阅读说明：本技术 表达异源靶向构建体的淋巴细胞 (Lymphocytes expressing heterologous targeting constructs ) 是由 奥利弗·努斯鲍默 伊斯特万·科瓦奇 艾琳·皮齐托拉 拉伊·梅塔 于 2019-03-25 设计创作，主要内容包括：本发明提供了包含异源靶向构建体的工程化的淋巴细胞(例如γδT细胞、NK细胞、NK样T细胞、工程化的先天淋巴样细胞或MAIT细胞),该异源靶向构建体缺乏能够激活表达该构建体的淋巴细胞的细胞内信号传导结构域。还提供了工程化的淋巴细胞(例如γδT细胞)的组合物以及使用所述工程化的淋巴细胞(例如γδT细胞,例如过继性T细胞疗法的一部分)的方法。(The invention provides engineered lymphocytes (e.g., γ δ T cells, NK-like T cells, engineered innate lymphoid cells, or MAIT cells) comprising a heterologous targeting construct that lacks an intracellular signaling domain capable of activating lymphocytes expressing the construct. Also provided are compositions of engineered lymphocytes (e.g., γ δ T cells) and methods of using the engineered lymphocytes (e.g., γ δ T cells, e.g., as part of an adoptive T cell therapy).)

1. an engineered γ - δ (γ δ) T cell comprising a heterologous targeting construct, wherein the heterologous targeting construct comprises an extracellular antigen binding domain and a transmembrane domain operably linked to the antigen binding domain, wherein the heterologous targeting construct lacks an intracellular domain capable of activating the engineered γ δ T cell.

2. The engineered γ δ T-cell of claim 1, further comprising a stalk domain operably linking the antigen binding domain to the transmembrane domain.

3. An engineered γ δ T-cell comprising a heterologous targeting construct, wherein the heterologous targeting construct comprises an antigen binding domain and a transmembrane domain, wherein the transmembrane domain is a terminal transmembrane domain that does not transmit signal 1 activation of the engineered γ δ T-cell.

4. The engineered γ δ T-cell according to claim 3, further comprising a stalk domain operably linking the antigen binding domain to the transmembrane domain.

5. An engineered γ δ T-cell comprising a heterologous targeting construct, wherein the heterologous targeting construct consists of an antigen binding domain, a stem domain operably linked to the antigen binding domain, and a transmembrane domain operably linked to the stem domain, wherein the heterologous targeting construct does not propagate signal 1 activation of the engineered γ δ T-cell.

6. The engineered γ δ T-cell according to any one of claims 3 to 5, wherein the transmembrane domain does not activate the engineered γ δ T-cell.

7. The engineered γ δ T-cell according to any one of claims 1 to 6, wherein the engineered γ δ T-cell is V δ 2 negative.

8. The engineered γ δ T-cell of claim 6, wherein the ν δ 2 negative γ δ T-cell is ν δ 1 positive.

9. The engineered γ δ T-cell according to any one of claims 1 to 8, wherein the antigen binding domain comprises a single chain variable fragment (scFv), a monoclonal antibody, a Fab fragment, a B-cell receptor, a T-cell receptor, an antibody scaffold, a receptor-specific ligand, or a ligand-specific receptor.

10. The engineered γ δ T-cell according to any one of claims 2 or 4 to 9, wherein the stem domain comprises one or more domains selected from: CD8 stem, IgG1 hinge, IgG1 hinge-CH₂Domain, IgG 1-hinge-CH₃Domain, IgG 1-hinge-CH₂-CH₃(ii) Domain, (G)₄S)₃Hinge, CD7 stem, IgD hinge-CH₂Domain, IgD hinge-CH₂-CH₃Domain, IgD hinge-CH₃Domain, IgG4 hinge, IgG4 hinge-CH₂Domain, IgG4 hinge-CH₂-CH₃Domain, IgG4 hinge-CH₃Domain or fcsri stem.

11. The engineered γ δ T-cell according to any one of claims 1 to 10, wherein the transmembrane domain comprises a CD8 transmembrane domain, a CD4 transmembrane domain, a CD3 ζ transmembrane domain, a CD28 transmembrane domain, a CD45 transmembrane domain, a CD5 transmembrane domain, a CD8 transmembrane domain, a CD9 transmembrane domain, a CD16 transmembrane domain, a CD22 transmembrane domain, a CD33 transmembrane domain, a CD37 transmembrane domain, a CD64 transmembrane domain, a CD80 domain, a CD86 transmembrane domain, a CD134 transmembrane domain, a CD137 transmembrane domain, a CD154 transmembrane domain, a CD7 transmembrane domain, a CD71 transmembrane domain, a CD18 transmembrane domain, a CD29 transmembrane domain, a CD11a transmembrane domain, a CD11b transmembrane domain, a CD11c transmembrane domain, a CD11 transmembrane domain 11d transmembrane domain, a CD94 transmembrane domain, a γ Fc 2D, or a NKG2 transmembrane domain.

12. The engineered γ δ T-cell according to any one of claims 1 to 11, wherein no more than 50% of the amino acids of the C-terminal transmembrane domain reside within the cell.

13. The engineered γ δ T-cell according to any one of claims 1 to 12, wherein the clustering of the heterologous targeting construct does not substantially activate the TCR pathway within the engineered γ δ T-cell upon binding of the antigen-binding domain to a target antigen.

14. The engineered γ δ T-cell of any one of claims 1 to 13, wherein the antigen-binding domain binds a tumor-associated antigen.

15. The engineered γ δ T-cell of claim 14, wherein the tumor-associated antigen is a protein or peptide antigen expressed on the surface of a tumor cell.

16. The engineered γ δ T-cell of claim 15, wherein the tumor-associated antigen is CD 19.

17. The engineered γ δ T-cell of claim 16, wherein the tumor-associated antigen is a carbohydrate expressed on the surface of a tumor cell.

18. The engineered γ δ T-cell of claim 14, wherein the tumor-associated antigen is a ganglioside expressed on the surface of a tumor cell.

19. The engineered γ δ T-cell of claim 18, wherein the ganglioside is GD 2.

20. The engineered γ δ T-cell of any one of claims 14 to 19, wherein the tumor-associated antigen is an immunosuppressive antigen.

21. The engineered γ δ T-cell of any one of claims 1 to 20, wherein the antigen-binding domain binds a target antigen expressed by a solid tumor cell.

22. The engineered γ δ T-cell according to any one of claims 1 to 21, wherein binding of the antigen-binding domain to a target antigen expressed on healthy cells triggers substantially less cytolysis by the engineered γ δ T-cell relative to a reference cell having a functional intracellular domain.

23. The engineered γ δ T-cell according to claim 22, wherein binding of the antigen-binding domain to a target antigen expressed on a healthy cell does not substantially trigger cytolysis by the engineered γ δ T-cell.

24. The engineered γ δ T-cell according to any one of claims 1 to 23, wherein binding of the antigen-binding domain to a target antigen expressed on a tumor cell or infected cell substantially triggers cytolysis by the engineered γ δ T-cell.

25. The engineered γ δ T-cell of claim 22, wherein the cytolysis is dependent on endogenous expression of NKG2D, NKp30, NKp44, NKp46, or DNAM1 by the engineered γ δ T-cell.

26. The engineered γ δ T-cell according to claim 24 or 25, wherein the cytolysis is characterized by one, two, three, four, five or all six of the responses selected from CD107 degranulation, granzyme release, perforin release, granulysin release, target cell killing, proliferation of γ δ T-cells and cytokine production.

27. An engineered NK cell or NK-like T cell comprising a heterologous targeting construct, wherein the heterologous targeting construct comprises an extracellular antigen binding domain and a transmembrane domain operably linked to the antigen binding domain, wherein the heterologous targeting construct lacks an intracellular domain capable of activating the engineered NK cell or NK-like T cell.

28. An engineered innate lymphoid cell comprising a heterologous targeting construct, wherein the heterologous targeting construct comprises an extracellular antigen binding domain and a transmembrane domain operably linked to the antigen binding domain, wherein the heterologous targeting construct lacks an intracellular domain capable of activating the engineered innate lymphoid cell.

29. An engineered mucosa-associated constant T (mait) cell comprising a heterologous targeting construct, wherein the heterologous targeting construct comprises an extracellular antigen binding domain and a transmembrane domain operably linked to the antigen binding domain, wherein the heterologous targeting construct lacks an intracellular domain capable of activating the engineered mucosa-associated constant T cell.

30. An isolated population of cells comprising at least ten engineered γ δ T-cells of any one of claims 1-26, engineered NK cells or NK-like T-cells of claim 27, engineered innate lymphoid cells of claim 28, or engineered MAIT-cells of claim 29.

31. The isolated population of cells of claim 30, wherein said engineered γ δ T-cells, engineered NK-cells or NK-like T-cells, engineered innate lymphoid cells, or engineered MAIT-cells comprise greater than 2% of the total number of cells in the isolated population of cells.

32. An isolated population of cells comprising the population of engineered γ δ T-cells of any one of claims 1-26, the population of engineered NK cells or NK-like T-cells of claim 27, the population of engineered innate lymphoid cells of claim 28, or the population of engineered MAIT-cells of claim 29, wherein the population comprises greater than 2% of the total number of cells in the isolated population of cells.

33. The isolated population of cells of claim 31 or 32, comprising at least ten engineered γ δ T-cells of any one of claims 1 to 26 and/or at least ten engineered NK cells or NK-like T-cells of claim 27 and/or at least ten engineered innate lymphoid cells of claim 28 and/or at least ten engineered MAIT cells of claim 29.

34. A γ δ T cell comprising a heterologous polynucleotide encoding a heterologous targeting construct, wherein the heterologous targeting construct comprises an extracellular antigen binding domain and a transmembrane domain operably linked to the antigen binding domain, wherein the heterologous targeting construct lacks an intracellular domain capable of activating the engineered γ δ T cell.

35. A γ δ T cell comprising a heterologous polynucleotide encoding a targeting construct, wherein the heterologous targeting construct comprises an antigen binding domain and a transmembrane domain, wherein the transmembrane domain is a terminal transmembrane domain that is not involved in signal 1 activation of the engineered γ δ T cell.

36. The engineered γ δ T-cell according to any one of claims 1 to 26, the engineered NK-cell or NK-like T-cell according to claim 27, the engineered innate lymphoid cell according to claim 28, the engineered MAIT-cell according to claim 29, the isolated population of cells according to any one of claims 30 to 33, or the γ δ T-cell comprising a heterologous polynucleotide according to claim 34 or 35, for use in a method of treating a subject by adoptive T-cell therapy, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of the engineered γ δ T-cell according to any one of claims 1 to 24, the engineered NK-cell or NK-like T-cell of claim 25, the engineered innate lymphoid cell of claim 26, the engineered MAIT-cell of claim 27, the engineered γ δ T-cell of claim 28, the engineered innate lymphoid cell of claim 28, the engineered T-cell of claim, The isolated population of cells of any one of claims 28 to 31 or the γ δ T-cell of claim 32 or 33 comprising a heterologous polynucleotide.

37. The engineered γ δ T-cell, engineered NK-cell or NK-like T-cell, engineered innate lymphoid cell, engineered MAIT-cell, isolated population of cells, or γ δ T-cell comprising a heterologous polynucleotide for use according to claim 36, wherein the subject is a human.

38. The engineered γ δ T-cell, engineered NK-cell or NK-like T-cell, engineered innate lymphoid cell, engineered MAIT-cell, isolated population of cells, or γ δ T-cell comprising a heterologous polynucleotide for use according to claim 37, wherein the human is a human cancer patient.

39. The engineered γ δ T-cell, engineered NK-cell or NK-like T-cell, engineered innate lymphoid cell, engineered MAIT-cell, isolated population of cells, or γ δ T-cell comprising a heterologous polynucleotide for use according to claim 38, wherein the human cancer patient is receiving solid tumor therapy.

40. The engineered γ δ T-cell, engineered NK-cell or NK-like T-cell, engineered innate lymphoid cell, engineered MAIT-cell, isolated population of cells, or γ δ T-cell comprising a heterologous polynucleotide for use according to claim 37, wherein the human is a human patient being treated for a viral infection.

41. A method of treating a subject by adoptive T cell therapy, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of the engineered γ δ T-cell of any one of claims 1 to 26, the engineered NK-cell or NK-like T-cell of claim 27, the engineered innate lymphoid cell of claim 28, the engineered MAIT cell of claim 29, the isolated population of cells of any one of claims 30 to 33, or the γ δ T-cell comprising a heterologous polynucleotide of claim 34 or 35.

42. The method of claim 41, wherein the subject is a human.

43. The method of claim 42, wherein the human is a human cancer patient.

44. The method of claim 43, wherein the human cancer patient is receiving solid tumor therapy.

45. The method of claim 42, wherein the human is a human patient being treated for a viral infection.

Background

Cancer is a group of diseases involving abnormal cell growth that has the potential to metastasize to other parts of the body. The diversity of cancer types is well known, and the genetic makeup of many types of cancer can vary greatly between patients. This difference places a difficult burden on determining effective therapeutic strategies to target certain cancers. In particular, there is a need to develop personalized treatment strategies for any given cancer target. Therefore, there is growing interest in T cell immunotherapy based on the fact that we can exploit the cellular recognition of the immune system and destroy the recognition of foreign or pathogenic cells. To date, T cell immunotherapy involves engineering α β T cells to express Chimeric Antigen Receptors (CARs). Such CAR T cells can recognize a cancer target based on the expression of a target antigen (e.g., a tumor-associated antigen) recognized by the chimeric antigen receptor. Upon binding to its target antigen, one or more intracellular domains of the CAR transmit signal 1 activation and/or signal 2 activation (co-stimulation) to activate the CAR T cells, triggering degranulation and lysis of the target cells. However, there are still various problems with this CAR T cell approach. For example, CAR T cells risk developing off-target cytotoxicity because healthy cells moderately express the target antigen. Accordingly, there is a need in the art for improved methods to engineer these powerful components of the immune system while enhancing the safety and effectiveness of the treatment.

Disclosure of Invention

The invention provides alternative methods of CAR T cells. In particular, featured herein are heterologous targeting constructs that lack a functional intracellular domain that is capable of activating a cell expressing it. When expressed on lymphocytes that have innate effector function and/or are not MHC-restricted (e.g., γ δ T cells, NK-like T cells, innate lymphoid cells, and engineered mucosa-associated constant T (mait) cells), engineered lymphocytes can exhibit enhanced specificity for diseased cells by avoiding aberrant TCR activation following binding to low levels of target antigens on healthy cells.

In a first aspect, the invention provides an engineered γ - δ (γ δ) T cell comprising a heterologous targeting construct, wherein the heterologous targeting construct comprises an extracellular antigen binding domain and a transmembrane domain operably linked to the antigen binding domain, wherein the heterologous targeting construct lacks an intracellular domain capable of activating the engineered γ δ T cell (e.g., if present, the intracellular domain does not transmit signal 1 activation nor signal 2 co-stimulation). In some embodiments, the heterologous targeting construct further comprises a stem domain (talk domain) operably linking the antigen binding domain to the transmembrane domain.

