor-expressing T cells, is administered to the subject as a single dose, or only once within a period of 2 weeks, 1 month, 3 months, 6 months, 1 year, or longer.

[0152] In the context of adoptive cell therapy, the administration of a given “dose” includes administration as a single composition and / or a single continuous dose of the given amount or number of cells, for example, as a single injection or continuous infusion, and also includes administration of the given amount or number of cells as divided doses provided in multiple individual compositions or infusions, or as multiple compositions, over a specified period, for example, up to three days. Thus, in some contexts, the dose is a specified number of cells administered as a single or continuous dose, run or initiated at a single point in time. However, in some contexts, the dose is administered over a period of up to three days, for example, by repeated injections or infusions once daily for three or two days, or by repeated infusions throughout the day.

[0153] In certain embodiments, the number and / or concentration of cells indicates the number of recombinant receptor (e.g., CAR) expressing cells. In other embodiments, the number and / or concentration of cells indicates the number or concentration of T cells administered.

[0154] In some embodiments, the subject receives repeated doses, e.g., two or more doses or consecutive repeated doses of cells. In some embodiments, two doses are administered to the subject. In some embodiments, the subject receives consecutive doses, e.g., the 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, consecutive repeated doses are administered so that one or more additional doses are administered following the administration of the consecutive dose. In some embodiments, the number of cells administered to the subject in the additional doses is the same as or similar to the first dose and / or consecutive dose. In some embodiments, one or more additional doses are greater than the previous doses.

[0155] In some aspects, the dose size is determined based on one or more criteria, such as the response of the subject to prior treatment, e.g., chemotherapy; the disease burden of the subject, e.g., tumor cell volume, tumor mass, tumor size, or degree, extent, or type of metastasis; the stage of the disease; and / or the likelihood or incidence of the subject exhibiting a toxic outcome, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and / or a host immune response against the cells; and / or the recombinant receptor being administered.

[0156] In some aspects, the time between the initial dose and the subsequent dose is approximately 9 to 35 days, approximately 14 to 28 days, or 15 to 27 days. In some aspects, the subsequent dose is administered more than approximately 14 days but less than approximately 28 days after the initial dose. In some aspects, the time between the initial dose and the subsequent dose is approximately 21 days. In some aspects, one or more additional doses, e.g., subsequent doses, are administered following the administration of the subsequent dose. In some aspects, one or more additional subsequent doses are administered at least 14 days after the previous dose but less than approximately 28 days after the previous dose. In some aspects, the additional dose is administered less than approximately 14 days after the previous dose, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days after the previous dose. In some aspects, the dose is not administered more than approximately 14 days after the previous dose, and / or the dose is not administered more than approximately 28 days after the previous dose.

[0157] In some aspects, the dose of cells is generally large enough to be effective in reducing the disease burden.

[0158] In certain embodiments, the number and / or concentration of cells refers to the number of recombinant receptor (e.g., CAR) expressing cells. In other embodiments, the number and / or concentration of cells refers to the number or concentration of all cells, T cells, or peripheral blood mononuclear cells (PBMCs) administered.

[0159] In some embodiments, the method also comprises administering one or more additional doses of chimeric antigen receptor (CAR)-expressing cells and / or administering lymphocyte depletion therapy, and / or repeating one or more steps of the method. In some embodiments, the one or more additional doses are the same as the initial dose. In some embodiments, the one or more additional doses differ from the initial dose, for example, being greater than the initial dose, e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 times or more, or about 2, 3, 4, 5, 6, 7, 8, 9 or 10 times or more, or less than the initial dose, e.g., greater than, e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 times or more. In some embodiments, the administration of one or more additional doses is determined based on the subject's response to the initial treatment or any prior treatment, the subject's disease burden, e.g., tumor cell volume, tumor mass, tumor size, or degree, extent, or type of metastasis, disease stage, and / or the subject's likelihood or incidence of toxic outcomes, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and / or host immune response against cells, and / or the recombinant receptor administered.

[0160] B. Efficacy, efficacy, and survival In some embodiments, administration by the method provided effectively treats subjects despite their resistance to other treatments. 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 this 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 subjects treated according to this method achieve objective response (OR). In some embodiments, at least 50% or at least about 50%, at least 60% or at least about 60%, at least 70% or at least about 70%, at least 80% or at least about 80%, or at least 90% or at least about 90% of subjects treated according to this method achieve CR and / or objective response (OR). In some embodiments, criteria used to evaluate an effective treatment include 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).

[0161] 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), progression-free survival (PFS) and / or overall survival (OS) of 3 months, 6 months or 12 months or about 3 months, 6 months or 12 months or about 13 months or about 14 months; on average, subjects treated according to the methods show a median PFS or OS of 6 months, 12 months or 18 months or about 6 months, 12 months or 18 months; and / or subjects show PFS or OS of at least 6 months, 12 months, 18 months or longer after treatment, or at least about 6 months, 12 months, 18 months or longer.

[0162] In some embodiments, subjects treated according to the provided method exhibit 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 having a best overall response (BOR) of up to 12 months, up to 18 months, up to 24 months, up to 36 months, or longer.

[0163] In some contexts, the response rate in subjects, such as those with NHL, is based on the Lugano criteria (Cheson et al., (2014) JCO 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323-338; Cheson, BD (2015) Chin Clin Oncol 4(1):5). In some contexts, clinical, hematological, and / or molecular methods are used to assess the response. In some contexts, the response assessed using the Lugano criteria is accompanied, as appropriate, by the use of positron emission tomography (PET)-computed tomography (CT) and / or CT. Assessment with PET-CT may further include the use of fluorodeoxyglucose (FDG) for FDG-accumulating lymphoma. In some contexts, when PET-CT is used to assess the response in FDG-accumulating histology, a 5-point scale may be used. In some respects, the 5-point scale is based on the following criteria: 1, no uptake beyond background; 2, uptake ≤ mediastinum; 3, uptake > mediastinum but ≤ liver; 4, moderate uptake > liver; 5, markedly higher uptake than liver and / or new lesions; X, new uptake areas not likely to be associated with lymphoma.

[0164] In some contexts, complete response, as defined by the Lugano criteria, involves metabolic and radiological complete response at various measurable sites. In some contexts, these sites include lymph nodes and extra-lymphatic sites, where CR is described using PET-CT as a score of 1, 2, or 3 on a 5-point scale indicating the presence or absence of residual mass. In some contexts, in the Waldeyer's ring or extra-lymphatic sites with high physiological uptake or activation in the spleen or medullary region (e.g., using chemotherapy or bone marrow colony-stimulating factors), uptake may be greater than in the mediastinum and / or liver. In this situation, metabolic complete response can be inferred even if the tissue has high physiological uptake, provided that the uptake at the site of initial infiltration does not exceed that around normal tissue. In some aspects, response is evaluated using CT within the lymph nodes, in which case CR should indicate no extra-lymphatic disease sites, and the target lymph node / lymph node mass should have regressed to a maximum transverse diameter (LDi) of ≤1.5 cm. Further evaluation sites include the bone marrow, in which case PET-CT-based evaluation should show no signs of intramedullary FDG-accumulating disease, CT-based evaluation should show normal morphological structure, and if uncertain, it should be indicated as IHC-negative. Further evaluation sites may include organ hypertrophy, which should have regressed to normal. In some cases, unmeasured lesions and new lesions are evaluated, which should be absent in cases of complete response (Cheson et al., (2014) JCO 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323-338; Cheson, BD (2015) Chin Clin Oncol 4(1):5).

[0165] In some cases, a partial response (PR; also known in some cases as partial remission) as defined using the Lugano criteria involves metabolic and / or radiological partial responses at various measurable sites. In some cases, such sites include lymph nodes and extra-lymphatic sites, in which case a PR is described as a score of 4 or 5, with reduced uptake and a reduction in residual mass of any size compared to baseline, as measured by PET-CT. Intermediately, such findings may indicate a response to the disease. At the end of treatment, such findings may indicate residual lesions. In some cases, response is assessed using CT in the lymph nodes, in which case a PR is described as a ≥50% reduction in SPD in up to six measurable target lymph nodes and extra-lymphatic sites. If the lesion is too small to be measured by CT, 5 mm × 5 mm is assigned as the default value; if the lesion is no longer visible, this value is 0 mm × 0 mm; and for lymph nodes >5 mm × 5 mm but smaller than normal, the actual measurement is used for the calculation. Further evaluation sites include the bone marrow, in which case PET-CT-based evaluation should show residual uptake that is higher than normal intramedullary uptake but reduced compared to baseline (expanded uptake equivalent to acceptable chemotherapy response changes). In some cases, if persistent intramedullary lesional changes are observed in the presence of lymph node response, further evaluation by MRI or biopsy or interval scanning should be considered. In some cases, further evaluation of organ hypertrophy may be considered, in which case the spleen must be more than normal in length and have regressed by >50%. In some cases, unmeasured lesions and new lesions should be evaluated, which should be absent / normal, regressed and not enlarged in the case of PR. No response / stable disease (SD) or disease progression (PD) may also be measured using PET-CT and / or CT-based evaluation. (Cheson et al., (2014) JCO 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323-338; Cheson, BD (2015) Chin Clin Oncol 4(1):5).

[0166] In some respects, progression-free survival (PFS) is described as the length of time a subject lives with the disease, such as cancer, during and after treatment, without the disease worsening. In some respects, objective response (OR) is described as a measurable response. In some respects, objective response rate (ORR; also known in some cases as overall response rate) is described as the proportion of patients who achieve complete response (CR) or partial response (PR). In some respects, overall survival (OS) is described as the length of time a subject diagnosed with the disease, such as cancer, is still alive from either the date of diagnosis or the start of treatment. In some respects, event-free survival (EFS) is described as the length of time a subject remains free from specific complications or events after the completion of treatment for cancer, which was intended to prevent or delay such complications. Such events may include cancer relapse or specific symptoms, such as bone pain due to cancer spreading to the bone, or death.

[0167] In some embodiments, the measurement of duration of response (DOR) includes the time from the recording of tumor response to disease progression. In some embodiments, parameters for evaluating response may include sustained response, such as a response that persists for a certain period after the initiation of treatment. In some embodiments, sustained 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 the initiation of treatment. In some embodiments, the response is sustained for more than 3 months or more than 6 months.

[0168] In some contexts, the RECIST criteria are used to determine objective tumor reduction; in some contexts, objective tumor reduction in solid tumors. (Eisenhauer et al., European Journal of Cancer 45(2009)228-247). In some contexts, the RECIST criteria are used to determine objective tumor reduction against target lesions. In some cases, a complete response as determined using the RECIST criteria is described as the disappearance of all target lesions, and if there are pathological lymph nodes (target or non-target), this must have a short-axis reduction of <10 mm. In other contexts, a partial response as determined using the RECIST criteria is described as a reduction of at least 30% of the sum of diameters of target lesions relative to the baseline sum of diameters. In other contexts, disease progression (PD) is described as an increase of at least 20% of the sum of diameters of target lesions relative to the minimum sum of diameters at the time of study (including cases where the baseline sum of diameters is the minimum sum of diameters at the time of study). In addition to a relative increase of 20%, the sum of diameters must also show an absolute increase of at least 5 mm (in some cases, the appearance of one or more new lesions is also considered progression). In other cases, stable disease (SD) is described as neither a reduction sufficient to be considered partial response (PR) nor an increase sufficient to be considered progressive disease (PD), relative to the minimum sum of diameters at the time of examination.

[0169] In some embodiments, survival rates for subjects with follicular lymphoma (FL) are generally based on scoring systems developed by the Italian Lymphoma Intergroup (ILI) and / or the International Follicular Lymphoma Prognostic Factor Project (IFLPFP), as described above (Luminari et al., (2012) Rev. Brad. Hematol. Hemoter., 34:54-59). In some embodiments, the severity of the disease, e.g., FL, can be assessed by the Ann Arbor staging system, tumor volume, large mass lesions, and the number of nodal or extra-lymph node sites of disease and / or bone marrow infiltration, as described above.

[0170] In several aspects, administration by the method provided and / or administration with the manufactured articles or compositions provided generally reduces or suppresses the growth or burden of the disease or condition in question. For example, if the disease or condition is a tumor, the method generally reduces tumor size, volume, metastasis, the percentage of blast cells in the bone marrow or molecularly detectable cancer, and / or improves other symptoms associated with prognosis or survival rate or tumor burden.

[0171] Disease burden may include the total number of diseased cells in the subject, or in the subject's organs, tissues, or bodily fluids, for example, in the organ or tissue where a tumor is present, or in another site where metastasis may be suggested. For example, tumor cells may be detected and / or quantified in the blood or bone marrow in the context of certain hematopoietic malignancies. In some embodiments, disease burden may include tumor mass, the number or extent of metastases, and / or the proportion of blast cells present in the bone marrow.

[0172] In some contexts, 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 certain contexts, flow cytometry is used to detect or monitor the presence of cancer cells in bone marrow. In some contexts, multi-parameter immunological detection by flow cytometry is used to detect cancer cells (see, e.g., Coustan-Smith et al., (1998) Lancet 351:550-554). In some contexts, multi-parameter 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 may be used to detect cancer cells. The antigens used for detection are selected based on the type of cancer being detected (Foon and Todd (1986) Blood 68:1-31).

[0173] In some cases, bone marrow is collected by bone marrow aspirate or bone marrow biopsy material, and lymphocytes are isolated for analysis. Monoclonal and / or polyclonal antibodies conjugated with fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), phycoerythrin, peridinin chlorophyll protein, or biotin) may be used to detect epitopes on isolated lymphocytes, such as terminal deoxynucleotidyltransferase (TdT), CD3, CD10, CD11c, CD13, CD14, CD33, CD19, CD20, CD21, CD22, CD23, CD34, CD45, CD56, CD79b, IgM, and / or KORSA3544. Labeled cells are then detected using flow cytometry, such as multi-parameter flow cytometry or mass cytometry, and multiple epitopes may be detected.

[0174] Lymphoid cells can be identified, gated based on light scattering dot plots, and then secondary gated to identify cell populations exhibiting immunophenotypic characteristics of interest. Exemplary epitopes are shown in Table 2 below. Other immunological classifications of leukemia and lymphoma have been presented by Foon and Todd (Blood (1986) 68(1):1-31). In some aspects, flow cytometry evaluation of MRD can be performed by quantifying hepatic lymphocytes with one or more CLL immunophenotypes (e.g., low forward / lateral scattering; CD3). neg ;CD5 + ;CD14 neg ;CD19 + ;CD23 + ;CD45 + ;CD56 neg ).

