Methods for improving the efficiency of depletion of TCRαβ+ cells

Abstract

Provided herein are improved methods for robust depletion of TCR+ cells and for generating populations of TCR- cells, which may be beneficial for minimizing the risk of GvHD in patients undergoing allogeneic CAR T cell therapy. Provided herein are methods for increasing the efficiency of depleting TCR+ cells from cell populations such that the residual levels of TCR+ cells present in cell populations in which endogenous TCR expression has been reduced or eliminated are significantly reduced. Related kits and cell populations are also provided.

Description

[Technical field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Application No. 62 / 821,768, filed March 21, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to methods for depleting cells expressing endogenous TCRs (e.g., TCRαβ) from a population of engineered immune cells, including cells containing chimeric antigen receptors (CARs). [Background technology]

[0003] Adoptive transfer of immune cells genetically modified to recognize malignant tumor-associated antigens has shown promise as a new approach to treat cancer (see, e.g., Brenner et al., Current Opinion in Immunology, 22(2):251-257(2010); Rosenberg et al., Nature Reviews Cancer, 8(4):299-308(2008)). Immune cells can be genetically modified to express chimeric antigen receptors (CARs) (see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2):720-724(1993); Sadelain et al., Curr. Opin. Immunol, 21(2):215-223(2009), which are incorporated herein by reference). Immune cells that make up CARs, such as CAR-T cells (CAR-T), are engineered to confer antigen specificity and to retain or enhance their ability to recognize and kill target cells, such as cancer cells. However, the potential for developing graft-versus-host disease (GvHD) or host-versus-graft disease (HvGD) represents a serious safety or efficacy obstacle to the widespread use of engineered allogeneic CAR-T cells for cancer therapy. Thus, there is a need to develop effective methods to reduce the risk of GvHD and HvGD, especially for allogeneic therapies. Summary of the Invention

[0004] Described herein are improved methods for depleting TCR+ (TCRαβ+) cells from populations of immune cells, methods for generating populations of immune cells depleted of TCR+ cells, kits containing reagents therefor, highly pure populations of TCR- (TCRαβ-) cells, and therapeutic methods using populations of cells prepared using the disclosed methods. For example, described herein are methods particularly suited for TCR+ cell depletion that can be used to produce cells useful for allogeneic cell therapy by reducing the risk of GvHD, and can be used in therapies that use chimeric antigen receptors (e.g., allogeneic CAR-T cell therapy).

[0005] In one aspect, a method of producing a population of immune cells depleted of immune cells expressing endogenous TCR is provided, in one embodiment the method comprises labeling a population of immune cells with an anti-TCR antibody and an anti-CD3 antibody, and separating the anti-TCR antibody labeled immune cells and the anti-CD3 antibody labeled immune cells from the population of immune cells. The method can further comprise labeling the population of immune cells with an anti-CD52 antibody, and depleting the anti-CD52 antibody labeled immune cells from the population of immune cells. Additionally, the method can further comprise labeling the population of immune cells with an anti-CD52 antibody, and separating the anti-CD52 antibody labeled immune cells from unlabeled immune cells.

[0006] In another aspect, a method is provided for depleting cells expressing endogenous TCR from a population of immune cells, which in one embodiment comprises labeling a population of immune cells with an anti-TCR antibody and an anti-CD3 antibody, separating the anti-TCR antibody labeled immune cells and the anti-CD3 antibody labeled immune cells from unlabeled immune cells, and collecting the unlabeled immune cells, thereby obtaining a population of immune cells depleted of cells expressing endogenous TCR.

[0007] In a further aspect, there is provided a method of producing a population of immune cells depleted of immune cells expressing endogenous TCR, in one embodiment the method comprises labelling a population of immune cells with an anti-TCR antibody and an anti-CD52 antibody, and separating the anti-TCR antibody labelled immune cells and the anti-CD52 antibody labelled immune cells from the population of immune cells.

[0008] In yet another aspect, a method is provided for depleting cells expressing endogenous TCR from a population of immune cells, which in one embodiment comprises labeling a population of immune cells with an anti-TCR antibody and an anti-CD52 antibody, separating the anti-TCR antibody labeled immune cells and the anti-CD52 antibody labeled immune cells from unlabeled immune cells, and collecting the unlabeled immune cells, thereby obtaining a population of immune cells depleted of cells expressing endogenous TCR.

[0009] In some embodiments, the anti-TCR antibody, anti-CD3 antibody, and / or anti-CD52 antibody are biotin conjugates, and the method may further comprise contacting the labeled cells with an agent that specifically recognizes biotin. The agent that specifically recognizes biotin may be selected from the group consisting of anti-biotin antibody, avidin, streptavidin, and may be bound 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, anti-CD3 antibody, and / or anti-CD52 antibody may be directly bound to magnetic beads, agarose beads, sonic particles, plastic well plates, glass well plates, ceramic well plates, columns, cell culture bags, or membranes.

[0010] In methods that employ separation, separation can be achieved using either magnetic separation or sonic separation.

[0011] In some embodiments, the immune cells of the disclosed methods are allogeneic immune cells, which may generally be genetically modified to reduce or eliminate expression of endogenous TCR or endogenous CD52, or both endogenous TCR and endogenous CD52, where reducing expression of endogenous TCR, or endogenous CD52, or both endogenous TCR and endogenous CD52 is accomplished using TALENs, CRISPR / Cas9, zinc finger nucleases (ZFNs), MegaTALs, meganucleases, Cpf1, homologous recombination, or single-stranded oligodeoxynucleotides (ssODNs).

[0012] The immune cells of the disclosed methods can be engineered immune cells that express a chimeric antigen receptor.

[0013] In some embodiments, the population of cells depleted of cells expressing endogenous TCR comprises 1.0% or less TCR+ cells, 0.9% or less TCR+ cells, 0.8% or less TCR+ cells, 0.7% or less TCR+ cells, 0.6% or less TCR+ cells, 0.5% or less TCR+ cells, 0.4% or less TCR+ cells, 0.3% or less TCR+ cells, 0.2% or less TCR+ cells, or 0.1% or less TCR+ cells. In some embodiments, the population of cells depleted of cells expressing endogenous TCR comprises 0.01-0.001% TCR+ cells immediately after depletion. In some embodiments, a population of cells depleted of cells expressing endogenous TCR comprises 0.1-0.01% TCR+ cells after 1-10 days of culture after depletion, in some embodiments, a population of cells depleted of cells expressing endogenous TCR comprises less than 0.1-1.0% TCR+ cells after 1 day of culture after depletion. In some embodiments, a population of cells depleted of cells expressing endogenous TCR comprises less than 0.1-1.0% TCR+ cells after 10 days of culture after depletion.

[0014] In an embodiment of the disclosed methods, the immune cell is a T cell.

[0015] In another aspect, a population of immune cells depleted of immune cells expressing endogenous TCRs produced by the disclosed methods is provided.

[0016] In one aspect, a population of engineered T cells is provided that comprises at least 99% TCR- cells, a population of engineered T cells is provided that comprises at least 99.9% TCR- cells, a population of engineered T cells is provided that comprises at least 99.99% TCR- cells, a population of engineered T cells is provided that comprises at least 99.999% TCR- cells, In some embodiments, the population of T cells expresses a chimeric antigen receptor.

[0017] In another aspect, a kit is provided for depleting cells expressing endogenous TCR from a population of immune cells comprising an anti-TCR antibody and an anti-CD3 antibody. The kit can further comprise an anti-CD52 antibody. In another aspect, a kit is provided for depleting cells expressing endogenous TCR from a population of immune cells comprising an anti-TCR antibody and an anti-CD52 antibody. In the disclosed kit, the anti-TCR antibody, the anti-CD3 antibody, and / or the anti-CD52 antibody are conjugated to an agent that specifically recognizes biotin, and the agent that specifically recognizes biotin can be selected from the group consisting of an anti-biotin antibody, streptavidin, and avidin. In some embodiments, the agent can be conjugated to a magnetic bead, an agarose bead, an acoustic particle, a plastic well plate, a glass well plate, a ceramic well plate, a column, a cell culture bag, or a membrane.

[0018] In the disclosed kits, the anti-TCR antibody, anti-CD3 antibody, and / or anti-CD52 antibody are directly bound to a support, which is one of magnetic beads, agarose beads, sonic particles, plastic well plates, glass well plates, ceramic well plates, columns, cell culture bags, or membranes.

[0019] Further provided are methods of treating a solid cancer or a hematological cancer in a subject in need thereof, comprising administering to the subject a therapeutic amount of a population of immune cells depleted of immune cells expressing endogenous TCR prepared using the disclosed methods, or a therapeutic amount of a population of engineered T cells prepared using the disclosed methods.

[0020] The methods provided herein represent a significant advance in allogeneic therapy. As mentioned above, also provided is a method of treating a solid cancer or hematological cancer in a subject in need thereof, comprising administering to the subject a population of immune cells depleted of immune cells expressing endogenous TCR, prepared using any of the disclosed methods provided herein. [Brief description of the drawings]

[0021] [Figure 1] Figure 1 shows a schematic diagram of the allogeneic CAR T manufacturing process with different unit operation scenarios. [Figure 2A] FIG. 2A shows the frequency of remaining TCR+ cells in the depleted cell population at each day after depletion, with arrows indicating the fold change in TCR+% for different depletion methods (using different antibody combinations) compared to the control method (using anti-TCR antibody only) at day 2 after depletion compared to day 0 after depletion. [Figure 2B] FIG. 2B shows the frequency of remaining TCR+ cells in the depleted cell population, with arrows indicating the fold change in TCR+% at day 9 post-depletion compared to day 0 post-depletion between the different depletion methods when compared to the control method. [Figure 3A] FIG. 3A shows flow cytometry plots of TCR+ cell depletion efficiency using different antibodies as measured by anti-TCR antibody staining on days 0 and 1 post-depletion. [Figure 3B] FIG. 3B shows the frequency of remaining TCR+ cells in the depleted cell population at days 0 and 1 post-depletion in a numerical bar format. [Figure 4]FIG. 4 shows flow cytometry plots showing the remaining TCR+ and CD3+ frequencies in the depleted populations detected by single or double staining with anti-TCR and / or anti-CD3 antibodies on days 0 and 1 post-depletion. [Figure 5A] FIG. 5A shows flow cytometry plots of TCR+, CD3+, CD3+ / TCR-, and CD3 / TCR+ frequencies of depleted cell populations measured by single or double staining with anti-TCR and / or anti-CD3 antibodies at day 1 post-depletion before and after freezing. [Figure 5B] FIG. 5B shows in a numerical bar format the frequency of TCR+ cells in the depleted cell populations measured by anti-TCR antibody staining at day 1 post-depletion before and after freezing. [Figure 5C] FIG. 5C shows in a numerical bar format the CD3+ cell frequency in the depleted cell populations measured by anti-CD3 antibody staining at day 1 after depletion before and after freezing. [Figure 5D] FIG. 5D shows in a numerical bar format the CD3+ / TCR- cell frequencies of the depleted cell populations measured by double staining with anti-TCR and anti-CD3 antibodies 1 day after depletion before and after freezing. [Figure 5E] FIG. 5E shows in a numerical bar format the CD3+ / TCR+ cell frequency of the depleted cell population measured by double staining with anti-TCR and anti-CD3 antibodies 1 day after depletion before and after freezing. [Figure 6] FIG. 6 shows flow cytometry plots of TCR+ frequency using anti-TCR antibody staining over a 10 day culture period after depletion. [Figure 7] FIG. 7 shows flow cytometry plots of CD3+ frequency using anti-CD3 antibody staining over a 10 day culture period after depletion. [Figure 8] FIG. 8 shows flow cytometry plots of TCR+ and CD3+ dual frequencies using both anti-TCR and anti-CD3 antibody staining over a 10-day culture period after depletion. [Figure 9A] FIG. 9A shows the frequency of TCR+ cells using anti-TCR antibody staining during the culture period after depletion in a numerical bar format. [Figure 9B]FIG. 9B shows the CD3+ cell frequency using anti-CD3 antibody staining in a numerical bar format during the culture period after depletion. [Figure 9C] FIG. 9C shows the CD3+ / TCR- cell frequency using both anti-TCR and anti-CD3 antibody staining in a numerical bar format during the culture period after depletion. [Figure 9D] FIG. 9D shows the CD3+ / TCR+ cell frequency using both anti-TCR and anti-CD3 antibody staining in a numerical bar format during the culture period after depletion. [Figure 10A] FIG. 10A shows the cell proliferation status in terms of viable cell density over a 10 day culture period after depletion. [Figure 10B] FIG. 10B shows the cell proliferation status in terms of cell viability over a 10 day culture period after depletion. [Figure 10C] FIG. 10C shows the cell proliferation status in terms of cell diameter over a 10 day culture period after depletion. [Figure 10D] FIG. 10D shows the cell proliferation status in terms of total cell proliferation fold over a 10 day culture period after depletion. [Figure 11A] FIG. 11A shows flow cytometry plots demonstrating CD52 depletion efficiency by using different antibodies as measured by anti-CD52 staining at days 0 and 1 post-depletion. [Figure 11B] FIG. 11B shows in a numerical bar format the frequency of remaining CD52+ cells in the depleted cell population as measured by anti-CD52 antibody staining on days 0 and 1 after depletion. [Figure 12] FIG. 12 shows flow cytometry plots of remaining CD52+ cell frequency over a 10-day culture period after depletion, as monitored by anti-CD52 antibody staining. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Provided herein are improved methods for robust TCRαβ+ cell depletion that can minimize GVHD risk in patients undergoing allogeneic CAR T cell therapy, and cell populations prepared using the disclosed methods. In one aspect, the disclosure provides methods to increase the efficiency of TCR+ cell depletion to significantly reduce residual levels of TCR+ cells in a TCR− cell population.

