Compositions and methods comprising engineered chimeric antigen receptors and CAR regulators

Abstract

A method for preparing a cell composition is provided, comprising the step of transducing a population of cells with a mixture of at least two viral vectors, wherein at least one vector comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR); and wherein at least one vector comprises a nucleic acid encoding an activity regulator that modulates the activity of the CAR, cells expressing the CAR, or target cells. [Solution] The present invention also provides cell compositions prepared by this method and their use in the treatment of diseases such as cancer.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to a viral vector composition, its use for transduction into cells, and a cell composition prepared by such a method. [Background technology]

[0002] Background of the Invention Tumor heterogeneity refers to the finding that different tumor cells may exhibit different morphological and phenotypic profiles (including cell morphology, gene expression, metabolism, motility, proliferation, and metastatic potential).

[0003] Heterogeneity occurs between patients, between tumors (intertumor heterogeneity), and within tumors (intratumor heterogeneity). Multiple types of heterogeneity are observed among tumor cells, and these types stem from both genetic and non-genetic variability.

[0004] Heterogeneity among tumor cells can be further amplified by heterogeneity within the tumor microenvironment. Different locations within the tumor (e.g., available oxygen) result in different selective pressures on tumor cells, leading to a wider range of dominant subclones in different spatial regions of the tumor. Furthermore, the influence of the microenvironment on clonal dominance may explain the heterogeneity between primary and metastatic tumors observed in many patients, as well as the intertumor heterogeneity observed among patients with the same type of tumor.

[0005] The heterogeneity of cancer cells presents a significant challenge in designing effective treatment strategies.

[0006] For example, heterogeneous tumors may exhibit different sensitivities to cytotoxic drugs among different clonal populations. This is due to clonal interactions that can inhibit or alter therapeutic efficacy.

[0007] Even when drugs are administered to heterogeneous tumors, it is rare to kill all tumor cells. The initial heterogeneous tumor population can act as a barrier, resulting in the survival of a small number of drug-resistant cells (if any). This allows the resistant tumor population to replicate and grow new tumors through branching evolutionary mechanisms (see above). As a result, the reaggregated tumors are heterogeneous and resistant to the initial drug treatment. Furthermore, these reaggregated tumors may recur in a more malignant form.

[0008] Chimeric antigen receptor (CAR) Chimeric antigen receptors are proteins that combine the specificity of, for example, a monoclonal antibody (mAb) with the effector function of a T cell. Their typical form is a type I transmembrane domain protein, possessing an antigen-recognizing amino terminus, a spacer, a compound end domain that transmits T cell survival and activation signals, and a transmembrane domain to which all are connected (see Figure 1A).

[0009] The most common form of these molecules is a fusion of a monoclonal antibody-derived single-chain variable fragment (scFv) that recognizes a target antigen, fused with a signaling endodomain via a spacer and transmembrane domain. Such molecules activate T cells in response to scFv recognition of their target. When T cells express such CARs, they recognize and kill target cells expressing the target antigen. Several CARs have been developed for tumor-associated antigens, and adoptive transfer approaches using such CAR-expressing T cells are currently in clinical trials for the treatment of various cancers.

[0010] For CAR treatment to be successful, tumor cells must express the target antigen. In heterogeneous tumors, especially solid tumors, antigen expression is heterogeneous, and it may not be possible to find a single target antigen expressed by all cancer cells.

[0011] Furthermore, new data from CAR T-cell studies in B-cell malignancies demonstrate that a common resistance mechanism to this class of therapeutics is the emergence of tumors with lost or downregulated target antigens. When antigen loss or avoidance occurs due to low antigen levels, the target antigen may be expressed in an even more heterogeneous manner, potentially creating a greater barrier to treating solid tumors. Potential approaches to overcome this challenge include making CAR T-cells multispecific and manipulating them to respond to low levels of target antigens, and more efficiently inducing a native antitumor immune response as a result of a CAR-induced inflammatory response.

[0012] Clinical research on CAR T cells has shown that CAR T cells engraft, expand and proliferate, It has been confirmed that survival and persistence are essential for clinical activity, particularly for sustained response. A key reason why CAR-T cells, especially those used for treating solid tumors, may not survive adequately in vivo is that they have difficulty overcoming the unfavorable tumor microenvironment. In particular, CAR-T cells may not be able to engraft and proliferate within the tumor bed of solid tumors.

[0013] The survival and activity of CAR T cells can be enhanced by administering cytokines or by manipulating CAR T cells to secrete or express cytokines, toxins, or other factors. However, these approaches have the following limitations: systemic administration of cytokines can be toxic; constitutive production of cytokines can lead to uncontrolled proliferation and transformation.

[0014] Therefore, an alternative CAR treatment approach is needed to address the problems commonly encountered with CAR-T cell therapy, particularly the heterogeneity between patients and between tumor cells and tumor cell sites within the same patient. [Brief explanation of the drawing]

[0015] [Figure 1]Schematic diagram showing classical chimeric antigen receptors. (a) Basic scheme of chimeric antigen receptors; (b) First-generation receptors; (c) Second-generation receptors; (d) Third-generation receptors.

[0016] [Figure 2] Schematic diagram showing the mechanisms of a) T cell activation and b) T cell inhibition in vivo.

[0017] [Figure 3] Schematic diagram showing the JAK-STAT signaling pathway (activated by α-interferon).

[0018] [Figure 4] Different binding domain formats of chimeric antigen receptors. (a) Fab CAR format; (b) dAb CAR format; (c) scFv CAR format

[0019] [Figure 5] Schematic diagram showing the differences in transduced cell compositions obtained by transduction with a single vector (A) co-expressing two genes; and a mixture of two vectors (B) each expressing a single gene. When cells are transduced with a single vector having a bicistronic cassette, any successfully transduced cell expresses both transgenes in approximately 1:1 stoichiometric amounts (A). However, when cells are transduced with multiple vectors, a more heterogeneous population is obtained: transduced cells can express only the first transgene, only the second transgene, or both transgenes. In cells expressing both transgenes, the relative expression levels of gene A and gene B are completely indeterminate (B).

[0020] [Figure 6]Scatter plots showing the cases when T cells are transduced with a mixture of vectors (A and B), when a certain proportion of cells are not transduced, when a certain proportion of cells are transduced with vector A only, when a certain proportion of cells are transduced with vector B only, and when a certain proportion of cells are transduced with vectors A and B. In this study, cells were transduced with a mixture of vectors, one of which expresses an anti-CD19 CAR and one of which expresses a CD22 CAR.

[0021] [Figure 7] Schematic diagram showing molecules expressed by vectors used in the dual vector composition described in Example 3. Vector 1 expresses a chimeric antigen receptor (CAR) with an antigen-binding domain that binds to GD2 (GD2 CAR), a constitutively active cytokine receptor (CCR), and a selection / suicide gene (RQR8). Vector 2 expresses the same GD2 CAR, a dominant negative SHP-2 (ΔSHP2); a dominant negative transforming growth factor (TGF) βII receptor (ΔTGFbRII), and the same selection / suicide gene (RQR8).

[0022] [Figure 8] Investigation of the ability of singly and doubly transduced T cell populations to kill GD2-expressing (SupT1 GD2) target cells and non-expressing (SupT1 NT) target cells.

[0023] [Figure 9] Investigation of the proliferation of singly and doubly transduced T cell populations after culturing for 7 days in cytokine-free complete cell culture medium without further antigen stimulation.

[0024] [Figure 10-1] Investigation of the ability of singly and doubly transduced T cell populations to kill GD2-expressing (SupT1 GD2) target cells and non-expressing (SupT1 NT) target cells in the presence or absence of TGFβ. [Figure 10-2]Investigation of the ability of single and double transduced T cell populations to kill GD2-expressing (SupT1 GD2) and non-expressing (SupT1 NT) target cells in the presence or absence of TGFβ.

[0025] [Figure 11-1] Investigation of cytokine production (IFNγ) from single and double transduced T cell populations after co-culturing GD2-expressing (SupT1 GD2) target cells and non-expressing (SupT1 NT) target cells in the presence or absence of TGFβ. [Figure 11-2] Investigation of cytokine production (IFNγ) from single and double transduced T cell populations after co-culturing GD2-expressing (SupT1 GD2) target cells and non-expressing (SupT1 NT) target cells in the presence or absence of TGFβ.

[0026] [Figure 12A]Figure 12. Results of an in vivo assay investigating the antitumor activity of T cells transduced with a dual-vector composition via intravenous administration in an established neuroblastoma xenograft model of NSG mice. 1 × 10⁶ CHLA-255 Fluc cells were intravenously injected into female NSG mice. The xenografts were immobilized for 15 days until stable engraftment could be detected by BLI. CAR-T cells were generated by transduction of cells with either a single vector expressing GD2 CAR (GD2 CAR) or with the dual-vector composition described in Example 3 and illustrated in Figure 7 (GD2 CAR + IL7 CCR / GD2 CAR + dSHP2 + dTGFbRII). CAR T cells were intravenously administered at a dose of 3 × 10⁶ CAR T cells / mouse. The quantified bioluminescence signal of CHLA-255 Fluc was plotted over time as total flux (photons / second). A. Graph showing the time course of fluorescence signals for mice administered with CAR-T cells expressing only GD2 CAR (GD2 CAR), non-transduced T cells (NT), or buffer only (PBS). B. Ventral images of mice obtained at -1, 2, 7, 10, and 14 days after administration of CAR-T cells expressing only GD2 CAR (GD2 CAR), non-transduced T cells (NT), or buffer only (PBS). C. Graph showing the time course of fluorescence signals for mice administered with the dual vector composition described in Example 3 and illustrated in Figure 7 (GD2 CAR + IL7 CCR / GD2 CAR + dSHP2 + dTGFbRII), non-transduced T cells (NT), or cells transduced with buffer only (PBS). D. Dual vector composition (GD2 CAR+IL7 CCR / GD2 CAR+dSHP2+dTGFbRII) described in Example 3 and illustrated in Figure 7; ventral images of mice obtained 1, 2, 7, 10, and 14 days after administration of untransduced T cells (NTs) or cells transduced with buffer alone (PBS). [Figure 12B]Figure 12. Results of an in vivo assay investigating the antitumor activity of T cells transduced with a dual-vector composition via intravenous administration in an established neuroblastoma xenograft model of NSG mice. 1 × 10⁶ CHLA-255 Fluc cells were intravenously injected into female NSG mice. The xenografts were immobilized for 15 days until stable engraftment could be detected by BLI. CAR-T cells were generated by transduction of cells with either a single vector expressing GD2 CAR (GD2 CAR) or with the dual-vector composition described in Example 3 and illustrated in Figure 7 (GD2 CAR + IL7 CCR / GD2 CAR + dSHP2 + dTGFbRII). CAR T cells were intravenously administered at a dose of 3 × 10⁶ CAR T cells / mouse. The quantified bioluminescence signal of CHLA-255 Fluc was plotted over time as total flux (photons / second). A. Graph showing the time course of fluorescence signals for mice administered with CAR-T cells expressing only GD2 CAR (GD2 CAR), non-transduced T cells (NT), or buffer only (PBS). B. Ventral images of mice obtained at -1, 2, 7, 10, and 14 days after administration of CAR-T cells expressing only GD2 CAR (GD2 CAR), non-transduced T cells (NT), or buffer only (PBS). C. Graph showing the time course of fluorescence signals for mice administered with the dual vector composition described in Example 3 and illustrated in Figure 7 (GD2 CAR + IL7 CCR / GD2 CAR + dSHP2 + dTGFbRII), non-transduced T cells (NT), or cells transduced with buffer only (PBS). D. Dual vector composition (GD2 CAR+IL7 CCR / GD2 CAR+dSHP2+dTGFbRII) described in Example 3 and illustrated in Figure 7; ventral images of mice obtained 1, 2, 7, 10, and 14 days after administration of untransduced T cells (NTs) or cells transduced with buffer alone (PBS). [Figure 12C]Figure 12. Results of an in vivo assay investigating the antitumor activity of T cells transduced with a dual-vector composition via intravenous administration in an established neuroblastoma xenograft model of NSG mice. 1 × 10⁶ CHLA-255 Fluc cells were intravenously injected into female NSG mice. The xenografts were immobilized for 15 days until stable engraftment could be detected by BLI. CAR-T cells were generated by transduction of cells with either a single vector expressing GD2 CAR (GD2 CAR) or with the dual-vector composition described in Example 3 and illustrated in Figure 7 (GD2 CAR + IL7 CCR / GD2 CAR + dSHP2 + dTGFbRII). CAR T cells were intravenously administered at a dose of 3 × 10⁶ CAR T cells / mouse. The quantified bioluminescence signal of CHLA-255 Fluc was plotted over time as total flux (photons / second). A. Graph showing the time course of fluorescence signals for mice administered with CAR-T cells expressing only GD2 CAR (GD2 CAR), non-transduced T cells (NT), or buffer only (PBS). B. Ventral images of mice obtained at -1, 2, 7, 10, and 14 days after administration of CAR-T cells expressing only GD2 CAR (GD2 CAR), non-transduced T cells (NT), or buffer only (PBS). C. Graph showing the time course of fluorescence signals for mice administered with the dual vector composition described in Example 3 and illustrated in Figure 7 (GD2 CAR + IL7 CCR / GD2 CAR + dSHP2 + dTGFbRII), non-transduced T cells (NT), or cells transduced with buffer only (PBS). D. Dual vector composition (GD2 CAR+IL7 CCR / GD2 CAR+dSHP2+dTGFbRII) described in Example 3 and illustrated in Figure 7; ventral images of mice obtained 1, 2, 7, 10, and 14 days after administration of untransduced T cells (NTs) or cells transduced with buffer alone (PBS). [Figure 12D]Figure 12. Results of an in vivo assay investigating the antitumor activity of T cells transduced with a dual-vector composition via intravenous administration in an established neuroblastoma xenograft model of NSG mice. 1 × 10⁶ CHLA-255 Fluc cells were intravenously injected into female NSG mice. The xenografts were immobilized for 15 days until stable engraftment could be detected by BLI. CAR-T cells were generated by transduction of cells with either a single vector expressing GD2 CAR (GD2 CAR) or with the dual-vector composition described in Example 3 and illustrated in Figure 7 (GD2 CAR + IL7 CCR / GD2 CAR + dSHP2 + dTGFbRII). CAR T cells were intravenously administered at a dose of 3 × 10⁶ CAR T cells / mouse. The quantified bioluminescence signal of CHLA-255 Fluc was plotted over time as total flux (photons / second). A. Graph showing the time course of fluorescence signals for mice administered with CAR-T cells expressing only GD2 CAR (GD2 CAR), non-transduced T cells (NT), or buffer only (PBS). B. Ventral images of mice obtained at -1, 2, 7, 10, and 14 days after administration of CAR-T cells expressing only GD2 CAR (GD2 CAR), non-transduced T cells (NT), or buffer only (PBS). C. Graph showing the time course of fluorescence signals for mice administered with the dual vector composition described in Example 3 and illustrated in Figure 7 (GD2 CAR + IL7 CCR / GD2 CAR + dSHP2 + dTGFbRII), non-transduced T cells (NT), or cells transduced with buffer only (PBS). D. Dual vector composition (GD2 CAR+IL7 CCR / GD2 CAR+dSHP2+dTGFbRII) described in Example 3 and illustrated in Figure 7; ventral images of mice obtained 1, 2, 7, 10, and 14 days after administration of untransduced T cells (NTs) or cells transduced with buffer alone (PBS).

[0027] [Figure 13A]Figure 13. (A) Schematic diagram showing molecules expressed by the vectors used in the triple vector composition "AUTO7" described in Example 6. Vector A expresses truncated SHP2 (dSHP2); safety switch RQR8; anti-PSMA CAR (7A12-28z) based on the novel humanized binder 7A12; and dominant-negative TGFβRII (dnTBRII). Vector B expresses constitutively active IL-7 receptor (CCR_IL7). Vector C expresses RapaCasp9 suicide gene (RapaCasp9); CD19 (dCD19); and IL-12 module (flexiIL-12). "2A" is a co-expression sequence based on the FMDV 2A peptide. (B) Another configuration of the triple vector composition. The components are as shown in (A) above. dNGFR is a truncated nerve growth factor receptor used as a marker protein. [Figure 13B] Figure 13. (A) Schematic diagram showing molecules expressed by the vectors used in the triple vector composition "AUTO7" described in Example 6. Vector A expresses truncated SHP2 (dSHP2); safety switch RQR8; anti-PSMA CAR (7A12-28z) based on the novel humanized binder 7A12; and dominant-negative TGFβRII (dnTBRII). Vector B expresses constitutively active IL-7 receptor (CCR_IL7). Vector C expresses RapaCasp9 suicide gene (RapaCasp9); CD19 (dCD19); and IL-12 module (flexiIL-12). "2A" is a co-expression sequence based on the FMDV 2A peptide. (B) Another configuration of the triple vector composition. The components are as shown in (A) above. dNGFR is a truncated nerve growth factor receptor used as a marker protein.

[0028] [Figure 14]Results of FACS-based killing (FBK) assays investigating the ability of single, double, and triple transduced T cells to kill PSMA-expressing target cells. A: FBK after 24-hour incubation using cell fluorescence analysis to indicate target cell viability. B: Secretion of IL-2 and IFNγ by CAR T cells, measured by detecting the supernatant collected from the co-culture after 24 hours by ELISA. AUTO7 was investigated as a single transduced product using vector A ("AUTO7 / A"), a double transduced product using vectors A and B ("AUTO7 / AB"), or a triple transduced product using vectors A, B, and C ("AUTO7 / ABC"). AUTO7 was tested against second-generation CARs ("parents") developed using the same anti-PSMA binder 7A12. SupT1 target cells modified to express human PSMA antigen at different levels (SupT1-PSMAhigh, SupT1-PSMAlow) were used as target cells. Unmodified SupT1 cells (SupT1-NT) were used as a negative control. CAR T cells were co-cultured with target cells in an effector:target ratio of 1:2.

[0029] [Figure 15A] Figure 15. Results of FACS-based FBK assays investigating the ability of single, double, and triple transduced T cells to kill PSMA-expressing target cells after culture in cytokine-free complete cell culture medium. After 7 days of culture under "starvation conditions," CAR T cells were co-cultured with SupT1-PSMAhigh and SupT1-PSMAlow target cells (or SupT1-NT cells as a negative control) in effector:target ratios of 1:2 and 1:8. A: FBK after 24-hour incubation using cell fluorescence analysis to indicate target cell viability. B: Secretion of IL-2 and IFNγ by CAR T cells, measured by detecting the supernatant collected from the co-culture after 24 hours by ELISA. [Figure 15B]Figure 15. Results of FACS-based FBK assays investigating the ability of single, double, and triple transduced T cells to kill PSMA-expressing target cells after culture in cytokine-free complete cell culture medium. After 7 days of culture under "starvation conditions," CAR T cells were co-cultured with SupT1-PSMAhigh and SupT1-PSMAlow target cells (or SupT1-NT cells as a negative control) in effector:target ratios of 1:2 and 1:8. A: FBK after 24-hour incubation using cell fluorescence analysis to indicate target cell viability. B: Secretion of IL-2 and IFNγ by CAR T cells, measured by detecting the supernatant collected from the co-culture after 24 hours by ELISA.

[0030] [Figure 16] Results of FACS-based killing (FBK) assays investigating the ability of monotransduced, duplexed, and triplicate T cells to kill PSMA-expressing target cells in the presence or absence of TGFβ. CAR T cells were co-cultured for 7 days with SupT1-PSMAhigh and SupT1-PSMAlow targets in 1:2 and 1:8 (E:T) ratios, either in the presence or absence of 10 ng / ml TGFβ1 (SupT1-NT was used as a control). Target cell killing was quantified by FACS and normalized to target only.

[0031] [Figure 17] Results of FACS-based cell killing (FBK) assays investigating the ability of single, double, and triple transduced T cells to kill PSMA-expressing target cells after repeated restimulation with target cells. Transduced T cells were co-cultured with SupT1-PSMAhigh or SupT1-PSMAlow target cells in a 1:1 (E:T) ratio. CAR T cells were re-stimulated with 5 × 10⁴ SupT1 cells every 7 days. After quantifying target cell killing by FACS, each group was re-stimulated.

[0032] [Figure 18A-1]Figure 18. In vivo assay investigating the antitumor activity of T cells transduced with a triple vector composition via intravenous administration in a prostate cancer xenograft model of NSG mice. 5 × 10⁶ PSMA-positive PC3 human cell line cells were injected into the flank of female NSG mice. The xenografts were immobilized for 3 weeks until stable engraftment could be detected by palpation and calipas measurement. CAR T cells were administered intravenously at a dose of 1 × 10⁶ CAR T cells / mouse. Calipas measurements were performed 2-3 times per week. A: Data for mice administered cells produced by single transduction using vector A ("AUTO7 / A"), double transduction using vectors A and B ("AUTO7 / AB"), triple transduction using vectors A, B, and C ("AUTO7 / ABC"); or cells produced by second-generation CARs ("parents") developed using the same anti-PSMA binder 7A12; B: Summary of data shown in Figure 18A. [Figure 18A-2] Figure 18. In vivo assay investigating the antitumor activity of T cells transduced with a triple vector composition via intravenous administration in a prostate cancer xenograft model of NSG mice. 5 × 10⁶ PSMA-positive PC3 human cell line cells were injected into the flank of female NSG mice. The xenografts were immobilized for 3 weeks until stable engraftment could be detected by palpation and calipas measurement. CAR T cells were administered intravenously at a dose of 1 × 10⁶ CAR T cells / mouse. Calipas measurements were performed 2-3 times per week. A: Data for mice administered cells produced by single transduction using vector A ("AUTO7 / A"), double transduction using vectors A and B ("AUTO7 / AB"), triple transduction using vectors A, B, and C ("AUTO7 / ABC"); or cells produced by second-generation CARs ("parents") developed using the same anti-PSMA binder 7A12; B: Summary of data shown in Figure 18A. [Figure 18B]Figure 18. In vivo assay investigating the antitumor activity of T cells transduced with a triple vector composition via intravenous administration in a prostate cancer xenograft model of NSG mice. 5 × 10⁶ PSMA-positive PC3 human cell line cells were injected into the flank of female NSG mice. The xenografts were immobilized for 3 weeks until stable engraftment could be detected by palpation and calipas measurement. CAR T cells were administered intravenously at a dose of 1 × 10⁶ CAR T cells / mouse. Calipas measurements were performed 2-3 times per week. A: Data for mice administered cells produced by single transduction using vector A ("AUTO7 / A"), double transduction using vectors A and B ("AUTO7 / AB"), triple transduction using vectors A, B, and C ("AUTO7 / ABC"); or cells produced by second-generation CARs ("parents") developed using the same anti-PSMA binder 7A12; B: Summary of data shown in Figure 18A. [Overview of the Initiative]

[0033] Summary of the embodiments of the invention The inventors have developed a combination approach to address the problem of heterogeneity in tumor cells and the microenvironment in CAR therapy.

[0034] When cells are transduced simultaneously with multiple vectors, the resulting product is a mixture of cells transduced individually and in combination. For example, when cells are transduced with two vectors (a vector containing transgene A and a vector containing transgene B), the transduced cells are a mixture of cells expressing only A; cells expressing only B; and cells expressing both A and B (Figure 5B). For cells transduced with three vectors, each containing a transgene, the resulting transduced cells are a mixture of: A only; B only; C only; A and B; A and C; B and C; and cells expressing A, B, and C.

