Methods of manufacturing engineered t cells from whole blood samples

Abstract

This invention discloses a method for manufacturing engineered T cells expressing a heterologous protein, wherein T cells are isolated from a whole blood sample, and the heterologous protein is, for example, a chimeric antigen receptor (CAR).

Description

Technical Field

[0001] This disclosure generally relates to methods and compositions for manufacturing engineered T cells expressing heterologous proteins, such as chimeric antigen receptors (CARs).

[0002] Incorporated by reference

[0003] This patent application incorporates by reference the material included in an amino acid and / or nucleotide sequence listing prepared in accordance with the ST.26 standard, contained in an .xml file named “KYVA-001-01WO-Sequence-Listing.xml”, prepared on November 1, 2024 and filed together with this application. Background Technology

[0004] Chimeric antigen receptor (CAR) T-cell therapy holds transformative potential in treating many life-altering diseases and conditions, such as cancer and immune disorders. As an immunotherapy, CAR T-cell therapy demonstrates promising capabilities by selectively targeting and eliminating the root cause of disease while providing a durable response to prevent relapse.

[0005] Despite the potential of CAR T cells, several key challenges remain to their commercial viability. Currently, the manufacture of CAR T cells involves time-consuming in vitro cell culture procedures, which are expensive and lead to high product variability. For example, prolonged in vitro culture is associated with phenotypic changes (differentiation) that are not yet fully characterized and may impair therapeutic efficacy; these changes become increasingly pronounced as the manufacturing process lengthens.

[0006] Current methods for manufacturing CAR T cells involve several steps, including lengthy in vitro culture, which is costly in terms of both economics and patient outcomes. This high cost begins with the initial step of collecting T cells to engineer into CAR-T cells. Essentially, to obtain a sufficient number of donor T cells for downstream manufacturing, methods for manufacturing CAR T cells rely on leukocyte ablation steps and / or peripheral blood mononuclear cell (PBMC) separation processes on ablation samples to produce a product from which T cells can be isolated.

[0007] This initial requirement introduces significant delays and costs in the production of CAR T-cell products. Leukoablation itself is a time-consuming procedure requiring experienced technicians and specialized facilities. In addition to the general cost of the procedure, scheduling leukoablation is also challenging for many patients, particularly those with certain conditions who may require T-cell depletion therapy, which can hinder the availability of sufficient T-cells. Consequently, patients are forced to postpone T-cell depletion therapy or abandon CAR T-cell therapy altogether. Furthermore, avoiding leukoablation, if possible, completely eliminates the risks inherent in central venous catheter placement.

[0008] Eliminating the requirement for leukocyte separation / PBMC separation in the manufacturing process will improve the efficacy and outcomes of CAR T-cell therapy.

[0009] Therefore, unless a reliable and economical manufacturing process for producing safe and effective T cells is developed, the potential of CAR T cell therapy will never translate into clinical application, as economic and logistical bottlenecks will hinder its development and delivery. Summary of the Invention

[0010] This document discloses methods for manufacturing engineered T cells expressing heterologous proteins such as chimeric antigen receptors (CARs). Exemplary methods for manufacturing the CAR T cells of this disclosure may include obtaining T cells from whole blood of a subject (e.g., a donor or patient). In some aspects, T cells are obtained from peripheral blood mononuclear cells (PBMCs) isolated from whole blood. In a more preferred aspect, the inventors have found, surprisingly, that a sufficient number of T cells for CAR T cell manufacturing can be obtained directly from whole blood without a leukocyte separation step or a PBMC separation step. This includes cases where it is used as starting material in the CAR-T cell manufacturing process of this disclosure (which lasts 6 to 9 days, typically about 8 days), as well as rapid methods that can produce engineered cells in about two to three days after receiving a whole blood sample containing donor T cells.

[0011] Furthermore, some methods of this disclosure include simultaneous T cell isolation and activation steps. Therefore, in some methods for manufacturing the CAR T cells of this disclosure, T cells are isolated from whole blood or PBMCs and activated simultaneously. Simultaneous activation / isolation not only reduces overall manufacturing time but also enables the isolation of a sufficient number of T cells with high purity to manufacture CAR T cells from a very small whole blood sample (e.g., a sample of 100 mL or less).

[0012] Some exemplary methods for manufacturing the CAR T cells of this disclosure are “rapid” processes that produce CAR T cells significantly faster than other processes. These shortened processes of this disclosure typically take two to three days in total, with a brief culture after T cell isolation / activation prior to transduction, typically less than or significantly less than one day, and a similar brief culture after transduction prior to harvesting the desired CAR T cell product. Despite the small starting whole blood sample, the lack of leukocyte ablation or other similar T cell isolation steps, and the brief culture steps, these methods of this disclosure are able to harvest large quantities of CAR T cells, all within 2-3 days of receiving the starting sample. Surprisingly, the functional activity of the final CAR T cell product produced by these two- to three-day manufacturing processes is higher than that of other 6- to 9-day processes. CAR T cells manufactured using the methods of this disclosure exhibit excellent T cell expansion in the final CAR T cell product.

[0013] Furthermore, due to the simplified process of this disclosure, cells prepared using the methods of this disclosure unexpectedly exhibit more favorable phenotypes. The degree of cell differentiation is significantly lower than that of cells produced using longer processes. When compared to longer processes, cells generated using the shortened method of this disclosure include higher levels of stem cell memory T cells (Tscm), which typically constitute only 2-3% of circulating T cells and are associated with long-term defensive immunity, antitumor activity, and self-renewal. Moreover, these cells express the introduced CAR at a very high percentage, producing a pure final product that exhibits targeted cytotoxicity and low T cell exhaustion. Even more surprisingly, the shortened manufacturing process produces a large number of pluripotent cells that simultaneously secrete multiple sets of cytokines, chemokines, and / or cytotoxic granules. Due to this pluripotent behavior, these cells have been shown to provide a more effective immune response, which is exactly what is expected of therapeutic CAR T cell products.

[0014] Similarly, some methods of this disclosure, using longer manufacturing times (e.g., 6 to 9 days) and whole blood starting samples (instead of the ablation samples used in previous methods), are still able to produce cells with similar phenotypes to those produced by methods using ablation starting materials. Therefore, the methods of this disclosure can alleviate manufacturing bottlenecks associated with obtaining ablation samples while providing cell products with comparable or better therapeutic efficacy.

[0015] Therefore, in some aspects, this disclosure provides a method for generating an engineered population of T cells expressing a heterologous protein (e.g., a chimeric antigen receptor). An exemplary method may include the steps of: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a carrier, thereby isolating and activating T cells from the whole blood sample; after binding the T cells, contacting the activated T cells with nucleic acid encoding the heterologous protein; culturing the nucleic acid-contacted T cells in a serum-free medium; and harvesting the cultured T cells, wherein the harvested T cells express the heterologous protein.

[0016] In some aspects, this disclosure provides a rapid method for generating a population of engineered T cells expressing a heterologous protein. An exemplary rapid method may include the steps of: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a vector, thereby isolating and activating T cells from the whole blood sample; contacting the activated T cells with nucleic acid encoding the heterologous protein between 10 and 25 hours after T cell binding; culturing the nucleic acid-contacted T cells in a serum-free medium for a period between 24 and 60 hours; and harvesting the cultured T cells, wherein the harvested T cells express the heterologous protein.

[0017] In the preferred methods of this disclosure, T cells are isolated directly from whole blood samples without the need for intermediate T cell separation steps and / or leukocyte separation. In some methods of this disclosure, prior to the binding step, the method includes separating peripheral blood mononuclear cells (PBMCs) containing T cells from a whole blood sample without a leukocyte separation step.

[0018] In a preferred method of this disclosure, the contact step occurs between 12 and 21 hours after the bonding step. In another preferred method of this disclosure, the contact step occurs between 15 and 19 hours after the bonding step. In some preferred aspects, the contact step occurs between 17 and 19 hours after the bonding step. In some methods, the contact step occurs approximately 18 hours after the bonding step.

[0019] In some methods of this disclosure, the cultivation step lasts for a period of 29 to 59 hours. In a preferred method of this disclosure, the cultivation step lasts for a period of 36 to 52 hours. In a more preferred aspect, the cultivation step lasts for a period of 46 to 50 hours. In some aspects, the cultivation step lasts for a period of approximately 48 hours.

[0020] In some aspects, this disclosure provides a method for generating an engineered population of T cells expressing a heterologous protein. An exemplary method may include the steps of: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a carrier, thereby isolating and activating T cells from the whole blood sample; contacting the activated T cells with nucleic acid encoding a heterologous protein between 20 and 28 hours after T cell binding; culturing the nucleic acid-contacted T cells in a serum-free medium for a period between 4 and 9 days; and harvesting the cultured T cells, wherein the harvested T cells express the heterologous protein.

[0021] In alternative methods of this disclosure, the contact step occurs between approximately 20 and 28 hours after the binding step, and the culture step lasts for a period of 4 to 9 days. In some aspects, the contact step occurs between approximately 22 and 26 hours after the binding step. In some aspects, the contact step occurs between approximately 23 and 25 hours after the binding step. In some aspects, the contact step occurs approximately 24 hours after the binding step. In some aspects, the culture step lasts for a period of approximately 5 to 7 days. In some aspects, the culture step lasts for a period of approximately 6 days. In some aspects, the culture step lasts for a period of approximately 8 days.

[0022] In the exemplary method of this disclosure, the harvested T cells comprise between approximately 10% and 60% CD45RO- / CCR7+ T cells (Tnscm). In a more preferred method, the harvested T cells comprise between approximately 15% and 60% CD45RO- / CCR7+ T cells (Tnscm). In some aspects, the harvested T cells comprise at least 18% Tnscm. In some aspects, the harvested T cells comprise at least 22% Tnscm. In some aspects, the harvested T cells comprise at least 25% Tnscm.

[0023] Tnscm may include naive T cells (Tn) and stem cell memory T cells (Tscm). In a preferred aspect, the Tnscm contains more Tscm than Tn. The Tnscm may contain at least 1.5 times more Tscm than Tn; at least twice more Tscm than Tn; at least three times more Tscm than Tn; at least five times more Tscm than Tn; at least ten times more Tscm than Tn; and / or at least fifty times more Tscm than Tn.

[0024] In some methods of this disclosure, one or more anti-CD3 antibodies and one or more anti-CD28 antibodies are attached to the same carrier. In some methods, one or more anti-CD3 antibodies and / or one or more anti-CD28 antibodies are attached to the carrier via cleavable linkers. Linkers can be, for example, enzymatically cleavable, hydrolyzable, redox-cleavable, phosphate-based, acid-based, ester-based, peptide-based, disulfide-based, nitrobenzyl-based, methoxymethyl-based, and / or photocleavable linkers. Therefore, the methods of this disclosure also include contacting activated T cells with a stimulant of the cleavable linker to release T cells from the surface.

[0025] In some aspects, the surface is a solid surface. Preferably, the solid surface is a bead, pore, chip, or microfluidic channel. More preferably, the solid surface is a bead. In some aspects, the surface comprises a polymer. In some aspects, the polymer is a hydrogel. In some methods, the surface comprises a polymer scaffold.

[0026] In a preferred method of this disclosure, the harvested T cells comprise a population representing at least 6% of the harvested T cells and are pluripotent T cells upon activation based on a specific target. In some aspects, pluripotent T cells and / or a portion of their population simultaneously secrete granzyme B and TNFb. In some aspects, pluripotent T cells and / or a portion of their population simultaneously secrete granzyme B and IFNg. In a preferred aspect, at least 1% of the harvested T cells and / or a portion of their population simultaneously secrete granzyme B and IFNg. In some aspects, pluripotent T cells and / or a portion of their population simultaneously secrete MIP-1a and MIP-1b. In some methods, pluripotent T cells and / or a portion of their population simultaneously secrete IFNg and granzyme B.