[0023] As used herein, the terms "a" and "an" are used to mean one or more. For example, reference to a "cell" or an "antibody" means "one or more cells" or "one or more antibodies."

[0024] As used herein, the term "TCR-depleted" when used in reference to a population of cells produced using the methods provided herein means a population of cells that contains fewer cells that express endogenous TCR α / β heterodimers than the population of cells collected from a donor. For example, a TCR-depleted cell population (a cell population depleted of TCR α / β) can contain 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than 1% cells that express endogenous TCR α / β complexes.

[0025] As used herein, the terms "TCR+" and "TCR" when used with respect to a cell or population of cells (including populations produced using the methods provided in this disclosure) refer to cells that express at least an endogenous TCR α / β heterodimer, although one or more components of the CD3 complex may or may not be expressed on the cell surface.

[0026] As used herein, the term "TCR-" when used with respect to a cell or population of cells (including populations produced using the methods provided in this 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.

[0027] As used herein, the terms "anti-TCR antibody" and "anti-TCR," used interchangeably, refer to an antibody that binds to and recognizes only the human TCR alpha chain, or only the human TCR beta chain, or the human TCR alpha / beta heterodimer. Anti-TCR antibodies may be murine, human, or humanized antibodies.

[0028] As used herein, the terms "anti-CD3 antibody" and "anti-CD3", 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, or the CD3 epsilon chain or human CD3 complex that includes two or more components of the normal human epsilon / epsilon / zeta / zeta / gamma / delta CD3 complex. The anti-CD3 antibody may be a murine, human, or humanized antibody.

[0029] The terms "patient" and "subject" are used interchangeably and include human and non-human animal subjects, as well as subjects with a formally diagnosed disorder, subjects without a formally recognized disorder, subjects undergoing medical treatment, subjects at risk for developing a disorder, and the like.

[0030] The terms "treat" and "treatment" include curative treatment, prophylactic treatment, and applications that reduce the risk that a subject will develop a disorder or other risk factor. Treatment does not require a complete cure of the disorder, but encompasses embodiments that reduce symptoms or underlying risk factors. The term "prevent" does not require 100% elimination of the likelihood of an event. Rather, it indicates a decreased likelihood of the occurrence of the event in the presence of a given compound or method.

[0031] Engineered TCR-cell-based allogeneic CAR-T products are a typical example of a strategy for generating next-generation CAR-T therapeutics. However, potential immune responses such as graft-versus-host disease (GvHD) risk and host-versus-graft disease (HvGD) are among the serious safety or efficacy concerns of allogeneic CAR-T cell therapy. GvHD results from donor-derived T cells recognizing HLA mismatches via T cell αβ receptor (TCRαβ) and potentially attacking patient tissues. GVHD and HvGD can be serious and even fatal, because even in the environment of an HLA-matched donor, small mismatches can still trigger immune responses.

[0032] Current methods for removing TCR+ cells (depleting TCRαβ+ cells), such as CliniMACS Prodigy® and TCR Reagent Kits, can separate TCR+ cells (TCRαβ+ cells) from TCR- cells (TCRαβ- cells) using only anti-TCR antibodies, achieving greater than 99% TCR- cell purity in the final drug product (see, for example, Radestad et al., (2014) J Immunol Res, Vol 2014:578741). However, residual levels of less than 1% TCR+ cells present in TCR- in the final drug product may still represent a risk of GvHD associated with allogeneic CAR-T cell therapy, especially at high dose levels.

[0033] The present disclosure provides methods for producing a population of engineered immune cells that are depleted of immune cells expressing endogenous TCR, the disclosed methods comprising labeling a population of immune cells with an anti-TCR antibody and an anti-CD3 antibody, and separating the anti-TCR antibody labeled immune cells and the anti-CD3 antibody labeled immune cells from the population of engineered immune cells.

[0034] The present disclosure also provides a method for depleting a population of immune cells of cells expressing an endogenous TCR, the disclosed method comprising labeling a population of immune cells with an anti-TCR antibody and an anti-CD3 antibody, separating the anti-TCR antibody-labeled immune cells and the anti-CD3 antibody-labeled immune cells from unlabeled immune cells, and collecting the unlabeled immune cells, thereby obtaining a population of immune cells depleted of cells expressing an endogenous TCR. This method can also be adapted to include a population of immune cells labeled with an anti-TCR antibody and one or more antibodies directed to other targets of interest other than CD3 (e.g., MHC1, MHC2, CD52, or PD1).

[0035] In one aspect, the disclosure provides a method of depleting TCRαβ+ cells comprising contacting a population of immune cells with a TCR antibody and a CD3 antibody. This method can also be adapted to include a population of immune cells labeled with an anti-TCR antibody and one or more antibodies directed to other targets of interest besides CD3 (e.g., MHC1, MHC2, CD52, or PD1).

[0036] The present disclosure provides methods for producing a population of engineered immune cells that are depleted of immune cells expressing endogenous TCR, the disclosed methods comprising labeling a population of immune cells with an anti-TCR antibody and an anti-CD52 antibody, and separating the anti-TCR antibody labeled immune cells and the anti-CD52 antibody labeled immune cells from the population of engineered immune cells.

[0037] The present disclosure also provides a method for depleting cells expressing endogenous TCR from a population of immune cells, the disclosed method comprising labeling a population of immune cells with an anti-TCR antibody and an anti-CD52 antibody, separating the anti-TCR antibody labeled immune cells and the anti-CD52 antibody labeled immune cells from unlabeled immune cells, and collecting the unlabeled immune cells, thereby obtaining a population of immune cells depleted of cells expressing endogenous TCR.

[0038] In another aspect, the present disclosure provides methods for depleting TCRαβ+ cells, comprising contacting a population of immune cells with an anti-TCR antibody and a CD52 antibody. The effectiveness of these methods, as shown in the examples, was unexpected given the fact that CD52 and TCR are not physically associated with each other in human cells, and, without wishing to be bound by any theory, it is understood that CD52 and TCR are not directly related biologically. Thus, in another aspect of the present disclosure, the disclosed methods can include the use of an anti-TCR antibody and another antibody directed to a target of interest not normally associated with the TCR.

[0039] In yet another aspect, the disclosure provides a method of depleting TCRαβ+ cells comprising contacting a population of immune cells with an anti-TCR antibody, an anti-CD3 antibody, and an anti-CD52 antibody.

[0040] The methods described herein are particularly useful for the production of engineered TCRαβ-cell-based allogeneic CAR T products, applying anti-TCR and anti-CD3 antibodies together for depletion of TCRαβ+ cells. Prior to this disclosure, depletion of TCR+ cells to a level of 99.9%-99.99% TCR-cell purity, leaving 0.1%-0.01% residual TCR+ cells in engineered T cells (not unmodified T cell products), was very difficult to achieve on a clinical scale and was a challenge in developing allogeneic therapies. The present disclosure provides a solution to this problem.

[0041] Achieving these kinds of low levels of residual TCR+ cells (e.g., in the range of 0.1%-0.01% TCR+ cells measured 1 day after depletion) represents a significant advancement for engineered allogeneic CAR T products (e.g., TCRα / β-CAR T cells) and will enhance clinical benefit for patients by reducing the likelihood of GvHD.

[0042] Effective methods of reducing the risk of GvHD (and HvGD) while enhancing anti-tumor efficacy are particularly useful for the production of effective, safe, and highly pure engineered allogeneic immune cells (e.g., allogeneic CAR-T cells expressing CARs targeted against cancer antigens). In some embodiments, the present disclosure provides methods for producing allogeneic CAR-T cells and products that reduce the risk of or prevent the development of GvHD and / or HvGD. In some embodiments, the engineered allogeneic CAR-T cells have enhanced anti-tumor activity against solid tumors or hematological cancers. In some embodiments, the CAR-T cells comprise a population of cells that have been modified to reduce or eliminate expression of one or more endogenous TCR, CD52, MHC1, MHC2, or PD1 gene expression. Thus, in some embodiments, the CAR-T cells comprise a population that is depleted of cells that are TCR+, CD52+, MHC1+, MHC2+, and / or PD1+.

[0043] Provided herein are methods for depleting a population of cells expressing one or more of endogenously expressed TCR, CD3, CD52, MHC1, MHC2, or PD1 (e.g., cells that are TCR+, CD3+, CD52+, MHC1+, MHC2+, and / or PD1+) from a population of engineered immune cells, and kits containing antibody reagents therefor.

[0044] 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 that is responsible for the recognition of antigens presented to the T cell by MHC molecules, potentially resulting in activation of the T cell and an immune response to the antigen. Human T cell receptors are heterodimers consisting of two transmembrane heterodimeric glycoprotein chains, an α chain and a β chain (with a smaller proportion of γδ chains), each of which has two domains linked by a disulfide bond. The short cytoplasmic tail of the TCR prevents it from directly signaling when it binds to a peptide-MHC complex. Instead, the TCR is associated with a group of signaling molecules collectively referred to as CD3, which transmit an intracellular signal when the TCR binds to a peptide-MHC complex.

[0045] CD3 is composed of one γ and δ and two ε molecules, all of which have 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 transmembrane region of the TCR. The TCR-CD3 receptor complex is completed by two other invariant proteins, ζ and η, which form dimers linked by disulfide bonds. Thus, on the T cell surface, the TCR-CD3 complex is expressed as an αβ (or γδ) heterodimer in association with the CD3γε dimer and the CD3δε dimer with the intracellular ζζ homodimer or ζη heterodimer.

[0046] Without wishing to be bound by theory, some researchers believe that if TCR expression is disrupted, CD3 surface expression may also be ineffective. However, as described herein, it has been unexpectedly discovered that residual CD3 surface expression may still indicate residual TCR+ cells, and that TCR+ depletion efficiency can be improved by using anti-CD3 antibodies in combination with anti-TCR antibodies.

[0047] Methods for selecting and depleting immune cells As described herein, in one aspect, the disclosure provides a method for depleting cells expressing endogenous TCR α / β dimers from a population of immune cells, the method comprising labeling the population of cells with an anti-TCR antibody and an anti-CD3 antibody (which labeling can be performed sequentially or simultaneously in a single operation), separating the anti-TCR-labeled and anti-CD3-labeled cells from unlabeled cells, and collecting the unlabeled cells, thereby obtaining a population of cells depleted of cells expressing endogenous TCR.

[0048] First, engineered immune cells modified to lack endogenous TCRs for the TCR alpha or TCR beta genes are exposed to a TCR depletion reagent. In some embodiments, the TCR depletion reagent comprises an antibody that targets a TCR alpha polypeptide, a TCR beta polypeptide, or a TCR alpha / beta heterodimer endogenously expressed on the surface of the immune cells. As described herein, TCR depletion using an anti-TCR antibody in combination with an anti-CD3 antibody (and / or optionally an anti-CD52 antibody) antibody unexpectedly increases the efficiency of TCR+ depletion compared to depletion performed with an anti-TCR antibody alone.