[0175] (Table 2) Exemplary immunophenotypes and cytogenetic characteristics TIFF2026113543000027.tif200165+: >90% of cases are positive + / -: More than 50% of cases are positive. - / +: Less than 50% of cases are positive. -: <10% of cases are positive Pan-B markers: e.g., CD19, CD20, CD79a sIG: Surface immunoglobulin cyIg: Intracellular immunoglobulin

[0176] In some cases, deep sequencing of the immunoglobulin heavy chain (IGH) locus of collected B cells may be used to detect minimal residual disease (MRD). The presence of specific clonal IgG rearrangements may serve as a marker for detecting the presence of B-cell malignancies, such as CLL or NHL, and / or the persistence of their malignant cells. In some cases, cells, such as populations containing B cells or suspected populations containing B cells, are collected and isolated from blood. In some cases, cells are collected and isolated from bone marrow, such as from bone marrow aspirate or bone marrow biopsy material, and / or other biological samples. In some cases, polymerase chain reaction (PCR) amplification of complementarity-determining region 3 (CDR3) is performed using primers against highly conserved sequences in the V and J regions of the locus, and this may be used to identify clonal cell populations for the purpose of assessing minimal residual disease. Other methods for detecting clonal populations may be used, such as single-cell sequencing approaches, which provide information about the number of clonal populations, such as cells of a specific lineage and / or cells expressing a specific variable chain, such as a variable heavy chain or its binding site. In some cases, IGH DNA is amplified using degenerate primers or primers that recognize variable region chains shared between different cell clones, such as those that recognize the consensus V region and the degenerate consensus J region of the IGH sequence. An example sequence of the V region is The example degenerate consensus sequence for the J region is TIFF2026113543000028.tif4128. The filename is TIFF2026113543000029.tif4128.

[0177] In some cases, the PCR product or sequencing result is specific to the rearranged allele and functions as a clonal marker for MRD detection. After PCR amplification of the CDR3 region, the PCR product can be sequenced to obtain patient-specific oligonucleotides constructed as probes for allelic-specific PCR for sensitive detection of MRD after treatment of B-cell malignancies with CAR-T cell therapy, e.g., CD19 CAR-T cell therapy. In cases where PCR products are not generated using consensus primers, V-region family-specific primers for framework region 1 may be used instead.

[0178] In some aspects, the persistent presence of detectable IGH sequences after treatment, corresponding to PCR-detectable tumor cells, e.g., B-cell malignancies, e.g., cells of NHL or CLL, e.g., malignant or clonal IGH sequences, is associated with a high risk of relapse. In some aspects, patients who are negative for malignant IGH sequences after treatment (even in some aspects, in situations where there is disease progression or only a slight partial response, e.g., other criteria indicating the persistent presence of lymphadenopathy, e.g., other criteria that may be associated with the absence of disease or complete response) may have a higher PFS likelihood or be considered to be in the stage of CR, sustained CR, or extended survival compared to patients with persistent malignant IGH sequences. In some aspects, such prognostic and staging determinations are particularly important for treatments in which the disappearance of malignant cells is observed within a short period after administration of therapy, for example, in comparison with the disappearance of other clinical symptoms, e.g., lymph node size or other staging criteria. For example, in some such situations, the absence of detectable IGH or minimal residual disease in a sample, e.g., bone marrow, may be a preferred readout for the likelihood of response or response or its durability compared to other available staging or prognostic approaches. In some situations, MRD results, e.g., information from IGH deep sequencing, may provide information on whether further intervention is necessary or unnecessary. For example, in some situations and in other embodiments provided, subjects deemed negative for malignant IGH may, in some situations, not be treated further, or a certain dose of the therapy offered may not be administered further, or the subject may be administered a lower or reduced dose. Conversely, subjects showing MRD by IGH deep sequencing may, or may be designated, be treated further, e.g., with the same or a higher dose of the therapy initially administered, or with further treatment. In some cases, the disease or condition persists after the initial dose, and / or the initial dose is insufficient to eradicate the disease or condition.

[0179] In some embodiments, the method reduces the disease or condition burden, such as the number of tumor cells, tumor size, patient survival, or event-free survival, to a greater degree and for a longer duration than the reduction observed by equivalent methods using alternative dosing regimens, such as those in which the subject receives one or more alternative therapeutic agents, and / or in which the subject does not receive a certain dose of a cell and / or lymphocyte depleting agent in the provided product or composition. In some embodiments, the disease or condition burden of the subject is detected, evaluated, or measured. In some aspects, the disease burden may be detected by detecting the total number of diseased or disease-associated cells, such as tumor cells, in the subject or in the subject's organs, tissues, or fluids, such as blood or serum. In some aspects, the subject's survival rate, survival rate within a specific period, degree of survival, presence or duration of event-free survival, or asymptomatic survival or recurrence-free survival rate is evaluated. In some embodiments, if symptoms of the disease or condition are present, they are evaluated. In some embodiments, the measurement of the burden of disease or pathological condition is specified.

[0180] In some embodiments, the event-free survival rate or overall survival rate of subjects is improved by the present method compared to other methods, such as a method in which subjects receive one or more alternative therapeutic agents and / or a method and / or a method in which subjects do not receive a certain dose of a cell and / or lymphocyte depletion agent in the manufactured article or composition provided. For example, in some embodiments, the event-free survival rate or probability of event-free survival at 6 months post-administration of subjects treated by the present method is approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or approximately 95%. In some aspects, the overall survival rate is approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or approximately 95%. In some embodiments, subjects treated with this method exhibit event-free survival, recurrence-free survival, or overall survival of 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, for example, to more than 6 months or approximately more than 6 months or at least 1 year, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

[0181] In some embodiments, the probability of relapse after treatment by the present method is lower than that of other methods, such as a subject receiving one or more alternative therapeutic agents and / or a subject not receiving a certain dose of a cell and / or lymphocyte depleting agent by the provided method and / or the provided manufactured article or composition. For example, in some embodiments, the probability of relapse at 6 months after the first dose is less than approximately 80%, less than approximately 70%, less than approximately 60%, less than approximately 50%, less than approximately 40%, less than approximately 30%, less than approximately 20%, or less than approximately 10%.

[0182] In some cases, the pharmacokinetics of administered cells, such as adoptive cells, are investigated to assess the availability of the administered cells, such as bioavailability. Methods for investigating the pharmacokinetics of adoptive cells may include obtaining peripheral blood from the subject to which the manipulated cells were administered and examining the number or proportion of manipulated cells in the peripheral blood. Approaches for selecting and / or isolating cells may include the use of chimeric antigen receptor (CAR) specific antibodies (e.g., Brentjens et al., Sci.Transl.Med. 2013 Mar;5(177):177ra38) Protein L (Zheng et al., J.Transl.Med. 2012 Feb;10:29), epitope tags directly introduced into specific sites of CARs, such as Strep-Tag sequences, where the CAR is directly evaluated using a conjugate reagent for Strep-Tag (Liu et al. (2016) Nature Biotechnology, 34:430; International Patent Application Publication No. 2015095895), and monoclonal antibodies that specifically conjugate to CAR polypeptides (see International Patent Application Publication No. 2014190273). Exogenous marker genes may be used in connection with manipulative cell therapy to enable cell detection or selection in some cases, and to promote cell suicide in others. In some cases, truncated epidermal growth factor receptor (EGFRt) may be co-expressed in transduced cells together with the transgene of the target of interest (CAR) (see, e.g., U.S. Patent No. 8,802,374). EGFRt may include an epitope recognized by the antibody cetuximab (Erbitux®) or an EGFRt construct and other recombinant receptors, such as chimeric antigen receptors (CARs), or / or other therapeutic anti-EGFR antibodies or binding molecules that can be used to identify or select cells manipulated by such receptors, or / or to eliminate or isolate cells expressing such receptors. (See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April;34(4):430-434).

[0183] In some embodiments, CARs in biological samples obtained from patients, for example, blood + The number of T cells can be measured a certain period after administration of cell therapy, for example, to investigate the pharmacokinetics of cells. In some embodiments, CARs that are detectable in the blood of the subject, or detectable in most of the subjects thus treated by this method, are available. + T cells, optionally CAR + CD8 + T cells and / or CARs + CD4 + The number of T cells is more than 1 cell per μL, more than 5 cells per μL, or more than 10 cells per μL.

[0184] C. Toxicity In some embodiments, the methods provided include, for example, alternative cell therapy, for example, alternative CAR + The administration of T cell compositions and / or alternative cell administrations, for example, compared to cell administration without administration at a specified ratio, are designed to include, or include, characteristics associated with toxicity, toxicity outcomes or symptoms, toxicity-promoting profiles, toxicity-promoting factors or characteristics, such as cytokine release syndrome (CRS) or neurotoxicity (NT), or characteristics that result in a reduced rate and / or degree of symptoms or outcomes exhibiting cytokine release syndrome (CRS) or neurotoxicity (NT). Cytokine release syndrome (CRS) and neurotoxicity may be graded according to the American Society for Transplantation and Cell Therapy (ASTCT) Consensus Grading System (see, for example, Lee et al. Biol Blood Marrow Transplant. 2019 Apr;25(4):625-38).

[0185] In some cases, poorly differentiated engineered T cells (e.g., CCR7) are administered as part of the method provided herein. + CD45RA + CD27 + CCR7 + , or CD62L- CCR7 + While a high proportion of engineered T cells with naive-like or central memory phenotypes (such as those selected from the phenotypes) is expected to be more active than cells in a more differentiated state, findings have shown that the safety of cell therapy can be successfully managed. In some aspects, robust efficacy and high safety can be obtained by providing the composition at lower doses compared to cell compositions produced by processes in which cells are more differentiated, such as processes involving cell expansion and proliferation. In some aspects, it has been found that the provided anti-BCMA CAR composition can be administered even at high doses of cells while maintaining low toxicity, such as severe cytokine release syndrome (CRS) or severe neurotoxicity. Therefore, the methods provided in some aspects are alternative cell therapies, such as alternative CARs having engineered T cells in a more differentiated state than those administered herein. + Compared to methods involving the administration of T cell compositions, high doses of engineered T cells (e.g., 50 × 10) 6 More than 100 CAR-expressing T cells, for example, 100 × 10⁶ 6 Or approximately 100 x 10 6 This includes the administration of individual CAR-expressing T cells.

[0186] In some embodiments, the methods provided do not result in a higher rate or likelihood of toxicity or toxic outcomes compared to, for example, certain other cell therapies, or the rate or likelihood of toxicity or toxic outcomes, such as neurotoxicity (NT) or cytokine release syndrome (CRS), is reduced. 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 38 degrees Celsius or at least about 38 degrees Celsius for three days or more, and plasma CRP levels of at least 20 mg / dL or at least about 20 mg / dL. In some embodiments, more than 30% or about 30%, 35%, 40%, 50%, 55%, 60% or more of subjects treated according to the methods provided do not exhibit any grade of CRS or any grade of neurotoxicity. In some embodiments, less than 50% of treated subjects (e.g., at least 60%, at least 70%, at least 80%, at least 90%, or more of treated subjects) exhibit cytokine release syndrome (CRS) of grade 2 or higher and / or neurotoxicity of grade 2 or higher. In some embodiments, at least 50% of subjects treated according to this method (e.g., at least 60%, at least 70%, at least 80%, at least 90%, or more of treated subjects) do not exhibit severe toxic outcomes (e.g., severe CRS or severe neurotoxicity), e.g., no grade 3 or higher neurotoxicity, and / or no severe CRS, or within a certain period after treatment, e.g., within one week, two weeks, or one month after cell administration. In some embodiments, parameters evaluated to investigate specific toxicities include adverse events (AEs), dose-limiting toxicity (DLTs), CRS, and NTs.

[0187] Adoptive T-cell therapy, such as treatment with T cells expressing chimeric antigen receptors, may induce toxic effects or outcomes, such as cytokine release syndrome and neurotoxicity. In some cases, such effects or outcomes coexist with high levels of circulating cytokines, which may be the cause of the observed toxicity.

[0188] In some cases, the toxic outcome is cytokine release syndrome (CRS) or severe CRS (sCRS), or is associated with cytokine release syndrome (CRS) or severe CRS (sCRS), or exhibits cytokine release syndrome (CRS) or severe CRS (sCRS). CRS, such as sCRS, can occur in some cases after adoptive T-cell therapy and after administration of other biological agents to the subject. 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); see Xu et al., Cancer Letters 343 (2014) 172-78.

[0189] Typically, CRS is caused by an excessive systemic immune response mediated by, for example, T cells, B cells, NK cells, monocytes, and / or macrophages. These cells may release large amounts of inflammatory mediators, such as cytokines and chemokines. Cytokines can trigger an acute inflammatory response and / or induce endothelial organ damage that may lead to microvascular leakage, heart failure, or death. Life-threatening severe CRS may progress to pulmonary infiltration and lung injury, renal failure, or disseminated intravascular coagulation. Other life-threatening severe toxicities may include cardiotoxicity, respiratory distress, neurotoxicity, and / or hepatic failure. In some aspects, fever, particularly high fever (≥38.5°C or ≥101.3°F), is associated with CRS or its risk. In some cases, the characteristics or symptoms of CRS closely resemble those of infection. In some aspects, infection should also be considered in subjects presenting with CRS symptoms, and culture-based monitoring and empirical antibiotic therapy may be administered. Other symptoms associated with CRS may include cardiac dysfunction, adult respiratory distress syndrome, renal and / or hepatic failure, coagulation disorders, disseminated intravascular coagulation, and capillary leak syndrome.

[0190] CRS may be treated with anti-inflammatory therapies, such as anti-IL-6 therapies, such as anti-IL-6 antibodies, such as tocilizumab, or antibiotics or other agents as described herein. The outcomes, signs, and symptoms of CRS are well known and include those described herein. In some embodiments, if a particular dosage regimen or administration results in or does not result in a given CRS-related outcome, sign, or symptom, then a particular outcome, sign, and symptom, and / or its quartile or degree, may be specified.

[0191] In the context of CAR-expressing cell administration, CRS typically occurs 6–20 days after infusion of CAR-expressing cells. See Xu et al., Cancer Letters 343(2014)172–78. In some cases, CRS occurs before 6 days or after 20 days after CAR T cell infusion. The onset and timing of CRS may be related to baseline cytokine levels or tumor volume at the time of infusion. Generally, CRS is accompanied by elevated serum levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and / or interleukin (IL)-2. Other cytokines that can be rapidly induced in CRS are IL-1β, IL-6, IL-8, and IL-10.

[0192] Exemplary outcomes associated with CRS include fever, rigidity, chills, hypotension, dyspnea, acute respiratory distress syndrome (ARDS), encephalopathy, elevated ALT / AST, renal failure, cardiac dysfunction, hypoxia, neurological ataxia, and death. Neurological complications include delirium, seizure-like activity, confusion, dysphagia, aphasia, and / or blunting. Other CRS-related outcomes include fatigue, nausea, headache, seizures, tachycardia, myalgia, rash, acute vasoleap syndrome, hepatic dysfunction, and renal failure. In some aspects, CRS is associated with elevated levels of one or more factors, such as serum ferritin, d-dimer, aminotransferase, lactate dehydrogenase, and triglycerides, or with hypofibrinogenemia or hepatosplenomegaly. Other exemplary signs or symptoms associated with CRS include hemodynamic instability, febrile neutropenia, elevated serum C-reactive protein (CRP), changes in coagulation parameters (e.g., international normalized ratio (INR), prothrombin time (PTI), and / or fibrinogen), changes in cardiac and other organ function, and / or changes in absolute neutrophil count (ANC).

