Methods of transducing cells using viral vectors for expression of Chimeric Antigen Receptors (CARs) or transgenic T Cell Receptors (TCRs)
阅读说明：本技术 使用用于表达嵌合抗原受体(car)或转基因t细胞受体(tcr)的病毒载体转导细胞的方法 (Methods of transducing cells using viral vectors for expression of Chimeric Antigen Receptors (CARs) or transgenic T Cell Receptors (TCRs) ) 是由 R.贝斯威克 E.陈 C.佩蒂科尼 J.福克纳 E.科索普劳 M.普勒 于 2019-01-08 设计创作，主要内容包括：本发明提供了一种用病毒载体转导细胞的方法,该病毒载体包含编码针对靶抗原的嵌合抗原受体(CAR)或转基因T细胞受体(TCR)的核酸,其中细胞包含表达靶抗原的细胞亚群和不表达靶抗原的细胞亚群,该方法包括以感染复数(MOI)转导细胞的步骤,使得与表达靶抗原的细胞亚群相比,不表达靶抗原的细胞亚群以更大的程度被转导。本发明还提供了通过此类方法制备的细胞组合物及其在治疗和/或预防疾病中的用途。(The invention provides a method of transducing a cell with a viral vector comprising a nucleic acid encoding a Chimeric Antigen Receptor (CAR) or a transgenic T Cell Receptor (TCR) against a target antigen, wherein the cell comprises a subpopulation of cells that express the target antigen and a subpopulation of cells that do not express the target antigen, the method comprising the step of transducing the cell at a multiplicity of infection (MOI) such that the subpopulation of cells that do not express the target antigen is transduced to a greater extent than the subpopulation of cells that express the target antigen. The invention also provides cellular compositions prepared by such methods and their use in the treatment and/or prevention of disease.)
1. A method of transducing a cell with a viral vector comprising a nucleic acid encoding a Chimeric Antigen Receptor (CAR) or a transgenic T Cell Receptor (TCR) against a target antigen, wherein the cell comprises a subpopulation of cells that express the target antigen and a subpopulation of cells that do not express the target antigen,
the method comprises the step of transducing the cells at a multiplicity of infection (MOI) such that the subpopulation of cells that do not express the target antigen is transduced to a greater extent than the subpopulation of cells that express the target antigen.
2. The method according to claim 1, wherein at least 5% of the subpopulations of cells that do not express the target antigen are transduced, and less than 1% of the subpopulations of cells that express the target antigen are transduced.
3. The method according to claim 1, wherein at least 10% of the subpopulations of cells that do not express the target antigen are transduced, and less than 1% of the subpopulations of cells that express the target antigen are transduced.
4. The method according to claim 1, wherein at least 15% of the subpopulations of cells that do not express the target antigen are transduced, and less than 1% of the subpopulations of cells that express the target antigen are transduced.
5. The method according to claim 1, wherein at least 20% of the subpopulations of cells that do not express the target antigen are transduced, and less than 1% of the subpopulations of cells that express the target antigen are transduced.
6. The method according to claim 1, wherein at least 30% of the subpopulations of cells that do not express the target antigen are transduced, and less than 1% of the subpopulations of cells that express the target antigen are transduced.
7. The method according to claim 1, wherein at least 20% of the subpopulations of cells that do not express the target antigen are transduced, and the subpopulations of cells that express the target antigen are not significantly transduced.
8. The method according to claim 1, wherein the MOI is selected to achieve about 10-50% transduction of the subpopulation of cells that do not express the target antigen.
9. The method according to claim 1, wherein said cells are transduced with the RD 114-pseudotyped viral vector at an MOI ranging from 0.2 to 1.0.
10. The method according to claim 1, wherein the cell is transduced with a GALV-pseudotyped viral vector at an MOI in the range of 1.0 to 2.0.
11. The method according to claim 1, wherein said cell is transduced with a VSVG-pseudotyped viral vector at an MOI ranging from 5.0 to 10.0.
12. The method according to any one of the preceding claims, wherein the target antigen is TCR beta constant region 1(TRBC 1).
13. The method according to any one of claims 1 to 11, wherein the target antigen is TCR beta constant region 2(TRBC 2).
14. The method according to any of the preceding claims, comprising the steps of:
(i) providing an initial population of cells;
(ii) depleting the starting population of cells from cells expressing the target antigen; and
(iii) transducing the target antigen-depleted cells with a viral vector expressing the CAR or TCR at a multiplicity of infection (MOI) such that cells not expressing the target antigen are transduced to a greater extent than the residual cells expressing the target antigen.
15. The method according to claim 14, wherein less than 10% of the genetically modified cells express the CAR or the target antigen of the transgenic TCR.
16. The method according to claim 14, wherein less than 5% of the genetically modified cells express the CAR or the target antigen of the transgenic TCR.
17. The method according to claim 14, wherein less than 1% of the genetically modified cells express the CAR or the target antigen of the transgenic TCR.
18. A cell composition prepared by transducing cells according to the method of any one of the preceding claims.
19. A pharmaceutical composition comprising a cell transduced by the method according to any one of claims 1 to 17.
20. A composition according to claim 18 or 19 for use in the treatment and/or prevention of a disease.
21. A method for the treatment and/or prevention of a disease comprising the step of administering a composition according to claim 18 or 19 to a subject in need thereof.
22. The method according to claim 21, comprising the steps of:
(i) providing a sample comprising a starting population of cells;
(ii) depleting the starting population of cells from cells expressing the target antigen;
(iii) transducing the target antigen-depleted cells with a viral vector expressing the CAR or TCR at a multiplicity of infection (MOI) such that cells not expressing the target antigen are transduced to a greater extent than residual cells expressing the target antigen; and
(iv) (iv) administering the cells from (iii) to the subject.
23. The method according to claim 21 or 22, wherein the disease is cancer.
24. The method according to claim 23, wherein the disease is a T cell leukemia or lymphoma.
25. Use of a composition according to claim 18 or 19 for the preparation of a medicament for the treatment and/or prevention of a disease.
Technical Field
The present invention relates to methods of transducing a cell with a viral vector comprising a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) or a transgenic T Cell Receptor (TCR) against a target antigen. The cells to be transduced comprise a proportion of cells expressing the CAR or the target antigen of the transgenic TCR. The method includes the step of transducing cells at a multiplicity of infection (MOI) such that cells that do not express the target antigen are transduced to a greater extent than cells that express the target antigen. The invention further relates to cell compositions prepared by the transduction methods of the invention and their use in the treatment and/or prevention of disease.
Background
Adoptive Cell Therapy (ACT) is a personalized therapy that involves administering to a subject immune cells that have activity against specific disease-associated antigens. ACT using naturally occurring tumor-reactive lymphocytes or tumor-infiltrating lymphocytes (TILs) mediates durable, complete regression in patients with melanoma. However, melanoma appears to be the only cancer that can reproducibly produce TIL cultures capable of specific anti-tumor recognition and reactivity.
Subsequent approaches have attempted to more broadly apply ACT to the treatment of other diseases and cancers by genetically engineering cells to express anti-tumor receptors. For example, a TCR consists of one α chain and one β chain. These receptors recognize antigens that have been processed and presented by MHC molecules. Normal circulating lymphocytes transduced with a retrovirus encoding a TCR recognizing MART-1 melanoma-melanocyte antigen have been shown to mediate tumor regression.
Another approach is to administer lymphocytes that are gene-expressed to express the CAR. CARs are artificial receptors that can be constructed by linking the variable regions of the antibody heavy and light chains to separate intracellular signaling chains or the intracellular signaling chains in combination with other signaling moieties. CARs recognize antigens presented on the surface of tumor cells, but need not be MHC restricted. For example, CARs directed against the B cell antigen CD19 have been shown to mediate regression of advanced B cell lymphomas.
Materials essential in the manufacture of genetically modified cell therapy are starting materials, i.e. cells to be genetically modified. These cells may be obtained from the patient in the case of autologous therapy, or from different donors in the case of allogeneic therapy. These cells may be obtained from peripheral blood or white blood cell collections (leukapheresate), for example, from the patient to be treated (autologous) or from a different donor (allogeneic).
CAR-expressing T cells can be prepared by transducing a T cell-containing cell sample, such as a leukocyte collection, with a viral vector. The goal of most research protocols is to achieve maximum transduction, and therefore a saturating (or excess) amount of vector is typically used in the transduction step.
There remains a need for improved methods of transducing cells with viral vectors that provide reproducible efficiency and/or enhanced safety, and/or that avoid the manufacture of cell populations and/or pharmaceutical compositions that are unsuitable for administration to a patient, thereby preventing subsequent treatment delays.
Drawings
Figure 1-Fluorescence Activated Cell Sorting (FACS) plots showing TRBC1 cell depletion. Representative depletion with unconjugated antibody/anti-Ms IgG microbeads or biotinylated antibody/anti-biotin microbeads was performed as indicated. The graph shows the input from 2 without exhaustion.1x107TRBC1+ cell content in the depleted TRBC 1-out (depleted column flow-through) and TRBC1+ out (column capture) samples performed above.
Figure 2-graph showing TRBC1 depletion efficiency. The mean data show (A) comparison of pre-depletion (input) and post-depletion (TRBC 1-output) TRBC1+ cell content. (B) Percentage of input cell subsets recovered from depletion (TRBC 1-export) (n ═ 13).
Figure 3-graph showing TRBC1CAR transduction comparison. Data show the level of transduction achieved using retronectin (marker +/CD3+ cells) at the end of the procedure (undiluted n ═ 15; depleted n ═ 11; 8 pairs of matches.
Figure 4-graph showing transduction efficiency v TRBC1+ cell content. Data correlating transduction in non-depleted cells with TRBC1+ cell content at the time of transduction (n-11). TRBC1+ cell content indicating failure of transduction.
Figure 5-graph showing TRBC1 CAR-transduction process comparison. The data show (a) final CD3 and TRBC1 cell content with cell viability (undiluted n-12; depleted n-15; 12 pairs matched. (B) Post-transduction culture amplifications (undigested n-7; depleted n-5; 4-pair matched. paired t-test n-4).
Figure 6-graph showing depletion phenotype in transduced (marker +) CD8+ T cell subsets. The averaged data shows the percentage of cells expressing multiple depletion markers (Lag3, PD1, and Tim3) (undiluted n-12; depleted n-15; 12 pairs matched.
