Methods and systems for production of engineered t-cells

Abstract

The present application provides methods and systems for improved production of cells (e.g., engineered immune cells) susceptible to fratricide. In some cases, production of engineered immune cells susceptible to fratricide that express chimeric antigen receptor (CAR) molecules capable of binding a cell surface molecule expressed by other immune cells that also express the same CAR molecules can limit viability and yield of the engineered immune cells. As described herein, methods and systems allowing control over parameters such as metabolite concentration and cell clump formation during culture can in some cases be used to increase yield and / or viability in the production of cells susceptible to fratricide. Also provided herein is an engineered immune cell, a population of engineered immune cells, and / or a pharmaceutical composition comprising the engineered immune cell or the population of engineered immune cells produced using the methods or systems described herein.

Description

METHODS AND SYSTEMS FOR PRODUCTION OF ENGINEERED T-CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 421,886, filed November 2, 2022, the entire content of which is incorporated herein by reference in its entirety.BACKGROUND

[0002] T-cells can be engineered to express a chimeric antigen receptor (CAR) capable of recognizing a molecule expressed on the surface of cancerous T-cells; however, CAR-T cells targeting a self-antigen are prone to killing other CAR-T cells (e.g., fratricide). Reducing cell surface expression of the target antigen can reduce fratricide, but some fratricide remains so long as there is any expression of the target. This can reduce the yield, viability, and activity of the CAR-T cells. Accordingly, new and improved methods and systems are needed for manufacturing CAR-T cells susceptible to fratricide.INCORPORATION BY REFERENCE

[0003] All publications, patents, and patent applications mentioned in this specification, including but not limited to US 10,765,699, US 10,550,183, and US 20210395779 Al, are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.SUMMARY

[0004] Described herein are methods and systems for the manufacture of a population of cells comprising a binding domain and a target-binding molecule, for example, for increasing cell production efficiency.

[0005] In one aspect the disclosure provides, a cell population comprising: at least 108immune cells of which: at least 70% are viable and at least 50% express a chimeric antigen receptor (CAR) comprising a binding domain that binds to a cell surface polypeptide expressed by the immune cells linked to an activation domain.

[0006] In some embodiments, at least 80% of the at least 108immune cells are viable.

[0007] In some embodiments, at least 60% of the at least 108immune cells express theCAR.

[0008] In some embodiments, at least 50% of the immune cells have reduced expressionof the cell surface polypeptide. In some embodiments, the cell surface polypeptide is CD2, CD3, CD5, CD7, CD8, or CD38.

[0009] In some embodiments, the immune cells having reduced expression of the cell surface polypeptide comprise a chimeric targeting polypeptide that comprises a binding domain that binds to the cell surface polypeptide linked to an intracellular localizing domain. In some embodiments, the intracellular localizing domain is an ER retention signal, a Golgi retention signal or a PEST sequence. In some embodiments, the ER retention signal comprises a KKXX sequence wherein X is any amino acid. In some embodiments, the ER retention signal comprises a KDEL sequence. In some embodiments, the chimeric targeting polypeptide further comprises a spacer sequence between the binding domain and the KDEL sequence.

[0010] In some embodiments, the immune cells are T-cells. In some embodiments, the immune cells are NK cells. In some embodiments, the immune cells are frozen.

[0011] In one aspect, the disclosure provides, a pharmaceutical composition comprising the cell population described herein.

[0012] In one aspect, the disclosure provides, a method comprising: incubating cells susceptible to fratricide in a culture medium without continuous agitation in a vessel comprising a gas-permeable membrane, wherein at least 70% of the cells are viable after 5 days of incubation.

[0013] In some embodiments, the cells comprise a chimeric antigen receptor (CAR) comprising: (i) a binding domain that binds to a cell surface polypeptide expressed by the cells, and (ii) an activation domain that activates the cells. In some embodiments, the cells have reduced expression of the cell surface polypeptide compared to healthy human cells of a cell type that is the same cell type as the cells. In some embodiments, the cells comprise a non-natural modification of a gene encoding the polypeptide.

[0014] In some embodiments, the cells further comprise a chimeric targeting polypeptide that comprises a domain that binds to the cell surface polypeptide linked to an intracellular localizing domain. In some embodiments, the cells susceptible to fratricide comprise immune cells. In some embodiments, the immune cells comprise T-cells. In some embodiments, the immune cells comprise NK-cells.

[0015] In some embodiments, the activation domain induces cytotoxic activity of the immune cells. In some embodiments, the cell surface polypeptide is CD7.

[0016] In some embodiments, the method further comprises replacing at least a portion of the culture medium in the vessel. In some embodiments, the replacing is executed every 1 to3 days and wherein the replacing comprises replacing at least 75% of the culture medium in the vessel. In some embodiments, the vessel contains 100 mL to 1 L of the culture medium.

[0017] In some embodiments, a concentration in the culture medium of a metabolite produced by the cells is maintained below a threshold level for a time interval. In some embodiments the metabolite is lactate and / or ammonium. In some embodiments, the threshold is about 1.2 mM-14 mM. In some embodiments, the time interval is at least 7 days of culture. In some embodiments, the method further comprises, disrupting clumps of the cells, e.g., mechanically disrupting the clumps of the cells.

[0018] In some embodiments, the replacing and the disrupting are executed at regular intervals. In some embodiments, disrupting is performed every 1 to 3 days. In some embodiments, the method further comprises mixing the culture medium in the vessel.

[0019] In some embodiments, the method further comprises activating the cells. In some embodiments, the activating comprises contacting cells with an antibody that binds to CD3 or CD28. In some embodiments, the activating comprises contacting the cells with antibodies that bind to CD3 and CD28. In some embodiments, the cells are transduced cells, and the method further comprises, prior to incubating, transducing starting cells with a viral vector to generate the transduced cells. In some embodiments, the viral vector comprises a nucleotide sequence encoding the CAR or the chimeric targeting polypeptide. In some embodiments, the viral vector comprises a nucleotide sequence encoding the CAR and the chimeric targeting polypeptide.

[0020] In some embodiments, the starting cells were obtained from a human subject. In some embodiments, the human subject is a healthy human subject. In some embodiments, the human subject is a human subject diagnosed with a cancer, e.g., T-cell lymphoma or leukemia. In some embodiments, the human subject had been diagnosed with an autoimmune disease.

[0021] In some embodiments, at least 20% of the starting cells are CD4 positive. In some embodiments, at least 80% of the starting cells are CD4 positive. In some embodiments, the starting cells are enriched for CD4 positive cells. In some embodiments, at least 20% of the starting cells are CD8 positive. In some embodiments, at least 80% of the starting cells are CD8 positive. In some embodiments, the starting cells are enriched for CD8 positive cells. In some embodiments, at least 20% of the starting cells are CD3 positive. In some embodiments, at least 80% of the starting cells are CD3 positive. In some embodiments, the starting cells are enriched for CD3 positive cells. In some embodiments, at least 20% of the starting cells are CD56 positive. In some embodiments, at least 80% of the starting cells areCD56 positive. In some embodiments, the starting cells are enriched for CD56 positive cells.

[0022] In some embodiments, the method further comprises selecting the cells susceptible to fratricide from the transduced cells prior to the incubating. In some embodiments, the selecting comprises isolating cells that express the CAR, and / or isolating cells that express CD4 or CD8 and / or isolating cells that express CD25 and CD69 after activation.

[0023] In some embodiments, at least 80% of the selected cells are positive for surface expression of CD3. In some embodiments, at least 80% of the selected cells have reduced surface expression of the cell surface polypeptide compared to an unmodified cell of the same cell type from a healthy human subject. In some embodiments, at least 80% of the selected cells are negative for surface expression of CD56. In some embodiments, the selecting comprises an affinity-based selection, e.g., fluorescence-activated cell sorting or magnetic- activated cell sorting.

[0024] In some embodiments, the incubating results in a 30 to 40-fold increase in the quantity of cells. In some embodiments, the method further comprises harvesting at least 1 E8 of the cells sensitive to fratricide after a time period of incubation. In some embodiments, the method further comprises harvesting at least 1 x 108of the cells sensitive to fratricide after no more than 11 days of incubation. In some embodiments, at least 50% of the harvested cells comprise the CAR and have reduced expression of the cell surface polypeptide. In some embodiments, at least 70% of the harvested cells are viable. In some embodiments, at least 70% of harvested cells expressing the CAR are viable. In some embodiments, at least 70% of harvested cells having reduced expression of the cell surface polypeptide are viable.

[0025] In some embodiments, the method further comprises freezing the harvested cells, optionally wherein at least 70% of the frozen cells are viable.

[0026] In some aspects, the disclosure provides a system comprising: a vessel comprising a gas permeable membrane and a culture medium; and cells susceptible to fratricide contacting a surface of the gas permeable membrane.

[0027] In some embodiments, the cells susceptible to fratricide comprise a chimeric antigen receptor (CAR) comprising: (i) a binding domain that binds to a cell surface polypeptide expressed by the cells susceptible to fratricide, and (ii) an activation domain that activates the portion of the cells susceptible to fratricide. In some embodiments, the cells susceptible to fratricide have reduced expression of the cell surface polypeptide compared to cells of the same lineage isolated from a healthy human donor. In some embodiments, the cells susceptible to fratricide comprise a non-natural modification of a gene encoding thepolypeptide. In some embodiments, the cells susceptible to fratricide comprise a chimeric targeting polypeptide comprising a domain that binds to the cell surface polypeptide linked to an intracellular localizing domain.

[0028] In some embodiments, the system comprises a closed culture component. In some embodiments, the system further comprises a liquid handler configured to exchange the culture medium. In some embodiments, the system further comprises a device configured to disrupt clumps of cells. In some embodiments, the system further comprises a controller configured to cause the liquid handler to replace at least a portion of the culture medium in the vessel or cause the device to disrupt clumps of cells. In some embodiments, the system further comprises a sensor configured to analyze the culture medium optionally wherein the sensor can measure a nutrient, a metabolite, e.g., a lactate or an ammonium, or a pH. In some embodiments, the controller is configured to operate the liquid handler based at least in part on a measurement made by the sensor.

[0029] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure”, “Fig ”, and “FIGURE” herein) of which:

[0031] FIG. 1 provides an illustrative schematic of a manufacturing process to manufacture fratricide-susceptible immune cells as described herein, in some embodiments.

[0032] FIG. 2 shows a culture vessel with a gas permeable membrane at the base of the vessel, a harvest line depicted by a curved tube, a medium removal line depicted by the longer straight tube, a sampling line depicted by the shorter straight tube, and an air filter for an attachment depicted by the round head attached to a tube coming out of the top of the vessel.

[0033] FIG. 3 depicts the nucleic acid sequence (SEQ ID NO: 1) of an exemplary construct comprising an MSCV promoter, an anti-human CD7 (TH69) CAR, P2A selfcleaving peptide, and an anti-human CD7 (TH69) protein expression blocker (PEBL). The MSCV promoter is double underlined, a restriction enzyme site and Kozak sequence are between the MSCV promoter and CAR, an anti-human CD7 (TH69) CAR is in bold, a P2A is in normal font, and anti-human CD7 (TH69) PEBL is single underlined.

[0034] FIG. 4 depicts a flowchart of an embodiment of the methods described herein, including, but not limited, to providing a population of cells, incubating the cells and a culture medium, controlling a concentration of metabolites in the medium, and disrupting cell clumps.

[0035] FIG. 5 depicts a flowchart of an embodiment of the methods described herein, including, but not limited, to providing a population of cells, selecting a subpopulation of cells, activating the cells, viral transduction of the cells, incubating the cells and a culture medium, controlling a concentration of metabolites in the medium, and disrupting cell clumps.

[0036] FIG. 6 shows expression of CAR and CD7 by primary T-cells transduced with MSCV-CD7CAR-P2A-CD7PEBL lentivirus and analyzed by flow cytometry at 3 days, 6 days, and 9 days post transduction.

[0037] FIGs. 7A-7D show cell count, viability and cell type after CliniMACS Prodigy® runs. FIG. 7A depicts a graph of total viable cells over time showing the effect of run conditions IPR1, IPR2, and Run 1 on total viable cells during manufacturing. FIG. 7B depicts a graph of viability over time showing the effect of run conditions IPR1, IPR2, and Run 1 on percentage viability of cells in culture during manufacturing. FIG. 7C shows a graph of percentage of CD3+CD56-CAR+CD7- cells during manufacturing. CD3 is a T-cell marker; CD56 is an NK cell marker; CAR is the anti-CD7 CAR; CD7- indicates activity of the anti-CD7 PEBL. FIG. 7D shows a graph of percentage of CD3+CD56-CD7- cells during manufacturing.

[0038] FIGs. 8A-8D show cell expansion and viability obtained during manufacturing using a G-Rex vessel at 10M and 100M scale. Arms G1-G3 are at a 10M scale, and Arms G4-G5 are at a 100M scale. Arm 1 is a bench-scale control. Arm G1 was performed in a G- Rex 10M vessel, utilizing a top-up feeding strategy (as opposed to 75% medium exchanges used in other experiments), under the same conditions as Run 1; Arm G3 was also performed in a G-Rex 10M vessel, but 75% of the culture medium was exchanged at days 8 and 11 in Arm G3. Arm G2 was performed without any sampling or resuspension of culture mediumfrom day 4 until harvest.

[0039] FIG. 8A is a graph of fold change over time showing the effects of run conditions of Arm 1 and Arms G1-G5 on fold change in total viable cell numbers relative to day 0. FIG. 8B is a graph of viability over time showing the effects of run conditions of Arm 1 and Arms G1-G5 on percentage viability of cells in culture during manufacturing. FIG 8C shows a graph of percentage of CD3+CD56-CD7- cells during manufacturing. FIG 8D shows a graph of the percentage of CD3+CD56-CAR+CD7- cells during manufacturing, which are T-cells expressing a T-cell marker (CD3), and a chimeric antigen receptor targeting CD7, and not expressing a NK cell marker (CD56) or CD7.

[0040] FIGs. 9A-9D show metabolite concentration in mmol / L and pH during use of a G-Rex vessel at 10M and 100M scale for manufacturing CAR+CD7- T-cells. Arms G1-G3 are at a 10M scale, and Arms G4-G5 are at a 100M scale. Arm 1 is a bench-scale control. FIG. 9A shows a graph of lactate concentration, FIG. 9B shows a graph of glucose concentration, FIG. 9C shows a graph of ammonium concentration, and FIG. 9D shows a graph of pH over the days in culture during manufacturing.DETAILED DESCRIPTION

[0041] Described herein are methods and systems for improved manufacturing of cells (e.g., engineered immune cells expressing a chimeric antigen receptor (CAR) and, in some cases, a protein expression blocker (PEBL)) and methods of use thereof. In some cases, engineered cells (e.g., cells engineered to express a CAR and / or a PEBL) can suffer from decreased viability during production. In some cases, engineered cells (e.g., expressing a CAR and / or a PEBL) can be more metabolically active than non-engineered cells of the same species and / or cell type. Metabolites produced by cells in culture and accumulated over the course of a manufacturing process in culture medium can affect culture medium pH and can decrease viability and / or yield of the engineered cells. Controlling pH and / or the concentration of metabolites in culture during manufacture can be important for improving viability and / or yield of the engineered cell population (see, 406, 512. FIG. 4, FIG. 5). In some cases, careful planning of cell culture medium exchange can help to control pH and concentration of metabolites in culture during manufacture. In some cases, the use of a gas-permeable membrane for culturing engineered cells described herein can aid in providing oxygen to engineered cells during production without continuous agitation, which can improve viability and / or yield of the engineered cells, in various embodiments. In some cases, the use of a gas-permeable membrane for culturingengineered cells described herein can also aid in controlling pH and nutrient concentrations for engineered cells during production, which can improve viability and / or yield of the engineered cells, in various embodiments. In some cases, engineered cells can form clumps during manufacture. In some cases, clumping of engineered cells during manufacture can lead to decreased rates of viral transduction and thus decreased rates of expression of desired proteins. Clumping of engineered cells during manufacture can be decreased by disruption of clumps of engineered cells during manufacture. In some cases, mechanical agitation of engineered cells can decrease viability of the engineered cells. In some cases, limiting the exposure of the cells to excessive mechanical agitation can improve viability of the engineered cells. In some cases, a method or system can comprise mixing (e.g., during culture medium exchanges) but not agitating the cells, which can improve viability and / or yield of engineered cell populations, in some cases.

[0042] Described herein are compositions and methods for manufacturing T cells that simultaneously express a chimeric antigen receptor targeting CD7 (e.g., CAR) and a CD7 protein expression blocker (e.g. PEBL) to result in high viability and yield. Compositions include populations of immune cells which comprise a receptor with a binding domain that binds to CD7 expressed on the surface of the T cells, an activation domain that activates the T cells, and a protein expression blocker (PEBL) that reduces cell surface expression of CD7.METHODS

[0043] Methods for manufacturing a population of cells described herein can comprise providing a population of cells (see, 402, 502; FIG. 4, FIG. 5). In some cases, providing a population of cells can comprise obtaining a blood sample or portion thereof (e.g., by blood draw or apheresis) from a subject (e.g., a healthy or affected subject), which may be referred to in some cases as a donor. The subject (e.g., donor) can be a human subject. For instance, the human subject (e.g., donor) can be a healthy human subject or an affected human subject. An affected subject (e.g., an affected human donor) can have or be at risk of having one or more conditions (e.g., one or more clinically defined diseases), affecting an immune cell, such as a cancer (e.g., T-cell lymphoma or T-cell leukemia), a viral infection, or an autoimmune disease. In some cases, an affected subject may have been diagnosed as having one or more conditions (e.g., one or more medically defined diseases), such as a cancer, a viral infection, or an autoimmune disease. In some cases, the subject (e.g., donor) can be anon-human primate. In some cases, the subject (e.g., donor) can be a mammal. In some cases, the subject (e.g., donor) can be a vertebrate. A population of cells can be fresh, frozen, or thawed. In some cases, providing a population of cells can comprise obtaining a previously prepared population of cells (e.g., a population of frozen cells or a population of thawed cells). In some cases, providing a population of cells can comprise thawing a previously prepared population of cells. In some cases, a previously prepared population of cells can comprise an enriched apheresis product (e.g., which may comprise cells collected through extraction of white blood cells, including but not limited to CD4-positive (CD4+), CD8- positive (CD8+), or CD3-positive (CD3+) T cells from a healthy individual). In some cases, an enriched apheresis product comprises NK cells. In some cases, providing a population of cells can comprise producing (e.g., expanding and / or differentiating) cells in culture. In some cases, a previously prepared population of cells can comprise or consist of a subpopulation of cells from a sample collected from a donor or subject or a sample produced in culture (e.g., mononuclear cells, immune cells, hematopoietic stem cells, precursor cells, T-cells, B-cells, or natural killer (NK) cells).

[0044] A method (e.g., 400, 500) for manufacturing a population of cells described herein can comprise selecting at least a portion of a population of cells (e.g., a population of cells provided as described herein, for instance from a healthy or affected human donor), for instance, as shown in step 504. In some cases, selecting at least a portion of a population of cells can comprise isolating one or more cell types of interest from the population of cells (e.g., for further processing and / or for use as a treatment). In some cases, isolating one or more cell types of interest can comprise performing one or more affinity -based cell separation techniques (for instance, fluorescence-activated cell sorting (FACS) and / or magnetic-activated cell sorting (MACS)), e.g., on the population of cells. In some cases, selecting at least a portion of a population of cells can comprise depleting the population of cells of one or more cell types. For instance, selecting a population of cells can comprise lysing a cell type (e.g., red blood cells) present in the population of cells. In some cases, depleting the population of cells of one or more cell types can comprise using density gradient centrifugation and / or affinity-based selection techniques to remove the one or more cell types from the population of cells. In some cases, selecting at least a portion of a population of cells can comprise isolating or enriching for cells expressing one or more cell surface molecules (e.g., cell surface markers) of interest, such as CD2, CD3, CD4, CD7, CD8, CD25 and / or CD69. For example, FACS or MACS (or another affinity-based selection technique) can be used to select (e.g., isolate or enrich) a population of cells expressing CD4and / or CD8. In some cases, MACS can be used to isolate or enrich a population of CD4 / CD8 double-positive cells. In some cases, selecting at least a portion of a population of cells can comprise isolating or enriching a population of cells expressing two or more molecules of interest (e.g., CD2, CD3, CD4, CD7, CD8, CD25, or CD69) at the same time (e.g., using a plurality of MACS or FACS affinity probes capable of binding one or more of the molecules of interest in a single selection assay) or sequentially. In some cases, selecting at least a portion of a population of cells can comprise isolating or enriching for cells expressing a chimeric antigen receptor (CAR). In some cases, selecting at least a portion of a population of cells can comprise selecting a population of cells susceptible to fratricide (e.g., a cell, such as an immune cell like a T-cell or an NK cell, that expresses a CAR capable of binding a molecule, for example a surface marker such as CD7, expressed by another cell (e.g., another T-cell) expressing a CAR capable of binding the same molecule). In some cases, a population of NK cells may be enriched. The enriched population of NK cells can express CD56.