[0049] Anti-TCR, anti-CD3 or anti-CD52 antibodies (or other antibodies directed to the target of the TCR depletion procedure) can be conjugated to biotin to further facilitate labeling and / or separation using magnetic depletion reagents such as magnetic depletion agents, e.g., magnetic microbeads (typically, but not necessarily, nanoparticles with a diameter of about 50 nm) or secondary antibodies (e.g., anti-biotin antibodies) bound directly or indirectly to any other surface, e.g., agarose beads, sonic particles, plastic well plates, glass well plates, ceramic well plates, columns, cell culture bags, or membranes. The use of magnetic microbeads facilitates the separation of TCR+ cells from TCR- cells, and upon contact with a magnetic column, the TCR+ cells can be retained on the column while unlabeled TCR- cells pass through to a collection bag. Sonic particles facilitate the separation of TCR+ from TCR- cells when exposed to sonic waves. Although anti-biotin antibodies are provided in the context of the disclosed methods, other biotin binding partners, such as streptavidin, avidin, and other proteins that recognize biotin, can be used in place of anti-biotin antibodies in all of the methods provided herein.

[0050] In some embodiments, the provided cells can be optionally sorted for other cell surface markers. For example, a subset of the population of immune cells can include engineered immune cells expressing an antigen-specific CAR that itself comprises one or more epitopes specific for one or more monoclonal antibodies (e.g., exemplary mimotope sequences, see, e.g., International Publication No. WO 2016 / 120216, incorporated herein by reference). The method includes contacting the population of immune cells with a monoclonal antibody specific for the 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.

[0051] In some embodiments, the monoclonal antibody specific for the epitope is optionally conjugated to a fluorescent molecule, in which the step of selecting cells that bind to the monoclonal antibody can be performed by fluorescence activated cell sorting (FACS).

[0052] In some embodiments, the monoclonal antibody specific for the epitope is optionally bound to a magnetic particle, in which embodiment, the step of selecting cells that bind to the monoclonal antibody can be performed by magnetic activated cell sorting (MACS).

[0053] In some embodiments, the monoclonal antibody used in the method for selecting immune cells expressing a CAR is selected from the group consisting of alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, aviximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, bevacimab, certolizumab pegol, daclizumab ... In some embodiments, the monoclonal antibody is selected from rituximab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10, and / or ustekinumab. In some embodiments, the monoclonal antibody is rituximab. In other embodiments, the monoclonal antibody is QBEND-10.

[0054] Flow cytometry can be used to quantify specific cell types within a cell population. In general, flow cytometry is a method for quantifying cellular components or structural features primarily by optical means. Quantifying structural features can distinguish different cell types, so that flow cytometry and cell sorting can be used to count and sort cells of different phenotypes in a mixture.

[0055] Flow cytometry analysis involves two major steps: (1) labeling a 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 a population. In some embodiments, the method of labeling a cell type involves binding a labeled antibody to a marker expressed by a particular cell type. The antibody can be directly labeled with a fluorescent compound or indirectly labeled, for example, using a fluorescently labeled secondary antibody that recognizes the primary antibody.

[0056] In some embodiments of the disclosed method, sorting or separation of TCR+ cells, optionally expressing a CAR, from TCR- cells can be achieved using magnetic activated cell sorting (MACS). MACS is a method that uses superparamagnetic nanoparticles and columns to separate different cell populations according to their surface antigens (CD molecules), allowing for highly pure cell populations to be obtained. Cells in single cell suspension can be magnetically labeled with microbeads. The sample is applied to a column made of ferromagnetic material, which is covered with a coating that does not destroy the cells, allowing for rapid and gentle separation of the cells. Magnetically labeled cells are retained in the column, while unlabeled cells pass through the column. The flow-through fraction can be collected as the unlabeled cell fraction. After a washing step, the column is removed from the separator and the magnetically labeled cells are eluted from the column.

[0057] A detailed protocol for purifying specific cell populations, such as T cells, is described in Basu S et al. (2010) (Basu S, Campbell HM, Dittel BN, Ray A. Purification of specific cell population by fluorescence activated cell sorting (FACS). J. Vis. Exp. (41): 1546).

[0058] In some embodiments of the disclosed method, sorting or separating TCR+ cells, optionally expressing a CAR, from TCR- cells may be accomplished using sonic separation instead of magnetic-based separation methods. Without wishing to be bound by theory, sonic separation is understood to separate components of a mixture by three-dimensional standing waves. In the context of the disclosed method, antibodies such as anti-TCR antibodies, anti-CD52 antibodies, or anti-CD3 antibodies can be bound to a surface, such as sonic particles. The sonic particles can be beads. In one embodiment, cells are exposed to sonic particles carrying one or more of anti-TCR antibodies, anti-CD52 antibodies, or anti-CD3 antibodies, allowing the sonic particles to bind to any cells expressing the target of interest. The cells are then placed in an acoustic chamber and exposed to sonic waves. Given the different properties of cells associated with beads and cells not labeled with antibody bead particles, the sonic waves can separate the labeled and unlabeled cells, allowing the labeled cells (e.g., TCR+ cells or CD52+ cells) to be collected while fending off the labeled cells.

[0059] immune cells Cells suitable for the TCR+ cell depletion methods described herein include immune cells.

[0060] Prior to in vitro manipulation or genetic modification (e.g., as described herein), cells (e.g., immune cells) for use in the methods described herein can be obtained from a subject. Cells can be obtained from many non-limiting subject-based sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines available and known to those of skill in the art can be used. In some embodiments, cells can be derived from healthy donors or patients diagnosed with cancer. In some embodiments, cells can be part of a mixed population of cells exhibiting different phenotypic characteristics.

[0061] In some embodiments, the immune cells are obtained from the subject who will ultimately receive the engineered immune cells (i.e., autologous therapy). In some embodiments, the immune cells are obtained from a donor who is a different individual than the subject who will ultimately receive the engineered immune cells (i.e., allogeneic therapy).

[0062] Cells can be obtained from an individual's circulating blood by apheresis. The apheresis product typically includes lymphocytes, such as T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, cells collected by apheresis can be washed to remove the plasma fraction and placed into an appropriate buffer or medium for further processing.

[0063] Using the methods described herein, PBMCs can be used directly for genetic modification to generate engineered immune cells (such as CARs or TCRs). In certain embodiments, after isolating PBMCs, T lymphocytes can optionally be further isolated to generate a population containing only T cells, and both cytotoxic and helper T lymphocytes can be further classified into subpopulations of naive, memory, and effector T cells, either before or after genetic modification and / or expansion.

[0064] In certain embodiments, T cells can be isolated from PBMCs by lysing red blood cells and depleting monocytes, for example, using centrifugation through a PERCOLL® gradient. Specific subpopulations of T cells, such as CD28+ T cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, CD25+ T cells, CD62L+ T cells, CD5+ T cells, CD45RA-, CCR7+ T cells, CD95+ T cells, IL2Rβ+ T cells, and CD45RO+ T cells, can be further isolated by positive or negative selection methods known in the art. For example, enrichment of T cell populations by negative selection can be achieved with a combination of antibodies directed against surface markers unique to the negatively selected cells. One method used herein is sorting and / or selection of cells by negative magnetic immunoadhesion reaction or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present disclosure.

[0065] In some embodiments, the population of T cells is enriched for CD8+ cells, either before or after applying the depletion methods provided herein.

[0066] In some embodiments, the population of T cells is enriched for CD4+ cells, either before or after applying the depletion methods provided herein.

[0067] In some embodiments, the populations or CD4+ cells and / or CD8+ cells can be further classified into naive cells, stem cell-like 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, expression of phenotypic markers of naive T cells includes CD45RA+, CD95-, IL2Rβ-, CCR7+, and CD62L+. In some embodiments, expression of phenotypic markers of stem cell-like memory T cells includes CD45RA+, CD95+, IL2Rβ+, CCR7+, and CD62L+. In some embodiments, expression of phenotypic markers of central memory T cells includes CD45RO+, CD95+, IL2Rβ+, CCR7+, and CD62L+. In some embodiments, expression of phenotypic markers of effector memory T cells includes CD45RO+, CD95+, IL2Rβ+, CCR7-, and CD62L-. In some embodiments, expression of phenotypic markers of T effector cells includes CD45RA+, CD95+, IL2Rβ+, CCR7-, and CD62L-. Thus, CD4+ and / or CD8+ T helper cells can be classified into naive cells, stem cell-like memory cells, central memory cells, effector memory cells, and T effector cells by identifying cell populations bearing cell surface antigens. In certain embodiments, the disclosed methods can be used to improve depletion of one or more of these subsets of T cells.

[0068] It will be understood that PBMCs can further include other cytotoxic lymphocytes such as NK cells and NK T cells. An expression vector carrying a coding sequence for a chimeric receptor as disclosed herein can be introduced into a population of human donor T cells, NK cells or NK T cells. Standard procedures can be used to cryopreserve T cells expressing CARs for storage and / or preparation for use in human subjects. In one embodiment, in vitro transduction, culture, and / or expansion of T cells is performed in the absence of non-human animal derived products such as fetal bovine serum and fetal bovine serum. In various embodiments, the cryopreservation medium can include, for example, CryoStor® CS2, CS5, or CS10, or other medium containing DMSO, or medium without DMSO.

[0069] Engineered immune cells The TCR+ cell depletion methods described herein may be particularly useful in creating immune cell therapies to treat cancer, including therapies comprised of engineered immune cells (e.g., CAR-T cells).

[0070] The genetically engineered immune cells can be allogeneic or autologous, and the disclosed methods can be applied to either autologous or allogeneic treatments.

[0071] In some embodiments, the engineered immune cells (or populations thereof) are or include T cells (e.g., inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, helper T lymphocytes, tumor infiltrating lymphocytes (TIL)), NK cells, NK-T cells, TCR expressing cells, dendritic cells, killer dendritic cells, mast cells, or macrophages. In some embodiments, the engineered immune cells may be from the group consisting of CD4+ T lymphocytes, CD8+ T lymphocytes, and combinations thereof. In some exemplary embodiments, the engineered immune cells are T cells. The T cells may also be gamma / delta T cells.

[0072] In some embodiments, the engineered immune cells may be derived from, for example, but not limited to, stem cells. The stem cells may be adult stem cells, non-human embryonic stem cells, more specifically, non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells (iPSCs), totipotent stem cells, or hematopoietic stem cells. The stem cells may be CD34+ or CD34-.

[0073] 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 cells are human cells. In some embodiments, the cells are transfected or transduced with a nucleic acid vector using a method selected from the group consisting of viral (lentiviral or gammaretroviral) transduction, electroporation, sonoporation, biolistics (e.g., gene guns), lipid transfection, polymer transfection, nanoparticles, or polyplexes. In some embodiments, the cells are T cells reprogrammed from non-T cells. In some embodiments, the cells are T cells reprogrammed from T cells.

[0074] Binding agents (including antibodies and fragments thereof) In some embodiments, the disclosed methods include 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 may also include a binding agent.

[0075] As used herein, the term "antibody" refers to a polypeptide that contains sufficient standard immunoglobulin sequence elements to confer specific binding to a particular target antigen. As is known in the art, intact antibodies produced in nature are tetrameric agents of about 150 kD consisting of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that are linked together in what is commonly referred to as a "Y-shaped" structure. Each heavy chain is composed of at least four domains (each about 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 (located at the base of the stem of the Y). A short region known as the "switch" connects the variable and constant regions of the heavy chain. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other within the intact antibody. Each light chain is composed of two domains: an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain. Those of skill in the art are familiar with the structural and sequence elements of antibodies and will recognize that there is some flexibility in identifying "variable" and "constant" regions in the sequences provided, and that the definition of the "boundaries" between such domains is therefore flexible, such that different representations of the same antibody chain sequence may exhibit such boundaries at positions that are shifted by one or several residues, for example, compared to one different representation of the same antibody chain sequence.

