Methods and systems for producing 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, the production of engineered immune cells susceptible to fratricide that express a chimeric antigen receptor (CAR) molecule capable of binding to a cell surface molecule expressed by other immune cells that also express the same CAR molecule can limit the viability and yield of the engineered immune cells. As described herein, methods and systems that allow for control of parameters such as metabolite concentrations and cell clumping during culture can, in some cases, be used to increase the yield and / or viability in the production of cells susceptible to fratricide. Also provided herein are engineered immune cells, populations of engineered immune cells, and / or pharmaceutical compositions comprising engineered immune cells or populations of engineered immune cells produced using the methods or systems described herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 421,886, filed November 2, 2022, the entire contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] background

[0002] T cells can be engineered to express chimeric antigen receptors (CARs) that can recognize molecules expressed on the surface of cancerous T cells; however, CAR-T cells that target self-antigens tend to kill other CAR-T cells (e.g., fratricide). Although fratricide can be reduced by reducing cell surface expression of the target antigen, some fratricide remains as long as any expression of the target is present. This can reduce the yield, viability, and activity of CAR-T cells. Therefore, there is a need for new and improved methods and systems for producing CAR-T cells that are susceptible to fratricide.

[0003] Incorporation by Reference

[0003] All publications, patents, and patent applications mentioned in this specification, including but not limited to U.S. Patent No. 10,765,699, U.S. Patent No. 10,550,183, and U.S. Patent No. 20210395779A1, are incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Summary of the Invention [Problem to be solved by the invention]

[0004] overview

[0004] Described herein are methods and systems for producing a population of cells that contain a binding domain and a target binding molecule, for example, to increase cell production efficiency. [Means for solving the problem]

[0005] In one aspect, the present disclosure provides a cell population comprising at least 10 8 The present invention provides a cell population comprising immune cells, wherein at least 70% of the immune cells 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 cell linked to an activation domain.

[0006] In some embodiments, at least 10 8 At least 80% of the immune cells are viable.

[0007] In some embodiments, at least 10 8 At least 60% of the immune cells express the CAR.

[0008] In some embodiments, at least 50% of the immune cells have reduced expression of a 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 a cell surface polypeptide comprise a chimeric targeting polypeptide comprising a binding domain that binds to the cell surface polypeptide linked to an intracellular localization domain. In some embodiments, the intracellular localization 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, where 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]

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

[0012]

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

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

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

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

[0016] In some embodiments, the method further includes replacing at least a portion of the culture medium in the container. In some embodiments, the replacing is performed every 1 to 3 days, and the replacing includes replacing at least 75% of the culture medium in the container. In some embodiments, the container contains 100 mL to 1 L of culture medium.

[0017] In some embodiments, the 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 to 14 mM. In some embodiments, the time interval is at least 7 days of culture. In some embodiments, the method further includes disrupting cell clumps, e.g., mechanically disrupting the cell clumps.

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

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

[0020] In some embodiments, the starting cells are 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 cancer, such as T-cell lymphoma or leukemia. In some embodiments, the human subject is 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 are CD56 positive. In some embodiments, the starting cells are enriched for CD56 positive cells.

[0022] In some embodiments, the method further comprises selecting cells susceptible to fratricide from the transduced cells prior to incubating. In some embodiments, selecting comprises isolating cells that express a 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 a cell surface polypeptide compared to unmodified cells 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 affinity-based selection, e.g., fluorescence-activated cell sorting or magnetic-activated cell sorting.

[0024] In some embodiments, the incubation results in a 30-40 fold increase in the amount of cells. In some embodiments, the method further comprises harvesting at least 1×10 cells susceptible to fratricide after the incubation period. In some embodiments, the method results in at least 1×10 cells susceptible to fratricide after 11 days or less of incubation. 8 The method further comprises harvesting the cells. In some embodiments, at least 50% of the harvested cells comprise a CAR and have reduced expression of a cell surface polypeptide. In some embodiments, at least 70% of the harvested cells are viable. In some embodiments, at least 70% of the harvested cells that express a CAR are viable. In some embodiments, at least 70% of the harvested cells that have reduced expression of a cell surface polypeptide are viable.

[0025]

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

[0026]

[0026] In some aspects, the present disclosure provides a system comprising a container having a gas-permeable membrane and a culture medium, and cells susceptible to fratricide in contact with the surface of the gas-permeable membrane.

[0027] In some embodiments, 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 a portion of the cells susceptible to fratricide. In some embodiments, 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, cells susceptible to fratricide comprise a non-natural modification of the gene encoding the polypeptide. In some embodiments, cells susceptible to fratricide comprise a chimeric targeting polypeptide comprising a domain that binds to a cell surface polypeptide linked to a subcellular localization 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 break up cell clumps. 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 to cause the device to break up cell clumps. In some embodiments, the system further comprises a sensor configured to analyze the culture medium, optionally the sensor can measure nutrients, metabolites such as lactate or ammonium, or pH. In some embodiments, the controller is configured to operate the liquid handler based at least in part on measurements made by the sensor.

[0029]

[0029] Although additional aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, only illustrative embodiments of the present disclosure are shown and described herein. As will be understood, the present disclosure is capable of other different embodiments, and its several details can be modified in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description should be regarded as illustrative in nature, and not as limiting.

[0030] BRIEF DESCRIPTION OF THE DRAWINGS 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 referred to herein as "Figure," "Fig.", and "FIGURE"). [Brief explanation of the drawings]

[0031] [Figure 1]

[0031] Figure 1 provides an illustrative schematic of a manufacturing process for producing immune cells susceptible to fratricide as described herein in some embodiments. [Figure 2]

[0032] FIG. 1 shows a culture vessel with a gas permeable membrane at the bottom of the vessel, a harvest line represented by a curved tube, a medium removal line represented by a long straight tube, a sampling line represented by a short straight tube, and an air filter for attachment represented by a rounded head attached to a tube emerging from the top of the vessel. [Figure 3]

[0033] 1 shows the nucleic acid sequence (SEQ ID NO: 1) of an exemplary construct comprising an MSCV promoter, an anti-human CD7 (TH69) CAR, a P2A self-cleaving peptide, and an anti-human CD7 (TH69) protein expression blocker (PEBL). The MSCV promoter is double underlined, the restriction enzyme site and Kozak sequence are between the MSCV promoter and the CAR, the anti-human CD7 (TH69) CAR is in bold, P2A is in regular font, and the anti-human CD7 (TH69) PEBL is single underlined. [Figure 4]

[0034] 1 is a flowchart of an embodiment of a method described herein, including, but not limited to, providing a population of cells, incubating the cells and culture medium, controlling the concentration of metabolites in the medium, and disrupting cell clumps. [Figure 5]

[0035] 1 is a flowchart of an embodiment of a method described herein, including, but not limited to, providing a population of cells, selecting a subpopulation of cells, activating the cells, virally transducing the cells, incubating the cells and culture medium, controlling the concentration of metabolites in the medium, and disrupting cell clumps. [Figure 6]

[0036] FIG. 1 shows CAR and CD7 expression by primary T cells transduced with MSCV-CD7CAR-P2A-CD7PEBL lentivirus and analyzed by flow cytometry 3, 6, and 9 days after transduction. [Figure 7A]

[0037] Figures 7A-7D show cell number, viability, and cell type after a CliniMACS Prodigy® run. Figure 7A is a graph of total viable cells over time showing the effect of run conditions IPR1, IPR2, and Run 1 on total viable cells during production. [Figure 7B]

[0037] Figure 1 is a graph of viability over time showing the effect of run conditions IPR1, IPR2, and Run 1 on the percentage of viable cells in cultures during production. [Figure 7C]

[0037] Figure 1 is a graph of the percentage of CD3+CD56-CAR+CD7- cells during production. CD3 is a T cell marker, CD56 is an NK cell marker, CAR is an anti-CD7 CAR, and CD7- indicates anti-CD7 PEBL activity. [Figure 7D]

[0037] Graph of percentage of CD3+CD56-CD7- cells during production. [Figure 8A]

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

[0039] FIG. 8A is a graph of fold change over time showing the effect of run conditions for Arm 1 and Arms G1-G5 on the fold change in total viable cell number relative to day 0. [Figure 8B]

[0039] Figure 1 is a graph of viability over time showing the effect of run conditions for Arm 1 and Arms G1-G5 on the percentage of cells that survive in cultures during production. [Figure 8C]

[0039] Graph of percentage of CD3+CD56-CD7- cells during production. [Figure 8D]

[0039] This is a graph of the percentage of CD3+CD56-CAR+CD7- cells in production, which are T cells that express the T cell marker (CD3) and a chimeric antigen receptor that targets CD7 and does not express the NK cell marker (CD56) or CD7. [Figure 9A]

[0040] Figures 9A-9D show metabolite concentrations in mmol / L and pH during use of G-Rex vessels at 10M and 100M scales to manufacture CAR+CD7- T cells. Arms G1-G3 are 10M scales, and arms G4-G5 are 100M scales. Arm 1 is the bench-scale control. Figure 9A shows a graph of lactate concentration. [Figure 9B]

[0040] A graph of glucose concentration is shown. [Figure 9C]

[0040] A graph of ammonium concentration is shown. [Figure 9D]

[0040] A graph of pH over days of culture during production is shown. DETAILED DESCRIPTION OF THE INVENTION

[0032] Detailed Description

[0041] Described herein are methods and systems for improved production of cells (e.g., engineered immune cells expressing a chimeric antigen receptor (CAR) and, optionally, a protein expression blocker (PEBL)), and methods for their use. In some cases, engineered cells (e.g., cells engineered to express a CAR and / or a PEBL) may suffer from reduced viability during production. In some cases, engineered cells (e.g., expressing a CAR and / or a PEBL) may be more metabolically active than unengineered cells of the same species and / or cell type. Metabolites produced by cells in culture and accumulated in the culture medium over the course of the manufacturing process can affect the pH of the culture medium, potentially reducing the viability and / or yield of the engineered cells. Controlling the pH and / or concentration of metabolites in the culture during manufacturing can be important for improving the viability and / or yield of engineered cell populations (see 406, 512, Figures 4 and 5). In some cases, carefully scheduling cell culture medium changes can help control the pH and concentration of metabolites in the culture during manufacturing. In some cases, the use of a gas-permeable membrane for culturing the engineered cells described herein can help provide oxygen to the engineered cells during production without continuous agitation, which can improve the viability and / or yield of the engineered cells in various embodiments. In some cases, the use of a gas-permeable membrane for culturing the engineered cells described herein can also help control the pH and nutrient concentration of the engineered cells during production, which can improve the viability and / or yield of the engineered cells in various embodiments. In some cases, the engineered cells may form clumps during production. In some cases, clumping of the engineered cells during production can result in a reduced viral transduction rate and therefore a reduced expression rate of the desired protein. Clumping of the engineered cells during production can be reduced by disintegrating the engineered cell clumps during production. In some cases, mechanical agitation of the engineered cells can reduce the viability of the engineered cells. In some cases, limiting the exposure of the cells to excessive mechanical agitation can improve the viability of the engineered cells.In some cases, the method or system may involve mixing the cells (e.g., during culture medium changes) but not stirring, which may in some cases improve the viability and / or yield of the engineered cell population.

[0033]

[0042] Described herein are compositions and methods for producing T cells that simultaneously express a chimeric antigen receptor that targets CD7 (e.g., a CAR) and a CD7 protein expression blocker (e.g., PEBL) to result in high viability and yield. The composition includes a population of immune cells that contain a receptor with a binding domain that binds to CD7 expressed on the surface of the T cell, an activation domain that activates the T cell, and a protein expression blocker (PEBL) that has reduced cell surface expression of CD7.

[0034] method

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

[0035]

[0044] Methods for producing a population of cells described herein (e.g., 400, 500) can include, for example, selecting at least a portion of a population of cells (e.g., a population of cells provided, e.g., from a healthy or diseased human donor, as described herein), as shown in step 504. In some cases, selecting at least a portion of the population of cells can include isolating one or more cell types of interest from the population of cells (e.g., for further processing and / or use as a therapy). In some cases, isolating one or more cell types of interest can include, for example, performing one or more affinity-based cell separation techniques (e.g., fluorescence-activated cell sorting (FACS) and / or magnetic-activated cell sorting (MACS)) on the population of cells. In some cases, selecting at least a portion of the population of cells can include depleting the population of cells of one or more cell types. For example, selecting a population of cells can include lysing a cell type (e.g., red blood cells) present in the population of cells. In some cases, depleting a population of cells of one or more cell types can include removing one or more cell types from a population of cells using density gradient centrifugation and / or affinity-based selection techniques. In some cases, selecting at least a portion of a population of cells can include isolating or enriching cells that express 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 that express CD4 and / 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 the population of cells may involve isolating or enriching a population of cells that express two or more molecules of interest (e.g., CD2, CD3, CD4, CD7, CD8, CD25, or CD69) simultaneously (e.g., using multiple MACS or FACS affinity probes that can bind to one or more of the molecules of interest in a single selection assay) or sequentially.In some cases, selecting at least a portion of the population of cells may include isolating or enriching cells that express a chimeric antigen receptor (CAR). In some cases, selecting at least a portion of the population of cells may include selecting a population of cells susceptible to fratricide (e.g., cells such as immune cells such as T cells or NK cells that express a CAR capable of binding to a molecule, e.g., a surface marker such as CD7, expressed by another cell (e.g., another T cell) that expresses a CAR capable of binding to the same molecule). In some cases, a population of NK cells may be enriched. The enriched population of NK cells may express CD56.

[0036]

[0045] In some cases, selecting at least a portion of the population of cells can be performed before or after one or more other steps described herein. For example, a step including selecting at least a portion of the population of cells can be performed before incubating all or a portion of the population of cells in culture medium. In some cases, a step including selecting at least a portion of the 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 the population of cells can include isolating cells that express CD25 and / or CD69 after activation of all or a portion of the population of cells.

[0037]

[0046] In some cases, a second population of cells can be produced by selecting at least a portion of the first 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 express one or more molecules of interest (e.g., CD2, CD3, CD4, CD7, CD8, CD25, CD69, or CAR). In some cases, at least a portion of the first population of cells can be selected to 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 CAR). In some cases, at least 80% of the second population (e.g., cells selected during the selection steps described herein) can be positive for surface expression of a polypeptide of interest such as CD2, CD3, CD4, CD7, CD8, CD25, CD69, and / or CAR. In some cases, at least 85% of the second population (e.g., cells selected during the selection steps described herein) may 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 the selection steps described herein) may 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 the selection steps described herein) may 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 include selecting or enriching cells from a population such that 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 are positive for expression (e.g., surface expression) of CD3 (e.g., compared to unmodified cells of the same cell type and species, e.g., unmodified human T cells).