[0045] In some cases, selecting at least a portion of a population of cells can occur before or after one or more other steps described herein. For instance, a step comprising selecting at least a portion of a population of cells can occur prior to incubating all or a portion of the population of cells in culture medium. In some cases, a step comprising selecting at least a portion of a population of cells can be performed after activating all or a portion of the population of cells, e.g., as described herein. For example, selecting at least a portion of a population of cells can comprise isolating cells that express CD25 and / or CD69 after activation of all or a portion of the population of cells.

[0046] In some cases, selecting at least a portion of a first population of cells can produce a second population of cells wherein at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% of the second population of cells expresses one or more molecules of interest (e.g., CD2, CD3, CD4, CD7, CD8, CD25, CD69, or a CAR). In some cases, selecting at least a portion of a first population of cells can produce a second population of cells wherein at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% of the second population of cells are positive for surface expression of one or more molecules of interest (e.g., one or more polypeptides, such as CD2, CD3, CD4, CD7, CD8, CD25, CD69, and / or a CAR). In some cases, at least 80% of the second population (e.g., cells selected during a selection step described herein) can be positive for surface expression of a polypeptide of interest, such as CD2, CD3, CD4, CD7, CD8, CD25, CD69, and / or a CAR. In some cases, at least 85% of thesecond population (e.g., cells selected during a selection step described herein) can be positive for surface expression of a polypeptide of interest, such as CD2, CD3, CD4, CD7, CD8, CD25, CD69, and / or a CAR. In some cases, at least 90% of the second population (e.g., cells selected during a selection step described herein) can be positive for surface expression of a polypeptide of interest, such as CD2, CD3, CD4, CD7, CD8, CD25, CD69, and / or a CAR. In some cases, at least 95% of the second population (e.g., cells selected during a selection step described herein) can be positive for surface expression of a polypeptide of interest, such as a polypeptide of interest, such as CD2, CD3, CD4, CD7, CD8, CD25, CD69, and / or a CAR. In some cases, at least 97% of the second population (e.g., cells selected during a selection step described herein) can be positive for surface expression of a polypeptide of interest, such as CD2, CD3, CD4, CD7, CD8, CD25, CD69, and / or a CAR. In some cases, at least 100% of the second population (e.g., cells selected during a selection step described herein) can be positive for surface expression of a polypeptide of interest, such as CD2, CD3, CD4, CD7, CD8, CD25, CD69, and / or a CAR. In some cases, a method or system described herein can comprise selection or enrichment of cells from a population, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the cells be positive for expression (e.g., surface expression) of CD3 (e.g., compared to an unmodified cell of the same cell type and same species, such as an unmodified human T-cell).

[0047] In some cases, one or more cells of a second population of cells (e.g., cells selected during a selection step described herein) can have reduced expression (e.g., reduced surface expression) of one or more molecules of interest (e.g., one or more cell surface polypeptides, such as CD2, CD3, CD4, CD7, CD8, and / or CD56), for example, compared to an unmodified cell of the same cell type, e.g., from a healthy human subject. In some cases, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% of the cell population can be negative for surface expression of one or more cell surface polypeptides selected from CD2, CD3, CD4, CD7, CD8, and / or CD56 (e.g., compared to an unmodified cell of the same type, such as T-cells, of the same species). For instance, a method or system described herein can comprise selection or enrichment of cells from a population, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the cells have reduced expression (e.g., surface expression) of CD7 (e.g., compared to an unmodified cell of the same cell type and same species, such as an unmodified human T-cell).

[0048] In some cases, reducing expression (e.g., cell surface expression) of one or moremolecules of interest can comprise preventing a polypeptide (e.g., a cell surface polypeptide) produced in a cell from being trafficked to or expressed on the surface of the cell. In some cases, this can be accomplished by expressing a protein expression blocker (PEBL), as described herein, which may comprise a binding domain capable of binding a molecule of interest and a localization domain, which may comprise a retention sequence (e.g., Golgi retention sequence, ER retention sequence (which may comprise a KDEL sequence or a KKXX sequence), PEST sequence, etc.). In some cases, reducing the expression (e.g., cell surface expression) of one or more molecules of interest in a cell can comprise destroying or sequestering an RNA molecule or polypeptide in a cell using, for example, siRNA, shRNA, or miRNA capable of binding and / or facilitating an RNA interference (RNAi) pathway with respect to a molecule of interest. In some cases, reducing the expression (e.g., cell surface expression) of one or more molecules of interest in a cell can comprise modifying a DNA sequence encoding the molecule or its expression in the cell (e.g., through genetic engineering of the cell’s DNA, for example, using an endonuclease-based system, such as a TALEN-based, Cas-based, and / or CRISPR-enabled system, or using a homologous recombination system). In some cases, reducing the expression (e.g., cell surface expression) of one or more molecules of interest in a cell can comprise introducing (e.g., via a genetic engineering technique) a dominant negative allele of a gene of interest (e.g., which may produce or affect the function of one or more molecules of interest) into the cell.

[0049] In some embodiments, methods include selecting one or more cells from a cell population. In some embodiments, one or more cells (e.g., one or more immune cells, such as T-cells) expressing CD4, CD8, or CD3 can be selected (e.g., for incubation or use). In some embodiments, T cells can be derived from an enriched apheresis product (e.g., leukopak) collected, for example, through extraction of white blood cells, including but not limited to CD4-positive, CD8-positive, and / or CD3-positive T cells from a healthy individual. In some embodiments, frozen human primary peripheral blood mononuclear cells (PBMCs) can be processed for use, for example, after thawing, allowing to recover overnight in culture, and culturing, for instance, in cell culture media supplemented with 3% human AB serum or serum-free cell culture media. Cells can be extracted via affinity-based separation techniques, for example, using a device configured to perform magnetic separation of cells (e.g. magnetic-activated cell separation (MACS), for example using a CliniMACS Prodigy system). In some cases, methods described herein can comprise enriching a cell population for CD4-positive and CD8-positve T-cells using CD4 Microbeads and CD8 Microbeads in an affinity-based separation technique, such as MACS. CD3 Microbeads can be used (e.g., inaddition to one or more other separation criteria) during a selection process of a method or system described herein.

[0050] Manufacturing a population of cells can comprise activating cells, e.g., before incubating the population of cells (see, e.g., 506, FIG. 5). Activating the initial population of cells can increase expression of one or more cell surface markers selected from CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD56, or CD69. In some cases, activating all or a portion of a cell population can comprise contacting one or more cells of the cell population with an antibody that binds to CD3 or CD28. Selection of the population of cells can be based at least in part on cell expression of CD25 and CD69 after stimulation. In some cases, activating a cell population can comprise contacting the cell with an antibody against CD3 and / or CD28 or an activation reagent such as TransAct™ (a colloidal polymeric nanomatrix covalently attached to humanized recombinant agonists against human CD3 and CD28). Manufacturing a population of cells can comprise activating (e.g., stimulating) the initial population of cells prior to (or, in some embodiments, after) selecting the population of cells. Manufacturing a population of cells can comprise activating cells before incubating the population of cells. Cells can be activated with a stimulation reagent on the first day of incubation (e.g. Day 0). The stimulation reagent can be designed to activate and / or expand T cells.Activating may comprise contacting a cell with an antibody that binds to CD3 and / or CD28. Activating may comprise contacting a cell with a plurality of antibodies that bind to CD3 and / or CD28.

[0051] Manufacturing a population of cells can comprise transducing cells, e.g., before incubating the population of cells. Transducing the starting cells can create a population of transduced cells. The starting cells can be transduced with a viral vector comprising a nucleotide sequence encoding a CAR and / or a chimeric targeting polypeptide. The transduced starting cells may then comprise a CAR and / or a chimeric targeting polypeptide. Following transduction with the viral vector, the transduced starting cells can be incubated. In some cases, the viral vector can comprise a nucleotide sequence encoding the CAR or the chimeric targeting polypeptide described herein. In some cases, the viral vector can comprise a nucleotide sequence encoding the CAR and the chimeric targeting polypeptide described herein.

[0052] In some cases, a method or system described herein can comprise incubating one or more cells (e.g., a population of cells). Incubating a population of cells can comprise culturing the population of cells in a culture vessel (e.g., a culture vessel comprising a gas-permeable membrane). In some cases, a population of cells can beincubated (e.g., cultured) in contact with a surface of a gas-permeable membrane of a culture vessel. As described herein, incubating a population of cells in a culture vessel comprising a gas-permeable membrane can improve oxygen delivery to the cells and / or carbon dioxide elimination from the cells, thereby improving viability and / or yield of the population of cells or a portion thereof (e.g., a population of cells that expresses a chimeric antigen receptor (CAR) and exhibits reduced cell surface expression of a cell surface polypeptide, such as (e.g., human) CD2, CD3, CD4, CD5, CD7, CD8, CD52, or major histocompatibility complex (MHC)). For instance, incubating a population of cells in contact with a surface of a gas-permeable membrane can improve viability and / or yield of the population of cells or a portion thereof (e.g., a population of cells that expresses a chimeric antigen receptor (CAR) and exhibits reduced cell surface expression of a cell surface polypeptide, such as (e.g., human) CD2, CD3, CD4, CD5, CD7, CD8, CD52, or major histocompatibility complex (MHC)).

[0053] In some cases, a population of cells described herein can comprise or consist of one or more cells susceptible to fratricide. A cell (or population thereof) susceptible to fratricide can be a cell (or population thereof, such as an immune cell) that expresses a receptor (e.g., a CAR) capable of binding to a cell surface polypeptide (e.g., a cell surface protein, such as CD7) expressed at the cell surface of the same species and type of cell (e.g., a cell that also expresses a CAR capable of recognizing the cell surface polypeptide (e.g., a human immune cell (e.g., NK or T-cell) that also expresses CD7), and wherein the receptor activates the immune cell for cytotoxicity when it binds to the cell surface polypeptide. Fratricide can be highest when the cell (or population thereof) expresses high levels of the cell surface polypeptide, but susceptibility to fratricide can still exist when the cell surface polypeptide is expressed at the cell surface in low levels. In some cases, a method or system described herein can comprise incubating a population comprising or consisting of one or more cells susceptible to fratricide. In some cases, a population of cells comprising or consisting of one or more cells susceptible to fratricide can be harvested from culture, frozen, thawed, and / or administered to a patient in need of treatment for a condition (e.g., treatment for a cancer). In some cases, a cell used in a method or system described herein can have reduced cell surface expression of the cell surface polypeptide.

[0054] Incubating a population of cells can comprise removing an activation reagent (e.g., a stimulation reagent). The stimulation reagent can be removed to control proliferation, activation, and / or expansion of T cells. The stimulation reagent can beremoved on day 1, on day 2, on day 3, on day 4, on day 5, from day 1 to day 3, from day 2 to day 3, or from day 3 to day 4 after the beginning of incubation. The optimal day to remove the stimulation reagent, to increase one or more of: the total number of viable cells, the percentage of viable cells, or the percentage of cells expressing desired proteins, can be day 2 after the first day of incubation.

[0055] In some embodiments, T cells can be seeded in vessels with gas-permeable membranes, and activated and expanded with a stimulation reagent (e.g. TransAct™). In some embodiments, lentiviral vectors can be transduced into the cells at MOI 10. In some embodiments, cells can be expanded until day 11. In some embodiments, there are two or more medium exchanges during the manufacturing process to minimize accumulation of byproducts (e.g. metabolites), for example, lactate and ammonium, in cultures.

[0056] Manufacturing cells can comprise incubating a population of cells, wherein the population of cells can be a population of T cells. The population of cells can comprise cells derived from a human subject. The population of cells can be a population of T cells comprised of cells derived from a human subject. The population of cells can comprise a receptor. The receptor can comprise a chimeric antigen receptor (CAR). The receptor can comprise a binding domain. The binding domain can bind to a polypeptide expressed on the surface of at least a portion of the population of cells. The polypeptide can comprise a CD7 binding domain. The receptor can comprise an activation domain. The activation domain can induce cytotoxic activity in the population of (e.g., engineered immune) cells. The population of cells can comprise a target-binding molecule. The target-binding molecule can be linked to a localizing domain. The target-binding molecule can comprise an scFv. The target binding molecule linked to a localizing domain can reduce cell surface expression of a target polypeptide. The target polypeptide can comprise CD7. The target-binding molecule linked to a localizing domain can comprise a protein expression blocker (PEBL). The population of cells can comprise T cells expressing a CAR and a PEBL (e.g. PC ART cells).

[0057] The population of cells can be incubated with a culture medium. The population of cells and culture medium can be incubated in a vessel with a gas permeable membrane. The population of cells and culture medium can be incubated in a closed system vessel. The population of cells incubated on a gas permeable membrane can obtain oxygen and release carbon dioxide through the gas permeable membrane, eliminating the need to agitate the culture medium to provide oxygen to the cells. Incubating the population of cells and culture medium in a closed system with a gaspermeable vessel can increase one or more of: the total number of viable cells, the percentage of viable cells, or the percentage of cells expressing desired proteins relative to other methods of incubating (e.g. cells susceptible to fratricide). The desired cells can be CD3+CD56- cells, CD3+CD56-CD7- cells, and / or CD3+CD56-CAR+CD7-. The CAR can be an anti-CD7 CAR.

[0058] Incubating a population of cells can comprise seeding cells. The seeded cells can comprise CD4+ and / or CD 8+ cells.

[0059] Cells can be incubated in a culture medium. Culture medium can be made on the first day of incubation (Day 0) for use throughout the incubation period. Culture medium can be made fresh on each day that medium is added to vessel or exchanged during incubation of cells.

[0060] In some cases, a population of cells described herein can be incubated in culture medium having a pH of between about 6.5 to 7.4. Culture medium can have a pH of from 6.5 to 6.7, from 6.5 to 6.9, from 6.5 to 7.1, from 6.5 to 7.4, from 6.7 to 6.9, from 6.7 to 7.1, from 6.7 to 7.4, from 6.9 to 7.1, from 6.9 to 7.4, or from 7.1 to 7.4. Culture medium can have a pH of less than about 6.5. Culture medium can have a pH of greater than about 7.4. In some cases, controlling (e.g., maintaining) the pH at which a population of engineered cells are incubated can increase one or more of: the total number of viable cells, the percent of viable cells, or the percent of cells expressing desired proteins. For example, it can be beneficial to maintain cells at a pH above 7.2 (e.g., starting at or beyond day 5). In some cases, this can be accomplished by performing culture medium exchanges as described herein.

[0061] Culture medium can comprise a volume of between about 7 mL to 1 L. Culture medium can comprise a volume of between about 7 mL to 200 mL, 7 mL to 400 mL, 7 mL to 600 mL, 7 mL to 800 mL, 7 mL to 1 L, 200 mL to 400 mL, 200 mL to 600 mL, 200 mL to 800 mL, 200 mL to 1 L, 400 mL to 600 mL, 400 mL to 800 mL, 400 mL to 1 L, 600 mL to 800 mL, 600 mL to 1 L, or between about 800 mL to 1 L. Culture medium can comprise a volume of less than about 7 mL. Culture medium can comprise a volume of greater than about 1 L. Optimal culture medium can differ by the day from the start of incubation.Optimal culture medium on day 3 after seeding can be about 40 mL. Optimal culture medium on day 4 and onwards can be about 1 L. Incubating a population of cells with an optimal culture medium volume can increase one or more of: the total number of viable cells, the percent of viable cells, or the percent of cells expressing desired proteins.

[0062] Manufacturing a population of cells can comprise changing at least a portionof the culture medium. At least a portion of the culture medium can be changed every 1 to 3 days. The portion of the culture medium to be changed can be at least 10%, at least 25 %, at least 50%, at least 75%, at least 85%, at least 95%, 100%, from 10% to 25%, from 25% to 50%, from 50% to 75%, from 75% to 85%, from 85% to 95%, or from 95% to 100% of the culture medium in the closed system. In some cases, a vessel for culturing cells can contain about 10 mL, 15 mL, 25 mL, 50 mL, 75 mL, 100 mL, 500 mL, 1000 mL, 2000 mL, 10 mL to 100 mL, 100 mL to 500 mL, 500 mL to 1000 mL, or 1000 mL to 2000 mL of culture medium. In some cases, a larger volume of medium can reduce the frequency with which culture medium should be changed to maintain a concentration of metabolites in the culture medium, for instance, reducing the impact of increased metabolite production (e.g., versus non-engineered cells) in the manufacture of engineered cells described herein (e.g. cells susceptible to fratricide). Culture medium can be changed on any and / or every day of incubation. Culture medium can be changed on day 6 and / or 9 after the first day of incubation. Culture medium can be changed on days 6, 8, and or 10 after the first day of incubation. Frequent culture medium changes can minimize metabolites and improve control of a concentration of a substance in the culture medium. Frequent culture medium exchanges can disturb an otherwise-closed system and decrease one or more of the total number of viable cells, the percent of viable cells, or the percent of cells expressing desired proteins. An optimal culture medium exchange frequency can control a concentration of a substance in the culture medium while improving one or more of the total number of viable cells, the percent of viable cells, or the percent of cells expressing desired proteins. Optimal culture medium changes can occur on day 6 and day 9 after the first day of incubation when the harvest day is day 11. Optimal culture medium changes can change 75% of the culture medium in the closed system. In some cases, incubation may comprise 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or more. In some cases, harvesting cells sensitive to fratricide may occur after a time period of incubation. In some cases, harvesting cells sensitive to fratricide may occur after at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, or at least about 60 days of incubation.

[0063] Incubating a population of cells can comprise reducing the volume of the incubation mixture. For example, a method or system described herein can comprise reducing a volume of culture medium in which cells are incubated, e.g., in order to dilute or remove a reagent (such as an activation reagent like TransAct™), to concentrate the cell population in culture (e.g., reduce the overall volume of the medium in culture), or to change the culture medium (e.g., to control the concentration of a substance in the medium, such as ammonium or lactate). The incubation mixture can comprise desired cells, a stimulation reagent, and / or culture medium. In some cases, a volume of the incubation mixture can be reduced without opening the closed system. For instance, a volume of the incubation mixture can be reduced to at most 750 mb, at most 500 mb, at most 250 mb, at most 100 mb, at most 50 mb, or at most 10 mL without opening the closed system. Volume of the incubation mixture can be reduced (e.g., without opening the closed system) on day 3, day 5, day 7, day 8, day 9, day 10, day 11, or any day after day 11 following the first day of incubation of cells in culture. A volume of the incubation mixture can be reduced on harvest day.

[0064] Manufacturing a population of cells can comprise controlling a concentration of a substance. The concentration of the substance can be controlled in the culture medium. The substance can be a metabolite or byproduct of cell growth. The substance can be lactate or ammonium.

[0065] Lactate, which can be a metabolite of cell culture (e.g., culture of an immune cell, such as an NK cell or T-cell) can be controlled (e.g., maintained) at a threshold concentration level, e.g., 5 mM. In some cases, lactate can be controlled at a concentration of less than about 15 mM, less than about 12 mM, less than about 10 mM, less than about 8 mM, less than about 6 mM, less than about 5 mM, less than about 3 mM, less than about 1 mM, at most about 15 mM, at most about 12 mM, at most about 10 mM, at most about 8 mM, at most about 6 mM, at most about 5 mM, at most about 3 mM, at most about 1 mM, from 1 mM to 3 mM, from 3 mM to 5 mM, from 5 mM to 6 mM, from 6 mM to 8 mM, from 8 mM to 10 mM, from 10 mM to 12 mM, or from 12 mM to 15 mM. In some cases, lactate can be controlled at or below a threshold value of 15 mM, 12 mM, 10 mM, 8 mM, 6 mM, 5 mM, 3 mM, or 1 mM. Lactate can be controlled at a concentration of greater than about 15 mM. Lactate can be controlled at an optimal concentration of less than about 5 mM. Lactate can be controlled at an optimal concentration of less than about 5 mM, 4 mM, 3 mM, 2 mM, or less than about 1 mM. Controlling lactate at or below a threshold concentration can, in some cases, increase one or more of: the total number of viable cells, the percent of viablecells, or the percent of cells expressing desired proteins. Lactate can be controlled at a concentration for an interval of time in culture, for example, for at least 7 days of culture. Lactate can be controlled at a concentration for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more than 11 days of culture.

[0066] Ammonium concentration in a culture medium can be controlled at a threshold concentration level, e.g., 0.4 mM. In some cases, ammonium concentration in a culture medium can be controlled at a concentration of less than about 1 mM, less than about 0.5 mM, less than about 0.4 mM, less than about 0.3 mM, less than about 0.2 mM, at most about 1 mM, at most about 0.5 mM, at most about 0.4 mM, at most about 0.3 mM, at most about 0.2 mM, from 0.4 mM to 0.2 mM, from 0.5 mM to 0.2 mM, or from 1.0 mM to 0.2 mM. Controlling ammonium at or below a threshold concentration can increase one or more of: the total number of viable cells, the percent of viable cells, or the percent of cells expressing desired proteins. Ammonium can be controlled at a concentration for an interval of time in culture, for example, for at least 7 days of culture. Ammonium concentration in culture medium can be controlled (e.g., at or below a threshold value) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more than 11 days of culture.