[0193] In some embodiments, outcomes associated with CRS include: a persistent fever, e.g., a specified body temperature of, e.g., above 38 degrees Celsius or above approximately 38 degrees Celsius for two or more consecutive days, e.g., three or more days, e.g., four or more days, or at least three consecutive days; a fever of, e.g., above 38 degrees Celsius or above approximately 38 degrees Celsius; e.g., at least two cytokines (e.g., from the group consisting of interferon-gamma (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L, fractalkine and IL-5 and / or tumor necrosis factor alpha (TNFα)). An increase in cytokines, such as a maximum change multiplier of at least 75 or at least about 75 compared to pretreatment levels of at least two cytokines, or a maximum change multiplier of at least 250 or at least about 250 for at least one of such cytokines; and / or at least one clinical sign of toxicity, including hypotension (e.g., measured with at least one intravenous vasopressor); hypoxia (e.g., plasma oxygen (PO2) levels less than 90% or about 90%); and / or one or more neurological disorders (e.g., altered mental state, blunting, and seizures). In some embodiments, neurotoxicity (NT) may be observed concurrently with CRS.

[0194] Exemplary CRS-related outcomes include elevated or high serum levels of one or more factors, such as cytokines and chemokines associated with CRS, and other factors. Further exemplary outcomes include increased synthesis or secretion of one or more such factors. Such synthesis or secretion may be mediated by T cells or cells that interact with T cells, such as innate immune cells or B cells.

[0195] In some embodiments, CRS-related serofactors or CRS-related outcomes include inflammatory cytokines and / or chemokines, such as interferon-gamma (IFN-γ), TNF-α, IL-1β, 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)-1, tumor necrosis factor alpha (TNFα), IL-6 and IL-10, IL-1β, IL-8, IL-2, MIP-1, Flt-3L, fractalkines and / or IL-5. In some embodiments, the factor or outcome includes C-reactive protein (CRP). In addition to being an easily measurable risk factor for CRS in its early stages, CRP is also a marker of cell proliferation. In some embodiments, subjects who are measured to have high levels of CRP, e.g., ≥15 mg / dL, have CRS. In some embodiments, subjects who are measured to have high levels of CRP do not have CRS. In some embodiments, the measurement of CRS includes the measurement of CRP and another factor indicating CRS.

[0196] 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-γ, TNF-α, IL-2, IL-1β, IL-6, IL-7, IL-8, IL-10, IL-12, sIL-2Rα, granulocyte-macrophage colony-stimulating factor (GM-CSF), or macrophage inflammatory protein (MIP). In some embodiments, IFN-γ, TNF-α, and IL-6 are monitored.

[0197] CRS criteria that appear to correlate with the development of CRS have been developed to predict which patients are more likely to develop sCRS (see Davilla et al. Science translational medicine. 2014;6(224):224ra25). Factors include fever, hypoxia, hypotension, neurological changes, and elevated serum levels of inflammatory cytokines, such as a set of seven cytokines (IFNγ, IL-5, IL-6, IL-10, Flt-3L, fractalkine, and GM-CSF) whose treatment-induced elevations often correlate well with both pre-treatment tumor volume and sCRS symptoms. Other guidelines for the diagnosis and management of CRS are publicly available (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 aspects, the criteria for reflecting the CRS grade are detailed in Table 3 below.

[0198] (Table 3) Exemplary Grade Classification Criteria for CRS TIFF2026113543000030.tif130164

[0199] In some aspects, the criteria that reflect the CRS grade are detailed in Table 4 below.

[0200] (Table 4) Exemplary Grade Classification Criteria for CRS TIFF2026113543000031.tif66165

[0201] In some embodiments, treatment with high-dose vasopressors includes those listed in Table 5 below.

[0202] (Table 5) High-dose vasopressors (total dose required in ≥3 hours) TIFF2026113543000032.tif42165 aVASST clinical trial vasopressor equivalent formula: Norepinephrine equivalent dose = [Norepinephrine (μg / min)] + [Dopamine (μg / kg / min) ÷ 2] + [Epinephrine (μg / min)] + [Phenylephrine (μg / min) ÷ 10]

[0203] In some embodiments, the toxic outcome is 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 considered to have developed a “severe CRS” (“sCRS”) in response to or incidentally to the administration of cell therapy or a dose of such cells if, after administration, the subject has (1) a fever of at least 38 degrees Celsius for at least 3 days; (2) (a) at least two of the following seven cytokines: interferon-gamma (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L, fractalkine and IL-5, with a maximum change of at least 75 compared to the level immediately after administration, and / or (b) the following seven The cytokine group: cytokine elevation including at least one of interferon-gamma (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L, fractalkine, and IL-5 with at least one of the maximum change multipliers of 250 compared to the level immediately after administration; and (c) the presence of at least one clinical sign of toxicity, e.g., hypotension (requiring at least one intravenous vasopressor) or hypoxia (PO2 < 90%) or one or more neurological disorders (e.g., altered mental state, blunting, and / or seizures). In some embodiments, severe CRS includes Grade 3 or higher CRS, e.g., shown in Tables 3 and 4.

[0204] In some embodiments, the level of toxic outcomes, such as CRS-related outcomes, such as serum levels of CRS indicators, is measured by ELISA. In some embodiments, fever and / or C-reactive protein (CRP) levels may be measured. In some embodiments, subjects with fever and CRP ≥ 15 mg / dL may be considered at high risk for the development of severe CRS. In some embodiments, CRS-related serofactors or CRS-related outcomes include elevated levels and / or concentrations of inflammatory cytokines and / or chemokines, such as Flt-3L, fractalkines, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 beta (IL-1β), IL-2, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, interferon-gamma (IFN-γ), macrophage inflammatory protein (MIP)-1, MIP-1, sIL-2Rα, or tumor necrosis factor alpha (TNFα). In some embodiments, the factor or outcome includes C-reactive protein (CRP). In addition to being an easily measurable risk factor for CRS early on, CRP is also a marker of cell proliferation. In some embodiments, subjects who are measured to have high levels of CRP, e.g., ≥15 mg / dL, have CRS. In some embodiments, subjects who are measured to have high levels of CRP do not have CRS. In some embodiments, the measurement of CRS includes the measurement of CRP and another factor indicating CRS.

[0205] In some embodiments, outcomes associated with severe CRS or grade 3 or higher, e.g., grade 4 or higher CRS, include: persistent fever, e.g., a specified body temperature of, e.g., above 38 degrees Celsius or above approximately 38 degrees Celsius for two or more consecutive days, e.g., three or more days, e.g., four or more days, or at least three consecutive days; fever of, e.g., above 38 degrees Celsius or above approximately 38 degrees Celsius; elevated cytokine levels, e.g., at least two cytokines (e.g., interferon-gamma (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L, fractalkine, and IL-5); A maximum change multiplier of, for example, at least 75 or at least about 75 compared to the pre-treatment level of at least two of the group consisting of rabini and / or tumor necrosis factor alpha (TNFα), or a maximum change multiplier of at least 250 or at least about 250 for at least one of such cytokines; and / or at least one clinical sign of toxicity, e.g., hypotension (e.g., measured with at least one intravenous vasopressor); hypoxia (e.g., plasma oxygen (PO2) levels less than 90% or about 90%); and / or one or more neurological disorders (e.g., altered mental status, blunting, and seizures). In some embodiments, severe CRS includes CRS requiring management or treatment in an intensive care unit (ICU).

[0206] In some embodiments, CRS, e.g., severe CRS, encompasses a combination of (1) a persistent fever (at least 38 degrees Celsius for at least 3 days) and (2) a serum CRP level of at least 20 mg / dL or at least about 20 mg / dL. In some embodiments, CRS encompasses hypotension requiring the use of two or more vasopressors or respiratory failure requiring mechanical ventilation. In some embodiments, the dosage of the vasopressor is increased with the second or subsequent administration.

[0207] In some embodiments, severe CRS or Grade 3 CRS includes elevated alanine aminotransferase, elevated aspartate aminotransferase, chills, febrile neutropenia, headache, left ventricular dysfunction, cerebral disturbance, hydrocephalus and / or tremor.

[0208] Methods for measuring or detecting various outcomes may be specified.

[0209] In some aspects, toxic outcomes are neurotoxic or associated with neurotoxicity. In some embodiments, symptoms associated with the clinical risk of neurotoxicity include confusion, delirium, aphasia, expressive aphasia, hypotonia, myoclonus, apathy, altered mental status, convulsions, seizure-like activity, seizures (optionally confirmed by electroencephalography (EEG)), elevated beta-amyloid (Aβ) levels, elevated glutamate levels, and elevated oxygen radical levels. In some embodiments, neurotoxicity is graded based on severity (e.g., using a grade 1-5 scale (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)).

[0210] In some cases, neurological symptoms may be the earliest symptom of sCRS. In some embodiments, neurological symptoms begin to appear 5 to 7 days after cell therapy infusion. In some embodiments, the duration of nervous system changes may range from 3 to 19 days. In some embodiments, recovery of nervous system changes occurs after the disappearance of other symptoms of sCRS. In some embodiments, the timing or degree of disappearance of nervous system changes is not accelerated by treatment with anti-IL-6 and / or steroids.

[0211] In some embodiments, a subject is considered to have responded to or developed "severe neurotoxicity" as a side effect of cell therapy or the administration of doses thereof if, after administration, the subject exhibits, among other, symptoms of: 1) peripheral motor neuropathy, e.g., inflammation or degeneration of peripheral motor nerves; 2) peripheral sensory neuropathy, e.g., inflammation or degeneration of peripheral sensory nerves, paresthesia, e.g., misperception of sensation resulting in abnormal and unpleasant sensations; neuralgia, e.g., severe painful sensation along a nerve or group of nerves and / or paresthesia, e.g., abnormal skin sensation such as tingling without stimulation; numbness, pressure, cold, and warmth resulting from dysfunction of sensory nerve cells that restricts daily activities (e.g., bathing, dressing, eating, excretion, taking medication). In some embodiments, severe neurotoxicity includes, for example, Grade 3 or higher neurotoxicity as shown in Table 6.

[0212] (Table 6) Exemplary Grade Classification Criteria for Neurotoxicity TIFF2026113543000033.tif72164

[0213] In some embodiments, the present method reduces symptoms associated with CRS or neurotoxicity compared to other methods. In some aspects, the provided method reduces symptoms, outcomes, or factors associated with CRS, such as those associated with severe CRS or CRS of grade 3 or higher, compared to other methods. For example, subjects treated according to the present method may be subjects in which CRS, such as severe CRS or CRS of grade 3 or higher, or any of the symptoms, outcomes, or factors listed, for example, in Tables 3 and 4, are undetectable, and / or may have fewer such symptoms, outcomes, or factors. In some embodiments, subjects treated according to the present method may have fewer neurotoxic symptoms, such as weakness or numbness of the limbs, impaired memory, vision and / or cognitive impairment, uncontrollable obsessive-compulsive behaviors and / or compulsive behaviors, delusions, headaches, cognitive and behavioral problems, such as impaired motor control, cognitive impairment and autonomic dysfunction, and sexual dysfunction, compared to subjects treated by other methods. In some embodiments, subjects treated according to this method may have fewer symptoms associated with peripheral motor neuropathy, peripheral sensory neuropathy, paresthesia, neuralgia, or paresthesia.

[0214] In some embodiments, this method reduces outcomes associated with neurotoxicity, such as damage to the nervous system and / or brain, such as neuronal cell death. In some aspects, this method reduces levels of factors associated with neurotoxicity, such as beta-amyloid (Aβ), glutamates, and oxygen radicals.

[0215] In some embodiments, the toxic outcome is dose-limiting toxicity (DLT). In some embodiments, the toxic outcome is dose-limiting toxicity. In some embodiments, the toxic outcome is no dose-limiting toxicity. In some embodiments, dose-limiting toxicity (DLT) is defined as any toxicity of grade 3 or higher when evaluated according to any known or published guidelines for assessing specific toxicity, such as any of the above, for example, the Common Toxicity Criteria for Adverse Events (CTCAE) version 4.0 of the National Cancer Institute (NCI).

[0216] In some embodiments, the low rate, risk, or likelihood of occurrence of toxicity, e.g., CRS or neurotoxicity, or severe CRS or neurotoxicity, e.g., grade 3 or higher CRS or neurotoxicity, observed with the administration of a certain dose of T cells by the method provided and / or with the manufactured article or composition provided, makes it possible to administer cell therapy on an outpatient basis. In some embodiments, cell therapy according to the method provided and / or with the manufactured article or composition provided, e.g., T cells (e.g., CAR + The administration of T cells can be done on an outpatient basis or does not require hospitalization at a target hospital, such as an overnight stay.

[0217] In some cases, according to the method provided and / or the manufactured articles or compositions provided, cell therapy, for example, T cells (e.g., CAR + Subjects treated with doses of T cells, for example, subjects treated on an outpatient basis, will not receive any intervention to treat any toxicity prior to or with the administration of doses of the cells, unless they exhibit signs or symptoms of toxicity, such as neurotoxicity or CRS, or until they exhibit signs or symptoms of toxicity, such as neurotoxicity or CRS. Examples of agents for treating, delaying, reducing, or alleviating toxicity are described in Section IC.

[0218] In some aspects, cell therapy, such as T cells (e.g., CAR +If a subject receiving a dose of T cells, for example a subject treated on an outpatient basis, develops a fever, the subject is given treatment to reduce the fever, or is instructed to receive treatment to reduce the fever, or to be administered treatment to reduce the fever. In some embodiments, the subject's fever is characterized as being at a specific threshold body temperature or level, or above a specific threshold body temperature or level (or being measured as being at a specific threshold body temperature or level, or being measured as being above a specific threshold body temperature or level). In some aspects, the threshold body temperature is a temperature associated with at least a slight fever, at least a moderate fever, and / or at least a high fever. In some embodiments, the threshold body temperature is a specific body temperature or range. For example, the threshold body temperature could be 38, 39, 40, 41 or 42 degrees Celsius, or approximately 38, 39, 40, 41 or 42 degrees Celsius, or at least 38, 39, 40, 41 or 42 degrees Celsius, or at least approximately 38, 39, 40, 41 or 42 degrees Celsius, and / or range from 38 degrees Celsius or approximately 38 degrees Celsius to 39 degrees Celsius or approximately 39 degrees Celsius, range from 39 degrees Celsius or approximately 39 degrees Celsius to 40 degrees Celsius or approximately 40 degrees Celsius, range from 40 degrees Celsius or approximately 40 degrees Celsius to 41 degrees Celsius or approximately 41 degrees Celsius, or range from 41 degrees Celsius or approximately 41 degrees Celsius to 42 degrees Celsius or approximately 42 degrees Celsius.