In another aspect, the invention provides an engineered γ δ T-cell comprising a heterologous targeting construct, wherein the heterologous targeting construct comprises an antigen binding domain and a transmembrane domain, wherein the transmembrane domain is a terminal transmembrane domain (i.e., a transmembrane domain having an unconnected terminal end, such as the C-terminus, which is not linked to a peptide or protein). Thus, the terminal transmembrane domain is not linked to an intracellular domain, such as an intracellular signaling domain. The transmembrane domain does not propagate signal 1 activation. In some embodiments, the terminal transmembrane domain is not involved in an intracellular signaling pathway (e.g., a TCR pathway, e.g., a T cell signaling pathway, e.g., signal 2 costimulation). In other embodiments, the transmembrane domain may bind to an endogenous molecule, thereby propagating signal 2 co-stimulation. In some embodiments, the heterologous targeting construct further comprises a stalk domain operably linking the antigen binding domain to the transmembrane domain.

In some embodiments of any aspect of the invention, the transmembrane domain does not activate the engineered γ δ T-cell.

In another aspect, the invention provides an engineered γ δ T-cell comprising a heterologous targeting construct consisting of an antigen binding domain, a stalk domain operably linked to the antigen binding domain, and a transmembrane domain operably linked to the stalk domain.

In some embodiments of any aspect of the invention, the engineered γ δ T-cell is V δ 2 negative (e.g., V δ 2 negative γ δ T-cell is V δ 1 positive or double negative). In an alternative embodiment of any aspect of the invention, the engineered γ δ T-cell may be V δ 2 positive.

The antigen binding domain may include a single chain variable fragment (scFv), a monoclonal antibody, a Fab fragment, a B cell receptor, a T cell receptor, an antibody scaffold, a receptor-specific ligand, or a ligand-specific receptor (e.g., a receptor specific for a surface-expressed ligand). In some embodiments, the stem domain comprises one or more of the domains selected from the group consisting of: CD8 stem, IgG1 hinge-CH₂Domain, IgG 1-hinge-CH₃Domain, IgG 1-hinge-CH₂-CH₃(ii) Domain, (G)₄S)₃Hinge, IgG1 hinge, CD7 stem, IgD hinge-CH₂Domain, IgD hinge-CH₃Domain, IgD hinge-CH₂-CH₃Domain, IgG4 hinge, IgG4 hinge-CH₂Domain, IgG4 hinge-CH₃Domain, IgG4 hinge-CH₂-CH₃A domain or fcsri stem domain.

In some embodiments of any of the aspects of the invention, the transmembrane domain comprises a CD8 transmembrane domain, a CD4 transmembrane domain, a CD3 epsilon transmembrane domain, a CD3 zeta transmembrane domain, a CD28 transmembrane domain, a CD45 transmembrane domain, a CD5 transmembrane domain, a CD8 transmembrane domain, a CD9 transmembrane domain, a CD16 transmembrane domain, a CD22 transmembrane domain, a CD33 transmembrane domain, a CD37 transmembrane domain, a CD64 transmembrane domain, a CD80 transmembrane domain, a CD86 transmembrane domain, a CD134 domain, a CD137 transmembrane domain, a CD154 transmembrane domain, a CD7 transmembrane domain, a CD71 transmembrane domain, a CD18 transmembrane domain, a CD29 domain, a CD11a transmembrane domain, a CD11b transmembrane domain, a CD11 transmembrane domain, a CD b transmembrane domain, a nkr b transmembrane domain, or a NKG b transmembrane domain. In some embodiments, no more than 50% of the amino acids of the terminal transmembrane domain reside within the cell (e.g., no more than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the amino acids of the terminal transmembrane domain (e.g., the C-terminal transmembrane domain) reside within the cell).

In some embodiments of any aspect of the invention, the clustering of the heterologous targeting construct does not substantially activate the TCR pathway in the engineered γ δ T cell upon binding of the antigen binding domain to the target antigen.

In some embodiments of any aspect of the invention, the antigen binding domain binds to a tumor associated antigen. For example, the tumor-associated antigen may be a protein or peptide antigen (e.g., CD19) expressed on the surface of tumor cells. Alternatively, the tumor-associated antigen may be a carbohydrate expressed on the surface of a tumor cell. In some embodiments, the tumor-associated antigen is a ganglioside expressed on a tumor cell representation (e.g., GD 2). In some embodiments, the tumor-associated antigen is an immunosuppressive antigen. In one embodiment, the antigen binding domain binds to a target antigen expressed by a solid tumor cell.

In some of any of the preceding embodiments, binding of the antigen binding domain to a target antigen expressed on a healthy cell triggers a substantial reduction in cytolysis (e.g., at least 5% less, at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, or at least 95% less) by the engineered γ δ T cell relative to a reference cell having a functional intracellular domain (e.g., it does not substantially trigger cytolysis by the engineered γ δ T cell). In some embodiments, binding of the antigen binding domain to a target antigen expressed on tumor cells or infected cells significantly triggers cytolysis by the engineered γ δ T cells. The cell lysis may depend on the endogenous expression of NKG2D, NKp30, NKp44, NKp46, or DNAM1 by the engineered γ δ T cells. In some embodiments, the cytolysis is characterized by one, two, three, four, five, or all six of the responses selected from CD107 degranulation, granzyme release, perforin release, granulysin release, target cell killing, γ δ T cell proliferation, and cytokine production.

In another aspect, the invention provides an engineered NK cell or NK-like T cell having the heterologous targeting construct of any of the embodiments described herein. In some embodiments, the heterologous targeting construct comprises an extracellular antigen binding domain and a transmembrane domain operably linked to the antigen binding domain. The heterologous targeting construct lacks the intracellular domain of an engineered NK cell or NK-like T cell.

In another aspect, the present invention provides an engineered Innate Lymphoid Cell (ILC). Engineered ILCs include heterologous targeting constructs of any of the embodiments described herein. In some embodiments, the heterologous targeting construct comprises an extracellular antigen binding domain and a transmembrane domain operably linked to the antigen binding domain. The heterologous targeting construct lacks an intracellular domain capable of activating the engineered innate lymphoid cells.

In another aspect, the invention provides an engineered MAIT cell. Engineered MAIT cells include the heterologous targeting constructs of any of the embodiments described herein. In some embodiments, the heterologous targeting construct comprises an extracellular antigen binding domain and a transmembrane domain operably linked to the antigen binding domain. The heterologous targeting construct lacks an intracellular domain capable of activating the engineered MAIT cells.

In another aspect, the invention provides an isolated population of cells comprising at least ten engineered γ δ T cells, engineered NK cells or NK-like T cells, engineered innate lymphoid cells or engineered MAIT cells of any of the preceding embodiments. In some embodiments, the engineered γ δ T-cell, engineered NK-cell or NK-like T-cell, engineered innate lymphoid cell, or engineered MAIT-cell comprises greater than 2% (e.g., 2% to 100%, 10% to 95%, 20% to 90%, 30% to 80%, 40% to 70%, e.g., greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, 96%, 97%, 98%, or 99%) of the total number of cells in the isolated population of cells.

In another aspect, the invention provides an isolated population of cells comprising a plurality of engineered γ δ T cells, NK-like T cells, innate lymphoid cells or MAIT cells of any preceding embodiment. The population of engineered γ δ T cells, NK-like T cells, innate lymphoid cells, or MAIT cells can account for greater than 2% (e.g., 2% to 100%, 10% to 95%, 20% to 90%, 30% to 80%, 40% to 70%, e.g., greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, 96%, 97%, 98%, or 99%) of the total number of cells in the isolated population of cells. In some embodiments, the isolated population of cells comprises at least ten of the engineered γ δ T cells, NK-like T cells, innate lymphoid cells, or MAIT cells of any of the preceding embodiments.

In another aspect, the invention includes a γ δ T cell, NK-like T cell, innate lymphoid cell, or MAIT cell comprising a heterologous polynucleotide. The heterologous polynucleotide may encode a heterologous targeting construct comprising an extracellular antigen-binding domain and a transmembrane domain operably linked to the antigen-binding domain, wherein the heterologous targeting construct does not directly activate engineered γ δ T cells, NK-like T cells, innate lymphoid cells, or MAIT cells.

In yet another aspect, the invention provides a γ δ T cell, NK-like T cell, innate lymphoid cell MAIT cell comprising a heterologous polynucleotide encoding a targeting construct comprising an antigen binding domain and a terminal transmembrane domain.

An engineered γ δ T cell, NK-like T cell, innate lymphoid cell, or MAIT cell of any preceding embodiment; an isolated population of engineered γ δ T cells, NK-like T cells, innate lymphoid cells, or MAIT cells; or a γ δ T cell, NK-like T cell, innate lymphoid cell, or MAIT cell comprising the heterologous polynucleotide can be used in a method of treating a subject by adoptive cell therapy (e.g., for use in a method of treating a subject by adoptive cell therapy).

In another aspect, the invention provides a method of treating a subject by adoptive cell therapy (e.g., adoptive T cell therapy) comprising administering to a subject in need thereof a therapeutically effective amount of the engineered cell, isolated cell population, or cell of any of the foregoing embodiments.

In another aspect, the invention provides an engineered cell, isolated population of cells, or cell of any of the preceding embodiments for use in a method of treating a subject by adoptive cell therapy (e.g., adoptive T cell therapy), wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of the engineered cell, isolated population of cells, or cell of any of the preceding embodiments.

In some embodiments of any of the preceding aspects, the subject is a human. For example, the subject may be a human cancer patient (e.g., a human cancer patient undergoing treatment for a solid tumor). Alternatively, the human patient may be a human patient being treated for a viral infection.

Drawings

Figure 1 is a schematic diagram illustrating one embodiment of a classical Chimeric Antigen Receptor (CAR) and a heterologous targeting construct that does not include an intracellular domain.

Fig. 2A-2C are a series of schematic diagrams showing how to modify heterologous targeting constructs using various extracellular domains tailored to the desired target. Fig. 2A shows a generalized extracellular domain, which may be, for example, a B cell receptor, an antibody scaffold or mimetic, an scFv, mAb, Fab, or T cell receptor. Figure 2B shows the extracellular domain as a ligand-specific receptor. Figure 2C shows the extracellular domain as a receptor-specific ligand.

Fig. 3A and 3B are flow cytometry histograms. Figure 3A shows the expression of anti-CD 19 targeting constructs without the intracellular domain ("non-signaling or nsCAR") and full-length anti-CD 19CAR on transduced V δ 1 cells. FIG. 3B shows the expression of NCR (Natural cytotoxic receptor) NKp30 (left column), NKp44 (middle column) and NKG2D (right column) on untransduced (UTD; top row), non-signaling CD19CAR transduced (middle row) and CD19CAR transduced (bottom row) V.delta.1 cells.

FIGS. 4A-4C are graphs showing CD19 expression on Nalm-6 and B cells (FIG. 4A) and results of a 16 hour killing assay at a 1:1 effector to target ratio (FIGS. 4B and 4C). FIG. 4B shows killing of CD19+ Nalm-6 cells, and FIG. 4C shows killing of primary B-ALL cells. Two independent donors and experiments are shown.

Fig. 5A and 5B are graphs showing anti-GD 2 non-signaling CAR expression on V δ 1 cells (fig. 5A) and showing the 60 hour time course of Kelly cell line growth alone or in the presence of V δ 1 cells (fig. 5B). The data is represented as a change in the number of green object counts per image, which has been normalized with respect to the number of green object counts per image when time is zero. Each data point represents triplicate wells.

Detailed Description

Disclosed herein are compositions of engineered lymphocytes (e.g., lymphocytes with innate effector function, such as γ δ T cells, NK-like T cells, lymphoid cells, or mucosa-associated constant T cells) expressing heterologous targeting constructs. The heterologous targeting construct comprises an extracellular antigen-binding domain and a transmembrane domain operably linked to the antigen-binding domain (e.g., directly linked or linked through a stalk domain). These engineered lymphocytes (e.g., γ δ T cells) can be used to treat diseases such as cancer or viral infections. Because the heterologous constructs of the invention lack a functional intracellular domain capable of transmitting T cell activation, they rely on the endogenous MHC-independent activation pathway properties of γ δ T cells, which are lacking in α β T cells. Thus, the heterologous constructs described herein are designed for expression on the surface of lymphocytes, such as γ δ T cells (e.g., V δ 1 cells, V δ 2 cells, V δ 3 cells, V δ 5 cells, and V δ 8 cells).

Definition of

It should be understood that the aspects and embodiments of the invention described herein include, "comprise", "consist of" and "consist essentially of" aspects and embodiments. As used herein, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

As used herein, the term "about" refers to the usual error range for individual values as would be readily known to one skilled in the art. References herein to "about" a value or parameter include (and describe) embodiments that are directed to that value or parameter itself. In some cases, "about" encompasses a variation of + 20%, in some cases + 10%, in some cases + 5%, in some cases + 1%, or in some cases + 0.1% from the specified value, as such a variation is suitable for performing the disclosed method.

As used herein, the terms "substantial" and "substantially" refer to a qualitative condition that exhibits all or almost all of the range or degree of a feature or characteristic of interest. One of ordinary skill in the art of biology will appreciate that little, if any, biological and chemical phenomena proceed to completion and/or proceed to completion or to achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of integrity inherent in many biological and chemical phenomena. When describing a physical scenario, such as receptor/ligand interaction or cell/cell contact, the scenario is essential if its functional outcome can be detected by conventional means available to the person performing the method. For example, "substantial TCR activation" refers to a detectable level of TCR activation (e.g., a statistically significant degree of TCR activation) in a population of cells. In some embodiments, the EC of a TCR pathway agonist (e.g., an antibody, e.g., anti-CD 3, or lectin) when exposed to up to 0.1%, up to 0.5%, up to 1%, up to 5%, up to 10%, up to 20%, up to 30%, or up to 40% of the respective cell population₅₀The TCR is substantially activated.

As used herein, a "heterologous targeting construct" refers to a protein or a group of proteins (e.g., two or more proteins that dimerize to form a functional quaternary protein) that resides on a host cell (i.e., an engineered cell) and binds to a target molecule that is present on another cell, and which is not naturally expressed by the cell in which it resides. The heterologous targeting construct may be encoded by a polynucleotide that is expressed within the engineered cell.

As used herein, "activating" a T cell refers to initiating or amplifying a T Cell Receptor (TCR) pathway by propagating signal 1 activation or signal 2 activation. For example, a chimeric antigen receptor with a functional signaling 1T cell activation domain (e.g., CD3 ζ) or co-stimulatory domain (e.g., CD28,4-1BB, etc.) can "activate" its host T cell by aggregating in response to antigen binding. Heterologous targeting constructs lacking a functional intracellular domain may be unable to transmit signal 1 activation or signal 2 activation and thus unable to activate the TCR pathway. A heterologous targeting construct lacking a functional intracellular domain may be capable of "activating" a T cell expressing it when its transmembrane domain transmits co-stimulation, e.g. upon binding of NKG2D transmembrane domain to endogenous DAP10 or DAP 12. In an alternative embodiment, the invention features a heterologous targeting construct with a non-functional transmembrane domain, and which does not activate a T cell expressing it.