[0027] In the preferred method of this disclosure, the pluripotent T cells and / or a portion of the population thereof comprise two or more of the following: cells that simultaneously secrete granzyme B and TNFb and / or a portion of the pluripotent T cell population; cells that simultaneously secrete granzyme B and IFNg and / or a portion of the pluripotent T cell population; cells that simultaneously secrete MIP-1a and MIP-1b and / or a portion of the pluripotent T cell population; and cells that simultaneously secrete IFNg and granzyme B and / or a portion of the pluripotent T cell population.

[0028] In some methods of this disclosure, a portion of a pluripotent T cell and / or a pluripotent T cell population includes: cells that simultaneously secrete granzyme B and TNFb and / or a portion of a pluripotent T cell population; cells that simultaneously secrete granzyme B and IFNg and / or a portion of a pluripotent T cell population; cells that simultaneously secrete MIP-1a and MIP-1b and / or a portion of a pluripotent T cell population; and cells that simultaneously secrete IFNg and granzyme B and / or a portion of a pluripotent T cell population.

[0029] In a preferred method, the serum-free culture medium contains at least one cytokine. Preferably, the at least one cytokine contains one or more of IL-2, IL-21, IL-7, and IL-15. In some methods, the at least one cytokine contains one or more of IL-21, IL-7, and IL-15 and does not contain IL-2.

[0030] In some methods of this disclosure, the number of isolated T cells from whole blood samples is approximately 1 × 10⁻⁶. 6 With approximately 1×10 8 Between 100 total T cells, and the number of T cells harvested is approximately 1 × 10⁻⁶. 6 With approximately 5×10 8 Between. In some methods of this disclosure, the number of isolated T cells from whole blood samples is approximately 1.5 × 10⁻⁶. 7 With approximately 1×10 8 Between 100 total T cells, and the number of T cells harvested was approximately 7.5 × 10⁻⁶. 7 With approximately 1.5 × 10 8 Between. In some preferred methods, the number of isolated T cells from whole blood samples is approximately 5 × 10⁻⁶. 7 With approximately 7.5 × 10 7 Between 100 total T cells, and the number of T cells harvested was approximately 7.5 × 10⁻⁶. 7 With approximately 1.2 × 10 8 between.

[0031] In some respects, the culture step achieves an expansion of the harvested T cells between 1.0 and 4 times. In other respects, the culture step achieves an expansion of the harvested T cells between 1.2 and 4 times.

[0032] This disclosure also provides a method for generating an engineered T cell population expressing a heterologous protein, comprising the steps of: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a carrier, thereby isolating and activating T cells from the whole blood sample without performing an intermediate PBMC separation step or leukocyte separation step; contacting the activated T cells with nucleic acid encoding the heterologous protein; culturing the nucleic acid-contacted T cells in a serum-free medium; and harvesting the cultured T cells, wherein the harvested T cells express the heterologous protein.

[0033] In the exemplary method of this disclosure, the harvested T cells comprise between approximately 10% and 60% CD45RO- / CCR7+ T cells (Tnscm). In a more preferred method, the harvested T cells comprise between approximately 15% and 60% CD45RO- / CCR7+ T cells (Tnscm). In some aspects, the harvested T cells comprise at least 18% Tnscm. In some aspects, the harvested T cells comprise at least 22% Tnscm. In some aspects, the harvested T cells comprise at least 25% Tnscm.

[0034] Tnscm may include naive T cells (Tn) and stem cell memory T cells (Tscm). In a preferred aspect, the Tnscm contains more Tscm than Tn. The Tnscm may contain at least 1.5 times more Tscm than Tn; at least twice more Tscm than Tn; at least three times more Tscm than Tn; at least five times more Tscm than Tn; at least ten times more Tscm than Tn; and / or at least fifty times more Tscm than Tn.

[0035] In a preferred method of this disclosure, the contact step occurs between 12 and 21 hours after the bonding step. In another preferred method of this disclosure, the contact step occurs between 15 and 19 hours after the bonding step. In some preferred aspects, the contact step occurs between 17 and 19 hours after the bonding step. In some methods, the contact step occurs approximately 18 hours after the bonding step.

[0036] In some methods of this disclosure, the cultivation step lasts for a period of 29 to 59 hours. In a preferred method of this disclosure, the cultivation step lasts for a period of 36 to 52 hours. In a more preferred aspect, the cultivation step lasts for a period of 46 to 50 hours. In some aspects, the cultivation step lasts for a period of approximately 48 hours.

[0037] In some methods of this disclosure, one or more anti-CD3 antibodies and one or more anti-CD28 antibodies are attached to the same carrier. In some methods, one or more anti-CD3 antibodies and / or one or more anti-CD28 antibodies are attached to the carrier via cleavable linkers. Linkers can be, for example, enzymatically cleavable, hydrolyzable, redox-cleavable, phosphate-based, acid-based, ester-based, peptide-based, disulfide-based, nitrobenzyl-based, methoxymethyl-based, and / or photocleavable linkers. Therefore, the methods of this disclosure also include contacting activated T cells with a stimulant of the cleavable linker to release T cells from the surface.

[0038] In some aspects, the surface is a solid surface. Preferably, the solid surface is a bead, pore, chip, or microfluidic channel. More preferably, the solid surface is a bead. In some aspects, the surface comprises a polymer. In some aspects, the polymer is a hydrogel. In some methods, the surface comprises a polymer scaffold.

[0039] In a preferred method of this disclosure, the harvested T cells comprise a population representing at least 6% of the harvested T cells and are pluripotent T cells upon activation based on a specific target. In some aspects, pluripotent T cells and / or a portion of their population simultaneously secrete granzyme B and TNFb. In some aspects, pluripotent T cells and / or a portion of their population simultaneously secrete granzyme B and IFNg. In a preferred aspect, at least 1% of the harvested T cells and / or a portion of their population simultaneously secrete granzyme B and IFNg. In some aspects, pluripotent T cells and / or a portion of their population simultaneously secrete MIP-1a and MIP-1b. In some methods, pluripotent T cells and / or a portion of their population simultaneously secrete IFNg and granzyme B.

[0040] In the preferred method of this disclosure, the pluripotent T cells and / or a portion of the population thereof comprise two or more of the following: cells that simultaneously secrete granzyme B and TNFb and / or a portion of the pluripotent T cell population; cells that simultaneously secrete granzyme B and IFNg and / or a portion of the pluripotent T cell population; cells that simultaneously secrete MIP-1a and MIP-1b and / or a portion of the pluripotent T cell population; and cells that simultaneously secrete IFNg and granzyme B and / or a portion of the pluripotent T cell population.

[0041] In some methods of this disclosure, a portion of a pluripotent T cell and / or a pluripotent T cell population includes: cells that simultaneously secrete granzyme B and TNFb and / or a portion of a pluripotent T cell population; cells that simultaneously secrete granzyme B and IFNg and / or a portion of a pluripotent T cell population; cells that simultaneously secrete MIP-1a and MIP-1b and / or a portion of a pluripotent T cell population; and cells that simultaneously secrete IFNg and granzyme B and / or a portion of a pluripotent T cell population.

[0042] In a preferred method, the serum-free culture medium contains at least one cytokine. Preferably, the at least one cytokine contains one or more of IL-2, IL-21, IL-7, and IL-15. In some methods, the at least one cytokine contains one or more of IL-21, IL-7, and IL-15 and does not contain IL-2. In alternative methods, the serum-free culture medium does not contain any added cytokine.

[0043] In some methods of this disclosure, the number of isolated T cells from whole blood samples is approximately 1 × 10⁻⁶. 6 With approximately 1×10 8 Between 100 total T cells, and the number of T cells harvested was approximately 2.5 × 10⁻⁶. 7 With approximately 5×10 8 Between. In some methods of this disclosure, the number of isolated T cells from whole blood samples is approximately 1.5 × 10⁻⁶. 7 With approximately 1×10 8 Between 100 total T cells, and the number of T cells harvested was approximately 7.5 × 10⁻⁶. 7 With approximately 1.5 × 10 8 Between. In some preferred methods, the number of isolated T cells from whole blood samples is approximately 5 × 10⁻⁶. 7 With approximately 7.5 × 10 7 Between 100 total T cells, and the number of T cells harvested was approximately 7.5 × 10⁻⁶. 7 With approximately 1.2 × 10 8 between.

[0044] In some respects, the culture step achieves an expansion of the harvested T cells between 1.0 and 4 times. In other respects, the culture step achieves an expansion of the harvested T cells between 1.2 and 4 times.

[0045] An alternative method disclosed herein for generating an engineered T cell population expressing a heterologous protein may include the following steps: obtaining a whole blood sample from a donor; separating T cells from the whole blood sample by binding T cells from the whole blood sample to one or more anti-CD4 antibodies and one or more anti-CD8 antibodies attached to a carrier; contacting the activated T cells with nucleic acid encoding the heterologous protein between 10 and 25 hours after binding the T cells; culturing the nucleic acid-contacted T cells in a serum-free medium for a period between 24 and 60 hours; and harvesting the cultured T cells, wherein the harvested T cells express the heterologous protein.

[0046] In some respects, the method also includes the step of activating the isolated T cells.

[0047] In the preferred methods of this disclosure, T cells are isolated directly from whole blood samples without the need for intermediate T cell separation steps and / or leukocyte separation. In some methods of this disclosure, prior to the binding step, the method includes separating peripheral blood mononuclear cells (PBMCs) containing T cells from a whole blood sample without a leukocyte separation step.

[0048] In the exemplary method of this disclosure, the harvested T cells comprise between approximately 10% and 60% CD45RO- / CCR7+ T cells (Tnscm). In a more preferred method, the harvested T cells comprise between approximately 15% and 60% CD45RO- / CCR7+ T cells (Tnscm). In some aspects, the harvested T cells comprise at least 18% Tnscm. In some aspects, the harvested T cells comprise at least 22% Tnscm. In some aspects, the harvested T cells comprise at least 25% Tnscm.

[0049] Tnscm may include naive T cells (Tn) and stem cell memory T cells (Tscm). In a preferred aspect, the Tnscm contains more Tscm than Tn. The Tnscm may contain at least 1.5 times more Tscm than Tn; at least twice more Tscm than Tn; at least three times more Tscm than Tn; at least five times more Tscm than Tn; at least ten times more Tscm than Tn; and / or at least fifty times more Tscm than Tn.

[0050] In a preferred method of this disclosure, the contact step occurs between 12 and 21 hours after the bonding step. In another preferred method of this disclosure, the contact step occurs between 15 and 19 hours after the bonding step. In some preferred aspects, the contact step occurs between 17 and 19 hours after the bonding step. In some methods, the contact step occurs approximately 18 hours after the bonding step.

[0051] In some methods of this disclosure, the cultivation step lasts for a period of 29 to 59 hours. In a preferred method of this disclosure, the cultivation step lasts for a period of 36 to 52 hours. In a more preferred aspect, the cultivation step lasts for a period of 46 to 50 hours. In some aspects, the cultivation step lasts for a period of approximately 48 hours.

[0052] In some methods of this disclosure, one or more anti-CD4 antibodies and one or more anti-CD8 antibodies are attached to the same carrier. In some methods, one or more anti-CD8 antibodies and / or one or more anti-CD4 antibodies are attached to the carrier via cleavable linkers. Linkers can be, for example, enzymatically cleavable, hydrolyzable, redox-cleavable, phosphate-based, acid-based, ester-based, peptide-based, disulfide-based, nitrobenzyl-based, methoxymethyl-based, and / or photocleavable linkers. Therefore, the methods of this disclosure also include contacting activated T cells with a stimulant of the cleavable linker to release T cells from the surface.