[0219] In some embodiments, the treatment designed to reduce fever includes treatment with an antipyretic. The antipyretic may be any fever-reducing agent, e.g., a compound, composition, or component, e.g., one of any number of agents known to have an antipyretic effect, e.g., NSAIDs (e.g., ibuprofen, naproxen, ketoprofen, and nimeslide), salicylates, e.g., aspirin, choline salicylate, magnesium salicylate, and sodium salicylate, paracetamol, acetaminophen, metamizole, nabumetone, phenaxone, antipyrine, or fever reducers. In some embodiments, the antipyretic is acetaminophen. In some embodiments, acetaminophen may be administered orally or intravenously at a dose of 12.5 mg / kg every four hours or less. In some embodiments, the antipyretic is ibuprofen or aspirin, or comprises ibuprofen or aspirin.

[0220] In some embodiments, if the fever is persistent fever, the subject will be administered an alternative treatment to address the toxicity, e.g., one of the treatments described in Section IC. For subjects treated on an outpatient basis, if the subject has and / or is judged to have persistent fever, or will develop persistent fever, the subject will be instructed to return to the hospital. In some embodiments, a subject is deemed to have, and / or be judged to have, or considered to have, a persistent fever if the subject exhibits a fever at or above the applicable threshold body temperature, and after a specified treatment, for example, after a treatment designed to reduce fever, for example, after treatment with an antipyretic, for example, an NSAID or salicylate, for example, ibuprofen, acetaminophen or aspirin, the subject's fever or body temperature does not decrease, or decreases by a specified amount or by a specified amount (for example, not by more than 1°C, generally by about 0.5°C, 0.4°C, 0.3°C or 0.2°C, or no fluctuation greater than about 0.5°C, 0.4°C, 0.3°C or 0.2°C). For example, a subject is considered to have sustained fever if, after treatment with an antipyretic, such as acetaminophen, the subject's temperature does not decrease by 0.5°C, 0.4°C, 0.3°C, or 0.2°C, by more than 0.5°C, 0.4°C, 0.3°C, or 0.2°C, or by more than approximately 0.5°C, 0.4°C, 0.3°C, or 0.2°C, or by 1%, 2%, 3%, 4%, or 5%, or by approximately 1%, 2%, 3%, 4%, or 5%, and is judged to exhibit a fever of at least 38°C or at least approximately 38°C or 39°C. In some embodiments, the dosage of the antipyretic is, for example, a dosage that is normally effective in lowering a fever or a particular type of fever, such as a fever associated with a bacterial or viral infection, such as a localized or systemic infection, in the subject.

[0221] In some embodiments, a subject is considered to have and / or be judged to have or be deemed to have persistent fever if he or she exhibits a fever at or above the applicable threshold, and the fever or body temperature of the subject does not fluctuate by more than approximately 1°C or approximately 1°C, generally by about 0.5°C, 0.4°C, 0.3°C or 0.2°C, or by fluctuations greater than approximately 0.5°C, 0.4°C, 0.3°C or 0.2°C. The absence of a certain amount of such fluctuation or exceeding a certain amount is generally measured over a given period of time (for example, over a period of 24 hours, 12 hours, 8 hours, 6 hours, 3 hours or 1 hour, which may be measured from the first sign of fever or when the body temperature first exceeded the threshold). For example, in some embodiments, a subject is considered to have sustained fever if, over a period of 6 hours, 8 hours, 12 hours, or 24 hours, the subject exhibits a fever of at least 38 degrees Celsius, at least approximately 38 degrees Celsius, at least approximately 38 degrees Celsius, or at least approximately 38 degrees Celsius or 39 degrees Celsius, with no fluctuation of 0.5°C, 0.4°C, 0.3°C, or 0.2°C over a period of 12 hours, or 24 hours.

[0222] In some aspects, the fever is persistent fever; in some aspects, when the subject is determined to have persistent fever, initial treatment that may induce toxicity, such as cell therapy, such as T cell therapy, such as CAR therapy, is administered. + After the dose of T cells, treatment should be administered within 1, 2, 3, 4, 5, or 6 hours of such judgment or within 6 hours of the first such judgment or within 6 hours of

[0223] In some embodiments, one or more interventions or agents for treating toxicity, such as toxicity-targeted therapy, are administered at the time when the subject is judged or confirmed (e.g., initially judged or confirmed) to have sustained fever, as measured, for example, by any of the embodiments described above, or immediately after the time when the subject is judged or confirmed (e.g., initially judged or confirmed) to have sustained fever. In some embodiments, one or more toxicity-targeted therapies are administered within a specific period of such confirmation or judgment, for example, within 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, or 8 hours.

[0224] II. Cell therapy and cell manipulation In some embodiments, the cell therapy methods disclosed herein (e.g., T-cell therapy) involve administering engineered cells expressing recombinant receptors (e.g., CARs) designed to recognize and / or specifically bind to antigens associated with a disease or condition, e.g., r / r / B-cell NHL. In certain embodiments, the antigen bound to or recognized by the recombinant receptor (e.g., CAR) is CD19. In some embodiments, binding to the antigen results in a response to such antigen, e.g., an immune response. In some embodiments, the cells contain a recombinant receptor, e.g., a chimeric antigen receptor (CAR), or are engineered to contain a recombinant receptor, e.g., a chimeric antigen receptor (CAR). Recombinant receptors, e.g., CARs, generally include, in some aspects, an antigen-specific extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components via a linker and / or transmembrane domain. In some aspects, the engineered cells are provided as pharmaceutical compositions and pharmaceutical formulations suitable for administration to a subject, e.g., adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to a subject, e.g., a patient.

[0225] In some embodiments, cells contain one or more nucleic acids introduced by genetic engineering, thereby expressing recombinant or genetically engineered products of such nucleic acids. In some embodiments, gene introduction is carried out by first stimulating the cells, for example, by combining them with a stimulus that induces a response, such as proliferation, survival, and / or activation, as measured by the expression of cytokines or activation markers, and then transducing and expanding the activated cells in culture to a number sufficient for clinical application.

[0226] A. Chimeric antigen receptor In some embodiments of the methods and uses provided, engineered cells, such as T cells, express a chimeric receptor, e.g., a chimeric antigen receptor (CAR), comprising one or more domains having both a ligand-binding domain (e.g., an antibody or antibody fragment) that provides specificity to a desired antigen (e.g., a tumor antigen) and an intracellular signaling domain. In some embodiments, the intracellular signaling domain is an activating intracellular domain portion, e.g., a T cell activating domain, which delivers a primary activation signal. In some embodiments, the intracellular signaling domain includes, or further includes, a co-stimulatory signaling domain to enhance effector function. Upon specific binding to a molecule, e.g., an antigen, the receptor generally delivers an immunostimulatory signal, e.g., an ITAM transduction signal, into the cell, thereby promoting an immune response targeting a disease or pathological condition. In some embodiments, the chimeric receptor, when genetically engineered in immune cells, can modulate T cell activity, and in some cases can modulate T cell differentiation or homeostasis, thereby potentially resulting in genetically engineered cells with improved in vivo longevity, survival, and / or persistent presence for use, for example, in adoptive cell therapy methods.

[0227] Exemplary antigen receptors, such as CARs, and methods for manipulating and introducing such receptors into cells include, for example, International Patent Application Publications No. 200014257, No. 2013126726, No. 2012 / 129514, No. 2014031687, No. 2013 / 166321, No. 2013 / 071154, No. 2013 / 123061, and U.S. Patent Application Publications No. 2002131960, No. 2013287748, and No. 20130149337. As described in 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 No. 2537416, and / or Sadelain Examples include those described in 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, examples of antigen receptors include the CAR described in U.S. Patent No. 7,446,190 and the International Patent Application Publication No. 2014055668 A1.Examples of CARs include those disclosed in any of the aforementioned publications, such as WO2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, US: 7,446,190, US: 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, US No. 7,446,190, and US No. 8,389,282.

[0228] In some embodiments, engineered cells, such as T cells, express a recombinant receptor, such as a chimeric antigen receptor (CAR), which has 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, the antigen is a glycan or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on diseased or pathological cells, such as tumor cells or pathogenic cells, compared to normal cells or tissues or untargeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or on engineered cells.

[0229] Antigens targeted by the receptor in some embodiments include antigens associated with B-cell malignancies, such as any known B-cell markers. In some embodiments, the antigens targeted by the receptor are CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig kappa, Ig lambda, CD79a, CD79b, or CD30. In certain aspects, the antigen is CD19. In some embodiments, any of these antigens are antigens expressed on human B cells.

[0230] Chimeric receptors, such as CARs, generally contain an extracellular antigen-binding domain, which is one or more antigen-binding portions of an antibody molecule. In some embodiments, the antigen-binding domain is a part of the antibody molecule, generally the weight (V) of the antibody. H ) Chain variable region and / or light (V L ) A chain variable region, e.g., an scFv antibody fragment. In some embodiments, the antigen-binding domain is a single-domain antibody (sdAb), e.g., sdFv, nanobody, V H H and V NAR In some embodiments, the antigen-binding fragment includes an antibody-variable region linked by a flexible linker.

[0231] In some embodiments, an antibody or antigen-binding fragment (e.g., scFv or V) is used. H The domain specifically recognizes an antigen, such as CD19. In some embodiments, the antibody or antigen-binding fragment is derived from an antibody or antigen-binding fragment that specifically binds to CD19, or is a variant of an antibody or antigen-binding fragment that specifically binds to CD19. In some embodiments, the antigen is CD19. In some embodiments, scFv is derived from an antibody or antibody fragment that is specific to CD19. H and V LThis includes, in some embodiments, the antibody or antibody fragment that binds to CD19 is a mouse-derived antibody, e.g., FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Application Publication No. 2016 / 0152723.

[0232] In some embodiments, the antigen-binding domain is derived from V from FMC63. H and / or V L This includes, which in some aspects may be scFv. FMC63 generally represents a mouse monoclonal IgG1 antibody induced against Nalm-1 and Nalm-16 cells expressing human-derived CD19 (Ling, NR, et al. (1987). Leucocyte typing III. 302). In some embodiments, the FMC63 antibody includes the sequences of CDR-H1 and CDR-H2 shown at SEQ ID NO: 38 and 39, CDR-H3 shown at SEQ ID NO: 40 or 54, and CDR-L1 shown at SEQ ID NO: 35, CDR-L2 shown at SEQ ID NO: 36 or 55, and CDR-L3 shown at SEQ ID NO: 37 or 56. In some embodiments, the FMC63 antibody includes a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 41. H ) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO:42 L ) includes.

[0233] In some embodiments, scFv includes a variable light chain comprising the CDR-L1 sequence of SEQ ID NO:35, the CDR-L2 sequence of SEQ ID NO:36, and the CDR-L3 sequence of SEQ ID NO:37, and / or a variable heavy chain comprising the CDR-H1 sequence of SEQ ID NO:38, the CDR-H2 sequence of SEQ ID NO:39, and the CDR-H3 sequence of SEQ ID NO:40, or any variant of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to these. In some embodiments, scFv includes the heavy chain variable region of FMC63 shown in SEQ ID NO:41 and the light chain variable region of FMC63 shown in SEQ ID NO:42, or any variant of the aforementioned having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with respect to these. In some embodiments, the variable heavy chain and variable light chain are linked by a linker. In some embodiments, the linker is shown in SEQ ID NO:24. In some embodiments, scFv is V in order. H , linker, and V L It includes. In some ways, scFv is V in order. L , linker, and V H This includes. In some embodiments, the scFv is encoded by a nucleotide sequence shown at SEQ ID NO:25 or a sequence exhibiting 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 includes an amino acid sequence shown at SEQ ID NO:43 or a sequence exhibiting 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.

[0234] In some embodiments, the antigen-binding domain is derived from SJ25C1 V H and / or V L It includes, which in some aspects may be scFv. SJ25C1 is a mouse monoclonal IgG1 antibody induced against Nalm-1 and Nalm-16 cells expressing human-derived CD19 (Ling, NR, et al. (1987). Leucocyte typing III. 302). In some embodiments, the SJ25C1 antibody includes the sequences of CDR-H1, CDR-H2 and CDR-H3 shown at SEQ ID NO: 47-49, and CDR-L1, CDR-L2 and CDR-L3 shown at SEQ ID NO: 44-46, respectively. In some embodiments, the SJ25C1 antibody includes a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 50. H ) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO:51 L) includes. In some embodiments, svFv includes a variable light chain comprising the CDR-L1 sequence of SEQ ID NO:44, the CDR-L2 sequence of SEQ ID NO:45 and the CDR-L3 sequence of SEQ ID NO:46 and / or a variable heavy chain comprising the CDR-H1 sequence of SEQ ID NO:47, the CDR-H2 sequence of SEQ ID NO:48 and the CDR-H3 sequence of SEQ ID NO:49, or any variant of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to these. In some embodiments, scFv includes the heavy chain variable region of SJ25C1 shown in SEQ ID NO: 50 and the light chain variable region of SJ25C1 shown in SEQ ID NO: 51, or any variant of the aforementioned having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with respect to these. In some embodiments, the variable heavy chain and variable light chain are linked by a linker. In some embodiments, the linker is shown in SEQ ID NO: 52. In some embodiments, scFv is V in order. H , linker, and V L It includes. In some ways, scFv is V in order. L , linker, and V H This includes. In some embodiments, scFv includes the amino acid sequence shown in SEQ ID NO:53 or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO:53.

[0235] In this specification, the term "antibody" is used in its broadest sense, encompassing polyclonal antibodies and monoclonal antibodies, such as intact antibodies and functional (antigen-binding) antibody fragments, such as antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, and heavy-chain variable (V) fragments that can specifically bind to an antigen. H ) region, single-chain antibody fragment, e.g., single-chain variable fragment (scFv) and single-domain antibody (e.g., sdAb, sdFv, nanobody, V) H H or V NAR This term includes ) or fragments. This term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, e.g., intrabodies, peptidebodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecificity, e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise specified, the term “antibody” should be understood to encompass its functional antibody fragment. This term also encompasses intact or full-length antibodies, e.g., antibodies of any class or subclass, e.g., IgG and its subclasses, IgM, IgE, IgA, and IgD. In some aspects, a CAR is a bispecific CAR containing, for example, two antigen-binding domains with different specificities.

[0236] In some embodiments, antigen-binding proteins, antibodies, and their antigen-binding fragments specifically recognize the antigen of a full-length antibody. In some embodiments, the heavy and light chains of the antibody may be full-length or may be antigen-binding moieties (Fab, F(ab')2, Fv, or single-chain Fv fragments (scFv)). In other embodiments, the constant region of the antibody's heavy chain is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, and in particular from, for example, IgG1, IgG2, IgG3, and IgG4, more specifically from IgG1 (e.g., human IgG1). In yet another embodiment, the constant region of the antibody's light chain is selected from, for example, kappa or lambda, in particular from kappa.