Activation of the "T Cell Receptor (TCR) pathway" refers to the induction of proliferation or other consequences of activating T cells by TCR signaling. The TCR signaling pathway involves signal 1 activation, for example, sequential activation of Src-associated Protein Tyrosine Kinase (PTK), Lck and Fyn, and the zeta chain (TCR) -associated protein kinase of 70kDA (ZAP 70). These PTKs cause phosphorylation of polypeptides including the adaptor activator of T cells (LAT), which leads to downstream stimulation by extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear factor of activated T cells (NFAT). Signal 2 (i.e., co-activation) can enhance phosphorylation and enhance TCR activation, for example, by CD28, CD45, DAP10, or DAP 12. Thus, any molecule that targets a TCR or part of a costimulatory pathway can directly activate T cell signaling. Surface-binding molecules that simply contact T cells with target cells may facilitate other molecules to directly trigger T cell activation (e.g., heterologous targeting constructs), but these targeting molecules do not directly activate the TCR pathway.

TCR pathway agonists include antibodies (e.g., monoclonal antibodies, e.g., anti-TCR V.delta.1, anti-TCR. delta.TCS-1, anti-TCR PAN. gamma.delta.and anti-CD 3), lectins (e.g., plant lectins, e.g., concanavalin A, lectins from Phaseolus vulgaris (PHA-P), Phytolacca Americana (Phytolacca Americana), Triticum Triticum vulgaris (Triticum vulgari), lentils (Lens culinaris), Glycine max, Sophora japonica (Maackia amurensis), Pisum sativum (Pisum sativum) and Sambucus nigra (Sambucus nigra)), synthetic phosphoantigens (e.g., BrHPP (bromohydrin pyrophosphate), 2M3B1PP (2-methyl-3-butenyl-1-pyrophosphate), HMBPP ((E) -4-hydroxy-3-methyl-but-2-enyl pyrophosphate), or IPP (isopentenyl pyrophosphate)), and N-bisphosphonates (e.g., zoledronate). TCR pathway agonists include co-receptor agonists, including antibodies (e.g., monoclonal antibodies, e.g., anti-CD 2, anti-CD 6, anti-CD 9, anti-CD 28, anti-CD 43, anti-CD 94, anti-CD 160, anti-SLAM, anti-NKG 2D, anti-2B 4, anti-HLA-a, anti-HLA-B, anti-HLA-C, and anti-ICAM-3) and proteins (e.g., recombinant proteins, e.g., recombinant human proteins, e.g., CD7L, CD26, CD27L, CD30L, CD40L, OX40L, 4-1BBL, ICAM-1, fibronectin, hydrocortisone, and variants thereof, e.g., Fc-fusion proteins, e.g., CD 27L-Fc). TCR pathway agonists may be soluble or membrane-bound, and may, for example, be present on cells, such as artificial antigen presenting cells (aapcs), as well as MHC or HLA complexes. Suitable aapcs for activating T cell signaling are known in the art. Suitable methods for activating T cells by exogenous addition of TCR pathway agonists are well known in the art and are summarized in figure 1 of Deniger, et al (Deniger, et al.

By "exogenous TCR pathway agonists" is meant TCR pathway agonists that do not originate from non-hematopoietic tissue or its donor (i.e., they are exogenously added). Thus, it is to be understood that in some embodiments of the invention, TCR pathway agonists may be present in culture as a residual substance of non-hematopoietic tissue (e.g., soluble fibronectin or cell-bound ICAM-1). In some embodiments, the concentration of residual TCR pathway agonist is negligible and does not substantially activate T cells.

For a domain of a protein (e.g., a heterologous targeting construct) that is to be "operably linked" to another domain herein, it is meant that the domain resides on the same protein as the other domain, is directly adjacent to the other domain or is separated by one or more amino acids or domains. For example, in a heterologous targeting construct having an N-terminal antigen binding domain, the mid-stem domain can be said to be operably linked to a C-terminal transmembrane domain, an antigen binding domain, and a transmembrane domain. In a heterologous targeting construct having an N-terminal antigen binding domain immediately adjacent to a C-terminal transmembrane domain, the antigen binding domain and the transmembrane domain may also be said to be operably linked, but more specifically directly linked.

As used herein, "antibody scaffold" refers to a non-native antigen binding protein, peptide, or antibody fragment. Antibody scaffolds include Adnectins, affibodies, affilins, anticalins, atrimers, avimers, bicyclic peptides, centryrins, cysteine-junctions (cys-knots), DARPins, fynomers, Kunitz domains, Obodies, and Tn3 s. Antibody scaffolds are known in the art and are described, for example, in Vazquz-Lombardi et al, Drug Discovery Today,2015,20(10):1271-83, which is incorporated herein by reference in its entirety.

The term "antibody" is used in the broadest sense and specifically covers antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they possess the desired biological activity.

As used herein, the term "cytotoxicity" refers to the ability of an immune cell (e.g., a γ δ T cell) to kill other cells (e.g., target cells). Immune cells with cytotoxic functions release toxic proteins (e.g., perforin and granzyme) that can kill nearby cells.

As used herein, the term "degranulation" refers to a cellular process in which molecules, including antimicrobial and cytotoxic molecules, are released from cellular endocrine vesicles known as particles. Degranulation is part of the immune response of immune cells (e.g., cytotoxic T cells) against pathogens and invading microorganisms. The molecules released during degranulation vary from cell type to cell type and may include molecules intended to kill invading pathogens and microorganisms or to promote an immune response (e.g., inflammation).

As used herein, the term "innate lymphoid cell" refers to an innate lymphoid cell that lacks rearranged antigen receptors (e.g., antigen receptors expressed by T and B cells). The innate lymphoid cells include NK cells, type 1 innate lymphoid cells (ILC1), ILC1 cells, type 2 innate lymphoid cells (ILC2), type 3 innate lymphoid cells (ILC3), and the like.

As used herein, the terms "mucosa-associated constant T cells" and "MAIT cells" refer to innate like T cells that express a constant T cell receptor alpha (TCR α) chain and a diverse TCR β chain and can recognize a unique set of molecules in the context of the evolutionarily conserved major histocompatibility complex-associated molecule 1(MR 1).

As used herein, the term "NK cell" refers to a natural killer cell, an innate lymphoid cell that does not express TCR or CD3 and is positive for CD56 and CD161 expression. NK cells may also express natural cytotoxic receptors, such as NKp44 and NKp 46.

As used herein, the term "NK-like T cells" refers to natural killer-like T cells or natural killer T cells (NKT cells), which are innate-like lymphocytes that express functional and structural properties shared with T cells and NK cells, i.e., they express TCRs (e.g., α β TCRs), CD3, and CD 56. NK-like T cells recognize and react with glycolipids in the context of the MHC class I glycoprotein CD1d and upon activation can produce IFN- γ and IL-4.

As used herein, "non-hematopoietic cells" include stromal cells and epithelial cells. Stromal cells are non-hematopoietic connective tissue cells of any organ and support the function of the essential cells of that organ. Examples of stromal cells include fibroblasts, pericytes, mesenchymal cells, keratinocytes, endothelial cells, and non-hematologic tumor cells. Epithelial cells are non-hematopoietic cells that line the lumen and surface of systemic blood vessels and organs. They are generally squamous, columnar or cuboidal in shape, and may be arranged as a monolayer of cells, or as two or more layers of cells.

As used herein, "non-hematopoietic tissue resident γ δ T cells," "non-hematopoietic tissue derived," and "non-hematopoietic tissue native γ δ T cells" refer to γ δ T cells present in non-hematopoietic tissue upon tissue removal. Non-hematopoietic tissue resident γ δ T cells may be obtained from any suitable non-hematopoietic tissue of a human or non-human animal. Non-hematopoietic tissues are tissues other than blood or bone marrow. In some embodiments, γ δ T cells are not obtained from a particular type of biological fluid sample (e.g., blood or synovial fluid). Examples of such suitable nonhematopoietic tissues of humans or non-human animals include skin or a portion thereof (e.g., dermis or epidermis), gastrointestinal tract (e.g., gastrointestinal epithelium, colon, small intestine, stomach, appendix, cecum or rectum), breast tissue, lung (preferably wherein the tissue is not obtained by bronchoalveolar lavage), prostate, liver and pancreas. In some embodiments, the non-hematopoietic tissue-resident γ δ T cells may be derived from lymphoid tissues, such as thymus, spleen, or tonsil. γ δ T cells may also reside in human cancer tissues, such as breast and prostate. In some embodiments, the γ δ T cells are not obtained from human cancer tissue. Non-hematopoietic tissue samples can be obtained by standard techniques, e.g., by explantation (e.g., biopsy). Non-hematopoietic tissue resident γ δ T cells include, for example, V δ 1T cells, Double Negative (DN) T cells, V δ 2T cells, V δ 3T cells, and V δ 5T cells.

Any one or more of the above factors may be included in the amplification protocol in an amount effective to produce an expanded population of lymphocytes (e.g., γ δ T cells) that can be transfected with a nucleic acid encoding the heterologous targeting construct of the invention. As used herein, the phrase "an amount effective at … …" refers to an amount that results in a detectable result (e.g., an increased number of cells with a statistical significance relative to the starting population, e.g., p < 0.05). In the case where multiple factors are present at once, an effective amount refers to the combined effect of all factors (e.g., the combined effect of IL-2 and IL-15, or the combined effect of IL-2, IL-4, IL-15, and IL-21).

As used herein, an "expanded γ δ cell population" refers to a hematopoietic or non-hematopoietic cell population that includes γ δ T cells that have been cultured for a period of time and under conditions to induce expansion of γ δ cells (i.e., increase the number of γ δ cells). Likewise, as used herein, an "expanded V δ 1T cell population" refers to a hematopoietic or non-hematopoietic cell population comprising V δ 1T cells that have been cultured for a time and under conditions to induce expansion of the V δ 1T cells (i.e., increase the number of V δ 1T cells).

As used herein, "feeder cells" refer to any exogenous cells added to a culture to provide cell-to-cell surface contact to cells of non-hematopoietic tissue origin. Feeder cells may be primary cells (e.g., from tissue) or derived from a cell line. Feeder cells may be live or irradiated and include tumor cells, fibroblasts, B cells and other antigen presenting cells.

The term "marker" herein refers to a DNA, RNA, protein, carbohydrate, glycolipid, or cell-based molecular marker, the expression or presence of which in a patient sample can be detected by standard methods (or methods disclosed herein).

A cell or population of cells that "expresses" a marker of interest is a cell or population of cells in which the presence of mRNA encoding the protein or the substance of the protein itself (including fragments thereof) is determined. Expression of the marker can be detected by a variety of methods. For example, in some embodiments, expression of a marker refers to the surface density of the marker on a cell. For example, the Mean Fluorescence Intensity (MFI) to be used as a flow cytometer reading represents the density of markers on a population of cells. As will be appreciated by those skilled in the art, the MFI value depends on staining parameters (e.g., concentration, duration, and temperature) and fluorescent dye composition. However, when considered within the appropriate control range, MFI may be quantitative. For example, a population of cells expressing a marker can be said to express the marker if the MFI of the antibody directed against the marker is significantly higher relative to the MFI of an appropriate isotype control antibody on the same population of cells stained under the same conditions. Additionally or alternatively, using positive and negative gates according to conventional flow cytometry analysis methods, it can be said that a population of cells expresses markers cell by cell (e.g., by gating according to isotype or "fluorescence minus one" (FMO) controls). By this measure, a population can be said to "express" a marker if the number of cells positive for that marker is detected to be significantly above background (e.g., by gating on an isotype control).

As used herein, when expression of a certain population is expressed as a percentage of positive cells and the percentage is compared to the corresponding percentage of positive cells of a reference population, the percentage difference is the percentage of the parent population of each corresponding population. For example, if a marker is expressed on 10% of the cells of group a and the same marker is expressed on 1% of the cells of group B, it can be said that the frequency of marker positive cells of group a is 9% (i.e., 10% -1%, not 10% ÷ 1%) greater than that of group B. When the frequency is multiplied by the number of cells in the parent population, the difference in absolute cell number will be calculated. In the example given above, if there are 100 cells in group a and 10 cells in group B, then the number of cells in group a is 100 times that of group B (10% x 100) ÷ (1% x 10).

The expression level of the marker can be a nucleic acid expression level (e.g., a DNA expression level or an RNA expression level, such as an mRNA expression level). Any suitable method of determining the level of expression of a nucleic acid may be used. In some embodiments, the nucleic acid expression level is determined using qPCR, rtPCR, RNA-seq, multiplex qPCR or RT-qPCR, microarray analysis, Serial Analysis of Gene Expression (SAGE), MassARRAY technique, in situ hybridization (e.g., FISH), or a combination thereof.

As used herein, a "reference population" of cells refers to a population of cells corresponding to a target cell relative to which a phenotype of the target cell is measured. For example, the expression level of a marker on an isolated population of non-hematopoietic tissue-derived γ δ cells can be compared to the expression level of the same marker on hematopoietic tissue-derived γ δ T cells (e.g., blood-resident γ δ cells, e.g., blood-resident γ δ cells from the same donor or donor) or non-hematopoietic tissue-derived γ δ T cells expanded under different conditions (e.g., in the presence of substantial TCR activation, in the presence of exogenous TCR activators (e.g., anti-CD 3), or in substantial contact with stromal cells (e.g., fibroblasts)). A cluster may also be compared to its own early state. For example, the reference population can be an isolated population of cells prior to expansion. In this case, the amplified population is compared to its own composition prior to the amplification step, i.e., in this case its past composition is the reference population.

"cancer" refers to abnormal proliferation of malignant cancer cells and includes hematopoietic cancers (e.g., hematological malignancies, e.g., leukemias, such as Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), Chronic Eosinophilic Leukemia (CEL), myelodysplastic syndrome (MDS), Acute Lymphocytic Leukemia (ALL) and Chronic Lymphocytic Leukemia (CLL), lymphomas, such as Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL) and Multiple Myeloma (MM)), and solid cancers, such as sarcomas (e.g., soft tissue sarcoma, uterine sarcoma), skin cancers, melanomas (e.g., malignant melanoma), bladder cancers, brain cancers, breast cancers, uterine cancers, ovarian cancers, prostate cancers, lung cancers, large bowel cancers (e.g., large bowel gland cancers), cervical cancers, liver cancers (i.e., liver cancers), head and neck cancers (e.g., head and neck squamous cell cancers), Esophageal cancer, pancreatic cancer, renal cancer (e.g., renal cell carcinoma), adrenal cancer, gastric cancer (e.g., gastric adenocarcinoma), testicular cancer, cancer of the gallbladder and biliary tract, thyroid cancer, thymus cancer, bone cancer, brain cancer, biliary tract cancer, bladder cancer, cancer of bone and soft tissue, brain tumor, cervical cancer, colon cancer, glioma, embryonic cancer, endometrial cancer, esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumor, osteosarcoma, ovarian cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, rhabdomyosarcoma, skin cancer, testicular germ cell tumor, urothelial cancer, and uterine cancer. Cancer cells in cancer patients may be immunologically different from somatic cells in normal individuals (e.g., cancerous tumors may be immunogenic). For example, the cancer cells may be capable of eliciting a systemic immune response in a cancer patient against one or more antigens expressed by the cancer cells. The antigen that elicits the immune response may be a tumor antigen or may be shared by normal cells. A patient with cancer may exhibit at least one identifiable sign, symptom, or laboratory finding sufficient for cancer diagnosis according to clinical criteria known in the art. Examples of such clinical criteria can be found in medical textbooks, such AS Harrison's Principles of Internal Medicine (Longo DL, Fauci AS, Kasper DL, Hauser SL, Jameson J, Loscalzo J. eds.18e. New York, NY: McGraw-Hill; 2012). In some cases, diagnosing cancer in an individual may include identifying a particular cell type (e.g., cancer cells) in a sample of bodily fluid or tissue obtained from the individual.