[0076] An intact antibody tetramer is composed of two heavy-light chain dimers in which the heavy and light chains are linked together by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions together, so that the dimers are linked together to form a tetramer. Antibodies produced in nature are also usually glycosylated in the CH2 domain. Each domain of a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two β-sheets (e.g., three-, four-, or five-stranded sheets) packed together in a compressed antiparallel β-barrel. Each variable domain contains three hypervariable loops called "complement determining regions" (CDR1, CDR2, and CDR3) and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4). When a native antibody folds, the FR regions form a β-sheet that provides the structural framework for the domain, and the CDR loop regions from both the heavy and light chains are assembled in three-dimensional space to create a single hypervariable antigen-binding site located at the tip of a Y-structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. As is known in the art, the affinity and / or other binding attributes of the Fc region for the Fc receptor can be modulated by glycosylation or other modifications. In some embodiments, the antibodies produced and / or utilized according to the present invention comprise a glycosylated Fc domain with such glycosylation modified or engineered.

[0077] In the present disclosure, in certain embodiments, any polypeptide or complex of polypeptides that contains a sufficient immunoglobulin domain sequence as found in a natural antibody may be referred to and / or used as an "antibody," regardless of whether such a polypeptide is produced naturally (e.g., produced by an organism in response to an antigen) or it may be produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, an antibody is polyclonal, and in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, the antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art.

[0078] Additionally, the term "antibody" as used herein may alternatively refer to any of the constructs or formats known or developed in the art for utilizing the structural and functional features of antibodies. For example, in some embodiments, the antibodies utilized in the methods of the present disclosure may be intact IgA, IgG, IgE, or IgM antibodies; bi- or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab fragments, F(ab)2 fragments, Fd fragments, and isolated CDRs or sets thereof; single chain variable fragments (scFV); Fc fusion polypeptides; 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, masked antibodies (e.g., Probodies®); small modular immunopharmaceuticals (SMIPs), and / or other suitable antibodies (e.g., SMIPs), such as SMIPs, SMIPs, and / or other suitable antibodies (e.g., ... IP™); single chain or tandem diabodies (TandAb®); VHH; Anticalins®; Nanobodies® minibodies; BiTE®; ankyrin repeat proteins or DARPIN®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; microproteins; Fynomers®, Centyrins®; and KALBITOR®. In some embodiments, the antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if it were naturally produced. In some embodiments, the antibody may include a covalent modification (e.g., attachment of a glycan, attachment of a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or attachment of other pendant groups (e.g., polyethylene glycol, etc.).

[0079] 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 that contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements that are humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody agent" is used to alternatively refer to one or more of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies. For example, antibody agents utilized in accordance with the present invention include intact IgA, IgG, IgE or IgM antibodies; bi- or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; Fc fusion polypeptides; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g., Probodies®); small modular immunopharmaceuticals (SMIPs™); single chain or tandem diabodies (T andAb(registered trademark); VHH; Anticalins(registered trademark); Nanobodies(registered trademark); BiTE(registered trademark); Ankyrin repeat proteins or DARPIN(registered trademark); Avimers(registered trademark); DART; TCR-like antibodies; Adnectins(registered trademark); Affilins(registered trademark); Trans-bodies(registered trademark); Affibodies(registered trademark); TrimerX(registered trademark); microproteins; Fynomers(registered trademark), Centyrins(registered trademark); and KALBITOR(registered trademark).

[0080] The antibody or antibody agent used in carrying out the method of the present disclosure may 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, Fab', Fv, F(ab')2, dAb, and any combination thereof.

[0081] Antibodies and antibody agents include antibody fragments. Antibody fragments include portions of an intact antibody, such as the antigen-binding or variable regions of the intact antibody. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, diabodies, linear antibodies, multispecific formed from antibody fragments, antibodies, and scFv fragments, and other fragments. Antibodies also include, but are not limited to, polyclonal, monoclonal, chimeric dAb (domain antibodies), single chain, Fab, Fa, F(ab')2 fragments, and scFv. Antibodies can be whole antibodies, immunoglobulins, or antibody fragments. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli, Chinese Hamster Ovary (CHO) cells, or phage) known in the art.

[0082] In some embodiments, the antibody or antibody agent may 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 remaining portion of the heavy and / or light chain is derived from a different source or species. In one example, a chimeric antibody may include a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody may be a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. A chimeric antibody includes an antigen-binding fragment thereof.

[0083] In some embodiments, a chimeric antibody can be a humanized antibody (see, e.g., Almagro and Fransson, Front Biosci., 13:1619-1633 (2008); Riechmann et al., Nature, 332:323-329 (1988); Queen et al., Proc. Natl Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005); Padlan, Mol. Immunol, 28:489-498 (1991); Dall'Acqua et al., Methods, 36:43-60 (2005); Osbourn et al., Methods, 36:61-68 (2005); and Klimka et al., Br. J. Cancer, 83:252-260 (2000). Humanized antibodies are chimeric antibodies that contain amino acid residues from a non-human hypervariable region and amino acid residues from human FRs. In certain embodiments, a humanized antibody contains substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable regions (e.g., CDRs) corresponding to those of a non-human antibody and all or substantially all of the framework regions (FRs) corresponding to those of a human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody.

[0084] In some embodiments, the antibody or antibody agent provided herein is a human antibody. Human antibodies can be produced using a variety of techniques known in the art (see, for example, van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5:368-74 (2001), and Lonberg, Curr. Opin. Immunol, 20:450-459 (2008)). A human antibody can have an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody strictly excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be prepared using methods well known in the art.

[0085] Chimeric antigen receptors (CARs) 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 use in therapeutic applications such as allogeneic therapy. In some embodiments, the engineered immune cells include a CAR that includes 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). Upon binding to a target antigen, the CAR can activate the immune cell to attack and destroy the cell that bears that antigen (e.g., a cancer cell). The CAR can also incorporate a costimulatory domain, including all or part of a protein such as 4-1BB, CD28, or OX40, and / or a signaling domain such as CD3 zeta, to enhance its efficacy. See Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161:2791-2797; Song et al., Blood 119:696-706 (2012); Kalos et al., Sci. Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016); U.S. Patent Nos. 7,741,465 and 6,319,494.

[0086] The chimeric antigen receptors described herein comprise an extracellular domain, a transmembrane domain, optionally a hinge domain that connects the extracellular domain to the transmembrane domain, and an extracellular domain, wherein the extracellular domain comprises an antigen binding domain that specifically binds to a target.

[0087] 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 domain As described above, the CARs described herein comprise an antigen binding domain. As used herein, "antigen binding domain" refers to any polypeptide that binds to a specific target antigen. In some embodiments, the antigen binding domain is an 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 cancers). The disclosed methods can be used to deplete TCR+ cells that are also CAR+ cells, thereby providing a population of CAR+ / TCR- cells.

[0088] 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 described herein. In some embodiments, the antigen binding domain is a single chain variable fragment (scFv) comprising the light chain CDRs CDR1, CDR2, and CDR3, and the heavy chain CDRs CDR1, CDR2, and CDR3.

[0089] An antigen-binding domain is said to be "selective" if it binds more strongly to one target than it binds to another target.

[0090] 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, Claudin-18.2, Muc17, FAP alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, CD52, or CD34. 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 alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, CD52, or CD34.

[0091] In some embodiments, the cancer antigen is 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, an antigen of a cytomegalovirus (CMV)-infected cell (e.g., a cell surface antigen), epithelial glycoprotein (EGP), or a combination thereof. 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine protein kinase erb-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-13Ra2), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), 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 (WT-1).

[0092] In other embodiments, the disclosure relates to an isolated polynucleotide encoding any one 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.

[0093] In some embodiments, a CAR immune cell (e.g., a CAR-T cell) that can form a component of a cell population generated by performing the methods of the present disclosure comprises a polynucleotide encoding a safety switch polypeptide, such as, for example, RQR8. See, for example, International Publication No. WO2013153391A, which is incorporated herein by reference in its entirety. In a CAR immune cell (e.g., a CAR-T cell) that comprises a polynucleotide, the safety switch polypeptide can be expressed on the surface of the CAR immune cell (e.g., a CAR-T cell).

[0094] CAR hinge domain The extracellular domain of a CAR of the present disclosure can include a "hinge" domain (or hinge region). This term generally refers to any polypeptide that functions to link the transmembrane domain of a CAR to the extracellular antigen-binding domain of the CAR. Specifically, the use of a hinge domain can provide more flexibility and accessibility to the extracellular antigen-binding domain.

[0095] 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 all or part of CD8, CD4, CD28, 4-1BB, or the extracellular region of IgG (particularly the hinge region of IgG; it will be understood that the hinge region includes all or part of members of the immunoglobulin family, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof), or all or part of the heavy chain constant region of an antibody. Alternatively, the hinge domain may be a synthetic sequence corresponding to a naturally occurring sequence, or may be a completely synthetic hinge sequence. In some embodiments, the hinge domain is part of the human CD8 alpha chain (e.g., NP_001139345.1). In another specific embodiment, the hinge domain and transmembrane domain comprise a portion of the human CD8α chain. In some embodiments, the hinge domain of the CAR described herein comprises a subsequence of CD8α, IgG1, IgG4, PD-1, or FcγRIIIα, specifically the hinge region of either CD8α, IgG1, IgG4, PD-1, or FcγRIIIα. In some embodiments, the hinge domain comprises a human CD8α hinge, a human IgG1 hinge, a human IgG4, a human PD-1, or a human FcγRIIIα hinge. In some embodiments, the CAR disclosed herein comprises an scFv, a CD8α human hinge and transmembrane domain, a CD3ζ signaling domain, and a 4-1BB signaling domain.

[0096] CAR transmembrane domain The CARs provided herein are designed with a transmembrane domain fused to the extracellular domain of the CAR. It can also be fused to the intracellular domain of the CAR. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein and minimize interaction with other members of the receptor complex. In some embodiments, a short linker can form a bond between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.

[0097] The transmembrane region may be synthetic (not naturally occurring) or may be any of the following: CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death 1 (PD-1), inducible T cell costimulatory factor (ICOS), lymphocyte function associated antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor , MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The ligand may be a ligand that specifically binds to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, or CD83, or may be derived from any combination thereof (including or corresponding to a fragment thereof).

[0098] In some embodiments, the transmembrane domain in a CAR of the present disclosure is a CD8α transmembrane domain.

[0099] In some embodiments, the transmembrane domain in a CAR of the present disclosure is a CD28 transmembrane domain.

[0100] CAR intracellular domain The intracellular (cytoplasmic) domain of the CAR can activate at least one of the normal effector functions of the immune cell that contains the CAR. Effector functions of T cells can refer to, for example, cytolytic activity and helper activity, including secretion of cytokines.

[0101] It will be appreciated that suitable (e.g., activating) intracellular domains include, but are not limited to, CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death 1 (PD-1), inducible T cell costimulatory factor (ICOS), lymphocyte function associated antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, and the like. Proteins, cytokine receptors, integrins, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The signaling domains include those derived from (including or corresponding to) a fragment thereof, or a ligand that specifically binds to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0102] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include the 41BB / CD137 domain by itself, or can be combined with any other desired intracellular domain useful in the context of a CAR. The complete native amino acid sequence of 41BB / CD137 is set forth in NCBI Reference Sequence: NP_001552.2. The complete native 41BB / CD137 nucleic acid sequence is set forth in NCBI Reference Sequence: NM_001561.5.

[0103] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include a CD28 domain by itself or can be combined with any other desired intracellular domain useful in the context of the CAR of the present disclosure. The complete native amino acid sequence of CD28 is described in NCBI Reference Sequence: NP_006130.1. The complete native CD28 nucleic acid sequence is described in NCBI Reference Sequence: NM_006139.1.

[0104] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include all or a fragment of the CD3 zeta domain by itself, or can be combined with any other desired intracellular domain useful in the context of a CAR.

[0105] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure comprises a domain of a costimulatory molecule. In some embodiments, the intracellular signaling domain of a CAR comprises a portion of a costimulatory molecule selected from the group consisting of a fragment of 41BB (GenBank: AAA53133.) and CD28 (NP_006130.1).

[0106] Engineered immune cells containing CARs Provided herein are engineered immune cells that have been depleted of cells expressing an endogenous TCR, and populations of engineered immune cells that express a CAR (e.g., CAR-T cells or CAR+ cells).