[0053] In some aspects, the surface is a solid surface. Preferably, the solid surface is a bead, pore, chip, or microfluidic channel. More preferably, the solid surface is a bead. In some aspects, the surface comprises a polymer. In some aspects, the polymer is a hydrogel. In some methods, the surface comprises a polymer scaffold.

[0054] In a preferred method of this disclosure, the harvested T cells comprise a population representing at least 6% of the harvested T cells and are pluripotent T cells upon activation based on a specific target. In some aspects, pluripotent T cells and / or a portion of their population simultaneously secrete granzyme B and TNFb. In some aspects, pluripotent T cells and / or a portion of their population simultaneously secrete granzyme B and IFNg. In a preferred aspect, at least 1% of the harvested T cells and / or a portion of their population simultaneously secrete granzyme B and IFNg. In some aspects, pluripotent T cells and / or a portion of their population simultaneously secrete MIP-1a and MIP-1b. In some methods, pluripotent T cells and / or a portion of their population simultaneously secrete IFNg and granzyme B.

[0055] In the preferred method of this disclosure, the pluripotent T cells and / or a portion of the population thereof comprise two or more of the following: cells that simultaneously secrete granzyme B and TNFb and / or a portion of the pluripotent T cell population; cells that simultaneously secrete granzyme B and IFNg and / or a portion of the pluripotent T cell population; cells that simultaneously secrete MIP-1a and MIP-1b and / or a portion of the pluripotent T cell population; and cells that simultaneously secrete IFNg and granzyme B and / or a portion of the pluripotent T cell population.

[0056] In some methods of this disclosure, a portion of a pluripotent T cell and / or a pluripotent T cell population includes: cells that simultaneously secrete granzyme B and TNFb and / or a portion of a pluripotent T cell population; cells that simultaneously secrete granzyme B and IFNg and / or a portion of a pluripotent T cell population; cells that simultaneously secrete MIP-1a and MIP-1b and / or a portion of a pluripotent T cell population; and cells that simultaneously secrete IFNg and granzyme B and / or a portion of a pluripotent T cell population.

[0057] In a preferred method, the serum-free culture medium contains at least one cytokine. Preferably, the at least one cytokine contains one or more of IL-2, IL-21, IL-7, and IL-15. In some methods, the at least one cytokine contains one or more of IL-21, IL-7, and IL-15 and does not contain IL-2.

[0058] In some methods of this disclosure, the number of isolated T cells from whole blood samples is approximately 1 × 10⁻⁶. 6 With approximately 1×10 8 Between 100 total T cells, and the number of T cells harvested was approximately 2.5 × 10⁻⁶. 7 With approximately 5×10 8 Between. In some methods of this disclosure, the number of isolated T cells from whole blood samples is approximately 1.5 × 10⁻⁶. 7 With approximately 1×10 8 Between 100 total T cells, and the number of T cells harvested was approximately 7.5 × 10⁻⁶. 7 With approximately 1.5 × 10 8 Between. In some preferred methods, the number of isolated T cells from whole blood samples is approximately 5 × 10⁻⁶. 7 With approximately 7.5 × 10 7 Between 100 total T cells, and the number of T cells harvested was approximately 7.5 × 10⁻⁶. 7 With approximately 1.2 × 10 8 between.

[0059] In some respects, the culture step achieves an expansion of the harvested T cells between 1.0 and 4 times. In other respects, the culture step achieves an expansion of the harvested T cells between 1.2 and 4 times. Attached Figure Description

[0060] Figure 1 An exemplary workflow for methods of generating engineered T cells from whole blood samples is provided in this disclosure.

[0061] Figure 2It is a bar graph showing T cell expansion, which represents the increase in the number of CD3+ cells 72 hours after thawing relative to the number of CD3+ cells at thawing time, to compare the method of producing CAR T cells disclosed herein.

[0062] Figure 3 It is a graph depicting the percentage of CAR expression 72 hours after thawing to compare the method of producing CAR T cells disclosed herein.

[0063] Figure 4 A-4B shows the percentage of TNSCM (naive and stem cell memory T cells (CD45RO- / CCR7+)) at thaw. “UNTD” indicates the untransduced control.

[0064] Figures 5A-5D It describes the thawing process ( Figure 5A and 5C ) or 72 hours after thawing ( Figure 5B and 5D Bar graph of memory phenotypes of CAR+ cells. The "post-enrichment" condition was untransduced.

[0065] Figure 6 The percentage of cell lysis when NALM6 target cells are cultured together with CAR-T cells generated under conditions D and E is shown. "D" represents the KYV 6-day condition, "E" represents the KYV 8-day condition, and "UNTD" indicates the untransduced condition.

[0066] Figures 7A-7D This describes the use of the KYV 3-day v2 scheme (“C”) Figure 7A and 7C ) or KYV-9-day plan (“E”) Figure 7B and 7D The bar graph shows the secretion frequency of each of the 32 cytokines produced by the cells after overnight culture in the presence of CD19+ NALM6 target cells. "ut" corresponds to untransduced control cells that have undergone each step in either the 3-day or 9-day protocol.

[0067] Figure 8 This is a bar graph showing the versatility of CAR-T cells produced through process C.

[0068] Figure 9 It is a bar chart showing the percentage of cells produced by process C or E among all multifunctional cells that secrete the indicated cytokines.

[0069] Figure 10 This is a flowchart illustrating the experimental design and conditions for six CAR-T cell manufacturing processes.

[0070] Figure 11Data from the same donor study were provided to validate that the X-LAB system can achieve excellent PBMC enrichment from whole blood samples.

[0071] Figure 12 It provides data showing the percentage of CD3+, CD4+, and CD8+ cells (T cells) in samples, including whole blood samples.

[0072] Figure 13 The protocol used to evaluate the procedure for directly isolating T cells from blood is outlined.

[0073] Figure 14 Enrichment and amplification data are provided, comparing the methods of this disclosure for isolating T cells from whole blood for CAR T cell production.

[0074] Figure 15 The present disclosure provides CD3+ and CD4+ percentage data and compares the methods of isolating T cells from whole blood for CAR T cell production.

[0075] Figure 16 Enrichment and amplification data are provided, comparing the methods of this disclosure for isolating T cells from whole blood for CAR T cell production.

[0076] Figure 17 The data provided show that, compared with simulated (untransduced) control cells, cells generated from CAR T cells produced directly from whole blood using the 3-day manufacturing protocol of this disclosure exhibit a less differentiated phenotype.

[0077] Figure 18 Data demonstrating the target-specific cytotoxicity of CAR T cells generated using the methods of this disclosure are provided.

[0078] Figure 19 Results were provided on the release of target-dependent cytokines from CAR T cells derived from whole blood of healthy donors (HD) after 24 hours of in vitro co-culture with CD19+ NALM6 target cells at a specified E:T (effectant:target) ratio.

[0079] Figure 20 Data are shown for CD19-targeted CAR-T cells prepared using the methods of this disclosure and CD19+ NALM6 target cells co-cultured at a specified E:T ratio (CAR-T effector: NALM6 target), with the percentage of target cell killing measured by flow cytometry at each specified time point. At each time point, a new round of target cells was added to the co-culture to assess the sequentially repeated cytotoxicity of CAR-T over time.

[0080] Figure 21Data on target-specific amplification of CAR T cells produced using the methods of this disclosure are provided.

[0081] Figure 22 It provides the TBNK / memory phenotypes of cells generated from T cells isolated from whole blood, before and after enrichment.

[0082] Figure 23 The TBNK / memory phenotype of the final CAR T cell product (e.g., after expansion) produced using an 8-day process is provided.

[0083] Figure 24 It provides the TBNK / memory phenotypes of cells generated from T cells isolated from whole blood as starting material, and the pre- and post-enrichment materials.

[0084] Figure 25 It provides the TBNK / memory phenotypes of cells generated from T cells isolated from whole blood as starting material, and the pre- and post-enrichment materials.

[0085] Figure 26 The steps of the three-day method of this disclosure, starting from whole blood (WB) starting material (SM), are outlined.

[0086] Figure 27A The T-cell memory phenotype of cells produced using the three-day method of this disclosure is shown.

[0087] Figure 27B The cell lysis activity of cells produced using the 3-day method of this disclosure is demonstrated.

[0088] Figure 27C The results of the continuous re-excitation measurements are shown.

[0089] Figure 27D The 3-day process demonstrated that Ingenui-T cells successfully killed autologous primary B cells in a dose-dependent manner. Ingenui-T cells derived from whole blood or untransduced T cells were co-cultured with autologous (donor-matched) PBMCs at a specified effector-to-target (E:T) ratio, which represents the CAR ratio. + The ratio of T cells (effectants) to total PBMCs (targets). B cell survival, defined by the expression of CD19 or CD20 surface markers, was measured by flow cytometry at 48 hours. Data are presented as mean ± SD from three technical replicates for each condition, derived from one donor representing N=2 healthy donors. ****p<0.0001, matched E:T ratios were compared by two-way ANOVA (GraphPad Prism).

[0090] Figure 28AThis shows the fold expansion, viability, and T cell purity of CAR-T cells manufactured using KYV 3-day process with leukocyte separation material as the starting material. KYV 3-day conditions “A”, “B”, “C”, and “D” indicate different culture cytokines used. “NT” = untransduced. N = 4 healthy donors for each condition.

[0091] Figure 28B The yield and purity of CAR-T cells produced using fresh whole blood from n=5 donors in a KYV 3-day process are shown.

[0092] Figure 29A The percentage of CAR+ expression, analyzed by flow cytometry, is shown in CAR-T cells manufactured using a KYV 3-day process with leukocyte-isolated materials as starting material. At harvest, CAR expression was analyzed in total CD3+ T cells, or in CD4+ or CD8+ T cells. KYV 3-day conditions “A”, “B”, “C”, and “D” indicate the different culture cytokines used. N=4 healthy donors were used under each condition.

[0093] Figure 29B The percentage of CAR+ expression, analyzed by flow cytometry, is shown in CAR-T cells manufactured using a KYV 3-day process starting with freshly collected whole blood. CAR expression was also analyzed in total T cells. N=5 donors.

[0094] Figure 30A This shows the percentages of CD4+ and CD8+ in total CD3+ in CAR-T cells manufactured using KYV 3-day process with leukocyte-isolated material as the starting material. KYV 3-day conditions “C1”, “C2”, “C3”, and “C4” indicate the different culture cytokines used. N = 4 healthy donors for each condition. “NT” = untransduced control. “Conv 9 days” refers to donor-matched CAR-T cells manufactured using the standard 9-day culture process. “Aph SM” refers to leukocyte-isolated donor starting material prior to the KYV 3-day process.

[0095] Figure 30B This shows the percentages of CD4+ and CD8+ in total CD5+ in CAR-T cells manufactured using freshly collected whole blood as the starting material in a KYV 3-day process. N=7 donors were included. "WB SM" refers to donor whole blood starting material prior to the KYV 3-day process.

[0096] Figures 31A-31BThis shows the results of T cell memory phenotype analysis by flow cytometry in CAR-T cells manufactured in a KYV 3-day process using leukocyte-isolated material as the starting material. KYV 3-day conditions “C1”, “C2”, “C3”, and “C4” represent different culture cytokines used. N = 4 healthy donors for each condition. “NT” = untransduced control. “Conv 9 days” refers to donor-matched CAR-T cells manufactured in a standard 9-day culture process. “Aph SM” refers to leukocyte-isolated donor starting material prior to the KYV 3-day process. Tnaive = initial (CCR7+CD45RO-CD95-); Tscm = stem cell memory (CCR7+CD45RO-CD95+); Tcm = central memory (CCR7+CD45RO+); Tem = effector memory (CCR7-CD45RO+); Te = effector (CCR7-CD45RO-CD95+).