[0237] The terms "complementarity-determining region" and "CDR," synonymous with "hypervariable region" or "HVR," are known to in some cases represent discontinuous amino acid sequences within the variable region of an antibody that confer antigen specificity and / or binding affinity. Generally, each heavy chain variable region contains three CDRs (CDR-H1, CDR-H2, CDR-H3) and each light chain variable region contains three CDRs (CDR-L1, CDR-L2, CDR-L3). The terms "framework region" and "FR" are known to in some cases represent non-CDR portions of the variable regions of the heavy and light chains. Generally, each full-length heavy chain variable region contains four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) and each full-length light chain variable region contains four FRs (FR-L1, FR-L2, FR-L3, and FR-L4).

[0238] The exact amino acid sequence boundaries of a given CDR or FR can be defined using one of several well-known schemes, e.g., Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” 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 This can be easily determined using the numbering schemes described in Immunol, 2003 Jan;27(1):55-77 ("IMGT" numbering scheme); Honegger A and Plueckthun 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).

[0239] The boundaries of a given CDR or FR can differ depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Numbering in both the Kabat and Chothia schemes is based on the full sequence length of the most common antibody regions, with some antibodies having insertions represented by insertion letters, e.g., "30a", and deletions. In these two schemes, certain insertions and deletions ("indels") are located in different positions, resulting in the numbering differences. The Contact scheme is based on the analysis of the complex's crystal structure and is similar in many ways to the Chothia numbering scheme. The AbM scheme is a compromise between the Kabat and Chothia definitions, based on those used by Oxford Molecular's AbM antibody modeling software.

[0240] Table 7 below lists the exemplary boundary locations of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs; for example, FR-L1 is located before CDR-L1, FR-L2 is located between CDR-L1 and CDR-L2, and FR-L3 is located between CDR-L2 and CDR-L3. Note that in the Kabat numbering scheme shown, insertions are placed at H35A and H35B, so the endpoint of the Chothia CDR-H1 loop when numbered using the Kabat numbering method shown will differ between H32 and H34 depending on the length of the loop.

[0241] (Table 7) Boundaries of CDRs under various numbering schemes TIFF2026113543000034.tif471611 - Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. 2 - Al-Lazikani et al.,(1997)JMB 273,927-948

[0242] Therefore, unless otherwise specified, a given antibody or its region, for example, its variable region, the "CDR" or "complementary determining region," or an explicitly stated individual CDR (e.g., CDR-H1, CDR-H2, CDR-H3), should be understood to encompass a certain (or specific) complementary determining region as defined by the aforementioned scheme or any other known scheme. For example, a particular CDR (e.g., CDR-H3) is a given V H or V L Where it is stated that a region contains the amino acid sequence of a corresponding CDR, such CDR is understood to have the sequence of a corresponding CDR (e.g., CDR-H3) within a variable region defined by the aforementioned scheme or any other known scheme. In some embodiments, specific CDR sequences are explicitly shown. While the exemplary CDR sequences of the antibodies provided are described using various numbering schemes, it should be understood that the antibodies provided may contain CDRs described by any of the aforementioned other numbering schemes or other numbering schemes known to those skilled in the art.

[0243] Similarly, unless otherwise specified, a given antibody or its region, for example, its variable region FR or an explicitly stated individual FR (e.g., FR-H1, FR-H2, FR-H3, FR-H4), should be understood to encompass a certain (or specific) framework region defined by one of the known schemes. In some cases, the scheme for identifying a particular CDR, FR, or multiple FRs or CDRs is explicitly stated, such as a CDR defined by the Kabat, Chothia, AbM, or Contact method or another known scheme. In other cases, the specific amino acid sequence of the CDR or FR is given.

[0244] The term "variable region" or "variable domain" refers to a domain in the antibody heavy chain or light chain that is involved in the binding of an antibody to an antigen. The variable regions of the heavy and light chains of antibodies in their natural state (V, respectively) H and V L They generally have similar structures, and each domain contains four conserved framework regions (FRs) and three CDRs. (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007). A single V H Domain or V L The domain may be sufficient to confer specificity to antigen binding. Furthermore, an antibody that binds to a specific antigen may have a V derived from the antibody that binds to that antigen. H Domain or V L The domains were isolated, and each had complementary V L Domain or V H Domain libraries can be screened. See, for example, Portolano et al., J.Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0245] In particular, the antibodies provided are antibody fragments. An "antibody fragment" refers to a molecule other than the intact antibody, containing a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments, though not limited to them, include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabolic antibodies; linear antibodies; and heavy-chain variable (V) antibodies. H ) region, single-chain antibody molecule, e.g., scFv and single-domain V H Examples include single antibodies and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment, e.g., scFv, containing a heavy-chain variable region and / or a light-chain variable region.

[0246] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of antibodies in their natural state (V, respectively) H and V L ) generally have a similar structure, and each domain contains four conserved framework regions (FRs) and three CDRs. (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007). Single V H Domain or V L The domain may be sufficient to confer specificity to antigen binding. Furthermore, an antibody that binds to a specific antigen may have a V derived from the antibody that binds to that antigen. H Domain or V L The domains were isolated, and each had complementary V L Domain or V H Domain libraries can be screened. See, for example, Portolano et al., J.Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0247] A single-domain antibody (sdAb) is an antibody fragment comprising all or part of the heavy chain variable domain or all or part 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 the heavy chain domain of an antibody that specifically binds to an antigen, such as a targeted cell or disease, such as a cancer marker or cell surface antigen of tumor cells or cancer cells, such as any target antigen described herein or known. Exemplary single-domain antibodies include sdFv, nanobody, and V. H H or V NAR These are some examples.

[0248] Antibody fragments can be produced by various methods, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, such as a fragment containing components not found in nature, such as having two or more antibody regions or antibody chains linked by a synthetic linker, such as a peptide linker, and / or a fragment containing components that cannot be produced by enzymatic digestion of naturally occurring intact antibodies. In some embodiments, the antibody fragment is an scFv.

[0249] A “humanized” antibody is one 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 may optionally contain at least a portion of the antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody typically represents a variant of a non-human antibody that has been humanized to reduce immunogenicity against humans but retains the specificity and affinity of the non-human parent antibody. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues derived from a non-human antibody (e.g., an antibody from which CDR residues are derived) to restore or improve the specificity or affinity of the antibody, for example.

[0250] In some aspects, recombinant receptors, such as chimeric antigen receptors, comprise an extracellular component containing one or more ligand-(e.g., antigen-) binding domains, such as an antibody or a fragment thereof, and one or more intracellular signaling regions or intracellular signaling domains (also interchangeably referred to as cytoplasmic signaling domains or cytoplasmic signaling regions). In some aspects, recombinant receptors, such as CARs, further comprise a spacer and / or a transmembrane domain or transmembrane portion. In some aspects, the spacer and / or transmembrane domain may link the extracellular component containing the ligand-(e.g., antigen-) binding domain to the intracellular signaling region or intracellular signaling domain.

[0251] In some embodiments, recombinant receptors, such as CARs, are at least a portion of the constant region or a variant or modified form of an immunoglobulin, such as the hinge region, such as the IgG4 hinge region and / or C H 1 / C L and / or Fc regions, or at least a portion of the constant region of an immunoglobulin or its variant or modified form, e.g., hinge regions, e.g., IgG4 hinge region and / or C H 1 / C LThe recombinant receptor further comprises a spacer which may include an Fc region. In some embodiments, the recombinant receptor further comprises a spacer and / or a hinge region. In some embodiments, the constant region or constant parity is that of human IgG, e.g., IgG4 or IgG1. In some aspects, a portion of the constant region functions as a spacer region between the antigen recognition component, e.g., scFv, and the transmembrane domain. The spacer may be of a length that results in increased cellular responsiveness after antigen binding compared to the absence of a spacer. In some examples, the spacer is 12 or about 12 amino acids long, or 12 or fewer amino acids long. Examples of spacers include those having at least approximately 10–229 amino acids, approximately 10–200 amino acids, approximately 10–175 amino acids, approximately 10–150 amino acids, approximately 10–125 amino acids, approximately 10–100 amino acids, approximately 10–75 amino acids, approximately 10–50 amino acids, approximately 10–40 amino acids, approximately 10–30 amino acids, approximately 10–20 amino acids, or approximately 10–15 amino acids, encompassing any integer number between any endpoint of the listed ranges. In some embodiments, the spacer region has approximately 12 or fewer amino acids, approximately 119 or fewer amino acids, or approximately 229 or fewer amino acids. Examples of spacers include IgG4 hinges alone, IgG4 hinges linked to CH2 and CH3 domains, or IgG4 hinges linked to the CH3 domain. Examples of spacers, though not limited to them, include those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, Hudecek et al. (2015) Cancer Immunol Res. 3(2):125-135, or International Patent Application Publication No. 2014031687.

[0252] In some embodiments, the spacer includes only the hinge region of IgG, for example, only the hinge of IgG4 or IgG1, for example, only the hinge encoded by the sequence shown in SEQ ID NO:1 and SEQ ID NO:2. In some embodiments, the spacer is C H 2 and / or C H Ig hinges linked to 3 domains, for example C H 2 and / or C H It is an IgG4 hinge linked to 3 domains. In some embodiments, the spacer is C H 2 and C H Ig hinges linked to 3 domains, for example C H 2 and C H An IgG4 hinge linked to three domains, for example, as shown in SEQ ID NO:4. In some embodiments, the spacer is C H Ig hinges linked to only 3 domains, for example, C H An IgG4 hinge linked to only three domains, for example, as shown in SEQ ID NO:3. In some embodiments, the spacer is a glycine-serine rich sequence or other flexible linker, for example, a known flexible linker, or includes a glycine-serine rich sequence or other flexible linker, for example, a known flexible linker. In some embodiments, the constant region or constant portion is of IgD. In some embodiments, the spacer has the sequence shown in SEQ ID NO:5. In some embodiments, the spacer has an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with respect to any of SEQ ID NO: 1, 3, 4, and 5, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity.

[0253] In some aspects, the spacer is (a) containing all or part of an immunoglobulin hinge or a modified form thereof, or consisting of all or part of an immunoglobulin hinge or a modified form thereof, or containing about 15 or fewer amino acids and not containing the extracellular domain of CD28 or CD8; (b) containing all or part of an immunoglobulin hinge, optionally an IgG4 hinge or a modified form thereof, or consisting of all or part of an immunoglobulin hinge, optionally an IgG4 hinge or a modified form thereof, and / or containing about 15 or fewer amino acids and not containing the extracellular domain of CD28 or CD8; or (c) being 12 or about 12 amino acids long, and / or containing all or part of an immunoglobulin hinge, optionally IgG4 or a modified form thereof, or consisting of all or part of an immunoglobulin hinge, optionally IgG4 or a modified form thereof; or (d) SEQ ID NO: Consists of any of the aforementioned variants having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to the amino acid sequences shown in NO:1, 3-5, 27-34, or 24, or SEQ ID A polypeptide spacer comprising any of the above, having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to the amino acid sequences shown in NO:1, 3-5, 27-34, or 24, or (e) formula X1PPX2P, or consisting of formula X1PPX2P, where X1 is glycine, cysteine, or arginine, and X2 is cysteine ​​or threonine.

[0254] In some embodiments, the antigen receptor includes an intracellular domain directly or indirectly linked to an extracellular domain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain to an intracellular signaling domain. In some embodiments, the intracellular signaling domain includes an ITAM. For example, in some aspects, the antigen recognition domain (e.g., the extracellular domain) is generally linked to one or more intracellular signaling components, such as a signaling component that mimics activation by an antigen receptor complex (e.g., a TCR complex in the case of a CAR) and / or activation by signaling by another cell surface receptor. In some embodiments, the chimeric receptor includes a transmembrane domain linked or fused between an extracellular domain (e.g., scFv) and an intracellular signaling domain. Thus, in some embodiments, the antigen-binding component (e.g., an antibody) is linked to one or more transmembrane domains and an intracellular signaling domain.

[0255] In one embodiment, a transmembrane domain is used that naturally associates with one of the domains within a receptor, such as a CAR. In some cases, the transmembrane domain is selected or modified by amino acid substitutions so that such domain is not bound to transmembrane domains of the same or different surface membrane proteins in order to minimize interaction with other members of the receptor complex.

[0256] In some embodiments, the transmembrane domain originates from either a natural or synthetic source. When the source is natural, in some aspects the domain originates from any membrane-bound or transmembrane protein. Examples of transmembrane domains include those derived from the alpha, beta, or zeta chains 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 (i.e., including at least their transmembrane domains). Alternatively, in some embodiments the transmembrane domain is synthetic. In some aspects the synthetic transmembrane domain mainly contains hydrophobic residues, such as leucine and valine. In some aspects a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. In some embodiments, linkage is by linkers, spacers, and / or transmembrane domains. In some aspects, the transmembrane domain includes the transmembrane portion of CD28 or a variant thereof. The extracellular domain and the transmembrane can be linked directly or indirectly. In some embodiments, the extracellular domain and the transmembrane are linked by a spacer, for example, one of those described herein.

[0257] In some embodiments, the transmembrane domain of a receptor, e.g., CAR, is either the transmembrane domain of human CD28 or a variant thereof, e.g., the 27-amino acid transmembrane domain of human CD28 (accession number: P10747.1), or the amino acid sequence shown in SEQ ID NO:8, or a transmembrane domain containing an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with respect to SEQ ID NO:8, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity. In some embodiments, the transmembrane domain-containing portion of the recombinant receptor includes the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity thereto, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity thereto.

[0258] In some embodiments, recombinant receptors, such as CARs, include at least one intracellular signaling component, such as an intracellular signaling region or intracellular signaling domain. T cell activation is described in some aspects as being mediated by two types of cytoplasmic signaling sequences: one that initiates antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequence) and another that acts in an antigen-independent manner to produce a secondary stimulatory or co-stimulatory signal (secondary cytoplasmic signaling sequence). In some aspects, CARs include one or both of such signaling components. Among these, the intracellular signaling region mimics or approximates signals mediated by innate antigen receptors, signals mediated by such receptors in combination with co-stimulatory receptors, and / or signals mediated by co-stimulatory receptors alone. In some embodiments, a short-chain oligopeptide linker or polypeptide linker, such as a linker 2 to 10 amino acids long, such as one containing glycine and serine, such as a glycine-serine doublet, is present between the transmembrane domain and the cytoplasmic signaling domain of the CAR to form a linkage.

[0259] In some embodiments, upon ligation of a CAR, the cytoplasmic domain or intracellular signaling region of the CAR activates at least one of the normal effector functions or responses of immune cells, such as T cells engineered to express the CAR. For example, in some situations, the CAR induces T cell function, such as cytotoxic activity or T-helper activity, such as the secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling region of an antigen receptor component or co-stimulatory molecule is used in place of the intact immunostimulatory chain, for example, when effector function signals are transmitted. In some embodiments, an intracellular signaling region comprising, for example, one or more intracellular domains comprises the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects also comprises a co-receptor and / or any derivative or variant of such molecule that, in native context, acts in coordination with such receptor to initiate signaling after antigen receptor binding, as well as any synthetic sequence having the same functional capacity. In some embodiments, an intracellular signaling region comprising, for example, one or more intracellular domains comprises the cytoplasmic sequence of a region or domain involved in providing a co-stimulatory signal.