[0107] In some embodiments, the engineered immune cells comprise CAR T cells, each CAR T cell comprises an extracellular antigen binding domain and has reduced or eliminated expression of an endogenous TCR. In some embodiments, the population of engineered immune cells comprises a population of CAR T cells, each CAR T cell comprises two or more different extracellular antigen binding domains and has reduced or eliminated expression of an endogenous TCR. In some embodiments, the immune cells comprise a population of CARs, each CAR T cell comprises the same extracellular antigen binding domain and has reduced or eliminated expression of an endogenous TCR.

[0108] The engineered immune cells can be allogeneic or autologous.

[0109] In some embodiments, the engineered immune cell or population of engineered immune cells is a T cell (e.g., inflammatory T lymphocytes cytotoxic T lymphocytes, regulatory T lymphocytes, helper T lymphocytes, tumor infiltrating lymphocytes (TILs)), NK cell, NK-T cell, TCR expressing cell, dendritic cell, killer dendritic cell, mast cell, or B cell, and expresses a CAR. In some embodiments, the T cell may be derived from the group consisting of CD4+ T lymphocytes, CD8+ T lymphocytes, or a population comprising a combination of CD4+ T cells and CD8+ T cells.

[0110] In some embodiments, the engineered immune cells or populations of engineered immune cells produced using the disclosed methods may be derived from, for example and without limitation, stem cells. The stem cells may be adult stem cells or non-human embryonic stem cells, and more specifically, non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells.

[0111] In some embodiments, the engineered immune cells or populations of immune cells 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 cells are human cells. In some embodiments, the cells are transfected or transduced with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., gene guns), lipid transfection, polymer transfection, nanoparticles, viral transfection (e.g., retrovirus, lentivirus, AAV) or polyplexes.

[0112] In some embodiments, engineered immune cells expressing an antigen-specific CAR on their cell surface membrane comprise greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of stem cell-like memory cells and central memory cells.

[0113] In some embodiments, engineered immune cells expressing an antigen-specific CAR of the present disclosure on their cell surface membrane comprise 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%, about 30% to about 100%, about 30% to about 9% 0%, 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% from about 60%, from about 60% to about 100%, from about 60% to about 90%, from about 60% to about 80%, from about 60% to about 70%, from about 70% to about 90%, from about 70% to about 80%, from about 80% to about 100%, from about 80% to about 90%, from about 90% to about 100%, from about 25% to about 50%, from about 75% to about 100%, or from about 50% to about 75% of stem cell-like memory cells and central memory cells.

[0114] In some embodiments, engineered immune cells expressing an antigen-specific CAR on their cell surface membrane comprise greater than 10%, 20%, 30%, 40%, 50%, or 60% of stem cell-like memory cells and central memory cells.

[0115] In some embodiments, engineered immune cells expressing an antigen-specific CAR on their cell surface membrane comprise 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% of stem cell-like memory cells and central memory cells.

[0116] In some embodiments, engineered immune cells that express an antigen-specific CAR on their cell surface membrane are characterized in that the engineered immune cells are T CM Cells and T SCM The cells are combined to comprise at least about 60%, 65%, 70%, 75%, or 80% of the T CM Cells and / or T SCM In some embodiments, engineered immune cells that express an antigen-specific CAR on their cell surface membrane are enriched in T cells. CM Cells and T SCM Combine the cells and mix until they contain at least 70% T CM Cells and / or T SCM In some embodiments, engineered immune cells that express an antigen-specific CAR on their cell surface membrane are enriched in T cells. CM Cells and / or T SCM Combine the cells and mix until they contain at least about 75% T CM Cells and / or T SCM Rich in cells.

[0117] In some embodiments, engineered immune cells expressing an antigen-specific CAR of the present disclosure on their cell surface membrane are characterized in that the engineered immune cells express at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% T SCM To contain the cells, CM Cells and / or T SCM In some embodiments, engineered immune cells expressing an antigen-specific CAR of the present disclosure on their cell surface membrane are enriched in at least about 30%, 35%, 40%, or 45% T cells. SCM To contain the cells, CM Cells and / or TSCM Rich in cells.

[0118] In some embodiments, the engineered immune cells are inflammatory T lymphocytes expressing a CAR. In some embodiments, the engineered immune cells are cytotoxic T lymphocytes expressing a CAR. In some embodiments, the engineered immune cells are regulatory T lymphocytes expressing a CAR. In some embodiments, the engineered immune cells are helper T lymphocytes expressing a CAR.

[0119] In some embodiments, engineered immune cells according to the present disclosure may include one or more disrupted or inactivated genes. In some embodiments, the gene for a target antigen (e.g., EGFRvIII, Flt3, 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 alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, or CD34, C D70) and introduce a CAR targeting the same antigen (e.g., EGFRvIII, Flt3, 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 alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, or CD34, CD70 CAR) and induce activation of the CAR. As described herein, in some embodiments, the engineered immune cells according to the present disclosure comprise a disrupted or inactivated gene selected from the group consisting of MHC1(β2M), MHC2(CIITA), EGFRvIII, Flt3, 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 alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, or one selected from the group consisting of CD34, CD70, TCR alpha, and TCR beta, and / or express a CAR or a multi-chain CAR. In some embodiments, the cells comprise a multi-chain CAR.In some embodiments, the isolated cells comprise two disrupted or inactivated genes selected from the group consisting of CD52 and TCR alpha, CDR52 and TCR beta, PD-1 and TCR alpha, PD-1 and TCR beta, MHC-1 and TCR alpha, MHC-1 and TCR beta, MHC2 and TCR alpha, MHC2 and TCR beta, and / or express a CAR or a multi-chain CAR.

[0120] In some embodiments, the method includes disrupting or inactivating one or more genes by introducing into a cell an endonuclease that can selectively inactivate genes by selective DNA cleavage. In some embodiments, the endonuclease can be, for example, a zinc finger nuclease (ZFN), a megaTAL nuclease, a meganuclease, a transcription activator-like effector nuclease (TALE nuclease, or TALEN®), or a CRISPR (e.g., Cas9 or Cas12) endonuclease.

[0121] In some embodiments, the TCR+ cells or populations thereof are rendered non-functional within the cells by disrupting or inactivating the endogenous TCRα and / or TCRβ genes. The depletion methods of the present disclosure are particularly useful for removing remaining TCR+ T cells from a population of cells following inactivation of the TRCα gene. The engineered immune cells generated 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). Thus, 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), comprising administering to the patient an effective amount of engineered immune cells comprising a disrupted or inactivated TCRα and / or TCRβ gene.

[0122] The present disclosure provides methods for increasing the purity of a population of engineered immune cells that lack or have 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. Such populations can be the product of the disclosed methods.

[0123] In some embodiments, the population of engineered immune cells 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. Such populations can be the product of the disclosed methods.

[0124] In some embodiments, the population of engineered immune cells comprises about 0.01-0.001% TCR+ cells. Such a population can be the product of the disclosed methods.

[0125] The present disclosure also provides engineered immune cells comprising any of the CARs described herein, which may be characterized in conjunction with reduced or eliminated expression of one or more endogenous genes. In some embodiments, the CAR may be introduced into the immune cell as a transgene via a plasmid vector. In some embodiments, the plasmid vector may also include a selection marker, e.g., that allows for identification and / or selection of cells that have received the vector.

[0126] Manufacturing of TCR engineered immune cells and populations of TCR engineered immune cells (including CAR T cells) Provided herein is a method for depleting cells expressing endogenous TCR 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-CD3 antibodies, separating the anti-TCR and anti-CD3 labeled cells from unlabeled cells, and collecting the unlabeled cells facilitates obtaining a cell population that is depleted of cells expressing endogenous TCR. In various embodiments, separation can generally be achieved using magnetic beads, sonic particles, membranes (all of which can be bound to moieties recognized by the labeled cells), FACS, and other known separation methods. Cells produced using this method also form an aspect of the present disclosure.

[0127] Provided herein is a method for depleting cells expressing endogenous TCR from a population of immune cells (including engineered immune cells such as CAR+ cells). As described herein, the cell population is labeled with anti-TCR and anti-CD52 antibodies, and the anti-TCR and anti-CD52 labeled cells are separated from unlabeled cells, and the unlabeled cells are collected, facilitating the obtaining of a cell population that is depleted of cells expressing endogenous TCR. In various embodiments, separation can generally be achieved using magnetic beads, sonic particles, membranes (all of which can be bound 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.

[0128] Also provided herein is a method for depleting cells expressing endogenous TCR from a population of immune cells (including engineered immune cells such as CAR+ cells). As described herein, a population of cells is labeled with anti-TCR and anti-CD3 and anti-CD52 antibodies, and the anti-TCR, anti-CD3 and anti-CD52 labeled cells are separated from unlabeled cells, and the unlabeled cells are collected, facilitating the obtaining of a cell population depleted of cells expressing endogenous TCR. In various embodiments, separation may generally be accomplished using magnetic beads, sonic particles, membranes (all of which may be bound to a moiety recognized by the labeled cells), FACS, and other known separation methods. Cells produced using this method also form an aspect of the present disclosure.

[0129] Prior to in vitro manipulation or genetic modification of the engineered immune cells described herein, cells can be obtained from a subject. A population of cells expressing a CAR can be derived from an allogeneic or autologous process, as described herein, and can be depleted of endogenous TCRs, as described herein. Genetic modification of isolated immune cells

[0130] As described herein, immune cells such as T cells can be genetically modified using known methods prior to carrying out the methods provided herein, or the immune cells can be activated and expanded (or differentiated in the case of iPSCs or progenitor cells) in vitro prior to genetic modification. In some embodiments, immune cells are genetically modified to reduce or eliminate expression of endogenous TCR (TRAC), MHC1 (β2M), MHC2, PD1, and / or CD52. In some embodiments, expression of two or more endogenous proteins can be reduced or eliminated. For example, expression of TRAC and CD52 can be reduced or eliminated. In another example, expression of TRAC and a protein targeted by the transduced CAR can be reduced or eliminated. In some embodiments, cells are genetically modified using gene editing techniques (e.g., CRISPR / Cas9, zinc finger nucleases (ZFNs), TALEN®, MegaTALs, meganucleases) to reduce or eliminate expression of one or more endogenous proteins (e.g., TCRα, MHC1, MHC2, PD1, CD52). In another embodiment, immune cells such as T cells are genetically modified with a chimeric antigen receptor described herein (e.g., by transduction with a viral vector comprising one or more nucleotide sequences encoding a CAR or by using non-viral means) and then activated and / or expanded in vitro.

[0131] Certain methods for making the constructs and engineered immune cells of the present disclosure are described in International Publication No. WO 2015 / 120096 (PCT / US15 / 14520), the contents of which are incorporated herein by reference in their entirety.

[0132] In one embodiment, the present disclosure provides a method for preserving engineered cells expressing a CAR. This includes cryopreserving immune cells so that the cells remain viable upon thawing. A portion of the immune cells expressing a CAR can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients suffering from malignant tumors. If necessary, the cryopreserved transformed immune cells can be thawed, expanded, and expanded for more such cells. As shown by the examples and figures, a population of cells produced using the depletion methods provided herein can be cryopreserved and later thawed for therapeutic use.

[0133] Allogeneic CAR T cells The process of creating allogeneic CAR T therapy, or AlloCARs™, involves harvesting healthy, selected, screened, and tested T cells from healthy donors. The T cells are then engineered to express a CAR that recognizes a specific cell surface protein expressed on hematological or solid tumors (e.g., a target antigen such as 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 alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, CD52, or CD34). The allogeneic T cells are gene edited using gene editing tools such as TALEN®, zinc finger, CRISPR, or other gene editing technologies to reduce the risk of graft versus host disease (GvHD) and prevent allogeneic rejection (HvGD) when given to a patient different from the donor.

[0134] To avoid GvHD, expression of endogenous T cell receptor genes (e.g., TCRα, TCRβ) can be reduced or eliminated. Expression of endogenous MHC1 (β2M), MHC2 and / or PD1 can also be reduced or eliminated to prevent host immune cells from recognizing MHC1 and MHC2 on transplanted cells as foreign antigens to avoid HvGD. Endogenously expressed CD52 (cluster of differentiation 52) gene can also be reduced or eliminated to make CAR T products resistant to anti-CD52 antibody therapy. In this way, lymphodepletion of anti-CD52 antibody therapy can be used to suppress the host immune system and maintain the engraftment of CAR T to obtain full therapeutic effect. As described herein, engineered immune cells or populations thereof for use in allogeneic therapy can include multiple knockouts, at least for the purposes exemplified above.