[0097] Figure 31C This study shows the T cell memory phenotype analyzed within total CD3+ T cells in CAR-T cells manufactured using a KYV 3-day process starting with freshly collected whole blood, compared to a conventional 9-day process using leukocyte ablation material. N=4 donors were included. "WB SM" and "Aph SM" refer to donor whole blood or leukocyte ablation starting material prior to the culture process, respectively. Figures 31A-31B The analysis of T-cell memory subsets is defined in the literature.

[0098] Figure 32 The target-dependent cytotoxic activity of anti-CD19 CAR-T cells manufactured using the KYV 3-day process against CD19+ target cells is shown, measured by the percentage of cell lysis of CD19+ NALM6 target cells or CD19- CEM / C1 control cells after co-culturing with anti-CD19 CAR-T cells manufactured using the KYV 3-day process at a specified E:T (effectant:target) ratio. N=2 donors are illustrated. Cell lysis activity was measured by chemiluminescence assay and normalized relative to target cells alone (0:1).

[0099] Figure 33AThe results show the killing or growth of CD19+ NALM6 target cells during 120 hours of co-culture with anti-CD19 CAR-T cells manufactured using leukocyte ablation material in a KYV 3-day process and co-cultured at a specified E:T (effectant:target) ratio of 0.3:1 or 1:1. NALM6 growth was measured by fluorescence in an Incucyte-based imaging assay and normalized relative to time=0. KYV 3-day conditions “C1”, “C2”, “C3”, “C4” indicate the different culture cytokines used. “NT” = untransduced control T cells. A representative donor from n=4 is illustrated.

[0100] Figure 33B The results show the killing or growth of CD19+ NALM6 target cells during 120 hours of co-culture with anti-CD19 CAR-T cells manufactured from freshly collected whole blood in a KYV 3-day process, co-cultured at a specified E:T (effectant:target) ratio of 0.3:1 or 1:1. NALM6 growth was measured by fluorescence in an Incucyte-based imaging assay and normalized relative to time=0. “NT” = untransduced control T cells. “Conv 9 days” refers to donor-matched CAR-T cells manufactured from leukocyte separation starting material in a standard 9-day culture process. A representative donor from n=4 is illustrated.

[0101] Figure 34 This figure shows the CAR-mediated cytotoxic activity of anti-CD19 CAR-T cells manufactured using the KYV 3-day process against CD19+ primary human B cells, using the measured percentage of cell lysis of CD19+ primary human B cells co-cultured with anti-CD19 CAR-T cells (manufactured from freshly collected whole blood in the KYV 3-day process). The E:T (effectant:target) ratio represents the ratio of CAR+ T cells plated in the co-culture to total PBMCs (peripheral blood mononuclear cells). "Conv 9 days" refers to donor-matched CAR-T cells manufactured from leukocyte separation starting material in a standard 9-day culture process. Each figure shows one representative donor from n=5.

[0102] Figure 35This study demonstrates the target-dependent cytokine release of anti-CD19 CAR-T cells manufactured using the KYV 3-day process in response to CD19+ target cells. This was achieved by measuring IFN-γ production in anti-CD19 CAR-T cells (manufactured from freshly collected whole blood in the KYV 3-day process) co-cultured with CD19+ NALM6 target cells or CD19- CEMC1 control cells at a specified E:T (effectant:target) ratio. Culture supernatants were collected and analyzed by ELLA. The study also illustrates N=2 donors.

[0103] Figure 36A This study demonstrates the effector dose-dependent CAR-mediated cytokine release of anti-CD19 CAR-T cells manufactured using the KYV 3-day process in response to CD19+ target cells. Cytokine release was measured from anti-CD19 CAR-T cells (manufactured from leukocyte separation starting material in the KYV 3-day process) co-cultured with CD19+ NALM6 target cells at a specified E:T (effectant:target) ratio of 0.3:1 or 1:1. Culture supernatants were collected, and specified cytokines were analyzed by MSD. KYV 3-day conditions “C1”, “C2”, “C3”, and “C4” indicate different cultured cytokines used in the manufacturing process. “NT” = untransduced control T cells. N = 4 healthy donors for each condition.

[0104] Figure 36B The study demonstrated cytokine release from anti-CD19 CAR-T cells manufactured from freshly collected whole blood in a KYV 3-day process, co-cultured with CD19+ NALM6 target cells at a specified E:T (effectant:target) ratio of 0.3:1 or 1:1. Culture supernatants were collected and the specified cytokines were analyzed by MSD. "Conv 9 days" refers to donor-matched CAR-T cells manufactured from leukocyte-isolated starting material in a standard 9-day culture process. N=4 healthy donors were used under each condition.

[0105] Figure 36C The study demonstrated cytokine release from anti-CD19 CAR-T cells manufactured from freshly collected whole blood in a KYV 3-day process, co-cultured with CD19+ NALM6 target cells at a specified E:T (effectant:target) ratio of 0.3:1 or 1:1. Culture supernatants were collected and the specified cytokines were analyzed by MSD. "Conv 9 days" refers to donor-matched CAR-T cells manufactured from leukocyte-isolated starting material in a standard 9-day culture process. N=4 healthy donors were used under each condition.

[0106] Figure 37The results showed that anti-CD19 CAR-T cells manufactured by the KYV 3-day process exhibited long-term continuous cytotoxic activity in response to target cells expressing CD19+, compared to the conventional 9-day process. Figure 37 This study provides the duration of in vitro cytotoxicity of KYV 3-day or Conv 9-day anti-CD19 CAR T cells derived from healthy donors in a series of rechallenge assays against CD19+ NALM6 tumor cells. KYV 3-day CAR T cells were derived from leukocyte ablation starting material (“APH”, top panel) or freshly collected whole blood (“WB”, bottom panel). CAR T cells were co-cultured in triplicate with NALM6 target cells at a specified effector:target (E:T) ratio, and NALM6 cell viability was analyzed by flow cytometry every 2–3 days. For each individual replicate, the time (in days) required for loss of CAR-mediated cytotoxic activity was measured, defined as the assay time point at which >95% target cell viability was detected. Data represent n=4 donors.

[0107] Figure 38 This diagram shows the results of in vitro expansion of anti-CD19 CAR-T cells manufactured using the KYV 3-day process in response to CD19+ target cells, compared to the conventional 9-day process. The figure shows the in vitro expansion of KYV 3-day or conventional (“Conv”) 9-day anti-CD19 CAR T cells in response to repeated stimulation in co-culture with CD19+ REH target cells. KYV 3-day CAR T cells were derived from leukocyte ablation starting material (“APH”, Figure A, n=4) or other freshly collected starting material (“WB”, Figure B, n=3) and compared to donor-matched Conv 9-day CAR T cells derived from leukocyte ablation material. CAR T cells were co-cultured with mitomycin C-treated REH target cells at a 1:1 ratio, and cells were replated with new target cells every 3–4 days. The total fold increase of CAR+ T cells (gated by flow cytometry analysis) was measured on day 16.

[0108] Figure 39AThis study demonstrates the in vivo activity of anti-CD19 CAR-T cells manufactured using the KYV 3-day process compared to the conventional 9-day process in CD19+ NALM6-bearing NSG mice. The figure shows the mean NALM6 tumor growth in NSG mice treated with a specified dose of donor-matched anti-CD19 CAR T cells manufactured using either the KYV 3-day process or the conventional (“Conv”) 9-day process, both starting with aleukocyte ablation (“APH”) as the starting material. NALM6-luciferase tumor cells were intravenously injected into mice on day 7 prior to T cell transfer. A specified dose of CAR T cells was injected intravenously into mice on day 0. Tumor burden in each animal was measured twice weekly using IVIS bioluminescence imaging and is shown as total flux (photons / second). Data are presented as mean ± SEM for all animals in each group. Data represent two studies using n=2 independent donors.

[0109] Figure 39B Individual NALM6 tumor growth curves are shown in NSG mice treated with donor-matched anti-CD19 CAR T cells at a dose of 1e6 CAR+ T cells. The CAR T cells were manufactured using a KYV 3-day process derived from freshly collected whole blood (“WB”) or a conventional (“Conv”) 9-day process derived from leukocyte ablation starting material. Tumor cells were inoculated and, as... Figure 39A The mice were treated and analyzed. N=5 animals per group.

[0110] Figure 40 Two 9-day processes for manufacturing CAR T cells using fresh whole blood as the starting material are outlined in this disclosure.

[0111] Figures 41A-41E provide flow cytometry data to characterize CAR T cells generated using the 9-day method of this disclosure, starting with whole blood.

[0112] Figure 42 Cell lysis % data are provided to characterize CAR T cells generated using the 9-day method of this disclosure as the starting material from whole blood.

[0113] Figure 43 Cytokine secretion data are provided to characterize CAR T cells generated using the 9-day method of this disclosure as the starting material from whole blood. Detailed Implementation

[0114] This document discloses methods for manufacturing engineered T cells under certain conditions, said engineered T cells expressing heterologous proteins, such as chimeric antigen receptors (CARs). Similar methods for manufacturing engineered T cells containing heterologous genes, wherein the heterologous genes may or may not encode proteins, are also contemplated. Compositions suitable for use in conjunction with the disclosed methods are also disclosed.

[0115] Exemplary methods for manufacturing such cells (e.g., CAR T cells) of this disclosure may include obtaining T cells from whole blood of a subject (e.g., a donor or patient). In some aspects, T cells are obtained from peripheral blood mononuclear cells (PBMCs) isolated from whole blood. In a more preferred aspect, the inventors have found, surprisingly, that a sufficient number of T cells for CAR T cell manufacturing can be obtained directly from whole blood without the need for a leukocyte ablation step or a PBMC separation step.

[0116] The methods disclosed herein may include simultaneous T cell isolation and activation steps. In such methods, T cells can be isolated from whole blood or PBMCs and activated simultaneously. This reduces overall manufacturing time while ensuring the isolation of a sufficient number of T cells with high purity to manufacture CAR T cells from very small whole blood samples (e.g., samples of less than 100 mL of blood).

[0117] Exemplary methods for producing the CAR T cells of this disclosure employ a short culture period, typically less than or significantly less than one day, after T cell isolation / activation prior to transduction, and a similarly short culture period after transduction prior to harvesting the desired CAR T cell product. Therefore, the methods of this disclosure are capable of harvesting large quantities of CAR T cells, all within 2-3 days of receiving the starting sample.

[0118] Surprisingly, the final CAR T cell product produced by the method of this disclosure has been shown to be of higher quality than that produced by existing, more time-consuming processes. CAR T cells manufactured using the method of this disclosure exhibit superior T cell expansion in the final CAR T cell product; their phenotype is better due to the simplified process of this disclosure; and their differentiation level is lower than that produced by more time-consuming processes. Furthermore, the resulting CAR T cells include a very high percentage of stem cell memory T cells (Tscm), which typically account for only 2-3% of circulating T cells and are associated with long-term defensive immunity, anti-tumor activity, self-renewal, and immune regulation.

[0119] Cells prepared using the methods disclosed herein express the introduced CAR at a very high percentage, producing a pure final product with targeted cytotoxicity and low T cell exhaustion. Even more surprisingly, the shortened manufacturing process yields a large number of pluripotent cells that simultaneously secrete multiple groups of cytokines, chemokines, and / or cytotoxic granules. Due to this pluripotent behavior, these cells have been shown to provide a more effective immune response, which is exactly what is expected of therapeutic CAR T cell products.