Figure 7-bar and graph showing memory phenotype in transduced CD8+ T cell subsets. The averaged data showed an enhanced phenotype of the initial cell subset (CCR7+/CD45RA +) to identify CD62L +/CD27+ cells and to further differentiate undifferentiated cells (n ═ 3).
Figure 8-graph showing cytokine release assay results. The end of process data shows the concentrations of (a) granzyme B, (B) IL-2, (C) IFN-g and (D) TNF-a released into the culture (undigested n-5; depleted n-7; 5-pair matched.
Figure 9-shows cytotoxicity assay: graph of results of target cell killing. The data show the percentage of killed TRBC1+ Raji cells after 48 hours (a) relative to untransduced control cells (normalized for non-specific killing), (B) relative to TRBC1+ Raji cells cultured alone (normalized for normal cell death) (non-depleted n-5; depleted n-7).
Figure 10-shows cytotoxicity: graph of the results of cytokine release assay. The data show the concentrations of (a) granzyme B, (B) IL-2, (C) IFN-g and (D) TNF-a released after 48 hours (undiluted n-5; depleted n-7; 5-pair matched.
Figure 11-bar graph showing TRBC1+ cell transduction. The data show a comparison of transduction of undigested, TRBC1+ and depleted cells at different MOIs, (a) with TRBC1CAR, (B) with control CAR (n ═ 1; mean of duplicate wells).
Figure 12-FACS plots showing CD19+ cell depletion. Representative depletion using CD19+ microbeads and MACS selection column. The figure shows samples of the undigested input, CD19- (depleted column flow-through), and CD19+ output (column capture).
Figure 13-graph showing the percentage of B cells (i.e., CD20+) cultured on day 6 (D6) of the manufacturing process. Even in the non-depleted cultures, a low percentage of viable CD20+ cells (i.e. < 1%) were detected at the end of the culture. N is 2
Figure 14-graph showing transduction efficiency at the end of culture period day 7 (D7). % CAR + cells were measured from live CD3+ cells. Higher transduction was observed in CD 19-depleted cells compared to the non-depleted control. N is 4.
Figure 15-graph showing cytokine release into culture medium. The end of these procedures data show the concentrations of (A) granzyme B, (B) IFN-. gamma. (C) IL-2 and (D) TNF-. alpha.released into the culture. For all cytokines, the lowest concentration was observed in the depleted samples compared to the non-depleted ones. N is 4.
Summary of aspects of the invention
The present inventors provide a method for producing a genetically modified cell population comprising a CAR or a transgenic TCR.
The inventors have shown that when transducing a population of cells in which some cells express the target antigen of a CAR or TCR, the multiplicity of infection (MOI) can be modulated such that cells that do not express the target antigen are transduced to a greater extent than cells that express the target antigen.
Without wishing to be bound by theory, the inventors predict that this is due to receptor interference during the transduction process. For cell subsets expressing a target antigen, the inventors predicted that the viral vector would bind to the cell via a CAR or TCR that binds the target antigen rather than (or simultaneously with) binding to the cell via the viral envelope protein. At low MOI, this means that cells that do not express the target antigen are preferentially transduced. At high MOI, this effect is overcome since all receptors are saturated.
The goal of most viral transduction protocols is to achieve maximum transduction of the cells. Thus, most transduction protocols use an excess of vector. Thus, the existing protocols teach away from the methods of the present invention, which involve the deliberate use of a low MOI, such that cells that do not express the target antigen are preferentially transduced.
Thus, in a first aspect, the invention provides a method of transducing a cell with a viral vector comprising a nucleic acid encoding a Chimeric Antigen Receptor (CAR) or a transgenic T Cell Receptor (TCR) against a target antigen, wherein the cell comprises a subpopulation of cells that express the target antigen and a subpopulation of cells that do not express the target antigen,
the method comprises the step of transducing cells at a multiplicity of infection (MOI) such that a subpopulation of cells that does not express the target antigen is transduced to a greater extent than a subpopulation of cells that express the target antigen.
The population of genetically modified cells will have a higher ratio of cells that do not express the target antigen to cells that express the target antigen than the population of cells prior to transduction.
A population of cells can be transduced with a viral vector at an MOI such that cells that do not express the target antigen are transduced, while cells that express the target antigen are not transduced. In the case where transduced cells in a subpopulation are not detectable by flow cytometry, then the subpopulation of cells can be considered "untransduced".
In the methods of the invention, at least 5%, 10%, 20% or 30% of the subpopulations of cells that do not express the target antigen may be transduced, and less than 1% of the subpopulations of cells that express the target antigen may be transduced.
In the methods of the invention, at least 20% of the subpopulations of cells that do not express the target antigen may be transduced, and the subpopulations of cells that express the target antigen are not significantly transduced. In the case where transduced cells in a subpopulation are not detectable by flow cytometry, then the subpopulation of cells can be considered to be "not transduced" or "not significantly transduced".
The MOI may be selected to achieve about 10-50% transduction of a subpopulation of cells that do not express the target antigen.
In the methods of the invention, cells may be transduced with RD 114-pseudotyped viral vectors at an MOI in the range of 0.2 to 1.0.
In the methods of the invention, cells may be transduced with a GALV-pseudotyped viral vector at an MOI in the range of 1.0 to 2.0.
In the methods of the invention, cells can be transduced with a VSVG-pseudotyped viral vector at an MOI in the range of 5.0 to 10.0.
The target antigen of the CAR or TCR may be TCR beta constant region 1(TRBC 1).
The target antigen of the CAR or TCR may be TCR beta constant region 2(TRBC 2).
The method may comprise the steps of:
(i) providing an initial population of cells;
(ii) depleting the starting population of cells expressing the target antigen; and
(iii) transducing target antigen-depleted cells with a viral vector expressing the CAR or TCR at a multiplicity of infection (MOI) such that cells not expressing the target antigen are transduced to a greater extent than residual cells expressing the target antigen.
Less than 10%, 5% or 1% of the genetically modified cells can express the CAR or the target antigen of the transgenic TCR.
In a second aspect, the invention provides a cell composition prepared by transducing a cell according to the method of the first aspect of the invention.
In a third aspect, the present invention provides a pharmaceutical composition comprising a cell transduced by a method according to the first aspect of the invention.
In a fourth aspect, the present invention provides a composition according to the second or third aspect of the invention for use in the treatment and/or prevention of a disease.
In a fifth aspect, the present invention provides a method for the treatment and/or prevention of a disease, comprising the step of administering a composition according to the second or third aspect of the invention to a subject in need thereof.
The method may comprise the steps of:
(i) providing a sample comprising a starting population of cells;
(ii) depleting the starting population of cells expressing the target antigen;
(iii) transducing target antigen-depleted cells with a viral vector expressing a CAR or a TCR at a multiplicity of infection (MOI) such that cells not expressing the target antigen are transduced to a greater extent than residual cells expressing the target antigen; and
(iv) (iv) administering the cells from (iii) to the subject.
The disease may be cancer, such as T cell leukemia or lymphoma.
In a sixth aspect, there is provided the use of a pharmaceutical composition according to the third aspect of the invention in the manufacture of a medicament for the treatment and/or prophylaxis of a disease.
The method of the present invention provides several advantages. A transduced cell population with a low or negligible proportion of transduced cells expressing the target antigen means that killing behavior (fratricide) is reduced and the likelihood of cell differentiation and/or depletion is low. This means that the cells should have improved in vivo persistence after administration and/or improved cytolytic activity after administration.
Another advantage of the present invention is that the genetically modified cells produced by the method are more uniformly genetically modified. There are minor differences between the methods performed on cell populations from different donors and between cell preparations and/or pharmaceutical compositions.
Advantageously, the method according to the invention results in a population of genetically modified cells that are more pure and contain low or undetectable levels of cells expressing the target antigen. The method according to the invention minimizes or eliminates the risk of genetically modifying cells expressing the target antigen. This may provide an additional safety benefit in the case where the target cell (i.e. the cell expressing the target antigen) is a cancer cell.
Furthermore, the method according to the invention reduces the failure of transduction or transfection when there is a high proportion of cells expressing the target antigen in the source of the cells (e.g. the starting population of cells).
Additional aspects
Further aspects of the invention are presented in the following numbered paragraphs:
1. a method of making a genetically modified cell population comprising a Chimeric Antigen Receptor (CAR) or a transgenic T Cell Receptor (TCR), comprising:
(i) providing an initial population of cells;
(ii) depleting the starting population of cells expressing the target antigen; and
(iii) a nucleic acid sequence encoding a CAR or a transgenic TCR against a target antigen is introduced into cells of the depleted starting population.
2. A method according to paragraph 1, wherein the CAR or transgenic TCR is introduced into a cytolytic immune cell.
3. A method according to paragraph 1, wherein the starting population of cells comprises a white blood cell collection (leukapherenate).
4. A method according to any of the preceding paragraphs, wherein the starting population of cells comprises Peripheral Blood Mononuclear Cells (PBMCs).
5. A method according to any of the preceding paragraphs, wherein the depleted starting population comprises PBMCs.
6. A method according to any of the preceding paragraphs, wherein the depleted starting population comprises cytolytic immune cells.
7. A method according to any of the preceding paragraphs, wherein the depleted starting population comprises T cells.
8. The method according to any one of the preceding paragraphs, wherein the target antigen is TCR beta constant region 1(TRBC 1).
9. The method according to any of paragraphs 1 to 7, wherein the target antigen is TCR beta constant region 2(TRBC 2).
10. A method according to any of the preceding paragraphs, wherein the percentage of CAR or transgenic TCR target antigen positive cells in the genetically modified cell population is lower than in the starting population.
11. A method according to any of the preceding paragraphs, wherein less than 10% of the genetically modified cells express the CAR or the target antigen of the transgenic TCR.
12. A method according to any of the preceding paragraphs, wherein less than 5% of the genetically modified cells express the CAR or the target antigen of the transgenic TCR.
13. A method according to any of the preceding paragraphs, wherein less than 1% of the genetically modified cells express the CAR or the target antigen of the transgenic TCR.
14. The method according to any of the preceding paragraphs, wherein the genetically modified cell is prepared as a pharmaceutical composition.
15. A genetically modified cell obtainable by any one of the preceding paragraphs, comprising a CAR or a transgenic TCR.
16. A population of genetically modified cells according to paragraph 15.
17. A genetically modified cell population according to paragraph 16, wherein the cells are cytolytic immune cells.
18. The genetically modified cell population according to paragraph 16 or 17, wherein the cells are T cells.
19. The population of genetically modified cells according to any of paragraphs 16 to 18, wherein the genetically modified cells are less differentiated than genetically modified cells prepared without depleting cells expressing the CAR or target antigen of the transgenic TCR.