[0135] In some embodiments, endogenous expression of the gene encoding programmed cell death protein 1 (PD1), also known as CD279, can be reduced or eliminated to enhance anti-tumor efficacy.

[0136] Autologous CAR T cells Autologous chimeric antigen receptor (CAR) T cell therapy involves harvesting a patient's own cells (e.g., white blood cells, 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 the patient.

[0137] Pharmaceutical Compositions and Treatments Pharmaceutical compositions comprising populations of cells prepared using the disclosed methods can be useful for treating patients with cancer. A desired therapeutic amount of a population of engineered cells generated using the methods provided herein can generally be at least 2 cells (e.g., at least one CD8+ central memory T cell, or at least one CD4+ helper T cell subset, or one each of CD8+ and CD4+ cells), or more typically, 102 More than 10 cells, up to a maximum of 10 6 Pieces, up to 10 8 Pieces or 10 9 cells, 10 10 The number of cells will depend on the intended desired use of the composition and the type of cells contained therein. Desired cell densities are typically between 10 and 200 cells. 6 More than 10 cells / ml 7 A clinically relevant number of immune cells can be allocated for multiple infusions, which cumulatively amount to more than 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 pieces, 10 10 pieces, 10 11 Pieces or 10 12 In some applications of the cell populations produced using the methods of the present disclosure, the number of cells injected may be equal to or greater than 10 6 Pieces / kilogram (10 per patient) 6 ~10 11 Fewer numbers of cells can be administered, ranging from 100 to 150 cells per 1000 cells per day. CAR therapy can be administered multiple times at doses within this range. Cells can be autologous, allogeneic, or xenogeneic to the patient receiving therapy.

[0138] As described herein, the engineered immune cell population can be depleted of cells expressing endogenous TCR (e.g., the cells are TCR- cells and / or contain 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 levels of TCR+ cells can be obtained using the methods disclosed herein.

[0139] In some embodiments, the population of engineered immune cells 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 levels of TCR+ cells can be achieved using the methods disclosed herein.

[0140] In some embodiments, the population of engineered immune cells comprises about 0.01-0.001% TCR+ cells. Cell populations having the above levels of TCR+ cells can be obtained using the methods disclosed herein.

[0141] In some embodiments, the population of engineered immune cells comprises undetectable levels of TCR+ cells. Cell populations having the above levels of TCR+ cells can be obtained using the methods disclosed herein.

[0142] In some embodiments, the population of engineered 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 the above levels of TCR-cells can be obtained using the methods disclosed herein.

[0143] In some embodiments, the population of engineered immune cells comprises about 99.99-99.999% TCR- cells. Cell populations having these levels of TCR- cells above can be obtained using the methods disclosed herein.

[0144] The TCR-depleted CAR-expressing cell populations produced 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 other cell populations. The pharmaceutical compositions of the present disclosure can include a CAR-expressing TCR- cell population, such as the engineered T cells described herein. The compositions of the present disclosure are preferably formulated for injection or intravenous administration. treatment

[0145] The present disclosure provides a method for treating or preventing a disease (e.g., cancer) in a patient, comprising administering to a patient in need thereof an effective amount of a population of engineered immune cells (e.g., cells obtained from a healthy donor rather than from the patient) that contain a CAR that has been depleted of cells expressing an endogenous TCR (e.g., the cells are TCR- cells and / or contain residual levels of TCR+ cells).

[0146] In some embodiments, the population of engineered immune cells 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 levels of TCR+ cells can be obtained using the methods disclosed herein.

[0147] In some embodiments, the population of engineered immune cells 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 levels of TCR+ cells can be obtained using the methods disclosed herein.

[0148] In some embodiments, the engineered immune cell population comprises about 0.01-0.001% TCR+ cells. In some embodiments, the engineered immune cells comprise undetectable levels of TCR+ cells. Cell populations having these levels of TCR+ cells above can be obtained using the methods disclosed herein.

[0149] In some embodiments, the population of engineered 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 levels of TCR+ cells above can be obtained using the methods disclosed herein.

[0150] In some embodiments, the population of engineered immune cells comprises about 99.99-99.999% TCR- cells. Cell populations having these levels of TCR+ cells above can be obtained using the methods disclosed herein.

[0151] Provided herein are methods for treating diseases or disorders, including cancer. These methods reduce the likelihood that patients will face GvHD when treated with allogeneic therapy. In some embodiments, the disclosure relates to administering to a subject in need thereof an effective amount of a population of TCR-depleted engineered immune cells prepared using the methods of the disclosure. In some embodiments, the T cell-mediated immune response is directed against a target cell or cells expressing a cancer antigen. In some embodiments, the population of TCR-depleted engineered immune cells comprises cells expressing a chimeric antigen receptor (CAR). In some embodiments, the target cell is a solid or hematological 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 population of TCR-depleted engineered immune cells prepared using the methods described herein. In some aspects, the disclosure includes a method for treating or preventing a malignancy, comprising administering to a subject in need thereof an effective amount of a population of TCR-depleted engineered immune cells prepared using the methods provided herein, wherein the population of TCR-depleted immune cells comprises at least one chimeric antigen receptor and / or an isolated antigen binding domain described herein. In some embodiments, the population of TCR-depleted CAR-containing immune cells prepared using the methods of the disclosure can be used to treat a hematological malignancy or solid tumor.

[0152] In some embodiments, the population of TCR-depleted CAR-containing immune cells prepared using the methods of the present disclosure may be used to treat small cell lung cancer, melanoma, low-grade glioma, glioblastoma, medullary thyroid carcinoma, carcinoid, pancreatic intracellular neuroendocrine tumor, bladder and prostate, testicular cancer, and lung adenocarcinoma with neuroendocrine function. 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 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).

[0153] Also provided is a method for reducing the size of a tumor in a subject comprising administering to the subject a population of TCR-depleted engineered cells prepared using the methods of the present disclosure, wherein the cells comprise a chimeric antigen receptor comprising an antigen binding domain that binds to an antigen on the tumor.

[0154] In some embodiments, the subject has a solid tumor or a hematological malignancy such as lymphoma or leukemia (blood cancer). In some embodiments, a population of TCR-depleted engineered cells prepared using the disclosed methods are delivered to the tumor bed. In some embodiments, the cancer is present in the subject's bone marrow and a population of cells prepared using the disclosed methods are delivered thereto. In some embodiments, the cancer is present in the patient's immune cells or blood cells and a population of cells prepared using the disclosed methods are delivered thereto. In some embodiments, the engineered cells are autologous immune cells (e.g., autologous T cells). In some embodiments, the engineered cells are allogeneic immune cells (e.g., allogeneic T cells), for which the use of the disclosed methods is particularly beneficial.

[0155] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dosage" of a therapeutic agent (e.g., a population of TCR-depleted engineered CAR T cells generated using the methods of the present disclosure) is any amount that, when used alone or in combination with other therapeutic agents, protects a subject from developing a disease, or promotes regression of the disease as evidenced by a decrease in the severity of disease symptoms or an increase in the frequency and duration of disease-free periods, or prevention of functional impairment or disability due to the affliction of the disease. The efficacy of a therapeutic agent in promoting regression of a disease can be assessed using a variety of methods known to those of skill in the art, such as in human subjects in clinical trials or in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in an in vitro assay.

[0156] A desirable therapeutic amount of TCR-depleted engineered cells in a composition includes at least two cells (e.g., at least one CD8+ central memory T cell and at least one subset of CD4+ helper T cells, or at least two CD4+ cells or at least two CD8+ cells), or at least 10 2 More than 10 cells, up to a maximum of 10 6 Pieces, up to 10 8 Pieces or 10 9 cells, 10 10 The number of cells will depend on the intended desired use of the composition and the type of cells contained therein. It is noted that the methods provided herein can be used to generate populations of TCR-depleted engineered immune cells that contain only CD4+ cells, or only CD8+ cells, or both CD4+ and CD8+ cells. Desirable cell densities are typically between 10 and 200 cells. 6 More than 10 cells / ml, typically 7 More than 10 cells / ml, typically 8 Clinically relevant numbers of immune cells can be allocated for multiple infusions, which cumulatively amount to 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8pieces, 10 9 pieces, 10 10 pieces, 10 11 Pieces or 10 12 In some aspects of the present disclosure, the number of injected cells is equal to or exceeds 10 cells, particularly since all injected cells are redirected to a specific target antigen. 6 Pieces / kilogram (10 per patient) 6 ~10 11 Fewer numbers of cells can be administered, ranging from 100 to 150 cells / ml. CAR therapy can be administered multiple times at dosages within these ranges. Cells can be autologous, allogeneic, or xenogeneic to the patient receiving therapy.

[0157] In some embodiments, a therapeutically effective amount of TCR-depleted CAR T cells prepared using the methods of the present disclosure is about 1×10 5 cells / kg, approximately 2×10 5 cells / kg, approximately 3×10 5 cells / kg, approximately 4×10 5 cells / kg, approximately 5×10 5 cells / kg, approximately 6×10 5 cells / kg, approximately 7×10 5 cells / kg, approximately 8×10 5 cells / kg, approximately 9×10 5 cells / kg, 2×10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 cells / kg, or approximately 9 x 107 cells / kg.

[0158] In some embodiments, the target dose of CAR+ / TCR- T cells is 1×10 6 From 2 × 10 8 range of cells / kg, e.g., 2 x 10 6 Cells / kg. Of course, doses above or below this range may be appropriate for a particular subject, and appropriate dose levels can be determined by a medical provider as needed. Furthermore, cells can be administered multiple times in accordance with the present disclosure.

[0159] In some embodiments, upon administration to a patient, a population of TCR-depleted engineered immune cells prepared using the methods of the present disclosure that express any one of the antigen-specific CARs described herein on their cell surface can reduce, kill, or lyse the patient's endogenous antigen-expressing cells. In one embodiment, the percentage of reduction or lysis of antigen-expressing endogenous cells or cells of a cell line expressing an antigen by engineered immune cells expressing any one of the antigen-specific CARs described herein is at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%, or greater. In one embodiment, the percentage of reduction or lysis of antigen-expressing endogenous cells or cells of a cell line expressing an antigen by engineered immune cells expressing an antigen-specific CAR is about 5% to about 95%, about 10% 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 cells are endogenous antigen-expressing bone marrow cells.

[0160] A variety of additional therapeutic agents can be used in combination with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab. These compounds can be administered prior to, simultaneously with, or after administration of the population of TCR-depleted engineered immune cells prepared using the methods provided herein.

[0161] In some embodiments, a composition comprising a population of TCR-depleted CAR-expressing immune cells prepared using the methods provided herein can be administered prior to, concurrently with, or following a therapeutic regimen designed to prevent or treat cytokine release syndrome (CRS) or neurotoxicity (such as an anti-IL6 antibody).

[0162] In certain embodiments, the composition of the population of TCR-depleted CAR-containing immune cells 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. Among the cytokines are growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; Müllerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factor (NGF), such as NGF-beta; platelet growth factor; transforming factor, such as TGF-alpha and TGF-beta. Examples of cytokines include transforming growth factors (TGFs); insulin-like growth factors-I and -II; erythropoietin (EPO); bone morphogenetic factors; interferons, such as interferon-alpha, beta, and gamma; colony stimulating factors (CSFs), such as macrophage CSF (M-CSF); granulocyte macrophage CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, and IL-21; tumor necrosis factors, such as TNF-alpha and TNF-beta; and other polypeptide factors, including LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0163] Kits and Products The present disclosure provides kits that include a TCR depletion reagent that includes one or more of an anti-TCR antibody, an anti-CD3 antibody, and an anti-CD52 antibody. The kits can be used in the depletion methods provided herein.

[0164] In one embodiment, the kit provided herein comprises an anti-TCR antibody and an anti-CD3 antibody. In the kit, one or both of the anti-TCR and / or anti-CD3 antibodies can be optionally conjugated to biotin. In a specific embodiment, both the anti-TCR antibody and the anti-CD3 antibody are conjugated to biotin.