[0038]

[0047] In some cases, one or more cells of the second population of cells (e.g., cells selected during a selection step described herein) may 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), e.g., compared to unmodified cells 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 may 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 unmodified cells of the same type, such as T cells of the same species). For example, a method or system described herein can involve selecting or enriching cells from a population such that 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 non-engineered cells of the same cell type and species, e.g., unmodified human T cells).

[0039]

[0048] In some cases, reducing the expression (e.g., cell surface expression) of one or more molecules of interest can include preventing a polypeptide (e.g., a cell surface polypeptide) produced within the cell from being transported to or expressed on the surface of the cell. In some cases, this can be achieved by expressing a protein expression blocker (PEBL) described herein, which can include a binding domain capable of binding to the molecule of interest and a localization domain that can include a retention sequence (e.g., a Golgi retention sequence, an ER retention sequence (which can include a KDEL sequence or a KKXX sequence), a PEST sequence, etc.). In some cases, reducing the expression (e.g., cell surface expression) of one or more molecules of interest within the cell can include disrupting or sequestering an RNA molecule or polypeptide within the cell, for example, using siRNA, shRNA, or miRNA that can bind to and / or promote an RNA interference (RNAi) pathway against the 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 involve modifying the DNA sequence encoding the molecules or their expression in the cell (e.g., by genetic manipulation of the cell's DNA, e.g., 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 involve introducing into the cell (e.g., via genetic engineering techniques) a dominant-negative allele of a gene of interest (e.g., capable of producing or affecting the function of one or more molecules of interest).

[0040]

[0049] In some embodiments, the method includes selecting one or more cells from a cell population. In some embodiments, one or more cells expressing CD4, CD8, or CD3 (e.g., one or more immune cells such as T cells) can be selected (e.g., for incubation or use). In some embodiments, T cells can be derived from an enriched apheresis product (e.g., leukopacks) 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 healthy individuals. In some embodiments, frozen human primary peripheral blood mononuclear cells (PBMCs) can be processed for use, for example, after thawing, allowing them to recover overnight in culture, and culturing them in cell culture medium supplemented with, for example, 3% human AB serum or serum-free cell culture medium. Cells can be extracted by affinity-based separation techniques, for example, using a device configured to perform magnetic separation of cells (e.g., magnetic-activated cell separation (MACS) using, for example, the CliniMACS Prodigy system). In some cases, the methods described herein may include enriching the cell population for CD4- and CD8-positive T cells using CD4 and CD8 microbeads in an affinity-based separation technique such as MACS. CD3 microbeads may be used during the selection process of the methods or systems described herein (e.g., in addition to one or more other separation criteria).

[0041]

[0050] Producing a population of cells can include activating the cells, for example, before incubating the population of cells (see, e.g., 506, FIG. 5). Activating the initial population of cells can increase the 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 the cell population can include contacting one or more cells of the cell population with an antibody that binds CD3 or CD28. Selection of the population of cells can be based, at least in part, on cellular expression of CD25 and CD69 after stimulation. In some cases, activating the cell population can include contacting the cells with an activation reagent, such as an antibody against CD3 and / or CD28 or TransAct™ (a colloidal polymer nanomatrix covalently linked to a humanized recombinant agonist for human CD3 and CD28). Producing a population of cells can include activating (e.g., stimulating) the initial population of cells before (or in some embodiments after) selecting the population of cells. Producing a population of cells may include activating the cells prior to incubating the population of cells. The cells may be activated with a stimulatory reagent on the first day of incubation (e.g., day 0). The stimulatory reagent may be designed to activate and / or expand T cells. Activating may include contacting the cells with an antibody that binds CD3 and / or CD28. Activating may include contacting the cells with multiple antibodies that bind CD3 and / or CD28.

[0042]

[0051] Producing a population of cells can include, for example, transducing the cells before incubating the population of cells. A population of transduced cells can be created by transducing starting 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 can then comprise a CAR and / or a chimeric targeting polypeptide. After transduction with the viral vector, the transduced starting cells can be incubated. In some cases, the viral vector can comprise a nucleotide sequence encoding a CAR or a chimeric targeting polypeptide described herein. In some cases, the viral vector can comprise a nucleotide sequence encoding a CAR and a chimeric targeting polypeptide described herein.

[0043]

[0052] In some cases, a method or system described herein may include incubating one or more cells (e.g., a population of cells). Incubating the population of cells may include culturing the population of cells in a culture vessel (e.g., a culture vessel comprising a gas-permeable membrane). In some cases, the population of cells may be incubated (e.g., cultured) in contact with the surface of the gas-permeable membrane of the 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 release from the cells, thereby improving the viability and / or yield of the population of cells or a portion thereof (e.g., a population of cells that express a chimeric antigen receptor (CAR) and exhibit 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 example, the viability and / or yield of a population of cells, or a portion thereof (e.g., a population of cells that express a chimeric antigen receptor (CAR) and exhibit 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)) can be improved by incubating the population of cells in contact with the surface of a gas-permeable membrane.

[0044]

[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) that can bind to a cell surface polypeptide (e.g., a cell surface protein such as CD7) expressed on the cell surface of a cell of the same species and type (e.g., a cell that also expresses a CAR that can recognize the cell surface polypeptide (e.g., a human immune cell (NK or T cell) that also expresses CD7), which receptor, upon binding to the cell surface polypeptide, activates the immune cell for cytotoxicity. Siblicide can be highest when the cell (or population thereof) expresses high levels of the cell surface polypeptide, but can also be highest when the cell (or population thereof) expresses low levels of the cell surface polypeptide. If the polypeptide is expressed on the cell surface in a manner that is not specifically targeted to fratricide, susceptibility to fratricide may still be present. Optionally, a method or system described herein may include incubating a population comprising or consisting of one or more cells susceptible to fratricide. Optionally, a population of cells comprising or consisting of one or more cells susceptible to fratricide may be harvested from the culture, frozen, thawed, and / or administered to a patient in need of treatment for a condition (e.g., treatment of cancer). Optionally, the cells used in a method or system described herein may have reduced cell surface expression of a cell surface polypeptide.

[0045]

[0054] Incubating the population of cells can include removing an activation reagent (e.g., a stimulatory reagent). The stimulatory reagent can be removed to control the proliferation, activation, and / or expansion of T cells. The stimulatory reagent can be removed on day 1, day 2, day 3, day 4, day 5, day 1-3, day 2-3, or day 3-4 after the start of incubation. The optimal day to remove the stimulatory reagent to increase one or more of the total number of viable cells, the percentage of viable cells, or the percentage of cells expressing the desired protein can be two days after the first day of incubation.

[0046]

[0055] In some embodiments, T cells can be seeded in a vessel with a gas-permeable membrane and activated and expanded with a stimulatory reagent (e.g., TransAct™). In some embodiments, cells can be transduced with a lentiviral vector at an MOI of 10. In some embodiments, cells can be expanded up to day 11. In some embodiments, there are two or more medium changes during the production process to minimize the accumulation of by-products (e.g., metabolic products), such as lactate and ammonium, in the culture.

[0047]

[0056] Producing the cells can include incubating a population of cells, and the population of cells can be a population of T cells. The population of cells can include cells derived from a human subject. The population of cells can be a population of T cells composed of cells derived from a human subject. The population of cells can include a receptor. The receptor can include a chimeric antigen receptor (CAR). The receptor can include 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 include a CD7 binding domain. The receptor can include an activation domain. The activation domain can induce cytotoxic activity in the population of (e.g., engineered immune) cells. The population of cells can include a target binding molecule. The target binding molecule can be linked to a localization domain. The target binding molecule can include an scFv. The target binding molecule linked to the localization domain can reduce cell surface expression of the target polypeptide. The target polypeptide can include CD7. The target binding molecule linked to the localization domain can include a protein expression blocker (PEBL). The population of cells can include T cells that express CAR and PEBL (e.g., PCART cells).

[0048]

[0057] The population of cells can be incubated with culture medium. The population of cells and culture medium can be incubated in a container with a gas-permeable membrane. The population of cells and culture medium can be incubated in a closed system container. The population of cells incubated on the gas-permeable membrane can obtain oxygen and release carbon dioxide through the gas-permeable membrane, eliminating the need to stir the culture medium to provide oxygen to the cells. Incubating the population of cells and culture medium in a closed system with a gas-permeable container can increase one or more of the total number of viable cells, the percentage of viable cells, or the percentage of cells expressing a desired protein (e.g., cells susceptible to fratricide) relative to other incubation methods. 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.

[0049]

[0058] Incubating the population of cells can include seeding the cells. The seeded cells can include CD4+ and / or CD8+ cells.

[0050]

[0059] The cells can be incubated in culture medium. The culture medium can be made on the first day of culture (day 0) for use throughout the culture period. The culture medium can be made fresh daily, and the medium is added to the vessel or replaced during cell incubation.

[0051]

[0060] In some cases, the population of cells described herein can be incubated in a culture medium having a pH of about 6.5 to 7.4. The culture medium can have a pH of 6.5 to 6.7, 6.5 to 6.9, 6.5 to 7.1, 6.5 to 7.4, 6.7 to 6.9, 6.7 to 7.1, 6.7 to 7.4, 6.9 to 7.1, 6.9 to 7.4, or 7.1 to 7.4. The culture medium can have a pH less than about 6.5. The culture medium can have a pH greater than about 7.4. In some cases, controlling (e.g., maintaining) the pH at which the population of engineered cells is incubated can increase one or more of the total number of viable cells, the percentage of viable cells, or the percentage of cells expressing the desired protein. For example, it can be beneficial to maintain the cells at a pH greater than 7.2 (e.g., starting after day 5). In some cases, this can be achieved by performing a culture medium change as described herein.

[0052]

[0061] The culture medium may comprise a volume of about 7 mL to 1 L. The culture medium may comprise a volume of 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 about 800 mL to 1 L. The culture medium may comprise a volume less than about 7 mL. The culture medium may comprise a volume greater than about 1 L. The optimal culture medium may vary depending on the day since the start of incubation. The optimal culture medium on day 3 after seeding may be about 40 mL. The optimal culture medium on days 4 and beyond may be about 1 L. Incubating a population of cells in an optimal volume of culture medium can increase one or more of the total number of viable cells, the percentage of viable cells, or the percentage of cells expressing a desired protein.

[0053]

[0062] Producing a population of cells can include changing at least a portion of 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 that is changed can be at least 10%, at least 25%, at least 50%, at least 75%, at least 85%, at least 95%, 100%, 10% to 25%, 25% to 50%, 50% to 75%, 75% to 85%, 85% to 95%, or 95% to 100% of the culture medium in the closed system. In some cases, the container for culturing the cells can contain approximately 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 medium. In some cases, a larger volume of culture medium can reduce the frequency at which the culture medium must be changed to maintain the concentration of metabolites in the culture medium, for example, reducing the impact of increased metabolite production in the production of engineered cells described herein (e.g., cells susceptible to fratricide) (e.g., compared to unengineered cells). The culture medium can be changed on any day of incubation and / or every day. The culture medium can be changed after 6 and / or 9 days from the first day of incubation. The culture medium can be changed after 6, 8, and / or 10 days from the first day of incubation. Frequent changes of the culture medium can minimize metabolites and improve control of the concentration of substances in the culture medium. Frequent culture medium changes can disrupt an otherwise closed system and reduce one or more of the total number of viable cells, the percentage of viable cells, or the percentage of cells expressing the desired protein. An optimal culture medium change frequency can control the concentration of substances in the culture medium while improving one or more of the total number of viable cells, the percentage of viable cells, or the percentage of cells expressing the desired protein. Optimal culture medium changes can be performed 6 and 9 days after the first day of incubation if the harvest date is day 11. Optimal culture medium changes can be performed by changing 75% of the culture medium in a closed system.In some cases, incubation can include 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 of cells susceptible to fratricide can occur after the incubation period. In some cases, harvesting of cells susceptible to fratricide can occur after incubation for 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.

[0054]

[0063] Incubating a population of cells can include reducing the volume of the incubation mixture. For example, a method or system described herein can include reducing the volume of the culture medium in which the cells are incubated, e.g., to dilute or remove a reagent (e.g., an activation reagent such as TransAct™) to enrich the cell population in the culture (e.g., to reduce the total volume of the medium in the culture), or to modify the culture medium (e.g., to control the concentration of a substance in the medium, such as ammonium or lactate). The incubation mixture can include the desired cells, stimulation reagents, and / or culture medium. In some cases, the volume of the incubation mixture can be reduced without opening the closed system. For example, the volume of the incubation mixture can be reduced by up to 750 mL, up to 500 mL, up to 250 mL, up to 100 mL, up to 50 mL, or up to 10 mL without opening the closed system. The volume of the incubation mixture can be reduced (e.g., without opening the closed system) on any of the following days: 3, 5, 7, 8, 9, 10, 11, or 12 after the first day of incubation of the cells in culture. The volume of the incubation mixture can be reduced on the day of harvest.

[0055]

[0064] Producing a population of cells can include controlling the concentration of a substance. The concentration of the substance in the culture medium can be controlled. The substance can be a metabolic product or by-product of cell growth. The substance can be lactate or ammonium.

[0056]

[0065] Lactate, which may be a metabolic product of cell culture (e.g., culture of immune cells such as NK cells or T cells), 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, up to about 15 mM, up to about 12 mM, up to about 10 mM, up to about 8 mM, up to about 6 mM, up to about 5 mM, up to about 3 mM, up to about 1 mM, 1 mM to 3 mM, 3 mM to 5 mM, 5 mM to 6 mM, 6 mM to 8 mM, 8 mM to 10 mM, 10 mM to 12 mM, or 12 mM to 15 mM. In some cases, lactate can be controlled below a threshold 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 greater than about 15 mM. Lactate can be controlled at an optimal concentration less than about 5 mM. Lactate can be controlled at an optimal concentration less than about 5 mM, 4 mM, 3 mM, 2 mM, or 1 mM. Controlling lactate below a threshold concentration can, in some cases, increase one or more of the total number of viable cells, the percentage of viable cells, or the percentage of cells expressing a desired protein. Lactate can be controlled at a concentration for a period of time in culture, for example, 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 days, or more than 11 days of culture.