20. The population of genetically modified cells according to any of paragraphs 16 to 19, wherein the genetically modified cells have increased expression of CD27 and/or CD62L as compared to genetically modified cells prepared without depleting cells expressing the CAR or target antigen of the transgenic TCR.
21. The population of genetically modified cells according to any of paragraphs 16 to 20, wherein the genetically modified cells are more naive than genetically modified cells prepared without depleting cells expressing the CAR or the target antigen of the transgenic TCR.
22. The population of genetically modified cells according to any of paragraphs 16-21, wherein the genetically modified cells are depleted less than genetically modified cells prepared without depleting cells expressing the target antigen of the CAR or the transgenic TCR.
23. The population of genetically modified cells according to any of paragraphs 16-22, wherein the genetically modified cells have reduced expression of one or more depletion markers compared to genetically modified cells prepared without depleting cells expressing the CAR or the target antigen of the transgenic TCR.
24. The genetically modified cell population according to paragraph 23, wherein the one or more depletion markers are selected from the group consisting of: PD1, Lag3, and Tim 3.
25. A pharmaceutical composition comprising a population of genetically modified cells according to any one of paragraphs 16 to 24.
26. A pharmaceutical composition according to paragraph 25 for use in the treatment and/or prevention of a disease.
27. A method for the treatment and/or prevention of a disease comprising the step of administering a pharmaceutical composition according to paragraph 25 to a subject in need thereof.
28. A method according to paragraph 27, comprising the steps of:
(i) providing a sample comprising a starting population of cells;
(ii) depleting the starting population of cells expressing the target antigen;
(iii) introducing a nucleic acid sequence encoding a CAR or a transgenic TCR against a target antigen into cells in the depleted starting population; and
(iv) (iv) administering the cells from (iii) to the subject.
29. A method according to paragraph 28, wherein the cells are autologous.
30. A method according to paragraph 28, wherein the cells are allogeneic.
31. Use of a pharmaceutical composition according to paragraph 25 in the manufacture of a medicament for the treatment and/or prevention of a disease.
32. The pharmaceutical composition for use according to paragraph 26, the method according to any one of paragraphs 27 to 30 or the use according to paragraph 31, wherein the disease is cancer.
33. The pharmaceutical composition, method or use for use according to paragraph 31 or paragraph 32, wherein the disease is a hematological malignancy.
34. A pharmaceutical composition, method or use for use according to paragraph 32 or paragraph 33, wherein the disease is leukemia or lymphoma.
35. A kit, comprising:
(i) a first nucleic acid sequence encoding a CAR or a transgenic TCR; and
(ii) means for depleting cells expressing a CAR or a target antigen of a transgenic TCR.
36. A method of reducing the number of cells in a pharmaceutical composition that express a target antigen and express a CAR or a transgenic TCR against the target antigen, comprising:
providing an initial population of cells;
depleting the starting population of cells expressing the target antigen;
introducing a nucleic acid encoding a CAR or a transgenic TCR against a target antigen into cells of a depleted starting population; and
incorporating the cells into a pharmaceutical composition.
37. A method according to paragraph 36, wherein the number of cells expressing the target antigen and expressing the CAR or transgenic TCR against the target antigen is reduced compared to a pharmaceutical composition produced without depleting a starting population of cells expressing the target antigen.
38. The method according to paragraph 36 or 37, wherein the cells are expanded prior to incorporation into the pharmaceutical composition.
39. A method according to any of paragraphs 36 to 38, wherein the cells are activated prior to introduction of the nucleic acid encoding the CAR or transgenic TCR against the target antigen.
40. A method according to any of paragraphs 36 to 39, wherein the starting population of cells has been previously frozen and thawed prior to depletion of cells expressing the target antigen.
41. A method according to any of paragraphs 36 to 40, wherein the CAR or transgenic TCR is introduced into the cell by transduction and the multiplicity of infection is sufficient to transduce cells that do not express the target antigen but insufficient to transduce cells that express the target antigen.
40. A method according to any of paragraphs 36 to 41, wherein the percentage of cells expressing the target antigen in the pharmaceutical composition is lower than in the starting population of cells.
41. A method according to any of paragraphs 36 to 40, wherein less than 10% of the cells in the pharmaceutical composition express the target antigen.
42. A method according to any of paragraphs 36 to 41, wherein less than 5% of the cells in the pharmaceutical composition express the target antigen.
43. A method according to any of paragraphs 36 to 42, wherein less than 1% of the cells in the pharmaceutical composition express the target antigen.
44. A method according to any of paragraphs 36 to 43, wherein less than 5% of the cells in the pharmaceutical composition express the CAR or transgenic TCR directed against the target antigen and the target antigen.
45. A method according to any of paragraphs 36 to 43, wherein less than 1% of the cells in the pharmaceutical composition express the CAR or transgenic TCR directed against the target antigen and the target antigen.
Detailed Description
The invention provides methods for transducing cells with a viral vector comprising a nucleic acid sequence encoding a chimeric antigen receptor or a transgenic T cell receptor. The cells to be transduced comprise a proportion of cells expressing the CAR or the target antigen of the transgenic TCR. The method includes the step of transducing cells at a multiplicity of infection (MOI) such that cells that do not express the target antigen are transduced to a greater extent than cells that express the target antigen. The resulting cell compositions are useful in methods of treating and/or preventing disease. The genetically modified cells cause depletion of cells expressing the target antigen by administering the genetically modified cells to the subject.
Chimeric Antigen Receptor (CAR)
Classical Chimeric Antigen Receptors (CARs) are chimeric type I transmembrane proteins that link an extracellular antigen-recognition domain (binder) to an intracellular signaling domain (endodomain). The conjugate is typically a single chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it may be based on other forms that comprise an antibody-like antigen binding site. Spacer domains are often required to separate the conjugate from the membrane and allow it to assume the proper orientation. A common spacer domain used is the Fc of IgG 1. Depending on the antigen, more compact spacers may suffice, such as stems from CD8 a and even just IgG1 hinges. The transmembrane domain anchors the protein in the cell membrane and connects the spacer to the intracellular domain.
Early CAR designs had intracellular domains derived from the intracellular part of the gamma chain of FcR1 or CD3 ζ. As a result, these first generation receptors transmit an immune signal 1 that is sufficient to trigger killing of the associated target cells by T cells, but that does not fully activate the T cells for proliferation or survival. To overcome this limitation, a complex endodomain was constructed: the fusion of the intracellular portion of the T cell costimulatory molecule to the intracellular portion of CD3 ζ creates a second generation receptor capable of simultaneously transmitting activation and costimulatory signals upon antigen recognition. The most commonly used co-stimulatory domain is that of CD 28. This provides the most potent co-stimulatory signal-immune signal 2, which triggers T cell proliferation. Several receptors have also been described, including the intracellular domains of the TNF receptor family, such as the closely related OX40 and 41BB, which transmit survival signals. An even more potent third generation CAR has now been described, having an endodomain capable of transmitting activation, proliferation and survival signals.
The nucleic acid encoding the CAR can be transferred to a T cell using, for example, a retroviral vector. Lentiviral vectors may be employed. In this way, a large number of antigen-specific cells can be generated for adoptive cell transfer. When the CAR binds to the target antigen, this results in transmission of an activation signal to the T cell on which it is expressed. The CAR thus directs the specificity and cytotoxicity of T cells to tumor cells expressing the targeted antigen.
Thus, a CAR typically comprises: (i) an antigen binding domain; (ii) a spacer region; (iii) a transmembrane domain; and (iii) an intracellular domain comprising or associated with a signaling domain.
Antigen binding domains
The antigen binding domain is the antigen-recognizing portion of the CAR.
Many antigen binding domains are known in the art, including those based on antibodies, antibody mimetics, and antigen binding sites of T cell receptors. For example, the antigen binding domain may comprise: single chain variable fragments (scFv) derived from monoclonal antibodies; a natural ligand for a target antigen; a peptide having sufficient affinity for a target; a single domain antibody; artificial single compounds such as Darpin (designed ankyrin repeat); or a single chain derived from a T cell receptor.
The antigen binding domain may comprise a domain that is not based on the antigen binding site of an antibody. For example, the antigen binding domain may comprise a domain based on a protein/peptide that is a soluble ligand (e.g., a soluble peptide, such as a cytokine or chemokine) for a tumor cell surface receptor; or the extracellular domain of a membrane-anchored ligand or a receptor expressed on tumor cells that binds to the counterpart.
The antigen binding domain may be based on the natural ligand of the antigen.
The antigen binding domain may comprise affinity peptides from a combinatorial library or de novo designed affinity proteins/peptides.
Spacer domains
The CAR may comprise a spacer sequence to connect the antigen binding domain and the transmembrane domain and spatially separate the antigen binding domain from the endodomain. The flexible spacer allows the antigen binding domain to be oriented in different directions to facilitate antigen binding.
The spacer sequence may, for example, comprise an IgG1 Fc region, an IgG1 hinge or human CD8 stem or mouse CD8 stem. Alternatively, the spacer may comprise an alternative linker sequence having similar length and/or inter-domain spacer properties to the IgG1 Fc region, IgG1 hinge, or CD8 stem. The human IgG1 spacer may be altered to remove the Fc binding motif.
Transmembrane domain
The transmembrane domain is the sequence of the CAR that spans the membrane.
The transmembrane domain may be any protein structure that is thermodynamically stable in the membrane. This is typically an alpha helix comprising several hydrophobic residues. The transmembrane domain of any transmembrane protein may be used to supply the transmembrane portion of the invention.
The presence and span of transmembrane domains of proteins can be predicted by one skilled in the art using bioinformatic tools such as TMHMM algorithms (http:// www.cbs.dtu.dk/services/TMHMM-2.0 /). Furthermore, artificially designed TM domains may also be used (e.g. as described in US 7052906B1, which is incorporated herein by reference) given that the transmembrane domain of a protein is a relatively simple structure, i.e. a polypeptide sequence predicted to form a hydrophobic alpha helix of sufficient length to span the membrane.
The transmembrane domain may be derived from CD28, which provides good receptor stability.
Activating the intracellular domain
The intracellular domain is the signaling portion of the CAR. It may be part of or associated with the intracellular domain of the CAR. Upon antigen recognition, the receptor cluster, native CD45 and CD148 are excluded from synapses and signals are transmitted to cells. The most commonly used endodomain component is that of CD3-zeta containing 3 ITAMs. This transmits an activation signal to the T cell upon antigen binding. CD3-zeta may not provide a fully capable activation signal and may require additional costimulatory signaling. For example, chimeric CD28 and OX40 may be used with CD3-Zeta to transmit proliferation/survival signals, or all three may be used together.