As used herein, a "solid tumor" is a cancer of any body tissue other than the blood, bone marrow, or lymphatic system. Solid tumors can be further divided into solid tumors of epithelial origin and solid tumors of non-epithelial origin. Examples of solid epithelial tumors include tumors of the gastrointestinal tract, colon, breast, prostate, lung, kidney, liver, pancreas, ovary, head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gall bladder, lip, nasopharynx, skin, uterus, male genital organs, urinary organs, bladder, and skin. Solid tumors of non-epithelial origin include sarcomas, brain tumors and bone tumors.

A patient, subject, or individual suitable for treatment as described above can be a mammal, such as a rodent (e.g., guinea pig, hamster, rat, mouse), murine (e.g., mouse), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), primate, ape (e.g., monkey or ape), monkey (e.g., marmoset or baboon), ape (e.g., gorilla, chimpanzee, orangutan, or gibbon), or human.

In some embodiments, the patient, subject, or individual is a human. In other preferred embodiments, non-human mammals may be used, particularly those mammals conventionally used as models to demonstrate efficacy of human therapy (e.g., murine, primate, porcine, canine, or rabbit).

As used herein, "treatment" (and grammatical variations thereof, such as "treating" or "treatment") refers to clinical intervention (e.g., in veterinary applications) in a human or animal where some desired therapeutic effect is achieved, such as inhibition or delay of disease progression, and includes a decrease in rate of progression, cessation of rate of progression, improvement of disease, cure or remission (partial or total), prevention, delay, alleviation or prevention of one or more symptoms and/or indications of disease, or extension of the subject or patient's survival beyond that expected in the absence of treatment.

Treatment as a prophylactic measure (i.e., prophylactic) is also included. For example, a patient, subject, or individual susceptible to or at risk of developing or reoccurring cancer may be treated as described herein. Such treatment can prevent or delay the onset or recurrence of cancer in the patient, subject, or individual.

In particular, treatment may include inhibiting cancer growth (including complete remission of cancer) and/or inhibiting cancer metastasis. Cancer growth generally refers to any of a number of indicators that indicate changes in the cancer to a more developed form. Thus, indicators measuring inhibition of cancer growth include decreased survival of cancer cells, decreased tumor volume or morphology (as determined, for example, using Computed Tomography (CT), ultrasound examination, or other imaging methods), delayed tumor growth, disrupted tumor vasculature, improved performance of delayed-type hypersensitivity skin tests, increased cytolytic T lymphocyte activity, and decreased levels of tumor-specific antigens. Reducing immunosuppression in cancerous tumors in an individual may increase the ability of the individual to resist cancer growth (particularly growth of cancer already present in the subject) and/or reduce the propensity for cancer growth in the individual.

In some embodiments, expanded γ δ T cells (e.g., non-hematopoietic tissue-derived γ δ T cells, e.g., non-hematopoietic tissue-derived V δ 1T cells) are administered to delay the progression of the disease or slow the progression of the disease or disorder.

As used herein, "administering" refers to a method of administering a dose of a therapy (e.g., an adoptive T cell therapy, including, for example, non-hematopoietic tissue-derived γ δ T cells) or composition (e.g., a pharmaceutical composition, such as a pharmaceutical composition including non-hematopoietic tissue-derived γ δ T cells) to a patient. The compositions used in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, transdermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, intraorbitally, intravitreally (e.g., by intravitreal injection), by eye drop, orally, topically, transdermally, by inhalation, by injection, by implantation, by infusion, by continuous infusion, bathing target cells directly by local perfusion, by catheter, by lavage, milk fat, or lipid compositions. The compositions used in the methods described herein may also be administered systemically or locally. The method of administration may vary depending on various factors such as the therapeutic agent or composition being administered and the severity of the condition, disease or disorder being treated.

"therapeutically effective amount" refers to the amount of a therapeutic agent used to treat or prevent a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of a therapeutic agent (e.g., non-hematopoietic tissue-derived γ δ T) can reduce the number of cancer cells; reducing primary tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and/or relieve to some extent one or more symptoms associated with the disease. To some extent, the drug can prevent growth and/or kill existing cancer cells, which can inhibit cell growth and/or be cytotoxic. For cancer therapy, in vivo efficacy can be measured, for example, by assessing survival time, time to disease progression (TTP), remission rate (e.g., Complete Remission (CR) and Partial Remission (PR)), duration of response, and/or quality of life.

The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents, wherein at least a portion of the administrations overlap in time. Thus, concurrent administration includes an administration regimen in which administration of one or more additional agents is discontinued and administration of one or more agents is continued. For example, in some embodiments, the non-hematopoietic tissue-derived γ δ T cells and IL-2 may be administered simultaneously.

The term "pharmaceutical composition" refers to a formulation in a form effective for the biological activity of one or more active ingredients contained therein and free of additional ingredients having unacceptable toxicity to the patient to whom the formulation is administered.

As used herein, a "terminal transmembrane domain" refers to a transmembrane domain having an unattached terminal end (e.g., a C-terminus unattached to a peptide or protein). Thus, the terminal transmembrane domain is not linked to an intracellular domain, such as an intracellular signaling domain. In some embodiments, the terminal transmembrane domain is not involved in an intracellular signaling pathway (e.g., a T cell signaling pathway, such as signal 1 activation or signal 2 co-stimulation).

As used herein, the term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct that includes an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain that transmits an activation signal that activates a cell. In some embodiments, the CAR comprises an optional leader sequence at the N-terminus of the CAR fusion protein.

In the event of any conflict or inconsistency between a definition set forth herein and a definition provided in any reference incorporated by reference herein, the definition set forth herein shall prevail.

Gamma delta T cells and other innate lymphoid cells expressing heterologous targeting constructs

Lymphocytes such as γ δ T cells and other innate-like lymphocytes (e.g., innate lymphoid cells such as NK cells and NK-like T cells, and mucosa-associated constant T (mait) cells) are attractive vectors for the heterologous targeting constructs described herein, as they can be transduced with heterologous targeting constructs while retaining their innate-like ability to recognize pathogenic cells such as cancer cells and infected cells. Transduction may be performed using any suitable method known in the art or described herein, such as by electroporation, gene editing (e.g., by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), Zinc Finger Nuclease (ZFN) transfection), transposon delivery, and the like. In addition, the lack of MHC-dependent antigen recognition, for example by γ δ T cells, reduces the likelihood of graft versus host disease and enables them to target tumors expressing low levels of MHC. Likewise, independence of γ δ T cells from conventional signal 2 costimulation (e.g., by engagement of CD 28) enhances targeting of tumors expressing low levels of costimulatory receptor ligands.

In one aspect, the invention provides γ δ T cells, NK-like T cells, innate lymphoid cells and MAIT cells and cell populations thereof that express heterologous targeting constructs on the surface. Such γ δ T cells, NK-like T cells, innate lymphoid cells and MAIT cells engineered to express heterologous targeting constructs can be used to target a desired antigen through the antigen binding domain on the heterologous construct. In contrast to conventional Chimeric Antigen Receptor (CAR) systems used as part of conventional (e.g., α β) T cell adoptive immunotherapy regimens, the heterologous targeting constructs do not require intracellular domains to induce cytolysis or cytotoxicity because γ δ T cells respond to foreign pathogens independent of MHC receptors. Instead, γ δ T cells cause intrinsic target-specific cytolysis, and this response can be further enhanced by using heterologous constructs to improve and increase the contact time with target cells (e.g., tumors, such as solid tumors). γ δ T cells engineered with heterologous constructs can bind to target antigens, such as tumor-associated antigens, and induce cytotoxicity and/or cytolysis. This cytotoxicity may be mediated by endogenously expressing activating receptors (e.g., NKG2D, NKp30, NKp44, NKp46, and/or DNAM 1).

The heterologous targeting construct can provide an extracellular antigen binding domain and a transmembrane domain operably linked to the antigen binding domain. The stalk domain may further be included as part of a heterologous targeting construct to link the antigen binding domain to the transmembrane domain. In some embodiments, the heterologous targeting constructs provided herein lack the intracellular domain (fig. 1) and also lack the ability to activate TCR signaling (e.g., via signal 1 activation and/or signal 2 activation (i.e., co-stimulation)).

In some embodiments, the cytolysis is characterized by degranulation of γ δ T cells (e.g., CD107 degranulation), granzyme release of γ δ T cells, perforin release of γ δ T cells, target cell killing, proliferation of γ δ T cells, or cytokine production of γ δ T cells. One skilled in the art will recognize that various assays to measure these properties or activities may be used to assess the efficacy of engineered T cells, for example in the treatment of cancer.

Generally, degranulation is a prerequisite for cell lysis. Degranulated cells (lysosomal associated membrane protein 1, also known as CD107) can be recognized, for example, by surface expression of LAMP-1. CD107 is transiently expressed on the surface and rapidly internalizes after degranulation. In the inactivated state, CD107a resides in the cytoplasm of the cytolytic granular membrane. Upregulation can be measured by staining CD107 in the presence of monensin (to prevent acidification of antibody-labeled CD107 a-containing vesicles) (e.g., by FACS).

Assays for perforin and granzyme were also measured by FACS according to methods known in the art. Cytotoxic γ δ T cells kill their target through a particle or receptor mediated mechanism. Cytotoxic granules are secretory lysosomes pre-formed in the cytoplasm containing lytic proteins (perforin and granzyme). After the target cells are recognized, the lytic proteins are secreted by exocytosis. Thus, once the target cells are identified, the decrease in intracellular granzyme and/or perforin levels can be measured by FACS.

Cell killing assays can be used to monitor the effect of γ δ T cells expressing heterologous targeting constructs. Kinetic target cell lysis assays can be used to track the percent killing over time at various effector to target ratios. Endpoint target cell lysis assays (e.g., luciferase assays) can be used to track the percent killing at various effector to target ratios at a particular endpoint time. Immune synapse formation (e.g., as observed by live cell imaging) may be used to measure binding kinetics, target recognition (e.g., Ca flux in effector cells), lethal impact (e.g., as measured by propidium iodide blush in target cells), or target cell rounding.

In some embodiments, binding of the antigen binding domain to a target antigen expressed on a healthy cell does not substantially trigger cytolysis by the engineered γ δ T cell. In some embodiments, binding of the antigen binding domain to a target antigen expressed on a tumor cell or infected cell significantly triggers cytolysis by the engineered γ δ T cell.

In one aspect, the invention provides a cell (e.g., a γ δ T cell, an NK-like T cell, an innate lymphoid cell, or a MAIT cell) engineered to express a heterologous targeting construct, wherein the engineered cell exhibits anti-tumor properties. In one aspect, a cell is transfected (e.g., by nuclear transfection, electroporation, etc.) with a heterologous targeting construct and the heterologous targeting construct is expressed on the surface of the cell. The cells (e.g., γ δ T cells, NK-like T cells, innate lymphoid cells, or MAIT cells) are transduced with a viral vector encoding a heterologous targeting construct. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell can stably express the heterologous targeting construct. In another embodiment, the cells (e.g., γ δ T cells, NK-like T cells, innate lymphoid cells, or MAIT cells) are transfected (e.g., by nuclear transfection, electroporation, etc.) with a nucleic acid (e.g., mRNA, cDNA, DNA) encoding a heterologous targeting construct. In some embodiments, the cell can transiently express the heterologous targeting construct.

In one aspect, the invention provides a cell population (e.g., an isolated cell population) (e.g., at least 10, 10) of engineered γ δ T cells²、10³、10⁴、10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²Or 10¹³Individual cells), wherein at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or substantially all) of the population of cells are engineered γ δ T cells expressing a heterologous targeting construct.

Alternatively, the invention provides a cell population (e.g., an isolated cell population) (e.g., at least 10, 10) of engineered NK cells or NK-like T cells²、10³、10⁴、10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²Or 10¹³Individual NK cells or NK-like T cells), wherein at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or substantially all) of the population of cells is engineered to express a heterologous targeting construct.

Alternatively, the invention provides a cell population (e.g., an isolated cell population) (e.g., at least 10, 10) of engineered innate lymphoid cells²、10³、10⁴、10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²Or 10¹³Individual NK cells or NK-like T cells), wherein at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or substantially all) of the population of cells is engineered to express a heterologous targeting construct. Alternatively, the invention provides a population (e.g., an isolated population) of cells (e.g., at least 10, 10) of engineered MAIT cells²、10³、10⁴、10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²Or 10¹³Individual NK cells or NK-like T cells), wherein at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or substantially all) of the population of cells is engineered to express a heterologous targeting construct.

Heterologous targeting constructs

Various types of γ δ T cells, NK-like T cells, innate lymphoid cells, or MAIT cells can be modified to include heterologous targeting constructs to produce engineered γ δ T cells, NK-like T cells, innate lymphoid cells, or MAIT cells. The heterologous targeting construct comprises an extracellular antigen binding domain and a transmembrane domain. For example, the heterologous targeting construct can include an extracellular antigen-binding domain operably linked to a transmembrane domain by 1-1,000 amino acid residues (e.g., by 1-10, 10-20, 20-30, 30-40, 40-50, 50-100, 250, 500, or 500 amino acid residues). In some embodiments, the antigen binding domain is linked to the transmembrane domain by a stalk domain. In some embodiments, the extracellular antigen-binding domain, stalk domain, and transmembrane domain are operably linked in an N-to-C terminal orientation (e.g., N-antigen-binding domain-stalk domain-transmembrane domain-C). In some embodiments, the extracellular antigen-binding domain, stalk domain, and transmembrane domain are operably linked in an N-to-C terminal orientation.

In general, the heterologous targeting constructs disclosed herein do not have an antigen binding domain of an antibody specific for an intracellular signaling domain. Activation of innate lymphoid cells (e.g., γ δ T cells) can be mediated by heterologous targeting constructs that do not have functional intracellular domains, as compared to engineered α β T cells (e.g., CAR T cells) that are not functional without functional intracellular domains (Ghosh et al, nat. med.,23:242-251, 2017; whiting et al, mol. ther.,25:259-273, 2017; and Wilkie et al, j. biol. chem.,285:25538-25544, 2010). One skilled in the art will appreciate that the polypeptide may contain non-functional intracellular amino acid residues, for example as an extension of a transmembrane domain, which does not directly activate the engineered T cell. For example, in some aspects, the transmembrane domain may include additional residues for structural, stability, and/or expression purposes, or may have a non-functional intracellular domain. In some embodiments, no more than 50% (e.g., no more than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of the residues of the C-terminal transmembrane domain reside within the cell.