[0057]

[0066] The ammonium concentration in the culture medium can be controlled at a threshold concentration level, e.g., 0.4 mM. In some cases, the ammonium concentration in the 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, up to about 1 mM, up to about 0.5 mM, up to about 0.4 mM, up to about 0.3 mM, up to about 0.2 mM, 0.4 mM to 0.2 mM, 0.5 mM to 0.2 mM, or 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 percentage of viable cells, or the percentage of cells expressing the desired protein. Ammonium can be controlled at a concentration for a period of time in culture, e.g., at least 7 days of culture. The ammonium concentration in the culture medium can be controlled (e.g., below a threshold value) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 days, or for more than 11 days of culture.

[0058]

[0067] Manufacturing a population of cells (e.g., immune cells, e.g., engineered immune cells described herein) can include breaking up clumps of the population of cells (e.g., 408, 514; see Figures 4, 5). In some cases, agitation (e.g., mechanical agitation) of the cell culture (e.g., containing the cells) can be used to break up clumps of cells in the 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 with a gas-permeable membrane) without continuous agitation. In some cases, agitation can be applied in one or more discrete or periodic applications of agitation to the cells in culture. The population of cells can include T cells. In culture, the T cells can clump together. The clumping of the T cells can increase one or more of the transduction efficiency, the total number of viable cells, the percent of viable cells, or the percent of cells expressing the desired protein. The clumping of the T cells during manufacturing can be reduced by the disintegration of the T cells during manufacturing. Existing manufacturing methods can cause cell rupture, thereby reducing 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 the transduction efficiency, total number of viable cells, percent of viable cells, or percent of cells expressing a desired protein.

[0059]

[0068] In some cases, limiting exposure of the cells to excessive mechanical disruption can improve the viability of engineered cells. In some cases, the method or system can include mixing the cells to break up cell clumps, but not stirring the cells. The mixing can be mechanical mixing. The mixing can be gentle mixing. The mixing can be irregular mixing. In some cases, mixing engineered cells in culture too infrequently can lead to uncontrolled clumping. In some cases, excessively frequent clump disruption of engineered cells in culture can increase cell rupture. Clump disruption of engineered cells can be performed every 1 to 3 days. Clump disruption of engineered cells can be performed multiple times per day. Clump disruption of engineered cells can be performed at least every 3 days. Clump disruption of engineered cells can be performed at least three times over an 11-day incubation period. Clump disruption of engineered cells can be performed on days with medium changes. Performing cytodisruption of engineered cell clumps on days with medium changes can minimize interference with the closed system. A novel method for producing T cells without cell disruption is described, while minimizing interference and increasing one or more of the following: transduction efficiency, total number of viable cells, percent of viable cells, or percent of cells expressing a desired protein. Disruption of engineered cell clumps can be performed on days 6 and 9 of an 11-day incubation period, coinciding with a medium change.

[0060]

[0069] The manufacturing methods and systems described herein may be useful for determining the quantity (e.g., yield) of cells at one or more manufacturing stages. A cell count and viability (CCV) assay 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. A CCV assay may be used one or more times during the manufacturing process. A CCV assay may be used zero times during the manufacturing process. A CCV assay may be used on starting material. A CCV assay may be used on manufactured cells. A CCV assay may be used on concentrated apheresis product used as starting material. A CCV assay may be used on concentrated apheresis product on the first day of incubation (day 0). A CCV assay can provide the number and viability of cells used as starting material, which can affect downstream cell count and viability. A CCV assay may be used after seeding CD4 and CD8 cells into a gas-permeable container on the first day of incubation (day 0). A CCV assay can be used to determine the number of cells for lentiviral transduction on the day of transduction. A CCV assay can be used to determine the success rate of lentiviral transduction on day 3. A CCV assay can be used to measure the progress of manufacturing approximately halfway through the manufacturing process. A CCV assay can be used to measure the progress of manufacturing on day 6 of an 11-day manufacturing process. A CCV assay can be used on the harvest day. A CCV assay can be used on the harvest day of an 11-day manufacturing process. Harvest day CCV assays can include, but are not limited to, one or more of CCV for formulation density, CCV for target volume determination, CCV for volume adjustment, and CCV for the final formulation (e.g., final cell viability percentage and other metrics).

[0061]

[0070] The methods described herein, or steps thereof, can be carried out for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. Optionally, the manufacturing methods described herein can proceed, e.g., to 3, 4, 5, 6, 7, 8, 9, 10, or 11 days after the start of incubation, to increase the desired yield of one or more types. Optionally, the viability of the engineered cells at the time of harvest (e.g., 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%, and the yield of engineered cells at the time of harvest (e.g., day 11) is at least 10 cells. In some cases, the viability of the engineered cells at the time of harvest (e.g., day 11) may be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100%, and the yield of engineered cells at the time of harvest (e.g., day 11) is at least 10 cells. In some cases, the viability of the engineered cells at the time of harvest (e.g., day 11) may be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100%, and the yield of engineered cells at the time of harvest (e.g., day 11) is at least 10 cells.

[0062]

[0071] Producing a population of cells can include, for example, incubating the population of cells until the cells reach a desired yield (404, 510; Figures 4, 5). The desired yield can be measured by total viable cells, fold change in cell number, percent viability, or a surface marker profile CD3 above a threshold. + CD56 - Number of cells expressing the surface marker profile CD3 above a threshold + CD56 - CD7 - The number of cells expressing and / or the surface marker profile CD3 above a threshold + CD56 - CAR + CD7- In some cases, the viability of the engineered cells produced may include one or more of the following: a number of cells expressing a nucleotide sequence encoding ... In some cases, the viability of cells produced using the methods or systems described herein may 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 the methods or systems described herein may 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, the viability of thawed engineered cells may be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100%.

[0063]

[0072] The yield of (e.g., viable) cells at harvest can be at least 1E6 cells, at least 1E7 cells, at least 3E7 cells, at least 5E7 cells, at least 1E8 cells, or at least 5E8 cells. In some cases, the yield of (e.g., viable) cells at harvest can be between 1E6 cells and 1E7 cells, between 1E7 cells and 3E7 cells, between 3E7 cells and 5E7 cells, between 5E7 cells and 1E8 cells, or between 1E8 cells and 5E8 cells. An increased yield can indicate a more successful manufacturing process that can produce more desirable PCART cells or other T cells in each manufacturing run.

[0064]

[0073] In some embodiments, the methods described herein can include harvesting at least 1E8 cells susceptible to fratricide after incubation for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or less. In some cases, harvesting at least 1E8 cells occurs after incubation for 11 days or less. A percentage of the harvested at least 1E8 cells susceptible to fratricide can 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 can be viable. In some embodiments, a range of about 30% to about 95% of the harvested cells can be viable.In some embodiments, 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 4 ...40% 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 75%, about 30% to about 80%, about 40% 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 65%, about 40% to about 75%, about 40% to about 80%, about 40% to about 95%, about % 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 6 5%, 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 Viability may be in the range of 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%.

[0065]

[0074] A percentage of the harvested 1E8 cells susceptible 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 the CAR may be viable. In some embodiments, a range of about 30% to about 95% of the harvested cells expressing the CAR may be viable. In some embodiments, 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 4 ...40% to about 65%, about 40% 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 65%, about 40% to about 65%, about 40% to about 65%, about 40% 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 0%, 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% may be viable.

[0066]

[0075] A percentage of the harvested 1E8 cells susceptible 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 with reduced expression of a surface polypeptide may be viable. In some embodiments, a range of about 30% to about 95% of the harvested cells with reduced expression of a surface polypeptide may be viable. In some embodiments, 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%, or about 50% to about 60% of the collected cells have reduced expression of the surface polypeptide. 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 Viability may be in the range of 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%.

[0067]

[0076] The methods or systems described herein can include determining the fold change in cell number relative to the start of incubation (e.g., relative to day 0). In some cases, culturing (e.g., incubation) of engineered cells can result in a 0.2-1 fold, 1.0-2.0 fold, 2.0-5.0 fold, 5.0-10.0 fold, 10.0-20.0 fold, 20.0-30.0 fold, 30.0-40.0 fold, at least 0.2 fold, at least 5 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, or at least 40 fold increase in the amount of cultured cells (e.g., engineered cells). If incubation proceeds for longer than 11 days, the fold change can continue to increase. If incubation proceeds for 14 days, the fold change can be at least 68.61. An increase in yield can indicate a more successful manufacturing process, capable of producing more desirable PCART cells or other T cells in each manufacturing run.

[0068]

[0077] The methods or systems described herein can include determining percent cell viability. The minimum desired cell viability 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 indicate a more successful manufacturing process that can produce more desirable PCART cells or other T cells in each manufacturing run.

[0069]

[0078] The population of cells according to the methods or systems described herein may contain a minimum percentage of CD3 + CD56- cells (e.g., on the day of collection, e.g., day 11). + The percentage of CD56- cells can be at least 85%. + The percentage of CD56- cells may be less than about 87.5%. + The percentage of CD56- cells can be at least 99.7%. +The percentage of 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 population of cells produced. The population of cells according to the methods or systems described herein can have a minimum percentage of CD3 - CD56 + It may contain NK cells (e.g., on the day of collection, e.g., day 11). - CD56 + The percentage of NK cells can be at least 99.7%. - CD56 + The percentage of 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 population of produced cells. An increased yield can indicate a more successful manufacturing process that is able to produce more desirable PCART cells or other T cells in each manufacturing run.

[0070]

[0079] The population of cells according to the methods or systems described herein may contain a minimum percentage of CD3 + CD56 - CD7 - CD3 cells (e.g., on the day of collection, e.g., day 11). + CD56 - CD7 - The minimum percentage of cells may be at least 80%. + CD56 - CD7 - The minimum percentage of cells may be less than about 85%. + CD56 - CD7 - The percentage of cells can be at least 99.7%. + CD56 - CD7 -The minimum percentage of cells can be at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. An increased yield can indicate a more successful manufacturing process that is capable of producing more desirable PCART cells or other T cells in each manufacturing run.

[0071]

[0080] The population of cells according to the methods or systems described herein may contain a minimum percentage of CD3 + CD56 - CAR + CD7 - CD3 cells (e.g., on the day of collection, e.g., day 11). + CD56 - CAR + CD7 - The minimum percentage of cells may be at least 40%. + CD56 - CAR + CD7 - The percentage of cells may be less than about 80%. + CD56 - CAR + CD7 - The minimum percentage of cells may be at least 90%. + CD56 - CAR + CD7 - The minimum percentage of cells can be at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100%. An increased yield can indicate a more successful manufacturing process that is capable of producing more desirable PCART cells or other T cells in each manufacturing run.

[0072]

[0081] The desired production yield by day 11 after the start of incubation is limited by the percentage of cells expressing CD56, and CD3 + CD56-, CD3 + CD56 - CD7 - , and / or CD3 + CD56 - CAR + CD7 -The yield may include increasing the percentage of cells expressing CD56. The percentage of cells expressing CD56 may be up to about 12.5%. The percentage of cells expressing CD56 may be greater than 12.5%. The percentage of cells expressing CD56 may be up to about 0.3%. The percentage of cells expressing CD56 may be up to about 12.5%, 10.5%, 8.5%, 6.5%, 4.5%, 2.5%, 0.5%, or up to about 0.3%. A decrease in yield may indicate a more successful manufacturing process that is able to produce more desirable PCART cells or other T cells in each manufacturing run.

[0073]

[0082] The population of cells according to the methods or systems described herein may contain a minimum percentage of CD3 + CD3 cells (e.g., on the day of collection, e.g., day 11). + The percentage of cells may be at least 87.5%. + The percentage of cells may be less than about 87.5%. + The percentage of cells can be at least 99.7%. + The percentage of cells can be at least 87.5%, 89.5%, 91.5%, 93.5%, 95.5%, 97.5%, 99.5%, or at least 99.7%. An increased yield can indicate a more successful manufacturing process that is capable of producing more desired PCART cells or other T cells in each manufacturing run.

[0074]

[0083] The methods or systems described herein can include increasing the percentage of cells expressing CD4 in a population. The percentage of cells expressing CD4 can be at least about 12.5%. The percentage of cells expressing 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%.

[0075]

[0084] The methods or systems described herein can include increasing the percentage of cells expressing CD8 in a population. The percentage of cells expressing CD8 can be at least about 12.5%. The percentage of cells expressing 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%.

[0076]

[0085] Producing a population of cells can include, for example, transducing the population of cells with one or more vectors 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. Transduction with a viral (e.g., lentiviral) vector can include a multiplicity of infection (MOI) at which the vector is used relative to the number of cells in the population of cells to be transduced with the vector. The MOI can be about 10-30. The MOI can be about 10-20, 10-30, or 20-30. 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. An optimal MOI that increases one or more of the total number of surviving cells, the percent of surviving cells, or the percent of cells expressing the desired protein can be about 10.

[0077]

[0086] Incubating the population of cells may include transduction with a specific volume of transduction reagent, which may include a vector (e.g., a viral vector encoding one or more CAR and / or PEBL constructs). The transduction volume may be about 4 mL to 20 mL. The transduction volume may be 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 about 16 mL to 40 mL. The transduction volume may be less than about 4 mL. The transduction volume may be more than about 20 mL. An optimal transduction volume for increasing one or more of the total number of viable cells, the percentage of viable cells, or the percentage of cells expressing the desired protein may be about 4 mL. Incubating the population of cells may involve transduction with a specific volume per surface area of ​​a gas-permeable membrane in a container, for example, about 0.4 mL / cm. 2 , 0.3-0.5mL / cm 2 , 0.2-0.8mL / cm 2 , or 0.1 to 2.0 mL / cm 2 The method may include transfecting with a transfection reagent of

[0078]

[0087] Incubating the population of cells can include transduction on a specific day of incubation. Transduction can occur between days 3 and 4. The optimal day for transduction, which increases one or more of the total number of viable cells, the percentage of viable cells, or the percentage of cells expressing the desired protein, can be day 3.

[0079]

[0088] Flow cytometry assays can sort cells. Flow cytometry assays can analyze populations of cells. Flow cytometry assays can be used one or more times during the manufacturing process. Flow cytometry assays can be used zero times during the manufacturing process. Flow cytometry assays can be used to analyze starting material. 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. The day 6 flow cytometry assay will measure cell surface expression after transduction. Flow cytometry assays can be used on the day of harvest. Flow cytometry assays can be used on the day of harvest of an 11-day manufacturing process. Flow cytometry assays on the day of harvest may include, but are not limited to, flow cytometry assays for formulation density and / or target volume determination.