[0035] The present invention involves using a mixture of therapeutic CAR-T cell products, etc. Using a combination product provides a unique flexibility that enhances the product's ability to adapt to differences in target cells and the tumor microenvironment.

[0036] For example, a vector may encode a combination of different CARs (e.g., their antigen-binding domain and / or co-stimulatory domains may differ). Alternatively, one or more vectors may encode activity regulators that modulate the activity of CARs, CAR-expressing cells, or target cells. When a combined CAR T cell composition is administered in vivo, the cells migrate to different tumor sites in the body. Any CAR-T cell subpopulation expressing a particular combination of CAR(s) and activity regulator(s) will have an optimal ability to survive, persist, and kill target cells at the tumor site, thus possessing a selective advantage over other subpopulations in the product and overcoming these subpopulations. Thus, the CAR-T cell product can adapt to tumor heterogeneity between patients and between sites within the same patient.

[0037] Furthermore, by using this method to analyze patients and investigate which subpopulations of CAR-T cells exhibit the best survival and / or activity within the patient, it is possible to confirm which vector combinations are optimal for generating CAR-T cells for the treatment of a particular disease or subtype of disease.

[0038] Accordingly, in a first aspect, the present invention provides a method for preparing a cell composition, comprising the step of transducing a population of cells with a mixture of at least two viral vectors, wherein at least one vector comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR).

[0039] The method of the present invention can be equally applied to cells expressing the engineered T cell receptor. Furthermore, all of the following aspects and embodiments are applicable to engineered TCR-expressing cells.

[0040] The mixture may contain two, three, four, five, or more viral vectors.

[0041] Two or more viral vectors in the mixture may each contain a nucleic acid sequence encoding a CAR. The first CAR and the second CAR may have different antigen-binding domains and / or different spacers and / or different end domains.

[0042] A nucleic acid encoding one or more CARs in a viral vector may encode two or more CARs. For example, a nucleic acid may encode a CAR logic gate (such as an OR gate).

[0043] The present invention provides a method for preparing a cell composition, comprising the step of transducing a population of cells with a mixture of at least two viral vectors, wherein at least one vector comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR); and wherein at least one vector comprises a nucleic acid encoding an activity regulator that modulates the activity of the aforementioned CAR, a cell expressing the aforementioned CAR, or a target cell.

[0044] The technology of the present invention is equally applicable to adoptive cell therapy cells (such as tumor-infiltrating lymphocytes (TILs)) that do not express CARs or manipulated TCRs, insofar as they relate to the expression of activity regulators (may include multiple factors). Furthermore, all aspects and embodiments described below are generally applicable to therapeutic T cells (such as TILs), insofar as they relate to the expression of activity regulators (may include multiple factors).

[0045] One or more viral vectors in the mixture may contain nucleic acid sequences encoding both the CAR and the regulatory factor, such that cells transduced with this vector co-express the CAR and the regulatory factor.

[0046] Activity regulators that modulate CAR activity can influence the balance between phosphorylation and dephosphorylation at CAR-expressing cell:target cell synapses. For example, activity regulators may include kinase domains capable of phosphorylating the immune receptor-activated tyrosine motif (ITAM) in the CAR endodomain.

[0047] Alternatively, the regulatory factor may be able to recruit the kinase to the vicinity of the CAR (the site where the kinase can phosphorylate ITAM in the CAR end domain).

[0048] Activity regulators that modify the activity of CAR-expressing cells may be intracellular molecules or expressed on the cell surface.

[0049] In vivo, membrane-bound immunosuppressive receptors (such as CTLA4, PD-1, LAG-3, 2B4, or BTLA1) inhibit T cell activation. Activation modifiers can block or influence this inhibitory pathway.

[0050] The regulatory factors can be action factors such as antibodies that bind to inhibitory immune receptors or to ligands of inhibitory immune receptors.

[0051] Activity modifiers that block or mitigate inhibition mediated by inhibitory immune receptors (such as CTLA4, PD-1, LAG-3, 2B4, or BTLA1) can disrupt the balance of phosphorylation and dephosphorylation at T cell-target cell synapses, thereby favoring ITAM phosphorylation and potentially leading to T cell activation. For example, activity modifiers may block or mitigate ITAM phosphorylation in the endodmain of the inhibitory receptor(s), or block or mitigate ITAM dephosphorylation in the CAR signaling domains mediated by proteins such as SHP-1 and SHP-2.

[0052] The activity regulator may be a dominant-negative SHP-1 or SHP-2.

[0053] For example, the activity regulator may be a truncated protein lacking a phosphatase domain, but containing an SH2 domain derived from a protein (such as SHP-1 or SHP-2) that binds to a phosphorylated immunoreceptor-suppressive tyrosine motif (ITIM).

[0054] The activity regulators may be cytokines or chemokines such as IL12, flexiIL-12, GM-CSF, IL7, IL15, IL21, IL2, or CCL19.

[0055] Alternatively, the activity regulator may affect the cytokine / chemokine signaling pathway in CAR-expressing cells.

[0056] For example, an activity regulator may be a chimeric cytokine receptor containing a cytokine receptor endodomain. The exodomain may originate from a different cytokine receptor or not from a cytokine receptor at all. The exodomain can bind to a ligand (e.g., a tumor antigen or secretory factor). In the presence of a ligand, the two chains of the cytokine receptor endodomain can associate to initiate cytokine signaling.

[0057] The regulatory factor may be a constitutively active chimeric cytokine receptor. The regulatory factor may contain two chains in which two cytokine receptor endodomains spontaneously dimerize together in the presence of an activator (a chemical inducer of dimerization, i.e., CID).

[0058] Activity regulators may affect the JAK / STAT cytokine signaling pathway. Activity regulators may include inducibly or constitutively active signaling and transcriptional activators (STATs) or Janus kinases (JAKs).

[0059] The regulatory factors may be or may include adhesion molecules or transcription factors. Transcription factors may prevent or mitigate the differentiation and / or exhaustion of CAR-expressing cells.

[0060] The activity regulator of the present invention can regulate TGFβ signaling.

[0061] For example, a regulatory factor may block or reduce TGFβ binding to TGFβ receptors; a regulatory factor may compete with TGFβ or TGFβR for binding to TGFβR or TGFβ; or a regulatory factor may modulate downstream TGFβ signaling, for example, via SMAD. The regulatory factor may be a dominant-negative TGFβ receptor.

[0062] The activity regulator of the present invention can provide a co-stimulatory signal to T cells.

[0063] For example, an activity modulator could be a TNF receptor, a chimeric TNF receptor, or a TNF receptor ligand.

[0064] Activity regulators can modulate the activity of target cells (e.g., tumor cells).

[0065] The activating factor may be a toxin, a prodrug, or a prodrug-activating compound.

[0066] Activity regulators can be enzymes that, when expressed intracellularly or in combination, can synthesize small molecules. When such enzymes or combinations of enzymes are expressed in CAR-expressing cells, they can confer the ability to synthesize small molecules (such as small molecules toxic to tumor cells) on the aforementioned cells.

[0067] Alternatively, the regulatory factor may be an enzyme secreted by CAR-expressing cells. The regulatory factor may be one or more enzymes that, when secreted or expressed on the cell surface, deplete extracellular molecules of the manipulated cell, the aforementioned molecules being: (i) necessary for tumor cells to survive, proliferate, metastasize or exhibit chemoresistance, and / or (ii) It is detrimental to the survival, proliferation, or activity of the manipulated cells.

[0068] Enzymes can deplete, for example, amino acids or amino acid metabolites, nucleic acid bases (such as nucleosides or nucleotides), or lipids.

[0069] In the method of the present invention, the mixture of viral vectors may include at least one vector comprising a nucleic acid sequence encoding a dominant-negative SHP-1 or SHP-2; and at least one vector comprising a nucleic acid sequence encoding a dominant-negative transforming growth factor (TGF) β receptor.

[0070] A mixture of viral vectors may comprise two, three, four, five, or six viral vectors, at least one of which comprises a nucleic acid sequence encoding a CAR; and at least one of which comprises a nucleic acid sequence encoding an activity regulator.

[0071] The present invention may include the following steps: (i) Transducing a population of cells with a mixture of at least two viral vectors; and (ii) A step of selecting CAR-expressing cells from the aforementioned transduced cell population derived from step (i).

[0072] Alternatively, if each viral vector in the mixture contains a nucleic acid sequence encoding a CAR, it may not be necessary to select or purify CAR-expressing cells from the transduced cell population.

[0073] In a second aspect, the present invention provides a viral vector composition. The viral vector composition may comprise a mixture of two or more vectors. The vector composition may be suitable for use in the method of the first aspect of the present invention.

[0074] The viral vector composition may include a first vector and a second vector, both of which contain nucleic acid sequences encoding a chimeric antigen receptor (CAR).

[0075] The CAR expressed by the first vector may be identical to the CAR expressed by the second vector. For example, the CAR expressed by the first vector may have the same antigen-binding domain as the CAR expressed by the second vector.

[0076] Furthermore, the first vector and / or the second vector may express activity regulators that modulate the activity of CARs, cells expressing CARs, or target cells. If both the first and second vectors express activity regulators, they may express different activity regulators or different combinations of activity regulators.

[0077] For example, the first and second vectors may express one or more activity regulators selected from dominant-negative SHP-1 or SHP-2; dominant-negative transforming growth factor (TGF) β receptors; and constitutively active chimeric cytokine receptors.

[0078] In one configuration, the first vector may include a nucleic acid sequence encoding a dominant-negative SHP-1 or SHP-2 and a nucleic acid sequence encoding a dominant-negative transforming growth factor (TGF) β receptor; the second vector may include a nucleic acid sequence encoding a constitutively active chimeric cytokine receptor.

[0079] If the first and second vectors encode the same CAR, the CAR may have an antigen-binding domain that binds to disialoganglioside (GD2).

[0080] The first vector and / or the second vector may contain a nucleic acid sequence encoding a suicide gene.

[0081] Furthermore, the present invention provides cell compositions prepared by the method of the present invention or by transducing cells ex vivo with the vector composition of the present invention.

[0082] In a third aspect, the present invention provides a cell composition prepared by the method of the first aspect of the present invention or by transducing a population of cells with a viral vector composition of the second aspect of the present invention.

[0083] A fourth aspect provides a method for treating a disease in a subject, comprising the step of administering a cell composition according to a third aspect of the present invention to the subject.

[0084] A fifth aspect provides a cell composition according to a third aspect of the present invention for use in treating and / or preventing diseases.

[0085] A sixth aspect provides the use of a cell composition according to the third aspect of the present invention in the manufacture of a pharmaceutical for the treatment and / or prevention of disease.

[0086] A seventh aspect relates to a method for determining the optimal combination of components of CAR-expressing cells for treating a disease, comprising the following steps: (i) The step of administering the cell composition according to the second aspect of the present invention to a subject having the aforementioned disease; (ii) Monitoring the aforementioned patient or a sample derived from the aforementioned patient to determine which cell subpopulation in the aforementioned cell composition exhibits the highest level of engraftment and / or proliferation; and (iii) A step to analyze the phenotype / genotype of the aforementioned cells in the subpopulation and to identify and analyze the CAR(s) and / or regulatory factors(s) expressed by these aforementioned cells. This provides a method that includes [something].

[0087] Further aspects The present invention also provides further embodiments, which are organized into the following numbered paragraphs. 1. A nucleic acid construct comprising a nucleic acid sequence encoding a dominant-negative SHP-2 receptor and a nucleic acid sequence encoding a dominant-negative TGFβ receptor. 2. The nucleic acid construct described in paragraph 1 having the following structure: dnSHP-coexpr-dnTGFβR, or dnTGFβR-coexpr-dnSHP Here, dnSHP is a nucleic acid sequence that encodes dominant-negative SHP-2, "coexpr" is a nucleic acid sequence that can co-express two polypeptides as separate entities. "dnTGFβR" is a nucleic acid sequence that codes for the dominant-negative TGFβ receptor. 3. The nucleic acid construct described in paragraph 1, including the nucleic acid sequence encoding CAR. 4. The nucleic acid construct described in paragraph 3 having the following structure: CAR-coexpr1-dnSHP-coexpr2-dnTGFβR CAR-coexpr1-dnTGFβR-coexpr2-dnSHP dnTGFβR-coexpr1-CAR-coexpr2-dnSHP dnTGFβR-coexpr1-dnSHP-coexpr2-CAR dnSHP-coexpr1-dnTGFβR-coexpr2-CAR or dnSHP-coexpr1-CAR-coexpr2-dnTGFβR Here, dnSHP is a nucleic acid sequence that encodes dominant-negative SHP-2, "coexpr1" and "coexpr2" may be the same or different, and are nucleic acid sequences that can co-express three polypeptides as separate entities. "dnTGFβR" is a nucleic acid sequence that encodes a dominant-negative TGFβ receptor; "CAR" is a nucleic acid sequence that codes for a chimeric antigen receptor. 5. The nucleic acid construct described in paragraph 4 having the following structure: dnSHP-coexpr1-CAR-coexpr2-dnTGFβR. 6. A nucleic acid construct described in any of paragraphs 3-5, wherein the CAR binds to one of the following target antigens: CD19, CD22, BCMA, PSMA, CD79, GD2, or FCRL5. 7. A nucleic acid construct described in paragraph 3, comprising two bisistronic nucleic acid sequences encoding CARs. 8. The nucleic acid construct described in paragraph 7 having the following structure: dnSHP-coexpr1-CAR1-coexpr2-CAR2-coexpr3-dnTGFβR Here, "dnSHP" is a nucleic acid sequence that encodes dominant-negative SHP-2. "coexpr1," "coexpr2," and "coexpr3" may be the same or different nucleic acid sequences that can co-express four polypeptides as separate entities; "CAR1" is a nucleic acid sequence that encodes the first chimeric antigen receptor; "CAR2" is a nucleic acid sequence that encodes a second chimeric antigen receptor; "dnTGFβR" is a nucleic acid sequence that codes for the dominant-negative TGFβ receptor. 9. The nucleic acid construct described in paragraph 7 or 8, wherein one CAR binds to CD19 and the other CAR binds to CD22. 10. CAR is a nucleic acid construct described in paragraph 6 that binds to CD19 and has an antigen-binding domain including the following: a) Heavy chain variable region (VH) having a complementarity-determining region (CDR) with the following sequence: CDR1-GYAFSSS(Sequence ID 1); CDR2-YPGDED (Sequence ID 2) CDR3-SLLYGDYLDY(Sequence ID 3); and b) Light chain variable region (VL) having a CDR having the following sequence: CDR1-SASSSVSYMH(Sequence ID 4); CDR2-DTSKLAS (Sequence ID 5) CDR3-QQWNINPLT (Sequence ID 6). 11. The nucleic acid construct described in paragraph 10, wherein the antigen-binding domain comprises a VH domain having the sequence shown as Sequence ID No. 7 and a VL domain having the sequence shown as Sequence ID No. 8. 12. A nucleic acid construct described in any of the preceding paragraphs, including a nucleic acid sequence that codes for a suicide gene. 13. A vector containing a nucleic acid construct as described in any of the preceding paragraphs. 14. A vector kit comprising the first vector described in paragraph 13 and a second vector containing a nucleic acid sequence encoding a chimeric antigen receptor (CAR) or activity regulator. 15. A vector kit comprising a first vector containing a nucleic acid sequence encoding a dominant-negative SHP-2 receptor and a second vector containing a nucleic acid sequence encoding a dominant-negative TGFβ receptor. 16. A vector kit as described in paragraph 15, wherein the first vector contains a nucleic acid sequence encoding a first CAR, and the second vector contains a nucleic acid sequence encoding a second CAR. 17. A kit of the vectors described in paragraph 16, wherein the first and second CARs have the same target antigen. 18. A kit of the Vector described in paragraph 16, in which the first and second CARs are identical. 19. A vector kit comprising the first vector described in paragraph 12 and a second vector encoding a chimeric cytokine receptor (CCR). 20. A vector kit as described in paragraph 19, wherein the first vector contains a nucleic acid sequence encoding a first CAR, and the second vector contains a nucleic acid sequence encoding a second CAR. 21. A kit of the vectors described in paragraph 20, wherein the first and second CARs have the same target antigen. 22. A kit of the Vector described in paragraph 20, in which the first and second CARs are identical. 23. A vector kit as described in any of paragraphs 14-22, wherein the first vector contains a nucleic acid sequence encoding a first suicide gene, and the second vector contains a nucleic acid sequence encoding a second suicide gene. 24. A kit of the vector described in paragraph 23, in which the first and second suicide genes are induced by the same molecule. 25. A kit of the vector described in paragraph 23, in which the first and second suicide genes are induced by different molecules. 26. A kit of the vector described in any of paragraphs 23-25, wherein the vector has the following structure: Vector 1:CAR1-coexpr1-SG1-coepr2-dSHP2-coexpr3-dnTGFβR Vector 2: CAR2-coexpr4-SG2-coepr5-CCR Here, "CAR1" is a nucleic acid sequence that encodes the first chimeric antigen receptor; "coexpr1", "coexpr2", "coexpr3", "coexpr4", and "co-expr5" are nucleic acid sequences that can be identical or different, and can co-express seven polypeptides as individual entities; "SG1" is the nucleic acid sequence that codes for the first suicide gene; "dnSHP" is a nucleic acid sequence that encodes dominant-negative SHP-2; "dnTGFβR" is a nucleic acid sequence that encodes a dominant-negative TGFβ receptor; "CAR2" is a nucleic acid sequence that encodes a second chimeric antigen receptor, which may or may not be identical to CAR1; "SG2" is a nucleic acid sequence that codes for a first suicide gene, which may or may not be identical to SG1; "CCR" is a nucleic acid sequence that codes for a chimeric cytokine receptor. 27. A kit of the vectors described in any of paragraphs 14-26, including a third vector containing a nucleic acid sequence encoding a cytokine. 28. A kit of the vector described in paragraph 27, wherein the cytokine is IL-12 or Flexi-IL12. 29. A kit of the vector described in paragraph 28 having the following structure: Vector 1: dnSHP2-coexpr1-SG1-coepr2-CAR-coexpr3-dnTGFβR Vector 2: CCR Vector 3:SG2-coexpr4-flexiIL12 Here, "dnSHP" is a nucleic acid sequence that encodes dominant-negative SHP-2; "coexpr1", "coexpr2", "coexpr3", and "coexpr4" may be the same or different nucleic acid sequences that can co-express the six polypeptides on vectors 1 and 3 as separate entities; "SG1" is the nucleic acid sequence that codes for the first suicide gene; "CAR" is a nucleic acid sequence that codes for a chimeric antigen receptor; "dnTGFβR" is a nucleic acid sequence that encodes a dominant-negative TGFβ receptor; "CCR" is a nucleic acid sequence that codes for a chimeric cytokine receptor; "SG2" is a nucleic acid sequence that codes for a first suicide gene, which may or may not be identical to SG1; "flexiIL12" is the nucleic acid sequence that codes for flexi-IL-12. 30. A vector composition comprising the following mixtures: the vector described in paragraph 13 and at least one other viral vector; the first and second vectors as defined in any of paragraphs 14 to 26; or the first, second, and third vectors as defined in any of paragraphs 27 to 29. 31. A method for preparing a cell composition, comprising the step of transducing a population of cells with the vector described in paragraph 13, the vector kit described in any of paragraphs 14 to 29, or the vector composition described in paragraph 30. 32. Cells co-expressing dominant-negative SHP-2 and dominant-negative TGFβ receptors. Cells as described in paragraph 32, which also express 33.1 or more chimeric antigen receptors (CARs(or more)). 34. A cell described in paragraph 33, in which CAR(or CAR) is defined as in any of paragraphs 6-11. 35. A cell composition comprising multiple cells prepared by the method described in paragraph 31 or described in any of paragraphs 32 to 34. 36. A method for treating and / or preventing a disease, comprising the step of administering a cell composition described in paragraph 35 to a subject. 37. The method described in paragraph 36, wherein the disease is cancer. 38. Cell compositions described in paragraph 35 for use in the treatment and / or prevention of disease. 39. Use of cells described in any of paragraphs 32-34 in the manufacture of a medicament for the treatment and / or prevention of disease. 40. A nucleic acid construct comprising a nucleic acid sequence encoding a dominant-negative TGFβ receptor; a nucleic acid sequence encoding IL7; and a nucleic acid sequence encoding CCL19. 41. The nucleic acid construct described in paragraph 40 having the following structure: dnTGFβR-coexpr1-IL7-coexpr2-CCL19; dnTGFβR-coexpr1-CCL19-coexpr2-IL7; IL7-coexpr1-CCL19-coexpr2-dnTGFβR; IL7-coexpr1-dnTGFβR-CCL19-coexpr2; CCL19-coexpr1-IL7-coexpr2-dnTGFβR; or CCL19-coexpr1-dnTGFβR-coexpr2-IL7 Here, "dnTGFβR" is a nucleic acid sequence that encodes a dominant-negative TGFβ receptor; "IL7" is the nucleic acid sequence that codes for IL7. "CCL19" is the nucleic acid sequence that codes for CCL19. "coexpr1" and "coexpr2" may be the same or different nucleic acid sequences that can co-express three polypeptides as separate entities. 42. A nucleic acid construct described in paragraph 40, including a nucleic acid sequence that codes for CAR. 43. The nucleic acid construct described in paragraph 3 having the following structure: CAR-coexpr1-dnTGFβR-coexpr2-IL7-coexpr3-CCL19; Here, "dnTGFβR" is a nucleic acid sequence that encodes a dominant-negative TGFβ receptor; "IL7" is the nucleic acid sequence that codes for IL7. "CCL19" is the nucleic acid sequence that codes for CCL19. "CAR" is a nucleic acid sequence that codes for a chimeric antigen receptor. "coexpr1," "coexpr2," and "coexpr3" are nucleic acid sequences that may be identical or different, and can co-express four polypeptides as separate entities. 44. A nucleic acid construct described in any of paragraphs 3-5, wherein CAR is bound to GD2. 45. A vector containing a nucleic acid construct as described in any of paragraphs 40-44. 46. ​​A vector kit comprising a first vector containing nucleic acid sequences encoding IL7 and CCL19, and a second vector containing nucleic acid sequences encoding a dominant-negative TGFβ receptor. 47. A kit of the vectors described in paragraph 46, in which both vectors also contain nucleic acid sequences encoding chimeric antigen receptors (CARs). 48. A kit of the vectors described in paragraph 47, wherein the CAR coded by the first vector is identical to the CAR coded by the second vector. 49. The CAR is combined with the GD2, as described in paragraph 48 of the Vector kit. 50. A method for preparing a cell composition, comprising the step of transducing a population of cells with the vector described in paragraph 45 or a vector kit described in any of paragraphs 46-49. 51. Cells co-expressing dominant-negative TGFβ receptor, IL7, and CCL19. Cells as described in paragraph 51, which also express 52.1 or more chimeric antigen receptors (CARs(or CARs)). 53. A cell composition comprising a plurality of cells prepared by the method described in paragraph 46 or described in paragraph 51 or 52. 54. A method for treating and / or preventing a disease, comprising the step of administering a cell composition described in paragraph 49 to a subject. 55. The method described in paragraph 54, where the disease is cancer. 56. Cell compositions described in paragraph 53 for use in the treatment and / or prevention of disease. 57. Use of cells as described in paragraph 51 or 52 in the manufacture of a medicament for the treatment and / or prevention of disease.