[0260] In some aspects, CARs contain primary cytoplasmic signaling sequences that modulate the primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulative manner may include signaling motifs known as immune receptor-activated tyrosine motifs or ITAMs. Examples of ITAM-containing primary cytoplasmic signaling sequences include those derived from the CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta, and CD3 epsilon. In some embodiments, the cytoplasmic signaling molecules within a CAR contain a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3 zeta.

[0261] In some embodiments, the receptor includes intracellular components of the TCR complex that mediate T cell activation and cytotoxicity, such as the TCR CD3 chain, or the CD3 zeta chain. Thus, in some aspects, the antigen-binding moiety is linked to one or more cellular signaling modules. In some embodiments, the cellular signaling module includes the transmembrane domain of CD3, the intracellular signaling domain of CD3, and / or the transmembrane domain of other CDs. In some embodiments, the receptor, e.g., CAR, further includes one or more additional molecules, e.g., a portion of Fc receptor γ, CD8 alpha, CD8 beta, CD4, CD25, or CD16. For example, in some aspects, the CAR or other chimeric receptor includes a chimeric molecule of CD3-zeta (CD3-ζ) or Fc receptor γ with CD8 alpha, CD8 beta, CD4, CD25, or CD16.

[0262] In some embodiments, the intracellular (or cytoplasmic) signaling region comprises a human CD3 chain, optionally a CD3 zeta-stimulated signaling domain or a functional variant thereof, for example, the cytoplasmic domains of 112 AAs of human CD3ζ isoform 3 (accession number: P20963.2) or the CD3 zeta signaling domain described in U.S. Patent No. 7,446,190 or U.S. Patent No. 8,911,993. In some embodiments, the intracellular signaling region includes an amino acid sequence represented by SEQ ID NO: 13, 14, or 15, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with respect to SEQ ID NO: 13, 14, or 15, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity.

[0263] In the context of natural TCRs, complete activation generally requires not only TCR-mediated signaling but also co-stimulatory signals. Therefore, in some embodiments, components for generating secondary or co-stimulatory signals are also included in the CAR to promote complete activation. In other embodiments, the CAR does not include components for generating co-stimulatory signals. In some aspects, additional CARs are expressed within the same cell to provide components for generating secondary or co-stimulatory signals.

[0264] In some embodiments, the chimeric antigen receptor comprises the intracellular domain of a T cell costimulatory molecule. In some embodiments, the CAR comprises the signaling domain and / or transmembrane portion of a costimulatory receptor, e.g., CD28, 4-1BB, OX40 (CD134), CD27, DAP10, DAP12, ICOS, and / or other costimulatory receptors. In some embodiments, the CAR comprises the costimulatory region or domain of CD28 or 4-1BB, e.g., the costimulatory region or domain of human CD28 or human 4-1BB.

[0265] In some embodiments, the intracellular signaling region or intracellular signaling domain includes an intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, for example, a 41-amino acid domain and / or such domain having an LL-to-GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular signaling domain may include an amino acid sequence shown in SEQ ID NO: 10 or 11 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 10 or 11, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity. In some embodiments, the intracellular domain includes an intracellular costimulatory signaling domain of 4-1BB or a functional variant or a portion thereof, for example, a 42-amino acid cytoplasmic domain of human 4-1BB (accession number Q07011.1) or a functional variant or a portion thereof, for example, the amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with respect to SEQ ID NO:12, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity.

[0266] In some aspects, the same CAR contains both a primary (or activating) cytoplasmic signaling region and a co-stimulatory signaling component.

[0267] In some embodiments, an activating domain is included within one CAR, while a co-stimulatory component is provided by another CAR that recognizes a different antigen. In some embodiments, the CARs include an activating or stimulating CAR and a co-stimulatory CAR, both expressed on the same cell (see WO2014 / 055668). In some aspects, the cell contains one or more stimulating or activating CARs and / or co-stimulatory CARs. In some embodiments, the cell further includes an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), for example, a CAR that recognizes antigens other than those associated with a disease or condition and / or antigens other than those specific to a disease or condition, in which case the activating signal delivered via the disease-targeted CAR is attenuated or inhibited by the binding of the inhibitory CAR to its ligand, for example, reducing off-target effects.

[0268] In some embodiments, two receptors induce activating and inhibitory signals in a cell, respectively, so that ligation of one receptor to its antigen activates the cell or induces a response, while ligation of the second inhibitory receptor to its antigen induces a signal that suppresses or blunts the response. An example is the combination of an activating CAR and an inhibitory CAR (iCAR). Such a strategy may be used to reduce the likelihood of off-target effects in situations where, for example, the activating CAR binds to an antigen expressed in a disease or pathological condition but also expressed on normal cells, and the inhibitory receptor binds to a different antigen expressed on normal cells but not on cells in the disease or pathological condition.

[0269] In some aspects, the chimeric receptor is an inhibitory CAR (e.g., iCAR) or contains an inhibitory CAR (e.g., iCAR) and includes intracellular components that blunt or suppress immune responses, such as intracellular ITAM- and / or costimulatory-promoting responses. Examples of such intracellular signaling components are those found on immune checkpoint molecules, such as PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptor, EP2 / 4 adenosine receptor, e.g., A2AR. In some aspects, the engineered cell contains an inhibitory CAR containing a signaling domain of such an inhibitory molecule or a signaling domain derived from such an inhibitory molecule, and thus it performs the function of blunting the cell's response induced by, for example, an activating CAR and / or a costimulatory CAR.

[0270] In some cases, CARs are referred to as first, second, and / or third-generation CARs. In some aspects, first-generation CARs are those that only produce a CD3 chain-induced signal upon antigen binding; in some aspects, second-generation CARs produce such a signal along with a co-stimulatory signal, including an intracellular signaling domain derived from a co-stimulatory receptor, such as CD28 or CD137; and in some aspects, third-generation CARs include multiple co-stimulatory domains from different co-stimulatory receptors.

[0271] In some embodiments, a CAR includes one or more, e.g., two or more, co-stimulatory domains and activation domains, e.g., a primary activation domain, in its cytoplasmic portion. An exemplary CAR includes intracellular components of CD3-zeta, CD28, and 4-1BB.

[0272] In some embodiments, the antigen receptor further comprises a marker, and / or cells expressing a CAR or other antigen receptor further comprises a surrogate marker, such as a cell surface marker, which can be used to confirm that the cells express the receptor by transduction or manipulation. In some aspects, the marker may be CD34, NGFR, or all or part of the epidermal growth factor receptor (e.g., a truncated form), such as a truncated form of such a cell surface receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is functionally linked to a linker sequence, such as a cleavable linker sequence, such as a polynucleotide encoding T2A. For example, the marker and optionally the linker sequence may be any of those disclosed in International Patent Application Publication No. 2014031687. For example, the marker may optionally be a truncated EGFR (tEGFR) linked to a linker sequence, such as a T2A cleavable linker sequence.

[0273] Exemplary polypeptides of truncated EGFR (e.g., tEGFR) include the amino acid sequence shown in SEQ ID NO: 7 or 16, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with SEQ ID NO: 7 or 16. Exemplary T2A linker sequences include the amino acid sequence shown in SEQ ID NO: 6 or 17, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with SEQ ID NO: 6 or 17.

[0274] In some embodiments, the marker is a molecule, such as a cell surface protein or a portion thereof, that is not naturally found on T cells or on the T cell surface. In some embodiments, the molecule is a non-self molecule, such as a non-self protein, i.e., one that is not recognized as "self" by the immune system of the host into which the cell is adopted.

[0275] In some embodiments, the marker does not perform a therapeutic function and / or has no effect other than being used as a marker for selecting, for example, successfully engineered cells in genetic engineering. In other embodiments, the marker may be a therapeutic molecule or a molecule that exerts some desired effect in another way, such as a ligand that cells encounter in vivo, such as a co-stimulatory molecule or immune checkpoint molecule that enhances and / or blunts the response of cells that have been adopted and encountered the ligand.

[0276] In some embodiments, the chimeric antigen receptor comprises an extracellular component containing the antibody or fragment described herein. In some aspects, the chimeric antigen receptor comprises an extracellular component containing the antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment is scFv or single-domain V H The receptor contains an antibody, and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain contains the signaling domain of the zeta chain of the CD3-zeta (CD3ζ) chain. In some aspects, the CD3-zeta chain is a human CD3-zeta chain. In some aspects, the intracellular signaling region further contains CD28 and CD137 (4-1BB, TNFRSF9) costimulatory domains linked to the CD3 zeta intracellular domain. In some aspects, CD28 is human CD28. In some aspects, 4-1BB is human 4-1BB. In some aspects, the chimeric antigen receptor contains a transmembrane domain located between the extracellular domain and the intracellular signaling region. In some aspects, the transmembrane domain contains the transmembrane portion of CD28. The extracellular domain and the transmembrane domain may be linked directly or indirectly. In some aspects, the extracellular domain and the transmembrane domain are linked by a spacer, e.g., any of those described herein.

[0277] In some embodiments, the CAR comprises an antibody, e.g., an antibody fragment, a transmembrane portion of CD28 or a functional variant thereof, or a transmembrane domain containing a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD3 zeta or a functional variant thereof, with the signaling portion of CD28 or the functional variant thereof being the signaling portion. For example, in some embodiments, the CAR comprises, for example, an antibody specific to CD19, e.g., an antibody fragment, e.g., scFv, e.g., any of the above, a spacer, e.g., a portion of an immunoglobulin molecule, e.g., a spacer containing one or more constant regions of a hinge region and / or a heavy chain molecule, e.g., an Ig-hinge-containing spacer, a transmembrane domain containing all or part of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3 zeta signaling domain.

[0278] In some embodiments, the CAR comprises an antibody, e.g., an antibody fragment, a transmembrane portion of CD28 or a functional variant thereof, or a transmembrane domain containing a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of 4-1BB or a functional variant thereof and a signaling portion of CD3 zeta or a functional variant thereof. In some such embodiments, the receptor further comprises a spacer containing a portion of an Ig molecule, e.g., a portion of a human Ig molecule, e.g., an Ig hinge, e.g., an IgG4 hinge, e.g., a hinge-only spacer. In some embodiments, the CAR comprises, e.g., a CD19-specific antibody or fragment, e.g., scFv, e.g., any of the above, a spacer, e.g., any Ig-hinge-containing spacer, a CD28-derived transmembrane domain, a 4-1BB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.

[0279] In any particular embodiment of the method provided, the CAR comprises, in order from the N-terminus to the C-terminus, an extracellular antigen-binding domain which is an scFv shown as SEQ ID NO:43, a spacer shown as SEQ ID NO:1, a transmembrane domain shown as SEQ ID NO:8, a 4-1BB costimulatory signaling domain shown as SEQ ID NO:12, and a CD3-zeta (CD3ζ) chain signaling domain shown as SEQ ID NO:13.

[0280] B. Nucleic acids, vectors, and methods for genetic manipulation In some embodiments, cells, such as T cells, are genetically engineered to express recombinant receptors. In some embodiments, the engineering is carried out by introducing polynucleotides encoding recombinant receptors. We also provide polynucleotides encoding recombinant receptors, as well as vectors or constructs comprising such nucleic acids and / or polynucleotides.

[0281] In some cases, the nucleic acid sequence encoding a recombinant receptor includes a signal sequence encoding 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 an exemplary GMCSFR alpha chain signal peptide, encoded by the nucleotide sequence shown in SEQ ID NO: 65 and SEQ ID NO: 66. In some cases, the nucleic acid sequence encoding a recombinant receptor, such as a chimeric antigen receptor (CAR), includes a signal sequence encoding a signal peptide. Non-exclusive exemplary examples of signal peptides include, for example, the GMCSFR alpha chain signal peptide, encoded by the nucleotide sequence shown in SEQ ID NO: 65 and SEQ ID NO: 66, or the CD8 alpha signal peptide shown in SEQ ID NO: 67.

[0282] In some embodiments, the polynucleotide encoding the recombinant receptor includes at least one promoter functionally linked to regulate the expression of the recombinant receptor. In some examples, the polynucleotide includes two, three, or more promoters functionally linked to regulate the expression of the recombinant receptor.

[0283] In certain cases where a nucleic acid molecule encodes two or more different polypeptide chains, such as a recombinant receptor and a marker, each polypeptide chain may be encoded by a separate nucleic acid molecule. For example, two separate nucleic acids may be prepared, and each may be individually translocated or introduced into a cell for expression within that cell. In some embodiments, the nucleic acid encoding the recombinant receptor and the nucleic acid encoding the marker are functionally linked to the same promoter and optionally separated by a nucleic acid encoding an internal ribosome entry site (IRES) or a self-cleaving peptide or optionally a peptide that induces ribosome skipping, such as T2A, P2A, E2A, or F2A. In some embodiments, the nucleic acid encoding the marker and the nucleic acid encoding the recombinant receptor are functionally linked to two different promoters. In some embodiments, the nucleic acid encoding the marker and the nucleic acid encoding the recombinant receptor are located at different locations within the cell's genome or are inserted at different locations within the cell's genome. In some embodiments, the polynucleotide encoding the recombinant receptor is introduced into a composition containing cultured cells, for example, by transduction, transfection, or transformation with a retrovirus.

[0284] In some embodiments, for example, in which a polynucleotide comprises a first nucleic acid sequence and a second nucleic acid sequence, the coding sequences encoding each of the different polypeptide chains may be functionally linked to a promoter that may be the same or different. In some embodiments, the nucleic acid molecule may include a promoter that drives the expression of two or more different polypeptide chains. In some embodiments, such a nucleic acid molecule may be multicistronic (biscistronic or tricistronic, see, for example, U.S. Patent No. 6,060,273). In some embodiments, the transcription unit may be manipulated as a bicistronic unit containing an IRES (internal ribosome entry site), thereby enabling co-expression of gene products (e.g., one encoding a marker and the other a recombinant receptor) via a message from a single promoter. Alternatively, in some cases, a single promoter may instruct the expression of RNA containing two or three genes (e.g., one encoding a marker and the other a recombinant receptor) separated from each other by a sequence encoding a self-cleaving peptide (e.g., a 2A sequence) or a protease recognition site (e.g., furin) within a single open reading frame (ORF). Thus, the ORF encodes a single polypeptide that is processed into individual proteins either in translation (in the case of 2A) or post-translation. In some cases, the peptide, e.g., T2A, may cause the ribosome to skip the synthesis of the peptide bond at the C-terminus of the 2A element (ribosome skipping), resulting in separation between that end of the 2A sequence and the downstream neighboring peptide (e.g., de Felipe, Genetic Vaccines and Ther. 2:13 (2004) and de See Felipe et al. Traffic 5:616-626 (2004). Various 2A elements are known.Examples of 2A sequences that may be used in the methods and systems disclosed herein include, but are not limited to, 2A sequences derived from 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 rhinitis virus-1 (P2A, e.g., SEQ ID NO: 18 or 19), as described in U.S. Patent Application Publication No. 20070116690.