Antigen binding domains

The antigen binding domain may be an antibody or antibody fragment engineered to specifically bind a target. The antigen binding domain may take the form of various structures, such as a B cell receptor, an antibody scaffold or mimetic (e.g., an affibody, affilin, anticalin, aptamer, atrimer, DARPin, FN3 scaffold, fynomer, Kunitz domain, fibronectin, obdy, bicyclic peptide, cysteine-knot, etc.), single chain variable fragment (scFv), monoclonal antibody (mAb), antigen binding fragment (Fab), or T Cell Receptor (TCR) (fig. 2A). The antigen binding domain can bind to a target, such as a tumor associated antigen (TAA; e.g., a TAA expressed on a solid tumor). TAA can be, for example, a protein or peptide antigen expressed on the surface of tumor cells. Alternatively, TAAs include carbohydrates or gangliosides expressed on the surface of tumor cells. In some embodiments, the TAA is an immunosuppressive antigen. In some embodiments, the antigen binding domain is a ligand-specific receptor, as shown in figure 2B. In some embodiments, the antigen binding domain is a receptor specific ligand, as shown in figure 2C.

In one aspect, the target binding moiety of the heterologous targeting construct is an scFv. In one aspect, such antibody fragments are functional in that they retain equivalent binding affinity, e.g., they bind the same antigen with comparable efficacy as an IgG antibody that produces the antigen. Alternatively, they can be engineered to enhance or attenuate binding affinity as needed, e.g., to achieve optimal binding kinetics (e.g., affinity) based on the expression density of the target antigen. In one aspect, all of such antibody fragments are functional in that they provide a biological response that may include, but is not limited to, activating an immune response, inhibiting signal transduction from its target antigen, inhibition of kinase activity, and the like, as will be understood by those skilled in the art.

In one aspect, the antigen binding domain of the heterologous targeting construct is a murine scFv antibody fragment. In another aspect, the antigen binding domain of the heterologous targeting construct is a scFv antibody fragment that is humanized compared to the scFv murine sequence from which it was derived. Humanization of mouse scFv may be desirable in a clinical setting, where mouse specific residues may induce a human anti-mouse antigen (HAMA) response in patients receiving engineered T cell therapy.

In one aspect, the antigen binding domain portion of the heterologous targeting construct is encoded by a transgene whose sequence has been codon optimized for expression in mammalian cells. In one aspect, the entire heterologous targeting construct of the invention is encoded by a transgene having a sequence that is codon optimized for expression in mammalian cells. Codon optimization refers to the finding that the frequency of occurrence of synonymous codons (i.e., codons encoding the same amino acid) in the coding DNA is biased among different species. This codon degeneracy allows the same polypeptide to be encoded by multiple nucleotide sequences. Various codon optimization methods are known in the art and include, for example, the methods disclosed in U.S. Pat. nos. 5,786,464 and 6,114,148, both of which are incorporated herein by reference in their entirety.

In one aspect, the heterologous targeting construct of the invention comprises a target-specific binding member antigen binding domain. The choice of moiety depends on the type and amount of ligand that defines the surface of the target cell. For example, the antigen binding domain can be selected to recognize ligands that serve as cell surface markers on target cells associated with a particular disease state. Examples of cell surface markers that can serve as ligands for the antigen binding domain in the heterologous targeting construct of the invention include those associated with cancer as well as viral, bacterial, parasitic infections.

In one aspect, heterologous targeting construct-mediated T cell responses can be directed to an antigen of interest by engineering an antigen binding domain that specifically binds the desired antigen into the heterologous targeting construct. The antigen binding domain may be any domain that binds to an antigen, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and functional fragments thereof, including but not limited to single domain antibodies, such as the heavy chain variable domain (VH), light chain variable domain (VL), and variable domain of a camelid nanobody, as well as alternative scaffolds known in the art to function as antigen binding domains, such as recombinant fibronectin domains and the like. In certain instances, it is beneficial for the antigen binding domain to be derived from the same species in which the heterologous targeting construct will ultimately be used. For example, for use in humans, it may be beneficial for the antigen binding domain of the heterologous targeting construct to include human or humanized residues of the antigen binding domain of an antibody or antibody fragment.

In some embodiments of any aspect of the invention, the heterologous targeting construct comprises an antigen binding domain that is a humanized antibody or antigen binding fragment thereof. Humanized antibodies can be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (see, e.g., european patent No. EP 239,400; PCT publication No. WO 91/09967; and U.S. patent nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein by reference in its entirety), veneering or resurfacing (see, e.g., european patent nos. EP 592,106 and EP 519,596, each of which is incorporated herein by reference in its entirety), chain shuffling (see, e.g., U.S. patent No. 5,565,332, which is incorporated herein by reference in its entirety), and techniques disclosed, e.g., in U.S. patent application publication No. 2005/0042664, U.S. patent application publication No. 2005/0048617, U.S. patent nos. 6,407,213 and 5,766,886, and international publication No. WO 93/17105, each of which is incorporated herein by reference in its entirety. Typically, framework residues in the framework regions will be substituted with corresponding residues from a CDR donor antibody to alter (e.g., improve) antigen binding. These framework substitutions are identified by methods well known in the art, for example by modeling the interaction of the CDRs with framework residues to identify framework residues important for antigen binding and performing sequence comparisons to identify aberrant framework residues at specific positions. See, e.g., U.S. Pat. No. 5,585,089, which is incorporated by reference herein in its entirety.

One or more amino acid residues from a non-human source are present in a humanized antibody or antibody fragment. These non-human amino acid residues are often referred to as "import" residues, and are typically taken from an "import" variable domain. As provided herein, a humanized antibody or antibody fragment comprises one or more CDRs from a non-human immunoglobulin molecule and framework regions, wherein the amino acid residues comprising the framework are derived entirely or predominantly from a human germline. Various techniques for humanizing antibodies or antibody fragments are well known in the art and can be substantially as described in Jones et al, Nature,1986,321: 522-525; riechmann et al, Nature,1988,332: 323-; and Verhoeyen et al, Science,1988,239:1534-1536, each of which is incorporated herein by reference in its entirety, by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody, i.e., CDR grafting. In such humanized antibodies and antigen binding fragments thereof, substantially less than an entire human variable domain has been substituted with the corresponding sequence from a non-human species. Humanized antibodies are typically human antibodies in which some CDR residues and possibly some Framework (FR) residues are substituted by residues from analogous sites in rodent antibodies.

In some aspects, the heterologous targeting constructs of the invention, including antibody fragments, are partially humanized while retaining high affinity for the target antigen and other advantageous biological properties. According to one aspect of the invention, humanized antibodies and antibody fragments are prepared by analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. Computer software is available that illustrates and displays the three-dimensional conformational structure of a selected candidate immunoglobulin sequence. Examination of these displays allows analysis of the available role of the residues in the function of the candidate immunoglobulin sequence, e.g., analysis of residues that affect the ability of the candidate immunoglobulin to bind to the target antigen. In this manner, FR residues can be selected and combined from the recipient and import sequences to achieve desired antibody or antibody fragment properties, such as increased affinity for the target antigen. In general, CDR residues are directly and most substantially involved in affecting antigen binding.

In certain instances, scFv can be prepared according to methods known in the art (see, e.g., Bird et al, Science,1988,242:423- & 426 and Huston et al, Proc. Natl. Acad. Sci. USA,1988,85:5879- & 5883; each of which is incorporated herein by reference in its entirety). ScFv molecules can be produced by linking VH and VL regions together using a flexible polypeptide linker. The scFv molecule includes a linker (e.g., a Ser-Gly linker) of optimal length and/or amino acid composition. Linker length greatly affects how the scFv variable regions fold and interact. In fact, if shorter polypeptide linkers (e.g., 5-10 amino acids) are used, intra-strand folding is prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site. For examples of linker orientation and size see, e.g., Hollinger et al Proc Natl Acad. Sci. USA,1993,90: 6444-; U.S. publication nos. 2005/0100543, 2005/0175606, 2007/0014794 and international patent publication nos. WO 2006/020258 and WO 2007/024715, which are incorporated herein by reference.

The scFv may comprise a linker of at least 1,2, 3, 4,5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acid residues between its VL and VH regions. In some embodiments, the linker sequence comprises the amino acids glycine and serine. In another embodiment, the linker sequence comprises multiple sets of glycine and serine repeats, such as (GGGGS) n, where n is a positive integer equal to or greater than 1 (e.g., 1,2, 3, 4,5, or greater). Changes in linker length can retain or enhance activity, resulting in superior efficacy in binding and activity.

The cytolytic kinetics of the engineered γ δ T cells of the invention against target cells depends in part on the binding affinity of the antigen binding domain. Any of the antigen binding domains provided herein can be modified as needed to enhance or reduce binding affinity to a particular target according to known methods. In some embodiments, the antigen binding domain to its antigen binding affinity or dissociation constant (K)_D) Is 10^-4M to 10^-10M (measured, for example, by surface plasmon resonance, e.g., BIAcore, at standard physiological temperatures and pressures, e.g., 10^-4M to 10^-5M、10^-5M to 10^-6M、10^-6M to 10^-7M、10^-7M to 10^-8M、10^-8M to 10^-9M、10^-9M to 10^-10M, e.g. 10^-5M to 10^-9M、10^-5M to 10^-8M、10^-5M to 10^-7M、10^-5M to 10^-6M、10^-6M to 10^-10M、10^-6M to 10^-9M、10^-6M to 10^-8M、10^-6M to 10^-7M、10^-7M to 10^-10M、10^-7M to 10^-9M、10^-7M to 10^-8M、10^-8M to 10^-10M、10^-8M to 10^-9M, or 10^-9M to 10^-10M)。

In addition to the binding affinity of the antigen binding domain of engineered γ δ T cells to target cells, affinity interactions also play a role in efficient binding and subsequent lysis of target cells. The affinity is determined by (a) the binding affinity of the antigen-binding domain and (b) the number of interactions between the antigen-binding domain and the antigen along a given interface between the T cell and the target. In some embodiments, the engineered γ δ T-cell contains 10 on its surface²To 10⁶An antigen binding domain (e.g., 10)²To 10³、10³To 10⁴、10⁴To 10⁵、10⁵To 10⁶、10²To 10⁴、10²To 10⁵、10³To 10⁴、10³To 10⁵、10³To 10⁶、10⁴To 10⁵、10⁴To 10⁶Or 10⁵To 10⁶Individual antigen binding domains).

Stem domain

The heterologous targeting construct of the invention can include a stalk domain located between the transmembrane domain and the extracellular antigen-binding domain. In some embodiments, the stem domain comprises a stem from CD8, an IgG hinge-heavy Constant (CH) domain (e.g., IgG1 hinge-CH)₂Domain, IgG1 hinge-CH₃Domain or IgG1 hinge-CH₂-CH₃Domain), (G)₄S)₃Hinge, IgG1 hinge, CD7 stem, IgD hinge-CH₂Domain, IgD hinge-CH₃Domain, IgD hinge-CH₂-CH₃Domain, IgG4 hinge-CH₂Domain, IgG4 hinge-CH₃Domain, IgG4 hinge-CH₂-CH₃One or more of a domain or a selected domain in the fcsri stem. The stem may provide flexibility between the extracellular and transmembrane domains and facilitate target recognition. One skilled in the art will appreciate that the stem domain may include adjacent filamentsOne or more additional amino acids of the extracellular or transmembrane region, for example one or more amino acids associated with the extracellular or transmembrane region of the protein from which the stem is derived (e.g. 1,2, 3, 4,5,6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids of the extracellular or transmembrane region). The stem may optionally include one or more linkers, such as (GGGGS)_nOr GGGGSGGGGS (SEQ ID NO: 1).

Transmembrane domain

In various embodiments of any of the documents of the present invention, the heterologous targeting construct may be designed to include a transmembrane domain attached to an extracellular domain. The transmembrane domain may comprise one or more additional amino acids adjacent to the transmembrane region, for example one or more amino acids associated with the extracellular region from which the transmembrane protein is derived (e.g. 1,2, 3, 4,5,6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region from which the transmembrane protein is derived (e.g. 1,2, 3, 4,5,6, 7, 8, 9, 10 to up to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is a transmembrane domain associated with one of the other domains of the heterologous targeting construct. In certain instances, transmembrane domains may be selected or modified by amino acid substitutions to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interaction with other members of the receptor complex. Thus, in some cases, the transmembrane domain does not substantially propagate signal 1 activation and/or signal 2 activation (co-stimulation).

Alternatively, the transmembrane domain may be selected for its ability to bind to other proteins, induce clustering of other proteins, activate, phosphorylate, dephosphorylate, or otherwise interact with other proteins (e.g., endogenous proteins, such as endogenous membrane-associated proteins, e.g., transmembrane proteins). For example, in some embodiments, the transmembrane domain may be associated with a costimulatory protein, thereby directly activating a cell (e.g., a γ δ T cell) by transmitting a signal 2 costimulatory signal. In particular embodiments, the transmembrane domain is derived from the transmembrane portion of NKG2D, which can be associated with endogenously expressed DAP10 to propagate signal 2 activation (co-stimulation) of the host cell. In this case, the heterologous targeting construct does not have a functional intracellular domain capable of activating the cell. For example, the heterologous targeting construct may not have an intracellular domain, or it may contain an inert intracellular domain that does not transmit signal 1 or signal 2 activation. For example, a transmembrane domain that can transmit signal 2 by recruitment or association with an endogenous costimulatory molecule can be a terminal transmembrane domain (e.g., no additional domain attached to one of its termini).

In one aspect, the transmembrane domain is capable of heterodimerizing or homodimerizing with another heterologous targeting construct on the surface of a γ δ T cell. In various aspects, the amino acid sequence of the transmembrane domain may be modified or substituted in order to minimize interaction with the binding domain of a native binding partner present in the same engineered T cell.

The transmembrane domain may be derived from natural or recombinant sources. Where the source is natural, the domain may be derived from any membrane bound or transmembrane protein. Transmembrane domains particularly useful in the present invention may include at least transmembrane regions of the alpha, beta or zeta electric chain of, for example, T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD18, CD22, CD29, CD33, CD37, CD64, CD80, CD86, CD94, CD134, CD137, CD154, CD7, CD3 zeta, CD71, Fc gamma receptor (Fc γ R) or NKG 2D. In some embodiments, the transmembrane domain may include, for example, a CD8 transmembrane domain, a CD4 transmembrane domain, a CD3 zeta transmembrane domain, or a CD28 transmembrane domain. In some embodiments, the transmembrane domain is a terminal transmembrane domain (e.g., no additional domain attached to one of its termini).

In some cases, the transmembrane domain may be attached to an extracellular region of the heterologous targeting construct, such as an antigen-binding domain of the heterologous targeting construct, via a hinge (e.g., a hinge from a human protein). For example, in one embodiment, the hinge may be a human Ig (immunoglobulin) hinge, such as an IgG1 hinge, an IgG4 hinge, an IgD hinge, or a CD8 a hinge.