[0165] In one embodiment, the kit provided herein comprises an anti-TCR antibody and an anti-CD52 antibody. In the kit, one or both of the anti-TCR and / or anti-CD52 antibodies can be optionally conjugated to biotin. In a specific embodiment, both the anti-TCR antibody and the anti-CD52 antibody are conjugated to biotin.

[0166] In one embodiment, the kit provided herein comprises an anti-TCR antibody, an anti-CD3 antibody, and an anti-CD52 antibody. In the kit, one or all of the anti-TCR and / or anti-CD3 and / or anti-CD52 antibodies can be optionally conjugated to biotin. In a specific embodiment, the anti-TCR antibody, the anti-CD3 antibody, and the anti-CD52 antibody are all conjugated to biotin.

[0167] In another aspect, the kits provided herein can further include an anti-biotin antibody bound to magnetic nanomatrix microbeads, cell-sized beads, or other supports such as membranes, acoustic particles or beads, plastic plates or columns, etc. The anti-biotin antibody can be provided in a bound form or can be provided as a naked antibody, optionally with materials to aid in attachment to the magnetic nanomatrix microbeads, cell-sized beads, or other supports.

[0168] In certain embodiments, the kits provided herein comprise an anti-TCR antibody and an anti-CD3 antibody, where one or both of the anti-TCR and / or anti-CD3 antibodies can be directly bound to magnetic beads, membranes, sonic particles, plastic plates, or columns, as desired.

[0169] In certain embodiments, the kits provided herein comprise an anti-TCR antibody, an anti-CD3 antibody, and an anti-CD52 antibody, in which one or all of the anti-TCR and / or anti-CD3 and / or anti-CD52 antibodies can be directly bound to magnetic beads, membranes, sonic particles, plastic plates, or columns, as desired.

[0170] It should be noted that while the use of anti-biotin antibodies forms one embodiment of the methods disclosed herein, other means of capturing biotin-labeled moieties can be used, for example, streptavidin, avidin and other biotin-binding moieties can be used in place of anti-biotin antibodies in the disclosed methods.

[0171] The present disclosure also provides articles of manufacture that include any of the therapeutic compositions and kits described herein. Exemplary articles of manufacture include containers (e.g., sealed vials) that contain a therapeutic agent (e.g., a population of TCR-depleted cells, which may further include a CAR, prepared using the methods disclosed herein). EXAMPLES

[0172] As shown in the examples below, the disclosed combination antibody approach unexpectedly reduced residual TCR+ levels (%) from 0.1-1% to 0.1-0.01% (measured 1 day after depletion) compared to TCR antibody alone, resulting in a significant improvement in TCR+ cell depletion efficiency. These examples demonstrate the significant advantages provided by the disclosed depletion methods over current approaches for depleting TCR+ cells. These advantages may benefit patients undergoing allogeneic therapy in the form of patients being less likely to exhibit GvHD.

[0173] Example 1. Combination of anti-TCR and anti-CD3 antibodies improves the efficiency of depletion of TCR+ cells Using CAR engineered immune cells, the expression of endogenous TCR and CD52 genes was knocked out by electroporation of TALENs directed to the TRAC and CD52 genes. TCR and CD52 knockout cells were then exposed to TCR depletion reagents. As shown in Table 1, the cells were contacted with a primary anti-TCR antibody conjugated to biotin alone or in combination with an anti-CD3 or anti-CD52 antibody. A secondary anti-biotin antibody coupled to magnetic microbeads (nanoparticles with a diameter of about 50 nm) was then added to the primary antibody-labeled cells to bind the magnetic microbeads to the remaining TCR+ cells via the primary anti-TCR biotin moiety. The labeled cells were then applied to a magnetic column using a CliniMACS Prodigy® instrument. The TCR+ cells were retained in the magnetic column while the unlabeled TCR- cells passed through to a product collection bag. The TCR+ cells were later released from the column into a waste bag. The TCR+ cell depletion efficiency was investigated using various depletion methods provided in this disclosure, and the remaining TCR+ cells present in the TCR- cell population fraction were observed using anti-TCRα, anti-TCRβ and / or CD3 antibodies at various days after depletion. Figure 1 shows a schematic diagram of the allogeneic CART manufacturing process with different unit operation scenarios. [Table 1] [Table 2]

[0174] As shown in Figure 2A, the percentage of TCR+ cells present in the TCR-producing populations depleted using the various different depletion methods is substantially less than 1%. Figure 2A shows that the depletion method unexpectedly reduced TCR+ cell frequency by 204-fold compared to the control depletion method using a 1-fold lower TCR antibody concentration, as detected 2 days after depletion, with a comparable reduction observed when cells were treated with anti-TCR, anti-CD3 and anti-CD52 antibodies. Figure 2A also shows that the use of anti-TCR and anti-CD52 antibodies, as well as the use of 3-fold concentrations of anti-TCR antibodies, was less effective than the combination of anti-TCR and anti-CD3 antibodies, with 3-fold TCR antibody treatment showing a 6.2-fold reduction and the combination of anti-TCR and anti-CD52 showing a 2.7-fold reduction. Interestingly, Figure 2A shows that depletion using anti-CD52 and anti-TCR antibodies is more efficient than using anti-TCR antibodies alone. This result is surprising in light of the fact that, as described herein, CD52 has no biological association with the TCR or TCR complex, and therefore it was unexpected that a depletion method using these two antibodies together was more efficient at depleting TCR+ cells than simply using anti-TCR antibodies alone. The results shown in Figure 2A also unexpectedly show that simply increasing the concentration of anti-TCR antibodies does not produce significantly better results than using lower concentrations of anti-TCR antibodies in combination with anti-CD3 antibodies.

[0175] The depleted population was also examined 9 days after depletion, and the results are shown in FIG. 2B. FIG. 2B shows an 8-fold reduction in TCR+ cell frequency when anti-TCR and anti-CD3 antibodies were used in the depletion method, with comparable reductions observed when cells were treated with anti-TCR, anti-CD3, and anti-CD52 antibodies. FIG. 2B also shows that the use of anti-TCR and anti-CD52 antibodies, as well as a 3-fold concentration of anti-TCR antibody, was less effective than the combination of anti-TCR and anti-CD3 antibodies, with the 3-fold TCR antibody treatment showing a 5-fold reduction and the combination of anti-TCR and anti-CD52 antibodies showing a 2-fold reduction. These results indicate that simply increasing the concentration of anti-TCR antibody does not produce significantly better results than using a lower concentration of anti-TCR antibody in combination with anti-CD3 antibody.

[0176] The FACS plots in Figure 3A visualize the minimal presence of TCR+ cells when depletion is performed using anti-TCR and anti-CD3 antibodies compared to anti-TCR antibodies alone, or in combination with anti-CD3 and / or anti-CD52 antibodies, or increased (3-fold) concentrations of anti-TCR antibodies. Data was taken on days 0 and 1 post-depletion and compared to pre-depletion cell samples. The results further demonstrate that simply increasing the concentration of anti-TCR antibodies does not produce significantly better results than using lower concentrations of anti-TCR antibodies in combination with anti-CD3 antibodies across the days of the experiment.

[0177] The numerical bar graphs in Figure 3B show that the depletion method using anti-TCR and anti-CD3 antibodies reduced TCR+ cell frequency by 151-fold and 23- to 32-fold at days 0 and 1 post-depletion, respectively, versus a control depletion method using a low 1x TCR antibody concentration, with a comparable reduction of 70-fold observed when cells were treated with anti-TCR, anti-CD3 and anti-CD52 antibodies. It should be noted that current TCR depletion procedures simply use a standard 1x TCR antibody concentration, which is understood to be the current state of the art and differs from the disclosed method. Figure 3B also shows that anti-TCR and anti-CD52 antibodies, as well as the use of a 3x concentration of anti-TCR antibody, were less effective than the combined treatment of anti-TCR and anti-CD3 with 3x TCR antibody, showing a 19-fold and 7-fold reduction, and the combination of anti-TCR and anti-CD52 showing a 2.7-fold and 1.6-fold reduction. These results further show that simply increasing the concentration of anti-TCR antibody does not produce significantly better results than using a lower concentration of anti-TCR antibody in combination with anti-CD3 antibody across the days of the experiment.

[0178] Figure 4 is a FACS plot of data collected on days 0 and 1 after depletion, further demonstrating that the combination of anti-TCR and anti-CD3 antibodies results in a higher level of TCR depletion than either anti-TCR antibodies used alone at higher (3x) or lower (1x) concentrations, and anti-TCR antibodies used in combination with anti-CD52 antibodies. Remaining TCR+ cells after depletion were visualized by single staining with anti-TCR or anti-CD3, or double staining with anti-TCR / CD3, and were expected to stain positive for either TCR+ or CD3+, or CD3+ / TCR+. The small population of cells stained with CD3+ / TCR- reflects the remaining TCRγδ T cells present in the depleted TCR- cell population, as TCRγδ T cells are expected to be removed by anti-CD3 antibodies.

[0179] Example 2. Cultivation after depletion This example shows that the depleted immune cell populations retained the same low residual TCR+ levels during post-depletion culture. One day after depletion, the depleted cells were frozen and then thawed for post-depletion culture. The expression levels of TCR and CD3 were detected before freezing and immediately after thawing. As shown in Figures 5A-5E, there was no significant difference in either TCR or CD3 frequency due to the freeze-thaw cycle. FIG. 5A shows the frequency of TCR+ cells, CD3+ cells, CD3+ / TCR- cells, and CD3+ / TCR+ cells before and after freezing and thawing by FACS plot using anti-TCR or anti-CD3 single staining or anti-TCR / CD3 double staining; FIG. 5B shows the numerical frequency of TCR+ cells before and after freezing and thawing using anti-TCR antibody; FIG. 5C shows the numerical frequency of CD3+ cells before and after freezing and thawing using anti-CD3 antibody; FIG. 5D shows the numerical frequency of CD3+ / TCR- cells before and after freezing and thawing using anti-TCR / CD3 antibody; and FIG. 5E shows the numerical frequency of CD3+ / TCR+ cells before and after freezing and thawing using anti-TCR / CD3 antibody.

[0180] To determine whether the frequency of residual TCR+ cells in the population of depleted TCR- cells increases with culture time, depleted cells were cultured for 10 days after depletion. The frequencies of TCR and CD3 cells were detected using single staining with anti-TCR antibodies (Figure 6) and single staining with anti-CD3 antibodies (Figure 7), or double staining with anti-TCR / CD3 antibodies (Figure 8), every 2-3 days throughout the culture process. Specifically, the FACS plots in Figures 6, 7, and 8 show that, although for all depletion methods, there was a gradual trend in TCR+ cell frequency, CD3+ cell frequency, or CD3+ / TCR+ cell frequency, which peaked at about days 5-7 of the post-depletion culture period, cultures depleted using a combination of anti-TCR, anti-CD3, and / or anti-CD52 antibodies maintained similarly lower TCR+ or CD3+ cell frequencies over time than cultures treated with low or high concentrations of anti-TCR antibody alone, or with anti-TCR antibody in combination with anti-CD52 antibody.

[0181] Subsequently, the bar graphs in Figures 9A, 9B, 9C, and 9D numerically depict the frequency of TCR+, CD3+, CD3+ / TCR-, or CD3+ / TCR+ during the culture period following depletion.

[0182] In addition, cell proliferation status, including viable cell density (Figure 10A), viability (Figure 10B), cell diameter at each passage (Figure 10C), and total fold expansion (Figure 10D), was also monitored during the culture period after depletion. Overall, comparable cell proliferation characteristics were observed for all depletion methods studied.

[0183] The efficiency of CD52 depletion using different combinations of antibodies disclosed herein was also investigated. Specifically, anti-CD52 antibodies were used for CD52 depletion alone or in combination with other antibodies, and the effect of anti-TCR and / or anti-CD3 antibodies used for TCR+ cell depletion on the removal of CD52+ cells was also examined. The FACS plot in Figure 11A shows the frequency of remaining CD52+ cells after depletion at day 0 and day 1 after depletion. The bar graph in Figure 11B shows the numerical frequency of remaining CD52+ cells. Finally, the FACS plot in Figure 12 shows the frequency of remaining CD52+ cells after depletion for a 10-day culture period.