[0285] Any recombinant receptor described herein may be encoded in any combination or configuration by polynucleotides comprising one or more nucleic acid sequences encoding the recombinant receptor. For example, one, two, three or more polynucleotides may encode one, two, three or more different polypeptides, e.g., recombinant receptors. In some embodiments, one vector or construct comprises a nucleic acid sequence encoding a marker, and another vector or construct comprises 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 functionally linked to two different promoters. In some embodiments, the nucleic acid encoding the recombinant receptor is downstream of the nucleic acid encoding the marker.

[0286] In some embodiments, the vector skeleton includes nucleic acid sequences encoding one or more types of markers. In some embodiments, the one or more types of markers are transduction markers, surrogate markers, and / or selection markers.

[0287] In some embodiments, the marker is a transduction marker or a surrogate marker. Transduction markers or surrogate markers can be used to detect cells into which polynucleotides, such as polynucleotides encoding recombinant receptors, have been introduced. In some embodiments, the transduction marker can indicate or confirm the modification of cells. In some embodiments, the surrogate marker is a protein co-expressed on the cell surface together with a recombinant receptor, such as CAR. In certain embodiments, such a surrogate marker is a surface protein modified to have little or no activity. In certain embodiments, the surrogate marker is encoded by the same polynucleotide as that encoding the recombinant receptor. In some embodiments, the nucleic acid sequence encoding the recombinant receptor is optionally functionally linked to a marker-coding nucleic acid sequence separated by an internal ribosome entry site (IRES) or to a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome skipping, such as a 2A sequence, e.g., T2A, P2A, E2A, or F2A. Exogenous marker genes can be used in association with engineered cells to enable the detection or selection of cells in some cases, and in some cases to promote cell suicide.

[0288] Examples of surrogate markers include truncated cell surface polypeptides, such as non-functional truncated forms that do not transmit signals or signals normally transmitted by full-length cell surface polypeptides, or are unable to transmit signals or signals normally transmitted by full-length cell surface polypeptides, and / or do not internalize or are unable to internalize. Examples of truncated cell surface polypeptides include truncated growth factors or other receptors, such as truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal growth factor receptor (tEGFR, exemplary tEGFR sequences shown in SEQ ID NO: 7 or 16), or prostate-specific membrane antigen (PSMA) or modified forms thereof. tEGFR may include epitopes recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibodies or binding molecules that can be used to identify or select cells manipulated with tEGFR constructs and encoded exogenous proteins, and / or to eliminate or isolate 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 contexts, markers, such as surrogate markers, include all or part of CD34, NGFR, CD19, or truncated CD19, such as truncated non-human CD19, or epidermal growth factor receptor (e.g., tEGFR) (e.g., truncated form).

[0289] In some embodiments, the markers are fluorescent proteins, such as green fluorescent protein (GFP), fluorescence-enhanced green fluorescent protein (EGFP), such as superfold GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue-green fluorescent protein (BFP), fluorescence-enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), as well as their variants, such as species variants, monomeric variants, and codon-optimized and / or fluorescent forms of these fluorescent proteins. Enhanced variants or fluorescent proteins, such as green fluorescent protein (GFP), fluorescently enhanced green fluorescent protein (EGFP), such as superfold GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue-green fluorescent protein (BFP), fluorescently enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), as well as their variants, such as species variants, monomeric variants, and codon-optimized and / or fluorescently enhanced variants of these fluorescent proteins. In some embodiments, the marker is an enzyme, such as luciferase, the lacZ gene derived from Escherichia coli (E. coli), alkaline phosphatase, secretory embryonic alkaline phosphatase (SEAP), or chloramphenicol acetyltransferase (CAT), or includes an enzyme, such as luciferase, the lacZ gene derived from Escherichia coli, alkaline phosphatase, secretory embryonic alkaline phosphatase (SEAP), or chloramphenicol acetyltransferase (CAT). Exemplary luminescence reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or variants thereof.

[0290] In some embodiments, the marker is a selection marker. In some embodiments, the selection marker is a polypeptide that confers resistance to an exogenous drug or substance, or comprise...

Claims

1. A method for treating B-cell non-Hodgkin lymphoma (r / r B-cell NHL), the method comprising the step of administering to a subject having or suspected of having B-cell NHL a composition comprising engineered T cells expressing a CD19-targeting chimeric antigen receptor (CAR), The composition comprises CD4+ T cells expressing the CAR and CD8+ T cells expressing the CAR, The composition is 5 × 10 6 Or approximately 5 x 10 6 From a single CAR-expressing T cell, 100 × 10⁶ 6 Or approximately 100 x 10 6 It includes CAR-expressing T cells (including values ​​at both ends), At least 80% or at least about 80% of the cells in the composition are CD3 + It is a cell, and CAR in the composition + At least 80% or at least about 80% of T cells exhibit a naive-like phenotype or a central memory phenotype. method.

2. A method for treating B-cell non-Hodgkin lymphoma (r / r B-cell NHL), the method comprising the step of administering to a subject having or suspected of having B-cell NHL a composition comprising engineered T cells expressing a CD19-targeting chimeric antigen receptor (CAR), The composition comprises CD4+ T cells expressing the CAR and CD8+ T cells expressing the CAR, The composition comprises 5×10 6 or about 5×10 6 CAR-expressing T cells, and from 100×10 6 or about 100×10 6 CAR-expressing T cells (including the values at both ends). At least 80% or at least about 80% of the cells in the composition are CD3 + It is a cell, and CD4 in the composition + CAR + At least 50% or at least about 50% of T cells are CD27 + CCR7 + and / or the composition contains CD8 + CAR + At least 50% or at least about 50% of T cells are CD27 + CCR7 + That is, method.

3. The composition is a CD4 that expresses the CAR. + T cells and CD8 expressing the CAR + The method according to claim 1 or claim 2, comprising T cells in a ratio of approximately 1:2.5 to approximately 2.5:

1.

4. The composition is a CD4 that expresses the CAR. + T cells and CD8 expressing the CAR + The method according to any one of claims 1 to 3, comprising T cells in a ratio of approximately 1:2 to approximately 2:1, a ratio of approximately 1:1.5 to approximately 1.5:1, or a ratio of 1:1 or approximately 1:

1.

5. The composition is a CD4 that expresses the CAR. + T cells and CD8 expressing the CAR + The method according to any one of claims 1 to 3, comprising T cells in a ratio of approximately 1:1 to approximately 2.5:1, a ratio of approximately 1.5:1 to approximately 2:1, a ratio of 1.5:1 or approximately 1.5:1, or a ratio of 2:1 or approximately 2:

1.

6. The above composition is 5 × 10 6 Or approximately 5 x 10 6 From a single CAR-expressing T cell, 50 × 10 6 Or approximately 50 x 10 6 The method according to any one of claims 1 to 5, comprising CAR-expressing T cells (including values ​​at both ends).

7. The above composition is 5 × 10 6 Or approximately 5 x 10 6 From individual CAR-expressing T cells, 25 × 10⁶ 6 Or approximately 25 x 10 6 The method according to any one of claims 1 to 6, comprising CAR-expressing T cells (including values ​​at both ends).

8. The above composition is 5 × 10 6 Or approximately 5 x 10 6 From a single CAR-expressing T cell, 10 × 10 6 Or approximately 10 x 10 6 The method according to any one of claims 1 to 7, comprising CAR-expressing T cells (including values ​​at both ends).

9. The above composition is 10 × 10 6 Or approximately 10 x 10 6 From individual CAR-expressing T cells, 25 × 10⁶ 6 Or approximately 25 x 10 6 The method according to any one of claims 1 to 7, comprising CAR-expressing T cells (including values ​​at both ends).

10. The above composition is 5 × 10 6 Or approximately 5 x 10 6 The method according to any one of claims 1 to 8, comprising CAR-expressing T cells.

11. The above composition is 10 × 10 6 Or approximately 10 x 10 6 The method according to any one of claims 1 to 9, comprising CAR-expressing T cells.

12. The above composition is 25 × 10 6 Or approximately 25 x 10 6 The method according to any one of claims 1 to 7 and 9, comprising CAR-expressing T cells.

13. At least 90% or at least about 90% of the cells in the composition are CD3 + The method according to any one of claims 1 to 12, wherein the cell is a cell.

14. At least 91% or at least about 91%, at least 92% or at least about 92%, at least 93% or at least about 93%, at least 94% or at least about 94%, at least 95% or at least about 95%, or at least 96% or at least about 96% of the cells in the composition are CD3 + The method according to any one of claims 1 to 13, wherein the cell is a cell.

15. CAR in the composition + The method according to any one of claims 1 to 14, wherein 5% or about 5% to 30% or about 30% of T cells express an apoptosis marker, optionally annexin V or activated caspase 3.

16. CAR in the composition + The method according to any one of claims 1 to 15, wherein 10% or about 10% to 15% or about 15% of T cells express an apoptosis marker, optionally annexin V or activated caspase 3.

17. CAR in the composition + The method according to any one of claims 1 to 15, wherein 15% or about 15% to 20% or about 20% of T cells express an apoptosis marker, optionally annexin V or activated caspase 3.

18. CAR in the composition + The method according to any one of claims 1 to 15, wherein 20% or about 20% to 25% or about 25% of T cells express an apoptosis marker, optionally annexin V or activated caspase 3.

19. CAR in the composition + The method according to any one of claims 1 to 15, wherein 25% or about 25% to 30% or about 30% of T cells express an apoptosis marker, optionally annexin V or activated caspase 3.

20. CAR in the composition + The method according to any one of claims 1 to 15, wherein 5% or about 5%, 10% or about 10%, 15% or about 15%, 20% or about 20%, 25% or about 25%, or 30% or about 30% of T cells express an apoptosis marker, optionally annexin V or activated caspase 3.

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

22. CAR in the composition + The method according to any one of claims 1 to 21, wherein 80% or about 80% to 85% or about 85% of the T cells have a naive-like phenotype or a central memory phenotype.

23. CAR in the composition + The method according to any one of claims 1 to 21, wherein 85% or about 85% to 90% or about 90% of the T cells have a naive-like phenotype or a central memory phenotype.

24. CAR in the composition + The method according to any one of claims 1 to 21, wherein 90% or about 90% to 95% or about 95% of the T cells have a naive-like phenotype or a central memory phenotype.

25. CAR in the composition + The method according to any one of claims 1 to 21, wherein 95% or about 95% to 99% or about 99% of the T cells have a naive-like phenotype or a central memory phenotype.

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

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

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

29. The CAR in the composition that is naive-like or central memory phenotype, which accounts for at least 80% or at least about 80% + T cells, CCR7 + CD45RA + CD27 + CCR7 + , or CD62L - CCR7 + The method according to any one of claims 1 and 3 to 28, having a phenotype selected from among.

30. CAR in the composition + From 80% or about 80%, 85% or about 85%, 85% or about 85%, 90% or about 90%, 90% or about 90%, 95% or about 95%, 95% or about 95%, 99% or about 99%, CCR7 + CD45RA + CD27 + CCR7 + , or CD62L - CCR7 + The method according to any one of claims 1 to 29, wherein the phenotype is a naive-like phenotype or a central memory phenotype selected from the above.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

53. CAR in the composition + The method according to any one of claims 1 to 52, wherein the ratio of the number of integrated vector copies (iVCNs) to the total number of VCNs in T cells is less than 0.9 on average or about 0.

9.

54. CAR in the composition + The method according to any one of claims 1 to 53, wherein the ratio of the number of integrated vector copies (iVCNs) to the total number of VCNs in T cells is, on average, 0.9 or about 0.9 to 0.8 or about 0.

8.

55. CAR in the composition + The method according to any one of claims 1 to 53, wherein the ratio of the number of integrated vector copies (iVCNs) to the total number of VCNs in T cells is less than 0.8 on average or about 0.

8.

56. CAR in the composition + The method according to any one of claims 1 to 53 and 55, wherein the ratio of the number of integrated vector copies (iVCNs) to the total number of VCNs in T cells is, on average, 0.8 or about 0.8 to 0.7 or about 0.

7.

57. CAR in the composition + The method according to any one of claims 1 to 53 and 55, wherein the ratio of the number of integrated vector copies (iVCNs) to the total number of VCNs in T cells is, on average, 0.7 or about 0.7 to 0.6 or about 0.

6.

58. CAR in the composition + The method according to any one of claims 1 to 53 and 55, wherein the ratio of the number of integrated vector copies (iVCNs) to the total number of VCNs in T cells is, on average, 0.6 or about 0.6 to 0.5 or about 0.

5.

59. CAR in the composition + The method according to any one of claims 1 to 53 and 55, wherein the ratio of the number of integrated vector copies (iVCNs) to the total number of VCNs in T cells is, on average, 0.5 or about 0.5 to 0.4 or about 0.

4.

60. CAR in the composition + The method according to any one of claims 1 to 59, wherein the number of incorporated vector copies (iVCNs) of the T cells is, on average, 0.4 copies / diploid genome to 3.0 copies / diploid genome or approximately 0.4 copies / diploid genome to 3.0 copies / diploid genome (including the values ​​at both ends).

61. CAR in the composition + The method according to any one of claims 1 to 60, wherein the number of incorporated vector copies (iVCNs) of the T cells is, on average, 0.8 copies / diploid genome to 2.0 copies / diploid genome or approximately 0.8 copies / diploid genome to 2.0 copies / diploid genome (including the values ​​at both ends).

62. CAR in the composition + The method according to any one of claims 1 to 61, wherein the number of integrated vector copies (iVCNs) of the T cells is, on average, 0.8 copies / diploid genome to 1.0 copies / diploid genome or approximately 0.8 copies / diploid genome to 1.0 copies / diploid genome (including the values ​​at both ends).

63. CAR in the composition + The method according to any one of claims 1 to 61, wherein the number of incorporated vector copies (iVCNs) of the T cells is, on average, 1.0 copies / diploid genome to 1.5 copies / diploid genome or approximately 1.0 copies / diploid genome to 1.5 copies / diploid genome (including the values ​​at both ends).

64. CAR in the composition + The method according to any one of claims 1 to 61, wherein the number of integrated vector copies (iVCNs) of the T cells is, on average, 1.5 copies / diploid genome to 2.0 copies / diploid genome or approximately 1.5 copies / diploid genome to 2.0 copies / diploid genome (including the values ​​at both ends).