[0067] Manufacturing a population of cells (e.g., immune cells, for example engineered immune cells described herein) can comprise disrupting clumps of the population of cells (see, e.g., 408, 514; FIG. 4, FIG. 5). In some cases, agitation (e.g., mechanical agitation) of a cell culture (e.g., comprising cells) can be used to disrupt clumps of cells in a cell population. In some cases, agitation can be continuous during culture. In some cases, cells (e.g., cells susceptible to fratricide, as described herein) can be incubated in culture (e.g., in a vessel comprising a gas-permeable membrane) without continuous agitation. In some cases, agitation can be applied in one or more discrete or periodic applications of the agitation to the cells in culture. The population of cells can comprise T cells. In culture, T cells can clump together. T cell clumping can decrease one or more of: transduction efficiency, total number of viable cells, percent of viable cells, or percent of cells expressing desired proteins. Clumping of T cells during manufacture can be decreased by disruption of T cells during manufacture. Existing methods of manufacturing can cause cell rupture, thereby decreasing the amount of viable cells (e.g. cells susceptible to fratricide) created during the manufacturing process. Described herein are novel methods for manufacturing T cells without cell rupture while increasing one or more of: transduction efficiency, total number of viable cells, percent of viable cells, or percent of cells expressing desired proteins.

[0068] In some cases, limiting the exposure of the cells to excessive mechanical disruption can improve viability of the engineered cells. In some cases, a method or system can comprise mixing cells to disrupt clumps of cells but not agitating the cells. Mixing can be mechanical mixing. Mixing can be gentle mixing. Mixing can be infrequent mixing. In some cases, mixing engineered cells in culture too infrequently can lead to uncontrolled clumping. In some cases, disrupting clumps of engineered cells in culture too frequently can increase rupture of cells. Disruption of clumps of engineered cells can be performed every 1 to 3 days. Disruption of clumps of engineered cells can be performed multiple times a day. Disruption of clumps of engineered cells can be performed at least every 3 days. Disruption of clumps of engineered cells can be performed at least 3 times in an 11-day incubation period. Disruption of clumps of engineered cells can be performed on days with medium exchanges. Performing cell disruption of clumps of engineered cells on days with medium exchanges can minimize interference with the closed system. Minimizing interference can increase one or more of transduction efficiency, total number of viable cells, percent of viable cells, or percent of cells expressing desired proteins. Disruption of clumps of engineered cells can be performed on day 6 and day 9 of the 11-day incubation period, and can be at the same time as medium exchanges.

[0069] Methods and systems of manufacturing described herein can be useful in determining a quantity (e.g., yield) of cells at one or more stages of manufacturing. Cell count and viability (CCV) assays can be used to measure cell yield and viability, including but not limited to one or more of: the total number of viable cells or the percent of viable cells. CCV assays can be used one or more times during the manufacturing process. CCV assays can be used zero times during the manufacturing process. CCV assays can be used on starting materials. CCV assays can be used on manufactured cells. CCV assays can be used on an enriched apheresis product that is used as a starting material. CCV assays can be used on an enriched apheresis product on the first day of incubation (day 0). The CCV assay can provide the number and viability of cells used as starting material, which can affect downstream cell number and viability. CCV assays can be used once CD4 and CD8 cells are seeded in a gas permeable vessel on the first day of incubation (day 0). CCV assays can be used to determine the number of cells for lentiviral transduction on the transduction day. CCV assays can be used to determine the success rate of lentiviral transduction on day 3. CCV assays can be used to measure manufacturing progress approximately halfway through the manufacturing process. CCVassays can be used to measure manufacturing progress on day 6 of an 11-day manufacturing process. CCV assays can be used on harvest day. CCV assays can be used on harvest day of an 11-day manufacturing process. CCV assays on harvest day can include, but are not limited to, one or more of: a CCV for formulation density, a CCV for target volume determination, a CCV for volume adjustment, and a CCV for final formulation (e.g. final cell viability percentages and other metrics).

[0070] Methods described herein or steps thereof can be performed over at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some cases, a method of manufacturing described herein can proceed until, for instance, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, or day 11 after start of incubation to increase one or more types of desired yield. In some cases, viability of engineered cells at harvest (e.g., at day 11) can be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100%, wherein the yield of engineered cells at harvest (e.g., day 11) is at least 10A6 cells. In some cases, viability of engineered cells at harvest (e.g., at day 11) can be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100%, wherein the yield of engineered cells at harvest (e.g., day 11) is at least 10A7 cells. In some cases, viability of engineered cells at harvest (e.g., at day 11) can be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100%, wherein the yield of engineered cells at harvest (e.g., day 11) is at least 10A8 cells.

[0071] Manufacturing a population of cells can comprise incubating a population of cells, for example, until the cells reach a desired yield (404, 510; FIG. 4, FIG. 5). A desired yield can comprise one or more of: total viable cells, fold change in cell number, percent viability, number of cells expressing the surface marker profile CD3+CD56- at or above a threshold value, number of cells expressing the surface marker profile CD3+CD56‘ CD7" at or above a threshold value, and / or number of cells expressing the surface marker profile CD3+CD56'CAR+CD7‘ at or above a threshold value. In some cases, methods and systems described herein can result in increased viability of produced engineered cells. In some cases, the viability of cells produced using a method or system described herein can be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100%, for instance at the time of harvest (e.g., at day 11). In some cases, the viability of cells produced using a method or system described herein can be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100%, after 3 days of incubation in culture. In some cases, the viability of cells produced using a method or system described herein can be at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100%, after 5 days of incubation in culture. In some cases, the viability of cells produced using a method or system described herein can be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100%, after 7 days of incubation in culture. In some cases, the viability of cells produced using a method or system described herein can be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100%, after 10 days of incubation in culture. In some cases, viability of thawed engineered cells can be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100%.

[0072] A yield of (e.g., viable) cells at the time of harvest can be at least 1 E6 cells, at least 1 E7 cells, at least 3 E7 cells, at least 5 E7 cells, at least 1 E8 cells, or at least 5 E8 cells. In some cases, a yield of (e.g., viable) cells at the time of harvest can be from 1 E6 cells to 1 E7 cells, from 1 E7 cells to 3 E7 cells, from 3 E7 cells to 5 E7 cells, from 5 E7 cells to 1 E8 cells, or from 1 E8 cells to 5 E8 cells. An increased yield can be indicative of a more successful manufacturing process that can manufacture more desired PC ART cells or other T cells in each manufacturing run.

[0073] In some embodiments, methods described herein may comprise harvesting at least 1 E8 cells sensitive to fratricide after no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days of incubation. In some cases, the harvesting of at least 1 E8 cells occurs after no more than 11 days of incubation. A percentage of the harvested at least 1 E8 cells sensitive to fratricide may be viable. In some embodiments, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% of the harvested cells may be viable. In some embodiments, a range from about 30% to about 95% of the harvested cells may be viable. In some embodiments, a range from about 30% to about 40%, about 30% to about 45%, about 30% to about 50%, about 30% to about 55%, about 30% to about 60%, about 30% to about 65%, about 30% to about 70%, about 30% to about 75%, about 30% to about 80%, about 30% to about 90%, about 30% to about 95%, about 40% to about 45%, about 40% to about 50%, about 40% to about 55%, about 40% to about 60%, about 40% to about 65%, about 40% to about 70%, about 40% to about 75%, about 40% to about 80%, about 40% to about 90%, about 40% to about 95%, about 45% to about 50%, about 45% to about 55%, about 45% to about 60%, about 45% to about 65%, about 45% to about 70%, about 45% to about 75%, about 45% to about 80%, about 45% to about 90%, about 45%to about 95%, about 50% to about 55%, about 50% to about 60%, about 50% to about 65%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 55% to about 60%, about 55% to about 65%, about 55% to about 70%, about 55% to about 75%, about 55% to about 80%, about 55% to about 90%, about 55% to about 95%, about 60% to about 65%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 90%, about 60% to about 95%, about 65% to about 70%, about 65% to about 75%, about 65% to about 80%, about 65% to about 90%, about 65% to about 95%, about 70% to about 75%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 75% to about 80%, about 75% to about 90%, about 75% to about 95%, about 80% to about 90%, about 80% to about 95%, or about 90% to about 95% of the harvested cells may be viable.

[0074] A percentage of the harvested 1 E8 cells sensitive to fratricide may express a chimeric antigen receptor (CAR). In some embodiments, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% of the harvested cells expressing a CAR may be viable. In some embodiments, a range from about 30% to about 95% of the harvested cells expressing a CAR may be viable may be viable. In some embodiments, a range from about 30% to about 40%, about 30% to about 45%, about 30% to about 50%, about 30% to about 55%, about 30% to about 60%, about 30% to about 65%, about 30% to about 70%, about 30% to about 75%, about 30% to about 80%, about 30% to about 90%, about 30% to about 95%, about 40% to about 45%, about 40% to about 50%, about 40% to about 55%, about 40% to about 60%, about 40% to about 65%, about 40% to about 70%, about 40% to about 75%, about 40% to about 80%, about 40% to about 90%, about 40% to about 95%, about 45% to about 50%, about 45% to about 55%, about 45% to about 60%, about 45% to about 65%, about 45% to about 70%, about 45% to about 75%, about 45% to about 80%, about 45% to about 90%, about 45% to about 95%, about 50% to about 55%, about 50% to about 60%, about 50% to about 65%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 55% to about 60%, about 55% to about 65%, about 55% to about 70%, about 55% to about 75%, about 55% to about 80%, about 55% to about 90%, about 55% to about 95%, about 60% to about 65%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 90%, about 60% to about 95%, about 65% to about 70%, about 65% to about 75%, about 65% to about 80%, about65% to about 90%, about 65% to about 95%, about 70% to about 75%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 75% to about 80%, about 75% to about 90%, about 75% to about 95%, about 80% to about 90%, about 80% to about 95%, or about 90% to about 95% of the harvested cells expressing a CAR may be viable.

[0075] A percentage of the harvested 1 E8 cells sensitive to fratricide may have reduced expression of a surface polypeptide (e.g., CD7). In some embodiments, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% of the harvested cells having reduced expression of a surface polypeptide may be viable. In some embodiments, a range from about 30% to about 95% of the harvested cells having reduced expression of a surface polypeptide may be viable. In some embodiments, a range from about 30% to about 40%, about 30% to about 45%, about 30% to about 50%, about 30% to about 55%, about 30% to about 60%, about 30% to about 65%, about 30% to about 70%, about 30% to about 75%, about 30% to about 80%, about 30% to about 90%, about 30% to about 95%, about 40% to about 45%, about 40% to about 50%, about 40% to about 55%, about 40% to about 60%, about 40% to about 65%, about 40% to about 70%, about 40% to about 75%, about 40% to about 80%, about 40% to about 90%, about 40% to about 95%, about 45% to about 50%, about 45% to about 55%, about 45% to about 60%, about 45% to about 65%, about 45% to about 70%, about 45% to about 75%, about 45% to about 80%, about 45% to about 90%, about 45% to about 95%, about 50% to about 55%, about 50% to about 60%, about 50% to about 65%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 55% to about 60%, about 55% to about 65%, about 55% to about 70%, about 55% to about 75%, about 55% to about 80%, about 55% to about 90%, about 55% to about 95%, about 60% to about 65%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 90%, about 60% to about 95%, about 65% to about 70%, about 65% to about 75%, about 65% to about 80%, about 65% to about 90%, about 65% to about 95%, about 70% to about 75%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 75% to about 80%, about 75% to about 90%, about 75% to about 95%, about 80% to about 90%, about 80% to about 95%, or about 90% to about 95% of the harvested cells having reduced expression of a surface polypeptide may be viable.

[0076] A method or system described herein can comprise determining a fold changein cell number relative to the start of incubation (e.g., relative to day 0). In some cases, culture (e.g., incubation) of engineered cells can result in from 0.2 to 1-fold, 1.0 to 2.0- fold, 2.0 to 5.0-fold, 5.0 to 10.0-fold, 10.0 to 20.0-fold, 20.0 to 30.0-fold, 30.0 to 40.0- fold, at least a 0.2-fold, at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20- fold, at least a 25-fold, at least a 30-fold, or at least a 40-fold increase in the quantity of cultured cells (e.g., engineered cells). Fold change can continue to increase if incubation proceeds longer than 11 days. If incubation proceeds 14 days, fold change can be at least 68.61. An increased yield can be indicative of a more successful manufacturing process that can manufacture more desired PCART cells or other T cells in each manufacturing run.

[0077] A method or system described herein can comprise determining a percent viability of the cells. A minimum desired cell viability percentage for manufacturing can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%. An increased yield can be indicative of a more successful manufacturing process that can manufacture more desired PCART cells or other T cells in each manufacturing run.

[0078] A population of cells of a method or system described herein can comprise a minimum percentage of CD3+CD56- cells (e.g., on the day of harvest, for instance day 11). Percent CD3+CD56- cells can be at least 85%. Percent CD3+CD56- cells can be less than about 87.5%. Percent CD3+CD56- cells can be at least 99.7%. Percent CD3+CD56- cells can be at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97.5%, at least 99.5%, or 100% in the manufactured population of cells. A population of cells of a method or system described herein can comprise a minimum percentage of CD3'CD56+NK cells (e.g., on the day of harvest, for instance day 11). Percent CD3'CD56+NK cells can be at least 99.7%. Percent CD3'CD56+NK cells can be at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97.5%, at least 99.5%, or 100% in the manufactured population of cells. An increased yield can be indicative of a more successful manufacturing process that can manufacture more desired PCART cells or other T cells in each manufacturing run.

[0079] A population of cells of a method or system described herein can comprise a minimum percentage of CD3+CD56'CD7‘ cells (e.g., on the day of harvest, for instance day 11). The minimum percentage of CD3+CD56'CD7‘ cells can be at least 80%. The minimum percentage of CD3+CD56'CD7‘ cells can be less than about 85%. Percent CD3+CD56'CD7‘ cells can be at least 99.7%. The minimum percentage of CD3+CD56'CD7‘ cells can be at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. An increased yield can be indicative of a more successful manufacturing process that can manufacture more desiredPCART cells or other T cells in each manufacturing run.

[0080] A population of cells of a method or system described herein can comprise a minimum percentage of CD3+CD56'CAR+CD7‘ cells (e.g., on the day of harvest, for instance day 11). The minimum percentage of CD3+CD56'CAR+CD7‘ cells can be at least 40%. Percent CD3+CD56'CAR+CD7‘ cells can be less than about 80%. The minimum percentage of CD3+CD56'CAR+CD7‘ cells can be at least 90%. The minimum percentage of CD3+CD56'CAR+CD7‘ cells can be at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 100%. An increased yield can be indicative of a more successful manufacturing process that can manufacture more desired PCART cells or other T cells in each manufacturing run.

[0081] A desired manufacturing yield by day 11 after start of incubation can comprise limiting the percent of cells that express CD56 to increase the percent of CD3+CD56-, CD3+CD56'CD7‘, and / or CD3+CD56'CAR+CD7‘ cells. The percent of cells that express CD56 can be at most about 12.5%. The percent of cells that express CD56 can be greater than 12.5%. The percent of cells that express CD56 can be at most about 0.3%. The percent of cells that express CD56 can be at most about 12.5%, 10.5%, 8.5%, 6.5%, 4.5%, 2.5%, 0.5%, or at most about 0.3%. A decreased yield can be indicative of a more successful manufacturing process that can manufacture more desired PCART cells or other T cells in each manufacturing run.

[0082] A population of cells of a method or system described herein can comprise a minimum percentage of CD3+cells (e.g., on the day of harvest, for instance day 11). Percent CD3+cells can be at least 87.5%. Percent CD3+cells can be less than about 87.5%. Percent CD3+cells can be at least 99.7%. Percent CD3+cells can be at least 87.5%, 89.5%, 91.5%, 93.5%, 95.5%, 97.5%, 99.5%, or at least 99.7% in the manufactured population of cells. An increased yield can be indicative of a more successful manufacturing process that can manufacture more desired PCART cells or other T cells in each manufacturing run.

[0083] A method or system described herein can comprise increasing the percent of cells that express CD4 in the population. The percent of cells that express CD4 can be at least about 12.5%. The percent of cells that express CD4 can be at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 12.5%, at least 10.5%, at least 8.5%, at least 6.5%, at least 4.5%, at least 2.5%, at least 0.5%, or at least about 0.3%.

[0084] A method or system described herein can comprise increasing the percent of cells that express CD8 in the population. The percent of cells that express CD8 can be atleast about 12.5%. The percent of cells that express CD8 can be at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 12.5%, at least 10.5%, at least 8.5%, at least 6.5%, at least 4.5%, at least 2.5%, at least 0.5%, or at least about 0.3%.

[0085] Manufacturing a population of cells can comprise transduction with one or more vectors, e.g., before incubating the population of cells (see, e.g., 508, FIG. 5). The vector can be a viral vector. The viral vector can be an engineered viral vector. The engineered viral vector can be a lentiviral vector. The transduction with a viral (e.g., lentiviral) vector can comprise a multiplicity of infection (MOI) at which the vector is used with relation to the number of cells in the population of cells being transduced with the vector. The MOI can be between about 10 and 30. The MOI can be between about 10 to 20, 10 to 30, or 20 to 30. The MOI can be less than 10. The MOI can be at least 5, at least 10, or at least 30. The MOI can be greater than 30. The optimal MOI to increase one or more of: the total number of viable cells, the percent of viable cells, or the percent of cells expressing desired proteins, can be about 10.

[0086] Incubating a population of cells can comprise transduction at a certain volume of transduction reagents, which may include a vector (e.g., a viral vector encoding one or more CAR and / or PEBL constructs). The transduction volume can be between about 4 mL to 20 mb. The transduction volume can be between about 4 mL to 8 mL, 4 mL to 12 mL, 4 mL to 16 mL, 4 mL to 20 mL, 8 mL to 12 mL, 8 mL to 16 mL, 8 mL to 20 mL, 12 mL to 16 mL, 12 mL to 20 mL, or between about 16 mL to 40 mL. The transduction volume can be less than about 4 mL. The transduction volume can be greater than about 20 mL. The optimal transduction volume to increase one or more of: the total number of viable cells, the percent of viable cells, or the percent of cells expressing desired proteins, can be about 4 mL. Incubating a population of cells can comprise transduction at a certain volume of transduction reagents per surface area of a gas permeable membrane in a vessel, such as about 0.4 mL / cm2, 0.3-0.5 mL / cm2, 0.2-0.8 mL / cm2, or 0.1-2.0 mL / cm2.

[0087] Incubating a population of cells can comprise transduction on a certain day of incubation. Transduction can be done between day 3 and day 4. The optimal day for transduction can increase one or more of: the total number of viable cells, the percent of viable cells, or the percent of cells expressing desired proteins, can be day 3.

[0088] Flow cytometry assays can sort cells. Flow cytometry assays can analyze a population of cells. Flow cytometry assays can be used one or more times during themanufacturing process. Flow cytometry assays can be used zero times during the manufacturing process. Flow cytometry assays can be used to analyze starting materials. Flow cytometry assays can be used on CD4+ and CD8+ cells on the first day of incubation (day 0). Flow cytometry assays can be used on CD4+ and / or CD8+ cells before enrichment. Flow cytometry assays can be used on CD4+ and / or CD8+ cells after enrichment. Flow cytometry assays can be used to measure manufacturing progress on day 6 of an 11-day manufacturing process. Flow cytometry assays on day 6 would measure post-transduction cell surface expression. Flow cytometry assays can be used on harvest day. Flow cytometry assays can be used on harvest day of an 11-day manufacturing process. Flow cytometry assays on harvest day can include, but are not limited to, flow cytometry assays for formulation density and / or target volume determination.

[0089] Manufacturing can comprise steps after cell harvest. Steps after harvest can comprise one or more of the following: concentration, wash, and / or formulation in balanced crystalloid solutions (e.g. a PlasmaLyte solution, such as Plasma-Lyte 148); volume adjustment; dimethyl sulfoxide (DMSO) addition; quality control; and cry opreservation.

[0090] Cryopreservation can be conducted in controlled rate freezers (CRF). Various materials can be cryopreserved, including, but not limited to, vectors (e.g. lentiviral vectors), cells (e.g. T cells, including but not limited to T cells comprising PEBL and CAR), and / or engineered transduced cells. Cryopreserved cells can retain viability. Viability can be at least 60%, at least 65%, least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% post-thaw. Post-thaw viability can depend on cryodensities of cells. Cryodensities of cells can be between about 1.6 E6 cells / mL to 3.84 E7 cells / mL to achieve at least 70% post-thaw viability. Cryodensities of cells can be between about 1.6 E6 cells / mL to 5.6 E6 cells / mL, 1.6 E6 cells / mL to 9.6 E6 cells / mL, 1.6 E6 cells / mL to 3.6 E7 cells / mL, 1.6 E6 cells / mL to 3.84 E7 cells / mL, 5.6 E6 cells / mL to 9.6 E6 cells / mL, 5.6 E6 cells / mL to 3.6 E7 cells / mL, 5.6 E6 cells / mL to 3.84 E7 cells / mL, 9.6 E6 cells / mL to 3.6 E7 cells / mL, 9.6 E6 cells / mL to 3.84 E7 cells / mL, or between about 3.6 E7 cells / mL to 3.84 E7 cells / mL to achieve at least 70% post-thaw viability. Cryodensities of cells can be less than about 1.6 E6 cells / mL to achieve at least 70% post-thaw viability. Cryodensities of cells can be about 8 E5 cells / mL to achieve at least 70% post-thaw viability. Cryodensities of cells can be greater than about 3.84 E7 cells / mL to achieve at least 70% post-thaw viability.