[0120] definition

[0121] 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 the claimed subject belongs. Generally, the nomenclature used in conjunction with the techniques described herein is that which is well-known and commonly used in the art. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and do not limit any of the claimed subjects. Section headings used herein are for organizational purposes only and should not be construed as limiting the described subjects. As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “and,” and “the” include plural indicators. Thus, for example, references to “an antibody” include multiple antibodies, and in some embodiments, references to “an antibody” include multiple antibodies, etc.

[0122] As used herein, unless the context explicitly indicates otherwise, all numerical values ​​or ranges of values ​​include whole integers within or encompassing such ranges, as well as fractions of values ​​or integers within or encompassing such ranges. Thus, for example, references to the range 90-100% include 91%, 92%, 93%, 94%, 95%, 96%, 97%, etc., and 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc. In another instance, references to the range of 1-5,000 times include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times, and 1.1, 1.2, 1.3, 1.4, 1.5 times, 2.1, 2.2, 2.3, 2.4, 2.5 times, and so on.

[0123] As used in this article, “approximately” refers to a range that includes the number and extends from 10% below the number to 10% above the number. “Approximately” range refers to the range from 10% below the lower limit of the range to 10% above the upper limit of the range.

[0124] As used herein, when applied in the context of T cell activation, the terms “activate,” “activating,” and “activated” encompass a variety of related biological processes, such as the induction of intracellular signaling pathways associated with T cell activation, changes in cell surface marker expression, cytokine release, and proliferation. Typically, T cell activation occurs due to the binding of the T cell receptor complex or its functional portions (e.g., CD3) and co-stimulatory molecules on T cells (e.g., CD28) via the major histocompatibility complex (MHC) and co-stimulatory molecules on antigen-presenting cells, respectively. The induction of intracellular signaling cascades associated with T cell activation includes activation of the PI3K pathway, recruitment of proteins containing a PH domain (e.g., PDKI), and eventual cytokine production (e.g., IL-2). Changes in T cell surface marker expression occur due to activation, resulting in increased expression of one or more of CD69, CD71, CD25, CD137, HLA-DR, and CTLA-4. The production and secretion of cytokines, chemokines, and other proteins (e.g., IFNγ, granzyme B, IL-1β, and / or IL-2) can also be induced by T cell activation.

[0125] As used herein, the term "basal medium" refers to a culture medium containing the minimum components necessary for cell (e.g., T cells) survival. "Basal medium" typically comprises an aqueous solution of amino acids (e.g., glycine, arginine, asparagine, aspartic acid, cysteine, glutamine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and / or the L-racemate of valine), vitamins (e.g., biotin, choline chloride, D-calcium pantothenate, folic acid, nicotinamide, para-aminobenzoic acid, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, and / or i-inositol), salts (e.g., calcium nitrate, ferric nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium pyruvate, and / or sodium phosphate), a sugar source (e.g., D-glucose), and optionally a reducing agent (e.g., glutathione). Representative examples of basal media include, but are not limited to, RPMI 1640, Eagle's Minimal Essential Medium (EMEM), Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle (a-MEM), and Glasgow Minimal Essential Medium (Glasgow MEM). In most cases, "basal media" do not include protein additives (e.g., cytokines, growth factors, and / or albumin). In some embodiments, the pH of the "basal media" is between 7.0 and 7.4, such as 7.0, 7.1, 7.2, 7.3, or 7.4. In some embodiments, the "basal medium" has an isotonic concentration of 290 to 320 mOsmol (e.g., 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, or 320 mOsmol). Those skilled in the art will understand that mediums with other pH and isotonic concentrations can be used. In some embodiments, the "basal medium" is hypotonic, isotonic, or hypertonic.

[0126] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a chimeric receptor protein comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain that are specific for antigen binding. In some cases, the extracellular domain may contain an antigen-binding domain. In some cases, the transmembrane domain may contain a transmembrane domain derived from a native polypeptide obtained from a membrane-bound or transmembrane protein.

[0127] For example, transmembrane domains may include, but are not limited to, transmembrane domains derived from the α or β chain of T cell receptors, the CD3ζ chain, the CD28 polypeptide, or the CD8 polypeptide. In some cases, intracellular domains may comprise cytoplasmic signaling domains (e.g., any cytoplasmic signaling domains described herein) and one or more co-stimulatory domains (e.g., any exemplary co-stimulatory domains described herein).

[0128] As used herein, the terms “contact,” “contacting,” “contacted,” etc., encompass any method of exposing a composition (e.g., cells or cell populations, such as T cells) to another composition (e.g., polynucleotides) such that they can interact directly. Those skilled in the art will understand that an exemplary method of contacting a cell population with a reagent (e.g., a nucleic acid encoding a heterologous protein, such as a CAR) is by mixing an aqueous suspension of the cells with an aqueous solution or suspension of the reagent. Although not all individual cells in the population can immediately interact directly with the reagent, even if the reagent is provided in an excess amount relative to the cells, over time (e.g., 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours or more), according to this method, a considerable number of cells (e.g., at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the cells in the population) will directly interact with one molecule of the reagent.

[0129] As used herein, the term "cytotoxicity" refers to the ability of a cell (e.g., a T cell engineered according to the methods disclosed herein to express a chimeric antigen receptor (CAR)) to induce cell death (e.g., apoptosis or necrosis) in another cell (e.g., a target cell). For example, engineered T cells expressing a CAR can elicit a cytotoxic response against target cells expressing a target antigen. In some embodiments, binding between the antigen-binding domain of the CAR and the target antigen can generate T cell activation and killing of the target cell. Assays for detecting CAR-T cell-induced cytotoxicity include, but are not limited to, chromium release assays, bioluminescence assays (e.g., luciferase-mediated bioluminescence imaging), real-time impedance-based analyses, flow cytometry (e.g., in combination with viability dyes such as CTV), and CFSE / PI assays.

[0130] As used herein, the term "delivery medium" refers to any drug carrier, diluent, excipient, etc., generally intended for use in conjunction with the administration of a bioactive agent, including nucleic acids. For example, a delivery medium may include lipid-based or polymer-based transfer media for delivering nucleic acids, including but not limited to lipid nanoparticles, liposomes, polymer nanoparticles (nanocapsules or nanospheres), etc. In some embodiments, the delivery medium is a lipid nanoparticle. In the case of delivering nucleic acids to target cells, "delivery medium" may also include any carrier (e.g., a viral or non-viral vector) capable of delivering nucleic acids to target cells. In some embodiments, the delivery medium is a viral vector, such as a lentiviral vector.

[0131] As used herein, when referring to cells (e.g., T cells) that have been in contact with nucleic acids encoding heterologous proteins (e.g., CARs), the term "engineered" means that the nucleic acid encoding the heterologous protein or a fragment thereof is stably integrated into the cell's genome after contact.

[0132] As used herein, the term "harvest" and the like refers to the isolation and / or collection of cells or cell populations (e.g., T cells) after culturing cells under culture conditions. In some embodiments, harvesting includes altering one or more conditions, such as temperature, cell culture medium, and / or the availability of certain reagents (e.g., one or more reagents that activate CD3 and / or CD28, one or more cytokines, and / or polynucleotides encoding heterologous proteins), to terminate a step immediately preceding the harvesting step.

[0133] As used herein, the term "heterologous" refers to a nucleic acid or polypeptide sequence or domain that is not present in its natural form or amount in its natural environment. For example, in some embodiments, a heterologous nucleic acid (e.g., a gene) is not present between its adjacent flanking sequences, such as where a heterologous sequence is not found in nature to be coupled to a nucleic acid or polypeptide sequence occurring at one or both ends. In some embodiments, the heterologous protein is completely absent in natural cells prior to engineering for protein expression. In some embodiments, the heterologous protein has post-translational modifications different from those of proteins in natural cells prior to being engineered for protein expression. In some embodiments, the amount of the heterologous protein is much lower than that of proteins in natural cells prior to being engineered for protein expression.

[0134] As used herein, the term "sample" refers to a biological sample obtained from a subject (e.g., a human), such as a blood sample (e.g., a whole blood sample). In some embodiments, a sample is a blood sample processed by conventional methods to separate a desired blood fraction (e.g., serum or plasma) or one or more cell types of interest (e.g., peripheral blood mononuclear cells (PBMCs), lymphocytes such as T lymphocytes). For example, "sample" may refer to a leukocyte ablation sample obtained from a subject's blood. In one embodiment, "sample" refers to a whole blood sample obtained from a subject.

[0135] In some embodiments, methods for rapidly manufacturing engineered T cells containing heterologous genes or expressing heterologous proteins, such as chimeric antigen receptors (CARs), are disclosed herein. Compositions suitable for use in conjunction with the disclosed methods are also disclosed. The methods disclosed herein offer several advantages over existing CAR-T manufacturing methods, including producing more efficient CAR-T cells compared to CAR-T cells produced using longer manufacturing protocols, thereby facilitating the use of lower CAR-T cell doses for therapeutic purposes. Furthermore, the disclosed methods preserve the "primitiveness" of T cells (i.e., a less differentiated phenotype), resulting in CAR-T cells with higher proliferative potential. The shorter CAR-T manufacturing time resulting from the disclosed methods also helps to scale up CAR-T cell production, thereby reducing costs, decreasing "needle-to-needle" time (i.e., the time from harvesting patient T cells to delivering autologous engineered T cells back to the patient), and improving patient accessibility.

[0136] CAR-T cells

[0137] Chimeric antigen receptors (CARs) are artificially constructed hybrid receptor proteins or polypeptides containing antigen-binding domains, such as antigen-binding fragments of antibodies. These domains can take various forms, such as single-chain variable fragments (scFvs), and are linked to one or more intracellular signaling or activation domains (optionally including co-stimulatory domains) via transmembrane domains. Autologous T-cell-based therapies, such as T cells modified to express CARs, have demonstrated significant therapeutic benefits for patients with cancer.

[0138] Unwilling to be bound by specific theories or mechanisms, it is believed that by inducing an antigen-specific response against cells expressing target antigens, CARs provide one or more of the following benefits: targeting and destroying cells expressing target antigens, reducing or eliminating target cells, promoting immune cell infiltration into target tissues, and enhancing / prolonging anti-cancer responses. CAR-T cells can also be used to reduce autoimmune responses by targeting cells that mediate autoimmunity (e.g., B cells).

[0139] Despite promising unprecedented therapeutic prospects, CAR-T cell production faces substantial obstacles, including long manufacturing times relevant to the scope of this disclosure. Prolonged CAR-T cell manufacturing processes can adversely lead to batch losses, reduced in vivo CAR-T cell expansion and persistence, increased batch-to-batch variability in the final cell product, increased differentiation and heterogeneity of the final cell product, increased manufacturing costs, and delays in providing potentially life-saving treatments to patients. Therefore, this disclosure provides methods and compositions for the rapid manufacture of CAR-T cells. It should be understood that similar methods and compositions can be used for the rapid manufacture of T cells expressing other heterologous genes.

[0140] Methods for manufacturing engineered T cells

[0141] In some embodiments, methods for manufacturing T cell populations engineered to express heterologous proteins are disclosed herein. The methods disclosed herein enable the rapid manufacturing of engineered T cells expressing heterologous proteins, such as chimeric antigen receptors (CARs), under specified conditions.