65. The method according to any one of claims 1 to 64, wherein the B-cell NHL is selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), optionally unspecified DLBCL; transformed DLBCL, optionally transformed DLBCL from follicular lymphoma or marginal zone lymphoma; high-grade B-cell lymphoma (HGBCL), optionally HGBCL having rearrangement of MYC and BCL2 and / or BCL6 with DLBCL histological features; primary mediastinal large B-cell lymphoma (PMBCL); and follicular lymphoma (FL), optionally follicular lymphoma grade 3b (FL3B).

66. The method according to any one of claims 1 to 65, wherein at the time of administration of the composition comprising the manipulated T cells or immediately before such administration, the subject has relapsed after remission following treatment with (i) two or more prior therapies for B-cell NHL and / or (ii) autologous stem cell transplantation (ASCT) therapy, or has become resistant to said therapy.

67. The method according to claim 66, wherein two or more prior therapies for B-cell NHL comprise an anthracycline and a CD20 target agent, optionally comprising rituximab as the CD20 target agent.

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

69. The method according to any one of claims 1 to 68, further comprising the step of obtaining a leukocyte apheresis sample from a subject in order to produce the composition comprising manipulated T cells.

70. The method according to any one of claims 1 to 69, wherein the subject is pre-treated with lymphocyte depletion therapy before the administration.

71. The method according to any one of claims 1 to 69, further comprising the step of administering the composition containing the manipulated T cells to a target of lymphocyte depletion therapy, including the administration of fludarabine and / or cyclophosphamide, immediately before administration of the composition containing the manipulated T cells.

72. The method according to any one of claims 1 to 71, wherein the administration of the composition containing the manipulated T cells and / or the lymphocyte depletion therapy is performed on an outpatient basis.

73. The aforementioned lymphocyte depletion therapy involves administering 30 mg / m² daily. 2 Fludarabine for the target body surface area and 300 mg / m² daily 2 The method according to any one of claims 70 to 72, comprising administering cyclophosphamide to the target body surface area for 3 days.

74. The method according to any one of claims 70 to 73, wherein the composition comprising the manipulated T cells is administered for 48 hours or about 48 hours to 9 days or about 9 days (including the values ​​at both ends) after the completion of the lymphocyte depletion therapy.

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

76. The method according to claim 75, wherein the drug is an anti-IL-6 antibody, an anti-IL-6 receptor antibody, or a steroid, or comprises the same.

77. The method according to claim 75 or 76, wherein the drug is tocilizumab or methylprednisolone, or comprises the same.

78. The method according to any one of claims 1 to 77, wherein the T cells are self to the subject.

79. At least 35%, at least 40%, or at least 50% of subjects treated according to the above method achieve complete response (CR); At least 60%, 70%, 80%, 90%, or 95% of subjects who reach CR exhibit a sustained CR for 3 months or more than 3 months or 6 months or more than 6 months; and / or At least 60%, 70%, 80%, 90%, or 95% of subjects who achieved CR within 1 month and / or 3 months remain in response, remain in CR, and / or survive or survive without progression for 3 months or more and / or 6 months or more and / or 9 months or more than 9 months after achieving CR; and / or At least 50%, at least 60%, or at least 70% of subjects treated according to the above method achieve an objective response (OR); At least 60%, 70%, 80%, 90%, or 95% of those who reached OR exhibited a persistent OR for 3 months or more than 3 months or 6 months or more than 6 months; and / or At least 35%, at least 40%, or at least 50% of subjects who reached OR remained in response or were alive for 3 months or more and / or 6 months or more after reaching OR. The method according to any one of claims 1 to 78.

80. The aforementioned cells are self to the subject, and For the preparation of the aforementioned therapy, the minimum absolute number of lymphocytes (ALC) for apheresis is not required and / or specified; and / or The aforementioned cells, A process that can produce cell products for administration by the above method in at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of subjects having B-cell NHL. Made by The method according to any one of claims 1 to 79.

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

82. The method according to any one of claims 1 to 81, wherein more than 80% or approximately more than 80% of subjects treated according to the method do not exhibit grade 3 or higher cytokine release syndrome (CRS) and / or grade 3 or higher neurotoxicity.

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

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

85. More than 30%, 35%, 40%, or 50% of subjects treated according to the above method, or approximately 30%, 35%, 40%, or more than 50%, will not exhibit cytokine release syndrome (CRS) or neurotoxicity of any grade; and / or At least 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of subjects treated according to the above method, or at least about 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, will not develop CRS earlier than 3 days after the start of the administration and / or will not develop neurotoxicity earlier than 5 days after the start of the administration; and / or The median onset of neurotoxicity in subjects treated according to the method is either the median peak of CRS in subjects treated according to the method, or after the median peak, or the median time to disappearance of CRS in subjects treated according to the method, and / or the median onset of neurotoxicity in subjects treated according to the method is greater than 8, 9, 10, or 11 days, or approximately greater than 8, 9, 10, or 11 days. The method according to any one of claims 1 to 84.

86. At least 50% of subjects treated according to the above method achieve a complete response (CR); At least 70% of subjects treated according to the above method achieve an objective response (OR); and More than 50% or approximately 50% of subjects treated according to the above method did not exhibit cytokine release syndrome (CRS) or neurotoxicity of any grade; and More than 80% or approximately more than 80% of subjects treated according to the above method did not exhibit grade 3 or higher cytokine release syndrome (CRS) and / or grade 3 or higher neurotoxicity. The method according to any one of claims 1 to 85.

87. The CAR comprises a CD19-specific extracellular antigen-binding domain, a transmembrane domain, a cytoplasmic signaling domain derived from an optional 4-1BB costimulatory molecule, and an optional CD3 zeta-containing primary signaling ITAM-containing molecule; The CAR comprises, in order, a CD19-specific extracellular antigen-binding domain, 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 includes an extracellular antigen recognition domain that specifically binds to CD19, a transmembrane domain, and an intracellular signaling domain that is a CD3-zeta (CD3ζ) chain and a 4-1BB signaling domain. The method according to any one of claims 1 to 86.

88. The method according to any one of claims 1 to 87, wherein the CAR comprises a CD19-specific extracellular antigen-binding domain, a transmembrane domain, a cytoplasmic signaling domain derived from a 4-1BB costimulatory molecule, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule which is CD3 zeta.

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

90. scFv The amino acid sequence, The amino acid sequence, The amino acid sequence, The amino acid sequence, The amino acid sequence, The method according to claim 89, comprising the amino acid sequence.

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

92. The method according to any one of claims 89 to 91, wherein scFv is indicated as SEQ ID NO:

43.

93. The method according to any one of claims 87 to 92, wherein the cytoplasmic signaling domain derived from the co-stimulatory molecule is a 4-1BB signaling domain, and optionally, the cytoplasmic signaling domain derived from the co-stimulatory 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 higher sequence identity thereto.

94. The method according to any one of claims 87 to 93, wherein the cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule is a CD3 zeta signaling domain, and optionally, the cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule includes one having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to SEQ ID NO: 13, 14, or 15.

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

96. The spacer, A polypeptide spacer comprising an immunoglobulin hinge or a modified thereof, optionally all or part of an IgG4 hinge or a modified thereof, or consisting of an immunoglobulin hinge or a modified thereof, optionally all or part of an IgG4 hinge or a modified thereof. The method according to claim 95.

97. The method according to claim 95 or claim 96, wherein the spacer is 12 or approximately 12 amino acids long.

98. The method according to any one of claims 96 to 97, wherein the spacer includes or consists of the sequence of SEQ ID NO:1 or the sequence encoded by SEQ ID NO:

2.

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

100. The method according to any one of claims 87 to 99, wherein the transmembrane domain is a transmembrane domain derived from CD28, optionally comprising the sequence of amino acids shown in SEQ ID NO:8 or a transmembrane domain exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with respect to SEQ ID NO:8, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity.

101. The aforementioned CAR is structured as follows, from the N-terminus to the C-terminus: The extracellular antigen-binding domain, which is an scFv, is shown in SEQ ID NO:43; the spacer is shown in SEQ ID NO:1; the transmembrane domain is shown in SEQ ID NO:8; the 4-1BB costimulatory signaling domain is shown in SEQ ID NO:12; and the CD3-zeta (CD3ζ) chain signaling domain is shown in SEQ ID NO:

13. The method according to any one of claims 96 to 100, including the method described in any one of claims 96 to 100.

102. The composition containing the manipulated T cells is as follows: (i) Exposure step, in which an input composition containing primary T cells, and optionally an input composition containing autologous T cells selected from the subject, is exposed to a stimulating reagent containing an oligomeric particle reagent containing multiple streptavidin mutein molecules under conditions that stimulate T cells, thereby generating a stimulated population, wherein, The oligomer particle reagent comprises a first agent containing an anti-CD3 antibody or its antigen-binding fragment, and a second agent containing an anti-CD28 antibody or its antigen-binding fragment. The exposure stage; (ii) An introduction step, in which a heterologous polynucleotide encoding the CAR targeting CD19 is introduced into the T cells of the stimulated population, thereby generating a population of transformed cells; (iii) the step of incubating the population of transformed cells for up to 96 hours; and (iv) A collection step of collecting T cells from the transformed cell population, thereby preparing a composition of engineered cells, wherein the collection is performed at a time between 24 and 120 hours (including both values) after exposure to the stimulating agent has started. The method according to any one of claims 1 to 101, manufactured by a manufacturing process including the following:

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

104. The method according to claim 102 or claim 103, wherein each of the first and second agents comprises a streptavidin-binding peptide that reversibly binds the first and second agents to the oligomer particle reagent, and optionally the streptavidin-binding peptide comprises an amino acid sequence shown in any of SEQ ID NO: 78 to 82.

105. The method according to any one of claims 102 to 104, wherein the streptavidin mutein molecule is a tetramer of streptavidin mutein comprising the amino acid residues Val44-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47, and optionally the streptavidin mutein comprises the sequence shown in SEQ ID NO: 69, 84, 87, 88, 90, 85, or 59.

106. The method according to any one of claims 102 to 105, wherein the oligomer particle reagent comprises 1,000 to 5,000 streptavidin mutein tetramers (including the values ​​at both ends).

107. The method according to any one of claims 102 to 106, further comprising adding biotin or a biotin analog before the collection of the cells, after the incubation, or during the incubation.

108. The method according to any one of claims 102 to 107, wherein the collection is performed at a time between 48 and 120 hours (including both extreme values) after exposure to the irritating reagent has started.

109. The aforementioned collection, The integrated vector is detected within the genome, but before it reaches a stable number of integrated vector copies (iVCNs) per diploid genome. The method according to any one of claims 102 to 108, which is carried out in [the specified location].

110. The aforementioned collection, The point in time before the total number of viable cells at collection is more than three times or approximately three times the total number of viable cells in the stimulated population. The method according to any one of claims 102 to 109, which is performed in [the specified location].

111. The aforementioned collection, At the point when the total number of living cells at collection is three times or approximately three times, two times or approximately two times, or the same as or nearly the same as the total number of living cells in the stimulated population. The method according to any one of claims 102 to 110, which is carried out in [the specified location].

112. The aforementioned collection, CD27 + CCR7 + The proportion of cells is total T cells in the population of transformed cells, and total CD3 cells in the population of transformed cells. + T cells, all CD4 in the aforementioned population of transformed cells + All CD8 in the aforementioned population of T cells or transformed cells + At a time when T cells, or CAR-expressing cells in the aforementioned population of transformed cells, account for more than 50% or approximately more than 50% The method according to any one of claims 102 to 111, which is performed in [the specified location].

113. The aforementioned collection, CD45RA + CCR7 + and CD45RA - CCR7 + The proportion of cells is total T cells in the population of transformed cells, and total CD3 cells in the population of transformed cells. + T cells, all CD4 in the aforementioned population of transformed cells + T cells, or all CD8 + At a time when T cells, or CAR-expressing cells in the aforementioned population of transformed cells, account for more than 60% or approximately more than 60% The method according to any one of claims 102 to 112, which is carried out in [the specified location].

114. The cells in the administered composition are A manufacturing process for producing an output composition that (i) comprises engineered CD4+ T cells and engineered CD8+ T cells and (ii) exhibits predetermined properties. The process is manufactured, and the iteration of the manufacturing process is optionally carried out in multiple different individual subjects, resulting in the production of multiple output compositions from a human biological sample, wherein the predetermined characteristics of one of the output compositions are as follows: The average percentage of memory phenotypic cells in the multiple output compositions is approximately 40% to 65%, approximately 40% to 45%, approximately 45% to 50%, approximately 50% to 55%, approximately 55% to 60%, or approximately 60% to 65%; The average percentage of cells exhibiting the central memory phenotype in the multiple output compositions is approximately 40% to 65%, approximately 40% to 45%, approximately 45% to 50%, approximately 50% to 55%, approximately 55% to 60%, or approximately 60% to 65%; The average percentage of cells in the plurality of output compositions that are CD27+, CD28+, CCR7+, CD45RA-, CD45RO+, CD62L+, CD3+, CD95+, granzyme B-, and / or CD127+ is about 40% to about 65%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, or about 60% to about 65%; The average percentage of cells that are CCR7+ / CD45RA- or CCR7+ / CD45RO+ in the plurality of output compositions is approximately 40% to approximately 65%, approximately 40% to approximately 45%, approximately 45% to approximately 50%, approximately 50% to approximately 55%, approximately 55% to approximately 60%, or approximately 60% to approximately 65%; The average percentage of central memory CD4+ T cells in the manipulated CD4+ T cells of the multiple output compositions, optionally CAR+CD4+ T cells, is approximately 40% to 65%, approximately 40% to 45%, approximately 45% to 50%, approximately 50% to 55%, approximately 55% to 60%, or approximately 60% to 65%; The manipulated CD8+ T cells of the multiple output compositions, optionally having an average percentage of central memory CD8+ T cells in CAR+CD8+ T cells of about 40% to about 65%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, or about 60% to about 65%; and / or The average proportion of central memory T cells, optionally CD4+ central memory T cells, and optionally CD8+ central memory T cells in the manipulated T cells, optionally CAR+ T cells, of the multiple output compositions is approximately 40% to approximately 65%, approximately 40% to approximately 45%, approximately 45% to approximately 50%, approximately 50% to approximately 55%, approximately 55% to approximately 60%, or approximately 60% to approximately 65%. A method according to any one of claims 1 to 113, selected from the above.

115. The composition to be administered is A manufacturing process for producing an output composition exhibiting a predetermined characteristic, optionally, when performed on multiple different individual subjects, a manufacturing process for producing an output composition exhibiting a certain threshold number of CAR-expressing cells in the output composition in at least about 80%, about 90%, about 95%, about 97%, about 99%, about 100%, or 100% of the human biological sample. A method according to any one of claims 1 to 114, which is produced by...

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

117. The method according to any one of claims 1 to 116, wherein the B-cell NHL is relapsed and / or antitherapy-treated B-cell non-Hodgkin lymphoma (B-cell NHL).

118. A manufactured article comprising a composition containing genetically modified cells expressing a CD19-targeting chimeric antigen receptor (CAR), and instructions for administering the cell composition according to any one of claims 1 to 117.

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