Where the CAR comprises an activating endodomain, it may comprise the CD3-Zeta endodomain alone, the CD3-Zeta endodomain with either CD28 or OX40 endodomain, or the CD28 endodomain with OX40 and CD3-Zeta endodomain.
Any endodomain containing an ITAM motif can serve as an activating endodomain.
Transgenic T Cell Receptor (TCR)
T Cell Receptors (TCRs) are molecules present on the surface of T cells that are responsible for recognizing fragments of target antigens that are peptides bound to Major Histocompatibility Complex (MHC) molecules.
TCRs are heterodimers consisting of two distinct protein chains. In humans, in 95% of T cells, the TCR consists of the alpha (α) and beta (β) chains (encoded by TRA and TRB, respectively), while in 5% of T cells, the TCR consists of the gamma and delta (γ /) chains (encoded by TRG and TRD, respectively).
Each chain is composed of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is close to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region is bound to the peptide/MHC complex. The variable domains of the TCR alpha and beta chains each have three hypervariable or Complementarity Determining Regions (CDRs). CDR3 is the primary CDR responsible for recognition of the processed antigen. The constant domain of the TCR consists of a short linking sequence in which cysteine residues form a disulfide bond that forms the link between the two chains.
When the TCR is engaged with antigenic peptides and MHC (peptide/MHC), T lymphocytes are activated by signal transduction.
Unlike the target antigens to which conventional antibodies are directed, the antigens recognized by the TCR may include an entire array of potential intracellular proteins that are processed and delivered to the cell surface as peptide/MHC complexes.
TRA and TRB genes were artificially introduced by using a vector; or TRG and TRD genes into a cell, the cell can be engineered to express a heterologous (i.e., non-native) TCR molecule. Such "heterologous" TCRs may also be referred to herein as "transgenic TCRs". For example, the gene for the genetically modified TCR can be reintroduced into autologous T cells and transferred back into the patient for T cell adoptive therapy.
Target antigens
As used herein, a "target antigen" refers to an antigen to which a CAR or transgenic TCR is specific, i.e., an antigen to which the antigen-binding domain of a CAR or transgenic TCR is engineered to be specific.
The target antigen may be a disease-associated antigen.
Suitably, the target antigen may be associated with a chronic infection.
Suitably, the target antigen may be associated with autoimmunity.
The target antigen may be a tumor-associated antigen, such as a cancer-associated antigen.
Various target antigens are known, as shown in the following table. The antigen binding domain used in the present invention may be a domain capable of binding to the antigen shown therein.
Cells
The invention also relates to genetically modified cells comprising a CAR or a transgenic TCR obtainable (or obtained) by the method of the invention.
As used herein, a "starting population of cells" refers to a sample of cells that will be used to generate genetically modified cells comprising a CAR or a transgenic TCR.
The starting population of cells may be obtained from any source of blood cells or Peripheral Blood Mononuclear Cells (PBMCs). The source cells may be provided fresh or may be cryopreserved prior to use. The starting population of cells may be used without any further processing or may be used after an isolation or enrichment step. Methods for isolating or enriching leukocytes are known in the art. For example, separation can be by various methods such as density gradient, e.g., using Ficoll-Paque density gradient media; separation by magnetic beads, such as MACS Milteyni Biotec CD3, CD4, or CD8 beads; leukocytes or PBMCs are obtained from whole blood by elutriation or any other method. The separation or isolation of the cells may be automated or may be performed manually.
The starting population of cells may be from blood, e.g. from a peripheral blood sample or from a biopsy. The starting population of cells may be peripheral blood mononuclear cells. The starting population of cells may be a white blood cell collection (leukapherenate).
Suitably, the starting population of cells may be obtained from a subject (first party). Suitably, the starting population of cells may be obtained from a donor (second party). Suitably, the starting population of cells may be obtained from a donor (third party) that is an unrelated donor.
Alternatively, the cells may be derived from an inducible progenitor or embryonic progenitor cell that is differentiated ex vivo into, for example, a T cell. Alternatively, immortalized cell lines that retain their lytic function and can act as therapeutic agents can be used.
Suitably, the starting population may be whole blood obtained from the subject. Suitably, the starting population may be PBMCs obtained from the subject. Suitably, the starting population may be a collection of leukocytes obtained from the subject.
Suitably, the starting population may be whole blood obtained from a donor. Suitably, the starting population may be PBMCs obtained from a donor. Suitably, the starting population may be a collection of leukocytes obtained from a donor.
As used herein, a "depleted starting population" refers to a population of cells remaining after the starting population has been depleted of cells expressing a target antigen. In other words, the starting population has been depleted of cells expressing the target antigen, i.e., the depleted starting population is depleted of the target antigen.
As used herein, "depleted" means that a particular type of cell (e.g., a cell expressing a CAR or a transgenic TCR target antigen) has been selectively reduced in number or has been eliminated from a population of cells.
As used herein, "transduced cells" refers to a population of cells that have undergone a transduction process. The cell population may comprise a mixture of cells that have been successfully genetically modified and cells that have not been successfully genetically modified.
As used herein, "genetically modified cell" means a cell that has been modified to contain or express a CAR or a transgenic TCR. Methods for engineering cells are known in the art and include genetic modification of cells by transduction, such as retroviral or lentiviral transduction.
Suitably, the genetically modified cell is a cell whose genome has been modified by transduction. Suitably, the genetically modified cell is a cell whose genome has been modified by retroviral transduction. Suitably, the genetically modified cell is a cell whose genome has been modified by lentiviral transduction.
As used herein, the term "introduced" refers to a method for inserting foreign DNA or RNA into a cell, e.g., by transduction. Transduction is the process of introducing foreign DNA or RNA into a cell via a viral vector.
Genetically modified cells according to the invention can be generated by transduction with a viral vector introducing DNA or RNA encoding a CAR or a transgenic TCR.
Prior to introduction of the nucleic acid sequence encoding the CAR or transgenic TCR, the cells may be activated and/or expanded, for example, by treatment with an anti-CD 3 monoclonal antibody or both an anti-CD 3 and an anti-CD 28 monoclonal antibody.
The cell may be a cytolytic immune cell.
As used herein, a "cytolytic immune cell" is a cell that directly kills other cells. Cytolytic cells can kill cancer cells; virus-infected cells or other damaged cells. Cytolytic immune cells include T cells and Natural Killer (NK) cells.
Cytolytic immune cells may be T cells or T lymphocytes, which are types of lymphocytes that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes such as B cells and NK cells by the presence of TCRs on the cell surface. There are various types of T cells, as summarized below.
Helper T cells (TH cells) assist other leukocytes in immunological processes, including B cell maturation into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells are activated when they present a peptide antigen via MHC class II molecules on the surface of Antigen Presenting Cells (APCs). These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, TH9, or TFH, which secrete different cytokines to promote different types of immune responses.
Cytolytic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells, and are also involved in transplant rejection. CTLs express CD8 at their surface. CTLs can be termed CD8+ T cells. These cells recognize their target by binding to MHC class I associated antigens present on the surface of all nucleated cells. By modulating the secretion of IL-10, adenosine and other molecules by T cells, CD8+ cells can be inactivated to an anergic (anergic) state, which prevents autoimmune diseases, such as experimental autoimmune encephalomyelitis.
Memory T cells are a subset of antigen-specific T cells that persist long after the infection has resolved. They rapidly expand into a large number of effector T cells upon re-exposure to their cognate antigen, thereby providing the immune system with "memory" against past infections. Memory T cells comprise three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). The memory cells may be CD4+ or CD8 +. Memory T cells typically express the cell surface protein CD45 RO.
Regulatory T cells (Treg cells), previously known as suppressor T cells, are critical for maintaining immune tolerance. Their main role is to shut down T cell mediated immunity towards the end of the immune response, and to suppress autoreactive T cells that escape the process of negative selection in the thymus.
Two major types of CD4+ Treg cells have been described, namely naturally occurring Treg cells and adaptive or inducible Treg cells.
Naturally occurring Treg cells (also known as CD4+ CD25+ FoxP3+ Treg cells) are present in the thymus and have been associated with the interaction between developing T cells and myeloid (CD11c +) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP 3. Mutations in the FOXP3 gene can prevent the development of regulatory T cells, resulting in the fatal autoimmune disease IPEX.
As used herein, the term "natural Treg" means thymus-derived tregs. The natural tregs are CD4+ CD25+ FOXP3+ Helios + neuropilin 1 +. Ntregs have increased PD-1 (programmed cell death 1, pdcd1), neurofibrillary protein 1(Nrp1), Helios (Ikzf2), and CD73 expression compared to itrregs. Ntregs can be distinguished from itrregs based on the expression of the Helios protein or neurofibrillary protein 1(Nrp1) alone.
Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can be generated during a normal immune response.
Peripherally-generated tregs may be referred to as induced Treg (itreg) cells.
The term "inducible regulatory T cells" (iTreg) as used herein means CD4+ CD25+ FOXP3+ Helios-neurofibrin 1-T cells developed from mature CD4+ conventional T cells outside the thymus. For example, iTregs can be induced in vitro from CD4+ CD25-FOXP 3-cells in the presence of IL-2 and TGF- β.
The cell may be a T cell, such as a helper T cell, a cytolytic T cell, a memory T cell or a regulatory T cell (Treg).
The cells may be natural killer cells (NK cells), which are a type of cytolytic cells that form part of the innate immune system. NK cells provide a rapid response to innate signals from virus-infected cells in an MHC-independent manner.
NK cells (belonging to the innate lymphoid cell population) are defined as Large Granular Lymphocytes (LGL) and constitute the third cell to differentiate from common lymphoid progenitors that give rise to B and T lymphocytes. NK cells are known to differentiate and mature in bone marrow, lymph nodes, spleen, tonsils and thymus, where they enter the circulation.
The cells may be stem cells or progenitor cells.
As used herein, the term "stem cell" means an undifferentiated cell that is capable of producing more stem cells of the same type indefinitely, and from which other specialized cells can be produced by differentiation. Stem cells are pluripotent. The stem cells may be, for example, embryonic stem cells or adult stem cells.
As used herein, the term "progenitor cell" means a cell that is capable of differentiating to form one or more types of cells but has limited self-renewal in vitro.