[0142] T cells are fully mature white blood cells found in the thymus. They can recognize certain foreign antigens and play various roles in the immune system, including the activation and inactivation of other immune cells. Typically, T cells can be any type of T cell, such as cultured T cells (e.g., primary T cells), or T cells derived from cultured T cell lines (e.g., Jurkat, SupT1), or T cells obtained from mammals. T cells include, but are not limited to, naive T cells, stimulating T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or subsets thereof. T cells can be CD3+ cells. T cells can be CD4+, CD8+, or CD4+ and CD8+. For example, T cells can be CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 or Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral cells, including but not limited to peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, γδ T cells, etc. T cells can be T cells at any developmental stage. Other types of helper T cells include Th3 (Treg) cells, Th17 cells, Th9 cells, or Tfh cells. Other types of memory T cells include, for example, central memory T cells (Tcm cells) and effector memory T cells (TfH cells). EM Cells and T EMRA T cells can also refer to genetically modified T cells (e.g., engineered T cells), such as T cells that have been modified to express heterologous proteins (e.g., chimeric antigen receptors (CARs)). T cells can also differentiate from stem cells or progenitor cells.

[0143] In some embodiments, the disclosed method involves generating engineered T cells that express CARs with binding specificity to a target antigen. In some embodiments, the disclosed method is used to generate a population of engineered T cells from a starting cell population within a short timeframe (e.g., 2, 3, or 4 days). For example, the method disclosed herein can generate a population of engineered T cells expressing a heterologous protein (e.g., CAR) within approximately 48 hours (hr), 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 81 hours, 82 hours, 83 hours, 84 hours, 85 hours, 86 hours, 87 hours, 88 hours, 89 hours, 90 hours, 91 hours, 92 hours, 93 hours, 94 hours, 95 hours, or 96 hours.

[0144] In some preferred aspects, the method allows the generation of engineered T cells (e.g., T cells expressing CAR) in about 2-3 days.

[0145] Therefore, in some embodiments, the disclosed method includes the following steps: (1) optionally enriching / isolating a biological sample from a subject (e.g., a human subject) containing a T cell initiation population; (2) activating the T cell initiation population with a CD3-binding reagent and a co-stimulatory molecule-binding reagent (e.g., CD28 / CD3); (3) contacting the T cells with a polynucleotide containing a nucleic acid sequence encoding a heterologous protein (e.g., CAR) and culturing the T cells for a short period under conditions suitable for promoting the expression of the heterologous protein by the T cells; and (4) harvesting and optionally storing the engineered T cells for subsequent use (e.g., therapeutic use or quality control testing). Each of the foregoing steps is described in more detail in the following sections.

[0146] Figure 1 A schematic diagram of the method of this disclosure for generating CAR T cells from whole blood samples and / or enriched PBMCs is provided. As will be described in more detail, the steps of this workflow may include certain variations. One such variation may occur in the isolation and activation steps. Certain preferred methods of this disclosure combine these steps in a single, simultaneous isolation and activation step using a T cell activator (e.g., CD3 / CD28 beads) to isolate and activate T cells.

[0147] Sample preparation steps

[0148] In some embodiments, this document discloses methods for isolating a T-cell initiation population from a biological sample, such as a sample obtained from a subject (e.g., a human subject). Non-limiting examples of biological samples include cells, tissues (e.g., tissues obtained by biopsy), blood, serum, plasma, or any sample derived therefrom.

[0149] In a preferred embodiment, the sample is a whole blood sample obtained from the subject, from which T cells are isolated without using a separation / leukocyte separation step. In such methods, T cells can be directly isolated from whole blood in a single step and / or the whole blood undergoes a step of enriching peripheral blood mononuclear cells (PBMCs), from which T cells are subsequently isolated. In the preferred method, T cells are isolated directly from whole blood without using a separation / leukocyte separation step, and without using a step of separating or enriching PBMCs (from which T cells are obtained). Surprisingly, the method disclosed herein is able to obtain a sufficiently pure and large population of T cells directly from a small whole blood sample without a series of intermediate steps to isolate / enrich T cells from whole blood.

[0150] In some embodiments, the method includes obtaining a sample from a subject. In some embodiments, the method includes having already obtained a sample from a subject.

[0151] In some embodiments, the method of this disclosure includes obtaining a T-cell initiation population from a biological sample obtained from a subject. In some embodiments, the biological sample is a leukocyte ablation sample. In some embodiments, the biological sample is a whole blood sample. In some embodiments, the T-cell initiation population includes helper T (Th) cells, cytotoxic T (T) cells, and other T-cell initiation populations. C ) cells, memory T (T) M Th cells include Th1 cells, Th2 cells, Th17 cells, Th9 cells, Tfh cells, and / or Th22 cells. In some embodiments, Th cells include Th1 cells, Th2 cells, Th17 cells, Th9 cells, Tfh cells, and / or Th22 cells. M Cells include central memory T cells (T cells) CM Cellular, effector memory T (T) EM Cellular and tissue resident memory T (T) RM ) cells and virtual memory T (T VM In some embodiments, innate T-like cells include natural killer T (NKT) cells, mucosa-associated invariant T (MAIT) cells, and γδ T cells.

[0152] In some embodiments, the method includes isolating a T-cell initiation population from a sample. T-cell isolation may include preliminary purification of T cells from a mixture of plasma, lymphocytes, platelets, erythrocytes, monocytes, and granulocytes. Methods for isolating T cells from biological samples, such as whole blood samples, enriched PBMC samples, or leukocyte ablation samples, are well known. Exemplary methods may include panning, density gradient centrifugation, selective enrichment, etc. For example, the method may include obtaining or having obtained a biological sample, such as a fresh, refrigerated, frozen, or cryopreserved product or an alternative source of hematopoietic tissue, such as whole blood samples, bone marrow samples, or tumor or organ biopsy or resected material from a solid body (e.g., a laboratory, hospital, or healthcare provider) (e.g., a thymectomy), and performing the isolation steps described above to produce an enriched T-cell population (e.g., a T-cell initiation population) suitable for expressing heterologous proteins.

[0153] Surprisingly, the short two- to three-day manufacturing process of this disclosure enables the direct generation of CAR T cells from whole blood samples and / or PBMC-enriched samples without the use of initial leukocyte separation. Prior to the inventors' discovery, methods for generating CAR T cells (particularly methods for generating CAR T cells at sufficient throughput and speed) typically employed leukocyte separation to produce concentrated leukocyte samples, thus allowing for easy isolation of T cells. However, the inventors have demonstrated that the method of this disclosure can directly isolate a sufficient number of T cells from whole blood and / or PBMC-enriched whole blood to generate CAR T cells. This not only avoids the costs and potential complications associated with leukocyte separation but also reduces the time required to generate the desired CAR T cells, while expanding sample availability due to the reduced logistical complexity resulting from only a simple whole blood draw.

[0154] Furthermore, the purity of the initial T cell population can be increased by using one or more selection steps (e.g., negative selection or positive selection). Negative selection typically involves removing unwanted cell types from a mixed cell population in a sample using one or more reagents that selectively bind to unwanted cell types, while positive selection typically involves separating the desired cell population using one or more reagents that selectively bind to desired cell types. For example, enrichment of the T cell population by negative selection can be accomplished using a combination of antibodies targeting surface markers specific to the negatively selected cells. One approach is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry, which uses a mixture of monoclonal antibodies targeting cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody mixture may include antibodies against CD14, CD20, CDb, CD16, HLA-DR, and CDR.

[0155] On the other hand, the positive selection step can be used to specifically select a desired cell type, including in the methods of this disclosure, where this step can be used to directly isolate T cells from whole blood or PBMC samples. In some embodiments, positive selection of T cells may include incubating a mixed cell population containing T cells (e.g., whole blood / PBMC sample) with a reagent having a CD3-binding moiety (e.g., anti-CD3 antibody-conjugated beads) for a duration sufficient to positively select the desired T cells. In some embodiments, the time period is about 30 minutes. In some embodiments, the time period ranges from 30 minutes to 36 hours or longer, and all integer values ​​in between. In some embodiments, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the time period is 10 to 24 hours, such as 18 hours. In any case where T cells are scarce compared to other cell types, a longer incubation time may be used to isolate T cells.

[0156] In some embodiments, the T cell initiation population comprises CD8+ T cells (e.g., CD8+ cytotoxic T cells). In some embodiments, the T cell initiation population also comprises CD4+ T cells (e.g., CD4+ helper T cells). In some embodiments, the T cell initiation population comprises 1-10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 20-30%, 20-40%, 20-50%, 20-60%, 30-40%, 30-50%, or 30-60% of all T cells in the population that are CD8+ T cells (e.g., CD8+ cytotoxic T cells). In some embodiments, the T cell initiation population further comprises 1-10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 1-70%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 30-40%, 30-50%, 30-60%, or 30-70% of all T cells in the population that are CD4+ T cells (e.g., CD4+ helper T cells). In some embodiments, the T cell initiation population comprises CD8+ T cells (e.g., CD8+ cytotoxic T cells) and CD4+ T cells (e.g., CD4+ helper T cells) in a ratio of 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, or 1:2 to 2:1.

[0157] In some embodiments, a T-cell initiation population is generated to achieve the desired purity. For example, the T-cell initiation population may include at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more of the total number of cells in the population. The purity of the T-cell initiation population can be measured using conventional methods, such as fluorescence-assisted cell sorting (FACS), immunopanning, microarray-based methods, etc. Various known T-cell phenotyping methods can also be applied to further increase the purity of the T-cell initiation population.

[0158] Furthermore, one or more freeze-thaw cycles can be performed on the initial T cell population to enrich the desired cell types. For example, freeze-thaw cycles can improve the purity of the T cell population by further removing granulocytes and a certain degree of monocytes from the mixed cell population. Routine and conventional methods for freezing and thawing T cells can be used in conjunction with the methods disclosed herein. In some embodiments, after freezing, the frozen cells are thawed, washed, and allowed to stand at room temperature for, for example, one hour before activation using the disclosed methods.

[0159] In some embodiments, the viability of the T cell initiation population can be determined using known methods. For example, one or more known T cell identity and viability markers (e.g., dyes, antibodies, etc.) can be used to determine the T cell initiation population, wherein the overlap of signals indicating both T cell identity and viability indicates the viability of the T cell initiation population. In some embodiments, the initiation population comprises a percentage of live T cells that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more of the total number of T cells in the T cell initiation population.

[0160] In some embodiments, the exhaustion and / or activation status of the T cell initiation population can be determined. For example, the exhaustion status of the T cell initiation population can be tested using one or more (e.g., 1, 2, 3 or more) T cell exhaustion markers, including but not limited to overexpression of one or more of LAG-3, PD-1, PD-LI, TIM-3, 2B4, CD160, TIGIT, CTLA-4, VISTA, etc. The activation status of T cells in the initiation population can be assessed by testing the overexpression of one or more T cell activation markers (e.g., CD69, CD71, CD25, CD137, HLA-DR, CTLA-4, L2RA / CD25, IFNγ, TNFα, etc.). Other indicators of T cell activation include, but are not limited to, T cell proliferation and differentiation.

[0161] After isolating and enriching T cells from a T cell initiation population from a source biological sample, the T cell initiation population can be cultured under conditions suitable for maintaining the T cells in a quiescent state prior to activation. Such methods typically do not use activators (e.g., CD3 / CD28 beads) to isolate T cells, but instead use other T cell isolation methods known in the art, such as CD4 / CD8 beads.

[0162] However, in some preferred methods of this disclosure, T cells are activated simultaneously upon isolation, for example, by using CD3 / CD28 beads. In this case, there is no resting period prior to activation. However, transduction can occur after a brief culture following activation. In some aspects, transduction is performed for at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, at least 25 hours, at least 26 hours, at least 27 hours, at least 28 hours, at least 29 hours, at least 30 hours, or longer after activation.