[0080]

[0089] Manufacturing can include post-cell harvesting steps, which can include one or more of the following: concentration, washing, and / or formulation in a balanced crystalloid solution (e.g., a PlasmaLyte solution such as Plasma-Lyte 148); volume adjustment; dimethyl sulfoxide (DMSO) addition; quality control; and cryopreservation.

[0081]

[0090] Cryopreservation can be performed in a controlled-rate freezer (CRF). A variety of materials can be cryopreserved, including vectors (e.g., lentiviral vectors), cells (e.g., T cells, including but not limited to T cells containing PEBL and CAR), and / or engineered transduced cells. Cryopreserved cells can retain viability. Viability can be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% after thawing. Post-thaw viability can depend on the freezing density of the cells. The freezing density of cells can be approximately 1.6 E6 cells / mL to 3.84 E7 cells / mL to achieve at least 70% post-thaw viability. The freezing density of the cells can be about 1.6E6 cells / mL to 5.6E6 cells / mL, 1.6E6 cells / mL to 9.6E6 cells / mL, 1.6E6 cells / mL to 3.6E7 cells / mL, 1.6E6 cells / mL to 3.84E7 cells / mL, 5.6E6 cells / mL to 9.6E6 cells / mL, 5.6E6 cells / mL to 3.6E7 cells / mL, 5.6E6 cells / mL to 3.84E7 cells / mL, 9.6E6 cells / mL to 3.6E7 cells / mL, 9.6E6 cells / mL to 3.84E7 cells / mL, or about 3.6E7 cells / mL to 3.84E7 cells / mL to achieve at least 70% post-thaw viability. The freezing density of the cells can be less than about 1.6E6 cells / mL to achieve at least 70% post-thaw viability. The freezing density of the cells can be about 8E5 cells / mL to achieve at least 70% post-thaw viability. The freezing density of the cells can be greater than about 3.84E7 cells / mL to achieve at least 70% post-thaw viability.

[0082]

[0091] The cryopreserved preparation may include an equilibrium crystalloid solution. The cryopreserved preparation may include a cryopreservation medium. The cryopreservation medium may include 10% DMSO. Additional DMSO may be added to the cryopreservation mixture. The cryopreservation mixture may include a 50:50 mixture of an equilibrium crystalloid solution and a cryopreservation medium containing 10% DMSO. The final DMSO concentration may be 5%.

[0083] Cell administration

[0092] In some embodiments, provided are methods of treating an immune cell disorder in a subject in need thereof, comprising administering to the subject a therapeutic amount of engineered immune cells having any of the embodiments described herein, thereby treating the immune cell disorder in the subject in need thereof.

[0084]

[0093] In certain embodiments, the method comprises administering a therapeutic amount of engineered immune cells 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 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, the 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 engineered immune cells or a population thereof (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a nucleic acid construct comprising the sequence shown in Figure 3 (SEQ ID NO: 1) is administered to a subject with cancer.

[0085]

[0094] In another aspect, the present disclosure relates to the use of engineered immune cells comprising 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 localization domain for treating cancer, comprising administering a therapeutically effective amount of the engineered immune cells to a subject in need thereof. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are bicistronic.

[0086]

[0095] In another aspect, the present invention relates to the use of engineered immune cells comprising a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) and a nucleic acid comprising a nucleotide sequence encoding a target binding molecule (e.g., an scFv) linked to a localization domain, for treating an autoimmune disorder, comprising administering a therapeutically effective amount of the engineered immune cells to a subject in need thereof. In some cases, the autoimmune disorder can be amyotrophic lateral sclerosis (ALS), primary Sjögren's syndrome, sarcoidosis, type 1 diabetes, autoimmune hepatitis (e.g., type 1 or type 2), multiple sclerosis, Guillain-Barré syndrome and AMAN (axonal neuropathy), psoriasis, scleroderma, or ankylosing spondylitis (AS).

[0087]

[0096] In another aspect, the present invention also relates to the use of engineered immune cells comprising a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR). The engineered immune cells may reduce expression of the target of the CAR. The engineered immune cells may comprise a nucleic acid comprising a nucleotide sequence encoding a target binding molecule (e.g., an scFv) linked to a localization domain. A therapeutically effective amount of the engineered immune cells may be administered to a subject in need thereof to treat an infectious or viral disease affecting immune cells.

[0088]

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

[0089]

[0098] In some embodiments, the engineered immune cells are autologous to a subject in need of treatment, e.g., cancer treatment, autoimmune disease treatment, infectious disease treatment, graft-versus-host disease (GvHD) treatment, and transplant rejection treatment. In other embodiments, the engineered immune cells are allogeneic to the subject in need of treatment. The isolated engineered immune cells of the present invention can be "off-the-shelf" immune cells that can be administered to multiple subjects and provide a reduced risk of GvHD. In some embodiments, the engineered immune cells do not elicit a GvHD response when administered to multiple subjects (e.g., at least two or more subjects).

[0090]

[0099] In certain embodiments, the engineered immune cells are administered to the subject by infusion. Methods for injecting immune cells (e.g., allogeneic or autologous immune cells) are known in the art. A sufficient number of cells are administered to the recipient to ameliorate disease symptoms. Typically, 10 7 ~10 10 Dosage of cells is 10 for a single setting, e.g., 10 9 For pediatric patients, lower doses (e.g., 10 6 ~10 9 Injections can be performed in a single 10 9 as a cell dose or several 10 9The cells may also be administered in divided doses. The frequency of infusions may be daily, every 2 to 30 days, or at longer intervals as needed. The amount of infusion may generally be at least one infusion per subject, preferably at least three infusions as tolerated or until disease symptoms improve. Cells may be infused intravenously at a rate of 50 to 250 ml / hour. Other suitable modes of administration include intra-arterial infusion, intraperitoneal infusion, direct injection into the tumor and / or perfusion of the tumor bed after surgery, implantation at the tumor site in an artificial scaffold, and intrathecal administration. Methods for adapting the present invention to such delivery modes are readily available to those skilled in the art.

[0091]

[0100] In certain embodiments, the methods of treating cancer according to the present invention are combined with at least one other known cancer therapy, such as radiation therapy, chemotherapy, or other immunotherapy.

[0092]

[0101] In another aspect, there is also provided a use of engineered immune cells having any of the embodiments described herein for treating cancer, comprising administering a therapeutic amount of the engineered immune cells 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 stage T-cell precursor acute lymphoblastic leukemia (ETP-ALL).

[0093]

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

[0094] system

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

[0095]

[0104] The system may include a container for culturing a population of cells (e.g., a population of immune cells transduced with one or more viral vectors). In some cases, the container of the system may include a gas-permeable membrane for culturing the cells. In some cases, cells (e.g., cells susceptible to fratricide that may express a CAR) can be cultured in contact with the surface of the gas-permeable membrane of the system. The gas-permeable membrane surface may aid in the provision of nutrients and gas exchange to the cells during culture. As described herein, culture containers with gas-permeable membranes may be particularly useful for maintaining or improving cell viability and / or cell yield in the manufacture of engineered cells, for example, by facilitating gas transport to and from the cells. Gas-permeable containers may be an efficient method for expanding immune cells, with superior viable cell numbers, expansion folds, and viability compared to other similar methods for expanding immune cells. Gas-permeable containers can streamline the transduction and manipulation of T cells for therapeutic applications without compromising cell function. In some embodiments, the gas-permeable membrane is a silicone membrane that allows for the exchange of oxygen and carbon dioxide. In some embodiments, the container with the gas permeable membrane contains tubing for medium exchange, harvesting, and sampling that minimizes disruption of the cells in culture.

[0096]

[0105] In some embodiments, a system including a container with a gas-permeable membrane for cell culture and expansion can be a closed system or can include one or more closed system components. A closed system can reduce the possibility of cell contamination during production and streamline the process for cell production. Culture vessels with a 10 cm² gas-permeable membrane surface and a 100 mL maximum medium volume capacity (e.g., G-Rex® 6M and G-Rex® 10M G-Rex vessels; Wilson Wolf) or a 100 cm² gas-permeable membrane surface and a 1000 mL maximum medium volume (e.g., G-Rex® 100M and 100M-CS G-Rex vessels) can be used. In some cases, the culture vessel (e.g., with a 100 cm² gas-permeable membrane surface and a 1000 mL maximum medium volume) can be a closed system vessel with weldable tubing and dip tubes for medium exchange, harvesting, and sampling.

[0097]

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

[0098] Expression constructs

[0107] In some embodiments, the vector may comprise a plasmid. In other embodiments, the vector may comprise a sequence of nucleotides. The nucleotide sequence may encode a sequence of amino acids. The nucleotide sequence may encode a protein. The nucleotide sequence may encode a receptor. The nucleotide sequence may encode a chimeric antigen receptor (CAR). The nucleotide sequence may encode a protein linked to a receptor. The nucleotide sequence may encode a CAR linked to an scFv. The vector may encode PEBL. The nucleotide sequence may encode a chain of a desired surface-expressed molecule. The nucleotide sequence may encode one or more of CD3, CD4, CD7, CD8, or CD56.

[0099]

[0108] A cell population can be transduced with one or more viral vectors. In some cases, a cell population can be transduced with multiple 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 to at least a portion of a CD7 molecule, e.g., 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 to at least a portion of a CD7 molecule, e.g., CD7 produced in a 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 PEBL. When separate vectors are used, separate promoter elements are configured upstream of each gene such that each promoter transcribes mRNA for its proximally linked gene. In some embodiments, cells are transduced with a vector encoding PEBL before a vector encoding a CAR to reduce surface expression of the 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 PEBL simultaneously with or after a vector encoding a CAR.

[0100]

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

[0101]

[0110] To reduce self-killing (e.g., fratricide), CAR-T cells can express PEBL, which acts to reduce the expression of target antigens on the cell surface of CAR-Ts. To produce viable CAR-T cells, a protein expression blocker (PEBL) protein can be first expressed to bind to and sequester the target protein prior to subsequent expression of the CAR. Due to the pre-existing presence of the target antigen on the cell surface of the resulting engineered T cells, co-expression of CAR and PEBL can result in fratricide. In some cases, the pre-existing cell surface target antigen is less susceptible to sequestration by the newly expressed PEBL protein and can be recognized and targeted by the newly expressed CAR protein.

[0102]

[0111] The cells can be transduced with a CAR but not with PEBL. Cells expressing a CAR without PEBL can express a cell surface marker (e.g., CD7). The cells can be CAR+CD7+ cells. The cells can express a cell surface marker (e.g., CD7+ cells) without expressing a CAR or PEBL. In some cases, CAR+CD7+ cells can be more susceptible to fratricide compared to cells expressing the same CAR and PEBL that contain an antigen-binding domain for a cell surface marker (e.g., CD7). In some cases, after cell expansion and enrichment, the population of CAR+CD7- cells (e.g., engineered cells expressing CAR and PEBL) may have 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 100x, 200x, 300x, 400x, 500x, 1000x, 2000x, or 5000x more cells than the population of CAR+CD7+ cells.

[0103]

[0112] In some cases, CAR-CD7+ cells may be more susceptible to fratricide compared to cells expressing PEBL that contain an antigen-binding domain for the same CAR and cell surface marker (e.g., CD7). In some cases, after cell expansion and enrichment, the population of CAR+CD7- cells (e.g., engineered cells expressing a CAR and PEBL) may have 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 100x, 200x, 300x, 400x, 500x, 1000x, 2000x, or 5000x more cells than the population of CAR-CD7+ cells.

[0104]

[0113] In some cases, cells transduced with one or more viral vectors may be more fragile (e.g., more likely to die or less proliferate) than cells not transduced with the vectors. In some cases, this may mean that the transduced cells must be treated more gently during manufacturing than untransduced cells. Thus, in some cases, engineered cells (e.g., as described herein, which may be susceptible to fratricide, may express a CAR that targets a cell surface protein expressed by the cell, and / or may be transduced with a viral vector) may require methods and / or systems (e.g., as described herein) designed to manufacture such cells in order to obtain useful cell yields, cell viability percentages, and / or functional cell populations.

[0105]

[0114] The specific two gene vectors can induce expression of both PEBL and CAR proteins in T cells in a manner that allows the resulting engineered T cells to survive, expand, and kill target cells. The relative timing and level of expression of each gene in the identified two gene vectors may allow downregulation of the target antigen before the CAR can cause excessive fratricide in the engineered T cells.

[0106]

[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, for example, cloning and / or expression. For example, the nucleotide sequences may be provided as part of a plasmid to facilitate cloning into other plasmids and / or vectors, for example, for transfection into cells (e.g., immune cells). 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.

[0107]

[0116] Nucleic acids can be introduced (directly transduced) into cells using retroviral and lentiviral vector constructs. The term "lentiviral vector" can refer to vectors derived from at least a portion of the lentiviral genome, including, in particular, self-inactivating lentiviral vectors. Other examples of lentiviral vectors that can be used in the clinic include, but are not limited to, Oxford BioMedica's LENTIVECTOR® Gene Delivery Technology and Lentigen's LENTIMAX™ Vector System. Preclinical lentiviral vectors are also available. In other embodiments, nucleic acids can be directly transfected into cells. In yet other embodiments, nucleic acids can be electroporated into cells.

[0108]

[0117] In some embodiments, the vector used is derived from a retrovirus, such as a lentivirus. Such vectors allow for long-term stable integration of an exogenous polynucleotide (e.g., a transgene) and its propagation in daughter cells, making them suitable tools for achieving long-term gene transfer. Unlike vectors derived from oncoretroviruses, lentiviral vectors can transduce non-proliferating cells. Lentiviral vectors may also have low immunogenicity. In other embodiments, the vector is an adenoviral vector. In certain embodiments, the vector is a plasmid.

[0109]

[0118] In some cases, the system or method may include a viral vector encoding a CD7 CAR and a viral vector encoding CD7 PEBL. In some cases, a bicistronic vector encoding a CD7 CAR and CD7 PEBL can be used. Figure 3 shows a schematic diagram of an exemplary bicistronic construct comprising an MSCV promoter-anti-human CD7(TH69)CAR-P2A-anti-human CD7(TH69)PEBL.

[0110]

[0119] The nucleic acid containing the nucleotide sequence 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 localization domain. A single bicistronic construct can be prepared by inserting an internal ribosome entry site (IRES) or a 2A peptide coding region between two cDNAs encoding the chimeric antigen receptors (e.g., CARs) described herein and the binding molecule (e.g., scFvs). In some embodiments, the bicistronic construct contains a CAR upstream of a PEBL with an IRES or a 2A peptide coding region therebetween. In other embodiments, the bicistronic construct contains a PEBL upstream of a CAR with an IRES or a 2A peptide coding region therebetween. Alternatively, separate transduction of the individual constructs (e.g., CAR and PEBL) can be performed (simultaneously or sequentially).