[0091] Cryopreservation preparation can comprise a balanced crystalloid solution.Cryopreservation preparation can comprise a cryopreservation medium. The cryopreservation medium can comprise 10% DMSO. Additional DMSO can be added to the cry opreservation mixture. The cryopreservation mixture can comprise a 50:50 mixture of a balanced crystalloid solution and a cry opreservation medium comprising 10% DMSO. The final DMSO concentration can be 5%.Administration of Cells

[0092] In some embodiments, provided is a method of treating immune cell diseases in a subject in need thereof, comprising administering a therapeutic amount of an engineered immune cell having any of the embodiments described herein to the subject, thereby treating immune cell disease in a subject in need thereof.

[0093] In certain embodiments, the method comprises administering a therapeutic amount of an engineered immune cell comprising a bicistronic viral construct comprising a polynucleotide comprising a nucleic acid sequence encoding a CAR and a polynucleotide comprising a nucleic acid sequence encoding a PEBL. In various embodiments, the method comprises administering a therapeutic amount of any one of the engineered immune cells described herein comprising a recombinant retroviral vector comprising: (a) a first promoter operably linked to a first polynucleotide encoding a CD7 chimeric antigen receptor (CD7 CAR) as outlined herein; and (b) a second promoter operably linked to a second polynucleotide encoding a CD7 protein expression blocker (CD7 PEBL) as outlined herein. In some embodiments, a therapeutic amount of an engineered immune cell or a population thereof (e.g., engineered CD3+T cell, engineered CD4+ T cell, or engineered CD8+ T cell) comprising a nucleic acid construct comprising the sequence depicted in FIG. 3 (SEQ ID NO: 1) is administered to a subject having cancer.

[0094] In another aspect, the present disclosure relates to the use of an engineered immune cell that comprises a first nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) and a second nucleic acid comprising a nucleotide sequence encoding a single-chain variable fragment (scFv) linked to a localizing domain for treating cancer, comprising administering a therapeutically effective amount of the engineered immune cell to a subject in need thereof. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are bicistronic.

[0095] In other aspects, the present invention relates to the use of an engineered immune cell that comprises a nucleic acid comprising a nucleotide sequence encoding a chimericantigen receptor (CAR), and a nucleic acid comprising a nucleotide sequence encoding a target-binding molecule (e.g., scFv) linked to a localizing domain for treating an autoimmune disorder, comprising administering a therapeutically effective amount of the engineered immune cell to a subject in need thereof. In some cases, the autoimmune disease may be amyotrophic lateral sclerosis (ALS), primary Sjogren’s syndrome, sarcoidosis, type 1 diabetes, autoimmune hepatitis (e.g., type 1 or type 2), multiple sclerosis, Guillain-Barre syndrome and the AMAN (axonal & neuronal neuropathy), psoriasis, scleroderma, or ankylosing spondylitis (AS).

[0096] In other aspects, the present invention also relates to the use of an engineered immune cell that comprises a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR). The engineered immune cell may have reduced expression of the target of the CAR. The engineered immune cell may comprise a nucleic acid comprising a nucleotide sequence encoding a target-binding molecule (e.g., scFv) linked to a localizing domain. A therapeutically effective amount of the engineered immune cells may be administered to a subject in need thereof for treating an infectious disease or a viral disease affecting immune cells.

[0097] In certain embodiments, the cancer is a T cell malignancy, e.g., T cell leukemia or T cell lymphoma, such as T-cell acute lymphoblastic leukemia, T-cell prolymphocytic leukemia, T- cell large granular lymphocytic leukemia, enteropathy- associated T-cell lymphoma, hepatosplenic T-cell lymphoma, subcutaneous panniculitislike T-cell lymphoma, mycosis fungoides, Sezary syndrome, primary cutaneous gammadelta T-cell lymphoma, peripheral T- cell lymphoma not otherwise specified, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma. In certain embodiments, the T cell malignancy is early T-cell progenitor acute lymphoblastic leukemia (ETP -ALL).

[0098] In some embodiments, the engineered immune cell is autologous to the subject in need of treatment, e.g., cancer treatment, autoimmune disease treatment, infectious disease treatment, graft versus host disease (GVHD) treatment, and transplantation rejection treatment. In other embodiments, the engineered immune cell is allogeneic to the subject in need of treatment. The isolated engineered immune cell of the present invention can be an “off-the-shelf’ immune cell that can be administered to a plurality of subjects and provides a reduced risk of GVHD. In some embodiments, the engineered immune cell does not elicit a GVHD response upon administration to a plurality of subjects (e.g., at least two moresubjects).

[0099] In certain embodiments, the engineered immune cell is administered by infusion into the subject. Methods of infusing immune cells (e.g., allogeneic or autologous immune cells) are known in the art. A sufficient number of cells are administered to the recipient in order to ameliorate the symptoms of the disease. Typically, dosages of 107to IO10cells can be infused in a single setting, e.g., dosages of 109cells. Lower doses (e.g. 106to 109cells) can be infused for pediatric patients. Infusions can be administered either as a single 109cell dose or divided into several 109cell dosages. The frequency of infusions can be daily, every 2 to 30 days or even longer intervals if desired or indicated. The quantity of infusions can be generally at least 1 infusion per subject and preferably at least 3 infusions, as tolerated, or until the disease symptoms have been ameliorated. The cells can be infused intravenously at a rate of 50- 250 ml / hr. Other suitable modes of administration include intra-arterial infusion, intraperitoneal infusion, direct injection into tumor and / or perfusion of tumor bed after surgery, implantation at the tumor site in an artificial scaffold, intrathecal administration. Methods of adapting the present invention to such modes of delivery are readily available to one skilled in the art.

[0100] In certain embodiments, the method of treating cancer according to the present invention is combined with at least one other known cancer therapy, e.g., radiotherapy, chemotherapy, or other immunotherapy.

[0101] In other aspects, also provided is use of an engineered immune cell having any of the embodiments described herein for treating cancer, comprising administering a therapeutic amount of the engineered immune cell to a subject in need thereof. In certain embodiments, the cancer is a T cell malignancy. In certain embodiments, the T cell malignancy is early T-cell progenitor acute lymphoblastic leukemia (ETP -ALL).

[0102] In certain embodiments, the engineered immune cell is administered into the subject by intravenous infusion, intra-arterial infusion, intraperitoneal infusion, direct injection into tumor and / or perfusion of tumor bed after surgery, implantation at a tumor site in an artificial scaffold, intraocular administration, or intrathecal administration.SYSTEMS

[0103] Described herein are systems that can be useful in the manufacture of a population of cells, such as a population of cells comprising a CAR and / or a PEBL. In some cases,systems disclosed herein can be useful in improving cell yield and / or cell viability in the manufacture of a population of cells. For instance, systems described herein can be useful in culturing engineered cells, which may be more sensitive to changes in culture conditions than non-engineered cells of the same cell type and species. In some cases, systems described herein can be useful in producing populations of cells susceptible to fratricide (e.g., cells expressing a CAR molecule that targets a cell surface protein expressed by the cells - at any level). Systems described herein can be useful with methods and reagents described herein for the production of engineered cells (e.g., engineered immune cells expressing a CAR and / or a PEBL).

[0104] A system can comprise a vessel for culturing a population of cells (e.g., a population of immune cells transduced with one or more viral vectors). In some cases, a vessel of a system can comprise a gas permeable membrane for culturing the cells. In some cases, the cells (e.g., cells susceptible to fratricide, which may express a CAR) can be cultured in contact with a surface of the gas permeable membrane of a system. A gas permeable membrane surface can aid in providing nutrients and gas exchange for cells in culture. As described herein, a culture vessel having a gas permeable membrane can be especially helpful in maintaining or improving cell viability and / or cell yield in the manufacture of engineered cells, for instance, because it can facilitate gas transport to and from the cells. Gas permeable vessels can be an efficient method of expanding immune cells, with superior viable cell numbers, fold expansion, and viability than other similar methods for expanding immune cells. Gas permeable vessels can streamline transduction and engineering of T-cells without decreasing function of cells for therapeutic use. In some embodiments, the gas-permeable membrane is a silicone membrane that allows oxygen and carbon dioxide to be exchanged. In some embodiments, vessels with gas-permeable membranes include tubes for medium exchange, harvest, and sampling with minimal disruption of cells during culture.

[0105] In some embodiments, a system comprising a vessel with a gas-permeable membrane for cell culture and expansion can be a closed system or can comprise one or more closed system components. A closed system can reduce the likelihood of contamination of cells during manufacture and can streamline processes for the production of the cells. Culture vessels having a gas permeable membrane surface of 10 cmA2 and a maximum media volume capacity of 100 mL (e.g., G-Rex®6M and G-Rex®10M G-Rex vessels; Wilson Wolf) or a gas permeable membrane surface of 100 cmA2 and a maximum media volume of 1000 mL (e.g., G-Rex®100M and 100M-CS G-Rex vessels) can be used. In some cases, culturevessels (e.g., having a gas permeable membrane surface of 100 cmA2 and a maximum media volume of 1000 mL) can be closed system vessels, having weldable tubing and dip tubes for medium exchange, harvest, and sampling.

[0106] In some cases, a system can comprise a liquid handling system (e.g., liquid handler) for removing and replacing liquids, such as culture medium and other reagents described herein, from and into a culture vessel of a system. In some cases, a system can comprise a controller for operating a liquid handling system. In some cases, a controller can control a heater of the system, for example, wherein the heater is configured to maintain the culture vessel, medium, or cells within a desired range of temperatures for culture. In some cases, a controller can comprise a processor and a non-transitory memory with instructions stored on the memory that when executed by the processor can cause the processor to operate the liquid handling system, heater, or sensor. In some cases, the controller can be configured to operate the sensor to analyze one or more aspects of the culture system. For instance, a controller can be configured to operate the sensor to measure one or more parameters in the culture medium, such as a nutrient concentration, a metabolite concentration (e.g., ammonium concentration or lactate concentration), or a pH. In some cases, a controller of a system can be linked to an actuator configured to disrupt cell clumps in a culture system (e.g., by agitation or non-agitation methods, such as gentle mixing). In some cases, a controller can be configured to operate a liquid handler or a cell disruption mechanism based at least in part on a measurement made by the sensor. In some cases, a controller can be configured to operate a liquid handler or a cell disruption mechanism based on a measurement from the sensor that exceeds a threshold value. In some cases, a controller can be configured to operate a sensor of the system. In some cases, a controller can be configured to operate a liquid handler or a cell disruption mechanism based on a predetermined, periodic, irregular, or regular time interval. In some cases, a controller can be configured to operate a liquid handler or a cell disruption mechanism based on user input.Expression Constructs

[0107] In some embodiments, the vectors can comprise plasmids. In other embodiments, the vectors can comprise a sequence of nucleotides. Nucleotide sequences can encode a sequence of amino acids. Nucleotide sequences can encode proteins. Nucleotide sequences can encode receptors. Nucleotide sequences can encode chimeric antigen receptors (CAR). Nucleotide sequences can encode proteins linking to a receptor. Nucleotide sequences can encode CAR linked to scFv. Vectors can encode PEBL.Nucleotide sequences can encode strands of desired surface expression molecules. Nucleotide sequences can encode one or more of: CD3, CD4, CD7, CD8, or CD56.

[0108] A cell population can be transduced with one or more viral vectors. In some cases, a cell population can be transduced with a plurality of viral vectors. In some cases, a cell population can be transduced with a viral vector comprising a nucleotide sequence encoding a CAR described herein (e.g., a CAR having a binding domain capable of binding at least a portion of a CD7 molecule, for example, a CD7 cell surface molecule on a human cell). In some cases, a cell population can be transduced with a viral vector comprising a nucleotide sequence encoding a PEBL described herein (e.g., a PEBL having a binding domain capable of binding at least a portion of a CD7 molecule, for example, a CD7 produced in the transduced cell). In some cases, a cell population can be transduced with a vector comprising a nucleotide sequence encoding a CAR and a separate vector encoding a PEBL. When separate vectors are used, separate promoter elements are configured upstream of each gene such that each promoter transcribes the mRNA for its proximally linked gene. In some embodiments, cells are transduced with a vector encoding a PEBL before a vector encoding a CAR to reduce surface expression of a target gene before the CAR is expressed, thereby reducing CAR activation and cytotoxicity within the population of transduced cells. In other embodiments, cells are transduced with a vector encoding a PEBL at the same time as a vector encoding a CAR or after a vector encoding a CAR.

[0109] Viral vectors can comprise two or more genes expressed from a single construct. These vectors can employ either a bicistronic element or a two-promoter configuration. In the case of bicistronic vectors, a sequence element can be introduced between two genes that can enable the translation of two proteins from a single messenger RNA. Examples include, but are not limited to, an internal ribosome entry site sequences (IRES) and virally derived "codon skipping" peptide sequences such as P2A, T2A, F2A, E2A, and the like (sometimes referred to as a “self-cleaving” peptide). In the case of two promoter designed vectors, separate promoter elements can be configured upstream of each gene such that each promoter transcribes the mRNA for its proximally linked gene. An expression vector (e.g., construct) can contain a first promoter operably linked to a CAR and a second promoter operably linked to a PEBL.

[0110] To reduce self-killing (e.g., fratricide), the CAR-T cells can express a PEBL that serves to reduce the expression of the target antigen on the cell surface of the CAR-T. To produce viable CAR-T cells, first a protein expression blocker (PEBL) protein can be expressed to bind and sequester the target protein prior to the subsequent expression of theCAR. Due to the pre-existing presence of the target antigen on the cell surface of the resulting engineered T-cells, simultaneous expression of the CAR and the PEBL can result in fratricide. In some cases, the preexisting cell surface target antigens are not susceptible to sequestration by the newly expressed PEBL proteins, and can be recognized and targeted by the newly expressed CAR proteins.

[0111] Cells may be transduced with a CAR but not a PEBL. A cell expressing a CAR without a PEBL may express a cell surface marker (e.g., CD7). The cell may be a CAR+CD7+ cell. A cell may not express a CAR or a PEBL and express a cell surface marker (e.g., a CAR-CD7+ cell). In some cases, a CAR+CD7+ cell may be more susceptible to fratricide compared to a cell expressing the identical CAR and a PEBL comprising an antigen-binding domain for the cell surface marker (e.g., CD7). In some cases, following expansion and enrichment of cells, a population of CAR+CD7- cells (e.g., engineered cells expressing a CAR and PEBL) may have 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, lOOx, 200x, 300x, 400x, 500x, lOOOx, 2000x, or 5000x more cells than a population of CAR+CD7+ cells.

[0112] In some cases, a CAR-CD7+ cell may be more susceptible to fratricide compared to a cell expressing the identical CAR and a PEBL comprising an antigen-binding domain for the cell surface marker (e.g., CD7). In some cases, following expansion and enrichment of cells, a population of CAR+CD7- cells (e.g., engineered cells expressing a CAR and PEBL) may have 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, lOOx, 200x, 300x, 400x, 500x, lOOOx, 2000x, or 5000x more cells than a population of CAR-CD7+ cells.

[0113] In some cases, cells transduced with one or more viral vectors may become fragile (e.g., more susceptible to death or low proliferation) compared to cells not transduced with a vector. In some cases, this can mean that transduced cells must be processed more gently during manufacture than cells that have not been transduced. Thus, in some cases, engineered cells (e.g., as described herein, which may be susceptible to fratricide, which may express a CAR targeting a cell surface protein expressed by the cells, and / or which may have been transduced with a viral vector) may require methods and / or systems (e.g., as described herein) that are designed for manufacture of such cells in order to obtain useful cell yields, cell viability percentages, and / or functional cell populations.

[0114] Certain two gene vectors can direct expression of both a PEBL and a CAR protein in T cells in a manner such that the resulting engineered T cells survive, expand, and can kill target cells. The relative timing and level of expression of each gene in the identified two gene vectors can enable the down regulation of the target antigen before the CAR can cause undue fratricide to the engineered T cells.

[0115] In certain embodiments, the nucleotide sequence encoding the CAR and / or the nucleotide sequence encoding the PEBL further comprise sequences (e.g., plasmid or vector sequences) that allow, e.g., cloning and / or expression. For example, the nucleotide sequence can be provided as part of a plasmid for ease of cloning into other plasmids and / or vectors for, e.g., transfection into a cell (e.g., an immune cell). In certain embodiments, the nucleotide sequence encoding the CAR and the nucleotide sequence encoding the PEBL are provided on a single plasmid or vector. In certain embodiments, the nucleotide sequences are provided on separate plasmids or vectors.

[0116] Nucleic acids can be introduced (directly transduced) into a cell using retroviral and lenti viral vector constructs. The term “lenti viral vector” can refer to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector. Other examples of lentivirus vectors that may be used in the clinic, can include but are not limited to, e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available. In other embodiments, the nucleic acids can be directly transfected into a cell. In yet other embodiments, the nucleic acids can be electroporated into a cell.

[0117] In some embodiments, the vectors used are derived from retroviruses such as lentiviruses. Such vectors can be suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of an exogenous polynucleotide (e.g., transgene) and its propagation in daughter cells. Unlike vectors derived from onco-retroviruses, lentiviral vectors can transduce non-proliferating cells. Lentiviral vectors can also have low immunogenicity. In other embodiments, the vector is an adenoviral vector. In certain embodiments, the vector is a plasmid.

[0118] In some cases, a system or method can comprise a viral vector encoding a CD7 CAR and a viral vector encoding a CD7 PEBL. In some cases, a bicistronic vector can be used that encodes CD7 CAR and CD7 PEBL. FIG. 3 depicts a schematic of an exemplary bicistronic construct comprising an MSCV promoter-anti-human CD7 (TH69) CAR-P2A- anti-human CD7 (TH69) PEBL.

[0119] The nucleic acid comprising a nucleotide sequence to be introduced as a vector can be a single bicistronic construct containing a chimeric antigen receptor described herein and a binding molecule (e.g., scFv) linked to a localizing domain. A single bicistronic construct can be prepared by inserting an internal ribosomal entry site (IRES) or a 2A peptide-coding region site between the two cDNAs encoding the chimeric antigen receptor asdescribed herein (e.g., CAR) and the binding molecule (e.g., scFv). In some embodiments, the bicistronic construct includes a CAR upstream of a PEBL with an IRES or 2A peptide coding region between them. In other embodiments, the bicistronic construct includes a PEBL upstream of a CAR with an IRES or 2A peptide coding region between them.Alternatively, separate transductions (simultaneously or sequentially) of the individual constructs (e.g., CAR and PEBL) can be performed.

[0120] In some embodiments, the bicistronic vector comprises a nucleic acid sequence comprising from 5' end to 3' end: a promoter, a nucleic acid sequence encoding a CD7 CAR, a P2A sequence, and a nucleic acid sequence encoding a CD7 PEBL. In some instances, the bicistronic vector comprises a nucleic acid sequence comprising from 5' end to 3' end: aMSCV promoter, a nucleic acid sequence encoding a CD7 CAR, a nucleic acid sequence encoding a P2A peptide, and a nucleic acid sequence encoding a CD7 PEBL.

[0121] In some embodiments, single promoter bicistronic vectors were used to produce CD7 PEBL-CAR-T cells from different starting cells including bulk PBMCs, purified T cells comprising CD4+ and CD8+ T cells, and purified CD3+ T cells. In some embodiments, the cells described herein (e.g., CD7 CAR+ / CD7-neg T cells) were generated from purified CD4+ positively selected and CD8+ positively selected T cells and then transduced with CD7CAR-P2A-CD7PEBL lentivirus at MOI 10 to generate CD7-CAR+ T-cells. In some embodiments, static transduction was performed where lentiviruses were directly added to T- cells. In some embodiment, a complete media change was performed two days later to remove lentiviruses from cultures.

[0122] In some embodiments, a percentage of the starting cells may be positive for a cell surface marker (e.g., CD4, CD8, CD56, CD3, CD7, CD5, CD38, and / or CD2).

[0123] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% of the starting cells may be CD4 positive. In some embodiments, at least 20% of the starting cells are CD4 positive. In some embodiments, a range from about 10 % to about 90 % of the starting cells may be CD4 positive. In some embodiments, a range from about 10 % to about 20 %, about 10 % to about 25 %, about 10 % to about 30 %, about 10 % to about 35 %, about 10 % to about 40 %, about 10 % to about 45 %, about 10 % to about 50 %, about 10 % to about 60 %, about 10 % to about 70 %, about 10 % to about 80 %, about 10 % to about 90 %, about 20 % to about 25 %, about 20 % to about 30 %, about 20 % to about 35 %, about 20 % to about 40 %, about 20 % to about 45 %, about 20 % to about 50 %, about 20 % to about 60%, about 20 % to about 70 %, about 20 % to about 80 %, about 20 % to about 90 %, about 25 % to about 30 %, about 25 % to about 35 %, about 25 % to about 40 %, about 25 % to about 45 %, about 25 % to about 50 %, about 25 % to about 60 %, about 25 % to about 70 %, about 25 % to about 80 %, about 25 % to about 90 %, about 30 % to about 35 %, about 30 % to about 40 %, about 30 % to about 45 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 80 %, about 30 % to about 90 %, about 35 % to about 40 %, about 35 % to about 45 %, about 35 % to about 50 %, about 35 % to about 60 %, about 35 % to about 70 %, about 35 % to about 80 %, about 35 % to about 90 %, about 40 % to about 45 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 40 % to about 90 %, about 45 % to about 50 %, about 45 % to about 60 %, about 45 % to about 70 %, about 45 % to about 80 %, about 45 % to about 90 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 50 % to about 90 %, about 60 % to about 70 %, about 60 % to about 80 %, about 60 % to about 90 %, about 70 % to about 80 %, about 70 % to about 90 %, or about 80 % to about 90 % of the starting cells may be CD4 positive.