[0163] In alternative or other aspects, the activation and / or contact with an activating stimulant (e.g., CD3 / CD28 beads) may be carried out for a period of at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, at least 25 hours, at least 26 hours, at least 27 hours, at least 28 hours, at least 29 hours, at least 30 hours, or longer. Preferably, the time from isolation / activation to transduction is less than 30 hours, less than 25 hours, less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour. In a preferred aspect, the time from isolation / activation to transduction is about 12-24 hours, more preferably about 14-22 hours, more preferably about 16-22 hours, and even more preferably about 18 hours.

[0164] A nucleic acid vector encoding a heterologous protein (e.g., CAR) can be used to seed a T cell initiation population at the desired density to promote T cell transduction (and / or activation in some methods, if desired). For example, the T cell initiation population can be seeded at 1 × 10⁻⁶. 5 Cells / mL to 1×10 7 The cells were seeded into the culture at a concentration of approximately 1 × 10⁶ cells / mL. In some embodiments, the T cell initiation population was seeded at approximately 1 × 10⁶ cells / mL. 6 The cells were seeded into the culture at a concentration of 1 × 10⁶ cells / mL. In some embodiments, the T cell initiation population was seeded at 1 × 10⁶ cells / mL. 6 The cells were seeded into the culture at a concentration of approximately 100 cells / mL. In some embodiments, the T cell initiation population was seeded at approximately 2 × 10⁻⁶ cells / mL. 6 The cells were seeded in the culture at a concentration of 10 cells / mL. In some embodiments, the T cell initiation population was seeded at 10... 6 The cells were seeded into the culture at a concentration of approximately 5 × 10⁶ cells / mL. In some embodiments, the T cell initiation population was seeded at approximately 5 × 10⁶ cells / mL. 6 The cells were seeded into the culture at a concentration of 10 cells / mL. In some embodiments, the T cell initiation population was seeded at 5 × 10⁻⁶ cells / mL. 6 The cells were inoculated into the culture at a concentration of 1 cell / mL.

[0165] In some methods of this disclosure, the number of isolated T cells from whole blood samples is approximately 1 × 10⁻⁶. 6 With approximately 1×10 8 Between a total of 10,000 CAR T cells, and the number of CAR T cells harvested was approximately 1 × 10⁻⁶. 8 With approximately 5×10 8 Between. In some methods of this disclosure, the number of isolated T cells from whole blood samples is approximately 12.5 × 10⁻⁶. 7 With approximately 1×10 8 Between 100 total CAR T cells, and the number of harvested CAR T cells was approximately 7.5 × 10⁻⁶. 7 With approximately 1.5 × 10 8 Between. In some methods of this disclosure, the number of isolated T cells from whole blood samples is approximately 5 × 10⁻⁶. 7 With approximately 7.5 × 10 7 Between 100 total CAR T cells, and the number of CAR T cells harvested was approximately 7.5 × 10⁻⁶. 7 With approximately 1.2 × 10 8 between.

[0166] In some embodiments, prior to activation, the T-cell initiation population is cultured in a medium containing no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% serum (e.g., human serum). In some embodiments, prior to activation, the T-cell initiation population is cultured in a medium containing 2% serum. In some embodiments, prior to activation, the T-cell initiation population is cultured in a serum-free medium.

[0167] In some embodiments, prior to activation, a T cell initiation population is cultured in a medium containing one or more cytokines selected from the group consisting of IL-2, IL-7, IL-15, and IL-21. In some embodiments, the one or more cytokines is IL-2. In some embodiments, the one or more cytokines are IL-7 and IL-15. In some embodiments, the one or more cytokines are IL-2, IL-7, and IL-15. In some embodiments, the one or more cytokines are IL-21. In some embodiments, the one or more cytokines are IL-21, IL-7, and IL-15. In some embodiments, T cells are contacted with 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL of IL-2, alone or in combination with one or more other cytokines. In some embodiments, T cells are contacted with 100 ng / mL of IL-2. In some embodiments, T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL of IL-7, alone or in combination with one or more other cytokines. In some embodiments, T cells are contacted with 10 ng / mL IL-7. In some embodiments, T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL IL-15, alone or in combination with one or more other cytokines. In some embodiments, T cells are contacted with 10 ng / mL IL-15. In some embodiments, T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL IL-21, alone or in combination with one or more other cytokines. In some embodiments, a T cell initiation population is cultured prior to activation in the absence of any cytokines selected from IL-2, IL-7, IL-15, and IL-21. In some embodiments, the T cell initiation population is cultured in a cytokine-free medium prior to activation.

[0168] T-cell activation steps

[0169] In some embodiments, this document discloses a method for preparing engineered T cell populations expressing heterologous proteins (e.g., CARs), which includes the step of activating a T cell initiation population. In some embodiments, activation of the T cell initiation population includes contacting the T cell initiation population with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates co-stimulatory molecules on the surface of T cells. In some embodiments, the co-stimulatory molecules are CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, the agent stimulating the CD3 / TCR complex is an anti-CD3 antibody or its antigen-binding fragment (e.g., full-length IgG, Fab fragment, single-domain antibody, scFv, bifunctional antibody, trifunctional antibody, etc.). In some embodiments, the agent stimulating the CD3 / TCR complex is a small molecule or peptide ligand. In some embodiments, the co-stimulatory molecule is CD28. In some embodiments, the agent stimulating the co-stimulatory molecule is an anti-CD28 antibody or its antigen-binding fragment.

[0170] In some embodiments, the reagent stimulating the CD3 / TCR complex or the reagent stimulating the co-stimulatory molecule comprises beads (e.g., magnetic beads). In some embodiments, the reagent stimulating the CD3 / TCR complex or the reagent stimulating the co-stimulatory molecule is a solid surface (e.g., beads) comprising an anti-CD3 antibody and / or an anti-CD28 antibody covalently linked thereto. In some embodiments, the reagent stimulating the CD3 / TCR complex or the reagent stimulating the co-stimulatory molecule does not comprise beads. In some embodiments, the reagent stimulating the CD3 / TCR complex and the reagent stimulating the co-stimulatory molecule (e.g., CD28) comprise a first reagent stimulating CD3 and a second reagent stimulating the co-stimulatory molecule. In some embodiments, the reagent stimulating the CD3 / TCR complex and the reagent stimulating the co-stimulatory molecule (e.g., CD28) are the same reagent. In some embodiments, the reagent stimulating the CD3 / TCR complex and the reagent stimulating the co-stimulatory molecule (e.g., CD28) are bispecific antibodies that specifically bind to CD3 and CD28. In some embodiments, the reagents for stimulating the CD3 / TCR complex and the co-stimulatory molecules (e.g., CD28) are beads (e.g., magnetic beads) containing anti-CD3 and anti-CD28 antibodies covalently linked thereto. When the reagents for stimulating the CD3 / TCR complex and the co-stimulatory molecules (e.g., CD28) are beads, it should be understood that the beads may remain attached to T cells at the end of the activation step (i.e., at the start of the transfection step) and that the T cells may be separated from the beads prior to harvest due to the natural degradation of the protein portions of the reagents and / or by the use of cleavable adapters.

[0171] In some respects, stimulants (e.g., anti-CD3 antibodies) and co-stimulatory molecules (e.g., anti-CD28 antibodies) attach to the surface via cleavable linkers. In such methods, one or more of these activators can be selectively removed by providing the cells with stimuli that cleave the cleavable linkers.

[0172] For example, in some methods of this disclosure, stimulants (e.g., anti-CD3 antibodies) and / or co-stimulatory molecules (e.g., anti-CD28 antibodies) are attached to a vector via cleavable linkers. Similarly, in methods for isolating and activating T cells at different times, such as using CD4 / CD8 beads, anti-CD4 and anti-CD8 antibodies can also be surface-linked using cleavable linkers. In such methods, anti-CD4 / CD8 binding can be stopped by linker cleavage, after which the cells can be contacted with an activator, for example. In some methods of this disclosure, one or more anti-CD3 antibodies and one or more anti-CD28 antibodies are attached to the same vector. Alternatively, the antibodies bind to different vectors, such as different beads.

[0173] In a preferred method, one or more surface-bound anti-CD3 antibodies and / or one or more anti-CD28 antibodies are used to simultaneously isolate and activate T cells from a whole blood sample. In some aspects, the anti-CD3 and anti-CD28 antibodies are attached to a carrier via a cleavable linker. The linker can be, for example, an enzymatically cleavable, hydrolyzable, redox-cleavable, phosphate-based, acid-based, ester-based, peptide-based, disulfide-based, nitrobenzyl-based, methoxymethyl-based, and / or photocleavable linker. Therefore, the method of this disclosure also includes contacting the activated T cells with a stimulant of the cleavage linker, thereby releasing the T cells from the surface.

[0174] In some aspects, the surface is a solid surface. Preferably, the solid surface is a bead, a pore, a chip, or a microfluidic channel. More preferably, the solid surface is a bead.

[0175] In some aspects, the surface comprises a polymer. In some aspects, the polymer is a hydrogel. In some methods, the surface comprises a polymer scaffold. In some embodiments, the reagents stimulating the CD3 / TCR complex and / or the reagents stimulating the co-stimulatory molecules comprise anti-CD3 antibodies and / or anti-co-stimulatory molecule antibodies covalently linked to the colloidal polymer matrix (e.g., a nanomatrix). In some embodiments, the matrix comprises or is composed of a polymer (e.g., a biodegradable or biocompatible) inert material (e.g., a cell-free material). In some embodiments, the matrix consists of hydrophilic polymer chains that acquire maximum mobility in aqueous solution due to chain hydration. In some embodiments, the mobile matrix may be collagen, purified protein, purified peptide, polysaccharide, glycosaminoglycan, or an extracellular matrix composition. Polysaccharides may include, for example, cellulose ethers, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectin, xanthan gum, guar gum, or alginate. Other polymers may include polyesters, polyethers, polyacrylates, polyacrylamides, polyamines, polyethyleneimine, polyquaternium salt polymers, polyphosphazenes, polyvinyl alcohol, polyvinyl acetate, polyvinylpyrrolidone, block copolymers, or polyurethanes.

[0176] In some embodiments, the contact between the T cell initiation population and the reagent stimulating the CD3 / TCR complex and / or the reagent stimulating the co-stimulatory molecule is performed once at the start of the activation step. In some embodiments, the contact between the T cell initiation population and the reagent stimulating the CD3 / TCR complex and / or the reagent stimulating the co-stimulatory molecule is performed simultaneously with T cell dissociation. In some embodiments, the contact between the T cell initiation population and the reagent stimulating the CD3 / TCR complex and / or the reagent stimulating the co-stimulatory molecule is performed once at the start of the activation step, and once or more (e.g., 1, 2, 3 or more) throughout the duration of the activation step.

[0177] Without being bound by any theory, those skilled in the art will understand that the duration of binding between the reagents stimulating the CD3 / TCR complex and / or the reagents stimulating the co-stimulatory molecules and one or more cells in the T cell initiation population will depend on the specific reagents used, the concentration of the reagents, the concentration of cells seeded in the culture, and other factors.

[0178] In some embodiments, the activation step lasts for 12-24 hours (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours). In some embodiments, the activation step lasts for about 18 hours (e.g., 16, 17, 18, 19, or 20 hours). In some embodiments, the activation step lasts for 18 hours. In some embodiments, after the activation step, T cells remain associated with the reagent that stimulates the CD3 / TCR complex and / or the reagent that stimulates co-stimulatory molecules on the surface of T cells, such that activation can continue during subsequent steps. In some embodiments, activation effectively continues until the T cells dissociate from the reagent (e.g., by the natural degradation of the protein portion of the reagent and / or by the application of a stimulant that cleaves the cleavable linker).