[0088] In the paragraphs above and in the claims below, elements encoding polypeptides of nucleic acid constructs or vectors (such as "dnSHP", "dnTGFβR", "IL7", "CCL19", and "CAR") may be present in the construct in any order.

[0089] The following detailed description applies equally to the embodiments of the present invention described in the above paragraphs relating to the claims, insofar as it relates to nucleic acids and polypeptide sequences, polypeptide components, vectors, cells, methods, etc. [Modes for carrying out the invention]

[0090] Detailed explanation The present invention relates to a method for preparing a cell composition, comprising the step of transducing a population of cells with a mixture of at least two viral vectors.

[0091] Viral vectors can be, for example, retroviral vectors or lentiviral vectors.

[0092] Retroviruses are double-stranded RNA enveloped viruses whose primary characteristic is their ability to "reverse transcribe" their genome from RNA to DNA. Virians are 100–120 nm in diameter and contain a dimeric genome of identical RNA plus strands complexed with a nucleocapsid protein. The genome is encapsulated within a protein capsid that also contains enzyme proteins necessary for viral infection (i.e., reverse transcriptase, integrase, and protease). Matrix proteins form a layer outside the capsid core that interacts with the envelope (a lipid bilayer derived from the host cell membrane that surrounds the viral core particle). Viral envelope glycoproteins anchor on this bilayer and are responsible for recognizing specific receptors on the host cell and initiating the infection process. The envelope protein is composed of two subunits: a transmembrane (TM) subunit that anchors the protein within the lipid membrane and a surface (SU) subunit that binds to cell receptors.

[0093] Based on their genome structure, retroviruses are classified into simple retroviruses (such as MLV and murine leukemia virus) or complex retroviruses (such as HIV and EIAV). Retroviruses encode four genes: gag (group-specific antigen), pro (protease), pol (polymerase), and env (envelope). The gag sequence encodes three main structural proteins: matrix protein, nucleocapsid protein, and capsid protein. The pro sequence encodes a protease responsible for particle assembly, budding, and cleavage of Gag and Gag-Pol during maturation. The pol sequence encodes enzymes (reverse transcriptase and integrase; the former catalyzes the reverse transcription of the viral genome from RNA to DNA during infection, and the latter is responsible for the integration of proviral DNA into the host cell genome). The env sequence encodes both the SU and TM subunits of the envelope glycoprotein. Furthermore, retroviral genomes contain non-coding sequences such as: two LTRs (long-terminal repeats) (containing elements necessary to drive gene expression, reverse transcription, and integration into the host cell's chromosome); a sequence called a packaging signal (ψ) necessary for the specific packaging of viral RNA into newly formed virions; and polyprint lactates (PPTs) that function as sites for initiating positive-strand DNA synthesis during reverse transcription. In addition to gag, pro, pol, and env, complex retroviruses (such as lentiviruses) have accessory genes (including vif, vpr, vpu, nef, tat, and rev) that regulate viral gene expression, assembly of infection particles, and mediate viral replication in infected cells.

[0094] During the infection process, retroviruses first attach to specific cell surface receptors. Upon entering a susceptible host cell, the retroviral RNA genome is then copied into DNA by the virus-encoded reverse transcriptase contained within the parent virus. This DNA is transported to the host cell nucleus, where it is subsequently integrated into the host genome. At this stage, it is typically called a provirus. Proviruses remain stable within the host chromosome during cell division and are transcribed like other cellular proteins. Proviruses encode proteins and packaging mechanisms necessary for the creation of further viruses and can leave the cell through a process known as "budding."

[0095] When enveloped viruses, such as retroviruses and lentiviruses, bud from host cells, these viruses become part of the host cell's lipid membrane. Thus, host cell-derived membrane proteins become part of the retroviral particle. This invention utilizes this process to introduce a target protein into the envelope of a viral particle.

[0096] Viral vector Retroviruses and lentiviruses may be used as vectors or delivery systems for introducing nucleic acid sequences or multiple nucleic acid sequences into target cells. Transfer can be performed in vitro, ex vivo, or in vivo. When used in this manner, the virus is typically referred to as a viral vector.

[0097] Gamma-retroviral vectors, commonly known as retroviral vectors, were the first viral vectors used in clinical trials of gene therapy in 1990 and remain one of the most widely used. More recently, lentiviral vectors derived from complex retroviruses (such as human immunodeficiency virus (HIV)) have attracted attention because they can transduce non-dividing cells. The most attractive features of retroviral and lentiviral vectors as gene transfer tools include their ability to load large genes (up to 9kb), minimal immune response in patients, high transduction efficiency in vivo and in vitro, and the ability to sustainably modify the genetic content of target cells and maintain the expression of the delivered gene for extended periods.

[0098] Retroviral vectors can be based on any suitable retrovirus capable of delivering genetic information to eukaryotic cells. For example, retroviral vectors may be alpha-retroviral vectors, gamma-retroviral vectors, lentiviral vectors, or spuma-retroviral vectors. Such vectors are widely used in gene therapy and other gene delivery applications.

[0099] The viral vector of the present invention may be a retroviral vector (such as a gamma-retroviral vector). The viral vector may be based on human immunodeficiency virus.

[0100] The viral vector of the present invention may be a lentiviral vector. The vector may be based on a non-primate lentivirus (such as equine infectious anemia virus (EIAV)).

[0101] nucleic acid sequences and constructs In the mixture of viral vectors used in the method of the present invention, each vector may contain one or more nucleic acid sequences. For example, one or more vectors in the mixture may contain a nucleic acid construct containing multiple co-expressed nucleic acid sequences. The nucleic acid construct may be, for example, bicistronic or tricistronic. The nucleic acid construct may contain two, three, four, or five transgenes.

[0102] Nucleic acid sequences in a nucleic acid construct can be separated by “co-expression” sequences that can be individually expressed intracellularly or on the cell once two or more polypeptides have been translated.

[0103] A co-expression sequence may encode a cleavage site such that the nucleic acid construct produces and contains two or more polypeptides linked by the cleavage site(s). The cleavage site(s) may be self-cleavable such that, upon polypeptide production, they are immediately cleaved into individual polypeptides without requiring any external cleavage activity.

[0104] The cleavage site can be any sequence that allows two or more polypeptides to be separated.

[0105] The term "cleavage" is used herein for convenience, but depending on the cleavage site, polypeptides can be separated into individual entities by mechanisms other than classical cleavage. For example, for the foot-and-mouth disease virus (FMDV) 2A autocleavage peptide (see below), various models have been proposed to explain the following "cleavage" activity: proteolytic activity by host cell proteinases, autoprotein degradation, or translational effect (Donnelly et al. (2001) J. Gen. Virol. 82:1027-1041). The exact mechanism of such "cleavage" is not important to the purposes of the present invention, as long as the protein is expressed as an individual entity when the cleavage site is located between the nucleic acid sequences encoding the protein.

[0106] The cleavage site may be a furin cleavage site.

[0107] Furin is an enzyme belonging to the subtilisin-like proprotein convertase family. Members of this family are proprotein convertases that process potential precursor proteins into their biologically active products. Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at its paired basic amino acid processing site. Examples of furin substrates include proparathyroid hormone, transforming growth factor beta-1 precursor, proalbumin, pro-beta-secretase, type 1 membrane matrix metalloproteinase, pro-nerve growth factor beta subunit, and von Willebrand factor. Furin cleaves proteins immediately downstream of the basic amino acid target sequence (normatively, Arg-X-(Arg / Lys)-Arg') (SEQ ID NO: 58) and is enriched in the Golgi apparatus.

[0108] The cleavage site may be a site for cleavage of the tobacco eczema virus (TEV).

[0109] TEV proteases are highly sequence-specific cysteine ​​proteases and chymotrypsin-like proteases. TEV proteases are highly specific to their target cleavage site and are therefore frequently used to control the cleavage of fusion proteins both in vitro and in vivo. The consensus TEV cleavage site is ENLYFQ\S (where "\" indicates the peptide bond to be cleaved) (SEQ ID NO: 59). Mammalian cells (such as human cells) do not express TEV proteases. Therefore, in embodiments in which this nucleic acid construct contains a TEV cleavage site and is expressed in mammalian cells, the exogenous TEV protease must also be expressed in mammalian cells.

[0110] The cleavage site may encode a self-cleaving peptide.

[0111] A "self-cleaving peptide" refers to a peptide that, when a polypeptide containing a protein or self-cleaving peptide is produced, functions to be immediately "cleaved" or separated into individual and isolated first and second polypeptides without requiring any external cleavage activity.

[0112] Self-cleaving peptides can be 2A self-cleaving peptides derived from aphthoviruses or cardioviruses. Primary 2A / 2B cleavage in aphthoviruses and cardioviruses is mediated by 2A "cleavage" at its own C-terminus. In aphthoviruses (such as foot-and-mouth disease virus (FMDV) and equine rhinitis A virus), the 2A region is a short segment of about 18 amino acids that, together with the N-terminal residue (conserved proline residue) of protein 2B, exhibits an autonomous element capable of mediating its own C-terminus "cleavage" (Donelly et al. (2001) above).

[0113] "2A-like" sequences have been found in picornaviruses other than aftviruses or cardioviruses, "picornavirus-like" insect viruses, type C rotaviruses, and repetitive sequences in Trypanosoma spp and bacterial sequences (Donnelly et al. (2001) above).

[0114] The cleavage site may include a 2A-like sequence as shown in Sequence ID No. 9. Sequence ID 9 RAEGRGSLLTCGDVEENPGP

[0115] The present invention provides nucleic acid constructs comprising nucleic acid sequences encoding a dominant-negative SHP-1 or SHP-2 receptor and a dominant-negative TGFβ receptor receptor.

[0116] Dominant-negative SHP-1 or SHP-2 and TGFβ receptors are described in more detail below.

[0117] Nucleic acid constructs may have the following structures: dnSHP-coexpr-dnTGFβR, or dnTGFβR-coexpr-dnSHP Here, dnSHP is a nucleic acid sequence that encodes a dominant-negative SHP-1 or SHP-2. "coexpr" is a nucleic acid sequence that can co-express two polypeptides as separate entities. dnTGFβR is a dominant-negative TGFβ receptor.

[0118] Furthermore, the nucleic acid construct may contain a nucleic acid sequence that codes for CAR. In that case, the nucleic acid construct may have the following structure: CAR-coexpr1-dnSHP-coexpr2-dnTGFβR CAR-coexpr1-dnTGFβR-coexpr2-dnSHP dnTGFβR-coexpr1-CAR-coexpr2-dnSHP dnTGFβR-coexpr1-dnSHP-coexpr2-CAR dnSHP-coexpr1-dnTGFβR-coexpr2-CAR or dnSHP-coexpr1-CAR-coexpr2-dnTGFβR Here, dnSHP is a nucleic acid sequence that encodes dominant-negative SHP-2, "coexpr1" and "coexpr2" may be the same or different, and are nucleic acid sequences that can co-express three polypeptides as separate entities. dnTGFβR is a dominant-negative TGFβ receptor; CAR is a nucleic acid sequence that codes for a chimeric antigen receptor.

[0119] Suicide gene Furthermore, nucleic acid constructs may contain nucleic acids that encode suicide genes.

[0120] Since T cells engraft and are autonomous, a means of selectively deleting CAR T cells in a patient is desirable. Suicide genes are gene-encoding mechanisms that selectively destroy injected T cells when faced with unacceptable toxicity. Early clinical experience with suicide genes involved the use of herpesvirus thymidine kinase (HSV-TK) to make T cells ganciclovir-sensitive. HSV-TK is a highly effective suicide gene. However, the use of HSV-TK in highly immunosuppressed clinical situations, such as haplotype-matched stem cell transplantation, may be limited due to pre-formed immune responses. Inducible caspase 9 (iCasp9) is a suicide gene constructed by replacing the activating domain of caspase 9 with a modified FKBP12. iCasp9 is activated by a dimerization-inducing compound (CID) of the normally inactive small molecule. iCasp9 has recently been tested in haplotype-matched HSCT settings and can prevent (abort) GvHD. Since both iCasp9 and HSV-TK are intracellular proteins, when used as the sole transgene, it is possible to select transduced cells by co-expressing them with marker genes.

[0121] WO2016 / 135470 describes suicide genes, including caspase-9, that can be induced to dimerize using rapamycin or a rapamycin analog.

[0122] This suicide gene is sometimes called Rapcasp9 or Rapacasp9 and has the amino acid sequence shown as SEQ ID NO: 80. Sequence ID 80 (Rapcasp9) [ka]

[0123] WO2013 / 153391 describes a marker / suicide gene known as RQR8 that can be detected in expressing cells lysed with the antibody QBEnd10 and the therapeutic antibody rituximab.

[0124] The selection / suicide gene RQR8 has the amino acid sequence shown as sequence number 79. Sequence ID 79 (RQR8) CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV

[0125] By inducing a suicide gene in one or more vectors within the viral vector composition of the present invention, a certain proportion of transduced cells in a subject can be selectively eliminated.

[0126] For example, with respect to two vectors A and B, the transduced cells are a mixture of cells transduced by vector A alone, cells transduced by vector B alone, and cells transduced by both vectors A and B. If vector A expresses or co-expresses a suicide gene, activating the suicide gene will delete cells transduced by vector A alone or by both vectors A and B, but not cells transduced by vector B alone.

[0127] This is particularly useful when one vector in a mixture codes for a potentially dangerous or toxic gene. If a suicide gene is included on the vector cassette, and the patient is experiencing an unacceptable immunological or toxic event, the cells expressing the gene in question can be selectively deleted by inducing the suicide gene. Cells expressing other vector combinations that do not contain the potentially dangerous gene / suicide gene combination will not be deleted and their therapeutic effect can continue.

[0128] For example, a suicide gee may be included in a vector expressing an immunomodulatory cytokine (such as IL-12) or a constitutively active cytokine receptor (see below).

[0129] Viral vector composition The present invention provides a viral vector composition comprising a viral vector mixture. The composition is made by a simple mixture of two or more viral vectors. It can be manufactured. The composition may contain viral vectors between 2 and 10 (e.g., 2, 3, 4, 5, or 6 viral vectors).

[0130] Each viral vector in the mixture may contain one or more transgenes. Two or more viral vectors in the composition may have one or more duplicate transgenes. For example, two viral vectors in the composition may contain the same nucleic acid sequence encoding the same CAR, but may differ in the presence or type of the other nucleic acid sequence encoding the activity regulator(s).

[0131] One or more viral vectors in the composition may contain a nucleic acid sequence encoding a dominant-negative SHP-1 or SHP-2. One or more viral vectors in the composition may contain a nucleic acid sequence encoding a dominant-negative TGFβ receptor. One or more viral vectors in the composition may contain a nucleic acid sequence encoding a chimeric antigen receptor.

[0132] The viral vector composition may include a vector comprising a nucleic acid sequence encoding a dominant-negative SHP-1 or SHP-2 and a nucleic acid sequence encoding a dominant-negative TGFβ receptor.

[0133] A viral vector composition may contain multiple vectors, each encoding a different activity regulator(s) or combination of activity regulators.

[0134] Chimeric antigen receptor In the method of the present invention, at least one vector in the mixture of viral vectors may contain a nucleic acid sequence encoding a chimeric antigen receptor (CAR).

[0135] Chimeric antigen receptor (CAR) The CAR, schematically shown in Figure 1, is a chimeric type I transmembrane protein in which an extracellular antigen-recognition domain (binder) is connected to an intracellular signaling domain (endodomain). The binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but can be based on other forms including an antibody-like antigen-binding site. A spacer domain is usually required to separate the binder from the membrane and allow for proper orientation. A common spacer domain used is the Fc of IgG1. Smaller spacers, such as a stalk derived from CD8α depending on the antigen, or even just the IgG1 hinge, may be sufficient in some cases. The transmembrane domain anchors the protein within the cell membrane and connects the spacer to the endodomain.

[0136] Early CAR designs had endodomains derived from either the γ chain of FcεR1 or the intracellular portion of CD3ζ. Consequently, these first-generation receptors were sufficient to transmit immunological signal 1 and induce T cell killing of congeneral target cells, but they could not sufficiently activate T cells to proliferate and survive. To overcome this limitation, composite endodomains were constructed as follows: fusion of the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3ζ yields a second-generation receptor capable of simultaneously transmitting activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is the CD28 costimulatory domain, which delivers the most potent costimulatory signal (i.e., immunological signal 2 that induces T cell proliferation). Several other receptors have also been described, including those with TNF receptor family endodomains (such as the closely related OX40 and 41BB that transmit survival signals). Here we describe even more potent third-generation CARs with endodomains capable of transmitting activation, proliferation, and survival signals.

[0137] CAR-coding nucleic acids can be introduced into T cells, for example, using retroviral or lentiviral vectors, to generate cancer-specific T cells for adoptive cell transfer. When a CAR binds to a target antigen, this binding transmits an activation signal to the T cells expressing the CAR. Thus, the CAR directs the specificity and cytotoxicity of T cells towards tumor cells expressing the targeted antigen.

[0138] Tandem Car (TanCAR) Bispecific CARs, known as tandem CARs or TanCARs, are developed to simultaneously target two or more cancer-specific markers. In TanCARs, the extracellular domain contains two antigen-binding specificity sites linked by a linker in tandem. Thus, both of the two binding specificity sites (scFvs) are linked to a single transmembrane portion: one scFv is close to the membrane, and the other is distal. When a TanCAR binds to one or both of the target antigens, this transmits an activation signal to the cell expressing the TanCAR.

[0139] Grada et al (2013, Mol Ther Nucleic Acids 2:e105) describes a TanCAR containing a CD19-specific scFv, followed by a Gly-Ser linker, and then a HER2-specific scFv. The HER2-scFv was located near the membrane, while the CD19-scFv was located distally. The TanCAR was shown to induce distinct T-cell reactivity to each of the two tumor-limiting antigens. This arrangement was chosen because the respective lengths of HER2 (632aa / 125Å) and CD19 (280aa, 65Å) were helpful for spatial arrangement. It was also known that the HER2 scFv binds to the four most distal loops of HER2.

[0140] antigen-binding domain The antigen-binding domain is a part of the CAR that recognizes an antigen. Numerous antigen-binding domains are known in the field and include antigen-binding domains based on antigen-binding sites of antibodies, antibody mimetic compounds, and T cell receptors. For example, antigen-binding domains may include: single-chain variable fragments (scFv) derived from monoclonal antibodies; native ligands of target antigens; peptides with sufficient affinity for the target; single-domain antibodies; artificial single binders such as Darpin (engineered ankyrin repeat proteins); or single chains derived from T cell receptors.

[0141] In classical CARs, the antigen-binding domain contains a single-chain variable fragment (scFv) derived from a monoclonal antibody (see Figure 4c). CARs with a domain antibody (dAb) antigen-binding domain or a VHH antigen-binding domain have also been produced (see Figure 4b), or they contain, for example, a Fab fragment of a monoclonal antibody (see Figure 4a). FabCARs consist of two chains: a chain with an antibody-like light chain variable region (VL) and a constant region (CL); and a chain with a heavy chain variable region (VH) and a constant region (CH). One of the chains also contains a transmembrane domain and an intracellular signaling domain. The association between CL and CH assembles the receptor.

[0142] The two chains of Fab CAR may have the following general structure: VH-CH-spacer-transmembrane domain-intracellular signaling domain; and VL-CL or VL-CL-spacer-transmembrane domain-intracellular signaling domain; and VH-CH.

[0143] In Fab-type chimeric receptors, the antigen-binding domain consists of VH derived from one polypeptide chain and VL derived from the other polypeptide chain.

[0144] Polypeptide chains may contain linkers between the VH / VL domain and the CH / CL domain. These linkers exhibit mobility, spatially separating the VH / VL domain from the CH / CL domain. This could help in separating them.

[0145] The antigen-binding domain of CARs can bind to tumor-associated antigens. For example, various tumor-associated antigens (TAAs) are known, as shown in Table 1 below.

[0146] [Table 1]

[0147] Each CAR can bind to one of the following target antigens: CD19, CD22, BCMA, PSMA, GD2, CD79, or FCRL5.

[0148] CD19 The antigen-binding domain of the CAR that binds to CD19 may contain a sequence derived from one of the CD19 binders shown in Table 2.

[0149] [Table 2]

[0150] Alternatively, a CAR that binds to CD19 may have an antigen-binding domain including the following: a) Heavy chain variable region (VH) having a complementarity-determining region (CDR) with the following sequence: CDR1-GYAFSSS(Sequence ID 1); CDR2-YPGDED (Sequence ID 2) CDR3-SLLYGDYLDY(Sequence ID 3); and b) Light chain variable region (VL) having a CDR having the following sequence: CDR1-SASSSVSYMH(Sequence ID 4); CDR2-DTSKLAS (Sequence ID 5) CDR3-QQWNINPLT (Sequence ID 6).

[0151] The antigen-binding domain may include a VH domain having the sequence shown as Sequence ID No. 7, and a VL domain having the sequence shown as Sequence ID No. 8. Sequence ID 7-VH sequence QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSS Sequence ID 8-VL sequence QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKR

[0152] CD22 As described by Haso et al. (Blood;2013;121(7)), CARs that bind to CD22 may have antigenic domains derived from m971, HA22, or BL22.

[0153] Alternatively, a CAR that binds to CD22 may have an antigen-binding domain as described in UK Patent Application No. 1809773.3 (including, for example, the following): a) Heavy chain variable region (VH) having a complementarity-determining region (CDR) with the following sequence: CDR1-NFAMA (Sequence ID 10) CDR2-SISTGGGNTYYRDSVKG (Sequence ID 11) CDR3-QRNYYDGSYDYEGYTMDA(Sequence ID 12); and b) Light chain variable region (VL) having a complementarity-determining region (CDR) with the following sequence: CDR1-RSSQDIGNYLT (Sequence ID 13) CDR2-GAIKLED (Sequence ID 14) CDR3-LQSIQYP (Sequence ID 15)

[0154] The antigen-binding domain of the CD22 CAR may include a VH domain having the sequence shown as Sequence ID No. 16, and a VL domain having the sequence shown as Sequence ID No. 17. Sequence ID 16 EVQLVESGGGLVQPGRSLKLSCAASGFTFSNFAMAWVRQPPTKGLEWVASISTGGGNTYYRDSVKGRFTISRDDAKNTQYLQMDSLRSEDTATYYCARQRNYYDGSYDYEGYTMDAWGQGTSVTVSS Sequence ID 17 DIQMTQSPSSLSASLGDRVTITCRSSQDIGNYLTWFQQKVGRSPRRMIYGAIKLEDGVPSRFSGSRSGSDYSLTISSLESEDVADYQCLQSIQYPFTFGSGTKLEIK

[0155] BCMA Several BCMA-targeted CARs are in clinical development (including bb2121, LCAR-B38M, MCARH171, JCARH125, P-BCMA-101, FCARH143, bb21217, and CT053).

[0156] WO2015 / 052538 describes a BCMA-targeted CAR whose antigen-binding domain is derived from the growth-inducing ligand (APRIL), a natural ligand for BCMA.

[0157] British Patent Application No. 1815775.0 describes the use of 14 BCMA-binding domains in VH and VL domains and CARs.

[0158] PSMA T cells expressing CARs specific to prostate-specific membrane antigen (PSMA) are currently undergoing clinical trials for the treatment of prostate cancer (Junhans et al (2016) Prostate 76:1257-1270).