In one embodiment, the transmembrane domain is recombinant, in which case it will comprise predominantly hydrophobic residues, such as leucine and valine. In one aspect, triplets of phenylalanine, tryptophan, and valine can be found at each end of the recombinant transmembrane domain.

Alternatively, a shorter polypeptide linker (e.g., 2 to 10 amino acids in length) may form a link between the transmembrane domain and the cytoplasmic region of the heterologous targeting construct. An example of a suitable linker is a glycine-serine doublet. For example, in one aspect, the linker comprises an amino acid sequence (GGGGS)_nOr GGGGSGGGGS (SEQ ID NO: 1).

Method for harvesting and expanding gamma delta T cells

The engineered γ δ T cells of the present invention may be derived from any suitable autologous or allogeneic γ δ T cell or population thereof. In some embodiments, suitable γ δ T cells for use as a source of the engineered γ δ T cells herein include V δ 1 cells, V δ 2 cells, V δ 3 cells, V δ 5 cells, and V δ 8 cells. In some embodiments, the population of engineered γ δ T cells is derived from a population of V δ 1 cells, V δ 3 cells, V δ 5 cells, or V δ 8 cells.

For example, provided herein are methods of isolating and expanding V δ 1 cells from non-hematopoietic tissues (e.g., skin or intestine). For example, V δ 1 cells may be isolated from human skin biopsies as described in u.s.2018/0312808, which is incorporated herein by reference in its entirety and in particular methods of isolating V δ 1 cells from tissue.

In other embodiments, suitable γ δ T cells may be derived from blood (e.g., peripheral blood). Methods of isolating and expanding V δ 1 cells from blood include, for example, those disclosed in U.S. patent No. 9,499,788 and international patent publication No. WO 2016/198480, each of which is incorporated herein by reference in its entirety. In some embodiments, suitable γ δ T cells can be derived from tumor tissue (e.g., tumor infiltrating γ δ T cells). Alternatively, suitable γ δ T cells that can be engineered to express heterologous targeting constructs can be obtained from non-hematopoietic tissues according to the methods described below.

Isolation and expansion of gamma delta T cells from blood

In some embodiments, the engineered γ δ T-cells of the invention may be derived from the blood (e.g., peripheral blood) of a subject. For example, the engineered γ δ T cells may be derived from blood-derived V δ 2 cells or blood-derived V δ 1 cells.

In some embodiments, Peripheral Blood Mononuclear Cells (PBMCs) may be obtained from a subject according to any suitable method known in the art. PBMCs can be cultured for one to two weeks in the presence of an amino bisphosphonate (e.g., zoledronic acid), a synthetic phosphate antigen (e.g., bromohydrin pyrophosphate; BrHPP), 2M3B1PP, or 2-methyl-3-butene-1-pyrophosphate in the presence of IL-2 to produce an enriched V.delta.2 cell population. Alternatively, immobilized anti-TCR γ δ (e.g., pan-TCR γ δ) can induce preferential expansion of V δ 2 cells in the PBMC population in the presence of IL-2, e.g., for about 14 days. In some embodiments, preferential expansion of V δ 2 cells from PBMCs can be achieved upon culturing an immobilized anti-CD 3 antibody (e.g., OKT3) in the presence of IL-2 and IL-4. In some embodiments, the foregoing cultures are maintained for about 7 days prior to subculture in soluble anti-CD 3, IL-2 and IL-4. Alternatively, artificial antigen presenting cells may be used to promote preferential expansion of γ δ T cells (e.g., V δ 2 cells). For example, PBMC-derived γ δ T cells cultured in the presence of irradiated aapcs, IL-2 and/or IL-21 can be expanded to produce a γ δ T cell population that includes a high proportion of V δ 2 cells, a moderate proportion of V δ 1 cells and some double negative cells. In some embodiments of the foregoing methods, PBMCs may be pre-enriched or post-enriched (e.g., by using positive selection for TCR γ δ -specific agents or negative selection for TCR α β -specific agents). Such methods and other suitable methods for expanding γ δ T cells (e.g., V δ 2 cells) are described in detail by Deniger et al, Frontiers in Immunology 2014,5,636:1-10, which is incorporated herein by reference in its entirety.

In some embodiments, the V δ 1T cell may be engineered to express a heterologous targeting construct. Any suitable method of obtaining a V δ 1T cell population may be used. For example, Almeida et al (Clinical Cancer Research 2016,22, 23; 5795-. For example, in some embodiments, PBMCs are pre-enriched using magnetic bead sorting, which can produce greater than 90% γ δ T cells. These cells can be cultured in the presence of one or more factors (e.g., TCR agonists, co-receptor agonists, and/or cytokines, e.g., IL-4, IL-15, and/or IFN- γ) in a gas permeable bioreactor bag for up to 21 days or more. Variations of this method, as well as other methods of obtaining V δ 1T cells, are suitable as part of the present invention. For example, blood-derived V δ 1T cells may alternatively be obtained using methods such as described in U.S. patent No. 9,499,788 and international patent publication No. WO 2016/198480 (each of which is incorporated herein by reference in its entirety) and WO2017197347 and WO2016081518 (U.S. publication No. 20160175338) (each of which is incorporated herein by reference in its entirety).

Isolation and expansion of non-hematopoietic tissue resident γ δ T cells from non-hematopoietic tissue

The non-hematopoietic tissue-resident γ δ T cells obtained as described below may be suitable carriers for the heterologous targeting constructs herein, as they may exhibit good tumor penetration and retention capabilities. For example, more detailed methods of isolating and amplifying non-hematopoietic tissue resident γ δ T cells can be found in GB application No. 1707048.3(WO2018/202808) and international patent publication No. WO2017/072367 (U.S. publication No. 20180312808), each of which is incorporated herein by reference in its entirety.

Non-hematopoietic tissue resident γ δ T cells (e.g., skin-derived γ δ T cells and/or non-V δ 2T cells, e.g., V δ 1T cells and/or DN T cells) may be isolated from any human or non-human animal non-hematopoietic tissue that may be removed from a patient to obtain cells suitable for engineering according to the methods of the invention. In some embodiments, the non-hematopoietic tissue from which the γ δ T cells are derived and expanded is skin (e.g., human skin), which may be obtained by methods known in the art. In some embodiments, the skin is obtained by punch biopsy. Alternatively, the methods of isolating and expanding γ δ T cells provided herein can be applied to the gastrointestinal tract (e.g., colon), breast, lung, prostate, liver, spleen, and pancreas. γ δ T cells may also reside in human cancer tissues, such as breast or prostate tumors. In some embodiments, the γ δ T cells may be from human cancer tissue (e.g., solid tumor tissue). In other embodiments, the γ δ T cells may be from non-hematopoietic tissues other than human cancer tissues (e.g., tissues that do not have a substantial number of tumor cells). For example, γ δ T cells may be from a region of skin (e.g., healthy skin) that is separated from nearby or adjacent cancerous tissue.

The γ δ T cells that predominate in blood are predominantly V δ 2T cells, while the γ δ T cells that predominate in non-hematopoietic tissues are predominantly V δ 1T cells, whereby the V δ 1T cells comprise about 70-80% of the non-hematopoietic tissue resident γ δ T cell population. However, some V δ 2T cells are also found in non-hematopoietic tissues (e.g., the gut), where they may comprise about 10-20% of γ δ T cells. Some γ δ T cells residing in non-hematopoietic tissues express neither V δ 1 nor V δ 2 TCRs, and we named them Double Negative (DN) γ δ T cells. These DN γ δ T cells may be predominantly V δ 3-expressing cells, with a small number of V δ 5-expressing cells. Thus, the γ δ T cells that normally reside in non-hematopoietic tissues and are expanded by the methods of the invention are preferably non-V δ 2T cells, e.g., V δ 1T cells, and include a small number of DN γ δ T cells.

In some embodiments, a key step is the deliberate separation of non-hematopoietic tissue resident T cells (e.g., within a mixed lymphocyte population, which may, for example, include α β cells, Natural Killer (NK) cells, B cells, and γ δ 2 and non- γ δ 2T cells) from the non-hematopoietic cells (e.g., stromal cells, particularly fibroblasts) of the tissue from which the T cells were obtained, e.g., after days or weeks of culture. This allows preferential and rapid expansion of non-hematopoietic tissue-derived V δ 1T cells and DN γ δ T cells over the next days and weeks.

In general, non-hematopoietic tissue resident γ δ T cells are capable of spontaneous expansion upon removal of physical contact with stromal cells (e.g., skin fibroblasts). Thus, the scaffold-based culture methods described above can be used to induce such separations, resulting in the suppression of γ δ T cells to trigger expansion. Thus, in some embodiments, there is no substantial TCR pathway activity during the amplification step (e.g., no exogenous TCR pathway activator is included in the culture). In addition, the invention provides methods for expanding non-hematopoietic tissue resident γ δ T cells, wherein the methods do not involve contact with feeder cells, tumor cells, and/or antigen presenting cells.

The expansion protocol includes culturing non-hematopoietic tissue resident γ δ T cells in the presence of an effective mixture of biological factors to support efficient γ δ T cell expansion. In one embodiment, a method of expanding γ δ T cells comprises providing a population of γ δ T cells obtained from a non-hematopoietic tissue (e.g., an isolated population of non-hematopoietic tissue-derived γ δ T cells, e.g., isolated according to the methods described herein) and culturing the γ δ T cells in the presence of IL-2, IL-4, IL-15, and/or IL-21. These cytokines or analogs thereof can be cultured with the cells in an amount effective to produce an expanded population of γ δ T cells for a period of time (e.g., at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days, or longer, e.g., 5 days to 40 days, 7 days to 35 days, 14 days to 28 days, or about 21 days).

Expanded gamma delta T cell populations

The expanded γ δ T-cell population is greater in number than the γ δ T-cell population isolated prior to the expansion step (e.g., at least 2-fold, at least 3-fold, at least 4-fold in number, at least 5-fold in number, at least 6-fold in number, at least 7-fold in number, at least 8-fold in number, at least 9-fold in number, at least 10-fold in number, at least 15-fold in number, at least 20-fold in number, at least 25-fold in number, at least 30-fold in number, at least 35-fold in number, at least 40-fold in number, at least 50-fold in number, at least 60-fold in number, at least 70-fold in number, at least 80-fold in number, at least 90-fold in number, at least 100-fold in number, at least 200-fold in number, at least 300-fold in number, relative to the γ δ T-cell population isolated prior to the expansion step, At least 400 times in number, at least 500 times in number, at least 600 times in number, at least 700 times in number, at least 800 times in number, at least 900 times in number, at least 1,000 times in number, at least 5,000 times in number, at least 10,000 times in number, or more). Thus, large populations of γ δ T cells (e.g., skin-derived γ δ T cells and/or non-V δ 2T cells, e.g., V δ 1T cells and/or DN T cells) can be expanded at a higher rate. In some embodiments, the amplification steps described herein amplify γ δ T cells at a lower population doubling time, given by the following equation:

in view of the information provided herein, one of skill in the art will recognize that the present invention provides methods of expanding γ δ T cells (e.g., expanded and/or selected for engineering to heterologously express γ δ T cells or γ T cells) in a population doubling time of less than 5 days (e.g., less than 4.5 days, less than 4.0 days, less than 3.9 days, less than 3.8 days, less than 3.7 days, less than 3.6 days, less than 3.5 days, less than 3.2 days, less than 3.1 days, less than 3.0 days, less than 2.9 days, less than 2.8 days, less than 2.7 days, less than 2.6 days, less than 2.5 days, less than 2.4 days, less than 2.3 days, less than 2.2 days, less than 2.1 days, less than 2.0 days, less than 46 hours, less than 42 hours, less than 38 hours, less than 35 hours, less than 32 hours).

In some embodiments, γ δ T cells isolated and expanded by the methods provided herein (e.g., engineered γ δ T cells or γ δ T cells expanded and/or selected for engineering to express heterologous targeting constructs) can have a phenotype well suited for anti-tumor efficacy. In some embodiments, the expanded γ δ T-cell population has a higher average expression of CD27 than the reference population (e.g., the isolated γ δ T-cell population prior to the expanding step). In some embodiments, the expanded γ δ T-cell population has an average expression of CD27 that is at least 2-fold (e.g., at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 20,000-fold, or higher) that.

A unique portion of the expanded γ δ T cell population (e.g., engineered γ δ T cells or γ δ T cells expanded and/or selected for engineering to express heterologous targeting constructs) can up-regulate CD27, while another portion is CD27^{Is low in}Or CD27^-. In this case, CD27 in the population was expanded relative to the isolated γ δ T cell population⁺The frequency of the cells may be greater. For example, CD27 in the expanded γ δ T cell population relative to the isolated γ δ T cell population prior to expansion⁺The frequency of cells can be at least 5% higher (e.g., CD27 relative to the isolated γ δ T cell population prior to expansion)⁺The frequency of the cells is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%) higher. In some embodiments, CD27 in the population is expanded relative to an isolated γ δ T cell population⁺The number of cells may increase. For example, CD27 in expanded γ δ T cell populations⁺The number of cells is at least 2-fold greater in the isolated γ δ T cell population prior to expansion (e.g., CD27 relative to the isolated γ δ T cell population prior to expansion⁺The frequency of the cells is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%) higher.

In some embodiments, the expansion methods provided herein produce an expanded γ δ T cell population (e.g., engineered γ δ T cells or γ δ T cells expanded and/or selected for engineering to express the heterologous targeting construct) that has low expression of TIGIT relative to a reference population (e.g., an isolated γ δ T cell population prior to the expansion step). In some embodiments, the expanded γ δ T cell population has a lower TIGIT mean expression than the reference population (e.g., the isolated γ δ T cell population prior to the expansion step). In some embodiments, TIGIT average expression of the expanded γ δ T cell population is at least 10% lower than the isolated γ δ T cells (e.g., at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, or as much as 100% lower than the isolated γ δ T cell population).

A unique portion of the expanded γ δ T cell population (e.g., engineered γ δ T cells or γ δ T cells expanded and/or selected for engineering to express the heterologous targeting construct) can express TIGIT, e.g., high levels of TIGIT, while another portion is TIGIT^{Is low in}Or TIGIT^-. In this case, TIGIT in the population is expanded relative to the isolated γ δ T cell population⁺The cell frequency can be lower. For example, TIGIT in an expanded population of γ δ T cells relative to isolated γ δ T cells prior to expansion⁺The frequency of cells may be at least 5% lower (e.g., TIGIT relative to the isolated γ δ T cell population prior to expansion)⁺The frequency of the population of cells is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%) lower. In some embodiments, TIGIT in the expanded population is compared to the isolated γ δ T cell population prior to expansion⁺The number of cells may be smaller. For example, relative to TIGIT in the isolated γ δ T cell population prior to expansion⁺Number of cells, TIGIT in expanded γ δ T cell population⁺The cells may be at least 10% fewer (e.g., relative to TIGIT in the isolated γ δ T cell population prior to expansion)⁺Number of cells, TIGIT⁺At least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, or up to 100%).