Suitably, the cell may be any cell capable of differentiating into a cytolytic immune cell.
Suitably, the cell may be capable of differentiating into a T cell or an NK cell.
Suitably, the cell may be an Embryonic Stem Cell (ESC). Suitably, the cells may be hematopoietic stem cells or hematopoietic progenitor cells. Suitably, the cell may be an Induced Pluripotent Stem Cell (iPSC). Suitably, the cells may be obtained from umbilical cord blood. Suitably, the cells may be obtained from adult peripheral blood.
In some aspects, Hematopoietic Stem and Progenitor Cells (HSPCs) may be obtained from cord blood. Cord blood may be harvested according to techniques known in the art (e.g., U.S. patent nos. 7,147,626 and 7,131,958, which are incorporated herein by reference).
In one aspect, HSPCs may be obtained from pluripotent stem cell sources, e.g., induced pluripotent stem cells (ipscs) and Embryonic Stem Cells (ESCs).
As used herein, the term "hematopoietic stem and progenitor cells" or "HSPCs" refers to cells and populations of such cells that express the antigenic marker CD34(CD34 +). In certain embodiments, the term "HSPC" refers to cells identified by the presence of the antigenic marker CD34(CD34+) and the absence of the lineage (lin) marker. Cell populations comprising CD34+ and/or Lin (-) cells include hematopoietic stem cells and hematopoietic progenitor cells.
HSPCs may be obtained or isolated from the bone marrow of adults, including the femur, hip, ribs, sternum, and other bones. Bone marrow aspirate containing HSPCs can be obtained or isolated directly from the hip using a needle and syringe. Other sources of HSPCs include umbilical cord blood, placental blood, mobilized peripheral blood, wharton's jelly, placenta, fetal blood, fetal liver, or fetal spleen. In particular embodiments, harvesting a sufficient amount of HSPCs for therapeutic applications may require mobilization of stem and progenitor cells in a subject.
As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to a non-pluripotent cell that has been reprogrammed to a pluripotent state. Once the cells of the subject are reprogrammed to a pluripotent state, the cells can then be programmed to the desired cell type, such as hematopoietic stem or progenitor cells (HSC and HPC, respectively).
As used herein, the term "reprogramming" refers to a method of increasing the potency of a cell to a state of lower differentiation.
As used herein, the term "programming" refers to a method of reducing the potency of a cell or differentiating a cell to a more differentiated state.
The present invention provides a cellular composition prepared by transducing a starting population of cells (comprising a subpopulation of cells that express a target antigen and a subpopulation of cells that do not express the target antigen) with a viral vector at a multiplicity of infection (MOI) such that the subpopulation of cells that do not express the target antigen is transduced to a greater extent than the subpopulation of cells that express the target antigen.
The invention also provides a cell composition prepared as follows:
(i) providing an initial population of cells;
(ii) depleting the starting population of cells expressing the target antigen; and
transducing target antigen-depleted cells with a viral vector expressing the CAR or TCR at a multiplicity of infection (MOI) such that cells not expressing the target antigen are transduced to a greater extent than residual cells expressing the target antigen.
Prior to the transduction step, the population of cells may or may not be activated.
In one embodiment, (iii) introducing a nucleic acid sequence encoding a CAR or a transgenic TCR against a target antigen into cells of a depleted starting population is performed simultaneously with (ii) depleting the starting population of cells expressing the target antigen.
In one embodiment, (iii) introducing a nucleic acid sequence encoding a CAR or a transgenic TCR against a target antigen into cells of a depleted starting population is performed after (ii) depleting the starting population of cells expressing the target antigen.
Optionally, the method may comprise (iii) (introducing the nucleic acid sequence encoding the CAR or transgenic TCR against the target antigen into the cells of the depleted starting population) prior to (ii) (depleting the cells expressing the target antigen from the starting population), i.e. the nucleic acid sequence encoding the CAR or transgenic TCR against the target antigen may be introduced into the cells prior to depleting the cells expressing the target antigen in the cells.
Optionally, the method may further comprise isolating a sample containing the cells from the subject. The sample containing cells can be used as a starting population of cells.
Optionally, cells used in the invention can be activated and/or expanded prior to introduction of the nucleic acid sequence encoding the CAR or transgenic TCR.
Any method known in the art for activating and/or expanding cells may be used in the methods of the invention. For example, cells used in the invention, such as T cells, may be activated and/or expanded, e.g., by treatment with an anti-CD 3 monoclonal antibody or both anti-CD 3 and anti-CD 28 monoclonal antibodies.
Suitably, interleukin 7(IL-7) and/or interleukin 15(IL-15) may be used to expand cells, such as T cells, in vitro. Suitably, interleukin 2(IL-2) may be used for in vitro cell expansion.
NK cells for use in the present invention can be activated and/or expanded by treatment with cytokines such as interleukin 2(IL-2) and/or interleukin 15 (IL-15). Incubation with helper cells such as monocytes, B-lymphoblastoid cells or cell lines expressing stimulatory molecules can be used to provide additional signals for NK cell expansion.
As used herein, "activation" means that a cell has been stimulated, resulting in cell proliferation, differentiation, or initiation of effector function.
Methods for measuring cell activation are known in the art and include measuring expression of activation markers, such as CD69, CD25, CD38, or HLA-DR, or measuring intracellular cytokines, for example, by flow cytometry.
As used herein, "expand" means that a cell or population of cells has been induced to proliferate.
Expansion of a cell population can be measured, for example, by counting the number of cells present in the population. The phenotype of the cells can be determined by methods known in the art, such as flow cytometry.
In one aspect, a population of engineered cells (e.g., genetically modified cells) comprising a chimeric antigen receptor or a transgenic T cell receptor is generated according to the methods of the invention.
Suitably, the genetically modified cell or population of genetically modified cells according to the invention may be prepared by a method according to the invention.
In one aspect, the population of genetically modified cells according to the invention or the population of genetically modified cells obtainable (e.g., obtained) by the method according to the invention is less differentiated than a genetically modified cell that has not been prepared according to the method of the invention (i.e., has not been depleted of cells expressing the target antigen of the CAR or transgenic TCR).
As used herein, "differentiated" refers to the developmental stage of a particular cell within the linear progression of differentiation of that cell type. For example, CD4+ and CD8+ T cells can be classified into different memory subgroups based on their differentiation status. CD4+ and CD8+ T cells follow a progressive pathway from the differentiation of naive T cells into central memory and effector memory cell populations. The differentiation status of CD8+ T cells is inversely correlated with their proliferative and persisting capacity.
Preclinical studies have shown that improved anti-tumor responses are obtained when genetically modified T cells are in an early stage of differentiation (e.g., primary or central memory cells). The central memory cells have improved in vivo persistence compared to effector memory cells.
In one aspect, the population of genetically modified cells according to the invention or the population of genetically modified cells obtainable by the method according to the invention is more initial compared to genetically modified cells that have not been prepared according to the method of the invention (i.e. have not been depleted of cells expressing the CAR or the target antigen of the transgenic TCR).
As used herein, "naive" means cells that are not fully differentiated. The naive T cells may not encounter an antigen.
Naive T cells can be characterized by surface expression of L-select (CD62L), absence of activation markers CD25, CD44, or CD69, and absence of memory CD45RO isoforms, e.g., naive T cells can be CD62LHiCD25LoCD44LoCD69LoNaive T cells also express a functional IL-7 receptor, consisting of subunit IL-7 receptor- α 127 and common gamma chain, CD 132.
In one aspect, the initial cell subset can be defined as CCR7+/CD45RA + cells. Suitably, the starting cell subset may be further defined as CCR7+/CD45RA +/CD62L +/CD27+ cells.
Suitably, the genetically modified cell according to the invention or the genetically modified cell obtainable (e.g. obtained) by the method according to the invention may have increased expression of CD27 and/or CD62L compared to a genetically modified cell not prepared according to the method of the invention (i.e. prepared with a high MOI viral vector and/or without depleting cells expressing the CAR or the target antigen of the transgenic TCR).
Without wishing to be bound by theory, the population of genetically modified cells, which is more primitive or immature, is advantageous for use in therapy because the primitive cells exhibit enhanced persistence in vivo and enhanced cytolytic activity when compared to cells with a more differentiated phenotype.
Suitably, the method according to the invention can produce more initial or central memory cells than methods using high MOI and/or non-depleting cells expressing the target antigen of the CAR or transgenic TCR. Suitably, at least 75% of the genetically modified cells may be primary or central memory cells. Suitably, at least 80% of the genetically modified cells may be primary or central memory cells. Suitably, at least 85% of the genetically modified cells may be primary or central memory cells.
Suitably, the method according to the invention may produce fewer effector and effector memory cells than methods using high MOI and/or non-depleting cells expressing the target antigen of the CAR or transgenic TCR. Suitably, less than 25% of the genetically modified cells may be effector or effector memory cells. Suitably, less than 20% of the genetically modified cells may be effector or effector memory cells. Suitably, less than 15% of the genetically modified cells may be effector or effector memory cells.
In one aspect, the population of genetically modified cells according to the invention or the population of genetically modified cells obtainable (or obtained) by the method according to the invention is less depleted than genetically modified cells not prepared according to the method of the invention (i.e., prepared with a high MOI viral vector and/or without depleting cells expressing the CAR or the target antigen of the transgenic TCR).
As used herein, "depleted" or "depleted" means that the cell exhibits reduced effector function and/or an altered phenotype. Immune cell depletion describes a state of dysfunction of immune cells, usually in the case of tumors or chronic infections. Depletion can be accompanied by phenotypic changes, epigenetic modifications, and changes in transcriptional profiles.
Effector functions may include production of effector cytokines and direct cytotoxic activity.
Suitably, the population of genetically modified cells according to the invention or obtainable (or obtained) by the method according to the invention may have a reduced expression of one or more depletion markers compared to genetically modified cells not prepared according to the method of the invention (i.e. prepared with a high MOI viral vector and/or without depleting a target antigen expressing a CAR or a transgenic TCR).
For example, in the case of NK cells, the effector function may include the production of interferon gamma (IFN- γ). Other effector functions of NK cells include direct cytotoxic activity, such as activity dependent on perforin and granzyme, or target cell apoptosis induced by tumor necrosis factor alpha (TNF- α), Fas ligand (FasL) and TNF-related apoptosis inducing ligand (TRAIL).