[0159] GD2 CARs that bind to disialoganglioside (GD2) (sialic acid-containing sphingoglycolipids) have been developed. Such CARs can be obtained, for example, based on the GD2 binder 14g2a or huK666 as described in WO2015 / 132604.

[0160] CARs that bind to GD2 may have antigen-binding domains including the following: a) Heavy chain variable region (VH) having a complementarity-determining region (CDR) with the following sequence: CDR1-SYNIH(sequence number 71); CDR2-VIWAGGSTNYNSALMS (Sequence ID 72) CDR3-RSDDYSWFAY(sequence number 73); and b) Light chain variable region (VL) having a CDR having the following sequence: CDR1-RASSSVSSSYLH (Sequence ID 74); CDR2-STSNLAS (Sequence ID 75) CDR3-QQYSGYPIT (Sequence ID 76).

[0161] The GD2 binding domain may include a VH domain having the sequence shown as sequence number 77; and / or a VL domain having the sequence shown as sequence number 78. Sequence ID 77 (Humanized KM666 VH sequence) QVQLQESGPGLVKPSQTLSITCTVSGFSLASYNIHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLTISKDNSKNQVFLKMSSLTAADTAVYYCAKRSDDYSWFAYWGQGTLVTVSS Sequence ID 78 (Humanized KM666 VH sequence) ENQMTQSPSSLSASVGDRVTMTCRASSSVSSSYLHWYQQKSGKAPKVWIYSTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSGYPITFGQGTKVEIK

[0162] FCRL5 Commercially available monoclonal antibodies against FcRL5 (such as CD307e (ThermoFisher) and REA391 (Miltenyi Biotec)) are known.

[0163] WO2016090337 describes several scFv-type antigen-binding domains that bind to FcRL5.

[0164] British Patent Application No. 1815775.0 describes an anti-FCRL5 CAR.

[0165] CD79 Several anti-CD79 antibodies have been previously described (e.g., JCB117, SN8, CB3.1, 2F2 (polatuzumab)).

[0166] British Patent Application No. 1807870.9 describes various CD79 CARs.

[0167] If a viral vector composition contains more than one vector containing nucleic acid sequences encoding a CAR, the CAR may have different antigen-binding domains. A CAR may recognize different antigens, or a CAR may bind to the same antigen but have different antigen-binding domains. A CAR that binds to the same antigen but has different antigen-binding domains may bind to different epitopes of the antigen and / or have different affinities and / or on or off rates.

[0168] The affinity of a CAR for a target antigen and / or its on- and off-rate can affect the CAR's ability to kill target cells. For example, U.S. Patent Application Publication US 2018 / 0064785 reports that on-rate and off-rate of rapidly antibody-derived CARs allow CAR T cells to kill target cells more effectively and continuously. By administering a CAR-T cell composition containing multiple CARs for a target antigen to a patient, CARs with antigen-binding domains best suited to killing target cells in the patient or at a specific site in the patient receive activation / survival / proliferation signals and become dominant. The composition of the present invention provides flexibility in this regard and further allows subpopulations of CAR-T cells with different CARs to "win" at different sites within the same patient.

[0169] Intracellular T cell signaling domain (endodomain) CARs contain or can associate with an activating endodomain (the signaling portion of the CAR). After antigen recognition, receptor clusters and signals are transmitted to the cell. The most commonly used endodomain component is the CD3-zeta endodomain component containing three ITAMs. This transmits an activation signal to T cells after binding to the antigen. CD3-zeta may not provide a sufficiently qualified activation signal, and further co-stimulatory signaling may be required. For example, chimeric CD28 and OX40 can be used with CD3-zeta, or all three can be used together, to transmit proliferation / survival signals.

[0170] The CAR end domain may include the CD28 end domain as well as the OX40 and CD3-zeta end domains.

[0171] The end domain may include the following: (i) ITAM-containing end domains (such as CD3 zeta-derived end domains); and / or (ii) Co-stimulatory domains (such as CD28-derived endodomains); and / or (iii) Domains that transmit survival signals (e.g., TNF receptor family endodomains (OX-40 or 4-1BB, etc.)).

[0172] An endodomain containing an ITAM motif can act as an activating endodomain in the present invention. Several proteins are known to contain endodomains having one or more ITAM motifs. Examples of such proteins include CD3 epsilon chains, CD3 gamma chains, and CD3 delta chains. The ITAM motif can be readily recognized as tyrosine separated from leucine or isoleucine by any two other amino acids (thereby obtaining the signature YxxL / I (SEQ ID NO: 60)). Typically, though not always, two of these motifs are separated by 6-8 amino acids at the tail of the molecule (YxxL / Ix(6-8)YxxL / I). Therefore, those skilled in the art can readily find existing proteins containing one or more ITAMs to transmit an activation signal. Furthermore, given that the motif does not require a simple or complex secondary structure, those skilled in the art can design polypeptides containing artificial ITAMs to transmit an activation signal (see WO2000 / 063372 for synthetic signaling molecules).

[0173] Several systems have been described in which the antigen-recognition portion of a CAR resides on a separate molecule derived from the signaling portion (e.g., those described in WO015 / 150771; WO2016 / 124930; and WO2016 / 030691). One or more of the viral vectors used in the method of the present invention may encode such “splittered CARs.” Alternatively, one vector may contain a nucleic acid sequence encoding the antigen-recognition portion, and another vector may contain a nucleic acid sequence encoding the intracellular signaling domain.

[0174] If a viral vector composition contains more than one vector containing nucleic acid sequences encoding CARs, the CARs may have different end-domains or combinations of different end-domains. For example, one CAR may be a second-generation CAR and another may be a third-generation CAR. Alternatively, both CARs may be second-generation CARs but have different co-stimulatory domains. For example, different second-generation CAR signaling domains include: 41BB-CD3ζ;OX40-CD3ζ, and CD28-CD3ζ.

[0175] Signal peptide One or more nucleic acid sequences in the vector composition may encode a signal peptide such that, when a CAR or activity regulator is expressed inside the cell, the nascent protein is guided to the endoplasmic reticulum, then to the cell surface, and expressed (or secreted) on the cell surface.

[0176] The core of a signal peptide may contain a long, sequential chain of hydrophobic amino acids that tend to form a single alpha-helix. Signal peptides may begin with a short, positively charged amino acid chain, which is crucial for the proper topology of the polypeptide during translocation. This helps to enhance the signal peptide. At the end of a signal peptide, there is typically a sequence of amino acids that are recognized and cleaved by a signal peptidase. The signal peptidase can cleave the signal peptide either during or after migration to produce a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease.

[0177] Signal peptides can be located at the amino terminus of a molecule.

[0178] CAR may have the following general formula: Signal peptide - antigen-binding domain - spacer domain - transmembrane domain - intracellular T cell signaling domain (endodomain).

[0179] Spacer CARs include a spacer sequence that connects the antigen-binding domain to the transmembrane domain and spatially separates the antigen-binding domain from the endodomain. The movable spacer allows the antigen-binding domain to be oriented in different directions, enabling antigen binding.

[0180] The spacer sequence may include, for example, an IgG1 Fc region, an IgG1 hinge, or a CD8 stalk, or a combination thereof. The spacer may alternatively include an alternative sequence having similar length and / or domain spacing characteristics to the IgG1 Fc region, IgG1 hinge, or CD8 stalk.

[0181] If the viral vector composition contains more than one vector containing nucleic acid sequences encoding CARs, the CARs may have different spacers.

[0182] ORGATE The cell composition of the present invention may contain two or more CARs. This may be the result of transduction with two or more vectors, each containing a nucleic acid sequence encoding a CAR; or it may be the result of transduction with a single vector containing a nucleic acid construct encoding two or more CARs.

[0183] The CAR may be used in combination with one or more other activating or repressive chimeric antigen receptors. For example, the CAR of the present invention may be used in combination with one or more CARs in a "logic gate" such that the CAR combination can detect a specific expression pattern of at least two target antigens when expressed by cells such as T cells. If at least two target antigens are arbitrarily represented as antigen A and antigen B, there are three possible options: "OR gate" - T cells are triggered when either antigen A or antigen B is present on the target cell. "AND gate" - T cells are triggered when both antigen A and B are present on the target cell. "AND NOT GATE" - T cells are triggered when antigen A is present alone on the target cell, but not when both antigens A and B are present on the target cell.

[0184] Engineered T cells expressing these CAR combinations can be tuned to be highly specific to cancer cells based on the specific expression (or lack thereof) of two or more markers in cancer cells.

[0185] Such “logic gates” are described, for example, in WO2015 / 075469, WO2015 / 075470, and WO2015 / 075470.

[0186] An OR gate contains two or more activated CARs, each directed to a specific target antigen expressed by the target cell. The advantage of an OR gate is that the effectiveness is antigen A + antigen B, thus increasing the number of effectively targetable antigens on the target cell. This is particularly important for antigens expressed at fluctuating or low densities on the target cell, as the level of a single antigen may be below the threshold required for CAR-T cells to effectively target it. Furthermore, OR gates avoid antigen avoidance. For example, some lymphomas and leukemias become CD19-negative after CD19 is targeted: if this occurs, using an OR gate that targets CD19 in combination with another antigen provides a "backup" antigen. As described in WO2016 / 102965, the "backup" antigen can be CD22.

[0187] activity regulator In the method of the present invention, at least one vector in the viral vector mixture may contain a nucleic acid sequence encoding an activity regulator. If this is the case, at least a portion of the cells transduced with the CAR-expressing cell composition of the present invention will express one or more activity regulators. The activity regulators are molecules produced by CAR-expressing cells that modulate the activity of CARs, CAR-expressing cells, or target cells.

[0188] The activating factor can be an intracellular molecule expressed on the cell surface or secreted by CAR-expressing cells.

[0189] Regulation of CAR activity 1. Enhancement of ITAM phosphorylation During T cell activation in vivo (schematically shown in Fig. 2a), when an antigen is recognized by the T cell receptor (TCR), the immunoreceptor activation tyrosine motif (ITAM) on CD3ζ is phosphorylated. The phosphorylated ITAM is recognized by the ZAP70 SH2 domain and the T cell is activated.

[0190] T cell activation uses kinetic segregation to convert antigen recognition by the TCR into downstream activation signals. Briefly, in the basal state, signaling components on the T cell membrane are in dynamic homeostasis, so dephosphorylated ITAM is preferred over phosphorylated ITAM. This is due to the higher activity of the transmembrane CD45 / CD148 phosphatase than membrane-tethered kinases such as lck. When a T cell binds to a target cell by recognition of its cognate antigen by the T cell receptor (or CAR), a strong immunological synapse is formed. The juxtaposition of this T cell and the target membrane excludes CD45 / CD148 because its ectodomain is too large to fit into the synapse. When ITAM and kinases bound to the high concentration of T cell receptors in the synapse separate in the absence of phosphatase, phosphorylated ITAM reaches a dominant state. ZAP70 recognizes the threshold phosphorylated ITAM and transmits the T cell activation signal.

[0191] This process is essentially the same as the process during CAR-mediated T cell activation. The activating CAR contains one or more ITAMs within its intracellular signaling domain, usually because this signaling domain contains the endodomain of CD3ζ. When an antigen is recognized by the CAR, the ITAM(s) within the CAR signaling domain are phosphorylated, thereby activating the T cell.

[0192] As schematically shown in Figure 2b, phosphorylated ITAM is dephosphorylated by inhibitory immune receptors such as PD1. PD1 has ITIM in its endodomain, and ITIM is recognized by the SH2 domain of molecules such as PTPN6 (SHP-1) and SHP-2. During recognition, PTPN6 is recruited to the near-membrane region, and its phosphatase domain subsequently dephosphorylates the ITAM domain, inhibiting immune activation.

[0193] Activity regulators that can modify CAR activity may be able to phosphorylate ITAMs (or multiple ITAMs) within the CAR signaling domain, either directly or indirectly.

[0194] 1.1 Supply or mobilization of kinases For example, the activity regulator may be a membrane target molecule that contains a kinase domain or can recruit individual molecules containing kinase domains around the CAR. WO2018 / 193231 describes various molecules having such "phosphorylation-amplified endodomains".

[0195] Activity regulators that can directly phosphorylate ITAM may include tyrosine kinase domains (such as the kinase domains of the Src family kinases), including Fyn, Src, Lck, or their derivatives (such as Lck(Y505F)). The tyrosine kinase domains of Fyn, Src, Lck, and Lck(Y505) are shown below as Sequence IDs 18-21, respectively.

[0196] Fyn's tyrosine kinase domain (SEQ ID NO: 18) LQLIKRLGNGQFGEVWMGTWNGNTKVAIKTLKPGTMSPESFLEEAQIMKKLKHDKLVQLYAVVSEEPIYIVTEYMNKGSLLDFLKDGEGRALKLPNLVDMAAQVAAGMAYIERMNYIHRDLRSANIL VGNGLICKIADFGLARLIEDNEYTARQGAKFPIKWTAPERALYGRFTIKSDVWSFGILLTELVTKGRVPYPGMNNREVLEQVERGYRMPCPQDCPISLHELMIHCWKKDPEERPTFEYLQSFLEDYF

[0197] Src tyrosine kinase domain (SEQ ID NO: 19) LRLEVKLGQGCFGEVWMGTWNGTTRVAIKTLKPGTMSPEAFLQEAQVMKKLRHEKLVQLYAVVSEEPIYIVTEYMSKGSLLDFLKGETGKYLRLPQLVDMAAQIASGMAYVERMNYVHRDLRAANIL VGENLVCKVADFGLARLIEDNEYTARQGAKFPIKWTAPEAALYGRFTIKSDVWSFGILLTELTTKGRVPYPGMVNREVLDQVERGYRMPCPPECPESLHDLMCQCWRKEPEERPTFEYLQAFLEDYF

[0198] Lck tyrosine kinase domain (SEQ ID NO: 20): LKLVERLGAGQFGEVWMGYYNGHTKVAVKSLKQGSMSPDAFLAEANLMKQLQHQRLVRLYAVVTQEPIYIITEYMENGSLVDFLKTPSGIKLTINKLLDMAAQIAEGMAFIEERNYIHRDLRAANIL VSDTLSCKIADFGLARLIEDNEYTAREGAKFPIKWTAPEAINYGTFTIKSDVWSFGILLTEIVTHGRIPYPGMTNPEVIQNLERGYRMVRPDNCPEELYQLMRLCWKERPEDRPTFDYLRSVLEDFF

[0199] Tyrosine kinase domain of Lck_Y505F (SEQ ID NO: 21) LKLVERLGAGQFGEVWMGYYNGHTKVAVRSLKQGSMSPDAFLAEANLMKQLQHQRLVRLYAVVTQEPIYIITEYMENGSLVDFLKTPSGIKLTINKLLDMAAQIAEGMAFIEERNYIHRDLRAANIL VSDTLSCKIADFGLARLIEDNEYTAREGAKFPIKWTAPEAINYGTFTIKSDVWSFGILLTEIVTHGRIPYPGMTNPEVIQNLERGYRMVRPDNCPEELYQLMRLCWKERPEDRPTFDYLRSVLEDFF

[0200] Activity regulators that can indirectly phosphorylate ITAM may include intracellular domains of CD4 or CD8 coreceptors.

[0201] As described above, during T cell activation, the ITAM of CD3 (or CAR) is phosphorylated by Lck and then binds to ZAP70. After ZAP70 binds to CD3, the coreceptor CD4 or CD8 becomes associated with the TCR / CD3 complex and binds to the major compatibility complex (MHC). The association of the CD4 / CD8 coreceptor with the aforementioned complex stabilizes the TCR-MHC peptide (MHCp) interaction, allowing recruited / free Lck to continue phosphorylating the CD3 element, ZAP70, and many other downstream targets. .

[0202] The cytoplasmic tails of CD4 and CD8, along with other regulatory factors, amplify the signals generated by CARs through Lck recruitment, which are essential for the activation of numerous molecular components in the activated T cell signaling cascade. The sequences of the intracellular domains of human CD4 and CD8 are shown below as SEQ ID NOs. 22 and 23. CD4 cytoplasmic tail (SEQ ID NO: 22) CVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI CD8 cytoplasmic tail (SEQ ID NO: 23) LYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV

[0203] Activity regulators regulated by CAR activity can be tethered to the membrane. In this regard, such activity regulators may include transmembrane domain sequences or myristoylation sequences.

[0204] Regulation of the activity of CAR-T cells 1. Checkpoint Inhibition Activity regulators that can regulate CAR-expressing cell activity can block or reduce the inhibition of CAR-mediated T cell activation mediated by inhibitory immune receptors (such as CTLA4, PD-1, LAG-3, 2B4, or BTLA1) (described above and schematically shown in Figure 2b).

[0205] Activity regulators can be agents such as antibodies that bind to inhibitory immune receptors or bind to ligands of inhibitory immune receptors. Activity regulators can bind to CTLA4, PD-1, LAG-3, 2B4, or BTLA1, or can bind to ligands of CTLA4, PD-1, LAG-3, 2B4, or BTLA1.

[0206] PD-1 / PD-L1 In the context of cancer conditions, as described above, when PD-L1 on tumor cells interacts with PD-1 on T cells, T cell activation is reduced, and thus the immune system is prevented from attacking tumor cells. The use of inhibitors that block the interaction of PD-L1 with the PD-1 receptor can prevent cancer from evading the immune system in the aforementioned manner. Some PD-1 inhibitors and PD-L1 inhibitors are in clinical trials in hospitals for use in several cancer types, particularly in advanced melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, and Hodgkin lymphoma. Some such inhibitors are currently approved (including the PD1 inhibitors nivolumab and pembrolizumab and the PD-L1 inhibitors atezolizumab, avelumab, and durvalumab).

[0207] CTLA4 CTLA4 is a member of the immunoglobulin superfamily expressed by activated T cells and transmits inhibitory signals to T cells. CTLA4 is analogous to the T cell costimulatory protein (CD28), and both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. Because CTLA-4 binds to CD80 and CD86 with higher affinity and avidity than CD28, CTLA-4 can outperform CD28 due to its ligand. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulating signals.

[0208] Antagonist antibodies against CTLA4 include ipilimumab and tremelimumab.

[0209] LAG-3 Lymphocyte-activating gene 3, also known as LAG-3 and CD223, is an immune checkpoint receptor with diverse biological effects on T cell function.

[0210] Antibodies against LAG3 currently include relatimab, which is in Phase 1 clinical trials, and several other antibodies in preclinical development. LAG-3 may be a better checkpoint inhibitor target than CTLA-4 or PD-1 because, while antibodies against these two checkpoints activate only effector T cells and do not inhibit Treg activity, antagonist LAG-3 antibodies can both activate T effector cells (by downregulating LAG-3 inhibitory signals to pre-activated LAG-3+ cells) and inhibit inducible (i.e., antigen-specific) Treg suppressive activity. Combination therapies involving LAG-3 antibodies and CTLA-4 or PD-1 antibodies are also underway.

[0211] 1.2 Dominant-Negative SHP Activity modifiers that block or mitigate inhibition mediated by inhibitory immune receptors (such as CTLA4, PD-1, LAG-3, 2B4, or BTLA1) can disrupt the balance of phosphorylation and dephosphorylation at T cell-target cell synapses, thereby favoring ITAM phosphorylation and potentially leading to T cell activation. For example, activity modifiers may block or mitigate ITAM phosphorylation in the endodmain of the inhibitory receptor(s), or block or mitigate ITAM dephosphorylation in the CAR signaling domains mediated by proteins such as SHP-1 and SHP-2.

[0212] WO2016 / 193696 describes various different types of proteins that can modulate the balance of phosphorylation:dephosphorylation at T cell:target cell synapses. For example, the activity regulators may include truncated forms of SHP-1 or SHP-2 that contain one or both SH2 domains but lack a phosphatase domain. When expressed in CAR-T cells, these molecules act as dominant-negative versions of wild-type SHP-1 and SHP-2, competing with endogenous molecules for binding to phosphorylated ITIM.

[0213] The activity regulator may be a shortened protein containing an SH2 domain derived from a protein that binds to a phosphorylated immunoreceptor-suppressive tyrosine motif (ITIM) but lacks a phosphatase domain. The shortened protein may contain one or both SHP-1 SH2 domains but lack the SHP-1 phosphatase domain. Alternatively, the shortened protein may contain one or both SHP-2 SH2 domains but lack the SHP-2 phosphatase domain.

[0214] SHP-1 Src homology domain 2-domain phosphatase-1 (SHP-1) is a member of the protein tyrosine phosphatase family. It is also known as PTPN6.

[0215] The N-terminal region of SHP-1 contains two tandem SH2 domains that mediate the interaction between SHP-1 and its substrate. The C-terminal region contains a tyrosine-protein phosphatase domain.

[0216] SHP-1 can bind to several suppressive immune receptors or ITIM-containing receptors and propagate signals from them. Examples of such receptors include, but are not limited to, PD1, PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1, and KIR3DL3.

[0217] The human SHP-1 protein has UniProtKB accession number P29350.

[0218] The activity regulator contains, or may contain, the SHP-1 tandem SH2 domain shown below as Sequence ID No. 24.

[0219] SHP-1 SH2 complete domain (SEQ ID NO: 24) MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTS ERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYY

[0220] SHP-1 has two SH2 domains at the N-terminal residues 4-100 and 110-213 of the sequence. The activity regulator may contain one or both of the sequences shown as SEQ ID NOs. 25 and 26.

[0221] SHP-1 SH2 1 (Sequence ID 25) WFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPL

[0222] SHP-2 SH2 2 (Sequence ID 26) WYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPY

[0223] The activity regulator may include variants of SEQ ID NOs. 24, 25, or 26 having at least 80, 85, 90, 95, 98, or 99% sequence identity, provided that the variant sequence is an SH2 domain sequence with the required properties. In other words, the variant sequence should bind to at least one phosphorylated tyrosine residue in the cytoplasmic tail of PD1, PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1, or KIR3DL3 that can recruit SHP-1.

[0224] SHP-2 SHP-2, also known as PTPN11, PTP-1D, and PTP-2C, is a member of the protein tyrosine phosphatase (PTP) family. Similar to PTPN6, SHP-2 has a domain structure consisting of two tandem SH2 domains at its N-terminus, followed by a protein tyrosine phosphatase (PTP) domain. In the inactive state, the N-terminal SH2 domains bind to the PTP domain, blocking potential substrate access to the active site. Thus, SHP-2 is self-inhibited. Upon binding to a target phosphotyrosyl residue, the N-terminal SH2 domains are released from the PTP domain, catalytically activating the enzyme by mitigating self-inhibition.

[0225] Human SHP-2 has UniProtKB accession number P35235-1.

[0226] The regulatory factor contains, or may contain, the SHP-1 tandem SH2 domain shown below as Sequence ID No. 29. SHP-1 has two SH2 domains at residues 6-102 and 112-216 at the N-terminus of the sequence. The regulatory factor may contain one or both of the sequences shown as Sequence IDs No. 27 and 28.