In some embodiments, the expanded γ δ T cell population (e.g., engineered γ δ T cells or γ δ T cells expanded and/or selected for engineering to express heterologous targeting constructs) has a high number or frequency of CD27⁺Cell and low frequency TIGIT⁺A cell. In some embodiments, the expanded γ δ T cell population has a high frequency of CD27 relative to a reference population (e.g., relative to an isolated γ δ T cell population prior to expansion)⁺TIGIT^-A cell. For example, the expanded γ δ T cell population has CD27 relative to the isolated γ δ T cell population prior to expansion⁺TIGIT^-The frequency of cells is at least 5% greater (e.g., CD27 relative to the isolated γ δ T cell population prior to expansion⁺TIGIT^-The frequency of cells is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%) greater. In some embodiments, CD27 in the population is expanded relative to an isolated γ δ T cell population⁺TIGIT^-The number of cells can be increased. For example, CD27 in expanded γ δ T cell population⁺TIGIT^-The number of cells can be at least 2-fold greater in the isolated γ δ T cell population prior to expansion (e.g., CD27 relative to the isolated γ δ T cell population prior to expansion⁺TIGIT^-The frequency of cells is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%) greater.

In certain instances, CD27 in an expanded γ δ T cell population (e.g., engineered γ δ T cells or γ δ T cells expanded and/or selected for engineering to express heterologous targeting constructs) relative to a reference population⁺TIGIT mean expression on γ δ T cell populations was low. In some embodiments, the amplified CD27⁺The γ δ T cell population has a higher than reference population (e.g. isolated CD27 prior to the expansion step⁺γ δ T cell population) low TIGIT mean expression. In some embodimentsIn the formula, amplified CD27⁺TIGIT mean expression ratio of gamma delta T cell population isolated CD27⁺The γ δ T cell population is at least 10% lower (e.g., than isolated CD 27)⁺A γ δ T cell population is at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, or up to 100% lower).

Additionally or alternatively, TIGIT in an expanded γ δ T cell population (e.g., engineered γ δ T cells or γ δ T cells expanded and/or selected for engineering to express a heterologous targeting construct) relative to a reference population^-The median expression of CD27 was higher on γ δ T cell populations. For example, TIGIT relative to isolation prior to amplification^-Gamma delta T cell population, expanded TIGIT^-CD27 in gamma delta T cell population⁺The frequency of the cells may be at least 5% greater (e.g., relative to TIGIT isolated prior to amplification)^-Gamma delta T cell population, CD27⁺The frequency of cells is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%) greater. In some embodiments, with respect to a separate TIGIT^-Gamma delta T cell population, expansion of CD27 in the population⁺The number of cells can be increased. For example, amplified TIGIT^-CD27 possessed by gamma delta T cell population⁺TIGIT with cell number isolated prior to expansion^-At least 2-fold in a γ δ T cell population (e.g., relative to isolated TIGIT prior to expansion)^-Gamma delta T cell population, CD27⁺The frequency of cells is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% greater)

The increase or decrease in expression of other markers may additionally or alternatively be used to characterize one or more expanded γ δ T cell populations (e.g., engineered γ δ T cells or γ δ T cells expanded and/or selected for engineering to express heterologous targeting constructs), including CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, CD2, NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD 64. In some cases, the expanded γ δ T cell population has a greater, equal, or lower average expression of one or more markers selected from CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 relative to the isolated γ δ T cell population (e.g., prior to expansion). Additionally or alternatively, the expanded γ δ T-cell population may have a greater, equal, or lower frequency of cells expressing one or more markers selected from CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 relative to the isolated γ δ T-cell population. In some embodiments, the expanded γ δ T-cell population has a greater, equal, or lower average expression of one or more markers selected from NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 relative to the isolated γ δ T-cell population. The expanded population can similarly have a greater, equal, or lower frequency of cells expressing one or more markers selected from NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 relative to an isolated reference γ δ T cell population.

There are many basal media suitable for proliferation of γ δ T cells, particularly complete media such as AIM-V, Iscoves medium and RPMI-1640(Life Technologies). The medium may be supplemented with other medium factors such as serum, serum proteins and selective agents, e.g. antibiotics. For example, in some embodiments, RPMI-1640 medium contains 2mM glutamine, 10% FBS, 10mM HEPES, pH 7.2, 1% penicillin-streptomycin, sodium pyruvate (1 mM; Life Technologies), non-essential amino acids (e.g., 100. mu.M Gly, Ala, Asn, Asp, Glu, Pro, and Ser; 1 XMEM non-essential amino acids, Life Technologies), and 10. mu.l/L β -mercaptoethanol. Conveniently, in a suitable medium, in the presence of 5% CO₂The cells were cultured at 37 ℃ in a humidified atmosphere.

The γ δ T cells may be cultured in any suitable system as described herein, including stirred tank fermentors, airlift fermentors, roller bottles, culture bags or dishes, and other bioreactors, such as hollow fiber bioreactors. The use of such systems is well known in the art. General methods and techniques for culturing lymphocytes are well known in the art.

The methods described herein may include more than one selection step, such as more than one depletion step. Enrichment of T cell populations by negative selection can be achieved, for example, using a combination of antibodies to surface markers unique to negatively selected cells. One approach is cell sorting and/or selection via negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells.

Pharmaceutical compositions and methods of treatment

Engineered lymphocytes (e.g., γ δ T cells, NK-like T cells, innate lymphoid cells, or MAIT cells) described herein (e.g., engineered cells with heterologous targeting constructs (e.g., γ δ T cells)) can be used as a medicament, e.g., as an adoptive T cell therapy. The use herein relates to the transfer of lymphocytes (e.g. γ δ T cells) obtained by the method of the invention into a patient. The therapy may be autologous, i.e., lymphocytes (e.g., γ δ T cells) may be transferred back into the same patient from which they were obtained, or the therapy may be allogeneic, i.e., lymphocytes from one person (e.g., γ δ T cells) may be transferred into a different patient. Where allogenic transfer is involved, the lymphocytes (e.g., γ δ T cells) may be substantially free of α β T cells. For example, α β T cells can be depleted from a population of lymphocytes (e.g., γ δ T cells) after, e.g., amplification, using any suitable method known in the art (e.g., by negative selection, e.g., using magnetic beads).

In some embodiments where the γ δ T cells are engineered to express a heterologous targeting construct, the γ δ T cells are V δ 1 cells, V δ 2 cells, V δ 3 cells, V δ 5 cells, or V δ 8 cells. A method of treatment comprising: providing a sample of endogenous γ δ T cells from a patient; culturing the γ δ T cells in the sample in the presence of a vector carrying a polynucleotide encoding a heterologous targeting construct to produce a population of engineered γ δ T cells expressing the heterologous targeting construct (e.g., an expanded population of engineered γ δ T cells expressing the heterologous targeting construct); and administering the γ δ T cell population to the recipient patient. In some embodiments, the polynucleotide encoding the heterologous targeting construct is delivered into endogenous γ δ T cells by electroporation or any other suitable transfection method known in the art or described herein.

The patient or subject to be treated may be a human cancer patient (e.g., a human cancer patient who is receiving a solid tumor) or a virally infected patient (e.g., a patient infected with CMV or HIV). In certain instances, the patient has a solid tumor and/or is receiving a solid tumor therapy.

Since γ δ T cells are non-MHC restricted, they cannot recognize the host into which they are transferred as foreign material, which means that they are less likely to cause graft versus host disease. This means that they can be used "off the shelf and transferred to any recipient, for example for allogeneic adoptive T cell therapy.

In some embodiments, the γ δ T cells of the invention express NKG2D and respond to NKG2D ligands (e.g., MICA) that are closely associated with malignancy. They also express a cytotoxic profile without any activation and can therefore effectively kill tumor cells. For example, engineered γ δ T cells as obtained herein may express one or more (preferably all) of IFN- γ, TNF- α, GM-CSF, CCL4, IL-13, granulysin, granzymes A and B, and perforin without any activation, but may not express IL-17A.

The pharmaceutical composition may comprise engineered lymphocytes (e.g., γ δ T cells) as described herein and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffering agents, such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids, such as glycine; an antioxidant; chelating agents such as EDTA or glutathione, etc.; adjuvants (e.g., aluminum hydroxide); and a preservative. Cryopreservation solutions useful in the pharmaceutical compositions of the invention include, for example, DMSO. The compositions may be formulated, for example, for intravenous administration.

In one embodiment, the pharmaceutical composition is substantially free of (absent detectable levels of) contaminants, such as endotoxins or mycoplasma.

In certain instances, a therapeutically effective amount of an engineered lymphocyte (e.g., a γ δ T cell, an NK-like T cell, an innate lymphoid cell, or a MAIT cell) obtained by any of the above methods can be administered to a subject in a therapeutically effective amount (e.g., for treating cancer, e.g., for treating a solid tumor). In some cases, the therapeutically effective amount of engineered lymphocytes (e.g., γ δ T cells (e.g., engineered γ δ T cells, blood-derived T cells, e.g., V δ 1T cells, V δ 2T cells, and/or DN T cells), NK cells, NK-like T cells, innate lymphoid cells, or MAIT cells) is less than 10x 10¹²Individual cell/dose (e.g., less than 9x 10)¹²Less than 8 × 10 cells/dose¹²Less than 7x 10 per cell/dose¹²Less than 6x10 per cell/dose¹²Less than 5x10 per cell/dose¹²Less than 4x 10 per cell/dose¹²Less than 3x 10 per cell/dose¹²Less than 2x 10 per cell/dose¹²Less than 1x 10 per cell/dose¹²Less than 9x 10 cells/dose¹¹Less than 8x10 cells/dose¹¹Less than 7x 10 per cell/dose¹¹Less than 6x10 per cell/dose¹¹Less than 5x10 per cell/dose¹¹Less than 4x 10 per cell/dose¹¹Less than 3x 10 per cell/dose¹¹Less than 2x 10 per cell/dose¹¹Less than 1x 10 per cell/dose¹¹Less than 9x 10 cells/dose¹⁰Less than 7.5x 10 cells/dose¹⁰Less than 5x10 per cell/dose¹⁰Less than 2.5x 10 cells/dose¹⁰Less than 1x 10 per cell/dose¹⁰Less than 7 cells/dose.5x 10⁹Less than 5x10 per cell/dose⁹Less than 2.5x 10 cells/dose⁹Less than 1x 10 per cell/dose⁹Less than 7.5x 10 cells/dose⁸Less than 5x10 per cell/dose⁸Less than 2,5x 10 per cell/dose⁸Less than 1x 10 per cell/dose⁸Less than 7.5x 10 cells/dose⁷Less than 5x10 per cell/dose⁷Less than 2,5x 10 per cell/dose⁷Less than 1x 10 per cell/dose⁷Less than 7.5x 10 cells/dose⁶Less than 5x10 per cell/dose⁶Less than 2,5x 10 per cell/dose⁶Less than 1x 10 per cell/dose⁶Less than 7.5x 10 cells/dose⁵Less than 5x10 per cell/dose⁵Less than 2,5x 10 per cell/dose⁵Per cell/dose or less than 1x 10⁵Individual cells/dose).

In some embodiments, the therapeutically effective amount of engineered lymphocytes (e.g., γ δ T cells (e.g., engineered skin-derived γ δ T cells, engineered blood-derived γ δ T cells, e.g., V δ 1T cells and/or DN T cells), NK cells, NK-like T cells, innate lymphoid cells, or MAIT cells) is less than 10x 10 throughout the course of treatment¹²One cell (e.g., less than 9x 10 throughout the treatment period)¹²Less than 8x10 per cell¹²Less than 7x 10 per cell¹²Less than 6x10 per cell¹²Less than 5x10 per cell¹²Less than 4x 10 per cell¹²Less than 3x 10 per cell¹²Less than 2x 10 per cell¹²Less than 1x 10 per cell¹²Less than 9x 10 per cell¹¹Less than 8x10 per cell¹¹Less than 7x 10 per cell¹¹Less than 6x10 per cell¹¹Single cell, less than 5x10¹¹Less than 4x 10 per cell¹¹Less than 3x 10 per cell¹¹Less than 2x 10 per cell¹¹Less than 1x 10 per cell¹¹Less than 9x 10 per cell¹⁰Less than 7.5x 10 cells per cell¹⁰Less than 5x10 per cell¹⁰Less than 2.5x 10 per cell¹⁰Less than 1 cell per cellx 10¹⁰Less than 7.5x 10 cells per cell⁹Less than 5x10 per cell⁹Less than 2.5x 10 per cell⁹Less than 1x 10 per cell⁹Less than 7.5x 10 cells per cell⁸Less than 5x10 per cell⁸Less than 2,5x 10 per cell⁸Less than 1x 10 per cell⁸Less than 7.5x 10 cells per cell⁷Less than 5x10 per cell⁷Less than 2,5x 10 per cell⁷Less than 1x 10 per cell⁷Less than 7.5x 10 cells per cell⁶Less than 5x10 per cell⁶Less than 2,5x 10 per cell⁶Less than 1x 10 per cell⁶Less than 7.5x 10 cells per cell⁵Less than 5x10 per cell⁵Less than 2,5x 10 per cell⁵Single cell or less than 1x 10⁵Individual cells).

In some embodiments, a dose of engineered lymphocytes (e.g., γ δ T cells, NK-like T cells, innate lymphoid cells, or MAIT cells) as described herein comprises about 1x 10⁶、1.1x 10⁶、2x 10⁶、3.6x 10⁶、5x 10⁶、1x 10⁷、1.8x 10⁷、2x 10⁷、5x 10⁷、1x 10⁸、2x 10⁸Or 5x10⁸Individual cells/kg. In some embodiments, the dose of engineered lymphocytes (e.g., γ δ T cells (e.g., skin-derived γ δ T cells, blood-derived γ δ T cells, e.g., V δ 1T cells and/or DN T cells), NK cells, NK-like T cells, innate lymphoid cells, or MAIT cells) comprises at least about 1x 10⁶、1.1x 10⁶、2x 10⁶、3.6x 10⁶、5x 10⁶、1x 10⁷、1.8x 10⁷、2x 10⁷、5x 10⁷、1x 10⁸、2x 10⁸Or 5x10⁸Individual cells/kg. In some embodiments, the dose of engineered lymphocytes (e.g., γ δ T cells (e.g., skin-derived γ δ T cells, blood-derived γ δ T cells, e.g., V δ 1T cells and/or DN T cells), NK cells, NK-like T cells, innate lymphoid cells, or MAIT cells) comprises up to about 1x 10⁶、1.1x 10⁶、2x 10⁶、3.6x 10⁶、5x 10⁶、1x 10⁷、1.8x 10⁷、2x 10⁷、5x 10⁷、1x 10⁸、2x 10⁸、5x 10⁸Individual cells/kg. In some embodiments, the dose of engineered lymphocytes (e.g., γ δ T cells (e.g., skin-derived γ δ T cells, blood-derived γ δ T cells, e.g., V δ 1T cells and/or DN T cells), NK cells, NK-like T cells, innate lymphoid cells, or MAIT cells) comprises about 1.1x 10⁶-1.8x 10⁷Individual cells/kg. In some embodiments, the dose of engineered lymphocytes (e.g., γ δ T cells (e.g., skin-derived γ δ T cells, blood-derived γ δ T cells, e.g., V δ 1T cells and/or DN T cells), NK cells, NK-like T cells, innate lymphoid cells, or MAIT cells) comprises about 1x 10⁷、2x 10⁷、5x 10⁷、1x 10⁸、2x 10⁸、5x 10⁸、1x 10⁹、2x 10⁹Or 5x10⁹A cell. In some embodiments, the dose of engineered lymphocytes (e.g., γ δ T cells (e.g., skin-derived γ δ T cells, blood-derived γ δ T cells, e.g., V δ 1T cells and/or DN T cells), NK cells, NK-like T cells, innate lymphoid cells, or MAIT cells) comprises at least about 1x 10⁷、2x 10⁷、5x 10⁷、1x 10⁸、2x 10⁸、5x 10⁸、1x 10⁹、2x 10⁹Or 5x10⁹And (4) cells. In some embodiments, the dose of engineered lymphocytes (e.g., γ δ T cells (e.g., skin-derived γ δ T cells, blood-derived γ δ T cells, e.g., V δ 1T cells and/or DN T cells), NK cells, NK-like T cells, innate lymphoid cells, or MAIT cells) comprises up to about 1x 10⁷、2x 10⁷、5x10⁷、1x 10⁸、2x 10⁸、5x 10⁸、1x 10⁹、2x 10⁹Or 5x10⁹And (4) cells.