Suitably, depleted NK cells may produce a reduced amount of effector cytokines, such as IFN- γ, compared to non-depleted NK cells. Suitably, the depleted NK cells may have reduced cytolytic activity compared to non-depleted NK cells, and may for example produce a reduced amount of CD107a and/or granzyme B and/or perforin.
Suitably, the one or more depletion markers may be selected from the group consisting of: IFN-gamma, TNF-alpha, FasL, TRAIL, CD107a, granzyme B and perforin. Suitably, the one or more depletion markers may be selected from the group consisting of: IFN-gamma, TNF-alpha, FasL, TRAIL, CD107a, granzyme B and perforin, wherein the genetically modified cell is an NK cell.
Suitably, the one or more depletion markers may comprise reduced IFN- γ production. Suitably, the one or more depletion markers may comprise reduced TNF- α production. Suitably, the one or more depletion markers may comprise reduced FASL expression. Suitably, the one or more depletion markers may comprise reduced TRAIL expression. Suitably, the one or more depletion markers may comprise reduced expression of CD107 a. Suitably, the one or more depletion markers may comprise reduced production of granzyme B. Suitably, the one or more depletion markers may comprise reduced production of perforin.
For example, in the case of T cells, depletion can be defined by poor effector function, sustained expression of inhibitory receptors, and/or transcriptional state distinct from functional effector or memory T cells. For example, depleted T cells may express high levels of PD1, Tim3, Lag3, CD43(1B11), CD69, and inhibitory receptors, but low levels of CD62L and CD127, and reduced interleukin 2(IL-2), TNF-a, and IFN- γ production.
Suitably, the one or more depletion markers may comprise increased (e.g. high) expression of PD 1. Suitably, the one or more depletion markers may comprise increased (e.g. high) expression of Tim 3. Suitably, the one or more depletion markers may comprise increased (e.g. high) expression of lang 3. Suitably, the one or more depletion markers may comprise increased (e.g. high) expression of CD43(1B 11). Suitably, the one or more depletion markers may comprise increased (e.g. high) expression of CD 69. Suitably, the one or more depletion markers may comprise increased (e.g. high) expression of inhibitory receptors. Suitably, the one or more depletion markers may comprise reduced (e.g. low) expression of CD 62L. Suitably, the one or more depletion markers may comprise reduced (e.g. low) expression of CD 127. Suitably, the one or more depletion markers may comprise reduced (e.g. low) IL-2 production upon encountering the target. Suitably, the one or more depletion markers may comprise reduced (e.g. low) TNF-a production upon encountering the target. Suitably, the one or more depletion markers may comprise reduced (e.g. low) IFN- γ production upon encountering the target.
Suitably, the one or more depletion markers may be selected from the group consisting of: PD1, Lag3, and Tim 3. Suitably, the one or more depletion markers may comprise PD 1. Suitably, the one or more depletion markers may comprise lang 3. Suitably, the one or more depletion markers may comprise Tim 3. Suitably, the one or more depletion markers may be selected from the group consisting of: PD1, Lag3, and Tim3, wherein the genetically modified cell is a T cell.
In one aspect, more than 10%, more than 15%, more than 20%, more than 25%, more than 30% or more than 35% of the population of genetically modified cells according to the invention or of the population of genetically modified cells obtainable (or obtained) by the method according to the invention may be the initial (CCCR7+/CD45RA +) and CD62L +/CD27 +. Suitably, more than 10% of the genetically modified cells according to the invention may be naive (CCCR7+/CD45RA +) and CD62L +/CD27 +. Suitably, more than 15% of the genetically modified cells according to the invention may be naive (CCCR7+/CD45RA +) and CD62L +/CD27 +. Suitably, more than 20% of the genetically modified cells according to the invention may be naive (CCCR7+/CD45RA +) and CD62L +/CD27 +. Suitably, more than 30% of the genetically modified cells according to the invention may be naive (CCCR7+/CD45RA +) and CD62L +/CD27 +. Suitably, more than 40% of the genetically modified cells according to the invention may be naive (CCCR7+/CD45RA +) and CD62L +/CD27 +.
Suitably, the proportion of naive cells may be measured in the CD8+ T cell subset.
In one aspect, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5% of the population of genetically modified cells according to the invention or of the population of genetically modified cells obtainable (or obtained) by the method according to the invention may express a plurality of depletion markers. Suitably, less than 30% of the genetically modified cells according to the invention may exhibit multiple markers of depletion. Suitably, less than 25% of the genetically modified cells according to the invention may exhibit multiple markers of depletion. Suitably, less than 20% of the genetically modified cells according to the invention may exhibit multiple markers of depletion. Suitably, less than 15% of the genetically modified cells according to the invention may exhibit multiple markers of depletion.
Suitably, the plurality of depletion markers may be selected from increased (e.g. high levels) PD1, Tim3, Lag3, CD43(1B11), CD69 and inhibitory receptor expression as well as decreased (e.g. low levels) CD62L and CD127 expression and decreased (e.g. low) interleukin 2(IL-2), TNF-a and IFN- γ production. Suitably, the plurality of depletion markers may be selected from increased (e.g. high levels) expression of lang 3, PD1 and Tim 3.
In one aspect, a population of genetically modified cells according to the invention or a population of genetically modified cells obtainable (or obtained) by a method according to the invention has an increased level of transduction efficiency of a CAR or a transgenic TCR compared to genetically modified cells not prepared according to the method of the invention (i.e., prepared with a high MOI viral vector and/or without depleting cells expressing the CAR or the target antigen of the transgenic TCR).
Suitably, the level of transduction efficiency of the population of genetically modified cells according to the invention or obtainable (or obtained) by the method according to the invention may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% higher compared to the transduction efficiency of genetically modified cells not prepared according to the method of the invention (i.e. prepared with a high MOI viral vector and/or without depleting cells expressing the CAR or the target antigen of the transgenic TCR).
Suitably, the level of transduction efficiency of the population of genetically modified cells according to the invention or obtainable (or obtained) by the method according to the invention may be at least 50% higher compared to the transduction efficiency of genetically modified cells not prepared according to the method of the invention (i.e. prepared with a high MOI viral vector and/or without depleting cells expressing the CAR or the target antigen of the transgenic TCR). Suitably, the level of transduction efficiency of the population of genetically modified cells according to the invention or obtainable (or obtained) by the method according to the invention may be at least 60% higher compared to the transduction efficiency of genetically modified cells not prepared according to the method of the invention (i.e. prepared with a high MOI viral vector and/or without depleting cells expressing the CAR or the target antigen of the transgenic TCR). Suitably, the level of transduction efficiency of the population of genetically modified cells according to the invention or obtainable (or obtained) by the method according to the invention may be at least 70% higher compared to the transduction efficiency of genetically modified cells not prepared according to the method of the invention (i.e. prepared with a high MOI viral vector and/or without depleting cells expressing the CAR or the target antigen of the transgenic TCR). Suitably, the level of transduction efficiency of the population of genetically modified cells according to the invention or obtainable (or obtained) by the method according to the invention may be at least 80% higher compared to the transduction efficiency of genetically modified cells not prepared according to the method of the invention (i.e. prepared with a high MOI viral vector and/or without depleting cells expressing the CAR or the target antigen of the transgenic TCR). Suitably, the level of transduction efficiency of the population of genetically modified cells according to the invention or obtainable (or obtained) by the method according to the invention may be at least 90% higher compared to the transduction efficiency of genetically modified cells not prepared according to the method of the invention (i.e. prepared with a high MOI viral vector and/or without depleting cells expressing the CAR or the target antigen of the transgenic TCR).
In one aspect, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 60% of the population of transduced or transfected cells comprises a CAR or a transgenic TCR. Suitably, at least 40% of the transduced or transfected cell population comprises a CAR or a transgenic TCR. Suitably, at least 50% of the transduced or transfected cell population comprises a CAR or a transgenic TCR. Suitably, at least 60% of the transduced or transfected cell population comprises a CAR or a transgenic TCR.
Suitably, the percentage of cells that express the target antigen and that have been transduced or transfected to express the CAR or transgenic CAR is less than 5%. Suitably, the percentage of cells that express the target antigen and that have been transduced or transfected to express the CAR or transgenic CAR is less than 3%. Suitably, the percentage of cells that express the target antigen and that have been transduced or transfected to express the CAR or transgenic CAR is less than 2%. Suitably, the percentage of cells that express the target antigen and that have been transduced or transfected to express the CAR or transgenic CAR is less than 1%. Suitably, the percentage of cells that express the target antigen and that have been transduced or transfected to express the CAR or transgenic CAR is not detectable.
Viral vectors
Retroviruses and lentiviruses can be used as vectors or delivery systems for the transfer of a Nucleotide (NOI) or NOIs of interest into a target cell. The transfer may occur in vitro, ex vivo or in vivo. When used in this manner, the virus is often referred to as a viral vector.
Gamma retroviral vector was the first viral vector used in the gene therapy clinical trial in 1990, and has been commonly used so far. Recently, there has been increasing interest in lentiviral vectors derived from complex retroviruses, such as Human Immunodeficiency Virus (HIV), due to their ability to transduce non-dividing cells. The most attractive features of retroviral and lentiviral vectors as gene transfer tools include large genetic payloads (up to about 9kb), minimal patient immune responses, high transduction efficiency in vivo and in vitro, and the ability to permanently modify the genetic components of the target cell to maintain long-term expression of the delivered gene.
The retroviral or lentiviral vector used in the method of the invention may be based on any suitable retrovirus or lentivirus capable of delivering genetic information to a eukaryotic cell. For example, the retroviral vector may be an alpha retroviral vector, a gamma retroviral vector, or a spumaetroviral (spumaetroviral) vector. Lentiviral vectors can be based on Human Immunodeficiency Virus (HIV) or non-primate lentiviruses such as Equine Infectious Anemia Virus (EIAV).
The viral vector may comprise a heterologous viral envelope glycoprotein providing a pseudotype viral vector. For example, the viral envelope glycoprotein may be derived from feline endogenous virus (RD114), vesicular stomatitis virus (VSV-G), or Gibbon Ape Leukemia Virus (GALV).
Method of producing a composite material
Methods of transducing a cell with a viral vector comprising a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) or a transgenic T Cell Receptor (TCR) are provided.
Methods of preparing genetically modified cell populations for use in cell therapy are generally known in the art. A method of preparing a genetically modified cell for use in cell therapy may comprise some or all of the following steps:
the starting material may be first frozen, e.g., once obtained from a donor (e.g., a source of cells), a starting population of cells may be frozen. If frozen material is used, thawing and optionally a resting period may occur before the next step is performed. Alternatively, fresh starting material may be used. The starting material may be initially purified/enriched for leukocytes (e.g. Ficoll gradients) or for T cells.