[0227] The first SH2 domain of SHP-2 (SEQ ID NO: 27) WFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGAVTHIKIQNTGDYYDLYGGEKFATLAELVQYYMEHHGQLKEKNGDVIELKYPL

[0228] The second SH2 domain of SHP-2 (SEQ ID NO: 28) WFHGHLSGKEAEKLLTEKGKHGSFLVRESQSHPGDFVLSVRTGDDKGESNDGKSKVTHVMIRCQELKYDVGGGERFDSLTDLVEHYKKNPMVETLGTVLQLKQPL

[0229] Both SH2 domains of SHP-2 (SEQ ID NO: 29) WFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGAVTHIKIQNTGDYYDLYGGEKFATLAELVQYYMEHHGQLKEKNGDVIELKYPLNCADPTSE RWFHGHLSGKEAEKLLTEKGKHGSFLVRESQSHPGDFVLSVRTGDDKGESNDGKSKVTHVMIRCQELKYDVGGGERFDSLTDLVEHYKKNPMVETLGTVLQLKQPL

[0230] The activity regulator may include variants of SEQ ID NOs. 27, 28, or 29 having at least 80, 85, 90, 95, 98, or 99% sequence identity, provided that the variant sequence is an SH2 domain sequence with the required properties. In other words, the variant sequence should bind to at least one phosphorylated tyrosine residue in the cytoplasmic tail of PD1, PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1, or KIR3DL3 that is capable of recruiting SHP-2.

[0231] 3. Cytokines and cytokine signaling The activity regulators may be cytokines or chemokines. Cytokines can modulate the activity of CAR-expressing cells and / or modulate the tumor microenvironment.

[0232] The activity regulator may be a cytokine or chemokine selected from the following: IL12, flexiIL12, GM-CSF, IL7, IL15, IL21, IL2, and CCL19. In particular, this activator may be IL-7 or IL-12.

[0233] IL-7 is a cytokine that is important for the development of B cells and T cells. IL-7 stimulates the differentiation of pluripotent hematopoietic stem cells into lymphoid progenitor cells and stimulates the proliferation of all lymphoid cells (B cells, T cells, and NK cells).

[0234] Il-7 and hepatocyte growth factor (HGF) form a heterodimer and function as a pre-pro B cell growth stimulator. This cytokine has been found to be a cofactor of V(D)J rearrangement of the T cell receptor beta (TCRβ) during early T cell development. The amino acid sequence of human Il-7 is available from UniProt (accession number P13232).

[0235] Interleukin-12 (IL-12) is a potent immunomodulatory cytokine of particular interest for its role in modulating the tumor microenvironment and redirecting the immune response against cancer. Because IL-12 exhibits systemic toxicity, there is interest in methods for its local production. PCT / GB2018 / 052204 describes a construct that produces immunomodulatory cytokines such as IL-12 under the control of a promoter activated in the presence of environmental metabolites (such as kynurenine). Selective production of IL-12 in the presence of metabolites such as kynurenine allows for localized IL-12 production only when cells expressing CARs or TCRs are present in the tumor microenvironment.

[0236] Alternatively, immunomodulatory cytokines may be located downstream of a flame-slip motif or a translational readthrough motif. This provides a means to control cytokine expression and reduce the level of cytokine expression in relation to CAR.

[0237] A frame-slip motif (FSM) may contain repeats of the uracil, thymine, or guanine bases (such as the sequence UUUUUUU (sequence number 61)).

[0238] Furthermore, frame-slip motifs can contain stop codons. For example, FSM can contain one of the following sequences: UUUUUUUGA (Sequence ID 62) UUUUUUUAG (Sequence ID 63) UUUUUUUAA (Sequence ID 64).

[0239] A translation read-through motif (TRM) may contain the sequence STOP-CUAG or STOP-CAAUUA (where "STOP" is a stop codon). For example, a translation read-through motif may contain one of the following sequences: UGA-CUAG (Sequence ID 65) UAG-CUAG (Sequence No. 66) UAA-CUAG (Sequence ID 67) UGA-CAAUUA (Sequence ID 68) UAG-CAAUUA (Sequence ID 69) UAA-CAAUUA (Sequence ID 70).

[0240] IL-12 is a heterodimer cytokine encoded by two separate genes, IL-12A(p35) and IL-12B(p40). The active heterodimer (called "p70") is formed after protein synthesis. The regulatory factor for activity can be "flexi-IL12," which is a fusion between the IL-12α subunit and the IL-12β subunit linked by a linker. The stable flexi-IL-12 sequence is shown below as sequence number 81.

[0241] Sequence ID 81 (flexi-IL-12 sequence) [ka] [ka]

[0242] In sequence number 81, the serine-glycine linker is shown in underlined bold text.

[0243] The activity regulator of the present invention may be a cytokine that is selectively expressed in response to the presence of environmental metabolites in the cellular microenvironment. Environmental metabolites may activate aryl hydrocarbon receptors (AHRs). Environmental metabolites may be tryptophan metabolites such as kynureny.

[0244] Alternatively, the activator may influence the expression or activity of cytokines or chemokines. For example, the activator could be a dominant-negative version of a cytokine or chemokine. A dominant-negative version could be, for example, a mutant or shortened version of a cytokine / chemokine that binds to a receptor and competes with the wild-type cytokine / chemokine but does not induce cytokine / chemokine signaling.

[0245] For example, the activator could be a dominant-negative version of a cytokine receptor or chemokine receptor. The dominant-negative version could be, for example, a mutated or shortened version of a cytokine / chemokine receptor that binds to a cytokine and blocks its binding to the wild-type cytokine / chemokine receptor.

[0246] Alternatively, the activating factor may be an antibody or antibody fragment that blocks or otherwise modulates the signaling pathway of cytokines or chemokines.

[0247] The activity regulator may be a chimeric cytokine receptor containing a cytokine receptor endodomain.

[0248] The regulatory factors may include exodomains derived from immunosuppressive cytokines (such as IL-4) fused to endodomains derived from cytokines that enhance T cell proliferation (such as IL-7) (Leen et al (2014) Mol.Ther.22:1211-1220).

[0249] The activity regulators can be chemokines such as CCL19. Chemokine (CC motif) ligand 19 (CCL19) is a small cytokine belonging to the CC chemokine family, also known as the EBI1 ligand chemokine (ELC) and macrophage inflammatory protein-3-beta (MIP-3-beta). CCL19 manifests its effects on target cells by binding to the chemokine receptor CCR7. CCL19 attracts certain immune system cells (including dendritic cells, antigen-binding B cells, and CCR7+ central memory T cells). The amino acid sequence of human CCL19 is available from UniProt (accession number Q99731).

[0250] 3.1 Chimeric cytokine receptors Alternatively, the activity regulator may include a non-cytokine receptor exodomain. WO2017 / 029512 describes chimeric cytokine receptors (CCRs) including: an exodomain that binds to a ligand selected from tumor secretory factors, chemokines, and cell surface antigens; and a cytokine receptor endodomain.

[0251] Chimeric cytokine receptors may contain the following two polypeptides: (i) The first polypeptide including the following: (a) First antigen-binding domain that binds to the first epitope of the ligand (b) the first chain of the cytokine receptor endodomain; and (ii) A second polypeptide including the following: (a) A second antigen-binding domain that binds to a second epitope of the ligand. (b) A second chain of the cytokine-receptor endodomain.

[0252] Alternatively, a chimeric cytokine receptor containing the following two polypeptides: (i) The first polypeptide including the following: (a) Heavy chain variable domain (VH) (b) the first chain of the cytokine receptor endodomain; and (ii) A second polypeptide including the following: (a) Light chain variable domain (VL) (b) The second chain of the cytokine-receptor endodomain.

[0253] For example, a cytokine receptor endodomain may include the following: (i) IL-2 receptor β-chain endodomain (ii) IL-7 receptor α-chain endodomain; (iii) IL-15 receptor α-chain endodomain; or (iv) Common gamma chain receptor endodomain.

[0254] The cytokine receptor endodomain may include (i), (ii), or (iii); and (iv).

[0255] The cytokine receptor endodomain may include α-chain and β-chain endodomains derived from the granulocyte-macrophage colony-stimulating factor receptor (GMCSF-R).

[0256] The ligand may be a tumor secretory factor, for example, a tumor secretory factor selected from the following: prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), vascular endothelial growth factor (VEGF), and CA125.

[0257] The ligand may be a chemokine, for example, one selected from the following: CXCL12, CCL2, CCL4, CCL5, and CCL22.

[0258] The ligand can be a cell surface molecule (such as a transmembrane protein). For example, the ligand could be CD22.

[0259] Constitutively active chimeric cytokine receptors The regulatory factor may be a constitutively active chimeric cytokine receptor. The regulatory factor may contain two chains that spontaneously dimerize by combining two cytokine receptor endodomains in the presence of an activator (a chemical inducer of dimerization, i.e., CID).

[0260] Therefore, the activity regulator may include a dimerization domain and a cytokine receptor endodomain.

[0261] Dimerization can occur spontaneously, in which case the chimeric transmembrane protein is constitutively active. Alternatively, dimerization may occur only in the presence of a chemical inducer of dimerization (CID), in which case cytokine-type signaling occurs solely by the transmembrane protein in the presence of the CID.

[0262] Appropriate dimerization domains and CIDs are described in WO2015 / 150771 (the contents of which are incorporated herein by reference).

[0263] For example, one dimerized domain may contain the rapamycin-binding domain of FK-binding protein 12 (FKBP12), while the other may contain the FKBP12-rapamycin-binding (FRB) domain of mTOR; CID may be rapamycin or a derivative thereof.

[0264] One dimerized domain may contain the FK506 (tacrolimus) binding domain of FK-binding protein 12 (FKBP12), and the other dimerized domain may contain the cyclosporine binding domain of cyclophilin A; CID may be an FK506 / cyclosporine fusion or a derivative thereof.

[0265] One dimerized domain may contain an estrogen-binding domain (EBD), and the other dimerized domain may contain a streptavidin-binding domain; CID may be an estrone / biotin fusion protein or a derivative thereof.

[0266] One dimerized domain may contain a glucocorticoid-binding domain (GBD), and the other dimerized domain may contain a dihydrofolate reductase (DHFR)-binding domain; CID may be a dexamethasone / methotrexate fusion protein or a derivative thereof.

[0267] One dimerized domain may contain an O6-alkylguanine-DNA alkyltransferase (AGT) binding domain, and the other dimerized domain may contain a dihydrofolate reductase (DHFR) binding domain; CID may be an O6-benzylguanine derivative / methotrexer fusion protein or a derivative thereof.

[0268] One dimerized domain may contain a retinoic acid receptor domain, and the other dimerized domain may contain an ecodysone receptor domain; CID may be RSL1 or a derivative thereof.

[0269] When a dimerizing domain spontaneously undergoes heterodimerization, this domain may be based on the antibody's dimerizing domain. In particular, the dimerizing domain may include dimerized portions of the heavy chain constant domain (CH) and the light chain constant domain (CL). The "dimerized portion" of the constant domain is a part of the sequence that forms the interchain disulfide bond.

[0270] Chimeric cytokine receptors may contain the Fab portion of an antibody as an exodomain. In this regard, chimeric antigens may contain the following two polypeptides: (i) The first polypeptide including the following: (a) Heavy chain constant domain (CH) (b) the first chain of the cytokine receptor endodomain; and (ii) A second polypeptide including the following: (a) Light chain constant domain (CL) (b) The second chain of the cytokine-receptor endodomain.

[0271] The cytokine receptor endodomain may include the following: (i) IL-2 receptor β-chain endodomain (ii) IL-7 receptor α-chain endodomain; or (iii) IL-15 receptor α-chain endodomain; and / or (iv) Common gamma chain receptor endodomain.

[0272] The cytokine receptor endodomain may include α-chain and β-chain endodomains derived from the granulocyte-macrophage colony-stimulating factor receptor (GMCSF-R).

[0273] Constitutively active CCRs having the IL-2, IL-7, or GM-CSF receptor endodomains may have one of the following structures: Fab_CCR_IL2:HuLightKappa-IL2RgTM-IL2RgEndo-2A-HuCH1-IL2bTM-IL2RbENDO Fab_CCR_IL7:HuLightKappa-IL2RgTM-IL2RgEndo-2A-HuCH1-IL7RaTM-IL7RaENDO Fab_CCR_GMCSF: HuLightKappa-GMCSFRbTM-GMCSFRbEndo-2A-HuCH1-GMCSFRaTM-GMCSFRaENDO Here, HuLightKappa is a human κ light chain. IL2RgTM is a transmembrane domain derived from the human IL2R common gamma chain. IL2RgEndo is an end domain derived from the common gamma chain of human IL2R. 2A is a sequence that allows for the co-expression of two polypeptides, which may be self-cleaving peptides such as the 2A peptide. HuCH1 is human CH1, IL2b™ is a transmembrane domain derived from human IL-2R beta. IL2RbENDO is an endodomain derived from human IL2R beta. IL7RaTM is a transmembrane domain derived from human IL-7R alpha. IL7RaENDO is an endodomain derived from human IL-7R alpha. GMCSFRb™ is a transmembrane domain derived from the human GM-CSFR common beta chain. GMCSFRbEndo is an end domain derived from the GM-CSFR common beta chain. GMCSFRa™ is a transmembrane domain derived from human GF-CSFR alpha. GMCSFRaENDO is an endodomain derived from human GM-CSFR alpha.

[0274] Sequence numbers 30-43 represent the following sequences of components for constructing cytokine receptors that are constitutively active.

[0275] Sequence ID 30 (Human κ light chain) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0276] Sequence ID 31 (Human Hinge) EPKSCDKTHTCPPCPKDPK

[0277] Sequence ID 32 (Human CH1) STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV

[0278] Sequence ID 33 (Transmembrane domain derived from the common gamma chain of human IL2R): VVISVGSMGLIISLLCVYFWL

[0279] Sequence ID 34 (Transmembrane domain derived from human IL-2R beta) IPWLGHLLVGLSGAFGFIILVYLLI

[0280] Sequence ID 36 (Transmembrane domain derived from human IL-7R alpha) PILLTISILSFFSVALLVILACVLW

[0281] Sequence ID 37 (Transmembrane domain derived from human GF-CSFR alpha) NLGSVYIYVLLIVGTLVCGIVLGFLF

[0282] Sequence ID 38 (Transmembrane domain derived from human GM-CSFR common beta chain) VLALIVIFLTIAVLLAL

[0283] Sequence ID 39 (Endodomain derived from the common gamma chain of human IL2R) ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

[0284] Sequence ID 40 (Endodomain derived from human IL-2R beta) NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPL QPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0285] Sequence ID 41 (Endodomain derived from human IL-7R alpha) KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSSNQEEAYVTMSSFYQNEQ

[0286] Sequence ID 42 (Endodomain derived from human GM-CSFR alpha) KRFLRIQRLFPPVPQIKDKLNDNHEVEDEIIWEEFTPEEGKGYREEVLTVKEIT

[0287] Sequence ID 43 (Endodomain derived from the common beta chain of GM-CSFR) RFCGIYGYRLRRKWEEKIPNPSKSHLFQNGSAELWPPGSMSAFTSGSPPHQGPWGSRFPELEGVFPVGFGDSEVSPLTIEDPKHVCDPPSGPDTTPAASDLPTEQPPSP QPGPPAASHTPEKQASSFDFNGPYLGPPHSRSLPDILGQPEPPQEGGSQKSPPPGSLEYLCLPAGGQVQLVPLAQAMGPGQAVEVERRPSQGAAGSPSLESGGGPAPPA LGPRVGGQDQKDSPVAIPMSSGDTEDPGVASGYVSSADLVFTPNSGASSVSLVPSLGLPSDQTPSLCPGLASGPPGAPGPVKSGFEGYVELPPIEGRSPRRSPRNNPVPP EAKSPVLNPGERPADVSPTSPQPEGLLVLQQVGDYCFLPGLGPGPLSLRSKPSSPGPGPEIKNLDQAFQVKKPPGQAVPQVPVIQLFKALKQQDYLSLPPWEVNKPGEVC

[0288] The activity regulator may contain one or more variants from SEQ ID NOs: 30-43 having at least 80, 85, 90, 95, 98, or 99% sequence identity, provided that the variant sequence possesses the necessary properties. For example, the variant CH or CL sequence should retain the ability to form dimers with CL / CH-containing chains. Variant chains derived from the cytokine receptor endodomain should have the ability to induce cytokine-mediated signaling when coupled with the cytokine receptor reciprocal chain. We should maintain our strength.

[0289] 3.3 JAK / STAT Signaling and transcriptional activator (STAT) molecules are a family of transcription factors involved in cytokine-mediated signaling. STAT transcription factors are recruited to the cytoplasmic region of cell surface receptors and activated by phosphorylation. Upon activation, STAT transcription factors dimerize to form activated STAT molecules containing a first and second polypeptide, which then migrate into the cell nucleus, where they influence gene expression. STAT transcription factors play a role in regulating cell growth and differentiation. JAK-STAT signaling consists of three main components: (1) a cell membrane-permeable receptor, (2) Janus kinase (JAK) bound to the aforementioned receptor, and (3) a signaling and transcriptional activator (STAT) that transports the signal into the nucleus and DNA (see Figure 3).

[0290] By including constitutively active or inducibly active JAK or STAT molecules within cells, it is possible to enhance the engraftment and survival of CAR-expressing cells. International Patent Application No. PCT / GB2018 / 052583 describes various alternative configurations of constitutively active STAT molecules (Figure 4 in International Patent Application No. PCT / GB2018 / 052583) and inducibly active STAT molecules (Figure 5 in International Patent Application No. PCT / GB2018 / 052583).

[0291] As described in International Patent Application No. PCT / GB2018 / 052583, a constitutively active JAK molecule can be produced by expressing two JAK polypeptides that spontaneously dimerize or are linked by a linker to a constitutively active STAT molecule, as described below. Alternatively, a constitutively active JAK containing a gain-of-function mutation can be expressed.

[0292] The regulatory factors may be constitutively active or inducible signaling and transcriptional activator (STAT) molecules.

[0293] The cellular STAT molecule may contain a first STAT polypeptide containing a first dimerization domain (DD) and a second STAT polypeptide containing a second DD that binds to the first DD.

[0294] The first and second DDs of the cellular STAT molecule may include, for example, a leucine zipper domain; or a heavy chain constant region and a light chain constant region.

[0295] Inducible STAT molecules in cells can be inducibly activated in the presence of an activating factor that dimerizes the first and second DDs of the STAT molecule. For example, the first DD may contain FRB, the second DD may contain FKBP12, and the activating factor may be rapamycin.

[0296] Alternatively, the cellular STAT molecule may be inductively inactive in the presence of an activator that dissociates the first and second DDs of the STAT molecule, thereby inducing the deactivation of the STAT molecule. The first DD may include TetRB, the second DD may include TiP, and the activator may be tetracycline, doxycycline, or minocycline.

[0297] Constitutively active STATs may contain gain-of-function (GOF) mutations or may contain a first STAT polypeptide and a second STAT polypeptide linked by a linker sequence.

[0298] The cell may contain a membrane anchoring molecule comprising an anchoring domain and a first binding domain (BD), as well as a constitutively active STAT molecule comprising a second BD that specifically binds to the first BD. The binding of the first and second BDs can be disrupted by the presence of an activator such that the constitutively active STAT molecule is dissociated from the membrane anchoring molecule in the presence of the activator, thereby allowing the constitutively active STAT molecule to freely move into the nucleus.

[0299] The first and second DDs of the cellular STAT molecule; or the first BD of the cellular membrane anchoring molecule and the second BD of the cellular STAT molecule may contain a Tet repressor protein (TetR) and a transcription-inducing peptide (TiP), respectively; the activators may be tetracycline, doxycycline, or minocycline.

[0300] The cell may contain a) a CAR linked by a STAT release domain and a constitutively active STAT molecule, and b) a STAT release molecule that releases a constitutively active STAT molecule from the CAR via its STAT release domain only when the CAR recognizes a target antigen specific to the CAR, and as a result, the constitutively active STAT molecule freely moves to the nucleus upon release.

[0301] STAT-releasing molecules may include, for example, a CAR target domain that binds to a phosphorylated immune receptor-activated tyrosine motif (ITAM). For example, the CAR target domain may include one or more ZAP70 SH2 domains.

[0302] The STAT-releasing domain of the cell according to the present invention may include a protease cleavage site, and the STAT-releasing molecule of the cell may include a protease domain. As a result, when the CAR recognizes the target antigen, the protease domain cleaves the protease cleavage site, and the STAT molecule is released.

[0303] 4.Adhesion molecules Cell adhesion molecules (CAMs) are proteins present on the cell surface that are involved in cell adhesion, specifically in binding to other cells or the extracellular matrix (ECM).

[0304] These proteins are typically transmembrane receptors and consist of three domains: an intracellular domain that interacts with the cytoskeleton, a transmembrane domain, and an extracellular domain that interacts with either other homogeneous CAMs (allogeneic binding) or other CAMs or the extracellular matrix (heterogeneous binding).

[0305] Most CAMs belong to the following four protein families: the Ig (immunoglobulin) superfamily (IgSF CAM), integrins, cadherins, and selectins.

[0306] The activity regulators of the present invention may be, or may include, adhesion molecules that regulate the activity of CAR-expressing cells or TCR-expressing cells.

[0307] 5. Transcription factors The active factors of the present invention may be, or may include, transcription factors that modulate the activity of CAR-expressing cells or TCR-expressing cells.

[0308] Transcription factors are proteins that regulate the rate of transcription of genetic information from DNA to messenger RNA by binding to specific DNA sequences, thereby regulating the expression of genes that contain or are adjacent to the aforementioned sequences.

[0309] Transcription factors act by either promoting the recruitment of RNA polymerase (as activators) or blocking it (as repressors).

[0310] Transcription factors contain at least one DNA-binding domain (DBD) that attaches to either the enhancer or promoter region of DNA. Depending on the transcription factor, the transcription of neighboring genes is either upregulated or downregulated. Transcription factors also contain a transactivation domain (TAD) that has a binding site for other proteins, such as transcription coregulators.

[0311] Transcription factors utilize various mechanisms to regulate gene expression, including stabilizing or blocking the binding of RNA polymerase to DNA, or catalyzing the acetylation or deacetylation of histone proteins. Transcription factors may possess histone acetyltransferase (HAT) activity (acetylating histone proteins to reduce the association of DNA with histones, making DNA more transcribable and thus upregulating transcription). Alternatively, transcription factors may possess histone deacetylase (HDAC) activity (deacetylating histone proteins to enhance the association of DNA with histones, making DNA less transcribable and thus downregulating transcription). Another mechanism by which transcription factors can function is through the recruitment of coactivator or corepressor proteins into the transcription factor DNA complex.

[0312] Transcription can be constitutively active or conditionally active (i.e., requiring activation).

[0313] Transcription factors can be naturally occurring or artificial.

[0314] 5.1 Transcription Reprogramming For CAR-T cells to be effective, it is crucial that they survive and proliferate in vivo and exhibit resistance to excessive rapid differentiation and exhaustion. The survival and engraftment of CAR-T cells are related to the administration ratio of naive cells, central memory cells, and T stem cell memory T cells.

[0315] WO2018 / 115865 describes cells that co-express chimeric antigen receptors (CARs) and transcription factors. When transcription factors are expressed, cell differentiation and / or exhaustion may be prevented or mitigated in vitro and / or in vivo. Co-expression of CARs with transcription factors within cells makes it possible to prevent or mitigate cell differentiation and / or exhaustion. This increases the proportion of naive cells, central memory cells, and stem cell memory cells in cell compositions for immunotherapy, leading to more effective cell survival and proliferation in vivo.

[0316] The activity regulator of the present invention may be a transcription factor. This transcription factor may prevent or reduce cell differentiation and / or exhaustion.