[0111]

[0120] In some embodiments, the bicistronic vector comprises, from the 5' to the 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 examples, the bicistronic vector comprises, from the 5' to the 3' end: an MSCV 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. The nucleic acid sequence includes:

[0112]

[0121] In some embodiments, single promoter bicistronic vectors were used to generate CD7 PEBL-CAR T cells from different starting cells, including bulk PBMCs, purified T cells including 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 an MOI of 10 to generate CD7-CAR+ T cells. In some embodiments, static transduction was performed, where the lentivirus was added directly to the T cells. In some embodiments, a complete medium change was performed after 2 days to remove the lentivirus from the culture.

[0113]

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

[0114]

[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, between about 10% and about 90% of the starting cells may be CD4 positive. In some embodiments, 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 In some cases, the range of about 60% 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% may be CD4 positive.

[0115]

[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, between about 10% and about 90% of the starting cells may be CD8 positive. In some embodiments, 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 In some cases, the range of about 60% 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% may be CD8 positive.

[0116]

[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, between about 10% and about 90% of the starting cells may be CD56 positive. In some embodiments, 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%, Approximately 30% to approximately 50%, approximately 30% to approximately 60%, approximately 30% to approximately 70%, approximately 30% to approximately 80%, approximately 30% to approximately 90%, approximately 35% to approximately 40%, approximately 35% to approximately 45%, approximately 35% to approximately 50%, approximately 35% to approximately 60%, approximately 35% to approximately 70%, approximately 35% to approximately 80%, approximately 35% to approximately 90%, approximately 40% to approximately 45%, approximately 40% to approximately 50%, approximately 40% to approximately 60%, approximately 40% to approximately 70%, approximately 40% to approximately 80%, approximately 40% about 45% 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% may be CD56 positive.

[0117]

[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 starting cells are CD3 positive. In some embodiments, between about 10% and about 90% of the starting cells may be CD3 positive. In some embodiments, 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 In some cases, the range of about 60% 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% may be CD3 positive.

[0118]

[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, between about 10% and about 90% of the starting cells may be CD7 positive. In some embodiments, 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 In some cases, the range of about 60% 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% may be CD7 positive.

[0119]

[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, between about 10% and about 90% of the starting cells may be CD5 positive. In some embodiments, 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 In some cases, the range of about 60% 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% may be CD5 positive.

[0120]

[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, between about 10% and about 90% of the starting cells may be CD38 positive. In some embodiments, 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%, Approximately 30% to approximately 50%, approximately 30% to approximately 60%, approximately 30% to approximately 70%, approximately 30% to approximately 80%, approximately 30% to approximately 90%, approximately 35% to approximately 40%, approximately 35% to approximately 45%, approximately 35% to approximately 50%, approximately 35% to approximately 60%, approximately 35% to approximately 70%, approximately 35% to approximately 80%, approximately 35% to approximately 90%, approximately 40% to approximately 45%, approximately 40% to approximately 50%, approximately 40% to approximately 60%, approximately 40% to approximately 70%, approximately 40% to approximately 80%, approximately 40% about 45% 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% may be CD38 positive.

[0121]

[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, between about 10% and about 90% of the starting cells may be CD2 positive. In some embodiments, 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 In some cases, the range of about 60% 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% may be CD2 positive.

[0122]

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

[0123]

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

[0124] Culture medium

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

[0125] cell

[0134] The methods and systems described herein may include a population of cells. The population of cells (e.g., a population of engineered cells) may include one or more immune cells. In some cases, all or a portion of the population of cells may be immune cells. For example, all or a portion of the population of cells may be T cells. In some cases, all or a portion of the population of cells may be natural killer (NK) cells. In some cases, the population of cells (e.g., engineered cells) may express one or more cell surface polypeptides selected from CD2, CD3, CD4, CD7, CD8, CD28, and / or CD56. In some cases, activated cells may express CD3 and / or CD28. In some cases, cells (e.g., engineered cells) useful in the 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, cells (e.g., engineered cells) useful in the methods and systems described herein may express reduced amounts of one or more cell surface polypeptides selected from CD2, CD3, CD4, CD7, CD8, CD28, and / or CD56 (e.g., compared to non-engineered cells).

[0126]

[0135] In some cases, the cell (e.g., an engineered cell) may express a chimeric antigen receptor (CAR). In some cases, the cell may be engineered to express a CAR (e.g., as described herein), for example, by transducing the cell with a vector having a nucleic acid sequence encoding the CAR. In some cases, the CAR may include a binding domain that binds to a cell surface polypeptide of another cell. In some cases, the binding domain of the CAR may be linked to an activation domain. In some cases, the binding domain of a CAR expressed by a first (e.g., engineered immune) cell binds to a target cell surface polypeptide presented by a second cell, such that the activation domain of the CAR activates the first cell (e.g., induces an activated phenotype in the first cell, thereby inducing cytotoxic activity in the first cell) and can destroy the second cell. In some cases, for example, if a cell expressing a CAR also expresses a cell surface polypeptide, this can lead to fratricide between cells engineered to express a CAR. In some cases, a cell expressing a CAR that binds to a cell surface polypeptide that the cell itself can produce or express may be a cell that is 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 described herein (e.g., engineered immune cells) 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).

[0127]

[0136] In some cases, cells expressing a CAR can be further engineered to reduce expression of the cell surface antigen targeted by the CAR, for example, using PEBL, siRNA, gene disruption, or a dominant-negative allele. In some cases, the cells (e.g., engineered cells) can express a protein expression blocker molecule (PEBL). In some cases, the PEBL can comprise a chimeric targeting polypeptide, e.g., the chimeric targeting polypeptide comprises a binding domain that binds to a cell surface polypeptide (e.g., a cell surface polypeptide that the cell can produce). 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 in a population of cells (e.g., engineered immune cells) described herein can express PEBL (in addition to the CAR, which can be, for example, a CAR with a binding domain that recognizes the same cell surface polypeptide as the binding domain of PEBL). As described herein, cells may express reduced amounts of one or more cell surface polypeptides, such as CD7 (or not express one or more cell surface polypeptides) as a result of the cells expressing PEBL binding to one or more cell surface polypeptides (e.g., in the cytoplasm of the cell). In some cases, 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, PEBL can include an intracellular localization domain. In some cases, PEBL can prevent or reduce cell surface expression or localization of a cell surface polypeptide (e.g., CD7) by binding to the cell surface polypeptide within the cell (e.g., using the binding domain of PEBL) and retaining the cell surface polypeptide within the cell (e.g., using the intracellular localization domain of 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 described herein (e.g., engineered immune cells) can have reduced expression of a cell surface polypeptide (e.g., the cell surface polypeptide is recognized by the binding domain of a CAR expressed by the cells and / or by the binding domain of PEBL expressed by the same cells), e.g., compared to healthy human cells of the same cell type as the population of cells. In some cases, the cells with reduced expression of a cell surface polypeptide comprise a non-natural modification of a gene encoding the cell surface polypeptide.

[0128]

[0137] In some cases, a cell surface polypeptide to which the binding domain of PEBL expressed by a cell (e.g., an engineered cell) can bind can be the same cell surface polypeptide to which the binding domain of a CAR expressed by the same cell can bind. Thus, expression of a CAR and PEBL, which both recognize the same cell surface polypeptide in a population of cells (e.g., the engineered cells described herein), can help prevent or reduce the likelihood of fratricide between the population of cells, for example, by using PEBL to sequester the target of the CAR molecule within the cell. In some cases, this can improve the efficiency of the population of engineered cells in targeting and / or destroying non-engineered cells (e.g., immune cells (e.g., cancer cells) that express a cell surface polypeptide recognized by a CAR), for example, because the engineered cells are less likely to target and destroy each other.

[0129]

[0138] In some cases, the subcellular localization domain is selected from an ER retention signal, a Golgi apparatus retention signal, or a PEST signal. In some cases, the ER retention signal may comprise a KKXX sequence. In some cases, the ER retention signal may comprise a KDEL sequence. In some cases, the chimeric targeting polypeptide (e.g., PEBL) may comprise a spacer sequence between the binding domain and the subcellular localization signal (e.g., the ER retention signal), for example, between the binding domain and the KDEL sequence.

[0130] Chimeric Antigen Receptor

[0139] In some embodiments, the produced T cells may express a receptor. The receptor may be a chimeric antigen receptor (CAR). In certain aspects, the CAR binds to a molecule 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-CD3ζ CAR). In certain embodiments, the immune activating receptor comprises an antibody or antigen-binding fragment thereof (e.g., scFv) that binds to a molecule 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 the CD7 protein.

[0131]

[0140] In some embodiments, the 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.

[0132]

[0141] In some embodiments, the binding domain that binds to the 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). The scFv can be engineered to bind to any protein that is selectively expressed on the surface of a cell. The scFv can be engineered to bind to any protein that is selectively expressed on the surface of a cancer cell. The scFv can be engineered to bind to CD7. ScFv binding to cancer cells can induce activation of CAR T cells. Activation of CAR T cells can kill the cells. Activation of CAR T cells while bound to cancer cells can kill the cancer cells.

[0133]

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

[0134]

[0143] In some embodiments, the CAR comprises an activation domain. The activation domain can activate T cells for cytotoxicity. In some embodiments, the CAR comprises a stimulatory domain, a costimulatory domain, and a binding domain. In some embodiments, the stimulatory domain and the costimulatory domain can be the activation domain. The stimulatory domain and the costimulatory domain can be linked to the binding domain. The binding domain can be an antibody or an scFv. Binding of the scFv of the CAR to a cell that it has been engineered to target can trigger activation of the stimulatory domain and the costimulatory domain of the activation domain. The stimulatory domain can be linked to the costimulatory domain. The costimulatory domain can be linked to the binding domain.

[0135]

[0144] The CAR can include an intracellular signaling domain. The intracellular signaling domain can include a stimulatory region. The stimulatory region can be part of an activation domain. The stimulatory region can be derived from CD3ζ, FcεRIγ, DAP10, DAP12, or other molecules known to deliver activation signals in immune cells. In some embodiments, the stimulatory molecule (in this specific example, CD3ζ) can be replaced with another known stimulatory molecule.

[0136]

[0145] The CAR may comprise a costimulatory region (or domain). The intracellular signaling domain may further comprise a costimulatory region. The costimulatory region may be part of the activation domain. At least one costimulatory domain of the receptor may be a costimulatory molecule such as 4-1BB (also known as CD137), CD28 mutant, OX40, ICOS, CD27, GITR, HVEM, TIM-1, TIM-3, LFA-1, CD2, CD30, CD84, CRTAM, DR3, or SLAMF1. The costimulatory domain may be a functional signaling domain obtained from a protein selected from the group consisting of 4-1BB (also known as CD137), CD28 mutant, OX40, ICOS, CD27, GITR, HVEM, TIM-1, TIM-3, LFA-1, CD2, CD30, CD84, CRTAM, DR3, or SLAMF1. In other embodiments, the costimulatory molecule (in this specific example, 4-1BB) can also be varied with a different costimulatory molecule, for example, CD28.

[0137]

[0146] The CAR may include a linker. The CAR may include a linker between the stimulatory region and the costimulatory region. The linker can enhance the activation of the stimulatory region. The linker can promote the activation of the stimulatory region. The CAR may not include a linker between the stimulatory region and the costimulatory region.

[0138]

[0147] A CAR may comprise a linker between the binding domain (e.g., antigen-binding region or target-binding region) and the intracellular signaling domain. The linker between the binding domain and the intracellular signaling domain (e.g., stimulatory region or costimulatory region) may comprise a transmembrane domain. The transmembrane domain of a CAR may be derived from a single-pass membrane protein, including, but not limited to, CD8α, CD8p, 4-1BB, CD28, CD34, CD4, FcsRIγ, CD16 (e.g., CD16A or CD16B), OX40, CD3ζ, CD3δ, CD3γ, CD35, TCRα, CD32 (e.g., CD32A or CD32B), CD64 (e.g., CD64A, CD64B, or CD64C), VEGFR2, FAS, and FGFR2B. In some cases, the transmembrane protein is not CD8α. The transmembrane domain may also be a non-naturally occurring hydrophobic protein segment.

[0139]

[0148] The linker between the binding domain (e.g., antigen-binding region or target-binding region) and the intracellular signaling domain (e.g., stimulatory region or costimulatory region) can include a hinge domain. The hinge domain of a CAR can be derived from a protein such as CD8α or IgG. The hinge domain can be a fragment of the transmembrane domain or hinge domain of CD8α, or a non-naturally occurring peptide, such as a polypeptide consisting of hydrophilic residues of various lengths, or (GGGGS). n It can be a polypeptide (where n is, for example, an integer from 2 to 12, inclusive).

[0140]

[0149] The CARs described herein can include a linker between the costimulatory region and the binding domain, which can increase the specificity of the CAR.

[0141]

[0150] The engineered immune cells can be CAR-T cells that express a CAR against CD7 and have reduced or no surface expression of CD7. Coexpression of a chimeric antigen receptor (CAR) against CD7 and a protein expression blocker (PEBL) 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 engineered immune cells (e.g., engineered T cells) comprising a bicistronic construct comprising a polynucleotide sequence encoding an anti-CD7 CAR and a polynucleotide sequence encoding anti-CD7 PEBL. In some embodiments, the bicistronic vector can comprise a first nucleic acid sequence encoding a CAR and a second nucleic acid sequence encoding 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 the intracellular signaling domains of 4-IBB and CD3, and an antibody (e.g., a single-chain variable fragment or scFv) that specifically binds to CD7. In some embodiments, the CAR also comprises a CD8α hinge and transmembrane domain. In some embodiments, the anti-CD7 PEBL comprises an antibody that specifically binds to CD7 (e.g., an scFv) and an intracellular localization sequence. In certain embodiments, the anti-CD7 PEBL comprises an antibody that specifically binds to CD7 (e.g., an scFv), a CD8α hinge and transmembrane domain, and an intracellular localization sequence.

[0142]

[0151] In some embodiments, the engineered immune cells are engineered T cells (e.g., engineered cytotoxic T cells, engineered helper T cells, engineered regulatory T cells, engineered effector T cells, engineered memory T cells, engineered natural killer T cells, and engineered gamma delta T cells), engineered natural killer (NK) cells, engineered NK / T cells, engineered monocytes, engineered macrophages, or engineered dendritic cells. In some cases, the engineered immune cells are allogeneic cells. In other cases, the engineered immune cells are autologous cells.