[0124] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% of the starting cells may be CD8 positive. In some embodiments, at least 20% of the starting cells are CD8 positive. In some embodiments, a range from about 10 % to about 90 % of the starting cells may be CD8 positive. In some embodiments, a range from about 10 % to about 20 %, about 10 % to about 25 %, about 10 % to about 30 %, about 10 % to about 35 %, about 10 % to about 40 %, about 10 % to about 45 %, about 10 % to about 50 %, about 10 % to about 60 %, about 10 % to about 70 %, about 10 % to about 80 %, about 10 % to about 90 %, about 20 % to about 25 %, about 20 % to about 30 %, about 20 % to about 35 %, about 20 % to about 40 %, about 20 % to about 45 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 80 %, about 20 % to about 90 %, about 25 % to about 30 %, about 25 % to about 35 %, about 25 % to about 40 %, about 25 % to about 45 %, about 25 % to about 50 %, about 25 % to about 60 %, about 25 % to about 70 %, about 25 % to about 80 %, about 25 % to about 90 %, about 30 % to about 35 %, about 30 % to about 40 %, about 30 % to about 45 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 80 %, about 30 % to about 90 %, about 35 % to about 40 %, about 35 % to about 45 %, about 35 % to about 50 %, about 35 % to about 60 %, about 35 % to about 70 %, about 35 % to about 80 %, about 35 % to about 90 %, about 40 % to about 45 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about70 %, about 40 % to about 80 %, about 40 % to about 90 %, about 45 % to about 50 %, about 45 % to about 60 %, about 45 % to about 70 %, about 45 % to about 80 %, about 45 % to about 90 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 50 % to about 90 %, about 60 % to about 70 %, about 60 % to about 80 %, about 60 % to about 90 %, about 70 % to about 80 %, about 70 % to about 90 %, or about 80 % to about 90 % of the starting cells may be CD8 positive.

[0125] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% of the starting cells may be CD56 positive. In some embodiments, at least 20% of the starting cells are CD56 positive. In some embodiments, a range from about 10 % to about 90 % of the starting cells may be CD56 positive. In some embodiments, a range from about 10 % to about 20 %, about 10 % to about 25 %, about 10 % to about 30 %, about 10 % to about 35 %, about 10 % to about 40 %, about 10 % to about 45 %, about 10 % to about 50 %, about 10 % to about 60 %, about 10 % to about 70 %, about 10 % to about 80 %, about 10 % to about 90 %, about 20 % to about 25 %, about 20 % to about 30 %, about 20 % to about 35 %, about 20 % to about 40 %, about 20 % to about 45 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 80 %, about 20 % to about 90 %, about 25 % to about 30 %, about 25 % to about 35 %, about 25 % to about 40 %, about 25 % to about 45 %, about 25 % to about 50 %, about 25 % to about 60 %, about 25 % to about 70 %, about 25 % to about 80 %, about 25 % to about 90 %, about 30 % to about 35 %, about 30 % to about 40 %, about 30 % to about 45 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 80 %, about 30 % to about 90 %, about 35 % to about 40 %, about 35 % to about 45 %, about 35 % to about 50 %, about 35 % to about 60 %, about 35 % to about 70 %, about 35 % to about 80 %, about 35 % to about 90 %, about 40 % to about 45 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 40 % to about 90 %, about 45 % to about 50 %, about 45 % to about 60 %, about 45 % to about 70 %, about 45 % to about 80 %, about 45 % to about 90 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 50 % to about 90 %, about 60 % to about 70 %, about 60 % to about 80 %, about 60 % to about 90 %, about 70 % to about 80 %, about 70 % to about 90 %, or about 80 % to about 90 % of the starting cells may be CD56 positive.

[0126] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% of the starting cells may be CD3 positive. In some embodiments, at least 20% of the startingcells are CD3 positive. In some embodiments, a range from about 10 % to about 90 % of the starting cells may be CD3 positive. In some embodiments, a range from about 10 % to about 20 %, about 10 % to about 25 %, about 10 % to about 30 %, about 10 % to about 35 %, about 10 % to about 40 %, about 10 % to about 45 %, about 10 % to about 50 %, about 10 % to about 60 %, about 10 % to about 70 %, about 10 % to about 80 %, about 10 % to about 90 %, about 20 % to about 25 %, about 20 % to about 30 %, about 20 % to about 35 %, about 20 % to about 40 %, about 20 % to about 45 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 80 %, about 20 % to about 90 %, about 25 % to about 30 %, about 25 % to about 35 %, about 25 % to about 40 %, about 25 % to about 45 %, about 25 % to about 50 %, about 25 % to about 60 %, about 25 % to about 70 %, about 25 % to about 80 %, about 25 % to about 90 %, about 30 % to about 35 %, about 30 % to about 40 %, about 30 % to about 45 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 80 %, about 30 % to about 90 %, about 35 % to about 40 %, about 35 % to about 45 %, about 35 % to about 50 %, about 35 % to about 60 %, about 35 % to about 70 %, about 35 % to about 80 %, about 35 % to about 90 %, about 40 % to about 45 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 40 % to about 90 %, about 45 % to about 50 %, about 45 % to about 60 %, about 45 % to about 70 %, about 45 % to about 80 %, about 45 % to about 90 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 50 % to about 90 %, about 60 % to about 70 %, about 60 % to about 80 %, about 60 % to about 90 %, about 70 % to about 80 %, about 70 % to about 90 %, or about 80 % to about 90 % of the starting cells may be CD3 positive.

[0127] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% of the starting cells may be CD7 positive. In some embodiments, at least 20% of the starting cells are CD7 positive. In some embodiments, a range from about 10 % to about 90 % of the starting cells may be CD7 positive. In some embodiments, a range from about 10 % to about 20 %, about 10 % to about 25 %, about 10 % to about 30 %, about 10 % to about 35 %, about 10 % to about 40 %, about 10 % to about 45 %, about 10 % to about 50 %, about 10 % to about 60 %, about 10 % to about 70 %, about 10 % to about 80 %, about 10 % to about 90 %, about 20 % to about 25 %, about 20 % to about 30 %, about 20 % to about 35 %, about 20 % to about 40 %, about 20 % to about 45 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 80 %, about 20 % to about 90 %, about 25 % to about 30 %, about 25 % to about 35 %, about 25 % to about 40 %, about 25 % to about45 %, about 25 % to about 50 %, about 25 % to about 60 %, about 25 % to about 70 %, about 25 % to about 80 %, about 25 % to about 90 %, about 30 % to about 35 %, about 30 % to about 40 %, about 30 % to about 45 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 80 %, about 30 % to about 90 %, about 35 % to about 40 %, about 35 % to about 45 %, about 35 % to about 50 %, about 35 % to about 60 %, about 35 % to about 70 %, about 35 % to about 80 %, about 35 % to about 90 %, about 40 % to about 45 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 40 % to about 90 %, about 45 % to about 50 %, about 45 % to about 60 %, about 45 % to about 70 %, about 45 % to about 80 %, about 45 % to about 90 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 50 % to about 90 %, about 60 % to about 70 %, about 60 % to about 80 %, about 60 % to about 90 %, about 70 % to about 80 %, about 70 % to about 90 %, or about 80 % to about 90 % of the starting cells may be CD7 positive.

[0128] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% of the starting cells may be CD5 positive. In some embodiments, at least 20% of the starting cells are CD5 positive. In some embodiments, a range from about 10 % to about 90 % of the starting cells may be CD5 positive. In some embodiments, a range from about 10 % to about 20 %, about 10 % to about 25 %, about 10 % to about 30 %, about 10 % to about 35 %, about 10 % to about 40 %, about 10 % to about 45 %, about 10 % to about 50 %, about 10 % to about 60 %, about 10 % to about 70 %, about 10 % to about 80 %, about 10 % to about 90 %, about 20 % to about 25 %, about 20 % to about 30 %, about 20 % to about 35 %, about 20 % to about 40 %, about 20 % to about 45 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 80 %, about 20 % to about 90 %, about 25 % to about 30 %, about 25 % to about 35 %, about 25 % to about 40 %, about 25 % to about 45 %, about 25 % to about 50 %, about 25 % to about 60 %, about 25 % to about 70 %, about 25 % to about 80 %, about 25 % to about 90 %, about 30 % to about 35 %, about 30 % to about 40 %, about 30 % to about 45 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 80 %, about 30 % to about 90 %, about 35 % to about 40 %, about 35 % to about 45 %, about 35 % to about 50 %, about 35 % to about 60 %, about 35 % to about 70 %, about 35 % to about 80 %, about 35 % to about 90 %, about 40 % to about 45 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 40 % to about 90 %, about 45 % to about 50 %, about 45 % to about 60 %, about 45 % to about 70 %, about 45 % to about 80 %, about 45 % toabout 90 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 50 % to about 90 %, about 60 % to about 70 %, about 60 % to about 80 %, about 60 % to about 90 %, about 70 % to about 80 %, about 70 % to about 90 %, or about 80 % to about 90 % of the starting cells may be CD5 positive.

[0129] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% of the starting cells may be CD38 positive. In some embodiments, at least 20% of the starting cells are CD38 positive. In some embodiments, a range from about 10 % to about 90 % of the starting cells may be CD38 positive. In some embodiments, a range from about 10 % to about 20 %, about 10 % to about 25 %, about 10 % to about 30 %, about 10 % to about 35 %, about 10 % to about 40 %, about 10 % to about 45 %, about 10 % to about 50 %, about 10 % to about 60 %, about 10 % to about 70 %, about 10 % to about 80 %, about 10 % to about 90 %, about 20 % to about 25 %, about 20 % to about 30 %, about 20 % to about 35 %, about 20 % to about 40 %, about 20 % to about 45 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 80 %, about 20 % to about 90 %, about 25 % to about 30 %, about 25 % to about 35 %, about 25 % to about 40 %, about 25 % to about 45 %, about 25 % to about 50 %, about 25 % to about 60 %, about 25 % to about 70 %, about 25 % to about 80 %, about 25 % to about 90 %, about 30 % to about 35 %, about 30 % to about 40 %, about 30 % to about 45 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 80 %, about 30 % to about 90 %, about 35 % to about 40 %, about 35 % to about 45 %, about 35 % to about 50 %, about 35 % to about 60 %, about 35 % to about 70 %, about 35 % to about 80 %, about 35 % to about 90 %, about 40 % to about 45 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 40 % to about 90 %, about 45 % to about 50 %, about 45 % to about 60 %, about 45 % to about 70 %, about 45 % to about 80 %, about 45 % to about 90 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 50 % to about 90 %, about 60 % to about 70 %, about 60 % to about 80 %, about 60 % to about 90 %, about 70 % to about 80 %, about 70 % to about 90 %, or about 80 % to about 90 % of the starting cells may be CD38 positive.

[0130] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% of the starting cells may be CD2 positive. In some embodiments, at least 20% of the starting cells are CD2 positive. In some embodiments, a range from about 10 % to about 90 % of the starting cells may be CD2 positive. In some embodiments, a range from about 10 % to about20 %, about 10 % to about 25 %, about 10 % to about 30 %, about 10 % to about 35 %, about 10 % to about 40 %, about 10 % to about 45 %, about 10 % to about 50 %, about 10 % to about 60 %, about 10 % to about 70 %, about 10 % to about 80 %, about 10 % to about 90 %, about 20 % to about 25 %, about 20 % to about 30 %, about 20 % to about 35 %, about 20 % to about 40 %, about 20 % to about 45 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 80 %, about 20 % to about 90 %, about 25 % to about 30 %, about 25 % to about 35 %, about 25 % to about 40 %, about 25 % to about 45 %, about 25 % to about 50 %, about 25 % to about 60 %, about 25 % to about 70 %, about 25 % to about 80 %, about 25 % to about 90 %, about 30 % to about 35 %, about 30 % to about 40 %, about 30 % to about 45 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 80 %, about 30 % to about 90 %, about 35 % to about 40 %, about 35 % to about 45 %, about 35 % to about 50 %, about 35 % to about 60 %, about 35 % to about 70 %, about 35 % to about 80 %, about 35 % to about 90 %, about 40 % to about 45 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 40 % to about 90 %, about 45 % to about 50 %, about 45 % to about 60 %, about 45 % to about 70 %, about 45 % to about 80 %, about 45 % to about 90 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 50 % to about 90 %, about 60 % to about 70 %, about 60 % to about 80 %, about 60 % to about 90 %, about 70 % to about 80 %, about 70 % to about 90 %, or about 80 % to about 90 % of the starting cells may be CD2 positive.

[0131] In some embodiments, T cells were transduced with varying volumes of lentiviruses in the presence of 5pg / mL polybrene. In some embodiments, after transduction medium was removed, cells were treated with an endonuclease that nonspecifically cleaves DNA to release di-, tri- and oligonucleotide products with 5 '-phosphorylated and 3'- hydroxylated ends (1,2) in fresh culture media. In some embodiments, media was then replaced with fresh basal medium with 10% FBS. Transduced cells were then harvested.

[0132] In some embodiments, 293T cells were cotransfected with lentiviral transfer plasmid and packaging plasmids via a transfection reagent to generate lentiviral vectors. In some embodiments, the transfection reagent comprises cationic-lipid transfection reagents formulated for the transfection of DNA into eukaryotic cells. In some embodiments, 293T cells were cotransfected with retroviral transfer plasmids and pEQ and pRDF packaging plasmids using a transfection reagent.Culture Medium

[0133] In some embodiments, T cell populations described herein are cultured in medium. The medium can comprise sterile water. The medium can be GMP grade media. In some embodiments, the medium is a serum-free cell culture medium. In some embodiments, the serum-free cell culture medium is optimized for expansion of T cells (e.g. TexMACS medium). In some embodiments, T cells are maintained in a basal medium with 10% fetal bovine serum (FBS), penicillin, and streptomycin. In some embodiments, T cell populations can be cultured in the presence of IL-2. In some embodiments, T cell populations can be cultured in the presence of anti-CD3 antibody OKT3. In some embodiments, T cell populations can be cultured in the presence of T2 cells.Cells

[0134] Methods and systems described herein can comprise a population of cells. A population of cells (e.g., an engineered population of cells) can comprise one or more immune cells. In some cases, all or a portion of the population of cells can be immune cells. For example, all or a portion of the population of cells can be T-cells. In some cases, all or a portion of the population of cells can be natural killer (NK) cells. In some cases, the population of cells (e.g., engineered cells) can express one or more cell surface polypeptides selected from CD2, CD3, CD4, CD7, CD8, CD28, and / or CD56. In some cases, an activated cell can express CD3 and / or CD28. In some cases, a cell (e.g., an engineered cell) useful in methods and systems described herein may not express one or more cell surface polypeptides selected from CD2, CD3, CD4, CD7, CD8, CD28, and / or CD56. In some cases, a cell (e.g., an engineered cell) useful in methods and systems described herein may express a reduced amount of one or more cell surface polypeptides selected from CD2, CD3, CD4, CD7, CD8, CD28, and / or CD56 (e.g., compared to a cell that has not been engineered).

[0135] In some cases, a cell (e.g., an engineered cell) can express a chimeric antigen receptor (CAR). In some cases, a cell can be engineered to express a CAR, for instance, by transducing the cell with a vector having a nucleic acid sequence that encodes the CAR (e.g., as described herein). In some cases, a CAR can comprise a binding domain that binds to a cell surface polypeptide of another cell. In some cases, a binding domain of a CAR can be linked to an activation domain. In some cases, binding of the binding domain of a CAR expressed by a first (e.g., engineered immune) cell to a target cell surface polypeptide presented by a second cell can cause the activation domain of the CAR to activate the first cell (e.g., to induce cytotoxic activity in the first cell, forexample, by inducing an activated phenotype in the first cell) to destroy the second cell. In some cases, this can lead to fratricide among cells engineered to express the CAR, for example, if the cell expressing the CAR also expresses the cell surface polypeptide. In some cases, a cell expressing a CAR that binds to a cell surface polypeptide that the cell itself does or can express may be a cell susceptible to fratricide. In some cases, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70% at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% of the cells of a population of cells (e.g., engineered immune cells) described herein can express a CAR (e.g., a CAR having a binding domain that recognizes a cell surface polypeptide and an activation domain linked to the binding domain).

[0136] In some cases, a cell expressing a CAR can be further engineered for reduced expression of a cell surface antigen targeted by the CAR using, for example, a PEBL, siRNA, gene disruption, or a dominant-negative allele. In some cases, a cell (e.g., an engineered cell) can express a protein expression blocker molecule (PEBL). In some cases, a PEBL can comprise a chimeric targeting polypeptide, for example, wherein the chimeric targeting polypeptide comprises a binding domain that binds to a cell surface polypeptide (e.g., a cell surface polypeptide that the cell is capable of producing). In some cases, at least 50%, at least 60%, at least 65%, at least 70% at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% of the cells of a population of cells (e.g., engineered immune cells) described herein can express a PEBL (e.g., in addition to a CAR, which may be a CAR having a binding domain that recognizes the same cell surface polypeptide as the binding domain of the PEBL). As described herein, a cell may express a reduced amount of one or more cell surface polypeptides (or none of one or more cell surface polypeptides), such as CD7, as a result of the cell expressing a PEBL that binds to the one or more cell surface polypeptides (e.g., in the cytoplasm of the cell). In some cases, a PEBL can reduce or eliminate cell surface expression (or cell surface localization) of a cell surface polypeptide by binding to the cell surface polypeptide and sequestering the cell surface polypeptide within the cell. For example, a PEBL can comprise an intracellular localizing domain. In some cases, a PEBL can prevent or reduce cell surface expression or localization of a cell surface polypeptide (e.g., CD7) by binding the cell surface polypeptide within the cell (e.g., using a binding domain of the PEBL) and retaining the cell surface polypeptide within the cell (e.g., using the intracellular localizing domain ofthe PEBL). In some cases, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% of the cells of a population of cells (e.g., engineered immune cells) described herein can have reduced expression of a cell surface polypeptide (e.g., wherein the cell surface polypeptide is recognized by the binding domain of a CAR expressed by the cell and / or by the binding domain of a PEBL expressed by the same cell), for example, compared to healthy human cells of a cell type that is the same cell type as the population of cells. In some cases, the cells having reduced expression of a cell surface polypeptide comprise a non-natural modification of a gene encoding the cell surface polypeptide.

[0137] In some cases, the cell surface polypeptide that a binding domain of a PEBL expressed by a cell (e.g., engineered cell) can bind to may be the same cell surface polypeptide that a binding domain of a CAR expressed by the same cell can bind to. Thus, expression of a CAR and a PEBL that both recognize the same cell surface polypeptide in a population of cells (e.g., engineered cells described herein) can aid in preventing or reducing a likelihood of fratricide among the population of cells, for example, by sequestering the target of the CAR molecule within the cell using the PEBL. In some cases, this can improve the efficiency of a population of engineered cells in targeting and / or destroying cells that have not been engineered (e.g., immune cells (e.g. cancer cells) expressing the cell surface polypeptide recognized by the CAR), for example, because the engineered cells are less likely to target and destroy one another.

[0138] In some cases, the intracellular localizing domain is selected from an ER retention signal, a Golgi retention signal, or a PEST signal. In some cases, the ER retention signal can comprise a KKXX sequence. In some cases, the ER retention signal can comprise a KDEL sequence. In some cases, the chimeric targeting polypeptide (e.g., PEBL) can comprise a spacer sequence between the binding domain and the intracellular localizing signal (e.g., the ER retention signal), for instance, between the binding domain and the KDEL sequence.Chimeric Antigen Receptors

[0139] In some embodiments, manufactured T cells can express a receptor. The receptor can be a chimeric antigen receptor (CAR). In certain aspects, the CAR binds to molecules expressed on the surface of tumor cells, including but not limited to CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD33, CD45, CD52, CD38, CS-1, TIM3, CD123, mesothelin,folate receptor, HER2-neu, epidermal-growth factor receptor, and epidermal growth factor receptor. In some embodiments, the immune activating receptor is a CAR (e.g., anti-CD19-4- 1BB-CD3(^ CAR). In certain embodiments, the immune activating receptor comprises an antibody or antigen-binding fragment thereof (e.g., scFv) that binds to molecules expressed on the surface of tumor cells, including but not limited to, CD20, CD22, CD33, CD2, CD3, CD4, CD5, CD7, CD8, CD45, CD52, CD38, CS-1, TIM3, CD123, mesothelin, folate receptor, HER2-neu, epidermal-growth factor receptor, and epidermal growth factor receptor. In some embodiments, the chimeric antigen receptor targets CD7 protein.

[0140] In some embodiments, a CAR comprises a binding domain. In some embodiments, the binding domain can bind to a target epitope expressed on the surface of a T cell. The target epitope can be a polypeptide. The target epitope can be a cell surface polypeptide. The target epitope can be CD7. In some embodiments, the binding domain can bind to CD7 expressed on the surface of a T cell. The T cell can be isolated from a subject. The subject can be a human subject. The T cell can be isolated from a human subject by leukapheresis.

[0141] In some embodiments, the binding domain that binds to a marker can be an antibody. The antibody can be a single chain antibody. The single chain antibody can be a single-chain variable fragment (scFv). ScFv can be engineered to bind to any protein selectively expressed on the surface of a cell. ScFv can be engineered to bind to any protein selectively expressed on the surface of a cancer cell. ScFv can be engineered to bind to CD7. ScFv binding to a cancer cell can trigger activation of a CAR T cell. Activation of a CAR T cell can kill a cell. Activation of a CAR T cell while bound to a cancer cell can kill a cancer cell.

[0142] ScFv can bind a non-cancer cell expressing the target epitope (protein) that the scFv is engineered to bind. An scFv can bind a non-target cell expressing the target epitope. ScFv binding to a non-target cell expressing the target epitope can trigger activation of the CAR T cell for cytotoxic activity against the non-target cell. Activation of a CAR T cell while bound to a non-target cell can kill a non-target cell, for example, committing fratricide of other CAR T cells. Fratricide can decrease the number of CAR T cells. Decreasing the number of CAR T cells can decrease the number of CAR T cells that can bind and kill a target cell.

[0143] In some embodiments, a CAR comprises an activation domain. The activation domain can activate T cells for cytotoxicity. In some embodiments, a CAR includes a stimulatory region, a co-stimulatory region, and a binding domain. In some embodiments, thestimulatory region and co- stimulatory region can be the activation domain. The stimulatory region and co-stimulatory region can be linked to the binding domain. The binding domain can be an antibody or an scFv. Binding of the scFv of a CAR to a cell it is engineered to target can trigger activation of the stimulatory region and co-stimulatory region of the activation domain. The stimulatory region can be linked to the co-stimulatory region. The co- stimulatory region can be linked to the binding domain.

[0144] A CAR can comprise an intracellular signaling domain. The intracellular signaling domain can comprise a stimulatory region. The stimulatory region can be part of the activation domain. The stimulatory region can be derived from CD3(^, FcsRIy, DAP10, DAP12 or other molecules known to deliver activating signals in immune cells. In some embodiments, the stimulatory molecule (CD3(^, in this specific example), can be substituted with another known stimulatory molecule.

[0145] A CAR can comprise a co-stimulatory region (or domain). The intracellular signaling domain can further comprise the co-stimulatory region. The co-stimulatory region can be part of the activation domain. At least one co-stimulatory domain of the receptor can be a costimulatory molecule such as 4-1BB (also known as CD137), a CD28 variant, 0X40, ICOS, CD27, GITR, HVEM, TIM-1, TIM-3, LFA-1, CD2, CD30, CD84, CRTAM, DR3, SLAMF1. The co-stimulatory domain can be a functional signaling domain obtained from a protein selected from the group consisting of 4-1BB (also known as CD137), a CD28 variant, 0X40, ICOS, CD27, GITR, HVEM, TIM-1, TIM-3, LFA-1, CD2, CD30, CD84, CRTAM, DR3, SLAMF1. In other embodiments, the co-stimulatory molecule (4-1BB in this specific example) can also be varied with a different co-stimulatory molecule, e.g., CD28.

[0146] A CAR can comprise a linker. A CAR can comprise a linker between the stimulatory region and co-stimulatory region. The linker can strengthen activation of the stimulatory region. The linker can quicken activation of the stimulatory region. A CAR may not comprise a linker between the stimulatory region and co-stimulatory region.

[0147] A CAR can comprise a linker between the binding domain (e.g., antigen-binding region or target-binding region) and the intracellular signaling domain. A linker between the binding domain and the intracellular signaling domain (e.g., the stimulatory region or the co- stimulatory region) can comprise a transmembrane domain. The transmembrane domain of the CAR can be derived from a single-pass membrane protein, including, but not limited to, CD8a, CD8p, 4-1BB, CD28, CD34, CD4, FcsRIy, CD16 (e g., CD16A or CD16B), 0X40, CD3< CD35, CD3y, CD35, TCRa, CD32 (e.g, CD32A or CD32B), CD64 (e.g, CD64A, CD64B, or CD64C), VEGFR2, FAS, and FGFR2B. In some cases, the transmembraneprotein is not CD8a. The transmembrane domain can also be a non-naturally occurring hydrophobic protein segment.

[0148] A linker between the binding domain (e.g., the antigen-binding region or the target binding region) and the intracellular signaling domain (e.g., the stimulatory region or the co-stimulatory region) can comprise a hinge domain. The hinge domain of the CAR can be derived from a protein such as CD8a, or IgG. The hinge domain can be a fragment of the transmembrane or hinge domain of CD8a, or a non-naturally occurring peptide, such as a polypeptide consisting of hydrophilic residues of varying length, or a (GGGGS)npolypeptide, in which n is an integer of, e.g., 2-12, inclusive.

[0149] A CAR described herein can comprise a linker between the co-stimulatory region and the binding domain. The linker can increase the specificity of the CAR.

[0150] An engineered immune cell can be a CAR-T cell expressing a CAR directed against CD7 and having reduced or no surface expression of CD7. Co-expression of a chimeric antigen receptor (CAR) directed against CD7 and a protein expression blocker (PEBL) directed against CD7 in immune cells (e.g., T cells) can be achieved by using a bicistronic construct, such as a bicistronic viral vector. In some embodiments, the present invention relates to an engineered immune cell (e.g., an engineered T cell) comprising a bicistronic construct comprising a polynucleotide sequence encoding an anti-CD7 CAR and a polynucleotide sequence encoding an anti-CD7 PEBL. In some embodiments, the bicistronic vector may comprise a first nucleic acid sequence encoding the CAR and a second nucleic acid sequence encoding the PEBL, and the two nucleic acid sequences can be linked by a linker sequence. The linker sequence can encode a 2A sequence. In some embodiments, the CAR comprises intracellular signaling domains of 4-IBB and CD3, and an antibody (e.g., a single chain variable fragment or scFv) that specifically binds CD7. In some embodiments, the CAR also includes a CD8oc hinge and transmembrane domain. In some embodiments, the anti-CD7 PEBL comprises an antibody (e.g., a scFv) that specifically binds CD7 and an intracellular localization sequence. In certain embodiments, the anti-CD7 PEBL comprises an antibody (e.g., a scFv) that specifically binds CD7, CD8oc hinge and transmembrane domains, and an intracellular localization sequence.

[0151] In some embodiments, the engineered immune cell is an engineered T cell (e.g., engineered cytotoxic T cell, engineered helper T cell, engineered regulatory T cell, engineered effector T cell, engineered memory T cell, engineered natural killer T cell, andengineered gamma delta T cell), an engineered natural killer (NK) cell, an engineered NK / T cell, an engineered monocyte, an engineered macrophage, or an engineered dendritic cell. In some cases, the engineered immune cell is an allogeneic cell. In other cases, the engineered immune cell is an autologous cell.

[0152] In certain embodiments, the engineered immune cell proliferates at a substantially equal rate compared to a comparable immune cell.

[0153] The engineered cells of the present invention can be expanded in a culture media under specific conditions. In some embodiments, the engineered cells are cultured in the presence of IL-2. The engineered cells can be frozen (e.g., cryopreserved) according to a method recognized by one skilled in the art. Prior to administration to the patient, the engineered calls can be thawed and cultured. In other cases, the engineered calls can also be expanded prior to administration.

[0154] In some embodiments, a subject has a reduced likelihood of developing graft- versus-host-disease when the engineered immune cell is administered to the subject, wherein the engineered immune cell is allogeneic to said subject. The engineered immune cell can induce cytotoxicity of CD7-positive leukemic cells.Protein Expression Blockers (PEBLs)

[0155] In some embodiments, fratricide can be reduced by co-expressing a protein expression blocker (PEBL). In some embodiments, a PEBL reduces cell surface expression of a polypeptide. The polypeptide can be CD7. In some embodiments, a PEBL retains its target polypeptide within the cell, preventing its detection by a receptor on another T cell. In some embodiments, a PEBL comprises a localizing domain and a binding domain. In some embodiments, T cells expressing a PEBL are able to more efficiently treat T cell leukemia or T cell lymphoma due to decreased cell fratricide.

[0156] In some embodiments, the binding domain of a PEBL can comprise a molecule that specifically binds to a TCR complex protein such as TCRa, TCRP, CD35, CD3s, CD3y, and CD3^. In a further aspect, the PEBL molecule binds to a molecule selected from, e.g., CD2, CD4, CD5, CD7, CD8, CD30, CD38, CD45, CD52, or CD127. In some embodiments, the PEBL can bind to a molecule that is expressed on the surface of a cell including, but not limited to members of the CD1 family of glycoproteins, CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD28, CD30, CD38, CD45, CD45RA, CD45RO, CD52, CD56, CD57, CD99, CD127, and CD137.

[0157] In certain embodiments, the PEBL molecule binds to a target molecule expressedon the surface of an immune cell. In some embodiments, the PEBL molecule inhibits the activity or function of the target molecule. By way of example, as disclosed herein, PEBL molecule can be designed to bind to, e.g., TCRa, TCRP, CD3 (e.g., CD3s, CD3y, CD35, or CD3Q, CD7, CD45, hB2MG, KIR2DL1, KIR2DL2 / DL3, NKG2A, or NKG2D thereby downregulating cell surface expression of such molecules. Downregulation of such molecules can be achieved through, for example, localizing / targeting the molecules for degradation and / or internalization. In other embodiments, the PEBL molecule renders the target inactive (e.g., the target can no longer interact and / or bind to its cognate ligand or receptor).

[0158] A PEBL comprises a binding domain. A binding domain can increase specificity of PEBL. The binding domain can comprise an antibody. The antibody can be a single chain antibody. The single chain antibody can be a single-chain variable fragment (scFv). ScFv can be engineered to bind to any protein selectively expressed on the surface of a cell. ScFv can be engineered to bind to CD7. CD7 can be transduced into a cell. ScFv can be engineered to bind to CD7 that has been transduced into a cell. ScFv can be engineered to bind to CD7 that has been transduced into a T cell. ScFv can be engineered to bind to a newly synthesized surface protein in the endoplasmic reticulum (ER). ScFv can be engineered to bind to a newly synthesized CD7 in the endoplasmic reticulum (ER). When the scFv of PEBL binds to a newly synthesized surface protein in the endoplasmic reticulum (ER), PEBL can retain the newly synthesized surface protein in the ER. PEBL can keep a newly synthesized surface protein away from the cell surface. PEBL can keep CD7 away from the cell surface. Keeping CD7 away from the cell surface can significantly decrease the probability that the CAR’s scFv binds a non-target cell expressing CD7. Decreasing the probability that the CAR’s scFv binds an engineered T cell expressing CD7 can decrease activation of a CAR T cell while bound to a non-target cell. Decreasing activation of a CAR T cell while bound to a non-target cell can decrease killing of non-target cells, for example, decreasing fratricide of other CAR T cells. Decreasing fratricide can avoid decreasing the number of CAR T cells. Avoiding decrease of CAR T cells can avoid decreasing the number of CAR T cells that can bind and kill a target cell.

[0159] In some embodiments, a PEBL does not decrease CAR T cell targeting of cancer cells. In some cases, a target cell does not comprise a PEBL. In some cases, target cells that do not comprise a PEBL express CD7 on their surface instead of retaining it in the ER. CAR T cells can bind and activate destruction of a target cells that express CD7 on their surface.

[0160] In some embodiments, the scFv that is part of the PEBL molecule is not necessarily the same as the scFv that occurs in the context of, e.g., a chimeric antigenreceptor (CAR) or a similar antigen-binding (e.g., target-binding) signaling receptor. In some embodiments, the scFv that is part of the PEBL molecule is the same as the scFv that occurs in the context of, e.g., a chimeric antigen receptor (CAR) or a similar antigen-binding (e.g., target-binding) signaling receptor.

[0161] A PEBL can comprise a localizing domain. The localizing domain can direct the polypeptide that the binding domain binds to specific cellular compartments, such as the Golgi, endoplasmic reticulum (ER), proteasome, or cellular membrane, depending on the application. The localizing domain can be an ER localization sequence. An ER localization sequence can retain a newly synthesized surface protein in the ER. An ER localization sequence can keep a newly synthesized surface protein away from the cell surface. In some instances, the localizing domain comprises an amino acid sequence encoded by all or a portion of a nucleic acid sequence (see FIG. 3) of a viral vector transduced into a cell, as described herein.

[0162] In some embodiments, a PEBL can comprise one or more localizing domains. For example, a PEBL molecule can have at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten localizing domains linked together. When more than one localizing domain is used in a single PEBL molecule, each localizing domain can be linked with or without any intervening linker. In some instances, localization domains such as a CD8a transmembrane domain, KDEL motif, and a linker can be used in a single PEBL molecule. In some embodiments, localization domains do not comprise any intervening linkers. In other embodiments, various intervening linkers can be incorporated between some or all of the localization domains.

[0163] In some embodiments, the localizing domain comprises a retention domain. In certain embodiments, the localizing domain comprises a retention domain and a transmembrane domain. In some instances, the retention domain comprises an endoplasmic reticulum (ER) retention sequence, a Golgi retention sequence, or a proteasome localizing sequence. The retention domain can include an amino acid sequence that prevents or hinders a protein from being secreted by a cell. The retention domain can include an amino acid sequence that retains a protein in an intracellular compartment. In some cases, the retention domain can include an amino acid sequence that retains a protein in a cellular membrane such as a membrane of the ER or Golgi. For example, an ER or Golgi retention domain can comprise a KDEL sequence, KKD or KKE sequence, KKMP sequence, YQRL sequence, KXD or KXE sequence, or KKXX sequence, wherein X is any amino acid sequence. In someaspects, the localizing domain of PEBL can comprise a proteosome targeting sequence that comprises, e.g., a “PEST” motif- SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV.

[0164] The transmembrane domain of a PEBL (e.g. a PEBL comprising a KKXX ER retention domain) can comprise a transmembrane domain derived from CD8a, CD8P, 4-1BB, CD28, CD34, CD4, FcsRIy, CD16, 0X40, CD3< CD3s, CD3y, CD35, TCRa, CD32, CD64, VEGFR2, FAS, or FGFR2B. In certain embodiments, the transmembrane domain of the localizing domain is derived from CD8a. The transmembrane domain can be linked to a retention domain. In some embodiments, the transmembrane domain is linked to the retention domain by way of a linker.

[0165] A PEBL can comprise a linker. In some cases, a linker of a PEBL can couple a target-binding domain to a localizing domain (e.g. a KDEL ER retention domain). In some cases, a linker connecting a localizing domain and a target-binding domain of a PEBL can improve the localizing activity of the PEBL. Non-limiting examples of a linker can include (GS)n, (GGS)n, (Gly3Ser)n, (Gly2SerGly)n, (Gly2SerGly2)n, or (Gly4Ser)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker is (Gly4Ser)3 or (Gly4Ser)4. Variation in the linker length can retain or enhance activity, giving rise to improved retention.

[0166] The localizing domain can be located at the C-terminal region of the PEBL while the target-binding domain can be at the N-terminal region. In some embodiments, the PEBL from N-terminus to C-terminus comprises a signal / leader peptide, a binding domain, a linker, and a localizing domain. In other embodiments, the PEBL from N-terminus to C-terminus comprises a signal, a binding domain, a transmembrane domain, and a localizing domain. In certain embodiments, the PEBL from N-terminus to C-terminus comprises a signal peptide, a binding domain, and a localizing domain. In other embodiments, the PEBL from N-terminus to C-terminus comprises a binding domain and a localizing domain.

[0167] In some cases, one or more PEBLs can be encoded by a nucleic acid sequence of a vector (e.g., viral vector) described herein. In some cases, one or more PEBLs can be encoded by a nucleic acid sequence that also encodes one or more CARs. In some cases, a nucleic acid encoding a PEBL can be delivered to (e.g., transduced into) a cell via a vector (e.g., viral vector) that is different than a nucleic acid encoding a CAR that is also delivered to (e.g., transduced into) the cell (e.g., via a separate vector).DEFINITIONS

[0168] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to whichthe invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0169] It is also to be understood that the terminology used herein is only for the purpose of describing particular embodiments and is not intended to be limiting.

[0170] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0171] The term "about" and its grammatical equivalents in relation to a reference numerical value and its grammatical equivalents as used herein can include a range of values plus or minus 10% from that value. For example, the amount "about 10" includes amounts from 9 to 11. The term "about" in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.

[0172] As used herein, the term "nucleic acid" refers to a polymer comprising multiple nucleotide monomers (e.g., ribonucleotide monomers or deoxyribonucleotide monomers). "Nucleic acid" includes, for example, genomic DNA, cDNA, RNA, and DNA-RNA hybrid molecules. Nucleic acid molecules can be naturally occurring, recombinant, or synthetic. In addition, nucleic acid molecules can be single-stranded, double-stranded or triple-stranded. In certain embodiments, nucleic acid molecules can be modified. In the case of a double-stranded polymer, "nucleic acid" can refer to either or both strands of the molecule. Nucleic acids and polynucleotides as used herein are interchangeable.

[0173] The term "nucleotide sequence," in reference to a nucleic acid, refers to a contiguous series of nucleotides that are joined by covalent linkages, such as phosphorus linkages (e.g., phosphodiester, alkyl and aryl -phosphonate, phosphorothioate, phosphotri ester bonds), and / or non-phosphorus linkages (e.g., peptide and / or sulfamate bonds). In certain embodiments, the nucleotide sequence encoding, e.g., a target-binding molecule linked to a localizing domain is a heterologous sequence (e.g., a gene that is of a different species or cell type origin).

[0174] The terms "nucleotide" and "nucleotide monomer" refer to naturally occurring ribonucleotide or deoxyribonucleotide monomers, as well as non-naturally occurring derivatives and analogs thereof. Accordingly, nucleotides can include, for example,nucleotides comprising naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or deoxycytidine) and nucleotides comprising modified bases known in the art.

[0175] The term "operably linked" refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0176] The term "sequence identity" means that two nucleotide sequences or two amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least, e.g., 70% sequence identity, or at least 80% sequence identity, or at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity or more. For sequence comparison, typically one sequence acts as a reference sequence (e.g., parent sequence), to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0177] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. AppL Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mai. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'!. Acad Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel etal., Current Protocols in Molecular Biology). One example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul etal., J Mai. Biol. 215:403 (1990). Software for performing BLAST analyses is publicly available through the National Center forBiotechnology Information (publicly accessible through the National Institutes of Health NCBI internet server). Typically, default program parameters can be used to perform the sequence comparison, although customized parameters can also be used. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad Sci. USA 89: 10915 (1989)). As will be appreciated by those of skill in the art, in some aspects, the nucleic acid further comprises a plasmid sequence. The plasmid sequence can include, for example, one or more sequences of a promoter sequence, a selection marker sequence, or a locus-targeting sequence.

[0178] The term "promoter" or "promoter element" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0179] The term “ retroviral vector" can refer to a gammaretroviral vector. A retroviral vector may include, e.g., a promoter, a packaging signal, a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTR), and polynucleotides of interest, e.g., a polynucleotide encoding a CAR and a polynucleotide encoding aPEBL. A retroviral vector may lack viral structural genes such as gag, pol, and env. Exemplary retroviral (e.g., gammaretroviral) vectors include Murine Embryonic Stem Cell Virus (MESV), Murine Stem Cell Virus (MSCV), Murine Leukemia Virus (MLV), Spleen- Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Virus (MPSV), and vectors derived therefrom. Other gammaretroviral vectors are described, e.g., in Maetzig et al., Viruses, 2011; 3(6): 677-713.

[0180] The term "bicistronic expression" is typically achieved by operably linking the polynucleotides described herein to a promoter, and incorporating the bicistronic construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes.

[0181] Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0182] "Expression vector" refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.

[0183] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0184] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. Exemplary promoters include the immediate early cytomegalovirus (CMV), EF- la, ubiquitin C, or phosphoglycerokinase (PGK) promoters. A strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto can be used. Other constitutive promoter sequences may be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, theelongation factor-I Ovian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, and the like. In some embodiments, the promoter is an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0185] As used herein, "antibody" means an intact antibody or antigen-binding fragment of an antibody, including an intact antibody or antigen-binding fragment modified or engineered, or that is a human antibody. Examples of antibodies modified or engineered are chimeric antibodies, humanized antibodies, multiparatopic antibodies (e.g., biparatopic antibodies), and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, single chain antibodies (e.g, scFv), minibodies and diabodies.

[0186] The term "specifically (or selectively) binds" or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g, Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0187] In certain embodiments, the antibody that binds CD7 is a single-chain variable fragment antibody ("scFv antibody"). scFv refers to antibody fragments comprising theVH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun (1994) The Pharmacology Of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer- Verlag, New York, pp. 269-315. See also, PCT Publication No. WO 88 / 01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203. As would be appreciated by those of skill in the art, various suitable linkers can be designed and tested for optimal function, as provided in the art, and as disclosed herein.

[0188] As used herein, an "engineered" immune cell includes an immune cell that has been genetically modified as compared to a naturally-occurring immune cell. For example, an engineered T cell produced according to the present methods carries a nucleic acid comprising a nucleotide sequence that does not naturally occur in a T cell from which it was derived, such as the nucleic acids exemplified herein. In some embodiments, the engineered immune cell of the present invention includes a PEBL and a chimeric antigen receptor (CAR). Non-limiting examples of an illustrative CAR include a CAR that binds CD3, CD4, CD5, CD7, CD8, or other tumor associated antigens.

[0189] As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0190] As used herein, a "CD7 CAR+ / CD7-negative" T cell refers to a T cell expressing a chimeric antigen receptor against human CD7 and having low or no surface expression of endogenous CD7. In some embodiments, the low or no surface expression of endogenous CD7 is due to expression of a PEBL against human CD7 which reduces endogenous CD7 protein delivery to the surface of the T cell. In some instances, surface expression of CD7 can be determined using standard methods known to those in the art such as but not limited to immunocytochemistry, flow cytometry, or fluorescence- activated cell sorting (FACS).

[0191] The term "autologous" and its grammatical equivalents as used herein canrefer to as originating from the same being. For example, a sample (e.g., cells) can be removed, processed, and given back to the same subject (e.g., patient) at a later time. An autologous process is distinguished from an allogeneic process where the donor and the recipient are different subjects.

[0192] As used herein, the terms "treat," "treating," or "treatment," refer to counteracting a medical condition (e.g., a condition related to a T cell malignancy) to the extent that the medical condition is improved according to a clinically-acceptable standard.

[0193] As used herein, "subject" refers to a mammal (e.g., human, non-human primate, cow, sheep, goat, horse, dog, cat, rabbit, guinea pig, rat, mouse). In certain embodiments, the subject is a human. A "subject in need thereof refers to a subject (e.g., patient) who has, or is at risk for developing, a disease or condition that can be treated (e.g., improved, ameliorated, prevented) by inducing T cells to exert specific cytotoxicity against malignant T cells.

[0194] As defined herein, a "therapeutic amount," or “therapeutically effective amount” refers to an amount that, when administered to a subject, is sufficient to achieve a desired therapeutic effect (treats a condition related to a T cell malignancy) in the subject under the conditions of administration. An effective amount of the agent to be administered can be determined by a clinician of ordinary skill using the guidance provided herein and other methods known in the art, and is dependent on several factors including, for example, the particular agent chosen, the subject's age, sensitivity, tolerance to drugs and overall well-being.EXAMPLES

[0195] The following examples are illustrative of the embodiments described herein and are not to be interpreted as limiting the scope of this disclosure. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to be limiting. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of this disclosure.Example 1

[0196] This example shows a method for obtaining cellular reagents (e.g., starting cells) for use in methods and systems described herein.

[0197] An enriched apheresis product (e.g., a leukopak) comprising white blood cells extracted from peripheral blood from a healthy human subject was obtained. Healthy donorfull leukopaks (LP) were purchased from BioIVT with request to be delivered within 24 hours of collection at cold shipment temperatures (below 15 °C). The range of apheresis volumes collected varied between 116 - 279 mL. LPs were processed once delivered on site, within 24 hours of collection (only one instance resulted in delivery longer than 48 hours from collection due to shipper issues; the blood was not processed). Complete blood count (CBC) with differential was performed at the collection site. Eleven unique donors were used for the 10 PD runs and 2 confirmatory runs. Total white blood cell count and viability of the enriched apheresis product was determined using NC-200 Solution 17 blood lysis buffer (ChemoMetec) and a NucleoCounter® NC-200 analyzer (ChemoMetec).

[0198] Eight liters of TexMACS culture medium was prepared and supplemented with interleukin-2 (IL-2) for culture of cellular reagents. Phenol red-free, GMP grade TexMACS media (Miltenyi Biotec) and GMP grade IL-2 (Miltenyi Biotec) were used in all studies.Example 2

[0199] This example shows selection (e.g., enrichment) of cellular reagents for CD4 and CD8 expression, for example, as used in methods and systems described herein.

[0200] Enrichment of the apheresis product for CD4-positive and CD8-positive cells was accomplished with three cycles using a CliniMACS Prodigy® system to produce a CD4 / CD8 enriched cell population. Alternatively, a different cell selection technique, such as fluorescence-activated cell sorting (FACS), can be used to enrich or isolate cells expressing CD4 and / or CD8, for example; cell yield and / or cell viability may be affected if such an alternative technique is used.

[0201] GMP grade CliniMACS Prodigy® isolation reagents (CD4 microbeads, CD8 microbeads, and PBS / EDTA buffer) were used in all studies (Miltenyi Biotec). GMP grade TransAct™ (Miltenyi Biotec) was added. USP grade human serum albumin (HSA, Nova Biologies) was only used in the process to supplement PBS / EDTA buffer at a final concentration of 0.5% (v / v) and used in all studies. The TS 520 tubing set (Miltenyi Biotec) was used for all isolations.

[0202] For all studies, CD4 and CD8 T cells were selected using the CliniMACS Prodigy® “TCT Full Process” program (Miltenyi Biotec). The LP was loaded onto the CliniMACS Prodigy® and “TCT Full Process” program was selected using the two-vial option. The WBC density from the collection site CO A or XN-330 Sysmex reading was used along with the volume provided by the collection site for the total WBC input into the system. Since the actual T cell frequency was unknown at the time of LP receipt, T cellfrequency was input by setting the process to the theoretical maximum T cell enrichment capacity of the CliniMACS Prodigy® (3E9) divided by the total WBC count. Using this method, the maximum selection cycles were performed (three) and took approximately 3 hours. At the end of selection, either the culture was initiated in the CliniMACS Prodigy® CentriCult chamber (IPR1, IPR2, Run 1) or the process was ended and the enriched cells bag was heat sealed off and removed from the instrument for further processing.

[0203] For all runs, a portion of day 0 isolated cells were seeded in a 6 well plate to be used as a nontransduced or negative control for flow analysis.Example 3

[0204] This example shows activation of cellular reagents, for example, as used in methods and systems described herein.

[0205] The CD4 / CD8 enriched cell population was inoculated into two 100M-CS G-Rex vessels (Wilson Wolf) in 100 mL of culture medium with T Cell TransAct™, human stimulation reagent (Miltenyi Biotec).

[0206] The CD4 / CD8 enriched cell population was inoculated into two 100M-CS G-Rex vessels in 100 mL of culture medium with of T Cell TransAct™, human stimulation reagent (Miltenyi Biotec). GMP grade CliniMACS Prodigy® isolation reagents (CD4 microbeads, CD8 microbeads, and PBS / EDTA buffer) were used in all studies (Miltenyi Biotec).

[0207] GMP grade TransAct™ (Miltenyi Biotec) was added unless stated otherwise. USP grade human serum albumin (HSA, Nova Biologies) was only used in the process to supplement PBS / EDTA buffer at a final concentration of 0.5% (v / v) and used in all studies. The TS 520 tubing set (Miltenyi Biotec) was used for all isolations. TransAct™ reagent was removed from the culture system by dilution.Example 4

[0208] This example shows the use of a bicistronic expression vector in T-cells to express an anti-CD7 CAR and reduce CD7 expression. Cells were transduced with an MSCV promoter-anti-human CD7 (TH69) CAR-P2A-anti-human CD7 (TH69) PEBL (see, e.g., FIG. 3) lentivirus. Research grade lentiviral vector (LW) was stored at -80 °C until use.Two different lots were used during the studies. Lot X was used in IPR1 and supplied at 2.15 E8 infectious s particles (IP) / mL. Lot Y was used in IPR2. Lot Y was supplied at 1.53 E8 infectious particles (IP) / mL. Transduction generated a population of CD7 CAR + / CD7-neg T-cells. FIG. 6 shows expansion and enrichment of the CD7 CAR+ / CD7-neg T-cells overtime from day 0 to day 9 post-transduction. For example, at day 0, 10.9% of the cells were CD7 CAR-neg / CD7-neg cells, 0.016% were CD7 CAR+ / CD7-neg cells, 87.9% were CD7 CAR-neg / CD7+ cells, and 1.21% were CD7 CAR+ / CD7+ cells. At day 3, 24.1% of the cells were CD7 CAR-neg / CD7-neg cells, 17.7% were CD7 CAR+ / CD7-neg cells, 53.8% were CD7 CAR-neg / CD7+ cells, and 4.33% were CD7 CAR+ / CD7+ cells. At day 6, 27.5% of the cells were CD7 CAR-neg / CD7- cells, 63.7% were CD7 CAR+ / CD7-neg cells, 6.25% were CD7 CAR-neg / CD7+ cells, and 2.57% were CD7 CAR+ / CD7+ cells. At day 9, 16.1% of the cells were CD7 CAR-neg / CD7- neg cells, 83.7% were CD7 CAR+ / CD7- cells, 0.012% were CD7 CAR-neg / CD7+ cells, and 0.095% were CD7 CAR+ / CD7+ cells. The CAR+CD7+ cells observed on day 3 are highly susceptible to fratricide. The CAR-CD7+ cells observed on day 3 are also highly susceptible to fratricide.

[0209] This example demonstrates the generation and expansion of PEBL-CAR-T-cells produced using a CD7 CAR-P2A-CD7 PEBL bicistronic lentiviral vector. Such cells showed antigen-specific T-cell functional responses such as fFNy secretion and specific toxicity against CD7+ target cell lines. The PEBL-CAR-T-cells exhibited high percentage purity of CD7 negative, CAR+ T-cells.Example 5

[0210] This example shows cryopreservation of engineered cells, for example, as can be used in optimization of methods and systems described herein.

[0211] Extra isolated cells on day 0 were removed from the post-enrichment bag, centrifuged at 300 g for 10 minutes, and resuspended in a cry opreservation medium comprising 10% dimethyl sulfoxide (CryoStor® CS10 cryopreservation medium (BioLife Solutions)) to reach a cry opreservation density of 50 E6 cells / mL or 100 E6 cells / mL and cryopreserved in 1.8 mL cryovials at 1 mL / vial in a freezing container (CoolCell® freezing container (Coming®)) stored at -80 °C. After a minimum of one day, cells were transferred to liquid nitrogen storage.

[0212] When frozen (e.g., cryopreserved) cells were to be used, the freezing container was removed from liquid nitrogen storage and placed in a 37 °C water bath for 2-4 minutes. An appropriate volume cell mixture was removed and transferred to a culture tube and diluted in TexMACS medium at a ratio of 1 :3 (e.g., for 1.0 E6 cells / mL) or 1 :20 (for 4.07 E7 cells / mL). Optionally, thawed cells were analyzed for cell count, cell viability, and / or surface marker expression (e.g., by flow cytometry). Viability on day 0 post-thaw was greater than or equal to 85.0% (e.g., 85.0% to 90.0%, 85.0% to 95.0%, or 85.0% to 100.0%, for instance,85.5%). Viability on day 6 post-thaw was greater than or equal to 89.0% (e.g., 89.0% to 90.0%, for instance, 89.8%), and in some cases, viability on day 6 post-thaw was greater than or equal to 90.0% (e.g., 90.0% to 91.0%, 90.0% to 95.0%, or 90.0% to 100.0%, for instance, 90.1%).Example 6

[0213] This example shows evaluation of the CliniMACS Prodigy® system for manufacturing T cells transduced with an anti-CD7 CAR - anti-CD7 PEBL bicistronic vector.

[0214] Three runs (IPR1, IPR2, and Run 1) were performed to establish the baseline process using the CliniMACS Prodigy® (Table 2). Depending on the run, cells were seeded in 70 mL of medium after selection. Cells were activated on day 0 using TransAct™ added to the CentriCult chamber by using a syringe to remove the volume required and transferring to a transfer pack (IPR1 and IPR2) or attaching the entire TransAct™ vial to the appropriate line (Run 1).

[0215] In IPR1, the chamber temperature was initially set to 37° C for the first six days. A correction was made afterwards to set the chamber temperature to 39° C for an internal culture temperature of 37° C. The chamber temperature was set to 39° C for IPR2 and Run 1 at the start of the run. All three runs were set to 5% CO2 in the chamber. Satellite cultures using cells removed from the TCT process at different timepoints were grown in 6 well plates in a 37 °C, 5% CO2 incubator alongside each of the CliniMACS Prodigy® runs. Medium was prepared fresh every 2-3 days, as needed, for the runs. IL-2 for the process was reconstituted on day 0 and stored at 4 °C for up to two weeks and added to medium when prepared.

[0216] On day 2, a 50% medium exchange was performed by programming in a volume reduction to 35 mL followed by addition of 35 mL of fresh medium. A sample was taken for cell count and viability (CCV) analysis and lentiviral vector (LW) volume was determined using an MOI of 10. The appropriate volume of LVV was transferred to a 150 mL transfer pack and diluted further in fresh medium to bring the total volume to 10 mL. The transfer pack was welded onto the LVV addition line of the tubing set. The Prodigy® was programmed to perform LW transduction which introduced the contents of the transfer pack into the CentriCult unit. After LVV addition, the transfer pack and tubing were rinsed with 20 mL of fresh medium to bring the total culture volume to 100 mL.

[0217] On day 4, Culture Wash (Cycles: 1) was performed to remove the lentiviral vector and bring the total culture volume to 200 mL. Depending on the run, the shaker was activatedat different time points: in IPR1, the shaker was activated on day 12; in IPR2, the shaker was activated on day 5; in Run 1, the shaker was activated on day 8 if percent of CD7-negative cells was 85% or more. Medium exchanges were also performed at different timepoints and frequencies depending on the run: in IPR1, medium exchanges occurred on days 8, 10, 11, and 13; in IPR2, medium exchanges occurred on days 8 and 10; in Run 1, medium exchanges occurred on days 6, 8, 10, and 12. Samples for cell count and viability were taken on medium exchange days. Samples for flow analysis were taken post-transduction. For Run 1, samples were also taken from the culture on medium exchange days for metabolite analysis.

[0218] At harvest day, samples were taken for cell count and viability and flow analysis. The remaining cells were removed from the CentriCult chamber and pumped out into a target cells bag. The contents were transferred to a conical tube within a BSC, centrifuged at 300 g for 10 minutes, and cryopreserved in CS10 at 10 E6 cells / mL in 1.8 mL cryovials. If enough cells were available at harvest, cells were cryopreserved in the Thermo Scientific CRF.

[0219] Three runs were performed to establish a fully closed and automated baseline process to generate CD7 PCART cells using the CliniMACS Prodigy®.

[0220] Starting cell number, TransAct™ volume, chamber temperature, shaker settings, activation timepoint, feeding schedule and culture duration were varied amongst the three runs to achieve the target criteria (refer to Methods section for description of activity matrices for each run). However, none of the runs met the full set of target criteria.

[0221] In IPR1, cells were seeded and activated in the Prodigy® CentriCult chamber in 70 mL total volume. On day 2, there were 6.66 E7 total cells which were transduced. On day 5, the transduction efficiency of the Prodigy® culture was 25.2% CD3+CD56-CAR+CD7- and 40.5% CD3+CD56-CD7-, meeting day 5 target marker expression criteria. However, the total viable cell count of 7.00 E7 did not meet the cell expansion criteria. On day 6, it was noted that the chamber temperature had been set at 37 °C, when Miltenyi instructions were 39 °C due to a 2°C temperature offset, therefore the chamber temperature was adjusted from 37 °C to 39 °C, to follow vendor recommendation. Samples for CCV were taken from day 5 onwards and medium exchanges performed to maintain a minimum cell density of 5.0 E5 cells / mL until the culture volume reached 250 mL. On days 5 and 7, the volume was adjusted to obtain a density of 5.0 E5 cells / mL. From days 8 onward, the expansion of cells resulted in greater than 5.0 E5 cells / mL at the maximum 250 mL. Thus, 70% medium exchanges were performed on days 10, 11, and 13. By day 10, the % CD3+CD56-CAR+CD7-and % CD3+CD56-CD7- had increased to 67.5% and 97.3%, respectively suggesting that fratricide was ongoing. Since the cell expansion appeared to plateau from days 10-12, the shaker (Type1 setting) was activated to improve aeration of the culture for greater cell expansion. However, this only improved expansion by 1.3-fold on day 13 and it was hypothesized that the culture duration had been too long to observe any benefit from the shaker activation. Nonetheless, the total viable cells on day 14 for IPR1 was 3.30 E8, 86.1% viable, 42.4% CD3+CD56-CAR+CD7-, and 98.8% CD3+CD56-CD7- which did not meet the set target criteria, especially failing the cell expansion target.

[0222] In order to improve upon the results of IPR1, IPR2 was performed similarly from days 0 to 4 but the shaker was activated (Type 1 setting) on day 5 to provide sufficient aeration for early cell expansion. A portion of the Prodigy® culture (less than 4 mL) was removed during sampling and cultured in a 6 well plate as a static control arm on day 5. The day 2 total viable cell count of 8.05 E7 met the target criteria. The day 5 transduction efficiency was 38.3% CD3+CD56-CAR+CD7— and 45.7% CD3+CD56-CD7- but the total viable cell count of 1.34 E8 was lower than the target criteria. It was hypothesized that lower cell count was due to cells undergoing fratricide. By day 8, the total cell count had decreased by approximately 14% and the viability had also decreased by 10%. To maintain the minimum density of 5.0 E5 cells / mL, the volume was reduced to 236 mL. By day 9, however, the cell count decreased more than 50% from day 8 and the viability was also decreasing. It was hypothesized that the early activation of the shaker while cells were undergoing fratricide was detrimental to the health of the cells and causing the decrease. The shaker was deactivated on day 9. By day 11, the total cell count decreased further and the viability was 49%. Although the viability and cell count were low, the CD3+CD56- CAR+CD7- was 77.4% and CD3+CD56-CD7- was 87% which met the target criteria for CAR and CD7 expression. The bench-scale static control arm expanded 1.85-fold by day 11 from day 5 and viability was 78.8% which further suggested that activation of the shaker resulted in cell death.

[0223] Based on IPR1 and IPR2, adjustments were made for Run 1. To meet the final cell yield, the target starting cell number was increased to 2.0 E8. Following the standard Miltenyi TCT process, lentiviral vector removal was maintained on day 4 using the Culture Wash step and the subsequent medium exchanges were performed on days 6, 8, 10, and 12. Lastly, the shaker would be activated on day 8 based on the expression of CD3+CD56-CD7- being > 85%. This was based on the hypothesis that the cells are shear sensitive when the culture is undergoing fratricide. Also, the culture volume was limited to 200 mL while static to enable faster oxygen diffusion throughout the culture to improve expansion. With these changes the total cell yield was met by day 6; however, the transduction efficiency was thelowest observed from all the runs (4.2% CD3+CD56-CAR+CD7- and 9.3% CD3+CD56- CD7-). By day 8, total cell yield continued to increase but flow analysis revealed a decrease in CD3+CD56-CAR+CD7- to 1.4% suggesting that the nontransduced cells were expanding and fratricide was not taking place. Since the day 8 CD3+CD56-CD7- frequency did not meet the criteria, the shaker was not activated. The culture continued to expand to 7.25 E8 total cells on day 14 but by harvest the CD3+CD56-CAR+CD7-frequency was below 1%. Thus, Run 1 did not meet the target criteria for % CD3+CD56-CAR+CD7-, % CD3+CD56-CD7-, and CD3+CD56-CAR+CD7- yield.Table 1: Summary of IPR1, IPR2, and Run 1 viability and surface marker countsTable 2: Experimental conditions used in IPR1Table 3: Experimental conditions used in IPR2Table 4: Experimental conditions used in Run 1

[0224] Starting cell number, chamber temperature, shaker settings, activation timepoint, feeding schedule, and culture duration were varied amongst the three runs to achieve the target criteria set in Table 5. However, none of the runs met the full set of target criteria in Table 5, as shown in FIGs. 7A-7D.Table 5: Runs IPR1, IPR2, and Run 1

[0225] Target criteria for IPR1, IPR2, and Run 1 were: Total Viable Cells: 4.00 E8; Viability > 50%; Percent of CD3+CD56‘ cells > 90%; Percent of CD3+CD56 CD7' cells > 90%; Percent of CD3+CD56'CAR+CD7- cells > 60%.

[0226] The total viable cell count of 7.00 E7 did not meet the cell expansion criteria. Samples for cell count and viability were taken from day 5 onwards and medium exchanges performed to maintain a minimum cell density of 5.0 E5 cells / mL until the culture volume reached 250 mL. On days 5 and 7, the volume was adjusted to obtain a density of 5.0 E5 cells / mL. From days 8 onward, the expansion of cells resulted in greater than 5.0 E5 cells / mL at the maximum 250 mL. Thus, 70% medium exchanges were performed on days 10, 11, and 13. Cell expansion appeared to plateau from days 10-12, the shaker (Type 1 setting) was activated to improve aeration of the culture for greater cell expansion. However, this only improved expansion by 1.3-fold on day 13 and it was hypothesized that the culture duration had been too long to observe any benefit from the shaker activation. The total viable cells on day 14 for IPR1 was 3.30 E8, 86.1% viable, 42.4% CD3+CD56 CAR+CD7; and 98.8% CD3+CD56'CD7‘ which did not meet the set target criteria, especially failing the cell expansion target.

[0227] Overall, only IPR1 met close to the target criteria for total viable cells, viability, % CD3+CD56'CAR+CD7‘ cells, and % CD3+CD56'CD7‘ cells. Based on the three runs, the two main bottlenecks to achieving the CD3+CD56'CAR+CD7‘ target yield appear to be transduction efficiency and expansion. As observed in Run 1, the transduction efficiency was not high enough to achieve complete fratricide by the end of the culture. When transduction efficiency met the target criteria on day 5 as was observed in IPR1 and IPR2, expansion of the cells by harvest did not meet the target criteria. Activating the shaker early on in culture to improve expansion while cells were still undergoing fratricide was detrimental. The feeding strategies evaluated also did not improve expansion. The process could be improved to achieve a threshold under which the population of transduced cells could overtake the nontransduced cells during fratricide.Example 7

[0228] This example shows an evaluation of a cell culture system with a gas permeable membrane for manufacturing T cells transduced with an anti-CD7 CAR / CD7 PEBL bicistronic vector.

[0229] In Run 2, T-cells enriched by magnetically activated cell sorting were seeded into four mid-sized vessels with gas-permeable membranes (G-Rex 10M vessels; Wilson Wolf) and two large vessels with gas permeable membranes (G-Rex 100M vessels; Wilson Wolf). Cells were divided into “arms” by process to evaluate two feeding strategies: a top up feeding strategy (Arm Gl) and 75% medium exchanges performed on days 8 and 11 (Arm G3). Arms G1-G5 of Run 2 employed a culture vessel with a gas-permeable membrane for incubating cells, as opposed to Run 1, which did not use a vessel with a gas permeable membrane for culture. An arm was also set up (Arm G2) such that no sampling or resuspension of the culture occurred from day 4 onward until harvest, according to manufacturer’ s recommendations. Arms G4 and G5 employed larger culture medium volumes (G-Rex 100M scale versions, with a culture medium volume capacity of 1000 mL) of Arms Gl and G3 (which each used G-Rex 10M scale culture vessels, with a capacity of 100 mL of culture medium), respectively, where the volume of medium, number of cells, and volume of TransAct™ were multiplied by a factor of 10. Cells were activated on day 0 using TransAct™. On day 2, a 50% medium exchange was performed. For all arms, cells were transduced at an MOI of 10. The transduction volume was 10 mL in the 10M vessels and 100 mL in the 100M vessels. On day 4, a 75% medium exchange was performed for lentiviral vector removal by transferring the cultures to conical tubes, centrifugation of the cells, 75% removal of the supernatant, and top up with fresh medium to either 20 mL in the 10M vessels or 200 mL in the 100M vessels. On day 5, culture volumes were topped up to 50 mL in the 10M vessels or 500 mL in the 100M vessels. On day 8, depending on the arm, either a 75% medium exchange or a medium top up was performed. On day 11, depending on the arm, either IL-2 was added as a bolus or a 75% medium exchange was performed. IL-2 was added as a bolus to minimize disturbance to the cells for optimal expansion. On day 14, all cultures were ended. Samples were taken on days 2, 4, 5, 8, 11, and 14 for cell count and viability and metabolites for all arms except Arm G2, which was the minimal disturbance arm and only sampled on days 2, 4, and 14. Samples were taken on days 5, 8, 11, and 14 for flow cytometry for all arms except Arm G2, which was only sampled at harvest.Results

[0230] Due to the challenges with developing a fully closed and automated process with the Prodigy® system, gas permeable membrane vessels were evaluated as an alternative culture system and compared to the Prodigy® and bench-scale processes. The surface area of the 10M was approximated to be 1 / 1 Oth that of the Prodigy® chamber (100 cm2). Cells were transduced on day 2 and LVV was removed on day 4. A top up feeding strategy with minimal disturbance was employed. Care was taken to minimize the disturbance of the cells at the membrane where diffusion of oxygen takes place. On day 2, the G-Rex fold change relative to day 0 was 0.96 which was higher than the bench-scale and Prodigy® arms which were 0.70 and 0.53, respectively. On day 5, the transduction efficiencies of arms Gl, G3, G4, and G5 were similar and were within 7.4 - 9.3% CD3+CD56'CAR+CD7' and 11.0 - 15.0% CD3+CD56'CD7‘.

[0231] The percentage of CD3+CD56‘ CD7" T cells with reduced CD7 expression increased over time, reaching 100% by day 14 for all arms. FIG. 8C. The loss of CD7+ cells suggests that the anti-CD7 CAR T cells were killing the CD7+ cells (fratricide). The percentage of CD3+CD56'CAR+CD7‘ cells expressing the anti-CD7 CAR increased to 80%- 90% in some arms, but then decreased in those populations with the highest expression. FIG. 8D

[0232] As seen in FIG. 8C, the percentage of cells with CD3+CD56‘ CD7" and CD3+CD56'CAR+CD7‘ surface marker profiles were at or about 100% and 85%, respectively for Arms 1 at day 8, while all other Run 2 arms were below 60% for the same surface marker expression profiles at day 8. The bench-scale control process arm (Run 2 Arm 1) had the greatest CD3+CD56 CAR+CD7' (FIG. 8D) and CD3+CD56 CD7' (FIG. 8C) frequency at this timepoint, suggesting that the transduction efficiency in the G-Rex vessel could still be further optimized. For G-Rex arms Gl, G3, G4, and G5, the CD3+CD56'CAR+CD7‘ and CD7" frequencies continued to increase until day 11. As shown in FIG. 8D, Arms G4 and G5 also continued to increase in CD3+CD56'CAR+CD7‘ frequency until day 14. For all arms, a decrease in viability was observed from days 5 - 8, likely representative of fratricide occurring. The viabilities rebounded after day 11 until harvest. FIG. 8B.Table 6: Run 1 and Run 2 (Arms G1-G5) results summary* Fold change compared to Day 0

[0233] The 75% medium exchange arms (G3, G5) had up to 10-fold greater expansion compared to the medium top up arms (Gl, G4). The CD3+CD56'CAR+CD7‘ frequencies were similar at 10M scale between the two feed strategies but at 100M scale, the medium exchange arm had approximately 30% greater CD3+CD56'CAR+CD7‘ cells than the top up arm, suggesting at 100M scale medium exchange was better for both expansion and fratricide. The minimal disturbance arm (G2) had the lowest yield and second lowest % CD3+CD56‘ CAR+CD7'on harvest day suggesting that mixing the cells during culture assists with fratricide and subsequent expansion.Table 7: Cell manufacturing runs evaluating minimal disturbance vs media exchange in vesselwith gas-permeable membrane

[0234] As shown in FIGs. 9A-9D, the metabolite profile and pH were quite similar between G-Rex arms. The medium exchange arms (Arm G3 and G5) had lower levels of lactate and ammonium due to the removal of spent medium and replacement with fresh medium. The accumulation of lactate and ammonium and decrease in pH from days 11 to 14 suggest that an additional medium exchange past day 11 is required.

[0235] The two 100M scale arms were able to establish a baseline process in a G-Rex culture system amenable to closed system manufacturing.Example 8

[0236] This example describes an example of a manufacturing process using a vessel with a gas-permeable membrane.

[0237] The manufacturing process shown in FIG. 1 comprises magnetic isolation of CD4+ and CD8+ T-cells from fresh non-mobilized apheresis units, activation and culture of isolated T-cells in a vessel with gas-permeable membranes, lentiviral vector transduction, cell expansion with medium top up, and then medium exchanges.

[0238] Day 0 involved receiving leukopaks, conducting NC-200 Solution 17 count for total viable white blood cell count, preparing 8L of TexMACS media supplemented with IL-2 for the entire process, enriching CD4 and CD8 T-cells using 3 cycles of Prodigy®, conducting cell counts and viability testing for G-Rex inoculation, and cell activation.

[0239] Day 3 involved harvesting and pooling vessels for volume reduction to 50 mL using Sepax, performing cell count and viability measurement post-Sepax, adding lentiviralvector at MOI 10 based on post-Sepax cell count, re-inoculating cells back into the two G- Rex vessels, rinsing transfer pack with 30 mL of medium, rinsing tubing of each G-Rex vessel with 15 mL of medium from the transfer pack rinse, resulting in approximately 40 mL / vessel after re-inoculation.

[0240] On day 4, approximately 960 mL of fresh medium was added per vessel for a total of 1 L for lentiviral vector dilution.

[0241] Day 6 involved reducing culture volume to 250 mL, swirling the culture vessel to redistribute cells, withdrawing sample for cell count and viability measurement and flow assays, and adding 750 mL of fresh medium for 75% medium exchange for a total of 1 L.

[0242] Day 9 involved reducing culture volume to 250 mL, swirling the culture vessel to redistribute cells, withdrawing sample for cell count and viability testing and for a flow assay, and adding 750 mL of fresh medium for 75% medium exchange for a total of 1 L.

[0243] Day 11 involved reducing culture volume to 100-130 mL, swirling vessel to resuspend cells, withdrawing sample for cell count and viability measurement and flow assays, pooling cultures to perform concentration, wash, and formulation in Plasma-Lyte A to greater than 2X cryodensity using Sepax, filtering cells through 40 pm filter after Sepax, adjusting volume to meet 2X cryodensity, adding equivalent volume of CS10 to achieve IX cryodensity, filling cryopreservation bags and vials with cryopreservation mixture comprising cells, and cryopreserving cells using Planer CRF cryopreservation container.

[0244] This example shows a robust, closed system 12-day process for the manufacturing of fratricidal CAR T-cells. This process is amenable to clinical CAR-T manufacturing where: fresh or frozen apheresis starting material was processed to isolate CD4 and CD8 T-cells in a fully automated, closed system instrument and transduction conditions for a day 3 transduction process were optimized. Scale up to 100M G-Rex vessels was performed while meeting target criteria for yield and CD3+CD56'CAR+CD7‘ frequencies. The process shown here streamlines the manufacturing process and reduces suite time which included removal of TransAct™ on day 3 with transduction instead of day 2, dilution of lentiviral vector on day 4 with a medium top up instead of a medium exchange, minimal medium exchange timepoints, processing two vessels together on transduction and harvest instead of individually, and preparation of process medium and aliquoting on day 0 instead of preparing medium fresh throughout the process.Example 9

[0245] This example shows administration of engineered cells described herein to apatient in need of treatment, in accordance with methods and systems described herein.

[0246] A population of cells comprising engineered cells expressing a chimeric antigen receptor (CAR) and, optionally, a protein expression blocker (PEBL) produced according to methods described herein are prepared from freshly prepared or cryopreserved samples in accordance with methods described herein, for example using methods and reagents described in one or more of Examples 1-8. Cells are prepared as a pharmaceutically acceptable composition and administered to the patient in need of treatment in a therapeutically effective dose.

[0247] In this example, cryopreserved engineered human T-cells expressing a CD7 chimeric antigen receptor (CD7 CAR) and a CD7 protein expression blocker (CD7 PEBL), manufactured as described herein, are thawed and diluted in culture medium as described in Example 8 and resuspended in saline solution. Cells are administered via intravenous infusion to a subject having acute lymphocytic leukemia (ALL) cancer in an amount from 1 x 10A6 cells / kg to 10 x 10A6 cells / kg. Alternatively, engineered cells can be administered in an amount of less than 1 x 10A6 cells / kg or greater than 10 x 10A6 cells / kg.

[0248] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.-n -

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A cell population comprising: at least 108immune cells of which: at least 70% are viable and at least 50% express a chimeric antigen receptor (CAR) comprising a binding domain that binds to a cell surface polypeptide expressed by the immune cells linked to an activation domain.

2. The cell population of claim 1, wherein at least 80% of the at least 108immune cells are viable.

3. The cell population of claim 1 or 2, wherein at least 60% of the at least 108immune cells express the CAR.

4. The cell population of any one of claims 1-3, wherein at least 50% of the immune cells have reduced expression of the cell surface polypeptide.

5. The cell population of any one of claims 1-4, wherein the cell surface polypeptide is CD2, CD3, CD5, CD7, CD8, or CD38.

6. The cell population of claim 2 or 5, wherein the immune cells having reduced expression of the cell surface polypeptide comprise a chimeric targeting polypeptide that comprises a binding domain that binds to the cell surface polypeptide linked to an intracellular localizing domain.

7. The cell population of claim 6, wherein the intracellular localizing domain is an ER retention signal, a Golgi retention signal or a PEST sequence.

8. The cell population of claim 7, wherein the ER retention signal comprises a KKXX sequence, wherein X is any amino acid.

9. The cell population of claim 7, wherein the ER retention signal comprises a KDEL sequence.The cell population of claim 9, wherein the chimeric targeting polypeptide further comprises a spacer sequence between the binding domain and the KDEL sequence. The cell population of any one of claims 1-10, wherein the immune cells are T-cells. The cell population of any one of claims 1-10, wherein the immune cells are NK cells. The cell population of any one of claims 1-12, wherein the immune cells are frozen. A pharmaceutical formulation comprising the cell population of any one of claims 1-13. A method comprising: incubating cells susceptible to fratricide in a culture medium without continuous agitation in a vessel comprising a gas-permeable membrane, wherein at least 70% of the cells are viable after 5 days of incubation. The method of claim 15, wherein the cells comprise a chimeric antigen receptor (CAR) comprising:(i) a binding domain that binds to a cell surface polypeptide expressed by the cells, and(ii) an activation domain that activates the cells. The method of claim 16, wherein the cells have reduced expression of the cell surface polypeptide compared to healthy human cells of a cell type that is the same cell type as the cells. The method of claim 17, wherein the cells comprise a non-natural modification of a gene encoding the polypeptide. The method of claim 17, wherein the cells further comprise a chimeric targeting polypeptide that comprises a domain that binds to the cell surface polypeptide linked to an intracellular localizing domain.The method of any one of claims 15-19, wherein the cells susceptible to fratricide comprise immune cells. The method of claim 20, wherein the immune cells comprise T-cells. The method of claim 20, wherein the immune cells comprise NK cells. The method of any one of claims 16-22, wherein the activation domain induces cytotoxic activity of the immune cells. The method of any one of claims 16-23, wherein the cell surface polypeptide is CD7. The method of any one of claims 15-24, further comprising replacing at least a portion of the culture medium in the vessel. The method of claim 25, wherein the replacing is executed every 1 to 3 days. The method of any one of claims 25-26, wherein the replacing comprises replacing at least 75% of the culture medium in the vessel. The method of any one of claims 15-27, wherein the vessel contains 100 mL to 1 L of the culture medium. The method of any one of claims 15-28, wherein a concentration in the culture medium of a metabolite produced by the cells is maintained below a threshold level for a time interval. The method of claim 29, wherein the metabolite is lactate. The method of claim 30, wherein the threshold level is 14 mM. The method of claim 29, wherein the metabolite is ammonium. The method of claim 32, wherein the threshold level is 1.2 mM.The method of any one of claims 29-33, wherein the time interval is at least 7 days of culture. The method of any one of claims 15-34, further comprising disrupting clumps of the cells. The method of claim 35, wherein the disrupting comprises mechanically disrupting the clumps. The method of any one of claims 35-36, wherein the replacing and the disrupting are executed at regular intervals. The method of any one of claims 35-37, wherein disrupting is performed every 1 to 3 days. The method of any one of claims 15-38, further comprising mixing the culture medium in the vessel. The method of any one of claims 15-39, further comprising activating the cells. The method of claim 40, wherein the activating comprises contacting the cells with an antibody that binds to CD3 or CD28. The method of claim 40, wherein the activating comprises contacting the cells with antibodies that bind to CD3 and CD28. The method of any one of claims 15-42, wherein the cells are transduced cells, and the method further comprises, prior to incubating, transducing starting cells with a viral vector to generate the transduced cells. The method of claim 43, wherein the viral vector comprises a nucleotide sequence encoding the CAR or the chimeric targeting polypeptide.The method of claim 43, wherein the viral vector comprises a nucleotide sequence encoding the CAR and the chimeric targeting polypeptide. The method of any one of claims 43-44, wherein the starting cells were obtained from a human subject. The method of claim 46, wherein the human subject is a healthy human subject. The method of claim 46, wherein the human subject had been diagnosed with a cancer. The method of claim 48, wherein the cancer is a T-cell lymphoma or leukemia. The method of claim 46, wherein the human subject had been diagnosed with an autoimmune disease. The method of any one of claims 43-50, wherein at least 20% of the starting cells are CD4 positive. The method of claim 51, wherein at least 80% of the starting cells are CD4 positive. The method of any one of claims 43-52, wherein the starting cells are enriched for CD4 positive cells. The method of any one of claims 43-50, wherein at least 20% of the starting cells are CD8 positive. The method of claim 54, wherein at least 80% of the starting cells are CD8 positive. The method of any one of claims 43-55, wherein the starting cells are enriched for CD8 positive cells. The method of any one of claims 43-50, wherein at least 20% of the starting cells are CD3 positive. The method of claim 57, wherein at least 80% of the starting cells are CD3 positive.The method of any one of claims 43-58, wherein the starting cells are enriched for CD3 positive cells. The method of any one of claims 43-50, wherein at least 20% of the starting cells are CD56 positive. The method of claim 60, wherein at least 80% of the starting cells are CD56 positive. The method of any one of claims 43-61, wherein the starting cells are enriched for CD56 positive cells. The method of any one of claims 43-61, further comprising selecting the cells susceptible to fratricide from the transduced cells prior to the incubating. The method of claim 63, wherein the selecting comprises isolating cells that express the CAR. The method of claim 63 or claim 64, wherein the selecting comprises isolating cells that express CD4 or CD8. The method of any one of claims 63-65, wherein the selecting comprises isolating cells that express CD25 and CD69 after activation. The method of any one of claims 63-66, wherein at least 80% of the selected cells are positive for surface expression of CD3. The method of any one of claims 63-67, wherein at least 80% of the selected cells have reduced surface expression of the cell surface polypeptide compared to an unmodified cell of the same cell type from a healthy human subject. The method of any one of claims 63-68, wherein at least 80% of the selected cells are negative for surface expression of CD56.The method of any one of claims 63-69, wherein the selecting comprises an affinity -based selection. The method of claim 70, wherein the affinity-based selection comprises fluorescence- activated cell sorting or magnetic-activated cell sorting. The method of any one of claims 15-71, wherein the incubating results in a 30 to 40-fold increase in the quantity of cells. The method of any one of claims 15-72, further comprising harvesting at least 1 E8 of the cells sensitive to fratricide after a time period of incubation. The method of claim 73, further comprising harvesting at least 1 E8 of the cells sensitive to fratricide after no more than 11 days of incubation. The method of any one of claims 72-74, wherein at least 50% of the harvested cells comprise the CAR and have reduced expression of the cell surface polypeptide. The method of any one of claims 72-75, wherein at least 70% of the harvested cells are viable. The method of any one of claims 72-76, wherein at least 70% of harvested cells expressing the CAR are viable. The method of any one of claims 72-77, wherein at least 70% of harvested cells having reduced expression of the cell surface polypeptide are viable. The method of any one of claims 72-78, further comprising freezing the harvested cells. The method of claim 79, wherein at least 70% of the frozen cells are viable. A system comprising: a vessel comprising a gas permeable membrane and a culture medium; and cells susceptible to fratricide contacting a surface of the gas permeable membrane.. The system of claim 81, wherein the cells susceptible to fratricide comprise a chimeric antigen receptor (CAR) comprising:(i) a binding domain that binds to a cell surface polypeptide expressed by the cells susceptible to fratricide, and(ii) an activation domain that activates the portion of the cells susceptible to fratricide. . The system of claim 81 or claim 82, wherein the cells susceptible to fratricide have reduced expression of the cell surface polypeptide compared to cells of the same lineage isolated from a healthy human donor. . The system of claim 83, wherein the cells susceptible to fratricide comprise a non-natural modification of a gene encoding the polypeptide. . The system of claim 83, wherein the cells susceptible to fratricide comprise a chimeric targeting polypeptide comprising a domain that binds to the cell surface polypeptide linked to an intracellular localizing domain. . The system of any one of claims 81-85, wherein the system comprises a closed culture component. . The system of any one of claims 81-86, further comprising a liquid handler configured to exchange the culture medium. . The system of any one of claims 81-87, further comprising a device configured to disrupt clumps of cells. . The system of any one of claims 81-88, further comprising a controller configured to cause the liquid handler to replace at least a portion of the culture medium in the vessel or cause the device to disrupt clumps of cells. . The system of any one of claims 81-89, further comprising a sensor configured to analyze the culture medium.The system of claim 90, wherein the sensor can measure a nutrient, a metabolite, or a pH. The system of claim 91, wherein the metabolite is lactate or ammonium. The system of any one of claims 90-92, wherein the controller is configured to operate the liquid handler based at least in part on a measurement made by the sensor.

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