In one embodiment, the subject is administered 10 per kg of subject weight⁴To 10⁶An engineered lymphocyte (e.g., a γ δ T cell (e.g., a skin-derived γ δ T cell, a blood-derived γ δ T cell, e.g., a V δ 1T cell)And/or DN T cells), NK cells, NK-like T cells, innate lymphoid cells, or MAIT cells). In one embodiment, the subject receives an initial administration of a population of engineered lymphocytes (e.g., γ δ T cells, NK-like T cells, innate lymphoid cells, or MAIT cells (e.g., an initial administration of 10 per kg body weight of the subject)⁴To 10⁶Gamma delta T cells, NK-like T cells, innate lymphoid cells or MAIT cells, e.g. 10 per kg body weight of a subject⁴To 10⁵γ δ T cells, NK-like T cells, innate lymphoid cells, or MAIT cells)), and one or more (e.g., 2, 3, 4, or 5) subsequently administered engineered lymphocytes (e.g., γ δ T cells, NK-like T cells, innate lymphoid cells, or MAIT cells (e.g., one or more subsequently administered 10 per kg of subject body weight)⁴To 10⁶Engineered γ δ T cells, NK-like T cells, innate lymphoid cells or MAIT cells, e.g., 10 per kg subject weight⁴To 10⁵Engineered γ δ T cells)). In one embodiment, the one or more subsequent administrations are administered within less than 15 days (e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days) after the previous administration, e.g., less than 4, 3, or 2 days after the previous administration. In one embodiment, the subject receives a total of about 10 per kg of subject body weight during the entire at least three administrations of the population of γ δ T cells, NK-like T cells, innate lymphoid cells, or MAIT cells⁶Gamma delta T cells, NK-like T cells, innate lymphoid cells or MAIT cells, e.g., the subject receives an initial dose of 1x 10⁵Administering a second dose of 3x 10 gamma delta T cells, NK-like T cells, innate lymphoid cells or MAIT cells⁵Gamma delta T cells, NK-like T cells, innate lymphoid cells or MAIT cells, and a third administration of 6x10⁵γ δ T cells, NK-like T cells, innate lymphoid cells or MAIT cells, and for example each administration is administered within less than 4, 3 or 2 days after the previous administration.

In some embodiments, one or more additional therapeutic agents may be administered to the subject. The additional therapeutic agent may be selected from an immunotherapeutic agent, a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an anti-angiogenic agent, or a combination of two or more thereof. The additional therapeutic agent may be administered simultaneously with, prior to, or subsequent to the administration of the engineered lymphocytes (e.g., γ δ T cells, NK-like T cells, innate lymphoid cells, or MAIT cells). The additional therapeutic agent can be an immunotherapeutic agent that can act on a target in the subject (e.g., the subject's own immune system) and/or act on the transferred γ δ T cells, NK-like T cells, innate lymphoid cells, or MAIT cells.

The composition may be administered in any convenient manner. The compositions described herein can be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, by intravenous injection, or intraperitoneally, for example by intradermal or subcutaneous injection. Compositions of engineered lymphocytes (e.g., γ δ T cells, NK-like T cells, innate lymphoid cells, or MAIT cells) can be injected directly into a tumor, lymph node, or site of infection.

Examples

The following examples provide non-limiting methods of engineering γ δ T cells to express heterologous targeting constructs, non-limiting methods of functionally screening engineered γ δ T cells, and therapeutic methods using engineered γ δ T cells.

Example 1: functional characterization of engineered γ δ T cells expressing heterologous targeting constructs

Engineered V δ 1T cells with heterologous targeted receptors are functionally characterized in vitro by co-culturing with target cells (e.g., cancer cells, e.g., cells of a tumor cell line). Engineered V δ 1T cells were compared to three control cell types: (1) untransduced V δ 1T cells; (2) mimicking transduced V δ 1T cells expressing heterologous GFP; and (3) conventional Chimeric Antigen Receptor (CAR) transduced V δ 1T cells with functional intracellular signaling domains. Each group was co-cultured with at least two groups of target tumor cells: (A) a healthy cell group expressing normal levels of Tumor Associated Antigen (TAA), and (B) a tumor cell group expressing high levels of TAA. Various effector to target ratios (γ δ T cell to target cell ratio) were tested. Untransduced or mock-transduced V δ 1 cells were used as controls to identify the effects conferred by heterologous targeted receptors, and CAR T cells were used as controls to identify the effects of the lack of a functional intracellular domain configured to transmit signal 1 and/or signal 2 stimuli. The following measurements were performed:

1. proliferation assays were performed according to standard CFSE dilution protocols to quantify the effect of the interaction between the engineered γ δ T-cells and the target cells on the proliferation of the engineered γ δ T-cells, which is indicative of activation against the target cells. γ δ T cells expressing heterologous targeting constructs proliferate to a greater extent in response to cancer cells relative to healthy cells.

2. The CD107 degranulation assay was performed by quantifying the expression of the lysosomal associated membrane protein 1 (LAMP-1; i.e., CD107), LAMP-1 being transiently expressed on the surface of γ δ T cells after degranulation. Cells were stained at different time points to monitor the kinetics of degranulation. γ δ T cells expressing heterologous targeting constructs preferentially exhibit degranulation in response to cancer cells relative to healthy cells.

3. Perforin/granzyme assay was performed by staining for perforin and granzyme using FACS. Gamma delta T cells expressing heterologous targeting constructs preferentially express perforin and/or granzyme in response to cancer cells relative to healthy cells.

4. Cell lysis assays were performed to quantify the extent of target cell lysis (i.e., cell lysis). The kinetics of cell lysis was measured by the Incucyte or luciferase assay, expressed as the percentage of killing over a certain time, and the end point cell lysis was measured using the luciferase assay, expressed as the percentage of killing at a given time point. Gamma delta T cells expressing heterologous targeting constructs preferentially lyse cancer cells relative to healthy cells.

5. The formation of immune synapses was monitored by live cell imaging. The binding kinetics were monitored by observing the immunological synapses between γ δ T cells and target cells. In addition, calcium flux in γ δ T cells (indicative of recognition) and PI staining in target cells (determination of lethal hits) were observed. Target cell rounding was also observed. Binding kinetics and calcium flux are preferentially enhanced in γ δ T cells expressing heterologous targeting constructs when co-cultured with cancer cells relative to when cells are involved.

Example 2: peripheral blood-derived engineered gamma delta T cells expressing heterologous targeting constructs

One of the unique properties of V δ 1 γ δ T cells compared to conventional α β T cells is the selective killing of malignantly transformed cells while retaining healthy tissue, a process that can be mediated by the action of natural cytotoxic receptors. Current results indicate that the ability of V δ 1 cells to eradicate tumor cells can be further enhanced using heterologous targeting constructs lacking an intracellular signaling domain. Engineering V δ 1 cells using this construct maintains or even increases the cytotoxicity of these cells against malignantly transformed cells, while still retaining healthy cells. This approach overcomes the off-target tumor effects observed with conventional Chimeric Antigen Receptor (CAR) immunotherapy (e.g., B cell depletion following CD19 targeted CAR therapy).

Materials and methods

Peripheral blood gamma delta T-cell isolation and expansion

Blood-derived V δ 1 cells were generated from peripheral blood of healthy donors as described previously in u.s.2018/0169147 (incorporated herein by reference in its entirety, in particular its method of isolating V δ 1 cells from blood). Briefly, MACS-depleted α β T cells were resuspended in serum-free media supplemented with autologous plasma (CTS OpTsizer) and amplified in the presence of IL-4, IFN- γ, IL-21, IL-1 β, IL-15, and soluble OKT 3. Cells were transduced with lentiviral vectors encoding the constructs described below. Essentially, the full-length CAR construct includes a scFv binding region that targets the tumor antigen CD19 or GD2, a transmembrane domain, and an intracellular signaling domain (designed according to conventional CAR constructs). Non-signaling or "nsCAR" lacks the intracellular domain.

Other ways of obtaining Vd1 cells from blood are well known in the art, such as US9499788, WO2017197347, WO 2016081518. Alternatively, V δ 1 cells are isolated from human skin biopsies as described in u.s.2018/0312808, which is incorporated herein by reference in its entirety and in particular to a method of isolating V δ 1 cells from tissue. Skin-derived V δ 1 cells were transduced as above.

Flow cytometry analysis

Immunophenotyping was performed using a BD FACS Lyric flow cytometer. Cell surface marker expression was analyzed using PerCP-Vio700 anti-TCR a/b (Miltenyi), APC anti-TCR g/d (Miltenyi), VioBlue anti-TCR V δ 1(Miltenyi), PE anti-NKp 30(BioLegend), APC anti-NKp 44(BioLegend), percp.cy5.5 anti-NKG 2D (BioLegend). Conventional CD19CAR and non-signal CD19CAR construct expression was detected using FITC anti-STREP tag antibody (LSBio). Non-signal GD2 CAR expression was monitored using PE anti-FC antibody (BioLegend).

Cytotoxicity assays

Nalm-6(ATCC, CRL-1567) and primary B cells were labeled with CTV or CFSE and combined with T cells at an effector to target ratio of 1: 1. The culture was incubated at 37 ℃ for 16 hours. After incubation, sytoxaadvanced (invitrogen) and absolute counts were added to the wells and flow cytometer acquisitions were performed. Cytotoxicity was calculated as follows:

100- (sample count/max count) × 100

Wherein the maximum count is the number of target cells in the absence of any effector cells.

Live cell imaging

The neuroblastoma cell line Kelly (DSMZ ACC-355) expressing human GD2 was stably transduced using NucLight Green encoding a lentiviral vector (Essen BioScience) to enable automatic cell counting. Cell growth was monitored for 60 hours at 1 hour intervals using the Incucyte Zoom live cell imaging system (Essen Bioscience). The data is represented as a change in the ratio of the number of green object counts per image at a given point in time, which has been normalized with respect to the number of green object counts per image at zero. Each data point represents triplicate wells.

Comparison with α β T cell CAR

As described above, γ δ cells were engineered using full-length or non-signaling CAR constructs. Similarly, α β -derived T cells from blood or tissue are also engineered using full-length or non-signaling CAR constructs. Cytolytic activity of the engineered cells against healthy and malignant cells expressing the target antigen was measured to demonstrate on-target de-tumor cytotoxicity for each population.

Results

Transduction of blood-borne V δ 1 cells with lentiviral vectors encoding either full-length (CAR19) or non-signaling anti-CD 19 targeting constructs resulted in transduction efficiencies of greater than 90% (fig. 3A). There was no significant difference in the surface expression of the CAR molecules. Lentiviral transduction did not alter the immunophenotype and proliferative capacity of the transduced cells. FACS analysis of Untransduced (UTD) and transduced V.delta.1 cells did not show any significant differences in surface expression of key Natural Cytotoxic Receptor (NCR) molecules (NKp30, NKp44 and NKG 2D; FIG. 3B).

The V.delta.1 cells recognized and killed cells of the CD19 expressing acute lymphoblastic leukemia cell line NALM-6. Expression of full-length or non-signaling CD19CAR on V δ 1 cells resulted in a two-fold increase in target cell killing (fig. 4A and 4B; percent killing for donor 1 and donor 2 at 1:1 effector to target ratio was 21.4% (UTD) to 46% (nsCAR19) to 58% (CAR19) and 42.8% (UTD) to 83% (nsCAR19) to 88% (CAR19), respectively). Importantly, V δ 1 cells expressing non-signaling anti-CD 19CAR did not kill healthy human B cells (fig. 4C).

To further demonstrate the general applicability of the non-signaling CAR approach, V δ 1 cells were redirected to tumor cells expressing GD2 antigen. Transduction of blood-derived V δ 1 cells with GD 2-specific nsCAR molecules resulted in 54% transduction efficiency as measured by FACS (fig. 5A). Untransduced and nsCAR-transduced V δ 1 cells were co-cultured with a neuroblastoma cell line expressing GD2 (Kelly) at an effector to target ratio of 1: 1. Target cell killing was measured using live cell imaging (Incucyte, Essen Bioscience). Co-culture of Kelly cells with nsCAR-expressing V δ 1 cells resulted in a 40% reduction in total target cell numbers compared to culturing the target cells in the presence of untransduced V δ 1 cells (fig. 5B).

Example 3: treatment of cancer using gamma delta T cells engineered with heterologous targeting constructs

The heterologous targeting construct is synthesized using cloning and PCR methods known in the art. A protein fragment encoding an scFv targeting a Tumor Associated Antigen (TAA) was fused to the N-terminus of the stem domain, which was then fused to the N-terminus of the transmembrane domain of CD 8. The heterologous targeting construct is then cloned into a lentiviral vector.

The patient undergoes a leukapheresis procedure from which a blood sample is obtained and red blood cells are depleted. α β T cells were depleted using standard magnetic separation protocols. The remaining population comprising γ δ T cells is expanded using any suitable γ δ T cell expansion method known in the art or described herein. During amplification, cells are incubated with lentiviral vectors containing a polynucleotide encoding a heterologous targeting construct, and the polynucleotide is integrated into the genome of γ δ T cells by reverse transcription. The cells transduced with the lentivirus can express the heterologous targeting construct on the surface. The transduced cells expressing the heterologous targeting construct are then separated from the untransduced cells and collected for infusion as autologous or allogeneic therapy.

The cells were administered intravenously to the patient over a two hour period. Once weekly, intravenously for 12 weeks, and cancer symptoms were monitored.

Other embodiments

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specific embodiments thereof, it will be understood that the invention is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Other implementations are within the scope of the following claims. What is claimed is: