阅读说明：本技术 用于提高TCRαβ+细胞耗竭效率的方法 (Methods for increasing the efficiency of TCR α β + cell depletion ) 是由 倪亚瑾 宁红秀 J·M·李 M·W·伦纳德 于 2020-03-20 设计创作，主要内容包括：本文提供了用于稳健TCR+细胞耗竭和产生TCR-细胞群的改善方法,所述方法可以有益于最小化接受同种异体CAR T细胞疗法的患者的GvHD风险。本文提供了增加从细胞群中耗竭TCR+细胞的效率,以便显著降低存在于已经减少或消除了内源性TCR表达的细胞群中的TCR+细胞的任何残余水平的方法。还提供了相关联的试剂盒和细胞群。(Provided herein are improved methods for robust TCR + cell depletion and generation of TCR-cell populations that may be beneficial for minimizing GvHD risk in patients receiving allogeneic CAR T cell therapy. Provided herein are methods of increasing the efficiency of depleting TCR + cells from a population of cells, so as to significantly reduce any residual levels of TCR + cells present in a population of cells in which endogenous TCR expression has been reduced or eliminated. Associated kits and cell populations are also provided.)

1. A method of generating a population of immune cells depleted of immune cells expressing an endogenous TCR, the method comprising:

(a) labeling the immune cell population with an anti-TCR antibody and an anti-CD 3 antibody; and

(b) isolating anti-TCR antibody-labeled immune cells and anti-CD 3 antibody-labeled immune cells from the immune cell population.

2. A method of depleting cells expressing an endogenous TCR from a population of immune cells, the method comprising:

(a) labeling the immune cell population with an anti-TCR antibody and an anti-CD 3 antibody;

(b) separating the anti-TCR antibody-labeled immune cells and the anti-CD 3 antibody-labeled immune cells from the unlabeled immune cells; and

(c) collecting the unlabeled immune cells, and,

thereby obtaining a population of immune cells depleted of cells expressing endogenous TCR.

3. A method of generating a population of immune cells depleted of immune cells expressing an endogenous TCR, the method comprising:

(c) labeling the immune cell population with an anti-TCR antibody and an anti-CD 52 antibody; and

(d) isolating anti-TCR antibody-labeled immune cells and anti-CD 52 antibody-labeled immune cells from the immune cell population.

4. A method of depleting cells expressing an endogenous TCR from a population of immune cells, the method comprising:

(d) labeling the immune cell population with an anti-TCR antibody and an anti-CD 52 antibody;

(e) separating the anti-TCR antibody-labeled immune cells and the anti-CD 52 antibody-labeled immune cells from the unlabeled immune cells; and

(f) collecting the unlabeled immune cells, and,

thereby obtaining a population of immune cells depleted of cells expressing endogenous TCR.

5. The method of claim 1 or 2, further comprising: labeling the immune cell population with an anti-CD 52 antibody; and depleting anti-CD 52 antibody-labeled immune cells from the immune cell population.

6. The method of claim 1 or 2, further comprising: labeling the immune cell population with an anti-CD 52 antibody; and separating the anti-CD 52 antibody-labeled immune cells from the unlabeled immune cells.

7. The method of any one of claims 1-6, wherein the anti-TCR antibody, the anti-CD 3 antibody and/or the anti-CD 52 antibody is biotin-conjugated.

8. The method of claim 7, further comprising contacting the labeled cells with an agent that specifically recognizes biotin.

9. The method of claim 8, wherein the agent that specifically recognizes biotin is selected from the group consisting of: anti-biotin antibodies, avidin and streptavidin.

10. The method of claim 8 or 9, wherein the agent is conjugated to a magnetic bead, an agarose bead, a sonic particle, a plastic well plate, a glass well plate, a ceramic well plate, a column, a cell culture bag, or a membrane.

11. The method of any one of claims 1-6, wherein the anti-TCR antibody, the anti-CD 3 antibody, and/or the anti-CD 52 antibody is directly conjugated to a magnetic bead, an agarose bead, a sonic particle, a plastic well plate, a glass well plate, a ceramic well plate, a column, a cell culture bag, or a membrane.

12. The method of any one of claims 1 to 11, wherein the separation is achieved using one of magnetic separation or sonic separation.

13. The method of any one of the preceding claims, wherein the immune cells are allogeneic immune cells.

14. The method of any one of claims 1-13, wherein the immune cell is genetically modified to reduce or eliminate expression of an endogenous TCR.

15. The method of any one of claims 1-13, wherein the immune cell is genetically modified to reduce or eliminate expression of endogenous CD 52.

16. The method of any one of claims 1-13, wherein the immune cell is genetically modified to reduce or eliminate expression of both endogenous TCR and endogenous CD 52.

17. The method of any one of claims 14-16, wherein the reducing or eliminating expression of endogenous TCR or endogenous CD52 or both endogenous TCR and endogenous CD52 is achieved using TALENs, CRISPR/Cas9, Zinc Finger Nucleases (ZFNs), megatals, meganucleases, Cpf1, homologous recombination, or single-chain oligodeoxynucleotides (ssodns).

18. The method of any one of claims 1-17, wherein the immune cell is an engineered immune cell expressing a chimeric antigen receptor.

19. The method of any one of claims 1 to 18, wherein the population of cells depleted of cells expressing endogenous TCR comprises no more than 1.0% TCR + cells, no more than 0.9% TCR + cells, no more than 0.8% TCR + cells, no more than 0.7% TCR + cells, no more than 0.6% TCR + cells, no more than 0.5% TCR + cells, no more than 0.4% TCR + cells, no more than 0.3% TCR + cells, no more than 0.2% TCR + cells, or no more than 0.1% TCR + cells.

20. The method of any one of claims 1 to 19, wherein the population of cells depleted of cells expressing endogenous TCR comprises between 0.01% -0.001% TCR + cells immediately after depletion.

21. The method of any one of claims 1 to 19, wherein the population of cells depleted of cells expressing endogenous TCR comprises between 0.1% -0.01% TCR + cells after 1-10 days of culture after depletion.

22. The method of any one of claims 21, wherein the population of cells depleted of cells expressing an endogenous TCR comprises less than 0.1% -1.0% TCR + cells after 1 day of culture after depletion.

23. The method of any one of claims 21, wherein the population of cells depleted of cells expressing an endogenous TCR comprises less than 0.1% -1.0% TCR + cells after 10 days of culture after depletion.

24. The method of any one of the preceding claims, wherein the immune cell is a T cell.

25. A population of immune cells depleted of immune cells expressing endogenous TCRs, the population of immune cells produced by the method of any one of claims 1 to 24.

26. An engineered T cell population comprising at least 99% TCR-cells.

27. An engineered T cell population comprising at least 99.9% TCR-cells.

28. An engineered T cell population comprising at least 99.99% TCR-cells.

29. An engineered T cell population comprising at least 99.999% TCR-cells.

30. The engineered T cell population of any one of claims 26-29, wherein the T cell population expresses a chimeric antigen receptor.

31. A kit for depleting cells expressing endogenous TCR from a population of immune cells, the kit comprising an anti-TCR antibody and an anti-CD 3 antibody.

32. The kit of claim 31, further comprising an anti-CD 52 antibody.

33. A kit for depleting cells expressing endogenous TCR from a population of immune cells, the kit comprising an anti-TCR antibody and an anti-CD 52 antibody.

34. The kit of any one of claims 31-33, wherein the anti-TCR antibody, the anti-CD 3 antibody, and/or the anti-CD 52 antibody is conjugated to an agent that specifically recognizes biotin.

35. The kit of claim 34, wherein the agent that specifically recognizes biotin is selected from the group consisting of: anti-biotin antibodies, streptavidin, and avidin.

36. The kit of claim 34 or 35, wherein the agent is conjugated to a magnetic bead, an agarose bead, a sonic particle, a plastic well plate, a glass well plate, a ceramic well plate, a column, a cell culture bag, or a membrane.

37. The kit of any one of claims 31-35, wherein the anti-TCR antibody, the anti-CD 3 antibody, and/or the anti-CD 52 antibody is directly conjugated to a support.

38. The kit of claim 37, wherein the support is one of: magnetic beads, agarose beads, sonic particles, plastic well plates, glass well plates, ceramic well plates, columns, cell culture bags or membranes.

39. A method of treating a solid or hematologic cancer in a subject in need thereof, the method comprising administering to the subject a therapeutic amount of the population of immune cells according to any one of claims 19 to 23 depleted of immune cells expressing endogenous TCR or the population of engineered T cells according to any one of claims 26 to 30.

Technical Field

The present disclosure relates to methods for depleting cells expressing an endogenous TCR (e.g., TCR α β) from engineered immune cell populations, including those comprising Chimeric Antigen Receptors (CARs).

Background

Adoptive transfer of immune cells genetically modified to recognize malignancy-associated antigens has shown promise as a novel approach for treating Cancer (see, e.g., Brenner et al, Current Opinion in Immunology, 22 (2): 251-42 (2010); Rosenberg et al, natural Reviews of Cancer (Nature Reviews), 8 (4): 299-308 (2008)). Immune cells can be genetically modified to express Chimeric Antigen Receptors (CARs) (see, e.g., Eshhar et al, proceedings of the american national academy of sciences, inc. sci. usa, 90 (2): 720-724(1993) and Sadelain et al, new points of immunology (curr. opin. immunol.), 21 (2): 215-223(2009), which are incorporated herein by reference). Immune cells comprising CARs, such as CAR-T cells (CAR-T), are designed to confer antigen specificity to them, while retaining or enhancing their ability to recognize and kill target cells, such as cancer cells. However, the possibility of suffering from graft versus host disease (GvHD) or host versus graft disease (HvGD) is a major safety or efficacy hurdle for the widespread use of engineered allogeneic CAR-T cells in cancer therapy. Therefore, there is a need to develop effective methods to reduce the risk of GvHD and HvGD, especially for allotherapy.

Disclosure of Invention

Described herein are improved methods for depleting TCR + (TCR α β +) cells from a population of immune cells, methods for generating a TCR + cell depleted population of immune cells, kits comprising reagents for the methods, high purity TCR- (TCR α β -) cell populations, and methods of treatment employing cell populations prepared using the disclosed methods. For example, described herein are methods particularly suited for TCR + cell depletion that can be used to manufacture cells that can be used for allogeneic cell therapy by reducing the risk of GvHD and can be used for therapies employing chimeric antigen receptors (e.g., allogeneic CAR-T cell therapy).

In one aspect, a method of generating a population of immune cells depleted of immune cells expressing endogenous TCRs is provided, and in one embodiment, the method comprises: labeling the immune cell population with an anti-TCR antibody and an anti-CD 3 antibody; and isolating anti-TCR antibody-labeled immune cells and anti-CD 3 antibody-labeled immune cells from the population of immune cells. The method may further comprise: labeling the immune cell population with an anti-CD 52 antibody; and depleting anti-CD 52 antibody-labeled immune cells from the immune cell population. In addition, the method may further include: labeling the immune cell population with an anti-CD 52 antibody; and separating the anti-CD 52 antibody-labeled immune cells from the unlabeled immune cells.

In another aspect, a method of depleting cells expressing an endogenous TCR from a population of immune cells is provided, and in one embodiment, the method comprises: labeling the immune cell population with an anti-TCR antibody and an anti-CD 3 antibody; separating the anti-TCR antibody-labeled immune cells and the anti-CD 3 antibody-labeled immune cells from the unlabeled immune cells; and collecting the unlabeled immune cells, thereby obtaining a population of immune cells depleted of cells expressing endogenous TCRs.

In a further aspect, there is provided a method of generating a population of immune cells depleted of immune cells expressing an endogenous TCR, and in one embodiment, the method comprises: labeling the immune cell population with an anti-TCR antibody and an anti-CD 52 antibody; and isolating anti-TCR antibody-labeled immune cells and anti-CD 52 antibody-labeled immune cells from the population of immune cells.

In still a further aspect, there is provided a method of depleting cells expressing an endogenous TCR from a population of immune cells, and in one embodiment, the method comprises: labeling the immune cell population with an anti-TCR antibody and an anti-CD 52 antibody; separating the anti-TCR antibody-labeled immune cells and the anti-CD 52 antibody-labeled immune cells from the unlabeled immune cells; and collecting the unlabeled immune cells, thereby obtaining a population of immune cells depleted of cells expressing endogenous TCRs.

In embodiments, the anti-TCR antibody, the anti-CD 3 antibody, and/or the anti-CD 52 antibody is conjugated to biotin, and the method can further comprise contacting the labeled cells with an agent that specifically recognizes biotin. The agent specifically recognizing biotin may be selected from the group consisting of an anti-biotin antibody, avidin, and streptavidin, and may be conjugated to magnetic beads, agarose beads, sonic particles, plastic well plates, glass well plates, ceramic well plates, columns, cell culture bags, or membranes. In other embodiments, the anti-TCR antibody, the anti-CD 3 antibody, and/or the anti-CD 52 antibody is conjugated directly to a magnetic bead, an agarose bead, a sonic particle, a plastic well plate, a glass well plate, a ceramic well plate, a column, a cell culture bag, or a membrane.

In the method using separation, separation may be achieved using one of magnetic separation or acoustic separation.

In embodiments, the immune cells of the disclosed methods are allogeneic immune cells, and the immune cells can be genetically modified to reduce or eliminate expression of endogenous TCR, endogenous CD52, or both endogenous TCR and endogenous CD52, using TALENs, CRISPR/Cas9, Zinc Finger Nucleases (ZFNs), megatals, meganucleases, Cpf1, homologous recombination, or single-chain oligodeoxynucleotides (ssodns).

The immune cells of the disclosed methods can be engineered immune cells that express a chimeric antigen receptor.

In some embodiments, the population of cells depleted of cells expressing endogenous TCR comprises no more than 1.0% TCR + cells, no more than 0.9% TCR + cells, no more than 0.8% TCR + cells, no more than 0.7% TCR + cells, no more than 0.6% TCR + cells, no more than 0.5% TCR + cells, no more than 0.4% TCR + cells, no more than 0.3% TCR + cells, no more than 0.2% TCR + cells, or no more than 0.1% TCR + cells. In some embodiments, the population of cells depleted of cells expressing endogenous TCR comprises between 0.01% -0.001% TCR + cells immediately after depletion. In some embodiments, the population of cells depleted of cells expressing endogenous TCR comprises between 0.1% -0.01% TCR + cells after 1-10 days of culture after depletion; in some embodiments, the population of cells depleted of cells expressing an endogenous TCR comprises less than 0.1% -1.0% TCR + cells after 1 day of culture after depletion. In some embodiments, the population of cells depleted of cells expressing an endogenous TCR comprises less than 0.1% -1.0% TCR + cells after 10 days of culture after depletion.

In embodiments of the disclosed methods, the immune cell is a T cell.

In another aspect, there is provided a population of immune cells produced by the disclosed methods that are depleted of immune cells expressing endogenous TCRs.

In one aspect, an engineered T cell population comprising at least 99% TCR-cells is provided, an engineered T cell population comprising at least 99.9% TCR-cells is provided, an engineered T cell population comprising at least 99.99% TCR-cells is provided, and an engineered T cell population comprising at least 99.999% TCR-cells is provided. In embodiments, the population of T cells expresses a chimeric antigen receptor.

In another aspect, a kit for depleting cells expressing an endogenous TCR from a population of immune cells is provided, the kit comprising an anti-TCR antibody and an anti-CD 3 antibody. The kit may further comprise an anti-CD 52 antibody. In another aspect, a kit for depleting cells expressing an endogenous TCR from a population of immune cells is provided, the kit comprising an anti-TCR antibody and an anti-CD 52 antibody. In the disclosed kits, the anti-TCR antibody, the anti-CD 3 antibody, and/or the anti-CD 52 antibody is conjugated to an agent that specifically recognizes biotin; the agent specifically recognizing biotin may be selected from the group consisting of an anti-biotin antibody, streptavidin, and avidin. In embodiments, the agent may be conjugated to magnetic beads, agarose beads, sonic particles, plastic well plates, glass well plates, ceramic well plates, columns, cell culture bags, or membranes.

In the disclosed kits, the anti-TCR antibody, the anti-CD 3 antibody, and/or the anti-CD 52 antibody is directly conjugated to a support, and the support can be one of a magnetic bead, an agarose bead, a sonic particle, a plastic well plate, a glass well plate, a ceramic well plate, a column, a cell culture bag, or a membrane.

Further provided is a method of treating a solid or hematologic cancer in a subject in need thereof, the method comprising administering to the subject a therapeutic amount of a population of immune cells prepared using the disclosed methods or a population of engineered T cells prepared using the disclosed methods that deplete immune cells expressing endogenous TCRs.

The methods provided herein represent a significant advance in allotherapy. Thus, also provided is a method of treating a solid or hematologic cancer in a subject in need thereof, the method comprising administering to the subject a population of immune cells prepared using any of the disclosed methods provided herein that depletes immune cells expressing endogenous TCRs.

Drawings

Fig. 1 depicts a schematic diagram of an allogeneic CART manufacturing process in the context of different unit operation scenarios.

Figure 2A depicts residual TCR + cell frequency in the cell population depleted on each day after depletion, with arrows indicating fold change in TCR +% for different depletion methods (using different antibody combinations) compared to control methods (using anti-TCR antibody alone) on day 2 post depletion compared to day 0 post depletion.

Figure 2B depicts residual TCR + cell frequency in depleted cell populations, arrows indicating fold change in TCR +% between different depletion methods at 9 days post depletion compared to 0 days post depletion, when compared to control methods.

Figure 3A depicts a flow cytometry plot of TCR + cell depletion efficiency using different antibodies at day 0 and day 1 post-depletion, as measured by anti-TCR antibody staining.

Figure 3B depicts in a numerical column the residual TCR + cell frequency in the cell populations depleted on day 0 and day 1 post-depletion.

Figure 4 depicts a flow cytometry plot demonstrating residual TCR + and CD3+ frequencies in depleted populations detected by single or double staining with anti-TCR and/or anti-CD 3 antibodies on day 0 and day 1 post-depletion.

Figure 5A depicts flow cytometry plots of TCR + frequency, CD3+ frequency, CD3 +/TCR-frequency, and CD3/TCR + frequency in depleted cell populations, measured by single or double staining of anti-TCR antibody and/or anti-CD 3 antibody, before freezing and after freezing, on day 1 post-depletion.

Figure 5B depicts in a numerical column the TCR + cell frequency in the depleted cell population measured by anti-TCR antibody staining before and after freezing on day 1 post-depletion.

Figure 5C depicts in a numerical bar the frequency of CD3+ cells in the depleted cell population, measured by anti-CD 3 antibody staining before and after freezing, on day 1 post-depletion.

Figure 5D depicts in digital column fashion the CD3 +/TCR-cell frequency in the depleted cell population, measured by double staining with anti-TCR and anti-CD 3 antibodies, on day 1 post depletion, before freezing and after freezing.

Figure 5E depicts in digital column fashion the CD3+/TCR + cell frequency in the depleted cell population, measured by double staining with anti-TCR and anti-CD 3 antibodies, on day 1 post depletion, before freezing and after freezing.

Figure 6 depicts flow cytometry plots of TCR + frequency stained with anti-TCR antibody during post-exhaustion culture for 10 days.

Figure 7 depicts flow cytometry plots of CD3+ frequency of staining with anti-CD 3 antibody during 10 days post-depletion culture.

Figure 8 depicts flow cytometry plots of dual TCR + and CD3+ frequencies stained with anti-TCR and anti-CD 3 antibodies during post-exhaustion culture for 10 days. Figure 9A depicts in a digital column the TCR + cell frequency stained with anti-TCR antibody during post-depletion culture.

Figure 9B depicts in a digital bar fashion the frequency of CD3+ cells stained with anti-CD 3 antibody during post-depletion culture.

Figure 9C depicts in a numeric bar the CD3 +/TCR-cell frequency using both anti-TCR and anti-CD 3 antibodies to stain during post-depletion culture.

Figure 9D depicts in a digital bar fashion the frequency of CD3+/TCR + cells stained with anti-TCR and anti-CD 3 antibodies during post-depletion culture.

Fig. 10A depicts the cell growth state in terms of viable cell density during the 10 day post-depletion culture period.

Fig. 10B depicts the cell growth state in terms of cell viability during the post-depletion culture period of 10 days.

Fig. 10C depicts the cell growth state in terms of cell diameter during the 10 day post-depletion culture.

Figure 10D depicts the cell growth state in terms of total cell expansion fold during post-exhaustion culture for 10 days.

Figure 11A depicts a flow cytometry plot showing CD52 depletion efficiency with different antibodies at day 0 and day 1 post-depletion as measured by anti-CD 52 staining.

Figure 11B depicts in a numerical bar manner the residual CD52+ cell frequency in the depleted cell population on day 0 and day 1 post depletion as measured by anti-CD 52 antibody staining.

Figure 12 depicts flow cytometry plots of residual CD52+ cell frequency during 10 days post-depletion culture monitored by anti-CD 52 antibody staining.

Detailed Description

Provided herein are improved methods for robust TCR α/β + cell depletion that can minimize the risk of GVHD in patients receiving allogeneic CAR T cell therapy, as well as cell populations prepared using the disclosed methods. In one aspect, the present disclosure provides methods of increasing TCR + depletion efficiency to significantly reduce residual TCR + cell levels in a TCR-cell population.

As used herein, the term "a" is used to mean one or more. For example, reference to "a cell" or "an antibody" refers to "one or more cells" or "one or more antibodies".

As used herein, the term "TCR-depleted" when used in reference to a cell population generated using the methods provided by the present disclosure means a cell population that includes fewer cells expressing endogenous TCR α/β heterodimers as compared to a cell population collected from a donor. For example, the population of TCR-depleted cells (TCR α/β -depleted cells) may comprise 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less than 1% of cells expressing the endogenous TCR α/β complex.

As used herein, the terms "TCR +" and "TCR," when used in reference to a cell or population of cells (including populations generated using methods provided by the present disclosure), refer to cells that express at least endogenous TCR α/β heterodimers, although one or more components of the CD3 complex may or may not be expressed on the cell surface.

As used herein, the term "TCR", when used in reference to a cell or population of cells (including populations generated using methods provided by the present disclosure), refers to a cell that lacks at least the TCR α/β heterodimer, although one or more components of the CD3 complex may or may not be expressed on the cell surface.

As used herein, the terms "anti-TCR antibody" and "anti-TCR" used interchangeably refer to an antibody that binds and recognizes only the human TCR α chain, an antibody that binds and recognizes only the human TCR β chain, or an antibody that binds and recognizes the human TCR α/β heterodimer. The anti-TCR antibody can be murine, human, or humanized.

As used herein, the terms "anti-CD 3 antibody" and "anti-CD 3" used interchangeably refer to an antibody that binds to and recognizes and binds to the human CD3 gamma chain, human CD3 delta chain, CD3 zeta chain, CD3 epsilon chain, or a human CD3 complex that includes two or more components of the normal human epsilon/zeta/gamma/delta CD3 complex. The anti-CD 3 antibody may be a murine, human, or humanized antibody.

The terms "patient" and "subject" are used interchangeably and include human and non-human animal subjects as well as subjects having a formal diagnostic condition, subjects without a formal discrimination of the condition, subjects receiving medical care, subjects at risk of developing the condition, and the like.

The terms "treatment" and "treating" encompass therapeutic treatment, prophylactic treatment, and applications in which the risk of the subject suffering from the disorder or other risk factors is reduced. Treatment does not require a complete cure for the condition and encompasses embodiments that alleviate symptoms or potential risk factors. The term "prevention" does not require 100% elimination of the possibility of an event occurring. Conversely, the term "preventing" means that the likelihood of an event occurring in the presence of a compound or method has been reduced.

Engineered TCR-cell based allogeneic CAR-T products exemplify strategies for generating next generation CAR-T therapies. However, potential immune responses such as the risk of graft versus host disease (GvHD) and host versus graft disease (HvGD) represent one of the important safety or efficacy issues for allogeneic CAR-T cell therapy. GvHD is caused by donor-derived T cells that recognize HLA mismatches via the T cell α β receptor (TCR α β) and have the potential to attack patient tissues. GVHD and HvGD can be severe and even fatal, even in an HLA-matched donor setting, as slight mismatches can still elicit an immune response.

Such as CliniMACSAnd TCR kits currently used to deplete TCR + cells (TCR α β + cell depletion) only employ anti-TCR antibodies to deplete TCR + cells using anti-TCR antibodies: (TCR α β + cell depletion) ((TCR α β))TCR α β + cells) from TCR-cells (TCR α β -cells) and can reach 99% or higher TCR-cell purity in the final drug product (see, e.g., Radestad et al, (2014) journal of immunological research (J immunological Res), 2014: 578741 roll). However, residual TCR + cell levels of less than 1% that occur in TCR-final drug products may still carry the GvHD risk associated with allogeneic CAR-T cell therapy, particularly at high dose levels.

The present disclosure provides methods for generating an engineered immune cell population depleted of immune cells expressing endogenous TCRs, the disclosed methods comprising: labeling the immune cell population with an anti-TCR antibody and an anti-CD 3 antibody; and isolating anti-TCR antibody-labeled immune cells and anti-CD 3 antibody-labeled immune cells from the engineered immune cell population.

The present disclosure also provides a method of depleting cells expressing an endogenous TCR from a population of immune cells, the disclosed method comprising: labeling the immune cell population with an anti-TCR antibody and an anti-CD 3 antibody; separating the anti-TCR antibody-labeled immune cells and the anti-CD 3 antibody-labeled immune cells from the unlabeled immune cells; and collecting the unlabeled immune cells, thereby obtaining a population of immune cells depleted of cells expressing endogenous TCRs. The methods may also be applicable to populations of immune cells that include labeling with an anti-TCR antibody and one or more antibodies against any other target of interest other than CD3 (e.g., MHC1, MHC2, CD52, or PD 1).

In one aspect, the disclosure provides a method of depleting TCR α β + cells, the method comprising contacting a population of immune cells with a TCR antibody and a CD3 antibody. The methods may also be applicable to populations of immune cells that include labeling with an anti-TCR antibody and one or more antibodies against any other target of interest other than CD3 (e.g., MHC1, MHC2, CD52, or PD 1).

The present disclosure provides methods for generating an engineered immune cell population depleted of immune cells expressing endogenous TCRs, the disclosed methods comprising: labeling the immune cell population with an anti-TCR antibody and an anti-CD 52 antibody; and isolating anti-TCR antibody-labeled immune cells and anti-CD 52 antibody-labeled immune cells from the engineered immune cell population.

The present disclosure also provides a method of depleting cells expressing an endogenous TCR from a population of immune cells, the disclosed method comprising: labeling the immune cell population with an anti-TCR antibody and an anti-CD 52 antibody; separating the anti-TCR antibody-labeled immune cells and the anti-CD 52 antibody-labeled immune cells from the unlabeled immune cells; and collecting the unlabeled immune cells, thereby obtaining a population of immune cells depleted of cells expressing endogenous TCRs.

In another aspect, the disclosure provides a method of depleting TCR α β + cells, the method comprising contacting a population of immune cells with an anti-TCR antibody and a CD52 antibody. The effectiveness of these methods demonstrated in the examples is unexpected because CD52 and TCR are not physically associated with each other in human cells, and while not wishing to be bound by any theory, it is understood that CD52 and TCR are not biologically directly related. Thus, in another aspect of the disclosure, the disclosed methods can include the use of an anti-TCR antibody and another antibody directed against a target of interest that is not normally associated with a TCR.

In yet another aspect, the present disclosure provides a method of depleting TCR α β + cells, the method comprising contacting a population of immune cells with an anti-TCR antibody, an anti-CD 3 antibody, and an anti-CD 52 antibody.

The methods described herein use anti-TCR antibodies and anti-CD 3 antibodies together for TCR α β + cell depletion, which is particularly useful for generating engineered TCR α β -cell based allogeneic CAR T products. Prior to the present disclosure, depletion of engineered T cells (not unmodified T cell products) to 99.9% -99.99% TCR-cell purity levels on a clinical scale to leave 0.1% -0.01% TCR + cell remnants was extremely difficult and represents a challenge for the development of allogeneic therapies. The present disclosure provides a solution to this problem.

Achieving this low level of residual TCR + cells (e.g., TCR cells in the range of 0.1% -0.01% measured on day 1 post-depletion) represents a significant advance in engineered allogeneic CAR T products (e.g., TCR α/β -CAR T cells) and provides patients with improved clinical benefit by reducing the GvHD potential.

An effective method of reducing the risk of GvHD (and HvGD) and enhancing anti-tumor efficacy may be particularly useful for making effective, safe, and pure engineered allogeneic immune cells (e.g., allogeneic CAR-T cells expressing a CAR targeted to a cancer antigen). In some embodiments, the present disclosure provides methods for producing allogeneic CAR-T cells and products that reduce the risk of, or prevent progression of, GvHD and/or HvGD. In some embodiments, the engineered allogeneic CAR-T cells have enhanced anti-tumor activity against solid tumors or hematologic cancers. In some embodiments, the CAR-T cells comprise a population of cells modified to reduce or eliminate expression of one or more of endogenous TCR, CD52, MHC1, MHC2, or PD1 gene expression. Thus, in some embodiments, the CAR-T cells comprise a population depleted of TCR +, CD52+, MHC1+, MHC2+, and/or PD1+ cells.

Provided herein are methods of depleting a population of cells (e.g., TCR +, CD3+, CD52+, MHC1+, MHC2+, and/or PD1+ cells) expressing one or more of endogenously expressed TCR, CD3, CD52, MHC1, MHC2, or PD1 from a population of engineered immune cells, and kits comprising antibody reagents for use in the methods.

T cell receptor signaling

The T cell receptor complex (TCR as described herein) is an antigen receptor molecule on the surface of a T cell, responsible for recognizing antigens presented to the T cell by MHC molecules that may lead to T cell activation and an immune response to the antigen. The human T cell receptor is a heterodimer consisting of two transmembrane heterodimeric glycoprotein chains, an alpha chain and a beta chain, each having two disulfide-linked domains (with a small number of gamma delta chains as well). Since the cytoplasmic tail of the TCR is short, it cannot directly signal when bound to the peptide-MHC complex. In contrast, TCRs are associated with a group of signaling molecules collectively referred to as CD3 that transmit intracellular signals when the TCR is bound to a peptide-MHC complex.

CD3 consists of one gamma and delta molecule and two epsilon molecules, which all have some limited sequence homology with immunoglobulin domains in their extracellular domains. These molecules have a small cytoplasmic domain and a transmembrane domain with negatively charged residues. In the membrane, these negatively charged residues form salt bridges with positively charged residues in the TCR transmembrane region. The TCR-CD3 receptor complex is completed by two other invariant proteins ζ and η that form dimers linked by disulfide bonds. Thus, at the surface of T cells, the TCR-CD3 complex is expressed as an α β (or γ δ) heterodimer associated with CD3 γ epsilon and CD3 δ epsilon dimers having intracellular ζ ζ homodimers or ζ η heterodimers.

Without wishing to be bound by theory, some researchers believe that CD3 surface expression may also be abolished if TCR expression is disrupted. However, as described herein, it was unexpectedly found that residual CD3 surface expression can still be indicative of residual TCR + cells and TCR + depletion efficiency can be improved by using anti-CD 3 antibodies in combination with anti-TCR antibodies.

Method for sorting and depleting immune cells

As described herein, in one aspect, the present disclosure provides a method of depleting cells expressing endogenous TCR α/β dimers from a population of immune cells, the method comprising: labeling the cell population with an anti-TCR antibody and an anti-CD 3 antibody (the labeling can be performed sequentially or simultaneously in a single operation); anti-TCR and anti-CD 3 labeled cells were separated from unlabeled cells, thereby obtaining a population of cells depleted of cells expressing endogenous TCR.

Initially, engineered immune cells modified to be deficient in an endogenous TCR α or TCR β gene are exposed to a TCR-depleting agent. In some embodiments, the TCR-depleting agent comprises an antibody that targets a TCR α polypeptide, a TCR β polypeptide, or a TCR α/β heterodimer endogenously expressed on the surface of an immune cell. As described herein, TCR depletion using a combination of an anti-TCR antibody and an anti-CD 3 antibody (and/or optionally, an anti-CD 52 antibody) antibody unexpectedly increases TCR + depletion efficiency compared to depletion performed with an anti-TCR antibody alone.

An anti-TCR antibody, an anti-CD 3 antibody, or an anti-CD 52 antibody (or any other antibody directed to a target of interest for TCR depletion procedures) may be conjugated to biotin to facilitate further labeling and/or isolation using a secondary antibody (e.g., an anti-biotin antibody) conjugated directly or indirectly to a magnetic depletion agent, such as a magnetic depleting agent, such as magnetic microbeads (typically but not necessarily nanoparticles of about 50nm in diameter) or any other surface (such as agarose beads, sonic particles, plastic well plates, glass well plates, ceramic well plates, columns, cell culture bags or membranes). When magnetic microbeads are used, the microbeads facilitate the separation of TCR + cells from TCR-cells; when contacted with the magnetic column, TCR + cells may remain on the column while unlabeled TCR-cells pass through to the collection bag. When exposed to acoustic waves, the acoustic particles can facilitate the separation of TCR + from TCR-cells. Although anti-biotin antibodies are provided in the context of the disclosed methods, other biotin binding partners such as streptavidin, avidin, and other biotin-recognizing proteins can be employed in place of anti-biotin antibodies in all of the methods provided herein.

In some embodiments, the provided cells can optionally be sorted for other cell surface markers. For example, a subset of the immune cell population can include engineered immune cells that express an antigen-specific CAR that itself includes one or more epitopes specific for one or more monoclonal antibodies (e.g., exemplary mimotope sequences; see, e.g., WO2016/120216, incorporated herein by reference). The method comprises the following steps: contacting a population of immune cells with a monoclonal antibody specific for an epitope; and selecting immune cells that bind to the monoclonal antibody to obtain a population of cells enriched for engineered immune cells expressing the antigen-specific CAR.

In some embodiments, the monoclonal antibody specific for the epitope is optionally conjugated to a fluorophore. In this example, the step of selecting cells that bind to the monoclonal antibody can be accomplished by Fluorescence Activated Cell Sorting (FACS).

In some embodiments, the monoclonal antibody specific for the epitope is optionally conjugated to a magnetic particle. In this example, the step of selecting cells that bind to the monoclonal antibody can be accomplished by Magnetic Activated Cell Sorting (MACS).

In some embodiments, the monoclonal antibody used in the method for sorting immune cells expressing a CAR is selected from alemtuzumab (alemtuzumab), ibritumomab tiuxetan (ibritumomab tiuxetan), muruzumab-CD 3(muromonab-CD3), tositumomab (tositumomab), abciximab (abciximab), basiliximab (basiliximab), brentuximab (brentuximab vedotin), cetuximab (cetuximab), infliximab (infliximab), rituximab (rituximab), bevacizumab (bevacizumab), certolizumab (certolizumab pegol), daclizumab (daclizumab), eculizumab (eculizumab), efuzumab (rituzumab), eculizumab (gemtuzumab), gemtuzumab (daclizumab), eculizumab (rituzumab), rituzumab (rituximab), efuzumab), or (rituximab), or (rituximab), or (zevulizumab), or (rituximab), or (rituximab), or (daclizumab), or (e), or (e), or (e (daclizumab), or (e), or (e), or (e), or (e), or (daclizumab), or (e), or (e), or (e), or (e), or (e), or (e), or (e) or (e), or (e), or (, Belimumab (belimumab), connamab (canakinumab), denosumab (denosumab), golimumab (golimumab), yipima (ipilimumab), ofatumab (ofatumab), pertuzumab (panitumumab), QBEND-10 and/or eculizumab (usekinumab). In some embodiments, the mAb is rituximab. In another embodiment, the mAb is QBEND-10.

Flow cytometry can be used to quantify specific cell types in a cell population. In general, flow cytometry is a method for quantifying a component or structural feature of a cell, primarily by optical means. Since different cell types can be distinguished by quantifying structural features, flow cytometry and cell sorting can be used to count and sort cells of different phenotypes in a mixture.

Flow cytometry analysis involves two main steps: 1) labeling the selected cell type with one or more detectable markers, and 2) determining the number of labeled cells relative to the total number of cells in the population. In some embodiments, the method of labeling a cell type comprises binding a labeled antibody to a marker expressed by a particular cell type. The antibody may be directly labeled with a fluorescent compound or indirectly labeled using, for example, a fluorescently labeled secondary antibody that recognizes the primary antibody.

In some embodiments of the disclosed methods, sorting or isolating TCR + cells, optionally expressing a CAR, from TCR-cells can be achieved using Magnetically Activated Cell Sorting (MACS). Magnetic Activated Cell Sorting (MACS) is a method for separating various cell populations based on their surface antigens (CD molecules) by using superparamagnetic nanoparticles and columns. MACS can be used to obtain very pure cell populations. Cells in single cell suspensions can be magnetically labeled with microbeads. The sample is applied to a column consisting of ferromagnetic material covered with a coating that does not damage the cells, allowing a rapid and gentle separation of the cells. Unlabeled cells pass through the column, while magnetically labeled cells remain within the column. The flow-through can be collected as an unlabeled cell fraction. After the washing step, the column is removed from the separator and the magnetically labeled cells are eluted from the column.

Detailed protocols for the purification of specific cell populations, such as T cells, can be found in Basu S et al (2010). (Basu S, Campbell HM, Dittel BN, Ray A. "Purification of specific cell populations by Fluorescence Activated Cell Sorting (FACS)," journal of visual experiments (J.Vis. Exp.) (41): 1546).

In some embodiments of the disclosed methods, sorting or isolating TCR + cells, optionally expressing a CAR, from TCR-cells can be achieved using sonic separation instead of magnetic-based separation methods. While not wishing to be bound by theory, it is understood that sonic separation relies on three-dimensional standing waves to separate components of a mixture. In the context of the disclosed methods, antibodies, such as anti-TCR antibodies, anti-CD 52 antibodies, or anti-CD 3 antibodies, can be conjugated to a surface, such as sonic particles. The sonic particles may be beads. In embodiments, the cells are exposed to sonic particles bearing one or more of an anti-TCR antibody, an anti-CD 52 antibody, or an anti-CD 3 antibody, thereby associating the sonic particles with any cells expressing the target of interest. The cells were then placed in an acoustic chamber and exposed to acoustic waves. In view of the different properties of the bead-associated cells and the cells that are not labeled with antibody-bead particles, the acoustic waves separate the labeled cells from the unlabeled cells, which can be collected when the labeled cells (e.g., TCR + or CD52+ cells) are transferred away from the labeled cells.

Immune cell

Cells suitable for use in the TCR + cell depletion methods described herein comprise immune cells.

Cells (e.g., immune cells) for use in the methods described herein can be obtained from a subject prior to in vitro manipulation or genetic modification (e.g., as described herein). Cells can be obtained from a variety of non-limiting subject-based sources, including Peripheral Blood Mononuclear Cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines known to those of skill in the art may be used. In some embodiments, the cells may be derived from a healthy donor or a patient diagnosed with cancer. In some embodiments, the cells may be part of a mixed population of cells exhibiting different phenotypic characteristics.

In embodiments, the immune cells are obtained from a subject who will eventually receive engineered immune cells (i.e., autologous therapy). In embodiments, the immune cells are obtained from a donor that is a different individual than the subject that will receive the engineered immune cells (i.e., the allogeneic therapy).

Cells may be obtained from the circulating blood of an individual by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In certain embodiments, cells collected by apheresis may be washed to remove plasma fractions and placed in a suitable buffer or culture medium for subsequent processing.

PBMCs can be used directly for genetic modification to generate engineered immune cells (e.g., CARs or TCRs) using the methods described herein. In certain embodiments, after PBMC isolation, T lymphocytes may optionally be further isolated to generate populations comprising only T cells, and cytotoxic and helper T lymphocytes may be further sorted into naive, memory and effector T cell subsets, either before or after genetic modification and/or expansion.

In certain embodiments, T cells can be obtained by, for example, using a cell obtained byCentrifugation of the gradient lysed erythrocytes and depleted monocytes for separation from PBMCs. Specific subsets of T cells such as CD28+, CD3+, CD4+, CD8+, CD25+, CD62L +, CD5+, CD45RA-, CCR7+, CD95+, IL2R β +, and CD45RO + T cells can be further isolated by positive or negative selection techniques known in the art. For example, enrichment of a T cell population by negative selection can be accomplished by a combination of antibodies directed against surface markers specific to the negatively selected cells. One method for use herein is cell sorting and/or cell selection by negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically comprises antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD 8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present disclosure.

In some embodiments, the population of T cells is enriched for CD8+ cells prior to or after application of the depletion methods provided herein.

In some embodiments, the population of T cells is enriched for CD4+ cells prior to or after application of the depletion methods provided herein.

In some embodiments, the population or CD4+ and/or CD8+ cells can be further sorted into naive cells, stem cell memory cells, central memory cells, effector memory cells, and effector cells by identifying cell surface antigens associated with each of these types of cells. In some embodiments, the expression of phenotypic markers of naive T cells comprises CD45RA +, CD95-, IL2R β -, CCR7+, and CD62L +. In some embodiments, the expression of phenotypic markers for stem cell memory T cells comprises CD45RA +, CD95+, IL2R β +, CCR7+, and CD62L +. In some embodiments, the expression of the phenotypic marker of a central memory T cell comprises CD45RO +, CD95+, IL2R β +, CCR7+, and CD62L +. In some embodiments, expression of the phenotypic marker of effector memory T cells comprises CD45RO +, CD95+, IL2R β +, CCR7-, and CD 62L-. In some embodiments, expression of the phenotypic marker of a T effector cell comprises CD45RA +, CD95+, IL2R β +, CCR7-, and CD 62L-. Thus, by identifying cell populations with cell surface antigens, CD4+ and/or CD8+ T helper cells can be sorted into naive cells, stem cell memory cells, central memory cells, effector memory cells, and T effector cells. In particular embodiments, the disclosed methods can be used to enhance the depletion of one or more of these subsets of T cells.

It will be appreciated that PBMCs may further comprise other cytotoxic lymphocytes, such as NK cells or NK T cells. Expression vectors carrying the coding sequence of a chimeric acceptor as disclosed herein can be introduced into human donor T cells, NK cells, or a population of NK T cells. Standard procedures can be used for cryopreservation of CAR-expressing T cells for storage and/or preparation for use in a human subject. In one embodiment, the in vitro transduction, culture, and/or expansion of T cells is performed in the absence of non-human animal-derived products (e.g., calf serum and fetal bovine serum). In various embodiments, the cryopreservation media may include, for exampleCS2, CS5 or CS10 or other media including DMSO or media not including DMSO.

Engineered immune cells

The TCR + cell depletion methods described herein may be particularly useful in the manufacture of immune cell therapies (including therapies comprising engineered immune cells (e.g., CAR-T cells)) for the treatment of cancer.

The engineered immune cells can be allogeneic or autologous, and the disclosed methods can be applied to autologous or allogeneic therapy.

In some embodiments, the engineered immune cell (or population thereof) is or comprises a T cell (e.g., an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, a helper T lymphocyte, a Tumor Infiltrating Lymphocyte (TIL)), an NK cell, an NK-T cell, a TCR-expressing cell, a dendritic cell, a killer dendritic cell, a mast cell, or a macrophage. In some embodiments, the engineered immune cells may be derived from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes, or a combination thereof. In some exemplary embodiments, the engineered immune cell is a T cell. The T cells may also be gamma/delta T cells.

In some embodiments, the engineered immune cells may be derived from, for example, but not limited to, stem cells. The stem cell may be an adult stem cell, a non-human embryonic stem cell, more specifically a non-human stem cell, a cord blood stem cell, a progenitor cell, a bone marrow stem cell, an Induced Pluripotent Stem Cell (iPSC), a totipotent stem cell, or a hematopoietic stem cell. The stem cell may be CD34+ or CD 34-.

In some embodiments, the cells are obtained or prepared from peripheral blood. In some embodiments, the cells are obtained or prepared from Peripheral Blood Mononuclear Cells (PBMCs). In some embodiments, the cells are obtained or prepared from bone marrow. In some embodiments, the cell is a human cell. In some embodiments, the cell is transfected or transduced with the nucleic acid vector using a method selected from the group consisting of viral (lentivirus or gamma retrovirus) transduction, electroporation, sonoporation, biolistics (e.g., gene gun), lipofection, polymer transfection, nanoparticles, or polymeric complexes. In some embodiments, the cell is a T cell reprogrammed from a non-T cell. In some embodiments, the cell is a T cell reprogrammed from a T cell.

Binding agents (including antibodies and fragments thereof)

In embodiments, the disclosed methods comprise the use of an antibody or antigen binding agent (e.g., comprising an antigen binding domain or comprising an antibody or fragment thereof). As discussed below, in various embodiments, the engineered immune cells can further include a binding agent.

As used herein, the term "antibody" refers to a polypeptide comprising canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As known in the art, a naturally occurring intact antibody is a tetrameric agent of approximately 150kD comprising two identical heavy chain polypeptides (each about 50kD) and two identical light chain polypeptides (each about 25kD) associated with each other in what is commonly referred to as a "Y-shaped" structure. Each heavy chain comprises at least four domains (each approximately 110 amino acids long) -an amino-terminal Variable (VH) domain (located at the tip of the Y structure), followed by three constant domains: CHI, CH2 and carboxy terminal CH3 (at the base of the Y stem). A short region, called a "switch," connects the heavy chain variable and constant regions. The "hinge" connects the CH2 domain and the CH3 domain to the rest of the antibody. Two disulfide bonds in this hinge region link the two heavy chain polypeptides in the intact antibody to each other. Each light chain includes two domains, an amino-terminal Variable (VL) domain, followed by a carboxy-terminal Constant (CL) domain. The skilled person is well familiar with antibody structures and sequence elements, recognizes "variable" and "constant" regions in the provided sequences, and understands that the definition of "boundaries" between such domains may have some flexibility such that different presentations of the same antibody chain sequence may indicate such boundaries at positions where one or several residues are transferred, e.g. relative to different presentations of said same antibody chain sequence.

A complete antibody tetramer includes two heavy chain-light chain dimers in which the heavy and light chains are connected to each other by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge region to each other, such that the dimers are connected to each other and the tetramer is formed. Naturally occurring antibodies are also typically glycosylated on the CH2 domain. Each domain in a native antibody has a structure characterized as an "immunoglobulin fold" formed by two beta sheets (e.g., 3-chain, 4-chain, or 5-chain sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops (CDR1, CDR2 and CDR3) and four slightly invariant "framework" regions (FR1, FR2, FR3 and FR4) called "complementarity determining regions". When a natural antibody is folded, the FR regions form a beta sheet that provides the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are placed together in three-dimensional space such that they form a single hypervariable antigen-binding site located at the tip of the Y structure. The Fc region of a naturally occurring antibody binds to elements of a complementary system and also to receptors on effector cells, including, for example, effector cells that mediate cellular cytotoxicity. The affinity and/or other binding properties of the Fc region of an Fc receptor can be modulated by glycosylation or other modifications, as is known in the art. In some embodiments, antibodies produced and/or utilized according to the present invention comprise a glycosylated Fc domain, including Fc domains having such glycosylation modified or engineered.

For the purposes of this disclosure, in certain embodiments, any polypeptide or polypeptide complex comprising sufficient immunoglobulin domain sequence as found in a native antibody, whether such polypeptide is naturally-occurring (e.g., produced by an organism reacting with an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methods, may be referred to and/or used as an "antibody". In some embodiments, the antibody is polyclonal; in some embodiments, the antibody is monoclonal. In some embodiments, the antibody has a constant region sequence with the characteristics of a mouse, rabbit, primate, or human antibody. In some embodiments, the antibody sequence elements are humanized, primatized, chimeric, etc., as is known in the art.

Furthermore, as used herein, the term "antibody" may refer to any of the constructs or formats known or developed in the art for utilizing antibody structural and functional characteristics in alternative presentations. For example, in some embodiments, the antibodies utilized in the methods of the present disclosure are in a form selected from, but not limited to: a whole IgA antibody, an IgG antibody, an IgE antibody, or an IgM antibody; bispecific or multispecific antibodies (e.g.,etc.); antibody fragments such as Fab fragments, F (ab)2 fragments, Fd fragments, and isolated CDRs or collections thereof; single-chain variable fragment (scFV); a polypeptide-Fc fusion; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); camelid antibodies (also referred to herein as nanobodies or VHHs); shark antibodies, masking antibodies (e.g.,) (ii) a Small Modular Immunopharmaceuticals (SMIPs)^TM) (ii) a Single chain diabodies or tandem diabodiesVHH；A minibody;ankyrin repeat proteins orDART; a TCR-like antibody; a trace amount of protein;andin some embodiments, the antibody may lack the covalent modifications it would have if it were naturally occurring (e.g., attachment of a polysaccharide). In some embodiments, the antibody can contain covalent modifications (e.g., attachment of a polysaccharide), payloads (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.), or other side groups (e.g.E.g., polyethylene glycol, etc.).

As used herein, the term "antibody agent" generally refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex comprising sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, the antibody agent may comprise one or more constant region sequences characteristic of a mouse, rabbit, primate, or human antibody. In some embodiments, the antibody agent may comprise one or more sequence elements known in the art as humanized, primatized, chimeric, and the like. In many embodiments, the term "antibody agent" is used to refer to one or more of the constructs or formats known or developed in the art for utilizing antibody structural and functional characteristics in alternative presentations. For example, the antibody agents utilized according to the present invention are in a form selected from, but not limited to: a whole IgA antibody, an IgG antibody, an IgE antibody, or an IgM antibody; bispecific or multispecific antibodies (e.g.,etc.); antibody fragments such as Fab fragments, Fab 'fragments, F (ab') 2 fragments, Fd fragments and isolated CDRs or collections thereof; single-chain Fv; a polypeptide-Fc fusion; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); camelid (camelid) antibodies; the masking antibody (e.g.,) (ii) a Small Modular Immunopharmaceuticals (SMIPs)^TM) (ii) a Single chain diabodies or tandem diabodiesVHH；A minibody;ankyrin repeat proteins orDART; a TCR-like antibody; a trace amount of protein;and

antibodies or antibody agents used to perform the methods of the present disclosure can be single-chain or double-chain. In some embodiments, the antibody or antigen binding molecule is single chain. In certain embodiments, the antigen binding molecule is selected from the group consisting of: scFv, Fab ', Fv, F (ab')₂A dAb, and any combination thereof.

Antibodies and antibody agents comprise antibody fragments. Antibody fragments include a portion of an intact antibody, such as the antigen binding or variable region of an intact antibody. Antibody fragments include, but are not limited to, Fab '-SH, F (ab')₂Fv, diabodies, linear antibodies, multispecific and other fragments formed from antibody fragment antibodies and scFv fragments. Antibodies also include, but are not limited to, polyclonal, monoclonal, chimeric dAbs (domain antibodies), single chain, Fab, Fa, F (ab')₂Fragments and scFv. The antibody may be a whole antibody or an immunoglobulin or antibody fragment. Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies as known in the art and production by recombinant host cells (e.g., e.coli, Chinese Hamster Ovary (CHO) cells, or phage).

In some embodiments, the antibody or antibody agent can be a chimeric antibody (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). A chimeric antibody may be an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species. In one example, a chimeric antibody can include a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In another example, a chimeric antibody can be a "class switch" antibody, wherein the class or subclass has been altered from that of the parent antibody. Chimeric antibodies comprise antigen-binding fragments thereof.

In some embodiments, the chimeric antibody may be a humanized antibody (see, e.g., Almagro and Fransson, front of bioscience (Front. biosci.), 13: 1619-. Humanized antibodies are chimeric antibodies comprising amino acid residues from non-human hypervariable regions and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the Framework Regions (FRs) correspond to those of a human antibody. The humanized antibody may optionally include at least a portion of an antibody constant region derived from a human antibody.

In some embodiments, the antibodies or antibody agents provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art (see, e.g., van Dijk and van de Winkel, new pharmacology (curr. opin. pharmacol), 5: 368-74 (2001); and Lonberg, new immunology (curr. opin. immunol.), 20: 450-. The human antibody can be a human antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using a human antibody library or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies that include non-human antigen binding residues. Human antibodies can be made using methods well known in the art.

Chimeric Antigen Receptor (CAR)

As described herein, the disclosed methods can be used to deplete TCR + cells from a population of immune cells. The immune cells can be CAR + cells and engineered for therapeutic applications like xenogeneic therapy. In some embodiments, the engineered immune cell comprises a CAR comprising an extracellular antigen-binding domain. As used herein, a Chimeric Antigen Receptor (CAR) includes a protein that specifically recognizes a target antigen (e.g., a target antigen on a cancer cell; a cancer antigen). When bound to a target antigen, the CAR can activate immune cells to attack and destroy cells (e.g., cancer cells) bearing the antigen. The CAR may also incorporate a co-stimulatory domain comprising all or a portion of a protein (such as 4-1BB, CD28, or OX 40) and/or a signaling domain (such as CD3 ζ) in order to increase its potency. See Krause et al, journal of experimental medicine (j.exp.med.), vol 188, vol 4, 1998 (619-; finney et al, Journal of Immunology 1998, 161: 2791, 2797, Song et al, Blood 119: 696-706 (2012); kalos et al, "journal of science transformation medicine (sci. trans. med.)" 3: 95 (2011); porter et al, new england journal of medicine (n.engl.j.med.) 365: 725-33(2011), and Gross et al, "yearbook in pharmacology and toxicology (rev. pharmacol. toxicol.) 56: 59-83 (2016); U.S. patent nos. 7,741,465 and 6,319,494.

The chimeric antigen receptors described herein comprise an ectodomain, a transmembrane domain, a hinge domain that optionally connects the ectodomain to the transmembrane domain, and an endodomain, wherein the ectodomain comprises an antigen binding domain that specifically binds to a target.

In some embodiments, the antigen-specific CAR further comprises a safety switch and/or a monoclonal antibody-specific epitope. In some embodiments, the antigen-selective CAR comprises a leader peptide or a signal peptide.

CAR antigen binding domains

As discussed above, the CARs described herein include an antigen binding domain. As used herein, "antigen binding domain" refers to any polypeptide that binds to a specified target antigen. In some embodiments, the antigen binding domain is a scFv. In some embodiments, the antigen binding domain binds to an antigen on a tumor cell. In some embodiments, the antigen binding domain binds to an antigen on a cell involved in a hyperproliferative disease (e.g., Non-Hodgkin's Lymphoma), multiple myeloma or other solid or hematological cancer). The disclosed methods can be used to deplete TCR + cells, which are also CAR + cells, thereby providing a CAR +/TCR-cell population.

In some embodiments, the antigen binding domain of the CAR comprises a variable heavy chain, a variable light chain, and/or one or more CDRs as described herein. In some embodiments, the antigen binding domain is a single chain variable fragment (scFv) comprising the light chain CDR1, CDR2, and CDR3, and the heavy chain CDR1, CDR2, and CDR 3.

An antigen binding domain is said to be "selective" when it binds more tightly to one target than to a second target.

The antigen binding domain of the CAR selectively targets a cancer antigen. In some embodiments, the cancer antigen is selected from EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, tight junction protein (Claudin) -18.2, Muc17, FAP α, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, CD52, or CD 34. In some embodiments, the CAR comprises an antigen binding domain that targets EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, claudin-18.2, Muc17, FAP α, Ly6G6D, c6orf23, G6D, MEGT1 NG25, CD19, BCMA, FLT3, CD70, DLL3, CD52, or CD 34.

In some embodiments, the cancer antigen is selected from the group consisting of: carbonic Anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CDS, CD7, CDIO, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, antigens of Cytomegalovirus (CMV) infected cells (e.g., cell surface antigens), epithelial glycoprotein (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine protein kinase-B2, 3, 4, Folate Binding Protein (FBP), fetal acetylcholine receptor (AChR), folate receptor, ganglioside G2(GD2), ganglioside G3(GD3), human epidermal growth factor receptor 2(HER-2), human telomerase reverse transcriptase (hTERT), interleukin 13 receptor subunit alpha-2 (IL-13 receptor subunit 2), light chain kinase (Ra 19), and Lewis CA19. structural domain (KDCA) LI cell adhesion molecule (LICAM), melanoma antigen family A, 1(MAGE-AI), mucin 16(Muc-16), mucin 1(Muc-1), Mesothelin (MSLN), NKG2D ligand, cancer-testis antigen NY-ESO-1, carcinoembryonic antigen (h5T4), Prostate Stem Cell Antigen (PSCA), Prostate Specific Membrane Antigen (PSMA), tumor associated glycoprotein 72(TAG-72), vascular endothelial growth factor R2(VEGF-R2), and Wilms tumor protein (Wilms tumor protein) (WT-1).

In other embodiments, the disclosure relates to an isolated polynucleotide encoding any of the antigen binding domains described herein. In some embodiments, the disclosure relates to an isolated polynucleotide encoding a CAR. Also provided herein are vectors comprising the polynucleotides and methods of making the same.

In some embodiments, a CAR immune cell (e.g., a CAR-T cell) that can form a component of a cell population produced by practicing the methods of the present disclosure includes a polynucleotide encoding a safety switch polypeptide, such as RQR 8. See, for example, WO 2013153391a, which is hereby incorporated by reference in its entirety. In a CAR immune cell (e.g., CAR-T cell) comprising the polynucleotide, the safety switch polypeptide can be expressed at the surface of the CAR immune cell (e.g., CAR-T cell).

CAR hinge domain

The extracellular domain of the CARs of the present disclosure may include a "hinge" domain (or hinge region). The term generally refers to any polypeptide that functions to link a transmembrane domain in a CAR to an extracellular antigen-binding domain in a CAR. In particular, the hinge domain may be used to provide greater flexibility and accessibility to the extracellular antigen-binding domain.

The hinge domain may comprise up to 300 amino acids, in some embodiments, 10 to 100 amino acids, or in some embodiments, 25 to 50 amino acids. The hinge domain may be derived from all or part of a naturally occurring molecule, such as from all or part of the extracellular region of CD8, CD4, CD28, 4-1BB, or IgG (specifically, the hinge region of IgG; it is understood that the hinge region may contain all or part of the extracellular region of an immunoglobulin family member, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM or fragments thereof), or from all or part of an antibody heavy chain constant region. Alternatively, the hinge domain may be a synthetic sequence corresponding to a naturally occurring sequence, or may be a fully synthetic hinge sequence. In some embodiments, the hinge domain is part of the human CD8 a chain (e.g., NP _ 001139345.1). In another particular embodiment, the hinge and transmembrane domain comprise a portion of the human CD8 a chain. In some embodiments, the hinge domain of a CAR described herein comprises a subsequence of CD8 a, IgG1, IgG4, PD-1, or fcyriii a, specifically the hinge region of any one of CD8 a, IgG1, IgG4, PD-1, or fcyriii a. In some embodiments, the hinge domain comprises a human CD8 a hinge, a human IgG1 hinge, a human IgG4, a human PD-1, or a human Fc γ RIII a hinge. In some embodiments, the CARs disclosed herein include an scFv, a CD8 a human hinge and transmembrane domain, a CD3 ξ signaling domain, and a 4-1BB signaling domain.

CAR transmembrane domain

The CARs provided herein are designed to have a transmembrane domain fused to the extracellular domain of the CAR. The transmembrane domain can be similarly fused to the intracellular domain of the CAR. In some examples, 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, thereby minimizing interaction with other members of the receptor complex. In some embodiments, the short linker can form a linkage between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.

The transmembrane region may be synthetic (non-naturally occurring) or may be derived from (including or corresponding to fragments of): CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), induced T-cell costimulatory factor (ICOS), lymphocyte function-associated antigen 1(LFA-1, CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B3-H3), LIGHT, (TNFSF 3), NKG 23, Ig alpha (CD79 3), DAP-10, Fc gamma receptor, MHC 3-like molecule, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B3-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, LIFT, EM (LIFT 3), HVLA, NK3892 p 2, NKTR-2, NK3892, CD3, NKF 2, NK3892, CD3 alpha, CD-1, CD3 delta, CD3, CD-1, CD-gamma, CD-1, CD-3-gamma, CD3, and optionally, IL-2 Rgamma, IL-7 Ra, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, LFA-1, ITGAM, CD11, ITGAX, CD11, ITGB, CD, ITGB, LFA-1, ITGB, NKG2, TNFR, TRANCE/RANKL, DNAM (CD226), SLAMF (CD244, 2B), CD (tactile), CEACAM, CRT AM, Ly (CD229), CD160 (BY), PSGL, CD100(SEMA 4), CD, SLAMF (NTB-108), SLAMF (CD 150, IPO-3), BLAME (SLAMF), SELPLG (CD162), LTBR, SLP, LAT-76, PAG-19, CD19, or any combination thereof that specifically binds to any ligand.

In some embodiments, the transmembrane domain in the CAR of the present disclosure is a CD8 a transmembrane domain.

In some embodiments, the transmembrane domain in the CAR of the present disclosure is a CD28 transmembrane domain.

CAR intracellular domain

The intracellular (cytoplasmic) domain of the CAR can provide activation of at least one of the normal effector functions of an immune cell comprising the CAR. For example, effector function of a T cell may refer to cytolytic activity or helper activity, including secretion of cytokines.

It will be understood that suitable (e.g., activated) intracellular domains include, but are not limited to, signaling domains derived from (including or corresponding to all or fragments of): CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), induced T-cell costimulatory factor (ICOS), lymphocyte function-associated antigen 1(LFA-1, CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B3-H3), LIGHT, (TNFSF 3), NKG 23, Ig alpha (CD79 3), DAP-10, Fc gamma receptor, MHC 3-like molecule, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B3-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, LIFT, EM (LIFT 3), HVLA, NK3892 p 2, NKTR-2, NK3892, CD3, NKF 2, NK3892, CD3 alpha, CD-1, CD3 delta, CD3, CD-1, CD-gamma, CD-1, CD-3-gamma, CD3, and optionally, IL-2 Rgamma, IL-7 Ra, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, LFA-1, ITGAM, CD11, ITGAX, CD11, ITGB, CD, ITGB, LFA-1, ITGB, NKG2, TNFR, TRANCE/RANKL, DNAM (CD226), SLAMF (CD244, 2B), CD (tactile), CEACAM, CRTAM, Ly (CD229), CD160 (BY), PSGL, CD100(SEMA 4), CD, SLAMF (NTB-108), SLAM (AMF, CD150, IPO-3), BLAME (SLAMF), SELPLG (CD162), LTBR, SLP, PAG, SLP-76, LAT/DS, CD19, or any combination thereof that specifically binds to a ligand.

In some embodiments, the intracellular/cytoplasmic domain of the CAR can be designed to include the 41BB/CD137 domain alone or in combination with any other desired intracellular domain useful in the context of the CAR. NCBI reference sequence: the complete native amino acid sequence of 41BB/CD137 was described in NP-001552.2. NCBI reference sequence: the complete native 41BB/CD137 nucleic acid sequence is described in NM-001561.5.

In some embodiments, the intracellular/cytoplasmic domain of a CAR can be designed to include the CD28 domain alone or in combination with any other desired intracellular domain useful in the context of the CARs of the present disclosure. NCBI reference sequence: the complete native amino acid sequence of CD28 is described in NP _ 006130.1. NCBI reference sequence: the complete native CD28 nucleic acid sequence is described in NM — 006139.1.

In some embodiments, the intracellular/cytoplasmic domain of the CAR can be designed to include all or a fragment of the CD3 zeta domain alone or in combination with any other desired intracellular domain useful in the context of the CAR.

In some embodiments, the intracellular signaling domain of the CAR comprises a domain of a co-stimulatory molecule. In some embodiments, the intracellular signaling domain of the CAR comprises a portion of a co-stimulatory molecule selected from the group consisting of a fragment of 41BB (GenBank: AAA53133.) and CD28(NP _ 006130.1).

Engineered immune cells comprising a CAR

Provided herein are engineered immune cells (e.g., CAR-T cells or CAR + cells) and engineered immune cell populations that express a CAR that deplete cells that have expressed endogenous TCRs.

In some embodiments, the engineered immune cells comprise CAR T cells, each CAR T cell comprising an extracellular antigen-binding domain and having reduced or eliminated expression of an endogenous TCR. In some embodiments, the engineered immune cell population comprises a population of CAR T cells, each CAR T cell comprising two or more different extracellular antigen binding domains and having reduced or eliminated expression of an endogenous TCR. In some embodiments, the immune cell comprises a population of CARs, each CAR T cell comprising the same extracellular antigen-binding domain and having reduced or eliminated expression of an endogenous TCR.

The engineered immune cells may be allogeneic or autologous.

In some embodiments, the engineered immune cell or engineered immune cell population is a T cell (e.g., an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, a helper T lymphocyte, a Tumor Infiltrating Lymphocyte (TIL)), an NK cell, an NK-T cell, a TCR-expressing cell, a dendritic cell, a killer dendritic cell, a mast cell, or a B cell, and expresses a CAR. In some embodiments, the T cells may be derived from the group consisting of: CD4+ T lymphocytes, CD8+ T lymphocytes, or a population comprising a combination of CD4+ and CD8+ T cells.

In some embodiments, the engineered immune cells or engineered immune cell populations produced using the disclosed methods can be derived from, for example, but not limited to, stem cells. The stem cell may be an adult stem cell, a non-human embryonic stem cell, more specifically a non-human stem cell, a cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell, or a hematopoietic stem cell.

In some embodiments, the engineered immune cells or immune cell populations produced using the disclosed methods are obtained or prepared from peripheral blood. In some embodiments, the engineered immune cells are obtained or prepared from Peripheral Blood Mononuclear Cells (PBMCs). In some embodiments, the engineered immune cells are obtained or prepared from bone marrow. In some embodiments, the engineered immune cells are obtained or prepared from umbilical cord blood. In some embodiments, the cell is a human cell. In some embodiments, the cell is transfected or transduced with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., gene gun), lipofection, polymer transfection, nanoparticles, viral transfection (e.g., retrovirus, lentivirus, AAV), or a polymer complex.

In some embodiments, an engineered immune cell expressing an antigen-specific CAR at its cell surface membrane comprises a percentage of stem cell memory cells and central memory cells that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

In some embodiments, an engineered immune cell expressing an antigen-specific CAR of the disclosure at its cell surface membrane comprises a percentage of stem cell memory cells and central memory cells of about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 15% to about 100%, about 15% to about 90%, about 15% to about 80%, about 15% to about 70%, about 15% to about 60%, about 15% to about 50%, about 15% to about 40%, about 15% to about 30%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, or about 20% to about 30%, About 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 40%, about 40% to about 100%, about 40% to about 90%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 100%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 90%, about 70% to about 80%, about 80% to about 100%, about 80% to about 90%, about 90% to about 100%, about 25% to about 50%, about 75% to about 100%, or about 50% to about 75%.

In some embodiments, the engineered immune cells expressing the antigen-specific CAR at their cell surface membrane comprise a percentage of stem cell memory cells and central memory cells that is greater than 10%, 20%, 30%, 40%, 50%, or 60%.

In some embodiments, an engineered immune cell expressing an antigen-specific CAR at its cell surface membrane comprises a percentage of stem cell memory cells and central memory cells of about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 15% to about 50%, about 15% to about 40%, about 20% to about 60%, or about 20% to about 70%.

In some embodiments, an engineered immune cell expressing an antigen-specific CAR at its cell surface membrane is enriched for T_CMCells and/or T_SCMCells such that the engineered immune cells comprise at least about 60%, 65%, 70%, 75%, or 80% combined T_CMCells and T_SCMA cell. In some embodiments, an engineered immune cell expressing an antigen-specific CAR at its cell surface membrane is enriched for T_CMCells and/or T_SCMCells such that the engineered immune cells comprise at least about 70% combined T_CMCells and T_SCMA cell. In some embodiments, an engineered immune cell expressing an antigen-specific CAR at its cell surface membrane is enriched for T_CMCells and/or T_SCMCells such that the engineered immune cells comprise at least about 75% combined T_CMCells and/or T_SCMA cell.

In some embodiments, an engineered immune cell expressing an antigen-specific CAR of the disclosure at its cell surface membrane is enriched for T_CMCells and/or T_SCMCells such that the engineered immune cells comprise at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% T_SCMA cell. In some embodiments, an engineered immune cell expressing an antigen-specific CAR of the disclosure at its cell surface membrane is enriched for T_CMCells and/or T_SCMCells such that the engineered immune cells comprise at least about 30%, 35%, 40%, or 45% T_SCMA cell.

In some embodiments, the engineered immune cell is an inflammatory T lymphocyte that expresses the CAR. In some embodiments, the engineered immune cell is a cytotoxic T lymphocyte that expresses the CAR. In some embodiments, the engineered immune cell is a regulatory T lymphocyte that expresses the CAR. In some embodiments, the engineered immune cell is a helper T lymphocyte that expresses the CAR.

In some embodiments, an engineered immune cell according to the present disclosure may include one or more genes that are disrupted or inactivated. In some embodiments, a target antigen (e.g., EGFRvIII, Flt, WT-1, CD133, MHC-WT, TSPAN, MHC-PRAME, Liv, ADAM, CHRNA, LeY, NKGD2, CS, CD44v, ROR, tight junction protein-18.2, Muc, FAP α, Ly6G6, c6orf, G6, MEGT, NG, CD, BCMA, Flt, CD, DLL, or CD, CD) gene may be knocked out to introduce a CAR (e.g., EGFRvIII, Flt, WT-1, CD133, MHC-WT, TSPAN, MHC-PRAME, Liv, ADAM, CHRNA, LeY, NKGD2, CS, CD44v, ROR, tight junction protein-18.2, Muc, FAP α, lyorf 6G6, CAR, flg 6, dlt, CD) gene to induce activation or to avoid activation. As described herein, in some embodiments, an engineered immune cell according to the present disclosure comprises one disrupted or inactivated gene and/or expresses a CAR or a multi-chain CAR, the gene selected from the group consisting of: MHC1 (. beta.2M), MHC2(CIITA), EGFRvIH, Flt3, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, tight junction protein-18.2, Muc17, FAP α, LV6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3 or CD34, CD70, TCR α and TCR β. In some embodiments, the cell comprises a multi-chain CAR. In some embodiments, the isolated cell comprises two disrupted or inactivated genes and/or expresses a CAR or a multi-chain CAR, the genes selected from the group consisting of: CD52 and TCR α, CDR52 and TCR β, PD-1 and TCR α, PD-1 and TCR β, MHC-1 and TCR α, MHC-1 and TCR β, MHC2 and TCR α, MHC2 and TCR β.

In some embodiments, the method comprises disrupting or inactivating one or more genes by introducing into the cell an endonuclease capable of selectively inactivating the genes by selective DNA cleavage. In some embodiments, the endonuclease can be, for example, a Zinc Finger Nuclease (ZFN), megaTAL nuclease, meganuclease, or a meganucleaseNucleases, transcription activator-like effector nucleases (TALE-nucleases or) Or a CRISPR (e.g., Cas9 or Cas12) endonuclease.

In some embodiments, the TCR + cells or population thereof are rendered non-functional in the cells by disruption of or inactivation of endogenous TCR a and/or TCR β genes. The depletion methods of the present disclosure are particularly useful for removing any remaining TCR + T cells from the cell population after inactivation of the TRC α gene. The engineered immune cells produced using the methods disclosed herein can be used to treat a patient in need thereof by preventing or reducing host-versus-graft (HvGD) rejection and graft-versus-host disease (GvHD); accordingly, within the scope of the present disclosure is a method of treating a patient in need thereof by preventing or reducing host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD), the method comprising treating the patient by administering to the patient an effective amount of engineered immune cells comprising disrupted or inactivated TCR a and/or TCR β genes.

The present disclosure provides methods of increasing the purity of an engineered immune cell population that lacks endogenous TCR expression or has reduced endogenous TCR expression. In some embodiments, the engineered immune cells comprise less than 1.0% TCR + cells, less than 0.9% TCR + cells, less than 0.8% TCR + cells, less than 0.7% TCR + cells, less than 0.6% TCR + cells, less than 0.5% TCR + cells, less than 0.4% TCR + cells, less than 0.3% TCR + cells, less than 0.2% TCR + cells, or less than 0.1% TCR + cells. This population can be the product of the disclosed methods.

In some embodiments, the engineered immune cell population comprises less than 0.1% TCR + cells. In some embodiments, the engineered immune cells comprise less than 0.09% TCR + cells, less than 0.08% TCR + cells, less than 0.07% TCR + cells, less than 0.06% TCR + cells, less than 0.05% TCR + cells, less than 0.04% TCR + cells, less than 0.03% TCR + cells, less than 0.02% TCR + cells, less than 0.01% TCR + cells. This population can be the product of the disclosed methods.

In some embodiments, the engineered immune cell population comprises between about 0.01% -0.001% TCR + cells. This population can be the product of the disclosed methods.

The present disclosure also provides an engineered immune cell comprising any of the CARs described herein, and further characterized by reduced or eliminated expression of one or more endogenous genes. In some embodiments, the CAR can be introduced into the immune cell as a transgene through a plasmid vector. In some embodiments, the plasmid vector may also contain, for example, a selectable marker for identifying and/or selecting cells that receive the vector.

TCR-engineered immune cells and production of TCR-engineered immune cell populations (comprising CAR T cells)

Provided herein are methods of depleting cells expressing endogenous TCRs from a population of immune cells (including engineered immune cells, such as CAR + cells). As described herein, labeling the cell population with anti-TCR and anti-CD 3 antibodies, separating the anti-TCR and anti-CD 3 labeled cells from the unlabeled cells and collecting the unlabeled cells facilitates obtaining a cell population depleted of cells expressing endogenous TCR. In various embodiments, separation can be generally achieved using magnetic beads, sonic particles, membranes (all of which can be conjugated to moieties recognized by labeled cells), FACS, and other known separation methods. Cells produced using this method also form an aspect of the present disclosure.

Provided herein are methods of depleting cells expressing endogenous TCRs from a population of immune cells (including engineered immune cells, such as CAR + cells). As described herein, labeling the cell population with anti-TCR and anti-CD 52 antibodies, separating the anti-TCR and anti-CD 52 labeled cells from the unlabeled cells and collecting the unlabeled cells facilitates obtaining a cell population depleted of cells expressing endogenous TCR. In various embodiments, separation can be generally achieved using magnetic beads, sonic particles, membranes (all of which can be conjugated to moieties recognized by labeled cells), FACS, and other known separation methods. Cells produced using this method also form an aspect of the present disclosure.

Also provided herein are methods of depleting cells expressing endogenous TCRs from a population of immune cells (including engineered immune cells, such as CAR + cells). As described herein, labeling of the cell population with anti-TCR and anti-CD 3 antibodies and anti-CD 52 antibodies, separating anti-TCR, anti-CD 3-labeled, and anti-CD 52-labeled cells from unlabeled cells and collecting the unlabeled cells facilitates obtaining a cell population depleted of cells expressing endogenous TCR. In various embodiments, separation can be generally achieved using magnetic beads, sonic particles, membranes (all of which can be conjugated to moieties recognized by labeled cells), FACS, and other known separation methods. Cells produced using this method also form an aspect of the present disclosure.

The engineered immune cells described herein may be obtained from a subject prior to in vitro manipulation or genetic modification of the cells. The population of cells expressing the CAR may be derived from an allogeneic or autologous process as described herein, and may be depleted of endogenous TCRs as described herein.

Genetic modification of isolated immune cells

As described herein, immune cells, such as T cells, can be genetically modified using known methods prior to performing the methods provided herein, or immune cells can be activated and expanded (or differentiated in the case of ipscs or progenitor cells) in vitro prior to being genetically modified. In some embodiments, the immune cell is genetically modified to reduce or eliminate expression of endogenous tcr (trac), MHC1(β 2M), MHC2, PD1, and/or CD 52. In some embodiments, the expression of two or more endogenous proteins may be reduced or eliminated. For example, expression of TRAC and CD52 may be reduced or eliminated. In another example, expression of TRACs and proteins targeted by the transduced CAR may be reduced or eliminated. In some embodiments, gene editing techniques (e.g., CRISPR/Cas9, Zinc Finger Nucleases (ZFNs)),MegaTAL, meganuclease) to reduce or eliminate expression of one or more endogenous proteins (e.g., TCR α, MHC1, MHC2, PD1, CD 52). In another embodiment, an immune cell, such as a T cell, is genetically modified with a chimeric antigen receptor described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR or by transduction using non-viral means) and then activated and/or amplified in vitro.

Certain methods for making the constructs and engineered immune cells of the present disclosure are described in PCT application WO2015/120096(PCT/US15/14520), the contents of which are hereby incorporated by reference in their entirety.

In one embodiment, the present disclosure provides a method of storing a genetically engineered cell expressing a CAR. This involves cryopreservation of the immune cells so that they remain viable after thawing. A portion of the immune cells expressing the CAR can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients with malignancies. When desired, the cryopreserved transformed immune cells can be thawed, grown and expanded to obtain more such cells. As demonstrated by the examples and figures, cell populations produced using the depletion methods provided herein can be cryopreserved and later thawed for therapeutic applications.

Allogeneic CAR T cells

For the manufacture of allogeneic CAR T therapy or AlloCARs^TMThe process of (a) involves harvesting healthy, selected, screened and tested T cells from healthy donors. Next, T cells are engineered to express CARs that recognize certain cell surface proteins expressed in blood or solid tumors (e.g., target antigens such as EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, tight junction protein-18.2, Muc17, FAP α, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, CD52, or solid tumors (e.g., target antigens such as EGFRvIII, WT-1, CD20, CD23, CD38, CD33, CD133, CD 6338, MHC-pram-PRAME-18.2, Muc-2, Muc-c 6-orf 23, G6-b-6, c-6-b-CD 34). Allogeneic T cells use gene editing tools (e.g., gene editing toolsZinc fingers, CRISPR, or other gene editing technology) to reduce the risk of graft versus host disease (GvHD) and prevent allograft rejection (HvGD) when administered to a patient different from the donor.

Expression of endogenous T cell receptor genes (e.g., TCR α, TCR β) can be reduced or eliminated in order to avoid GvHD. Expression of endogenous MHCl (β 2M), MHC2, and/or PD1 may also be reduced or eliminated in order to avoid HvGD, thereby preventing the host immune cells from recognizing MHC1 and MHC2 on the transplanted cells as foreign antigens. Endogenously expressed CD52 (cluster of differentiation 52) genes can also be reduced or eliminated in order to render the CAR T product resistant to anti-CD 52 antibody therapy. Thus, anti-CD 52 antibody treatment lymphocyte depletion can be used to suppress the host immune system, allowing CAR T to remain implanted for its full therapeutic effect. As described herein, at least for the purposes of the above illustration, an engineered immune cell or population thereof for use in allotherapy may comprise multiple knockouts.

In some embodiments, endogenous expression of a gene encoding programmed cell death protein 1(PD1), also known as CD279, may be reduced or eliminated in order to enhance anti-tumor efficacy.

White body CAR T cells

Autologous Chimeric Antigen Receptor (CAR) T cell therapy involves harvesting the patient's own cells (e.g., leukocytes, including T cells) and genetically engineering the T cells to express a CAR that recognizes a target antigen expressed on the cell surface of one or more specific cancer cells and kills the cancer cells. The engineered cells are then cryopreserved and subsequently administered to a patient.

Pharmaceutical compositions and therapies

Pharmaceutical compositions comprising cell populations prepared using the disclosed methods can be used to treat patients with cancer. The desired therapeutic amount of engineered cells in a population generated using the methods provided herein is typically at least 2 cells(e.g., at least 1 CD8+ central memory T cell or at least 1 subset of CD4+ helper T cells or one of each of CD8+ and CD4+ cells), or more typically greater than 10²A cell, and up to 10⁶Up to and including 10⁸Or 10⁹A cell, and may exceed 10¹⁰And (4) cells. The number of cells will depend on the intended use of the composition and the cell type contained therein. Thus, the desired cell density is typically greater than 10⁶Individual cells/ml, and usually greater than 10⁷One cell/ml, typically 108 cells/ml or more. Clinically relevant numbers of immune cells can be assigned to a cumulative number equal to or exceeding 10⁵1, 10⁶1, 10⁷1, 10⁸1, 10⁹1, 10¹⁰1, 10¹¹Or 10¹²Multiple infusions of individual cells. In some applications of cell populations generated using the methods of the present disclosure, particularly because all infused cells will be redirected to a particular target antigen, an administration range of 10 may be used⁶Kg (10 per patient)⁶To 10¹¹Ones) of the cells. CAR therapy can be administered multiple times at doses within these ranges. For patients receiving therapy, the cells may be autologous, allogeneic or allogeneic.

As described herein, the engineered immune cell population can deplete cells expressing endogenous TCRs (e.g., the cells are TCR-and/or include residual levels of TCR + cells). In some embodiments, the engineered immune cells comprise less than 1.0% TCR + cells, less than 0.9% TCR + cells, less than 0.8% TCR + cells, less than 0.7% TCR + cells, less than 0.6% TCR + cells, less than 0.5% TCR + cells, less than 0.4% TCR + cells, less than 0.3% TCR + cells, less than 0.2% TCR + cells, or less than 0.1% TCR + cells. Cell populations having the above TCR + cell levels can be achieved using the methods disclosed herein.

In some embodiments, the engineered immune cell population comprises less than 0.09% TCR + cells, less than 0.08% TCR + cells, less than 0.07% TCR + cells, less than 0.06% TCR + cells, less than 0.05% TCR + cells, less than 0.04% TCR + cells, less than 0.03% TCR + cells, less than 0.02% TCR + cells, less than 0.01% TCR + cells. Cell populations having the above TCR + cell levels can be achieved using the methods disclosed herein.

In some embodiments, the engineered immune cell population comprises between about 0.01% -0.001% TCR + cells. Cell populations having the above TCR + cell levels can be achieved using the methods disclosed herein.

In some embodiments, the engineered immune cell population comprises undetectable levels of TCR + cells. Cell populations having the above TCR + cell levels can be achieved using the methods disclosed herein.

In some embodiments, the engineered population of immune cells comprises greater than 99% TCR-cells, greater than 99.9% TCR-cells, greater than 99.91% TCR-cells, greater than 99.92% TCR-cells, greater than 99.93% TCR-cells, greater than 99.94% TCR-cells, greater than 99.95% TCR-cells, greater than 99.96% TCR-cells, greater than 99.97% TCR-cells, or greater than 99.98% TCR-cells. Populations of cells having the above TCR-cell levels can be achieved using the methods disclosed herein.

In some embodiments, the engineered immune cell population comprises between about 99.99-99.999% TCR-cells. Cell populations having these TCR-cell levels described above can be achieved using the methods disclosed herein.

The TCR-depleted CAR-expressing cell population generated using the methods of the present disclosure can be administered alone or in combination with other components, such as IL-2 or other cytokines or cell groups. The pharmaceutical compositions of the present disclosure can include a population of CAR-expressing TCR-cells, such as engineered T cells described herein. The compositions of the present disclosure are preferably formulated for infusion or intravenous administration.

Method of treatment

The present disclosure provides methods for treating or preventing a disease (e.g., cancer) in a patient, the method comprising administering to a patient in need thereof an effective amount of an engineered immune cell population comprising a CAR (e.g., cells obtained not from the patient but from a healthy donor), wherein the engineered immune cell population depletes cells expressing endogenous TCR (e.g., the cells are TCR-and/or comprise residual levels of TCR + cells).

In some embodiments, the engineered immune cell population comprises less than 1.0% TCR + cells, less than 0.9% TCR + cells, less than 0.8% TCR + cells, less than 0.7% TCR + cells, less than 0.6% TCR + cells, less than 0.5% TCR + cells, less than 0.4% TCR + cells, less than 0.3% TCR + cells, less than 0.2% TCR + cells, or less than 0.1% TCR + cells. Cell populations having the above TCR + cell levels can be achieved using the methods disclosed herein.

In some embodiments, the engineered immune cell population comprises less than 0.09% TCR + cells, less than 0.08% TCR + cells, less than 0.07% TCR + cells, less than 0.06% TCR + cells, less than 0.05% TCR + cells, less than 0.04% TCR + cells, less than 0.03% TCR + cells, less than 0.02% TCR + cells, less than 0.01% TCR + cells. Cell populations having the above TCR + cell levels can be achieved using the methods disclosed herein.

In some embodiments, the engineered immune cell population comprises between about 0.01% -0.001% TCR + cells. In some embodiments, the engineered immune cells comprise undetectable levels of TCR + cells. Cell populations having these TCR + cell levels described above can be achieved using the methods disclosed herein.

In some embodiments, the engineered population of immune cells comprises greater than 99% TCR-cells, greater than 99.9% TCR-cells, greater than 99.91% TCR-cells, greater than 99.92% TCR-cells, greater than 99.93% TCR-cells, greater than 99.94% TCR-cells, greater than 99.95% TCR-cells, greater than 99.96% TCR-cells, greater than 99.97% TCR-cells, or greater than 99.98% TCR-cells. Cell populations having these TCR + cell levels described above can be achieved using the methods disclosed herein.

In some embodiments, the engineered immune cell population comprises between about 99.99-99.999% TCR-cells. Cell populations having these TCR + cell levels described above can be achieved using the methods disclosed herein.

Provided herein are methods for treating a disease or disorder comprising cancer. The method reduces the likelihood that a patient will face GvHD when treated with an allogeneic therapy. In some embodiments, the disclosure relates to administering to a subject in need thereof an effective amount of a TCR-depleted engineered immune cell population prepared using a method of the disclosure. In some embodiments, the T cell-mediated immune response is directed to one or more target cells expressing a cancer antigen. In some embodiments, the TCR-depleted engineered immune cell population comprises cells expressing a Chimeric Antigen Receptor (CAR). In some embodiments, the target cell is a solid or hematologic tumor cell. In some aspects, the disclosure includes a method for treating or preventing a malignancy, the method comprising administering to a subject in need thereof an effective amount of a TCR-depleted engineered immune cell population prepared using a method described herein. In some aspects, the disclosure includes a method for treating or preventing a malignancy, the method comprising administering to a subject in need thereof an effective amount of a TCR-depleted engineered immune cell population prepared using a method provided herein, wherein the TCR-depleted immune cell population comprises at least one chimeric antigen receptor and/or an isolated antigen binding domain as described herein. In some embodiments, TCR-depleted CAR-containing immune cell populations prepared using the methods of the present disclosure can be used to treat hematologic malignancies or solid tumors.

In some embodiments, TCR-depleted CAR-containing immune cell populations prepared using the methods of the present disclosure can be used to treat small cell lung cancer, melanoma, low-grade glioma, glioblastoma, medullary thyroid carcinoma, benign tumor, diffuse neuroendocrine tumor in pancreas, bladder, and prostate, testicular cancer, and lung adenocarcinoma with neuroendocrine features. In some embodiments, a population of TCR-depleted CAR-containing immune cells (e.g., CAR-T cells) prepared using the methods of the present disclosure are used to treat a solid tumor cancer. In some embodiments, the population of TCR-depleted CAR-containing immune cells (e.g., CAR-T cells) prepared using the methods of the present disclosure is used to treat non-hodgkin's lymphoma (NHL), Renal Cell Carcinoma (RCC), Acute Lymphocytic Leukemia (ALL), Multiple Myeloma (MM), or Acute Myeloid Leukemia (AML).

Also provided are methods for reducing the size of a tumor in a subject, the methods comprising administering to the subject a TCR-depleted engineered population of cells prepared using the methods of the disclosure, wherein the cells comprise a chimeric antigen receptor comprising an antigen binding domain that binds to an antigen on the tumor.

In some embodiments, the subject has a solid tumor or a hematological malignancy, such as lymphoma or leukemia (hematological cancer). In some embodiments, a TCR-depleted engineered cell population prepared using the methods of the present disclosure is delivered to a tumor bed. In some embodiments, the cancer is present in bone marrow of a subject, and a population of cells prepared using the methods of the present disclosure is delivered into the bone marrow. In some embodiments, the cancer is present in immune cells or blood cells of the patient, and the cell population prepared using the methods of the present disclosure is delivered into the immune cells or blood cells. In some embodiments, the engineered cells are autoimmune cells, such as autologous T cells. In some embodiments, the engineered cells are allogeneic immune cells, such as allogeneic T cells, for which use of the disclosed methods would be particularly beneficial.

A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a therapeutic agent (e.g., a population of TCR-depleted engineered CAR T cells produced using the methods of the present disclosure) is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes disease regression as evidenced by decreased severity of disease symptoms, increased frequency and duration of disease symptom-free periods, or prevention of damage or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to the skilled artisan, such as in human subjects during clinical trials, in animal model systems that can predict efficacy in humans, or by assaying the activity of the agent in an in vitro assay.

The desired therapeutic amount of TCR-depleted engineered cells in the composition includes at least 2 cells (e.g., at least one CD8+ central memory T cell or at least one CD4+ helper T cell subset or at least 2 CD4+ cells or at least 2 CD8+ cells), or more typically greater than 10²A cell, and up to 10⁶Up to and including 10⁸Or 10⁹A cell, and may exceed 10¹⁰And (4) cells. The number of cells will depend on the intended use of the composition and the cell type contained therein. It should be noted that the methods provided herein can be used to generate populations of engineered immune cells that comprise TCR depletion of CD4+ cells only, CD8+ cells only, or both CD4+ and CD8+ cells. Thus, the desired cell density is typically greater than 10⁶Individual cells/ml, and usually greater than 10⁷Individual cell/ml, usually 10⁸Individual cells/ml or greater. Clinically relevant numbers of immune cells can be assigned to a cumulative number equal to or exceeding 10⁵1, 10⁶1, 10⁷1, 10⁸1, 10⁹1, 10¹⁰1, 10¹¹Or 10¹²Multiple infusions of individual cells. In some aspects of the disclosure, particularly because all infused cells will be redirected to a particular target antigen, administration can range from 10⁶Kg (10 per patient)⁶To 10¹¹Ones) of the cells. CAR therapy can be administered multiple times at doses within these ranges. For patients receiving therapy, the cells may be autologous, allogeneic or allogeneic.

In some embodiments, a therapeutically effective amount of TCR-depleted CAR T cells prepared using the methods of the present disclosure is about 1 x 10⁵Individual cell/kg, about 2X 10⁵Individual cell/kg, about 3X 10⁵Individual cell/kg, about 4X 10⁵Individual cell/kg, about 5X 10⁵Individual cell/kg, about 6X 10⁵Individual cell/kg, about 7X 10⁵Individual cell/kg, about 8X 10⁵Per cell/kg,About 9X 10⁵Individual cell/kg, about 2X 10⁶Individual cell/kg, about 3X 10⁶Individual cell/kg, about 4X 10⁶Individual cell/kg, about 5X 10⁶Individual cell/kg, about 6X 10⁶Individual cell/kg, about 7X 10⁶Individual cell/kg, about 8X 10⁶Individual cell/kg, about 9X 10⁶Individual cell/kg, about 1X 10⁷Individual cell/kg, about 2X 10⁷Individual cell/kg, about 3X 10⁷Individual cell/kg, about 4X 10⁷Individual cell/kg, about 5X 10⁷Individual cell/kg, about 6X 10⁷Individual cell/kg, about 7X 10⁷Individual cell/kg, about 8X 10⁷Individual cell/kg or about 9X 10⁷Individual cells/kg.

In some embodiments, the target dose of CAR +/TCR-T cells is at 1X 10⁶Cell/kg to 2X 10⁸In the range of individual cells/kg, e.g. 2X 10⁶Individual cells/kg. It will be understood that doses above and below this range may be appropriate for certain subjects, and appropriate dosage levels may be determined as required by the healthcare provider. In addition, multiple doses of cells may be provided according to the present disclosure.

In some embodiments, a TCR-depleted engineered immune cell population prepared using the methods of the present disclosure that expresses any of the antigen-specific CARs described herein at its cell surface, can reduce, kill, or lyse endogenous antigen-expressing cells of a patient upon administration to the patient. In one embodiment, the percentage of cells that reduce or lyse the antigen-expressing endogenous cells or the antigen-expressing cell line that express any one of the antigen-specific CARs described herein is at least about or greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In one embodiment, the percentage of cells that express the antigen-specific CAR that engineered immune cells reduce or lyse the antigen-expressing endogenous cells or the antigen-expressing cell line is about 5% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 25% to about 75%, or about 25% to about 60%. In one embodiment, the endogenous antigen-expressing cell is an endogenous antigen-expressing myeloid cell.

A variety of additional therapeutic agents may be used in conjunction with the compositions described herein. For example, additional therapeutic agents that may be useful include PD-1 inhibitors, such as nivolumabPembrolizumab (pembrolizumab)Pembrolizumab, pidilizumab (pidilizumab), and atelizumab (atezolizumab); these compounds can be administered prior to, concurrently with, or subsequent to the administration of the TCR-depleted engineered immune cell population prepared using the methods provided herein.

In some embodiments, a composition comprising a TCR-depleted CAR-expressing immune cell population prepared using the methods provided herein can be administered prior to, concurrently with, or subsequent to a therapeutic regimen designed to prevent or treat Cytokine Release Syndrome (CRS) or neurotoxicity, such as an anti-IL 6 antibody.

In certain embodiments, a composition comprising a TCR-depleted CAR-containing immune cell population prepared using the methods provided herein can be administered to a subject in combination with a cytokine. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. The cell factor comprises growth hormone, such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; (ii) prorelaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), and Luteinizing Hormone (LH); hepatic Growth Factor (HGF); fibroblast Growth Factor (FGF); prolactin; placental lactogen; a secondary middle renal duct inhibitory substance; mouse gonadotropin-related peptides; a statin; an activin; vascular endothelial growth factor; an integrin; thrombopoietin (TPO); nerve Growth Factor (NGF), such as NGF-beta; platelet growth factor; transforming Growth Factors (TGF), such as TGF-alpha and TGF-beta; insulin-like growth factor-I and insulin-like growth factor-II; erythropoietin (EPO); an osteoinductive factor; interferons, such as interferon- α, interferon β, and interferon- γ; colony Stimulating Factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, IL-21, a tumor necrosis factor, such as TNF- α or TNF- β; and other polypeptide factors comprising LIF and Kit Ligand (KL). As used herein, the term cytokine encompasses proteins from natural sources or from recombinant cell culture as well as biologically active equivalents of the native sequence cytokines.

Kits and articles of manufacture

The present disclosure provides kits comprising TCR-depleting agents comprising one or more of anti-TCR antibodies, anti-CD 3 antibodies, and anti-CD 52 antibodies. The kits may be used in the depletion methods provided herein.

In one embodiment, the kits provided herein include an anti-TCR antibody and an anti-CD 3 antibody. In the kit, one or both of the anti-TCR antibody and/or the anti-CD 3 antibody can optionally be conjugated to biotin. In particular embodiments, both the anti-TCR antibody and the anti-CD 3 antibody are conjugated to biotin.

In one embodiment, the kits provided herein include an anti-TCR antibody and an anti-CD 52 antibody. In the kit, one or both of the anti-TCR antibody and/or the anti-CD 52 antibody can optionally be conjugated to biotin. In particular embodiments, both the anti-TCR antibody and the anti-CD 52 antibody are conjugated to biotin.

In one embodiment, the kits provided herein include an anti-TCR antibody, an anti-CD 3 antibody, and an anti-CD 52 antibody. In the kit, one or all of the anti-TCR antibody and/or anti-CD 3 antibody and/or anti-CD 52 antibody may optionally be conjugated to biotin. In particular embodiments, the anti-TCR antibody, the anti-CD 3 antibody, and the anti-CD 52 antibody are conjugated to biotin.

In another aspect, the kits provided herein can further comprise an anti-biotin antibody conjugated to a magnetic nanomatrix microbead, a cell-sized bead, or other support (e.g., a membrane, an acoustic particle or bead, a plastic plate, or a column). The anti-biotin antibody may be provided in conjugated form or optionally as a naked antibody along with materials that facilitate attachment of the antibody to magnetic nanomatrix microbeads, cell-sized beads or other supports.

In particular embodiments, the kits provided herein include an anti-TCR antibody and an anti-CD 3 antibody. In the kit, one or both of the anti-TCR antibody and/or the anti-CD 3 antibody can optionally be conjugated directly to a magnetic bead, a membrane, a sonic particle, a plastic plate, or a column.

In particular embodiments, the kits provided herein include anti-TCR antibodies, anti-CD 3 antibodies, and anti-CD 52 antibodies. In the kit, one or both of the anti-TCR antibody and/or anti-CD 3 antibody and/or anti-CD 52 antibody can optionally be conjugated directly to magnetic beads, membranes, sonic particles, plastic plates, or columns.

It should be noted that while the use of anti-biotin antibodies forms one embodiment of the methods disclosed herein, other means of capturing the biotin-labeled moiety may also be employed. For example, streptavidin, avidin, and other biotin binding moieties can be used in place of the anti-biotin antibodies in the disclosed methods.

The present disclosure also provides articles of manufacture comprising any of the therapeutic compositions and kits described herein. Examples of articles of manufacture include containers (e.g., sealed vials) containing a therapeutic agent (e.g., a TCR-depleted cell population prepared using the methods disclosed herein, which may further include a CAR).

Examples of the invention

As shown in the examples below, the disclosed combined antibody approach resulted in a significant increase in TCR + cell depletion efficiency, which unexpectedly reduced the residual TCR +% levels from 0.1% -1% to 0.1% -0.01% (measured on day 1 post-depletion) compared to TCR antibodies alone. These examples demonstrate that the disclosed depletion method provides significant advantages over current methods for depleting TCR + cells. These advantages may provide benefits to patients receiving allogeneic therapy in a form that reduces the likelihood that the patient will exhibit GvHD.

Example 1 combination of anti-TCR antibody and anti-CD 3 antibody increases TCR + cell depletion efficiency

Using immune cells engineered with CAR, by targeting TRAC and CD52 genesThe electroporation of (a) knocks out endogenous TCR and CD52 gene expression. The TCR and CD52 knockout cells are then exposed to a TCR-depleting agent. As shown in table 1, cells were contacted with primary anti-TCR antibody conjugated to biotin, either alone or in combination with anti-CD 3 antibody or anti-CD 52 antibody.

Next, a secondary anti-biotin antibody conjugated to magnetic microbeads (nanoparticles of about 50nm in diameter) was further added to the primary antibody-labeled cells to bind the magnetic microbeads to any residual TCR + cells through the primary anti-TCR biotin moiety. Then use CliniMACSThe instrument applies the labeled cells to a magnetic column. The TCR + cells are retained inside the magnetic column, while the unlabeled TCR-cells pass through to the product collection bag. The TCR + cells are then released from the column into a waste bag. Various depletion methods provided using the present disclosure explore TCR + cell depletion efficiency, and residual TCR + cells present in the TCR-cell population fraction were monitored at different days after depletion using anti-TCR α, anti-TCR β, and/or anti-CD 3 antibodies. Fig. 1 provides a schematic illustration of the allogeneic CART manufacturing process in the context of different unit operation scenarios.

TABLE 1 Experimental design of antibody conditions

TABLE 2 antibody information

As shown in fig. 2A, the percentage of TCR + cells present in the depleted TCR-product population using various depletion methods was substantially less than 1% or less. Figure 2A shows that when an anti-TCR antibody and an anti-CD 3 antibody were used together in the depletion method, a 204-fold unexpected decrease in TCR + cell frequency was detected on day 2 post-depletion compared to the control depletion method using a low 1 × TCR antibody concentration; equivalent reductions were also observed when cells were treated with anti-TCR antibody, anti-CD 3 antibody, and anti-CD 52 antibody. Figure 2A also shows that the effectiveness of using anti-TCR antibody and anti-CD 52 antibody and using a 3 x anti-TCR antibody concentration is lower than the combination of anti-TCR antibody and anti-CD 3 antibody, where 3 x TCR antibody treatment shows a 6.2-fold reduction and the combination of anti-TCR antibody and anti-CD 52 antibody shows a 2.7-fold reduction. Interestingly, figure 2A demonstrates that depletion using anti-CD 52 antibody and anti-TCR antibody is more effective than using anti-TCR antibody alone. This result is surprising because, as described herein, CD52 is not biologically associated with the TCR or TCR complex, and thus, unexpectedly, the depletion method using these two antibodies depletes TCR + cells more efficiently than using the anti-TCR antibody alone. The results shown in figure 2A also demonstrate that, unexpectedly, simply increasing the concentration of anti-TCR antibody did not produce significantly better results than using lower concentrations of anti-TCR antibody in combination with anti-CD 3 antibody.

Depleted populations were also studied on day 9 post-depletion and the results are shown in figure 2B. Figure 2B shows that TCR + cell frequency was reduced 8-fold when anti-TCR and anti-CD 3 antibodies were used in the depletion method; equivalent reductions were also observed when cells were treated with anti-TCR antibody, anti-CD 3 antibody, and anti-CD 52 antibody. Figure 2B also shows that the effectiveness of using anti-TCR antibody and anti-CD 52 antibody and using a 3 x anti-TCR antibody concentration is lower than the combination of anti-TCR antibody and anti-CD 3 antibody, where 3 x TCR antibody treatment shows a 5-fold reduction and the combination of anti-TCR and anti-CD 52 shows a 2-fold reduction. These results demonstrate that simply increasing the concentration of anti-TCR antibody does not produce significantly better results than using lower concentrations of anti-TCR antibody in combination with anti-CD 3 antibody.

The FACS plot of fig. 3A visualizes that there are minimal TCR + cells present when the depletion method is performed using anti-TCR antibody and anti-CD 3 antibody compared to anti-TCR antibody alone or in combination with anti-CD 3 antibody and/or anti-CD 52 antibody, or increased (3 ×) concentration of anti-TCR antibody. Data were taken at day 0 and day 1 post-depletion and compared to pre-depletion cell samples. The results further demonstrate that simply increasing the concentration of anti-TCR antibody did not produce significantly better results over the days of the study compared to using lower concentrations of anti-TCR antibody in combination with anti-CD 3 antibody.

Figure 3B is a digital bar graph showing that when anti-TCR antibody and anti-CD 3 antibody were used in the depletion method, the TCR + cell frequency decreased 151-fold and 23-32-fold at day 0 and day 1 after depletion, compared to the control depletion method using low 1 × TCR antibody concentration; an equivalent reduction of 70-fold was also observed when cells were treated with anti-TCR antibody, anti-CD 3 antibody, and anti-CD 52 antibody. It should be noted that current TCR depletion procedures only employ standard 1 × TCR antibody concentrations, which are the differences between the disclosed methods and are understood to be the current state of the art. Figure 3B also shows that the effectiveness of using anti-TCR antibody and anti-CD 52 antibody and using a 3 x anti-TCR antibody concentration is lower than the combination of anti-TCR antibody and anti-CD 3 antibody, where 3 x TCR antibody treatment shows 19-fold and 7-fold reduction, and the combination of anti-TCR and anti-CD 52 shows 2.7-fold and 1.6-fold reduction. These results further demonstrate that simply increasing the concentration of anti-TCR antibody did not produce significantly better results over the days of the study compared to using lower concentrations of anti-TCR antibody in combination with anti-CD 3 antibody.

Figure 4 is a FACS plot of data collected at day 0 and day 1 post-depletion and further demonstrates that the combination of anti-TCR antibody and anti-CD 3 antibody provides a higher level of TCR depletion compared to the use of high (3 x) or low (1 x) concentration of anti-TCR antibody alone and the use of anti-TCR antibody in combination with anti-CD 52 antibody. Residual TCR + cells after depletion were visualized by either single staining against TCR, anti-CD 3 or double staining against TCR/CD3 and were expected to stain positively for TCR +, CD3+ or CD3+/TCR +. The small cell population stained CD3 +/TCR-reflects that the remaining TCR γ δ T cells are present in the depleted TCR-cell population, as TCR γ δ T cells are expected to be removed by anti-CD 3 antibodies.

Example 2 post-depletion culture

This example demonstrates that the depleted immune cell population maintains the same low residual TCR + levels during post-depletion culture. On day 1 post-depletion, depleted cells were frozen and later thawed for post-depletion culture. TCR and CD3 expression levels were measured immediately before freezing and after thawing. As shown in fig. 5A-5E, there was no significant difference in both TCR frequency and CD3 frequency due to the freeze-thaw cycles; fig. 5A depicts the frequency of TCR +, CD3+, CD3+/TCR-, and CD3+/TCR + cells before and after thawing by FACS plotting using anti-TCR, anti-CD 3 single staining or anti-TCR/CD 3 double staining, fig. 5B numerically depicts the frequency of TCR + cells before and after thawing using anti-TCR antibodies, fig. 5C numerically depicts the frequency of CD3+ cells before and after thawing using anti-CD 3 antibodies, fig. 5D numerically depicts the frequency of CD3 +/TCR-cells before and after thawing using anti-TCR/CD 3 antibodies, and fig. 5E numerically depicts the frequency of CD3+/TCR + cells before and after thawing using anti-TCR/CD 3 antibodies.

To determine whether the residual TCR + cell frequency in the depleted TCR-cell population increased with increasing culture time, the depleted cells were cultured for 10 days after depletion. TCR and CD3 cell frequencies were examined by single staining with anti-TCR (fig. 6) and anti-CD 3 (fig. 7) antibodies or double staining with anti-TCR/CD 3 (fig. 8) antibodies every 2-3 days during the entire culture process. Specifically, the FACS plots of fig. 6, 7 and 8 depict the trend of increasing TCR +, CD3+, or CD3+/TCR + cell frequency during post-depletion culture for all depletion methods, followed by a peak around 5 to 7 days, maintaining the same lower TCR + or CD3+ cell frequency over time for cultures depleted using a combination of anti-TCR, anti-CD 3, and/or anti-CD 52 antibodies as compared to cultures treated with low or high concentrations of anti-TCR antibodies alone or with a combination of anti-TCR and anti-CD 52 antibodies.

Next, the bar graphs of FIGS. 9A, 9B, 9C and 9D numerically depict the TCR +, CD3+, CD3+/TCR-, or CD3+/TCR + frequencies during post-depletion culture.

In addition, cell growth status was also monitored during the post-depletion culture period, including viable cell density (fig. 10A), viability (fig. 10B), cell diameter (fig. 10C), and total fold expansion (fig. 10D) at each passage. Overall, comparable cell growth characteristics were observed for all the depletion methods studied.

The efficiency of depleting CD52 using different combinations of the antibodies disclosed herein was also investigated. Specifically, anti-CD 52 antibodies, alone or in combination with other antibodies, were used for CD52 depletion, and the effect of anti-TCR antibodies and/or anti-CD 3 antibodies for TCR + cell depletion on CD52+ cell depletion was also investigated. The FACS plots of fig. 11A show residual CD52+ cell frequency after depletion on day 0 and day 1 after depletion. The bar graph of fig. 11B numerically shows the residual CD52+ cell frequency. Finally, the FACS plot of fig. 12 shows the residual CD52+ cell frequency during the 10 day post-depletion culture.

In summary, the data from example 2 demonstrate that the use of both anti-TCR and anti-CD 3 antibodies enhances TCR + cell depletion efficiency. The use of anti-CD 3 antibody in addition to anti-TCR antibody more broadly eliminated CD3+/TCR + cells than anti-TCR antibody alone. This depletion mechanism provides improved TCR + cell depletion efficiency and can be a significant advantage for therapeutic allogeneic applications. The cells after depletion showed similar growth for all conditions during the post-depletion culture period, indicating that the depletion method did not affect cell viability and growth.