[0143]

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

[0144]

[0153] The engineered cells of the present invention can be expanded in a culture medium under certain 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 methods recognized by those skilled in the art. Before administration to a patient, the engineered cells can be thawed and cultured. In other cases, the engineered cells can be expanded before administration.

[0145]

[0154] In some embodiments, a subject has a reduced likelihood of developing graft-versus-host disease when the engineered immune cells are administered to the subject, and the engineered immune cells are allogeneic to the subject. The engineered immune cells are capable of inducing cytotoxicity of CD7-positive leukemia cells.

[0146] Protein Expression Blocker (PEBL)

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

[0147]

[0156] In some embodiments, the binding domain of PEBL can comprise a molecule that specifically binds to a TCR complex protein, such as TCRα, TCRβ, CD3δ, CD3ε, CD3γ, and CD3ζ. In a further aspect, the PEBL molecule binds to a molecule selected from, for example, CD2, CD4, CD5, CD7, CD8, CD30, CD38, CD45, CD52, or CD127. In some embodiments, PEBL can bind to molecules expressed on the surface of cells, including, but not limited to, CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD28, CD30, CD38, CD45, CD45RA, CD45RO, CD52, CD56, CD57, CD99, CD127, and CD137, which are members of the CD1 family of glycoproteins.

[0148]

[0157] In certain embodiments, PEBL molecules bind to target molecules expressed on the surface of immune cells. In some embodiments, PEBL molecules inhibit the activity or function of the target molecules. By way of example, as disclosed herein, PEBL molecules can be designed to bind, for example, TCRα, TCRβ, CD3 (e.g., CD3ε, CD3γ, CD3δ, or CD3ζ), CD7, CD45, hB2MG, KIR2DL1, KIR2DL2 / DL3, NKG2A, or NKG2D, thereby downregulating cell surface expression of such molecules. Downregulation of such molecules can be achieved, for example, by localizing / targeting the molecule for degradation and / or internalization. In other embodiments, PEBL molecules inactivate the target (e.g., the target can no longer interact with and / or bind to its cognate ligand or receptor).

[0149]

[0158] PEBL comprises a binding domain. The binding domain can increase the 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). The scFv can be engineered to bind to any protein selectively expressed on the surface of a cell. The scFv can be engineered to bind to CD7. CD7 can be transduced into a cell. The scFv can be engineered to bind to CD7 transduced into a cell. The scFv can be engineered to bind to CD7 transduced into a T cell. The scFv can be engineered to bind to newly synthesized surface proteins in the endoplasmic reticulum (ER). The scFv can be engineered to bind to newly synthesized CD7 in the endoplasmic reticulum (ER). When the scFv of PEBL binds to newly synthesized surface proteins in the endoplasmic reticulum (ER), PEBL can retain the newly synthesized surface proteins in the ER. PEBL can keep newly synthesized surface proteins away from the cell surface. PEBL can keep CD7 away from the cell surface. Keeping CD7 away from the cell surface can significantly reduce the probability that the CAR scFv will bind to non-target cells that express CD7. Reducing the probability that the CAR scFv will bind to engineered T cells that express CD7 can reduce the activation of CAR T cells while they are bound to non-target cells. Reducing the activation of CAR T cells while they are bound to non-target cells can reduce the killing of non-target cells, for example, reducing the fratricide of other CAR T cells. Reducing fratricide can avoid reducing the number of CAR T cells. Avoiding the reduction of CAR T cells can avoid reducing the number of CAR T cells that can bind to and kill target cells.

[0150]

[0159] In some embodiments, PEBL does not reduce CAR T cell targeting of cancer cells.In some cases, target cells do not contain PEBL.In some cases, target cells that do not contain PEBL express CD7 on their surface instead of being retained in the ER.CAR T cells can bind to target cells that express CD7 on their surface and activate their destruction.

[0151]

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

[0152]

[0161] The PEBL may comprise a localization domain. The localization domain can direct the polypeptide to a specific cellular compartment, such as the Golgi, the endoplasmic reticulum (ER), the proteasome, or the plasma membrane, depending on the application. The localization domain can be an ER localization sequence. The ER localization sequence can retain newly synthesized surface proteins in the ER. The ER localization sequence can keep newly synthesized surface proteins away from the cell surface. In some examples, the localization domain comprises an amino acid sequence encoded by all or part of the nucleic acid sequence (see Figure 3) of a viral vector transduced into a cell, as described herein.

[0153]

[0162] In some embodiments, PEBL can contain one or more localization 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 localization domains linked to one another. When more than one localization domain is used in a single PEBL molecule, each localization domain can be linked with or without an intervening linker. In some examples, a localization domain such as a CD8α transmembrane domain, a KDEL motif, and a linker can be used in a single PEBL molecule. In some embodiments, the localization domain does not contain any intervening linker. In other embodiments, various intervening linkers can be incorporated between some or all of the localization domains.

[0154]

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

[0155]

[0164] The transmembrane domain of PEBL (e.g., PEBL comprising a KKXX ER retention domain) can include a transmembrane domain derived from CD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16, OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, or FGFR2B. In certain embodiments, the transmembrane domain of the localization domain is derived from CD8α. The transmembrane domain can be linked to the retention domain. In some embodiments, the transmembrane domain is linked to the retention domain via a linker.

[0156]

[0165] PEBL may include a linker. In some cases, the linker of PEBL can couple the target binding domain to the localization domain (e.g., KDEL ER retention domain). In some cases, the linker connecting the localization domain and the target binding domain of PEBL can improve the localization activity of PEBL. Non-limiting examples of linkers include (GS) n , (GGS) n , (Gly3Ser) n , (Gly2SerGly) n , (Gly2SerGly2) n , or (Gly4Ser) n where 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 linker length can retain or enhance activity, resulting in improved retention.

[0157]

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

[0158]

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

[0159] definition

[0168] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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.

[0160]

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

[0161]

[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.

[0162]

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

[0163]

[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 natural, 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 one or both strands of the molecule. As used herein, nucleic acid and polynucleotide are interchangeable.

[0164]

[0173] The term "nucleotide sequence," with respect to nucleic acids, refers to a series of nucleotides linked by covalent bonds, such as phosphorus linkages (e.g., phosphodiester, alkyl and aryl-phosphonate, phosphorothioate, phosphotriester linkages), and / or non-phosphorus linkages (e.g., peptide and / or sulfamate linkages). In certain embodiments, for example, the nucleotide sequence encoding the target binding molecule linked to the localization domain is a heterologous sequence (e.g., a gene originating from a different species or cell type).

[0165]

[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. Thus, nucleotides can include, for example, nucleotides containing naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or deoxycytidine) as well as modified bases known in the art.

[0166]

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

[0167]

[0176] The term "sequence identity" means that two nucleotide sequences or two amino acid sequences share at least, for example, 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, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights. For sequence comparison, typically, one sequence serves as a reference sequence (e.g., parent sequence) to which a test sequence is 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 of the test sequence relative to the reference sequence based on the designated program parameters.

[0168]

[0177] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mal. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Nat'l. ACAD Sci. USA 85:2444 (1988), 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 visual inspection (see generally Ausubel et al., Current Protocols in Molecular Biology). One example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J Mal. Biol. 215:403 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (publicly accessible through the National Institutes of Health NCBI Internet server). Typically, default program parameters can be used to perform sequence comparisons, although customized parameters can also be used. For amino acid sequences, the BLASTP program uses as default a word length (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 one of skill in the art, in some embodiments, the nucleic acid further comprises plasmid sequences, which may include, for example, one or more of a promoter sequence, a selectable marker sequence, or a gene targeting sequence.

[0169]

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

[0170]

[0179] The term "retroviral vector" may refer to a gammaretroviral vector. Retroviral vectors may include, for example, a promoter, a packaging signal, a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a polynucleotide of interest, such as a polynucleotide encoding a CAR and a polynucleotide encoding a PEBL. Retroviral vectors may lack viral structural genes such as gag, pol, and env. Exemplary retroviral (e.g., gammaretroviral) vectors include mouse 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, for example, in Maetzig et al., Viruses, 2011;3(6):677-713.

[0171]

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

[0172]

[0181] Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence. Nucleic acids can be cloned into several types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0173]

[0182] An "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains 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.

[0174]

[0183] Furthermore, the expression vector can be provided to cells 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), as well as other virology and molecular biology manuals. Viruses 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).

[0175]

[0184] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30–110 bp upstream of the start site, although some promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, preserving promoter function when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by up to 50 bp before activity begins to decline. Depending on the promoter, individual elements may function cooperatively or independently to activate transcription. Exemplary promoters include the immediate-early cytomegalovirus (CMV), EF1a, ubiquitin C, or phosphoglycerokinase (PGK) promoters. Strong constitutive promoter sequences capable of driving high levels of expression of any operably linked polynucleotide sequence can be used. Other constitutive promoter sequences may also 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, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, elongation factor I, Ovian leukemia virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and the like. In some embodiments, the promoter is an inducible promoter, providing a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0176]

[0185] As used herein, "antibody" refers to an intact antibody or an antigen-binding fragment of an antibody, including an intact antibody or an antigen-binding fragment that has been engineered or modified, or is a human antibody. Examples of engineered or modified antibodies 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'), Fv, single-chain antibodies (e.g., scFv), minibodies, and diabodies.

[0177]

[0186] When referring to a protein or peptide, the terms "specifically (or selectively) bind" or "specifically (or selectively) immunoreactive" often refer to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biologics. Thus, under specified immunoassay conditions, a specified antibody binds to a particular protein at least twice above background, more typically 10-100 times above background. Specific binding to an antibody under such conditions requires that the antibody be selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only polyclonal antibodies that are specifically immunoreactive with a selected antigen and not with other proteins. This selection can be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats can 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).

[0178]

[0187] In certain embodiments, the antibody that binds to CD7 is a single-chain variable fragment antibody ("scFv antibody"). scFv refers to an antibody fragment comprising the VH 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 scFvs, 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 will be appreciated by those of skill in the art, a variety of suitable linkers can be designed and tested for optimal function, as provided in the art and as disclosed herein.

[0179]

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

[0180]

[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 that 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, the term simply refers to cells 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.

[0181]

[0190] As used herein, "CD7 CAR+ / CD7" T cells refer to T cells that express a chimeric antigen receptor for human CD7 and have 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 PEBL against human CD7, which reduces endogenous CD7 protein delivery to the surface of the T cell. In some examples, surface expression of CD7 can be determined using standard methods known to those skilled in the art, such as, but not limited to, immunocytochemistry, flow cytometry, or fluorescence-activated cell sorting (FACS).

[0182]

[0191] As used herein, the term "autologous" and its grammatical equivalents can refer to something that is derived from the same thing. For example, a sample (e.g., cells) can be removed, processed, and later returned to the same subject (e.g., patient). An autologous process is distinguished from an allogeneic process in which the donor and recipient are different subjects.

[0183]

[0192] As used herein, the terms "treat," "treating," or "treatment" refer to alleviating a medical condition (e.g., a condition associated with a T-cell malignancy) to the extent that the medical condition improves according to clinically accepted standards.

[0184]

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

[0185]

[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 in the subject (treat a condition associated with a T-cell malignancy) under the conditions of administration. The effective amount of an agent to be administered can be determined by an ordinary clinician using the guidance provided herein and other methods known in the art, and will depend on several factors, including, for example, the particular agent selected, the subject's age, sensitivity, drug tolerance, and overall well-being. [Example]

[0186] Example

[0195] The following examples illustrate embodiments described herein and should not be construed as limiting the scope of the present disclosure. To the extent that specific materials are mentioned, they are for illustrative purposes only and are not intended to be limiting. Those skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the present disclosure.

[0187] Example 1

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

[0188]

[0197] Concentrated apheresis products (e.g., leukopaks) containing leukocytes extracted from peripheral blood of healthy human subjects were obtained. Healthy donor leukopaks (LP) were purchased from BioIVT and requested to be delivered within 24 hours of collection at a low transport temperature (<15°C). The collected apheresis volumes ranged from 116 to 279 mL. LPS was delivered on-site and processed within 24 hours of collection (only one case resulted in delivery beyond 48 hours of collection due to a courier issue, in which the blood was not processed). A complete blood count (CBC) with differential was performed at the collection site. Eleven unique donors were used for 10 PD runs and two confirmatory runs. Total leukocyte count and viability of the concentrated apheresis products were determined using NC-200 Solution 17 blood lysis buffer (ChemoMetec) and a NucleoCounter® NC-200 analyzer (ChemoMetec).

[0189]

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

[0190] Example 2

[0199] This example illustrates the selection (eg, enrichment) of cell reagents for CD4 and CD8 expression for use, for example, with the methods and systems described herein.

[0191]

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

[0192]

[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 Biologics) was used in all studies, only in the process of supplementing the PBS / EDTA buffer at a final concentration of 0.5% (v / v). TS 520 tubing sets (Miltenyi Biotec) were used for all isolations.

[0193]

[0202] For all studies, CD4 and CD8 T cells were selected using the CliniMACS Prodigy® "TCT Full Process" program (Miltenyi Biotec). LPs were loaded into the CliniMACS Prodigy® and the "TCT Full Process" program was selected using the two-vial option. The WBC density from the collection site COA or XN-330 Sysmex readout was used along with the volume provided by the collection site for the total WBC input into the system. Because the actual T cell frequency was unknown at the time of LP receipt, the process entered T cell frequency by dividing the theoretical maximum T cell enrichment capacity of the CliniMACS Prodigy® (3E9) by the total WBC count. Using this method, a maximum selection cycle (3 times) was performed, taking approximately 3 hours. At the end of selection, culture was initiated in the CliniMACS Prodigy® CentriCult chamber (IPR1, IPR2, Run 1) or the process was terminated and the enriched cell bag was heat-sealed and removed from the instrument for further processing.

[0194]

[0203] For every run, a portion of the isolated cells on day 0 was seeded into a 6-well plate to serve as a non-transduced or negative control for flow analysis.

[0195] Example 3

[0204] This example illustrates activation of cellular reagents for use, for example, with the methods and systems described herein.

[0196]

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

[0197]

[0206] CD4 / CD8 enriched cell populations were seeded into two 100M-CS G-Rex vessels in 100 mL of culture medium containing 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).