[0317] The transcription factor could be an effector memory repressor (such as BLIMP-1).

[0318] Alternatively, the transcription factor could be a central memory repressor (such as BCL6 or Bach2).

[0319] The transcription factor may be or may include Bach2 or a modified version of Bach2 in which the phosphorylation capacity by ALK is reduced or removed. For example, a modified Bach2 may contain mutations at one or more of the following positions: Ser-535, Ser-509, Ser-520.

[0320] The transcription factor may be FOXO1, EOMES, Runx3, or CBF beta.

[0321] 6. Regulation of TGFβ signaling

[0322] Manipulated cells face a harsh microenvironment that limits adoptive immunotherapy. One of the main inhibitory mechanisms within the tumor microenvironment is transforming growth factor beta (TGFβ). The TGFβ signaling pathway plays a crucial role in signaling regulation that controls various cellular processes. TGFβ also plays a central role in regulating T cell homeostasis and cellular function. In particular, TGFβ signaling is involved in the immunosuppressive state of T cells (reduced proliferation and activation). TGFβ expression is associated with the immunosuppressive microenvironment of tumors.

[0323] It is well known that various cancerous tumor cells directly produce TGFβ. In addition to TGFβ production by cancer cells, TGFβ can also be produced by a wide variety of cancerous cells present in the tumor site (tumor-associated T cells, natural killer (NK) cells, macrophages, epithelial cells, and stromal cells, etc.).

[0324] Transforming growth factor beta receptors are a superfamily of serine / threonine kinase receptors. These receptors bind to members of the TGFβ superfamily of growth factor and cytokine signaling proteins. There are five type II receptors (active receptors) and seven type I receptors (signaling-type propagation receptors).

[0325] Co-receptors (also known as type III receptors) also exist. Each subfamily of the ligand TGFβ superfamily binds to type I and type II receptors.

[0326] The three transforming growth factors possess numerous activities. TGFβ1 and 2 are involved in cancer, and they can stimulate cancer stem cells, increase fibrotic / fibrogenic responses, and suppress immune recognition of tumors.

[0327] TGFβ1, 2, and 3 transmit signals by binding to the receptor TβRII, followed by association with TβRI, and in the case of TGFβ2, further association with TβRIII. This then leads to signal transmission via TβRI-mediated SMAD.

[0328] TGFβ is typically secreted in a prepro form. The "pre" is an N-terminal signal peptide that is cleaved upon entering the endoplasmic reticulum (ER). The "pro" is cleaved in the ER, but the covalent bond persists, forming a cage called a latent-associated peptide (LAP) around TGFβ. The cage opens in response to various proteases (particularly thrombin and metalloproteinases). The C-terminal region becomes a mature TGFβ molecule after the release of the TGFβ molecule from the pro region via proteolytic cleavage. The mature TGFβ protein dimerizes to produce an active homodimer.

[0329] TGFβ homodimers interact with LAP, derived from the N-terminal region of the TGFβ gene product, to form a small latent complex called the SLC. This complex remains intracellular until it binds to another protein (an extracellular matrix (ECM) protein called latent TGFβ-binding protein (LTBP)), where it forms a larger latent complex called the LLC. The LLC is secreted into the ECM. TGFβ is released from this complex into a biologically active form by several protease classes (including metalloproteinases and thrombin).

[0330] The activity regulator of the present invention can regulate TGFβ signaling.

[0331] For example, a regulatory factor may block or reduce TGFβ binding to the TGFβ receptor; a regulatory factor may compete with TGFβ or TGFβR for binding to TGFβR or TGFβ; or a regulatory factor may modulate downstream TGFβ signaling, for example, via SMAD.

[0332] The regulatory factors may be action factors such as antibodies that bind to TGFβ or the TGFβ receptor.

[0333] Fresolimubb is an immunomodulatory antibody that blocks TGFβ1-3. Fresolimubb has been tested in metastatic melanoma and high-grade glioma. Fresolimubb showed some enhancement of tumor-specific immune responses but failed to eradicate tumors. Other antibodies that bind to TGFβ include reldelimubab and metelimumab. Bedinger et al (2016) describe various human monoclonal antibodies that neutralize multiple TGFβ isotypes (MAbs 8(2):389-404).

[0334] Alternatively, the activity regulator may be a recombinant Fc-fusion protein containing a soluble ectodomain of either TβRII (TβRII-Fc) or type III receptor (beta-glycan). Soluble TβRII and soluble TβRIII (beta-glycan) function as decoy receptors that prevent TGF-β binding.

[0335] 6.1 Dominant-Negative TGFβ The regulatory factor may be a secreted factor that can bind to the transforming growth factor beta receptor (TβR) and disrupt the interaction between TβR and transforming growth factor beta (TGFβ).

[0336] The activity regulator could be dominant-negative TGFβ.

[0337] As used herein, “dominant-negative TGFβ” or dnTGFβ means that the secreted factor TGFβ acts antagonistically to wild-type TGFβ.

[0338] Dominant-negative TGFβ neutralizes the biological effects of wild-type TGFβ by inhibiting signal transduction induced by it.

[0339] The activity regulator could be a mutant TGFβ.

[0340] The mature protein of wild-type TGFβ2 is shown below as Sequence ID No. 44. ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS (Sequence ID 44).

[0341] When the amino acid numbers of mutant TGFβ are determined by alignment with SEQ ID NO: 44, one or more mutations may be found in amino acid residues W30, W32, L101, L51, Q67, and Y6, and the mutations are as follows: Amino acid residue 30 is mutated to N, R, K, D, Q, L, S, P, V, I, G, C, T, A, or E; and / or Amino acid residue 32 is mutated to A; and / or Amino acid residue 101 is mutated to A, E; and / or Amino acid residue 51 is mutated to Q, W, Y, A; and / or Amino acid residue 67 is mutated to H, F, Y, W, Y; and / or Amino acid residue 6 is mutated to A or a variant thereof.

[0342] Alternatively, the activity regulator may include abbreviated TGFβ polypeptides (such as monomeric TGFβ). Kim et al (2017) described an engineered TGFβ monomer that functions as a dominant-negative to block TGFβ signaling (J.Biol.Chem.doi:10.1074 / jbc.M116.768754).

[0343] Shortened TGFβ may lack the heel helix α3 (a structural motif essential for binding to TGFβ-type I receptors (TβRIs) but not necessarily required for binding to TβRII receptors).

[0344] The amino acid sequence of the TGFβ monomer is described in SEQ ID NO: 45. SEQ ID NO: 45 includes the signal peptide and the latent-associated peptide (LAP). MHYCVLSAFLILHLVTVALSLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFRKDLGWKWIHEPKGYNANFCAGACPYRASKSPSCVSQDLEPLTIVYYVGRKPKVEQLSNMIVKSCKCS (Sequence ID 45).

[0345] The activity regulator may have the amino acid sequence described in SEQ ID NO: 45 or a variant thereof. The variant TGFβ monomer may have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with respect to SEQ ID NO: 45, provided that the polypeptide provides a monomer that can bind to the transforming growth factor beta receptor (TβR) and disrupt its interaction with transforming growth factor beta (TGFβ).

[0346] 6.2 Dominant-Negative TGFβ Receptors The active TGFβ receptor (TβR) is a heterotetramer composed of two TGFβ receptor I (TβRI) and two TGFβ receptor II (TβRII). TGFβ1 is secreted in a latent form and is activated by multiple mechanisms. Upon activation, TGFβ1 forms a complex with TβRII and TβRI, and TβRI is phosphorylated and activated.

[0347] The activity regulator may be a dominant-negative TGFβ receptor. Dominant-negative TGFβ receptors may lack a kinase domain.

[0348] For example, the activity regulator may contain or be derived from the sequence shown as sequence number 46, which is the monomeric version of the TGF receptor II.

[0349] Sequence ID 46 (dnTGFβ RII) TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSS

[0350] Dominant-negative TGF-βRII (dnTGF-βRII) has been reported to enhance PSMA-targeted CAR-T cell proliferation, cytokine secretion, exhaustion tolerance, long-term in vivo survival, and tumor eradication in a mouse model of invasive human prostate cancer (Kloss et al (2018) Mol.Ther.26:1855-1866).

[0351] 6.3 SMAD As described above, the active TGFβ receptor (TβR) is a heterotetramer composed of two TGFβ receptor I (TβRI) and two TGFβ receptor II (TβRII). Signal transduction is initiated when activated TGF-β binds to the highly affinity transforming growth factor-β receptor-2 (TβRII). This binding requires the involvement of transforming growth factor-β receptor-3 (TβRIII) (also known as β-glycan, which alters the higher-order structure of TβRII to facilitate ligand-receptor binding), after which TGF-β receptor-1 / ALK-5 (TβR1) (serine / threonine kinase) is recruited to the TGF-β / TβRII complex, initiating signal transduction by phosphorylation of SMAD2 and SMAD3 (belonging to the receptor regulatory family of SMAD proteins). Phosphorylated SMAD2 and SMAD3 combine to form a heteromeric complex with SMAD4, which then translocates to the cell nucleus and interacts with various transcription factors, ultimately leading to a cellular response.

[0352] SMAD proteins are intracellular transcription factors that transmit extracellular signals from the membrane to the nucleus during TGFβ activation. Three types of SMADs have been identified: receptor-modulating SMADs (R-SMADs) (including SMAD2 and SMAD3), common mediator SMADs (co-SMADs) (including only SMAD4), and finally, inhibitory SMADs (I-SMADs) (including SMAD6 and SMAD7).

[0353] The SMAD protein consists of two globular domains linked by a linker region. The primary function of the N-terminal domain of SMAD (i.e., the "Mad homology 1" (MH1) domain) is to bind to DNA. The C-terminal domain (i.e., the MH2 domain) mediates protein-protein interactions with numerous regulator and effector proteins (including the TβR receptor), certain cytoplasmic anchor proteins, lineage-specific DNA-binding cofactors, and chromatin modifiers. In the presence of TGFβ, R-SMAD is phosphorylated by the TGFβ receptor. This phosphorylation targets two serine residues in the C-terminal sequence pSer-X-pSer of SMAD, generating an acidic tail that drives the formation of the SMAD transcription complex. Missense mutations in two conserved amino acids within the N-terminal portion of the MH2 domain have been identified in colorectal cancer patients. These two mutations result in dominant-negative behavior that counteracts the activity of the WT SMAD protein.

[0354] The regulatory factors may include SMAD signaling inhibitors such as garnicertib, which are being tested as monotherapy, in combination with alkylating agents such as lomustine or temozolomide for glioblastoma, and in other combinations.

[0355] Alternatively, the regulatory factors may be dominant-negative versions of the signal-truncate SMAD2, SMAD3, and SMAD4 that express only the MH2 domain. The regulatory factors may be: i) MH2, ii) MH2 lacking the truncated last 24aa, and iii) truncated SMAD2_MH2-linker-SMAD3_MH2. These dominant-negatives compete with wild-type SMAD proteins for the receptor docking domain and for binding to partner proteins, thus reducing or blocking TGFβ signaling.

[0356] dnSMAD may be selected from one or more of SMAD2, SMAD3, and / or SMAD4. dnSMAD lacks a functional MH1 domain.

[0357] The MH1 domain is the conserved MAD homology domain at the N-terminus of SMAD proteins. The MH1 domain can bind to DNA and negatively modulates the function of the MH2 domain.

[0358] The MH2 domain is the conserved MAD homology domain at the C-terminus of SMAD proteins. The MH2 domain contains a central β-sandwich along with a conserved loop-helix and can bind to phosphoserine residues. The MH2 domain mediates protein-protein interactions with regulator and effector proteins (including TβR receptors, cytoplasmic anchor proteins, lineage-specific DNA binding cofactors, and chromatin modifiers).

[0359] The regulatory factors may contain, essentially consist of, or be derived from wild-type MH2 domains derived from SMAD2, SMAD3, and SMAD4. The amino acid sequences of these MH2 domains are shown below as SEQ ID NOs. 47-49. WCSIAYYELNQRVGETFHASQPSLTVDGFTDPSNSERFCLGLLSNVNRNATVEMTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQMGSPSVRCSSMS (MH2 domain of sequence number 47-SMAD2) WCSISYYELNQRVGETFHASQPSMTVDGFTDPSNSERFCLGLLSNVNRNAAVELTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQMGSPSIRCSSVS (MH2 domain of sequence number 48-SMAD3) WCSIAYFEMDVQVGETFKVPSSCPIVTVDGYVDPSGGDRFCLGQLSNVHRTEAIERARLHIGKGVQLECKGEGDVWVRCLSDHAVFVQSYYLDREAGRAPGDAVHKIYPSAYIKVFDLRQCHRQMQQQAATAQAAAAAQAAAVAGNIPGPGSVGGIAPAISLSAAAGIGVDDLRRLCILRMSFVKGWGPDYPRQSIKETPCWIEIHLHRALQLLDEVLHTMPIADPQPLD (MH2 domain of sequence number 49-SMAD4)

[0360] The regulatory factor may include, or may be essentially derived from, one of the shortened versions of the MH2 domain outlined above, in which up to 24 amino acids are deleted from the C-terminus of the wild-type MH2 domain.

[0361] The regulatory factor may contain mutations in the MH2 domain that increase the binding affinity of dnSMAD to the phosphorylated TGFβ receptor. The regulatory factor may essentially consist of, or may consist of: the MH2 domain of the SMAD4 polypeptide containing mutations R497H, K507Q, and / or R515G (amino acid numbering corresponding to the numbering described in SEQ ID NO: 49); or the MH2 domain of SMAD3 containing mutations K378R and / or K314R (amino acid numbering corresponding to the numbering described in SEQ ID NO: 48).

[0362] The activity regulator may be a chimeric dnSMAD containing at least two dnSMAD polypeptides as defined above. The dnSMAD polypeptides of the chimeric dnSMAD may be linked by a linker domain.

[0363] The amino acid sequence of the chimeric dnSMAD, which includes the dnSMAD2 polypeptide and the dnSMAD3 polypeptide, is shown below as SEQ ID NO: 50. WCSIAYYELNQRVGETFHASQPSLTVDGFTDPSNSERFCLGLLSNVNRNATVEMTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQMLEYSGGGSGGGSLEWCSISYYELNQRVGETFHASQPSMTVDGFTDPSNSERFCLGLLSNVNRNAAVELTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQM (Sequence ID 50)

[0364] dnSMAD or chimeric dnSMAD may include sequences as shown below: SEQ ID NOs. 47-50; or variants having at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity to SEQ ID NOs. 47-50, provided that the variant sequence competes with the wild-type SMAD protein for receptor docking domain and partner protein binding, and retains the ability to mitigate or block TGFβ signaling.

[0365] 7. Metabolic enzymes The regulatory factors may be metabolic enzymes (such as AMP-activated protein kinase (AMPK) or isocitrate dehydrogenase (IDH)).

[0366] AMPK plays a role in cellular energy homeostasis, primarily by activating glucose and fatty acid uptake and oxidation when cellular energy is low.

[0367] AMPK is a heterotrimeric protein complex formed by α, β, and γ subunits. Each of these three subunits plays a specific role in both the stability and activity of AMPK. Specifically, the γ subunit contains four specific cystathionine β-synthase (CBS) domains that confer AMPK the ability to sensitively detect shifts in the AMP:ATP ratio. The four CBS domains create two AMP binding sites, commonly referred to as Bateman domains. When one AMP binds to a Bateman domain, the binding affinity of a second AMP to the other Bateman domain increases cooperatively. As AMP binds to both Bateman domains, the γ subunit undergoes a change in its higher-order structure, exposing a catalytic domain found on the α subunit. It is within this catalytic domain that AMPK becomes activated when threonine-172 is phosphorylated by upstream AMPK kinase (AMPKK). Furthermore, the α, β, and γ subunits can be found in different isoforms: the γ subunit can exist as either the γ1, γ2, or γ3 isoform; the β subunit can exist as either the β1 or β2 isoform; and the α subunit can exist as either the α1 or α2 isoform.

[0368] The following human genes encode the AMPK subunit: α-PRKAA1, PRKAA2 β-PRKAB1, PRKAB2 γ-PRKAG1, PRKAG2, PRKAG3.

[0369] The activity regulator may contain one or more AMPK subunits. The activity regulator may contain α, β, and γ subunits derived from AMPK.

[0370] IDH catalyzes the oxidative carboxylation of isocitrate, producing alpha-ketoglutarate (α-ketoglutarate) and CO2. This involves two processes: oxidation of isocitrate (a secondary alcohol) to oxalosuccinate (a ketone), followed by decarboxylation of the carboxyl group beta to the ketone (forming alpha-ketoglutarate). In humans, IDH exists in three isoforms: IDH3 catalyzes the third step of the citric acid cycle while converting NAD+ to NADH within mitochondria. Isoforms IDH1 and IDH2 catalyze the same reaction outside the environment of the citric acid cycle, using NADP+ instead of NAD+ as a cofactor. These are localized in the cytosol, as well as in mitochondria and peroxisomes.

[0371] The regulatory factors may be IHD1, IHD2, or IHD3. The amino acid sequences of human IDH1, 2, and 3 are available in the NCBI database under the following accession numbers: CAG38738.1(IDH1); NP_002159.2(IDH2, isoform 1); NP_001276839.1(IDH2, isoform 2); NP_001277043.1(IDH2, isoform 3); NP_689996.4(IDH3, isoform 1); NP_001274178.1(IDH3, isoform 2); NP_001339753.1(IDH3, isoform 3).

[0372] 8. Co-stimulatory signals The activity regulator of the present invention can provide a co-stimulatory signal to T cells.

[0373] For example, an activity modulator could be a TNF receptor, a chimeric TNF receptor, or a TNF receptor ligand.

[0374] TNF family costimulatory molecules provide survival and expansion signals to T cells during their development. These TNF receptors (TNFRs) are TN It sends signals via the F receptor-related factor (TRAF) second messenger.

[0375] TNFRSF9 (4-1BB), TNFRSF4 (OX40), TNFRSF5 (CD40), and TNFRSF14 (GITR) transmit survival signals to T cells. TNFRSF7 (CD27) and TNFRSF14 (HVEM) are expressed by naive T cells. Expression of OX40 and 4-1BB is induced in response to antigen stimulation, and these TNFRs have been proposed as markers of effector T cells. Although CD27 and GITR can be constitutively expressed by conventional T cells, their expression is also strongly upregulated after T cell activation, in parallel with the upregulation of OX40 and 4-1BB expression.

[0376] Induction or upregulation of OX40, 4-1BB, and GITR expression occurs within 24 hours of antigen recognition and activation by naive T cells, and even more rapidly in memory T cells; expression of these receptors can last for several hours or even several days.

[0377] The TNF receptor TNFRSF35 / death receptor 3 (D3R) is activated by TL1A, which is transiently upregulated by inflammatory tissue, and this interaction appears to be important in the later stages of T cell activity after an established immune response.

[0378] CD40 is not expressed by T cells, but CD40L and CD40 / CD40L are particularly important for B cell differentiation and expansion.

[0379] TNFRSF11A (RANK) is not expressed by T cells, but the RANK / RANK-L pathway is important for immune development, is also an important pathway for osteoclast activity, and is active in bone metastases.

[0380] TNFRSF12A(Fn14) is not expressed by T cells, but it is expressed with its ligand TWEAK in damaged or inflammatory tissues and in most cancers.

[0381] 8.1 Chimeric TNF receptors International Patent Application No. PCT / GB2018 / 053629 describes a chimeric TNF receptor comprising (a) a binding domain capable of binding to a TNFR ligand; and (b) a TNFR signaling domain.

[0382] The presence of chimeric TNFRs can decouple the strict temporal and / or spatial control of TNFR signaling, providing enhanced survival signals to manipulated cells (e.g., CAR T cells). Chimeric TNFRs can compensate for the lack of a complete physiological immune response in the tumor microenvironment. Chimeric TNFRs can be constructed so that antigen-binding domains associate, and therefore induce the necessary co-stimulatory signals in the tumor microenvironment.

[0383] The antigen-binding domain of a chimeric TNFR may include the ligand-binding domain of the TNFR. For example, the antigen-binding domain may include the ligand-binding domains of D3R, HVEM, CD27, CD40, RANK, or Fn14.

[0384] The signaling domain of a chimeric TNFR may be an activating signaling domain (such as one that can signal via TNFR-related factors (TRAFs)). For example, the activating signaling domain may include the signaling portion of the 4-1BB end domain, the OX40 end domain, or the GITR end domain.

[0385] The regulatory factors may be HVEM-41BB chimeras, CD27-41BB chimeras, RANK-41BB chimeras, or Fn14-41BB chimeras. Examples of appropriate amino acid sequences for these chimeric TNF receptors are shown below as SEQ ID NOs. 51-54, where the ectodomain is shown in regular letters, the transmembrane domain in bold, and the 41BB end-domain in italics.

[0386] [ka]

[0387] 8.2 TNF receptor ligands TNF-related cytokines (TNF family ligands) are type II transmembrane proteins (intracellular N-terminus) that have a short cytoplasmic tail (15-25 residues in length) and a larger extracellular region (approximately 50 amino acids) containing a signature TNF homologous domain where the receptor binding site is located.

[0388] Table 3 provides an overview of TNFRs and their ligands.

[0389] [Table 3-1] [Table 3-2]

[0390] The regulatory factors may be or may include TNF receptor ligands (such as CD40L (CD154), OX40L (CD134), or 41BBL). The amino acid sequences of these proteins are shown below as SEQ ID NOs. 55-57.

[0391] Sequence ID 55 (CD40L) MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEA SSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

[0392] Sequence ID 56 (OX40L) MERVQPLEENVGAARPRFERNKLLLVASVIQGLGLLLCFTYICLHFSALQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL

[0393] Sequence ID 57 (41BBL) MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYK EDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

[0394] Chimera RTK Receptor proteins—tyrosine kinases (RPTKs) constitute a family of intracellular signaling regulators that mediate embryonic development, cell growth, metabolism, and immune function. RPTKs are often dysregulated in cancer, promoting proliferation.

[0395] The proliferative capacity of CAR T cells does not correlate with different binders and structures, and the features that maximize proliferation are not well understood. CAR T cells are exposed to a harsh tumor microenvironment, and their proliferation can be favored by incorporating RPTKs in a dimeric manner. Expressing RTKs that can signal in the absence of homologous ligands disrupts the tumor's immune-deficient environment, improving CAR T cell survival signaling.

[0396] RTKs transmit signals via ligand-induced dimerization / oligomerization, and tyrosine residues in the kinase domain activation loop of their cytoplasmic tail are autophosphorylated. When RTKs are oligomerized via ligand, they are activated in two steps (increased catalytic activity and generation of a docking site for downstream signaling proteins).

[0397] Typically, RTKs transmit signals through homodimerization.

[0398] Autophosphorylation of RTKs can occur in either cis or trans orientation. Phosphorylated tyrosine residues constitute docking sites for numerous SH2-containing signaling molecules. Generally, all RTKs transmit signals via common downstream signaling proteins such as: PI3 kinase, Ras-Raf-MAPK, JNK, and PLCγ. Signaling is mediated by the JAK-STAT pathway.

[0399] The activity regulator may be a receptor tyrosine kinase (RTK) capable of signaling in the absence of a congener ligand. Such an RTK is described in UK Patent Application Publication No. 1803079.1.

[0400] RTKs may be overexpressed and / or mutated to enable signaling in the absence of their homologous ligands.

[0401] RTKs can be chimeric RTKs. Chimeric RTKs may contain an ectodomain or endodomain that mediates the dimerization or oligomerization of the chimeric RTK.

[0402] Domains mediating the dimerization or oligomerization of chimeric RTKs may contain disulfide bonds (for example, the aforementioned domains may be or may contain hinge domains).

[0403] Alternatively, the domain mediating the dimerization or oligomerization of chimeric RTKs may include a chemically manipulable dimerization or oligomerization domain. The dimerization or oligomerization of chimeric RTKs may be induced by the action factor.

[0404] Table 4 summarizes the human RTK subfamilies and RTKs. [Table 4]

[0405] Examples of UniProt accessions and associated amino acid sequences for human RTKs, as shown in Table 2, are provided in Table 5.

[0406] [Table 5-1] [Table 5-2] [Table 5-3]

[0407] Regulation of target cell activity

[0408] Activity regulators can modulate the activity of target cells (e.g., tumor cells).

[0409] For example, the activating factor could be a toxin (such as a toxin toxic to tumor cells). For instance, the activating factor could be diphtheria toxin, pseudomonas toxin, or Shigera toxin.

[0410] Alternatively, the activity regulator may be a prodrug or a prodrug activating compound. The activity regulator may also be a prodrug activating enzyme.

[0411] Prodrugs are widely used for the targeted delivery of cytotoxic compounds to cancer cells. Prodrugs are inactive or low-activity derivatives of drug molecules that regenerate their active form through enzymatic or chemical transformation.

[0412] In targeted cancer therapy, conventional chemotherapeutic agents lacking intrinsic target specificity are rationally modified to concentrate on tumor cells and redirect their cytotoxicity towards them. The usefulness of numerous conventional nonspecific chemotherapeutic agents (such as doxorubicin, paclitaxel, camptothecan, cisplatin, and their derivatives) has been significantly expanded by modifying them into prodrugs (particularly prodrugs with cell-targeting moieties).

[0413] The regulatory factor may be an enzyme that activates a separately administered specific inactive substrate (prodrug) into a cytotoxic product. The regulatory factor may be or may contain cytosine deaminase (CD), which converts the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), leading to so-called "thymine starvation death" by its downstream antimetabolite. Alternatively, the regulatory factor may be or may contain a cytosine deaminase / uracil phosphoribosyltransferase fusion (CD / UPRT; encoded by the Fcy::Fur gene), which is also used to produce 5-FU system antimetabolites. Other antimetabolite prodrugs include acyclovir and ganciclovir (activated to active triphosphates using recombinant thymidylate kinase) and nucleoside analogs such as 6-methyl-2'-deoxyriboside and 2-fluoro-2'-deoxyadenosine (converted to 6'-methylpurine and 2-fluoroadenine, respectively, by purine nucleoside phosphorylase). Human deoxycytidine kinase (DCK) and thymidylate kinase (tmpk) can monophosphorylate a range of (non-physiological) prodrugs (such as gemcitabine (dFdC), bromovinyl-deoxyuridine (BVdU), cytarabine (AraC), and 3'-azido-3'-deoxythymidine (AZT) monophosphate). Furthermore, a chimeric fusion of DCK with uridine monophosphate kinase (DCK::UMK) has been developed to directly activate gemcitabine to its cytotoxic diphosphate metabolite (dFdCDP) in pancreatic cancer. There are also "designer" prodrugs in which chemotherapeutic agents are derivatized into substrates for specific activating enzymes. Examples include the phenoxyacetamide conjugate of doxorubicin and melphalan (hydrolyzed by penicillin-V amidase), the dipiperidinyl conjugate of etoposide (VP-16) (hydrolyzed by recombinant carboxylesterase), and the cephalosporin conjugate of 5-FU (designed for hydrolysis by β-lactamase).

[0414] Activity regulators can be enzymes capable of cleaving prodrug conjugates (e.g., prodrug conjugates containing toxins). Similar to prodrug conjugates, many target toxins consist of a targeting moiety (e.g., an antibody in the case of an immunotoxin), a cleavable linker, and a drug (a cytotoxic enzyme). Moxetumomab pasdotoxin consists of a shortened exotoxin A derived from Pseudomonas aeruginosa (in which the innate receptor-binding domain (located at the N-terminus, 250 residues) is replaced by a single-chain variable fragment that targets the cell surface CD22 antigen). Cytotoxic activity is entirely conferred by the C-terminal segment (residues 405-613, called PE3). This conjugate is an inactive toxin in its current form: to be activated cytotoxicly, it requires cleavage between residues 279 and 280 by protease furin during endocytosis. Therefore, moxetumomab pasdotox is functionally a targeted prodrug conjugate, with residues 251-364 (domain II) derived from exotoxin A acting as a linker that releases cytotoxic PE3 upon cleavage.

[0415] 1. Biosynthesis of CAR T cells Activity regulators can be enzymes that can synthesize small molecules when expressed within a cell or in combination with other enzymes.

[0416] International Patent Application No. PCT / GB2018 / 053262 describes engineered cells encoding transgenic synthetic biological pathways in which engineered cells can produce small molecules, particularly therapeutic small molecules. Engineered cells may include: (i) chimeric antigen receptors (CARs) or transgenic T cell receptors (TCRs); and (ii) one or more engineered polynucleotides encoding one or more enzymes that, when expressed in combination within a cell, can synthesize therapeutic small molecules.

[0417] For example, there may be 1, 2, 3, 4, or 5 enzymes. One or more enzymes may be encoded in one or more open reading frames. One or more enzymes may be encoded in a single open reading frame. Appropriately, each enzyme may be separated by a cleavage site. The cleavage site may be a self-cleavage site (such as a sequence encoding an FMD-2A-like peptide).

[0418] Therapeutic small molecules may be, for example, cytotoxic molecules; cell proliferation inhibitors; agents that can induce tumor differentiation; or pro-inflammatory molecules. In particular, the small molecules may be violacein or its derivatives, or geraniol.

[0419] Factors that modulate the microenvironment of target cells Activity regulators can be agents that modulate the environment of target cells (e.g., tumor cells).

[0420] For example, the activating factor could be a cytokine (such as IL-7 or IL-12) or chemokine (such as CCL19) as discussed above. Alternatively, the activating factor could influence the expression or activity of the cytokine or chemokine discussed above.

[0421] 1.CAR-T cell secreted enzyme The immune microenvironment contains small molecule metabolites and nutrients that can alter the balance between tumor survival and / or progression and the immune response. Modification of the microenvironment can alter the balance to favor the immune response and / or improve the activity or efficacy of adaptive immunotherapy (such as the efficacy of engineered cells expressing CARs or transgenic TCRs).

[0422] The activity modulators related to this invention may modulate the levels of one or more metabolites or nutrients within the tumor microenvironment, potentially imbalanceing them to favor immune cells (such as T cells involved in the immune response) and / or to eliminate tumor cells.

[0423] For example, an activity regulator could be one or more enzymes that, when secreted or expressed on the cell surface, deplete extracellular molecules of the manipulated cell, the aforementioned molecules being: (i) necessary for tumor cells to survive, proliferate, metastasize or exhibit chemoresistance, and / or (ii) It is detrimental to the survival, proliferation, or activity of the manipulated cells.

[0424] British Patent Application No. 1820443.8 describes engineered cells that secrete or express such enzymes on their cell surface.

[0425] Enzymes can deplete amino acids or amino acid metabolites, nucleic acid bases (such as nucleosides or nucleotides), or lipids.

[0426] If the regulatory factor depletes nutrients or metabolites necessary for the growth or survival of tumor cells, immune cells (e.g., CAR-T cells) can be engineered to survive in the extracellular environment in the absence of the aforementioned molecule. For example, cells can be engineered to synthesize the aforementioned molecule or its precursor intracellularly.

[0427] cell composition Furthermore, the present invention provides a cell composition prepared by the method of the present invention.

[0428] The present invention provides a cell composition, which is prepared by transducing cells with multiple viral vectors such that the composition comprises a mixture of untransduced cells, single-transduced cells, and combined-transduced cells.

[0429] At least one vector in the mixture of viral vectors used in the method of the present invention contains a nucleic acid sequence encoding a CAR. Therefore, the cell composition may include a mixture of CAR-expressing cells transduced individually and in combination.

[0430] "Transduced in combination" means that cells are transduced by at least two viral vectors. For example, when cells are transduced by two vectors (a vector containing transgene A and a vector containing transgene B), the transduced cells will be a mixture of cells expressing only A; cells expressing only B; and cells expressing both A and B. In this situation, cells expressing both A and B are transduced in combination.

[0431] For cells transduced with three vectors, each containing a transgene, the resulting transduced cells are a mixture of the following: A only; B only; C only; A and B; A and C; B and C; and cells expressing A, B, and C. In this situation, three subpopulations expressing A and B; A and C; B and C; and cells expressing A, B, and C are transduced in combination.

[0432] The cell composition includes multiple subpopulations obtained by transduction with different vector combinations in a mixture of viral vectors.

[0433] The cell composition may include cytolytic immune cells (such as T cells and / or NK cells).

[0434] T cells, or T lymphocytes, are a type of lymphocyte that plays a central role in cellular immunity. They can be distinguished from other lymphocytes (such as B cells and natural killer cells (NK cells)) by the presence of T cell receptors (TCRs) on their cell surface. Various types of T cells exist, as summarized below.

[0435] Helper T cells (TH cells) assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and memory B cells, as well as the activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells become activated when peptide antigens are presented by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes (TH1, TH2, TH3, TH17, Th9, or TFH) that secrete different cytokines to facilitate different types of immune responses.

[0436] Cytolytic T cells (TC cells, or CTLs) destroy virus-infected cells and tumor cells and are also involved in graft rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of all nucleated cells. CD8+ cells can be inactivated into an anergic state via IL-10, adenosine, and other molecules secreted by regulatory T cells, thereby preventing autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0437] Memory T cells are a subset of antigen-specific T cells that are maintained over a long period after recovery from infection. Memory T cells rapidly expand into numerous effector T cells upon re-exposure to their congener antigen, thus "remembering" past infections in the immune system. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0438] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are essential for maintaining immune tolerance. Their main roles are to suspend T-cell immunity toward the termination of the immune response and to suppress autoreactive T cells that have evaded negative selection processes within the thymus.

[0439] Two main classes of CD4+ Treg cells (endogenous Treg cells and adaptive Treg cells) are described.

[0440] Endogenous Treg cells (also known as CD4+CD25+FoxP3+Treg cells) originate in the thymus and are involved in the interaction of developing T cells with both myeloid (CD11c+) dendritic cells and plasmacytoid (CD123+) dendritic cells activated by TSLP. Endogenous Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations in the FOXP3 gene can inhibit the development of regulatory T cells and can lead to the fatal autoimmune disease IPEX.

[0441] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can arise during a normal immune response.

[0442] Natural killer cells (or NK cells) are part of the innate immune system. NK cells respond rapidly to endogenous signals from virus-infected cells in an MHC-dependent manner.

[0443] NK cells (belonging to the congenital lymphoid cell group) are defined as large granular lymphocytes (LGLs) and constitute a third cell type differentiated from common lymphoid progenitor cells that produce B lymphocytes and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus before entering the circulation.

[0444] The cells of the present invention may be any of the cell types described above.

[0445] The cells to be introduced using the method of the present invention may be derived from a blood sample, for example, a leukapheresate. The cells may be or may include peripheral blood mononuclear cells (PBMCs).

[0446] Cells can be generated ex vivo in hematopoietic stem cell transplantation from the patient's own peripheral blood (first source), or from donor peripheral blood (second source) or peripheral blood from an unrelated donor (third source).

[0447] Alternatively, the cells may be derived from the ex vivo differentiation of induced progenitor cells or embryonic progenitor cells into, for example, T cells or NK cells. Or, immortalized T cell lines that retain lytic function and can act as therapeutic agents may be used.

[0448] Cells can be activated and / or proliferated by treatment with, for example, an anti-CD3 monoclonal antibody before being transduced with a nucleic acid encoding a molecule that yields the chimeric polypeptide of the first aspect of the present invention.

[0449] After transduction, cells can then be purified (e.g., selected) based on CAR expression. If a subpopulation of cells expressing the regulatory factor in the absence of CAR may exist after transduction, it may be desirable to select cells based on CAR expression. However, if each vector in the viral vector mixture contains a nucleic acid sequence encoding CAR, then no cell should be able to express the regulatory factor in the absence of CAR, and therefore, it may not be necessary to purify or select cells based on CAR expression.

[0450] Pharmaceutical composition The cell composition of the present invention, comprising a mixture of transduced CAR-expressing cells, both individually and in combination, can be administered to a patient as a pharmaceutical composition.

[0451] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and / or compounds. Such formulations may be, for example, in a form suitable for intravenous infusion.

[0452] Treatment method The present invention provides a method for treating a disease, comprising the step of administering the cell composition of the present invention (for example, in the pharmaceutical composition described above) to a subject.

[0453] The method for treating diseases relates to the therapeutic use of the cell composition of the present invention. The cell composition can be administered to a subject already having a disease or condition in order to alleviate, reduce or improve at least one symptom associated with the disease, and / or to delay, reduce, or block the progression of the disease.

[0454] The method for preventing disease relates to the prophylactic use of the cell composition of the present invention. Such cell composition can be administered to subjects who are not yet afflicted and / or who do not exhibit any symptoms of the disease, in order to prevent or impair the cause of the disease, or to reduce or prevent the onset of at least one symptom associated with the disease. Subjects may be predisposed to the disease or considered to be at risk of developing it.

[0455] This method may include the following steps: (i) A step of isolating the sample containing cells; (ii) Transducing such cells with a mixture of at least two viral vectors; (iii)(ii) A step of administering cells derived from (ii) to the subject.

[0456] The present invention also provides cell compositions for use in the treatment and / or prevention of diseases.

[0457] The present invention also relates to the use of the cell composition of the present invention in the manufacture of pharmaceuticals for the treatment of diseases.

[0458] Diseases to be treated by the method of the present invention may include cancerous diseases (such as bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell carcinoma), leukemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, and thyroid cancer).

[0459] The disease may be multiple myeloma (MM), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), neuroblastoma, T-cell acute lymphoblastic leukemia (T-ALL), or diffuse large B-cell lymphoma (DLBCL).

[0460] The disease may be plasmacytotoxicity, such as plasmacytoma, plasmacytoplasmia, multiple myeloma, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, solitary osteoplasmacytoma, extramedullary plasmacytoma, osteosclerosing myeloma, heavy chain disease, monoclonal gammaglobulinemia of undetermined significance, or smoldering multiple myeloma.

[0461] The cells of the composition of the present invention may be capable of killing target cells such as cancer cells. Target cells may be characterized by the presence of tumor-secreted ligands or chemokine ligands near the target cells. Target cells may be characterized by the presence of soluble ligands along with the expression of tumor-associated antigens (TAAs) on the target cell surface.

[0462] Different cell subpopulations within the compositions of the present invention may exhibit different levels of ability to kill target cells, both among different patients with the same disease and at different disease sites within a patient (e.g., tumor sites).

[0463] Operations based on results from CAR-expressing cells Furthermore, the present invention provides a method for determining the optimal combination of components of CAR-expressing cells for treating a disease.

[0464] As described above, the cell composition of the present invention comprises a mixture of CAR-expressing cells, some of which are transduced individually, and others which are transduced in combination. The number of different subpopulations depends on the number of viral vectors in the mixture used for transduction. For the three vectors A, B, and C, there should be the following seven transduced cell populations: populations transduced as follows: A only; B only; C only; A and B; A and C; B and C; and A, B, and C. For the four vectors A, B, C, and D, there should be the following 15 different subpopulations: A only; B only; C only; D only; A and B; A and C; A and D; B and C; B and D; C and D; A, B, and C; A, B, and D; A, C, and D; B, C, and D; and A, B, C, and D.

[0465] If each vector in the composition contains nucleic acids encoding a CAR and / or a regulatory factor, the cell composition is a mixture of transduced cell subpopulations, each expressing a different combination of CARs and regulatory factors.

[0466] For example, a mixture of viral vectors may express one or more CARs and multiple different activity regulators. CAR-expressing cells produced by transduction of cells with a mixture of vectors express multiple different combinations of activity regulators that can confer different properties to CAR-expressing cells with respect to target cell killing, survival, engraftment, resistance to checkpoint inhibition, and / or resistance to a harsh tumor microenvironment. In vivo, one subpopulation of cells expressing a particular combination of CARs and activity regulators best adapts to specific conditions within a patient or at a specific site in a patient. The aforementioned subpopulation receives the most effective activation signals, survival signals, and / or proliferation signals and overcomes other subpopulations in the cell composition.

[0467] By analyzing CAR-expressing cells in patients after administration, it is possible to determine which subpopulation best survives, engrafts, and kills target cells. By analyzing the phenotype or genotype of this subpopulation, it is possible to determine which combination of vectors within the subpopulation is successfully transduced during the preparation of the cell composition.

[0468] Using this information, homologous CAR-expressing cell compositions can be designed in which any transdextrin expresses the most successful combination of CARs and regulatory factors.

[0469] The method may include the following steps: (i) A step of administering a cell composition to a subject with a disease; (ii) Monitoring patient or patient-derived samples(s) to determine which cell subpopulations in the cell composition exhibit the highest levels of viability, engraftment, proliferation activation, and / or target cell killing; and (iii) A step of analyzing the phenotype / genotype of the aforementioned cells in the subpopulation to identify and analyze the CAR(s) and / or regulatory factors(s) expressed by the aforementioned cells.

[0470] Furthermore, the present invention provides a method for producing a CAR-expressing cell composition for use in treating a disease, comprising the steps of determining the optimal combination of components of CAR-expressing cells for treating the disease using the method described above, and then transducing the cells with a single vector expressing the identified combination of components. The resulting cell composition is uniform in the sense that all CAR-expressing cells express the same combination of components.

[0471] It is also possible to transduce cells with two or more vectors expressing identified combinations of components, and then select cells expressing all identified components to arrive at a homogeneous CAR-expressing cell composition.

[0472] Furthermore, the present invention provides a cell composition prepared by such a method.

[0473] The present invention will be further illustrated here by examples, which are intended to assist those skilled in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. [Examples]

[0474] Example 1 - Generation of CAR-T cell compositions transduced with multiple vectors We generated lentiviral vectors expressing either of the following: a) a second-generation anti-CD19 CAR (pCCL.PGK.aCD19cat-CD8STK-41BBZ) described in WO2016 / 139487, containing an anti-CD19 antigen-binding domain, a CD8 stalk spacer and a transmembrane domain, and a compound 4-1BB-CD3ζ endodomain, under the control of the PGK promoter; or b) an anti-CD22 CAR (pCCL.EF1a.aCD22_9A8-1-64_LH_scFv-CD8STK-41BBz) containing a second-generation endodomain containing the CDR, CD8 stalk spacer and CD3ζ, as shown above as SEQ ID NOs. 10-15, and a 4-1BB costimulatory domain, under the control of the EF1α promoter.

[0475] Two separate lentiviral supernatants were prepared and mixed 1:1 at a MOI of 2.5 + 2.5. The expression patterns of the two CARs were investigated by flow cytometry, staining for anti-CD19 CAR expression using an anti-idiotype antibody and for CD22 CAR expression using soluble CD22. The results are shown in Figure 6. After transduction with the lentiviral composition, the cells were a mixture of untransduced cells (46.5%); cells expressing only anti-CD19 CARs (23.1%); cells expressing only anti-CD22 CARs (11.1%); and cells expressing both CD19 and CD22 CARs (19.3%).

[0476] Example 2 - Expression of CARs (multiple extension modules) combined by transduction using multiple vectors Construct a vector containing a second-generation anti-CD19 CAR (Fmc63-41BBz) combined with a marker gene and one of the following extension modules expected to exhibit activity under specific conditions:

[0477] A shortened version of SHP2 possessing the dnSHP2-SH2 domain but lacking the phosphatase domain. The sequence is shown above as SEQ ID NO: 29. The shortened protein acts as a dominant-negative, competing with the wild-type protein for binding to phosphorylated ITIM on inhibitory immune receptors such as PD1.

[0478] dnTGFBRII is a dominant-negative TGFβ receptor lacking a kinase domain. Its sequence is shown above as SEQ ID NO: 46. dnTGFBRII competes with the wild-type TGF-β receptor for binding to TGF-β and downregulates TGFβ-mediated signaling.

[0479] CCRs are constitutively active chimeric cytokine receptors having IL-2, IL-7, or GM-CSF receptor endodomains, as described above. The sequences of components of such constitutively active CCRs are shown above as SEQ ID NOs: 30-43. In the presence of such receptors, cytokine signaling occurs constitutively (i.e., in the absence of the relevant cytokines).

[0480] Construct a retroviral vector containing the following cassette (GFP, mKate, and RQR8 are marker genes): Vector A-CAR+dnSHP+GFP Vector B-CAR + dnTBRII + mKate Vector C-CAR+CCR+RQR8

[0481] T cells are transduced with a mixture of two retroviruses encoding the two vectors described above in a 1:1 mixture with an MOI of 2.5:2.5. The resulting mixed population contains T cells expressing either the first vector, the second vector, or both vectors. Transduction is detected and measured by flow cytometry, which measures the expression of marker genes. For example, for cells transduced with a mixture of vectors A and B, transduction is detected and measured using the marker genes GFP and mKate.

[0482] To measure proliferation, transduced T cell populations were labeled with the dye Cell Trace Violet (CTV) (a fluorescent dye that is hydrolyzed and retained within cells). CTV is excited by a 405 nm (violet) laser, and its fluorescence can be detected in a Pacific Blue channel. T cells were placed in PBS at a rate of 2 × 10⁶ 6 Resuspend the cells in 1 ml / ml of medium and add 1 ul / ml of 5 mM CTV. Incubate the T cells with CTV at 37°C for 20 minutes. Then, quench the cells by adding 5V complete medium. After 5 minutes of incubation, wash the T cells and resuspend them in 2 ml of complete medium. Further incubation at room temperature for 10 minutes will induce acetic acid hydrolysis, allowing the dye to be retained.

[0483] Labeled T cells are co-cultured with CD-19-expressing target cells for 4 or 7 days. Target cells co-expressing the target antigen and PDL1 are used to investigate the function of the dnSHP2 extension module in the vector mixture containing vector A. T cells are co-cultured with target cells in the presence of soluble TGF-β to investigate the function of the dnTBRII extension module in the vector mixture containing vector B.

[0484] 5 x 10 per well 4The assay is performed in a 0.2 ml total volume 96-well plate using one transduced T cell and an equal number of target cells (1:1 ratio). At day 4 or 7, the T cells are analyzed by flow cytometry to measure the dilution of CTV that occurs as the T cells divide. The number of T cells present at the end of co-culture is calculated and expressed as a multiple compared to the number of T cells introduced.

[0485] The preferential expansion and proliferation within the module corresponding to the immunosuppression exerted by target cells is evaluated by the decrease in cell-trace violet dye and the increase in population-specific markers in the aforementioned population.

[0486] Example 3 - Generation of anti-GD2-CAR T cell products with an extended module by dual transduction using two separate retroviral vectors Background: Neuroblastoma is the most common extracranial solid cancer in children, and long-term survival rates are low in children with high-risk conditions.

[0487] An ongoing Phase I clinical study of GD2-targeted CART for refractory / recurrent neuroblastoma (NCT02761915) demonstrates activity against disseminated disease without inducing on-target / off-tumor toxicity. However, CART survival was limited, and clinical activity was transient and incomplete.