The starting material may then be activated, e.g. T cells may be activated. This can be done by any method known in the art, for example using soluble CD3/CD28 antibodies, CD3/CD28 beads (e.g., Dynabeads) or CD3/28 nanomatrix (e.g., TransAct). As understood in the art, the length of the activation step before proceeding to the next step may vary, for example from less than one hour to over 72 hours.
The activated cells can then be transduced with a viral vector (e.g., a retrovirus or lentivirus). This can be done in the presence of a transduction enhancer such as retronectin or polybrene, or by spin seeding or by simple incubation. Non-viral vectors may also be used for the gene modification step (e.g., using RNA electroporation or using transposition of DNA).
The cells may then undergo an expansion step lasting from hours to days, depending on the final dose of cells required. Generally, the more cells that are needed, the longer the amplification step.
At the end of the manufacturing process, the cells may be used fresh or, preferably, frozen prior to use.
Thus, the entire process may take 2 to 18 days. Typically, the entire process takes 6 to 10 days.
During this process, the cells may be cultured in a cell growth medium, which may contain additional supplements. Such supplements may be human serum, fetal bovine serum, human serum albumin and/or cytokines (e.g. IL2, IL7 and/or IL15, IL 21).
As used herein, "MOI"/"multiplicity of infection" means the number of infectious vector particles per cell used in transduction. For example, an MOI of 1 means 10410 is added into each cell4Infectious vector particles. The number of infectious particles is obtained by titration of the viral vector on a permissive cell line.
Suitably, the transduced cell type may be the same as the cell type used for the titration. In this case, an MOI of 1 results in an average number of vector integrations per cell of 1, as estimated by quantitative PCR or other suitable methods.
Cells can be transduced at low multiplicity of infection (MOI). For example, a lower MOI can be used in the methods of the invention to achieve a particular level of cell transduction compared to the MOI required to transduce cells prepared by methods that do not deplete target antigen positive cells.
Advantageously, transducing a population of cells at a lower MOI prevents transduction of cells expressing the target antigen while allowing transduction of cells not expressing the target antigen.
Suitably, the MOI is selected such that cells that do not express the target antigen are transduced, and cells that express the target antigen are not transduced. Suitably, the MOI is selected such that cells that do not express the target antigen are transduced to a greater extent than cells that express the target antigen. The population of genetically modified cells may have a higher ratio of cells that do not express the target antigen to cells that express the target antigen than the cells prior to transduction.
Suitably, the MOI is selected such that at least 5% of cells that do not express the target antigen are transduced, and cells that express the target antigen are not transduced.
Suitably, the MOI is selected such that at least 10% of cells that do not express the target antigen are transduced, and cells that express the target antigen are not transduced. Suitably, the MOI is selected such that at least 15% of cells that do not express the target antigen are transduced, and cells that express the target antigen are not transduced.
Suitably, the MOI is selected such that at least 20% of cells that do not express the target antigen are transduced, and cells that express the target antigen are not transduced.
Cells expressing the target antigen may be "untransduced" in that such cells are undetectable, or transduced at a minimal or very low level, such as 1% or less.
Suitably, the methods of the invention may advantageously enable transduction using low MOI. Suitably, the MOI required to achieve 20-30% transduction of target antigen-negative cells may be lower in the methods of the invention compared to a corresponding method in which target antigen-positive cells are not depleted.
Without wishing to be bound by theory, because the MOI required for transduction is low, the methods of the invention may provide a safety benefit for making genetically modified cells. Advantageously, by the methods of the invention, cells expressing the target antigen may be transduced at no or minimal/very low levels.
Suitably, the MOI may be selected to achieve about 10-50% transduction of cells that do not express the target antigen. Suitably, the MOI may be selected to achieve about 15-40% transduction of cells that do not express the target antigen. Suitably, the MOI may be selected to achieve about 20-30% transduction of cells that do not express the target antigen.
The range of MOIs that prevent transduction of cells expressing the target antigen while allowing transduction of cells that do not express the target antigen will depend on the envelope protein of the viral vector.
For viral vectors pseudotyped with RD114 envelope protein, the MOI may suitably be about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1 or about 1.2. Suitably, the MOI may range from about 0.1 to 1.2. Suitably, the MOI may range from about 0.2 to 1.0. Suitably, the MOI may range from about 0.3 to 0.8. Suitably, the MOI may range from about 0.4 to 0.6.
For viral vectors pseudotyped with RD114 envelope protein, suitably, the MOI to achieve about 20-30% transduction may be about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, or about 1.2. Suitably, the MOI to achieve about 20-30% transduction may range from about 0.1 to 1.2. Suitably, the MOI to achieve about 20-30% transduction may range from about 0.2 to 1.0. Suitably, the MOI to achieve about 20-30% transduction may range from about 0.3 to 0.8. Suitably, the MOI to achieve about 20-30% transduction may range from about 0.4 to 0.6.
For viral vectors pseudotyped with a GALV envelope protein, the MOI may suitably be about 0.5, 0.75, 1, 1.25, 1.5, 1.75, or about 2. Suitably, the MOI may range from about 0.5 to 2. Suitably, the MOI may range from about 0.8 to 1.8. Suitably, the MOI may range from about 1 to 2. Suitably, the MOI may range from about 1.4 to 1.6.
For viral vectors pseudotyped with a GALV envelope protein, suitably, the MOI to achieve about 20-30% transduction can be about 0.5, 0.75, 1, 1.25, 1.5, 1.75, or about 2. Suitably, the MOI to achieve about 20-30% transduction may range from about 0.5-2. Suitably, the MOI to achieve about 20-30% transduction may range from about 0.8 to 1.8. Suitably, the MOI to achieve about 20-30% transduction may range from about 1-2. Suitably, the MOI to achieve about 20-30% transduction may range from about 1.4 to 1.6.
For viral vectors pseudotyped with VSV-G, the MOI may suitably be about 3, 4, 5, 6, 7, 8, 9, or about 10. Suitably, the MOI may range from about 3 to 10. Suitably, the MOI may range from about 4 to 10. Suitably, the MOI may range from about 5 to 10. Suitably, the MOI may range from about 6 to 8.
For viral vectors pseudotyped with VSV-G, suitably, the MOI to achieve about 20-30% transduction can be about 3, 4, 5, 6, 7, 8, 9, or about 10. Suitably, the MOI to achieve about 20-30% transduction may range from about 3-10. Suitably, the MOI to achieve about 20-30% transduction may range from about 4-10. Suitably, the MOI to achieve about 20-30% transduction may range from about 5-10. Suitably, the MOI to achieve about 20-30% transduction may range from about 6-8.
Suitably, the percentage of CAR or transgenic TCR target antigen positive cells in the depleted starting population or genetically modified population of cells may be lower than in the starting population.
Suitably, the percentage of target antigen positive cells in the depleted starting population or the genetically modified population of cells may be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% when compared to the percentage of target antigen positive cells in the starting population. Suitably, the percentage of target antigen positive cells may be reduced by at least 90%. Suitably, the percentage of target antigen positive cells may be reduced by at least 95%. Suitably, the percentage of antigen positive cells may be reduced by at least 98%. Suitably, the percentage of antigen positive cells in the depleted starting population or in the genetically modified population of cells may be reduced by at least 99% when compared to the percentage of target antigen positive cells in the starting population.
Suitably, less than 40%, less than 30%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% of the depleted starting population may express a CAR or a target antigen of a transgenic TCR.
Suitably, less than 10% of the depleted starting population may express the CAR or target antigen of the transgenic TCR.
Suitably, less than 5% of the depleted starting population may express the CAR or target antigen of the transgenic TCR.
Suitably, less than 3% of the depleted starting population may express the CAR or target antigen of the transgenic TCR.
Suitably, less than 2% of the depleted starting population may express the CAR or target antigen of the transgenic TCR.
Suitably, less than 1% of the depleted starting population may express the CAR or target antigen of the transgenic TCR.
Suitably, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% of the genetically modified cells may express the CAR or the target antigen of the transgenic TCR.
Suitably, less than 10% of the genetically modified cells may express the CAR or the target antigen of the transgenic TCR.
Suitably, less than 5% of the genetically modified cells may express the CAR or the target antigen of the transgenic TCR.
Suitably, less than 1% of the genetically modified cells may express the CAR or the target antigen of the transgenic TCR.
Pharmaceutical composition
The invention also relates to a pharmaceutical composition comprising a genetically modified cell of the invention or a population of genetically modified cells according to the invention.
In one aspect, a pharmaceutical composition is provided comprising a population of genetically modified cells according to the invention or obtainable by a method according to the invention.
Suitably, the pharmaceutical composition may comprise cryopreserved genetically modified cells according to the invention or genetically modified cells obtainable by a method according to the invention.
The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more additional pharmaceutically active polypeptides and/or compounds. Such formulations may, for example, be in a form suitable for intravenous infusion.
Suitably, the starting population of cells may have been previously frozen. If frozen cells are used, the cells may be thawed and, optionally, may be allowed to recover in culture prior to processing.
The method may additionally comprise the step of enriching the starting population of leukocytes. Any method known in the art for isolating or enriching leukocytes can be used. For example, separation can be by various methods such as density gradient, e.g., using Ficoll-Paque density gradient media; separation by magnetic beads, such as MACS Milteyni Biotec CD3, CD4, or CD8 beads; leukocytes or PBMCs are obtained from whole blood by elutriation or any other method. The separation or isolation of the cells may be automated or may be performed manually.
Method of treatment
The genetically modified cells of the invention may be capable of killing a target cell, such as a cancer cell, a virus-infected cell, or other damaged cell.
The genetically modified cells of the invention can be used in therapy. The genetically modified cells of the invention can be used for the treatment and/or prevention of diseases. Suitably, the pharmaceutical composition comprising the genetically modified cell according to the invention may be used in therapy. Suitably, the pharmaceutical composition comprising the genetically modified cell according to the invention may be used for the treatment and/or prevention of a disease.
It will be appreciated that the target antigen of the CAR or transgenic TCR will be selected based on the desired therapy. For example, if the CAR or transgenic TCR is used to treat cancer, the target antigen of the CAR or transgenic TCR may be an antigen associated with cancer.
The genetically modified cells of the invention can be used to treat infections, such as viral infections.
The genetically modified cells of the invention may also be used to control pathogenic immune responses, for example in autoimmune diseases, allergies and graft-versus-host rejection.