[0198]

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

[0199] Example 4

[0208] This example demonstrates the use of a bicistronic expression vector in T cells to express an anti-CD7 CAR and reduce CD7 expression. Cells were transduced with MSCV promoter-anti-human CD7 (TH69) CAR-P2A-anti-human CD7 (TH69) PEBL (see, e.g., Figure 3) lentivirus. Research-grade lentiviral vectors (LVV) were stored at -80°C until use. Two different lots were used during the study. Lot X was used in IPR1 and delivered at 2.15 E8 infectious particles (IP) / mL. Lot Y was used in IPR2. Lot Y delivered at 1.53 E8 infectious particles (IP) / mL. Transduction generated a population of CD7 CAR+ / CD7-neg T cells. Figure 6 shows the expansion and enrichment of CD7 CAR+ / CD7-neg T cells over time from day 0 to day 9 post-transduction. For example, on 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. On 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. On 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. On 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 CD7+ cells observed on day 3 are also highly susceptible to fratricide.

[0200]

[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 exhibited antigen-specific T cell functional responses, such as IFNγ secretion, and specific toxicity against CD7+ target cell lines. PEBL-CAR-T cells exhibited a high percentage purity of CD7-negative, CAR+ T cells.

[0201] Example 5

[0210] This example demonstrates cryopreservation of engineered cells, which can be used, for example, in optimizing the methods and systems described herein.

[0202]

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

[0203]

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

[0204] Example 6

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

[0205]

[0214] Three runs (IPR1, IPR2, and Run 1) were performed to establish a 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™, which was added to the CentriCult chamber using a syringe to withdraw the required volume and transferred to a transfer pack (IPR1 and IPR2), or the entire TransAct™ vial was attached to the appropriate line (Run 1).

[0206]

[0215] For IPR1, the chamber temperature was set at 37°C for the first 6 days. Subsequently, the chamber temperature was set at 39°C, with a correction setting the internal culture temperature at 37°C. The chamber temperature was set at 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 time points were grown in 6-well plates in a 37°C, 5% CO2 incubator alongside each of the CliniMACS Prodigy® runs. Medium was prepared fresh for the runs every 2–3 days, as needed. IL-2 for the process was reconstituted on day 0, stored at 4°C for up to 2 weeks, and added to the medium once prepared.

[0207]

[0216] On day 2, a 50% medium exchange was performed by programming a volume reduction to 35 mL followed by the addition of 35 mL of fresh medium. Samples were taken for cell count and viability (CCV) analysis, and the lentiviral vector (LVV) volume was determined using an MOI of 10. The appropriate volume of LVV was transferred to a 150 mL transfer pack and further diluted with fresh medium to a total volume of 10 mL. The transfer pack was welded to the LVV addition line of the tubing set. The Prodigy® was programmed to perform LVV 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, bringing the total culture volume to 100 mL.

[0208]

[0217] On day 4, a culture wash (cycle: 1) was performed to remove the lentiviral vector and bring the total culture volume to 200 mL. The shaker was turned on at different times depending on the run: in IPR1, the shaker was turned on on day 12; in IPR2, the shaker was turned on on day 5; and in Run 1, the shaker was turned on on day 8 if the percentage of CD7-negative cells was 85% or higher. Medium changes were also performed at different times and frequencies depending on the run: in IPR1, medium changes were performed on days 8, 10, 11, and 13; in IPR2, medium changes were performed on days 8 and 10; and in Run 1, medium changes were performed on days 6, 8, 10, and 12. Samples for cell count and viability were obtained on the day of medium change. After transduction, samples were obtained for flow analysis. For Run 1, samples were obtained from the cultures for metabolite analysis on the day of medium change.

[0209]

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

[0210]

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

[0211]

[0220] Starting cell number, TransAct™ volume, chamber temperature, shaker settings, activation time point, feeding schedule, and culture duration were varied across three runs to achieve the target criteria (see Methods for a description of the activity matrix for each run). However, none of the runs met the complete set of target criteria.

[0212]

[0221] In IPR1, cells were seeded and activated in a Prodigy® CentriCult chamber in a total volume of 70 mL. On day 2, the total transduced cells were 6.66 E7. On day 5, the transduction efficiency of the Prodigy® culture was 25.2% CD3+CD56-CAR+CD7- and 40.5% CD3+CD56-CD7-, meeting the target marker expression criteria on day 5. 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 was set at 37°C when the Miltenyi instructions indicated 39°C due to a 2°C temperature offset. Therefore, the chamber temperature was adjusted from 37°C to 39°C to follow the vendor's recommendations. Samples for CCV were obtained from day 5 onwards, and medium changes were 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 day 8 onward, cell expansion resulted in >5.0 E5 cells / mL in a maximum of 250 mL. Therefore, 70% medium changes were performed on days 10, 11, and 13. By day 10, the %CD3+CD56-CAR+CD7- and %CD3+CD56-CD7- populations had increased to 67.5% and 97.3%, respectively, suggesting ongoing fratricide. Because cell expansion appeared to plateau between days 10 and 12, the shaker (type 1 setting) was activated to improve aeration of the culture for larger cell expansions. However, this only improved expansion by 1.3-fold on day 13, and it was assumed that the culture period was too long to observe any benefit of activating the shaker. Nevertheless, the total viable cells at day 14 for IPR1 was 3.30 E8, with 86.1% viable cells, 42.4% CD3+CD56-CAR+CD7-, and 98.8% CD3+CD56-CD7-, which did not meet the set target criteria, particularly failing the cell expansion target.

[0213]

[0222] To improve on the results of IPR1, IPR2 was performed similarly from days 0 to 4, except that the shaker was turned on on day 5 (Type 1 setting) to provide sufficient aeration for early cell expansion. A portion of the Prodigy® culture (<4 mL) was removed during sampling and cultured in a 6-well plate as a static control arm on day 5. The total viable cell count on day 2 was 8.05 E7, meeting the target criteria. The transduction efficiency on day 5 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 the lower cell count was due to cells undergoing fratricide. By day 8, the total cell count had decreased by approximately 14%, and viability had also decreased by 10%. The volume was reduced to 236 mL to maintain a minimum density of 5.0 E5 cells / mL. However, by day 9, cell numbers had decreased by more than 50% from day 8, and viability had also decreased. We hypothesized that early activation of the shaker while cells were undergoing fratricide was detrimental to cell health and caused the decline. The shaker was stopped on day 9. By day 11, total cell numbers had further decreased, and viability was 49%. Although viability and cell numbers were low, CD3+CD56-CAR+CD7- cells were 77.4% and CD3+CD56-CD7- cells were 87%, meeting the target criteria for CAR and CD7 expression. The bench-scale static control arm expanded 1.85-fold from day 5 to day 11, with a viability of 78.8%, further suggesting that activation of the shaker resulted in cell death.

[0214]

[0223] Based on IPR1 and IPR2, adjustments were made for Run 1. The target starting cell number was increased to 2.0E8 to meet the final cell yield. After the standard Miltenyi TCT process, a culture wash step was used to maintain lentiviral vector clearance on Day 4, with subsequent medium changes performed on Days 6, 8, 10, and 12. Finally, the shaker would be turned on on Day 8 based on a CD3+CD56-CD7- count of ≥85%. This was based on the assumption that cells are shear-sensitive when the culture is undergoing fratricide. Additionally, the culture volume was limited to 200 mL while static to allow for faster oxygen diffusion throughout the culture for improved expansion. With these changes, the total cell yield was met by Day 6; however, the lowest transduction efficiency of all runs was observed (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 non-transduced cells were expanding and that fratricide was not occurring. Because the CD3+CD56-CD7- frequency on day 8 did not meet criteria, the shaker was not started. The culture continued to expand to 7.25 E8 total cells on day 14, but by harvest, the CD3+CD56-CAR+CD7- frequency was less than 1%. Thus, Run 1 did not meet the target criteria for %CD3+CD56-CAR+CD7-, %CD3+CD56-CD7-, and %CD3+CD56-CAR+CD7- yields.

[0215] [Table 1]

[0216] [Table 2]

[0217] [Table 3]

[0218] [Table 4]

[0219]

[0224] Starting cell number, chamber temperature, shaker settings, start-up time point, feeding schedule, and culture duration were varied among the three runs to achieve the target criteria set out in Table 5. However, as shown in Figures 7A-7D, none of the runs met the complete set of target criteria in Table 5.

[0220] [Table 5]

[0221]

[0225] Target criteria for IPR1, IPR2, and Run 1 were: total viable cells: 4.00 E8; viability ≥ 50%; CD3 + CD56 - Percentage of cells ≥ 90%; CD3 + CD56 - CD7 - Percentage of cells ≥ 90%; CD3 + CD56 - CAR + CD7 - The percentage of cells was ≥ 60%.

[0222]

[0226] The total viable cell count of 7.00 E7 did not meet the cell expansion criteria. Samples for cell count and viability were obtained from day 5 onwards, and medium changes were 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 day 8 onwards, cell expansion resulted in >5.0 E5 cells / mL up to 250 mL. Therefore, 70% medium changes were performed on days 10, 11, and 13. Cell expansion appeared to plateau between days 10 and 12, and the shaker (type 1 setting) was activated to improve aeration of the culture for larger cell expansion. However, this only improved expansion by 1.3-fold on day 13, and it was assumed that the culture period was too long to observe any benefit of activating the shaker. The total viable cells on day 14 for IPR1 were 3.30 E8, 86.1% viable, and 42.4% CD3 + CD56 - CAR + CD7 - , and 98.8% CD3 + CD56 - CD7 - , which did not meet the established target criteria, in particular failed the cell expansion target.

[0223]

[0227] Overall, only IPR1 significantly improved the overall survival of total viable cells, viability, and %CD3 + CD56 - CAR + CD7 - cells, and %CD3 + CD56 - CD7 - Based on three runs, CD3 + CD56 - CAR + CD7 -The two main bottlenecks to achieving the target yield appear to be transduction efficiency and expansion. As observed in Run 1, the transduction efficiency was not high enough to achieve complete sibicide by the end of the culture. As observed in IPR1 and IPR2, when the transduction efficiency met the target criterion on day 5, expansion of cells by harvesting did not meet the target criterion. Starting the shaker early in the culture to improve expansion while cells were still undergoing sibicide was detrimental. The feeding strategies evaluated also did not improve expansion. This process could be improved to achieve a threshold where the population of transduced cells could overtake non-transduced cells during sibicide.

[0224] Example 7

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

[0225]

[0229] In Run 2, T cells enriched by magnetic-activated cell sorting were seeded into four medium-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 supplemental feeding strategy (arm G1) and 75% medium changes performed on days 8 and 11 (arm G3). Arms G1–G5 of Run 2 used culture vessels with gas-permeable membranes for cell incubation, in contrast to Run 1, which did not use vessels with gas-permeable membranes for culture. Following the manufacturer's recommendations, an arm was also set up to prevent sampling or resuspension of the culture from day 4 onward until harvest (arm G2). Arms G4 and G5 used larger culture medium volumes (G-Rex 100M scale version, with a 1000 mL culture medium volume capacity) than Arms G1 and G3 (each of which used a G-Rex 10M scale culture vessel and had a 100 mL culture medium volume capacity), respectively, and the medium volume, cell number, and TransAct™ volume were 10 times higher. On day 0, cells were activated using TransAct™. On day 2, a 50% medium change was performed. For all arms, cells were transduced at an MOI of 10. The transduction volume was 10 mL in the 10M vessel and 100 mL in the 100M vessel. On day 4, to remove the lentiviral vector, the culture was transferred to a conical tube, the cells were centrifuged, 75% of the supernatant was removed, and a 75% medium change was performed by replenishing either 20 mL in the 10M vessel or 200 mL in the 100M vessel with fresh medium. On day 5, the culture volume was replenished to 50 mL in 10 M vessels or 500 mL in 100 M vessels. On day 8, depending on the arm, either a 75% medium change or a medium replenishment was performed. On day 11, depending on the arm, either IL-2 was added as a bolus or a 75% medium change was performed. IL-2 was added as a bolus to minimize disturbance to the cells for optimal expansion. All cultures were terminated on day 14.Samples were taken on days 2, 4, 5, 8, 11, and 14 for cell counts and viability, and metabolites for all arms except arm G2, which was the minimally perturbed arm and was sampled only 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 sampled only at the time of harvest.

[0226] result

[0230] Due to the challenges of developing a fully closed and automated process using the Prodigy® system, a gas-permeable membrane vessel was evaluated as an alternative culture system and compared with the Prodigy® and bench-scale processes. The surface area of ​​the 10 M is 1 / 10 (100 cm) of the Prodigy® chamber. 2 ) cells were transduced on day 2 and the LVV was removed on day 4. A minimally disturbed feeding strategy was used. Care was taken to minimize disturbance of the cells at the membrane where oxygen diffusion occurs. 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 G1, G3, G4, and G5 were similar, with CD3 counts ranging from 7.4 to 9.3%. + CD56 - CAR + CD7 - and 11.0-15.0% CD3 + CD56 - CD7 - was within the range.

[0227]

[0231] CD3 with reduced CD7 expression + CD56 - CD7 - The percentage of T cells increased over time, reaching 100% by day 14 for all arms. Figure 8C. The loss of CD7+ cells suggests that anti-CD7 CAR T cells killed CD7+ cells (fratricide). CD3 T cells expressing anti-CD7 CAR + CD56 - CAR + CD7- The percentage of cells increased to 80%-90% in some arms, but then decreased in the population with the highest expression (Figure 8D).

[0228]

[0232] As can be seen in Figure 8C, CD3 + CD56 - CD7 - and CD3 + CD56 - CAR + CD7 - The percentage of cells with the surface marker profile was approximately 100% and 85%, respectively, for Arm 1 at day 8, while all other Run 2 arms had less than 60% for the same surface marker expression profile at day 8. The bench-scale control process arm (Run 2 Arm 1) had the highest CD3 expression at this time point. + CD56 - CAR + CD7 - (Figure 8D) and CD3 + CD56 - CD7 - (Figure 8C) suggesting that the transduction efficiency in G-Rex vessels can still be further optimized. For G-Rex arms G1, G3, G4, and G5, CD3 + CD56 - CAR + CD7 - and CD7 - The frequency continued to increase until day 11. As shown in Figure 8D, arms G4 and G5 also showed CD3 + CD56 - CAR + CD7 - The frequency continued to increase until day 14. For all arms, a decrease in viability, likely representing the occurrence of fratricide, was observed between days 5 and 8. Viability recovered until harvest after day 11. Figure 8B.