[0184] In summary, the data from Example 2 showed that the use of both anti-TCR and anti-CD3 antibodies improved the efficiency of TCR+ cell depletion. The use of anti-CD3 antibodies in addition to anti-TCR antibodies resulted in a greater depletion of CD3+ / TCR+ cells than the use of anti-TCR antibodies alone. This depletion mechanism improved the efficiency of TCR+ cell depletion and may be an important advantage for therapeutic allogeneic applications. The depleted cells showed similar proliferation in all conditions during the post-depletion culture period, indicating that the depletion method did not affect cell viability and proliferation. Yet another aspect of the present invention may be as follows. [1] A method for producing a population of immune cells depleted of immune cells expressing endogenous TCR, comprising: (a) labeling a population of immune cells with anti-TCR and anti-CD3 antibodies; and (b) separating anti-TCR antibody-labeled immune cells and anti-CD3 antibody-labeled immune cells from the population of immune cells. [2] A method for depleting cells expressing endogenous TCRs from a population of immune cells, comprising: (a) labeling the population of immune cells with an anti-TCR antibody and an anti-CD3 antibody; (b) separating the anti-TCR antibody-labeled immune cells and the anti-CD3 antibody-labeled immune cells from unlabeled immune cells; and (c) collecting the unlabeled immune cells; Thereby obtaining a population of immune cells that is depleted of cells expressing endogenous TCR. [3] A method for producing a population of immune cells depleted of immune cells expressing endogenous TCR, comprising: (c) labeling the population of immune cells with an anti-TCR antibody and an anti-CD52 antibody; and (d) separating anti-TCR antibody-labeled immune cells and anti-CD52 antibody-labeled immune cells from the population of immune cells. [4] A method for depleting cells expressing endogenous TCRs from a population of immune cells, comprising: (d) labeling the population of immune cells with an anti-TCR antibody and an anti-CD52 antibody; (e) separating the anti-TCR antibody-labeled immune cells and the anti-CD52 antibody-labeled immune cells from unlabeled immune cells; and (f) collecting the unlabeled immune cells; Thereby obtaining a population of immune cells that is depleted of cells expressing endogenous TCR. [5] The method according to [1] or [2], further comprising labeling the population of immune cells with an anti-CD52 antibody, and depleting the immune cells labeled with the anti-CD52 antibody from the population of immune cells. [6] The method according to [1] or [2], further comprising labeling the population of immune cells with an anti-CD52 antibody and separating the anti-CD52 antibody-labeled immune cells from unlabeled immune cells. [7] The method according to any one of [1] to [6] above, wherein the anti-TCR antibody, the anti-CD3 antibody, and / or the anti-CD52 antibody are biotin-conjugated. [8] The method according to [7], further comprising contacting the labeled cells with an agent that specifically recognizes biotin. [9] The method according to [8] above, wherein the agent that specifically recognizes biotin is selected from the group consisting of an anti-biotin antibody, avidin, and streptavidin.

[10] The method according to [8] or [9], wherein the agent is bound to a magnetic bead, an agarose bead, an acoustic 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 according to any one of [1] to [6] above, wherein the anti-TCR antibody, the anti-CD3 antibody, and / or the anti-CD52 antibody are directly bound to magnetic beads, agarose beads, sonic particles, a plastic well plate, a glass well plate, a ceramic well plate, a column, a cell culture bag, or a membrane.

[12] The method according to any one of [1] to

[11] above, wherein the separation is achieved using one of magnetic separation or sonic separation.

[13] The method according to any one of [1] to

[12] above, wherein the immune cells are allogeneic immune cells.

[14] The method according to any one of [1] to

[13] above, wherein the immune cells are genetically modified to reduce or eliminate expression of endogenous TCR.

[15] The method according to any one of [1] to

[13] above, wherein the immune cells are genetically modified to reduce or eliminate expression of endogenous CD52.

[16] The method according to any one of [1] to

[13] above, wherein the immune cells are genetically modified to reduce or eliminate expression of both endogenous TCR and endogenous CD52.

[17] The method according to any one of

[14] to

[16] above, wherein the reduction or elimination of the expression of endogenous TCR or endogenous CD52, or the expression of both endogenous TCR and endogenous CD52, is achieved using TALEN, CRISPR / Cas9, zinc finger nuclease (ZFN), MegaTAL, meganuclease, Cpf1, homologous recombination, or single-stranded oligodeoxynucleotide (ssODN).

[18] The method according to any one of [1] to

[17] above, wherein the immune cells are engineered immune cells that express a chimeric antigen receptor.

[19] The method according to any one of [1] to

[18] above, wherein the population of cells in which cells expressing endogenous TCR have been depleted comprises 1.0% or less TCR+ cells, 0.9% or less TCR+ cells, 0.8% or less TCR+ cells, 0.7% or less TCR+ cells, 0.6% or less TCR+ cells, 0.5% or less TCR+ cells, 0.4% or less TCR+ cells, 0.3% or less TCR+ cells, 0.2% or less TCR+ cells, or 0.1% or less TCR+ cells.

[20] The method according to any one of [1] to

[19] above, wherein the population of cells from which the cells expressing the endogenous TCR have been depleted contains 0.01 to 0.001% TCR+ cells immediately after depletion.

[21] The method according to any one of [1] to

[19] above, wherein the population of cells from which the cells expressing endogenous TCR have been depleted contains 0.1 to 0.01% TCR+ cells 1 to 10 days after depletion of the cells.

[22] The method according to

[21] , wherein the population of cells from which the cells expressing the endogenous TCR have been depleted contains less than 0.1 to 1.0% TCR+ cells after one day of culture after depletion.

[23] The method according to

[21] , wherein the population of cells from which the cells expressing the endogenous TCR have been depleted contains less than 0.1 to 1.0% TCR+ cells 10 days after depletion of the cells.

[24] The method according to any one of [1] to

[23] above, wherein the immune cells are T cells.

[25] A population of immune cells produced by the method according to any one of [1] to

[24] above, in which immune cells expressing endogenous TCR have been depleted.

[26] A population of engineered T cells containing at least 99% TCR- cells.

[27] A population of engineered T cells containing at least 99.9% TCR- cells.

[28] A population of engineered T cells containing at least 99.99% TCR- cells.

[29] A population of engineered T cells containing at least 99.999% TCR- cells.

[30] The population of engineered T cells described in any one of

[26] to

[29] above, wherein the population of T cells expresses a chimeric antigen receptor.

[31] A kit for depleting cells expressing endogenous TCR from a population of immune cells comprising an anti-TCR antibody and an anti-CD3 antibody.

[32] The kit according to

[31] , further comprising an anti-CD52 antibody.

[33] A kit for depleting cells expressing endogenous TCR from a population of immune cells, comprising an anti-TCR antibody and an anti-CD52 antibody.

[34] The kit according to any one of

[31] to

[33] , wherein the anti-TCR antibody, the anti-CD3 antibody, and / or the anti-CD52 antibody are bound to an agent that specifically recognizes biotin.

[35] The kit according to

[34] above, wherein the agent that specifically recognizes biotin is selected from the group consisting of an anti-biotin antibody, streptavidin, and avidin.

[36] The kit according to

[34] or

[35] , wherein the agent is bound to a magnetic bead, an agarose bead, an acoustic 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 according to any one of

[31] to

[35] , wherein the anti-TCR antibody, the anti-CD3 antibody, and / or the anti-CD52 antibody are directly bound to a support.

[38] The kit according to

[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 for treating a solid cancer or blood cancer in a subject in need of such treatment, comprising administering to the subject a therapeutic amount of a population of immune cells depleted of immune cells expressing an endogenous TCR described in any one of

[19] to

[23] above, or a population of engineered T cells described in any one of

[26] to

[30] above.

Claims

1. 1. A method for producing a population of immune cells depleted of immune cells expressing endogenous TCR α / β heterodimers, comprising: (a) labeling a population of immune cells with an anti-TCR antibody and an anti-CD3 antibody, wherein the anti-TCR antibody is an antibody that binds to and recognizes human TCR alpha, or human TCR beta, or a human TCR alpha / beta heterodimer; and (b) separating anti-TCR antibody labeled immune cells and anti-CD3 antibody labeled immune cells from the population of immune cells; This method produces a population of immune cells that contains 0.01% or less T cells that express endogenous α / β heterodimers.

2. 1. A method for depleting cells expressing endogenous TCR α / β heterodimers from a population of immune cells, comprising: (a) labeling the population of immune cells with an anti-TCR antibody and an anti-CD3 antibody, wherein the anti-TCR antibody is an antibody that binds to and recognizes human TCR alpha, or human TCR beta, or a human TCR alpha / beta heterodimer; (b) separating the anti-TCR antibody labeled immune cells and the anti-CD3 antibody labeled immune cells from unlabeled immune cells; and (c) collecting the unlabeled immune cells; This method results in a population of immune cells containing 0.01% or less T cells expressing endogenous α / β heterodimers.

3. 2. The method of claim 1, wherein the population of immune cells comprises 0.01% or less T cells expressing endogenous α / β heterodimers on day 2 or day 4 after depletion.

4. 3. The method of claim 2, wherein the population of immune cells comprises 0.01% or less T cells expressing endogenous α / β heterodimers on day 2 or day 4 after depletion.

5. 3. The method of claim 1 or 2, further comprising labeling the population of immune cells with an anti-CD52 antibody, and depleting the population of immune cells of the anti-CD52 antibody labeled immune cells.

6. 3. The method of claim 1 or 2, further comprising labeling the population of immune cells with an anti-CD52 antibody and separating the anti-CD52 antibody labeled immune cells from unlabeled immune cells.

7. The method of any one of claims 1 to 6, wherein the anti-TCR antibody, the anti-CD3 antibody, and / or the anti-CD52 antibody are biotin-conjugated.

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

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

10. 10. The method of claim 8 or 9, wherein the agent is bound 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 to 6, wherein the anti-TCR antibody, the anti-CD3 antibody, and / or the anti-CD52 antibody are directly bound to magnetic beads, agarose beads, sonic particles, plastic well plates, glass well plates, ceramic well plates, columns, cell culture bags, or membranes.

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 claims 1 to 12, wherein the immune cells are allogeneic immune cells.

14. The method of any one of claims 1 to 13, wherein the immune cells are genetically modified to be deficient in endogenous TCR α or TCR β genes to reduce or eliminate expression of endogenous TCR α / β heterodimers.

15. The method of any one of claims 1 to 14, wherein the immune cells are genetically modified to be deficient in the endogenous CD52 gene to reduce or eliminate expression of endogenous CD52.

16. The method of any one of claims 14 to 15, wherein the reduction or elimination of expression of endogenous TCR α / β heterodimer or endogenous CD52, or of both endogenous TCR α / β heterodimer and endogenous CD52, is achieved using TALENs, CRISPR / Cas9, zinc finger nucleases (ZFNs), MegaTALs, meganucleases, Cpf1, homologous recombination, or single-stranded oligodeoxynucleotides (ssODNs).

17. The method of any one of claims 1 to 16, wherein the immune cell is an engineered immune cell that expresses a chimeric antigen receptor.

18. The method of any one of claims 1 to 17, wherein the immune cell is a T cell.

19. 1. A kit for depleting cells expressing endogenous TCR α / β heterodimers from a population of immune cells, the population of immune cells comprising 0.01% or less T cells expressing endogenous α / β heterodimers; below: a) an anti-TCR antibody that binds to and recognizes human TCR alpha, or human TCR beta, or the human TCR alpha / beta heterodimer; and b-1) an anti-CD3 antibody, or b-2) an anti-CD3 antibody and an anti-CD52 antibody, Including the kit.

20. The kit of claim 19, wherein the anti-TCR antibody, the anti-CD3 antibody, and / or the anti-CD52 antibody are bound to an agent that specifically recognizes biotin, and the agent that specifically recognizes biotin may be optionally selected from the group consisting of an anti-biotin antibody, streptavidin, and avidin.

21. 21. The kit of claim 20, wherein the agent is bound 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.

22. The kit according to any one of claims 19 to 21, wherein the anti-TCR antibody, the anti-CD3 antibody, and / or the anti-CD52 antibody are directly bound to a support.

23. 23. The kit of claim 22, wherein the support is 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.

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