The present invention provides a method for treating and/or preventing a disease comprising the step of administering a cell composition prepared by the method of the present invention to a subject.
The present invention provides a method for treating and/or preventing a disease comprising the step of administering the pharmaceutical composition of the present invention to a subject.
The invention also provides a cell composition prepared by the method of the invention for use in the treatment and/or prevention of a disease.
The invention also provides a pharmaceutical composition of the invention for use in the treatment and/or prevention of a disease.
The invention also relates to the use of the genetically modified cell or cell composition according to the invention for the production of a medicament for the treatment and/or prevention of a disease.
Suitably, the treatment methods of the invention may involve administering a pharmaceutical composition of the invention to a subject.
Suitably, the present invention provides a method of treatment comprising:
(i) providing a sample comprising a starting population of cells;
(ii) depleting the starting population of cells expressing the target antigen;
(iii) transducing target antigen-depleted cells with a viral vector expressing a CAR or a TCR at a multiplicity of infection (MOI) such that cells not expressing the target antigen are transduced to a greater extent than residual cells expressing the target antigen; and
(iv) (iv) administering the cells from (iii) to the subject.
Suitably, the method may additionally comprise a cell expansion step prior to administration to the patient, e.g. the cells may be cultured prior to administration to the patient.
The genetically modified cell or the pharmaceutical composition of the present invention can be used for the treatment and/or prevention of cancerous diseases such as hematological malignancies, bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cells), lung cancer, melanoma, pancreatic cancer, prostate cancer and thyroid cancer, cancers of the oral cavity and pharynx, including cancers of the tongue, mouth and pharynx; cancers of the digestive system, including esophageal, gastric, and colorectal cancers; cancers of the liver and biliary (biliary tree) system, including hepatocellular carcinoma and cholangiocarcinoma; cancers of the respiratory system, including bronchial and laryngeal cancers; cancers of the bones and joints, including osteosarcomas; cancer of the skin, including melanoma; breast cancer; cancers of the reproductive tract, including uterine, ovarian and cervical cancers in females, prostate and testicular cancers in males; cancers of the renal tract (renal tract) including renal cell carcinoma and transitional cell carcinoma of the urethra (uterers) or bladder; brain cancer, including gliomas, glioblastoma multiforme, and medulloblastomas; cancers of the endocrine system, including thyroid cancer, adrenal cancer, and cancers associated with various endocrine neogenetic syndromes; and cancers of other and unspecified sites, including neuroblastoma.
Suitably, the genetically modified cell or the pharmaceutical composition of the invention may be used for the treatment and/or prevention of a hematological malignancy.
As used herein, "hematologic malignancy" refers to cancers that affect the blood and lymphatic system, and includes leukemias, lymphomas, myelomas, and related hematologic disorders.
Suitably, the genetically modified cell or the pharmaceutical composition of the invention may be used for the treatment and/or prevention of a hematological malignancy.
Suitably, the genetically modified cell or the pharmaceutical composition of the invention may be used for the treatment and/or prevention of acute and chronic myeloid or lymphoid leukemia, comprising: acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Acute Promyelocytic Leukemia (APL), B-cell or T-cell acute lymphoblastic leukemia (B-ALL or T-ALL, respectively), Chronic Lymphocytic Leukemia (CLL), Chronic Myeloid Leukemia (CML), chronic myelomonocytic leukemia (CMML), Hairy Cell Leukemia (HCL), and Large Granular Lymphocytic Leukemia (LGLL); lymphomas, including hodgkin lymphoma and non-hodgkin lymphoma (NHL), both low-grade NHL and high-grade NHL; myeloma (multiple myeloma (MM)) comprising: smoldering or asymptomatic myeloma and symptomatic myeloma and other conditions associated with hematological cancers, such as nonsense Monoclonal Gammopathy (MGUS), myelodysplastic syndrome (MDS), solitary plasmacytoma, and myeloproliferative neoplasms (MPN), which include Essential Thrombocythemia (ET), Myelofibrosis (MF), and Polycythemia Vera (PV).
Suitably, the genetically modified cell or the pharmaceutical composition of the invention may be used for the treatment and/or prevention of T-cell lymphoma. Suitably, the genetically modified cell or the pharmaceutical composition of the invention may be used for the treatment and/or prevention of T cell leukemia.
Methods for treating T cell lymphomas and/or leukemias involve therapeutic use of the genetically modified cells of the invention. The genetically modified cells can be administered to a subject with an existing disease of T-cell lymphoma and/or leukemia to alleviate, reduce, or ameliorate at least one symptom associated with the disease and/or slow, reduce, or block progression of the disease.
Suitably, the methods of the invention may be used to treat any lymphoma and/or leukemia associated with clonal expansion of cells expressing a T Cell Receptor (TCR) comprising a β constant region. Accordingly, the invention may relate to a method for treating a disease involving malignant T cells expressing a TCR comprising a TRBC (such as TRBC1 or TRBC 2).
Suitably, the methods of the invention may be used to treat a T cell lymphoma wherein malignant T cells express a TCR comprising TRBC. "lymphoma" is used herein according to its standard meaning to refer to cancer that typically develops in the lymph nodes but may also affect the spleen, bone marrow, blood, and other organs. Lymphomas are usually manifested as solid tumors of lymphoid cells. The primary symptom associated with lymphoma is lymphadenopathy, although secondary (B) symptoms may include fever, night sweats, weight loss, loss of appetite, fatigue, respiratory distress, and itching.
The methods of the invention may be used to treat T cell leukemia, wherein malignant T cells express TCRs comprising TRBC. "leukemia" is used herein according to its standard meaning to refer to cancers of the blood or bone marrow.
The following is an illustrative, non-exhaustive list of diseases that can be treated by the methods of the present invention.
Suitably, the T cell lymphoma or leukaemia may be peripheral T cell lymphoma non-specific (PTCL-NOS). Suitably, the T cell lymphoma or leukaemia may be angioimmunoblastic T cell lymphoma (AITL). Suitably, the T cell lymphoma or leukaemia may be Anaplastic Large Cell Lymphoma (ALCL). Suitably, the T cell lymphoma or leukemia may be a T cell lymphoma associated with intestinal diseases (EATL). Suitably, the T cell lymphoma or leukemia may be hepatosplenic T cell lymphoma (HSTL). Suitably, the T cell lymphoma or leukaemia may be extranodal NK/T cell lymphoma nasal. Suitably, the T cell lymphoma or leukaemia may be Cutaneous T Cell Lymphoma (CTCL). Suitably, the T cell lymphoma or leukemia may be primary skin (ALCL). Suitably, the T cell lymphoma or leukemia may be T cell prolymphocytic leukemia. Suitably, the T cell lymphoma or leukemia may be T cell acute lymphoblastic leukemia.
Treatment with the genetically modified cells of the invention or the pharmaceutical compositions according to the invention may help to prevent escape or release of tumor cells which usually occur with standard methods.
The term "treating" refers to administering a genetically modified cell, population of genetically modified cells, or pharmaceutical composition according to the invention to a subject with an existing disease or condition to alleviate, reduce, or ameliorate at least one symptom associated with the disease and/or slow, reduce, or block the progression of the disease.
As used herein, reference to "preventing" (or disease-preventing) refers to delaying or preventing the onset of disease symptoms. Prevention may be absolute (so that no disease occurs), or may be effective in only some individuals or for a limited time.
In a preferred embodiment of the invention, the subject of any of the methods described herein is a mammal, preferably a cat, dog, horse, donkey, sheep, pig, goat, cow, mouse, rat, rabbit or guinea pig. Preferably, the subject is a human.
Administration of
Administration of the pharmaceutical composition can be accomplished using any of a variety of routes that make the genetically modified cells contained in the pharmaceutical composition bioavailable to the subject. For example, the compositions may be administered by oral and parenteral routes, intraperitoneally, intravenously, subcutaneously, transdermally, intramuscularly, e.g., via local delivery by catheter or stent.
Suitably, the genetically modified cell according to the invention or the pharmaceutical composition according to the invention is administered intravenously.
One skilled in the art will appreciate that, for example, the route of delivery (e.g., oral versus intravenous versus subcutaneous, etc.) can affect the dosage and/or the desired dosage can affect the route of delivery. For example, where particularly high concentrations of an agent within a particular site or location are of interest, focused delivery may be desirable and/or useful. Other factors to be considered in optimizing the route and/or dosing regimen of a given treatment regimen may include, for example, the disease being treated (e.g., type or stage, etc.), the clinical condition of the subject (e.g., age, overall health, etc.), the presence or absence of a combination therapy, and other factors known to the practitioner.
The dosage is such that it is sufficient to stabilize or ameliorate the symptoms of the disease.
Typically, the physician will determine the actual dosage which will be most suitable for an individual subject, and this actual dosage will vary with the age, weight and response of the particular patient. The dose is such that it is sufficient to reduce or deplete the number of cells expressing the target antigen.
Use of
The invention also provides a pharmaceutical composition or a population of genetically modified cells according to the invention for use in the treatment of a disease. The pharmaceutical composition or the population of genetically modified cells may be any as defined above.
The invention also relates to the use of a population of genetically modified cells of the invention as defined above in the manufacture of a medicament for the treatment of a disease.
Reagent kit
The present invention also provides a kit comprising:
(i) a viral vector comprising a nucleic acid sequence encoding a CAR or a transgenic TCR; and
(ii) means for depleting cells expressing a CAR or a target antigen of a transgenic TCR.
As used herein, "means for depleting cells expressing a target antigen" refers to any product known in the art suitable for removing or separating particular cells from a mixed population of cells.
Suitably, the kit may comprise an antibody specific for the target antigen.
Suitably, the kit may comprise a depleting antibody. A "depleting antibody" is an antibody that binds to an antigen present on a target cell and mediates death of the target cell. Thus, administration of the depleting antibody to the population of cells results in a reduction in the number of cells in the population that express the target antigen.
Suitably, the kit may comprise an antibody coated solid matrix. Cells expressing the target antigen can be depleted by adsorption into an antibody-coated solid matrix.
Suitably, the kit may comprise a fluorescent antibody specific for the target antigen. Cells containing the target antigen can be isolated by flow cytometry.
Suitably, the kit may comprise an immunomagnetic product, i.e. an antibody specific for a target antigen attached to a magnetic nanoparticle or magnetic bead.
The invention will now be further described by way of examples, which are intended to assist those of ordinary skill in the art in carrying out the invention, and are not intended to limit the scope of the invention in any way.
Examples
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