[0229] [Table 6]

[0230] [Table 7]

[0231] [Table 8]

[0232]

[0233] The 75% medium exchange arms (G3, G5) had up to 10-fold greater expansion compared to the medium-fed arms (G1, G4). + CD56 - CAR + CD7 - The frequencies were similar between the two feeding strategies at the 10M scale, but at the 100M scale, the medium exchange arm had approximately 30% greater CD3 than the supplemented arm. + CD56 - CAR + CD7 - The least perturbed arm (G2) had the lowest yield and the second lowest %CD3 on the day of harvest, suggesting that medium exchange at the 100M scale was better for both expansion and fratricide. + CD56 - CAR + CD7 - , suggesting that mixing of cells in culture supports fratricide and subsequent expansion.

[0233] [Table 9]

[0234] As shown in Figures 9A-9D, metabolite profiles and pH were very similar between G-Rex arms. The medium exchange arms (arms G3 and G5) had lower lactate and ammonium levels due to removal of spent medium and replacement with fresh medium. The accumulation of lactate and ammonium and the decrease in pH between days 11 and 14 suggest the need for additional medium exchanges beyond day 11.

[0235]

[0235] Two 100M scale arms were able to establish a baseline process in the G-REX culture system suitable for closed system manufacturing.

[0236] Example 8

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

[0237]

[0237] The manufacturing process shown in Figure 1 involves magnetic isolation of CD4+ and CD8+ T cells from fresh, non-mobilized apheresis units, activation and culture of the isolated T cells in a container with a gas-permeable membrane, lentiviral vector transduction, cell expansion by medium replenishment, and then medium exchange.

[0238]

[0238] On day 0, the cells underwent leukocyte removal, NC-200 Solution 17 count for total viable leukocyte count, 8 L of TexMACS medium supplemented with IL-2 was prepared for the entire process, CD4 and CD8 T cells were enriched using 3 cycles of Prodigy®, cell count and viability testing was performed for G-Rex inoculation, and cell activation was performed.

[0239]

[0239] Day 3 involved harvesting and pooling the vessels to reduce the volume to 50 mL using Sepax, performing cell counts and viability measurements after Sepax, adding lentiviral vector based on post-Sepax cell counts at an MOI of 10, re-inoculating the cells into two G-Rex vessels, rinsing the transfer pack with 30 mL of media, and rinsing the tubing of each G-Rex vessel with 15 mL of media from the transfer pack rinse, obtaining approximately 40 mL per vessel after re-inoculation.

[0240]

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

[0241]

[0241] On day 6, the culture volume was reduced to 250 mL, the culture vessel was swirled to redistribute the cells, samples were removed for cell count and viability determination and flow assay, and 750 mL of fresh medium was added for a total of 1 L of 75% medium change.

[0242]

[0242] On day 9, the culture volume was reduced to 250 mL, the culture vessel was swirled to redistribute the cells, samples were removed for cell count and viability testing and flow assays, and 750 mL of fresh medium was added for a total of 1 L of 75% medium change.

[0243]

[0243] Day 11 involved reducing the culture volume to 100-130 mL, swirling the vessel to redistribute the cells, removing samples for cell count and viability determination and flow assay, pooling the cultures for concentration, washing, and formulation in Plasma-Lyte A using Sepax to >2x cryodensity, filtering the cells through a 40 μm filter after Sepax, adjusting the volume to meet 2x cryodensity, adding an equivalent volume of CS10 to achieve 1x cryodensity, filling cryopreservation bags and vials with the cell-containing cryopreservation mix, and cryopreserving the cells using a Planer CRF cryocontainer.

[0244] This example demonstrates a robust, closed-system, 12-day process for the manufacture of fratricidal CAR T cells. This process is suitable for clinical CAR-T manufacturing when fresh or frozen apheresis starting material is processed to isolate CD4 and CD8 T cells in a fully automated, closed-system instrument, and transduction conditions are optimized for the day 3 transduction process. Yield and CD3 + CD56 - CAR + CD7 -Scale-up of up to 100M G-Rex vessels was performed while meeting target criteria for frequency. The process presented here streamlines the manufacturing process and reduces sweet time, including removing TransAct™ on day 3 with transduction instead of day 2, diluting the lentiviral vector on day 4 with medium replenishment instead of medium change, minimal medium change points, processing two vessels together at transduction and harvest instead of separately, and preparing and aliquoting process medium on day 0 instead of preparing fresh medium throughout the process.

[0245] Example 9

[0245] This example demonstrates the administration of engineered cells described herein to a patient in need of treatment by the 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 the methods described herein, is prepared from a freshly prepared or cryopreserved sample according to the methods described herein, for example, using the methods and reagents described in one or more of Examples 1-8. The cells are formulated as a pharmaceutically acceptable composition and administered to a patient in need of treatment at 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), produced as described herein, are thawed, diluted with culture medium as described in Example 8, and resuspended in saline. The cells are administered by intravenous infusion to a subject with acute lymphocytic leukemia (ALL) cancer at a dose of 1 x 10 cells / kg to 10 x 10 cells / kg. Alternatively, the engineered cells can be administered at a dose less than 1 x 10 cells / kg or greater than 10 x 10 cells / kg.

[0248]

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

Claims

1. A cell population comprising: At least 10 ８ immune cells, At least 70% are viable, A population of cells, at least 50% of which express a chimeric antigen receptor (CAR) comprising a binding domain that binds to a cell surface polypeptide expressed by said immune cell linked to an activation domain.

2. The at least 10 ８ 2. The cell population of claim 1, wherein at least 80% of the immune cells are viable.

3. The at least 10 ８ 3. The cell population of claim 1 or 2, wherein at least 60% of the immune cells express the CAR.

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

6. 10. 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 comprising a binding domain that binds to the cell surface polypeptide linked to an intracellular localization domain.

7. The cell population of claim 6 , wherein the intracellular localization 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, where X is any amino acid.

9. The cell population of claim 7 , wherein the ER retention signal comprises a KDEL sequence.

10. 10. The cell population of claim 9, wherein the chimeric targeting polypeptide further comprises a spacer sequence between the binding domain and the KDEL sequence.

11. The cell population according to any one of claims 1 to 10, wherein the immune cells are T cells.

12. The cell population according to any one of claims 1 to 10, wherein the immune cells are NK cells.

13. The cell population according to any one of claims 1 to 12, wherein the immune cells are frozen.

14. A pharmaceutical preparation comprising the cell population of any one of claims 1 to 13.

15. incubating cells susceptible to fratricide in a culture medium without continuous agitation in a vessel equipped with a gas-permeable membrane; The method, wherein at least 70% of the cells are viable after 5 days of incubation.

16. The cells (i) a binding domain that binds to a cell surface polypeptide expressed by the cell; and (ii) an activation domain that activates the cell; and 16. The method of claim 15, comprising a chimeric antigen receptor (CAR) comprising:

17. 17. The method of claim 16, wherein the cell has reduced expression of the cell surface polypeptide compared to a healthy human cell of the same cell type as the cell.

18. 18. The method of claim 17, wherein the cell comprises a non-naturally occurring modification of the gene encoding the polypeptide.

19. 18. The method of claim 17, wherein the cell further comprises a chimeric targeting polypeptide comprising a domain that binds to the cell surface polypeptide linked to an intracellular localization domain.

20. The method of any one of claims 15 to 19, wherein the cells susceptible to fratricide comprise immune cells.

21. 21. The method of claim 20, wherein the immune cells comprise T cells.

22. 21. The method of claim 20, wherein the immune cells comprise NK cells.

23. The method of any one of claims 16 to 22, wherein the activation domain induces cytotoxic activity of the immune cell.

24. The method of any one of claims 16 to 23, wherein the cell surface polypeptide is CD7.

25. 25. The method of any one of claims 15 to 24, further comprising replacing at least a portion of the culture medium in the vessel.

26. 26. The method of claim 25, wherein said replacing is performed every 1 to 3 days.

27. 27. The method of claim 25 or 26, wherein said replacing comprises replacing at least 75% of the culture medium in said vessel.

28. 28. The method of any one of claims 15 to 27, wherein the vessel contains 100 mL to 1 L of the culture medium.

29. 29. The method of any one of claims 15 to 28, wherein the concentration in the culture medium of a metabolite produced by the cells is maintained below a threshold level for a period of time.

30. 30. The method of claim 29, wherein the metabolite is lactate.

31. 31. The method of claim 30, wherein the threshold level is 14 mM.

32. 30. The method of claim 29, wherein the metabolite is ammonium.

33. 33. The method of claim 32, wherein the threshold level is 1.2 mM.

34. The method of any one of claims 29 to 33, wherein the time interval is at least 7 days of culture.

35. 35. The method of any one of claims 15 to 34, further comprising disrupting the cell clumps.

36. 36. The method of claim 35, wherein said disintegrating comprises mechanically disintegrating said agglomerates.

37. 37. The method of claim 35 or 36, wherein the replacing and the disrupting are performed at regular intervals.

38. 38. The method of any one of claims 35 to 37, wherein the disintegrating is performed every 1 to 3 days.

39. 39. The method of any one of claims 15 to 38, further comprising mixing the culture medium in the vessel.

40. 40. The method of any one of claims 15 to 39, further comprising activating the cells.

41. 41. The method of claim 40, wherein said activating comprises contacting said cells with an antibody that binds to CD3 or CD28.

42. 41. The method of claim 40, wherein said activating comprises contacting said cells with a plurality of antibodies that bind to CD3 and CD28.

43. 43. The method of any one of claims 15 to 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.

44. 44. The method of Claim 43, wherein the viral vector comprises a nucleotide sequence encoding the CAR or the chimeric targeting polypeptide.

45. 44. The method of Claim 43, wherein the viral vector comprises a nucleotide sequence encoding the CAR and the chimeric targeting polypeptide.

46. 45. The method of claim 43 or 44, wherein the starting cells are obtained from a human subject.

47. 47. The method of claim 46, wherein the human subject is a healthy human subject.

48. 47. The method of claim 46, wherein the human subject has been diagnosed with cancer.

49. 49. The method of claim 48, wherein the cancer is a T-cell lymphoma or leukemia.

50. 47. The method of claim 46, wherein the human subject has been diagnosed with an autoimmune disease.

51. 51. The method of any one of claims 43 to 50, wherein at least 20% of the starting cells are CD4 positive.

52. 52. The method of claim 51, wherein at least 80% of the starting cells are CD4 positive.

53. 53. The method of any one of claims 43 to 52, wherein the starting cells are enriched for CD4 positive cells.

54. 51. The method of any one of claims 43 to 50, wherein at least 20% of the starting cells are CD8 positive.

55. 55. The method of claim 54, wherein at least 80% of the starting cells are CD8 positive.

56. 56. The method of any one of claims 43 to 55, wherein the starting cells are enriched for CD8 positive cells.

57. 51. The method of any one of claims 43 to 50, wherein at least 20% of the starting cells are CD3 positive.

58. 58. The method of claim 57, wherein at least 80% of the starting cells are CD3 positive.

59. 59. The method of any one of claims 43 to 58, wherein the starting cells are enriched for CD3 positive cells.

60. 51. The method of any one of claims 43 to 50, wherein at least 20% of the starting cells are CD56 positive.

61. 61. The method of claim 60, wherein at least 80% of the starting cells are CD56 positive.

62. 62. The method of any one of claims 43 to 61, wherein the starting cells are enriched for CD56 positive cells.

63. 62. The method of any one of claims 43 to 61, further comprising selecting from the transduced cells the cells susceptible to fratricide before said incubating.

64. 64. The method of claim 63, wherein said selecting comprises isolating cells that express said CAR.

65. 65. The method of claim 63 or 64, wherein said selecting comprises isolating cells that express CD4 or CD8.

66. 66. The method of any one of claims 63 to 65, wherein said selecting comprises isolating cells that express CD25 and CD69 after activation.

67. 67. The method of any one of claims 63 to 66, wherein at least 80% of the selected cells are positive for surface expression of CD3.

68. 68. 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 unmodified cells of the same cell type from a healthy human subject.

69. 69. The method of any one of claims 63 to 68, wherein at least 80% of the selected cells are negative for surface expression of CD56.

70. 70. The method of any one of claims 63 to 69, wherein said selecting comprises affinity-based selection.

71. 71. The method of claim 70, wherein the affinity-based selection comprises fluorescence-activated cell sorting or magnetic-activated cell sorting.

72. 72. The method of any one of claims 15 to 71, wherein the incubation increases the amount of cells by 30 to 40 times.

73. 73. The method of any one of claims 15 to 72, further comprising harvesting at least 1E8 of the cells susceptible to fratricide after the incubation period.

74. 74. The method of claim 73, further comprising harvesting at least 1E8 of the cells susceptible to fratricide after incubation for no more than 11 days.

75. 75. 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.

76. 76. The method of any one of claims 72 to 75, wherein at least 70% of the harvested cells are viable.

77. 77. The method of any one of claims 72-76, wherein at least 70% of the harvested cells expressing the CAR are viable.

78. 78. The method of any one of claims 72 to 77, wherein at least 70% of the harvested cells having reduced expression of the cell surface polypeptide are viable.

79. 79. The method of any one of claims 72 to 78, further comprising freezing the harvested cells.

80. 80. The method of claim 79, wherein at least 70% of the frozen cells are viable.

81. a container comprising a gas permeable membrane and a culture medium; and cells susceptible to fratricide in contact with the surface of said gas-permeable membrane. A system comprising:

82. said cells susceptible to fratricide, (i) a binding domain that binds to a cell surface polypeptide expressed by said cell susceptible to fratricide; (ii) an activation domain that activates a portion of said cells susceptible to fratricide; and 82. The system of claim 81 , comprising a chimeric antigen receptor (CAR) comprising:

83. The system of claim 81 or 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.

84. 84. The system of claim 83, wherein the cells susceptible to fratricide comprise a non-natural modification of the gene encoding the polypeptide.

85. 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 localization domain.

86. 86. The system of any one of claims 81 to 85, wherein the system comprises a closed culture component.

87. 87. The system of any one of claims 81 to 86, further comprising a liquid handler configured to exchange the culture medium.

88. 88. The system of any one of claims 81 to 87, further comprising a device configured to disrupt cell clumps.

89. 89. 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 to cause the device to break up cell clumps.

90. 90. The system of any one of claims 81 to 89, further comprising a sensor configured to analyze the culture medium.

91. 91. The system of claim 90, wherein the sensor is capable of measuring a nutrient, a metabolite, or pH.

92. 92. The system of claim 91, wherein the metabolic product is lactate or ammonium.

93. 93. The system of any one of claims 90 to 92, wherein the controller is configured to operate the liquid handler based at least in part on measurements made by the sensor.

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