[0488] Based on the GD2 CAR used in this study, the inventors developed a candidate next-generation T cell product called AUTO6NG. The AUTO6NG product consists of three distinct GD2-targeted CAR T cell populations produced by bitransduction of T cells using two distinct retroviral vectors. The first vector directs the expression of a GD2-targeted CAR co-expressed with either the constitutively signaling IL7 cytokine receptor (IL7R_CCR) or the constitutively signaling IL2 cytokine receptor (IL2R_CCR) (Product A), while the second vector is a tricistronic retroviral vector encoding the same GD2 CAR co-expressed with dominant-negative TGFbRII (dnTGFbRII) and abbreviated SHP2 (dSHP2) (Product B). dSHP2 confers resistance to inhibitory signals (such as those derived from PD1).

[0489] A vector design is schematically shown in Figure 7.

[0490] The GD2 CAR is as described in WO2015 / 132604 and has an antigen-binding domain having a VH domain with the sequence shown as Sequence ID No. 77 and a VL domain with the sequence shown as Sequence ID No. 78.

[0491] The constitutively signaling IL2 and IL7 cytokine receptors are as described in WO2017 / 029512. The IL2 CCR comprises a first polypeptide having an IL-2 receptor β-chain endodomain (SEQ ID NO: 40) and a second polypeptide containing a common γ-chain receptor endodomain (SEQ ID NO: 39); in contrast, the IL7 CCR comprises a first polypeptide having an IL-7 receptor α-chain endodomain (SEQ ID NO: 41) and a second polypeptide containing a common γ-chain receptor endodomain (SEQ ID NO: 39).

[0492] The selection / suicide gene RQR8 is as described in WO2013 / 153391 and has the sequence shown as Sequence ID No. 79.

[0493] Dominant-negative TGFbRII (dnTGFbRII) has the sequence shown above as sequence number 46.

[0494] The abbreviated SHP2 (dSHP2) has the sequence shown above as sequence number 29.

[0495] Example 4 - Investigation of cytotoxic activity of single and double transduced cells and the function of various vector expression elements in vitro. Human T cells were either duplexed with both vectors producing a mixture of products A / B / A+B(AUTO6NG), or duplexed with each vector producing either product A or B individually. Controls included untransduced cells (NTs) and cells expressing only GD2 CAR.

[0496] i) Cytotoxic assay Various effector cell types were co-cultured for 72 hours with GD2-expressing SupT1 target cells (SupT1 GD2) or controls (untransduced target cells (SupT1 NT)), and the lysis rate of target cells was analyzed by flow cytometry. The results are shown in Figure 8. All CAR-expressing effector cells were able to kill GD2-expressing target cells. Transduced T cells with dual vector compositions (product A / B / A+B) showed high efficacy against GD2-positive tumor cell lines in cytotoxicity assays, but no difference was observed compared to CAR T cells transduced with only one vector (product A or B).

[0497] ii) Verification of CCR The various transduced CAR T cells and control NT T cells described above were labeled with cell-trace violet (CTV) and cultured for 7 days in cytokine-free complete cell culture medium without antigen stimulation. In vitro survival was quantified as the percentage of proliferating cells with diluted CTV dye and the absolute number of CAR T cells after 7 days. The results are shown in Figure 9. T cells transduced with product A expressing either the constitutively signaling IL7 cytokine receptor (IL7R_CCR) or the constitutively signaling IL2 cytokine receptor (IL2R_CCR); or T cells transduced with product A+B, showed increased proliferation compared to untransduced cells (NT), cells transduced with a vector expressing only GD2 CAR, or cells transduced with a vector expressing only product B (GD2 CAR+dSHP2+dTGFbRII). When either IL2 or IL7R_CCR was expressed, modified T cells were conferred with exogenous cytokine-independent viability and homeostasis without autonomous growth of the T cells.

[0498] iii) Verification of dTGFbRII The various transduced CAR T cells and control NT T cells described above were co-cultured for 7 days with GD2-expressing SupT1 target cells (SupT1 GD2) or controls (non-transduced target cells (SupT1 NT)) in an E:T ratio of 1:2 or 1:8, in or without 10 ng / ml TGFβ. Target cell killing was analyzed by flow cytometry, and IFNγ secretion was analyzed by ELISA. The results are shown in Figures 10 and 11, respectively. CAR T cells transduced with product B expressing dnTGFbRII, or transduced with product A+B, showed resistance to TGFβ-mediated inhibition of target cell killing compared to CAR-T cells expressing only GD2 CAR or cells transduced with a vector expressing only either product A (GD2 CAR+IL2 CCR or GD2 CAR+IL7 CCR). CAR T cells transduced with product B or product A+B restored IFNγ secretion in the presence of TGFβ to levels comparable to those observed in the absence of TGFβ. In contrast, IFNγ secretion from CAR-T cells expressing only GD2 CAR, or cells transduced with vectors expressing only product A, was significantly inhibited in the presence of TGFβ. Therefore, T cells expressing dnTGFbRII demonstrated resistance to TGFβ-mediated immunosuppression in vitro.

[0499] Example 5 - Investigation of the antitumor activity of bitransfected CAR-T cell products in vivo in a xenograft model of neuroblastoma. The antitumor activity of T cells transduced with a dual-vector composition via intravenous administration was investigated using in vivo assays in an established neuroblastoma xenograft model of NSG mice. One million firefly luciferase-expressing CHLA-255 cells (CHLA-255 FFluc) were intravenously injected into 10-14 week old female NSG mice. The xenografts were immobilized for 15 days until stable engraftment could be detected by BLI. Transduction of cells with a single vector expressing GD2 CAR (GD2 CAR) or with the dual-vector composition described in Example 3 and illustrated in Figure 7 (GD2 CAR + IL7) was performed. CAR-T cells were generated by transduction using either CCR / GD2 (CAR+dSHP2+dTGFbRII). 10 × 10⁶ cells were introduced into mice. 6 10 CAR T cells (transduction efficiency 50%), 3 × 10 6 10 CAR T cells (transduction efficiency 50%), 20 × 10 6 Individual NT T cells (10 × 10 6 Intravenous injections of either a dose equivalent to a certain number of CAR T cells (total T cells) or PBS were administered. After 14 days, tumor growth was assessed by bioluminescence imaging twice weekly.

[0500] The results are shown in Figure 12. Intravenous delivery of CAR T cells expressing only the simple GD2 CAR did not have a significant effect on tumor growth (Figures 12A and B). In contrast, CAR T cells transduced with a dual-vector composition were delivered in 3 × 10⁶ cells. 6 pieces and 10 × 10 6 Intravenous delivery at both doses demonstrated potent antitumor activity and survival extension in NSG mice with established tumor burden (Figure 12C and D).

[0501] Example 6 - Generation of anti-PSMA CAR T cell products with an extended module by triple transduction using three separate retroviral vectors The inventors have developed a combination / multimodule CAR T therapy ("AUTO7") for the treatment of prostate cancer consisting of the following functional modules: 1) an anti-PSMA CAR having a second-generation CD28-CD3z compound endodomain (7A12-28z); 2) a safety switch (RapaCasp9, described in WO2016 / 135470, having the sequence shown as SEQ ID NO: 80 above); 3) a dominant-negative TGFβRII (dnTBRII) for inducing TGFβ1 resistance, having the sequence shown as SEQ ID NO: 46 above; 4) a truncated SHP2 (dnSHP2) for inhibiting the PD1 / PD-L1 pathway, having the sequence shown as SEQ ID NO: 29 above; 5) a constitutively active IL7 receptor (CCR_IL7) for inducing the above proliferation; and 6) a sorting-suicide gene having the sequence shown as SEQ ID NO: 79 above. Ultra-low secretion IL-12 (flexiIL-12) for lymphocyte recruitment / activation located downstream of a stop-skip sequence (SS) having the sequence shown as RQR8; and 7) Sequence ID 81. The vector design is shown in Figure 13A.

[0502] The first vector (A) encodes dnSHP2, the suicide gene RQR8, CAR, and dnTBRII; the second vector (B) encodes the constitutively active IL7 receptor; and the third vector (C) encodes the second suicide gene: Rapcasp9 and flexi-IL12.

[0503] Having different suicide genes on vectors A and C provides flexibility. If CAR-related toxicity is observed in a patient, it is possible to selectively remove CAR-expressing cells using the standard method for treating patients with rituximab (RQR8 screening / inducing apoptosis of CAR-expressing cells via the suicide gene). However, if toxicity thought to be related to IL-12 secretion is observed, cells transduced with vector C can be selectively destroyed by adding rapamycin or a rapamycin analog to induce apoptosis via the RapaCasp9 suicide gene. Since the therapeutic product contains cells transduced with a mixture of the three vectors, the therapeutic product is a combination product having different subpopulations of cells transduced with all various combinations of one, two, or all three vectors. This means that some CAR-expressing cells that induce the RapaCasp9 suicide gene should survive. For example, cells transduced with vector A alone or a combination of vectors A and B should not express RapaCasp9 and should not be affected by treatment with rapamycin. This means that it is possible to "modify" the therapeutic product in vivo, selectively deleting IL-12-expressing cells while preserving the CAR-expressing cell population necessary to maintain CAR-mediated antitumor effects.

[0504] AUTO7 was investigated as a single transduction product using vector A ("AUTO7 / A"), a double transduction product using vectors A and B ("AUTO7 / AB"), or a triple transduction product using vectors A, B, and C ("AUTO7 / ABC"). AUTO7 was tested against second-generation CARs ("parents") developed using the same anti-PSMA binder 7A12.

[0505] Example 7 - Investigation of cytotoxic activity of single, double, and triple transduced cells and the function of various vector expression elements in vitro. The ability of T cells transduced with the single, dual, and triple vector combinations described above to kill target cells was investigated using a FACS-based killing assay. SupT1 cells modified to express human PSMA antigen at different levels (SupT1-PSMAhigh, SupT1-PSMAlow) were used as target cells. Unmodified SupT1 cells (SupT1-NT) were used as a negative control. CAR T cells were co-cultured with target cells in an effector:target ratio of 1:2. FBK was assayed by cell fluorescence analysis after 24 hours of incubation, and the results are shown in Figure 14A. IL-2 and IFNγ secretion by CAR T cells was measured by detecting the supernatant collected from the co-culture after 24 hours using ELISA, and the results are shown in Figures 14B and 14C, respectively.

[0506] All cell compositions produced by single transduction using vector A ("AUTO7 / A"), double transduction using vectors A and B ("AUTO7 / AB"), or triple transduction using vectors A, B, and C ("AUTO7 / ABC") were found to be highly potent against PSMA-positive tumor cell lines in cytotoxicity assays. Cytotoxicity and cytokine release were compared to a second-generation assay developed using the same anti-PSMA binder 7A12 as a CAR-only control. It was comparable to what was recognized by CAR ("parent").

[0507] To investigate the ability of single, double, and triple transduced AUTO7 T cells to kill target cells after culture in the absence of IL-2, transduced T cells were cultured under starvation conditions for 7 days in a medium without IL-2 supernatant. After 7 days, CAR T cells were counted and plated with SupT1-PSMAhigh and SupT1-PSMAlow target cells (or SupT1-NT cells as a negative control). T cells were co-cultured with target cells in effector:target ratios of 1:2 and 1:8. FBK was assayed by cell fluorescence analysis 24 hours after incubation (Figure 15A). IL-2 and IFNγ secretion by CAR T cells was measured by ELISA after collecting the supernatant from the co-cultures described above 24 hours later (Figure 15B).

[0508] While cells expressing the control CAR and cells transduced with vector A alone inhibited cytotoxicity and cytokine release after culture under starvation conditions, this effect was less pronounced in cells transduced with dual or triple vector combinations (vector A+B or A, B+C). Vector B contains a gene expressing a constitutively active cytokine receptor: the IL7R_CCR module. Expression of this module results in cytokine-independent viability and proliferation without interfering with cytotoxicity; after starvation, cells transduced with AUTO7 / AB or AUTO7 / ABC were still shown to effectively kill PSMA-expressing target cells.

[0509] To investigate the ability of single, double, and triple transducible AUTO7 T cells to kill target cells in the presence of TGFβ, transducible T cells were co-cultured with SupT1-PSMAhigh and SupT1-PSMAlow targets in 1:2 and 1:8 (E:T) ratios, either in or out of the presence of 10 ng / ml TGFβ1, for 7 days (SupT1-NT was used as a control). Target cell killing was quantified by FACS and normalized to target only. The results are shown in Figure 16.

[0510] At an E:T ratio of 1:8, killing of target cells by T cells expressing only the CAR was inhibited by adding TGFβ1 to the culture medium. This inhibitory effect was reduced in cells transduced with any of the vector combinations A; A+B; or A, B+C. Vector A encodes a dnTGFβRII element that has previously been shown to block TGFβ-mediated inhibition of T cell signaling.

[0511] To investigate the ability of singly, doubly, and triply transduced AUTO7 T cells to kill target cells after repeated exposure to the target antigen, the transduced cells were co-cultured with SupT1-PSMAhigh target cells or SupT1-PSMAlow target cells at a 1:1 ratio (E:T), and the CAR T cells were restimulated every 7 days with 5×10 4 SupT1 cells. After quantification of target cell killing by FACS, each fresh CAR T cell was restimulated. The results are shown in Figure 17.

[0512] After two restimulations (week 2), cells transduced with the vector combination A+B or A+B+C killed significantly more target cells than cells transduced with only the CAR or vector A. After three restimulations (week 3), cells transduced with the vector combination A+B+C showed the best target cell killing.

[0513] Example 8 - Investigation of cytotoxic activity of single, double, and triple transduced cells and the function of various vector expression elements in vivo. An in vivo assay was used to investigate the anti-tumor activity of T cells transduced with single, dual, or triple vector compositions by intravenous administration in an established xenograft model of NSG mice.

[0514] Female NSG mice were injected subcutaneously with 5×10 6 PSMA-positive PC3 human cell lines. The xenografts were allowed to establish for 3 weeks until stable engraftment could be detected by palpation and measurement with calipers. CAR T cells were administered at 1×10 6CAR T cells were administered intravenously at a dose of one CAR T cell / mouse. Calypsoassimetry was performed 2-3 times per week. The results are shown in Figure 18.

[0515] Mice treated (parenterally) with T cells expressing only CAR showed delayed tumor growth compared to mice treated with non-transduced cells, but tumor growth was not controlled. Mice treated with T cells transduced with vector A encoding CAR, dnSHP2, and dnTGFβRII initially showed tumor growth (September 9), followed by a decrease in tumor size (September 23). At the end of the study, some resumption of tumor growth was observed (October 7).

[0516] Mice administered with T cells transduced with vectors A+B initially showed tumor growth (September 9-16), but subsequently experienced a reduction in tumor size, and this effect persisted throughout the study period. Triple-transduced AUTO7 CAR T cells completely eradicated tumors, with no signs of toxicity. Vector combinations, particularly A+B and A+B+C, demonstrate significantly improved tumor reduction and survival in mice in xenograft models compared to CAR alone.

[0517] The results demonstrate the feasibility and efficacy of multimodule AUTO7 products. Addition of the IL7R_CCR, ss_fIL12, dnTGFβRII, and dSHP2 modules to anti-PSMA CAR products enhanced T cell function through prolonged survival, proliferation, activation, and resistance to immunosuppression driven by TGFβ1 and PD1 / PDL1.

[0518] method Binder generation The PSMA binder was generated by CDR grafting of anti-PSMA antibodies derived from genetically vaccinated rats.

[0519] cell line PC3 cells and SupT1 cell lines (NT and PSMA+) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% GlutaMAX. T cells were isolated from peripheral blood mononuclear cells (PBMCs) and maintained in RPMI-1640 medium supplemented with 10% FBS, 1% GlutaMAX, and 100 U / mL IL-2.

[0520] Trait introduction Retroviruses were generated by transiently transfecting HEK293 T cells with GeneJuice containing RDF plasmid (RD114 envelope), gag / pol plasmid, and CAR plasmid. Retrovirus supernatant was collected at 48 and 72 hours. T cells were stimulated in TC-treated T175 flasks with 0.5 μg / mL anti-CD3 and anti-CD28 antibodies, and 100 U / mL The cells were retained in IL-2. Untreated TC 6-well plates were coated with Retronectin and incubated at 4°C for 24 hours, after which T cells were transduced. A total of 3 ml of viral supernatant / multiple supernatants were plated, followed by 1 ml of activated T cells (concentration 1 × 10⁶). 6 Cells ( / ml) were added, followed by 100 U / mL of IL-2, followed by centrifugation at 1000 × g at room temperature for 40 minutes, and incubation at 37°C and 5% CO2 for 2-3 days.

[0521] The human T cells were as follows: • Triple transduction using vectors A, B, and C to produce a mixture of products (AUTO7 / ABC) • Double transduction using vectors A and B, producing a mixture of products (AUTO7 / AB). • Transduced with a single trait via vector A, producing a single product (AUTO7 / A).

[0522] Cytotoxic assay CAR T cells were co-cultured with SupT1-NT and SupT1-PSMA in TC-treated 96-well plates with an effector:target ratio (E:T ratio) of 1:2 or 1:8. After 24 hours of co-culture, effector T cells and target cells were identified by readings by staining with anti-CD3-PeCy7 and Qben10-APC, and dead cells were eliminated using SYTOX Blue7-AAD dead cell stain. Cytotoxicity readings were evaluated by flow cytometry.

[0523] Cytotoxic assay in the presence of TGF CAR T cells were co-cultured in 96-well plates treated with TC at effector:target ratio (E:T ratio) of 1:2 or 1:8 with SupT1-NT, SupT1-PSMAhigh, and SupT1-PSMAlow. TGFβ1 was added at a concentration of 10 ng / ml on day 0, and cytotoxicity readings were evaluated by flow cytometry on day 7.

[0524] in vitro restimulation assay CAR T cells were co-cultured with SupT1-PSMAhigh or SupT1-PSMAlow target cells in a 1:1 ratio (E:T). Every 7 days, 5 × 10⁶ CAR T cells were cultured. 4 Individual SupT1 cells were restimulated. Cytotoxicity was evaluated by the FBK assay as described above, and the target:effector ratio after restimulation was increased by cell fluorescence analysis. The supernatant was collected and cytokine release was evaluated.

[0525] Cytokine ELISA Human IL-2 ELISA MAX® Deluxe Kit and Human IFN-γ The cytokine secretion levels in the supernatant of co-cultures collected from cytotoxic assays were evaluated using the ELISA MAX® Deluxe kit.

[0526] in vivo experiment 5 x 10 6One PSMA-positive PC3 human cell line was injected into the flank of female NSG mice. The xenografts were immobilized for 3 weeks until stable engraftment could be detected by palpation and caliper measurement. Human PBMCs were generated by single, double, or triple transduction. CAR T cells were introduced at a rate of 1 × 10⁶. 6 CAR T cells were administered intravenously at a dose of one CAR T cell / mouse. Measurements were taken with a caliper 2-3 times per week, and the animals were followed until the completion of the animal protocol.

[0527] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the methods and systems described in the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention is described in conjunction with certain preferred embodiments, it should be understood that the invention described in the claims should not be unduly limited to such specific embodiments. In fact, various modifications of the style of description for carrying out the invention that will be apparent to those skilled in the art in molecular biology or related fields are intended to fall within the scope of the following claims. In certain embodiments, for example, the following are provided: (Item 1) A method for preparing a cell composition, comprising the step of transducing a population of cells with a mixture of at least two viral vectors, wherein at least one vector comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR); and wherein at least one vector comprises a nucleic acid encoding an activity regulator that modulates the activity of the CAR, a cell expressing the CAR, or a target cell. (Item 2) The method according to item 1, wherein the activity regulator is a dominant-negative SHP-1 or SHP-2. (Item 3) The method according to item 1, wherein the activity regulator is a dominant-negative transforming growth factor (TGF) β receptor. (Item 4) The method according to item 1, wherein the activity regulator is a constitutively active chimeric cytokine receptor. (Item 5) The method according to item 1, wherein in the mixture of viral vectors, at least one vector comprises a nucleic acid sequence encoding a dominant-negative SHP-1 or SHP-2; and at least one vector comprises a nucleic acid sequence encoding a dominant-negative transforming growth factor (TGF) β receptor. (Item 6) The method according to item 1, wherein the mixture of viral vectors comprises two, three, four, five, or six viral vectors, at least one of which comprises a nucleic acid sequence encoding a CAR; and at least one of which comprises a nucleic acid sequence encoding an activity regulator. (Item 7) The following steps: (i) Transducing a population of cells with a mixture of at least two viral vectors; and (ii) step of selecting CAR-expressing cells from the population of transduced cells derived from step (i) A method for preparing a cell composition according to any of the above items, including the above. (Item 8) The method according to any one of items 1 to 5, wherein each of the viral vectors in the mixture comprises a nucleic acid sequence encoding a CAR. (Item 9) A viral vector composition comprising a viral vector mixture as defined in any of items 1 to 8. (Item 10) The viral vector composition according to item 9, comprising a first vector and a second vector, both comprising nucleic acid sequences encoding a chimeric antigen receptor (CAR). (Item 11) The viral vector composition according to item 10, wherein the nucleic acid sequence of the first vector and the nucleic acid sequence of the second vector encode the same CAR. (Item 12) The viral vector composition according to item 10 or 11, wherein both the first vector and the second vector also include nucleic acids encoding the CAR, cells expressing the CAR, or activity modulators that regulate the activity of target cells. (Item 13) The viral vector composition according to item 12, wherein the activity regulator is selected from dominant-negative SHP-1 or SHP-2; dominant-negative transforming growth factor (TGF) β receptor; and constitutively active chimeric cytokine receptor. (Item 14) The viral vector composition according to item 13, wherein the first vector comprises a nucleic acid sequence encoding a dominant-negative SHP-1 or SHP-2 and a nucleic acid sequence encoding a dominant-negative transforming growth factor (TGF) β receptor; and the second vector comprises a nucleic acid sequence encoding a constitutively active chimeric cytokine receptor. (Item 15) A viral vector composition according to any one of items 11 to 14, wherein the first and second vectors encode the same CAR, and the CAR has an antigen-binding domain that binds to disialoganglioside (GD2). (Item 16) A viral vector composition according to any one of items 10 to 15, wherein the first vector and / or the second vector comprises a nucleic acid sequence encoding a suicide gene. (Item 17) A cell composition prepared by any of the methods described in items 1 to 8, or by transducing cells with any of the vector compositions described in items 9 to 16. (Item 18) A method for treating a disease of a subject, comprising the step of administering a cell composition described in item 17 to the subject. (Item 19) A cell composition as described in item 17, for use in the treatment and / or prevention of disease. (Item 20) Use of the cell composition described in item 17 in the manufacture of a pharmaceutical product for the treatment and / or prevention of disease. (Item 21) A method for determining the optimal combination of components of CAR-expressing cells to treat a disease, comprising the following steps: (i) The step of administering the cell composition described in item 17 to a subject having the disease; (ii) Monitoring the patient or a sample derived from the patient to determine which cell subpopulation in the cell composition exhibits the highest level of engraftment and / or proliferation; and (iii) A step of analyzing the phenotype / genotype of the cells in the subpopulation to identify and analyze the CAR(s) and / or regulatory factors(s) expressed by the cells. Methods that include...

Claims

[Claim 1] The object, method, or system described herein and in the drawings.

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