[0179] In some embodiments, during the activation step, the T cell starter population is cultured in a medium containing no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% serum.

[0180] In some embodiments, during the activation step, the T cell initiation population is cultured in a medium containing 2% serum. In some embodiments, during the activation step, the T cell initiation population is cultured in a medium containing basal medium and serum (e.g., 2% serum). In some embodiments, the medium does not additionally contain any added cytokines or growth factors other than proteins derived from serum. In some embodiments, the medium does not additionally contain any added proteins (e.g., soluble proteins) other than proteins derived from serum.

[0181] In some embodiments, the T cell initiation population is cultured in serum-free medium during the activation step. In some embodiments, the T cell initiation population is cultured in basal medium without serum supplementation during the activation step. In some embodiments, the basal medium does not contain any cytokines or growth factors. In some embodiments, the basal medium does not contain any added proteins. It should be understood that cells in the culture medium may secrete proteins during cell culture. It should also be understood that reagents stimulating the CD3 / TCR complex and / or reagents stimulating co-stimulatory molecules may naturally degrade over time, releasing fragments into the culture medium. These proteins are not considered "added proteins".

[0182] In some embodiments, during the activation step, a T cell initiation population is cultured in a medium containing one or more cytokines selected from the group consisting of IL-2, IL-7, IL-15, and IL-21. In some embodiments, the one or more cytokines is IL-2. In some embodiments, the one or more cytokines are IL-7 and IL-15. In some embodiments, the one or more cytokines are IL-2, IL-7, and IL-15. In some embodiments, the one or more cytokines are IL-21. In some embodiments, the one or more cytokines are IL-21, IL-7, and IL-15. In some embodiments, T cells are contacted with IL-2 alone or in combination with one or more other cytokines at concentrations of 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL.

[0183] In some embodiments, T cells are contacted with 100 ng / mL IL-2. In some embodiments, T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL IL-7, alone or in combination with one or more other cytokines. In some embodiments, T cells are contacted with 10 ng / mL IL-7. In some embodiments, T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL IL-15, alone or in combination with one or more other cytokines. In some embodiments, T cells are contacted with 10 ng / mL IL-15. In some embodiments, T cells are contacted with IL-21 alone or in combination with one or more other cytokines at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL. In some embodiments, a T cell initiation population is cultured in the absence of any cytokines selected from IL-2, IL-7, IL-15, and IL-21 prior to activation. Serum-free medium (e.g., basal medium) or serum-supplemented medium may be provided in the presence or absence of the cytokines and combinations thereof disclosed herein.

[0184] In a preferred method, the culture medium is a serum-free medium and contains at least one cytokine. Preferably, the at least one cytokine contains one or more of IL-2, IL-21, IL-7, and IL-15. In some methods, the at least one cytokine contains one or more of IL-21, IL-7, and IL-15 and does not contain IL-2.

[0185] In some embodiments, the T cell initiation population is cultured in cytokine-free medium or without the addition of cytokines prior to activation. In other embodiments, the contact of the T cell initiation population with reagents that stimulate the CD3 / TCR complex and / or reagents that stimulate co-stimulatory molecules (e.g., CD28) occurs simultaneously with the contact of the cell population with one or more cytokines. In some embodiments, the contact of the T cell initiation population with reagents that stimulate the CD3 / TCR complex and / or reagents that stimulate co-stimulatory molecules (e.g., CD28) occurs for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours or longer prior to contact with one or more cytokines.

[0186] Transfection step: Delivering heterologous nucleic acids into T cells

[0187] In some embodiments, this document discloses methods for generating engineered T cell populations expressing heterologous proteins (e.g., CARs), including delivering nucleic acids encoding the heterologous protein to the T cells after an activation step. This document also discloses polynucleotides encoding heterologous proteins (e.g., CARs).

[0188] In some embodiments, the delivery of nucleic acid encoding a heterologous protein to T cells occurs no later than about 18 hours after the activation step (e.g., no later than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hour or less). In some embodiments, the delivery of nucleic acid encoding a heterologous protein to T cells occurs about 18 hours after the activation step (e.g., 16, 17, 18, 19, or 20 hours). In some embodiments, the delivery of nucleic acid encoding a heterologous protein to T cells occurs 18 hours after the activation step. Where transfection begins after the activation step has started, the cells at the end of the activation step (i.e., at the start of the transfection step) are referred to herein as the “T cell activated population,” although at least a portion of the cells in the population may not yet be fully activated, and further activation may occur during the transfection step. In other embodiments, the delivery of nucleic acid encoding a heterologous protein to the initial T cell population occurs in parallel with (e.g., simultaneously with) the activation step.

[0189] In some embodiments, the disclosed method includes contacting a population of T cells with an effective amount of polynucleotide encoding a heterologous protein after the activation step, such that the cells exhibit stable expression of the heterologous protein for at least 1 hour (hr), 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, or longer.

[0190] In some embodiments, the polynucleotide encoding a heterologous protein (e.g., CAR) comprises a codon-optimized nucleic acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 55, 65, 75, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 or more) nucleotide differences compared to the parental (i.e., non-codon-optimized) nucleic acid. Nucleic acid sequences can be codon optimized based on various principles, such as the principle that the frequency of synonymous codons (e.g., codons encoding the same amino acid) varies across different species. This codon degeneracy allows the same polypeptide to be encoded by multiple nucleotide sequences. This process can be performed on any of the sequences described in this specification to enhance expression or stability. Codon optimization can be performed using conventional methods. Sequences around translation initiation sites can be converted into shared Kozak sequences using known methods.

[0191] Nucleic acid vector

[0192] In addition to achieving high transcription and translation rates, stable expression of foreign genes (e.g., polynucleotides encoding heterologous polypeptides or functional fragments thereof) in mammalian cells can be achieved by integrating polynucleotides containing the gene into the nuclear genome of mammalian cells. Various vectors have been developed for the delivery and integration of polynucleotides encoding foreign proteins into the nuclear DNA of mammalian cells. Expression vectors used in the compositions and methods described herein contain polynucleotide sequences encoding heterologous proteins (e.g., CARs), as well as additional sequence elements, such as those for expressing these reagents and / or integrating these polynucleotide sequences into the genome of mammalian cells. Some vectors that can be used to express heterologous proteins include plasmids containing regulatory sequences (e.g., promoter and enhancer regions) that guide gene transcription. Other useful vectors for expressing heterologous proteins contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA transcribed from the genes. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (ORES), and polyadenylation signal sites to guide efficient transcription of the gene carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers are genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, and nourseothricin.

[0193] Expression vectors used in the compositions and methods described herein can express heterologous proteins (e.g., CARs) from monocistronic or polycistronic expression cassettes. Monocistronic expression cassettes contain a polynucleotide sequence encoding a single gene. The host cells described herein can be transfected with a variety of vectors, such as each vector containing a monocistronic expression cassette, or with a single vector containing more than one monocistronic expression cassette. Polycistronic expression cassettes can be used to simultaneously express two or more proteins from a single transcript. Polycistronic expression cassettes can include bicistronic or tricistronic expression cassettes, which can be used to produce two or three proteins separately from a single transcript, and can include an IRES sequence to recruit ribosomes to initiate translation from a region of mRNA excluding the 5' cap. Alternatively, foot-and-mouth disease virus 2A (FMDV 2A) polynucleotides can be used to express two or more genes (e.g., 2 genes, 3 genes, or more) and can be used in polycistronic expression cassettes to produce equimolar levels of multiple genes from the same transcript. FMDV 2A mediates co-translational cleavage events, which isolate proteins linked by 2A sequences, and multiple 2A sequences can be used in a single vector. 2A-like sequences from other viruses can also be used in the compositions and methods described herein, including 2A-like sequences from equine rhinitis A virus (E2A), porcine chevron virus-1 (P2A), and tussock moth virus (T2A).

[0194] Viral vector

[0195] Viral genomes provide a rich source of vectors for the efficient delivery of exogenous genes into mammalian cells. Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the nuclear genome of mammalian cells via general or specific transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Examples of viral vectors are retroviruses (e.g., retroviral vectors, such as lentiviral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles virus and Sendai virus), positive-strand RNA viruses such as piconemaviruses and alphaviruses, and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types I and II, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia virus, modified vaccinia Ankara (MVA), fowlpox virus, and canarypox virus). Other viruses include, for example, Norwalkvirus, tunica albuginea virus, flavivirus, reovirus, papillomavirus, hepatotropic DNA virus, human papillomavirus, human foamy virus, and hepatitis virus. Examples of retroviruses are: avian leukosis sarcoma virus, avian type C virus, mammalian type C virus, type B virus, type D virus, cancer retrovirus, HTLV-BLV group, alpha retrovirus, gamma retrovirus, and foamy virus. Other examples include murine leukosis virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukosis virus, feline leukosis virus, feline sarcoma virus, avian leukosis virus, human T-cell leukosis virus, baboon endogenous virus, gibbon leukosis virus, Mason-Fischer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses.

[0196] Exemplary lentiviral vectors that may be used according to this disclosure include vectors derived from human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), jembrana disease virus (JDV), equine infectious anemia virus (EIAV), and caprine arthritis encephalitis virus (CAEV).

[0197] Retroviral vectors are typically constructed by deleting most of the sequence encoding the structural genes of the virus and replacing it with a gene of interest or an expression cassette of interest (e.g., engineered nucleic acid as described herein). Most commonly, the structural genes (i.e., gag, pol, and env) are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with a suitable restriction endonuclease, or in some cases, digestion with a Bal 31 exonuclease, to produce a fragment containing the appropriate portion of the packaging signal. Thus, in some embodiments, the minimal retroviral vector comprises, from 5' to 3', a 5' long terminal repeat (LTR), a packaging signal, an optional exogenous promoter and / or enhancer, the exogenous gene of interest (or engineered nucleic acid), and a 3' LTR. In some embodiments, if no exogenous promoter is provided, gene expression may be driven by the 5' LTR, which is a weak promoter and requires the presence of Tat to activate expression. In many embodiments, the structural genes may be provided in a separate vector used to prepare lentiviruses, resulting in viral particles with replication defects. Specifically, for lentiviruses, the packaging system can contain a single packaging vector encoding the Gag, POL, Rev, and Tat genes, and a third separate vector encoding the envelope protein Env (typically VSV-G, due to its widespread infectivity). To improve the safety of the packaging system, the packaging vectors can be separated, with one vector expressing Rev and another expressing Gag and POL. Tat can also be removed from the packaging system using a retroviral vector containing a chimeric 5' LTR, where the U3 region of the 5' LTR is replaced by a heterologous regulatory element.

[0198] Nucleic acids (e.g., genes) to be packaged into retroviruses (e.g., lentiviruses) can be incorporated into the proviral backbone in several general ways. The most straightforward construction involves replacing the structural genes of the retrovirus with a single gene, which is then transcribed under the control of viral regulatory sequences within the LTR. Retroviral vectors capable of introducing more than one gene into target cells have also been constructed. Typically, in these vectors, one gene is regulated by the viral LTR, while a second gene is expressed via splicing or regulated by its own internal promoter.

[0199] Therefore, the nucleic acid (e.g., a gene) to be packaged into the retrovirus is flanked by 5' and 3' LTRs, which are used to promote transcription and polyadenylation of viral particle RNA, respectively. The term "long terminal repeat" or "(LTR)" refers to a base-pair domain located at the end of retroviral DNA, which in its natural sequence is a direct repeat sequence and contains U3, R, and U5 regions. LTRs typically provide essential functions for retroviral gene expression (e.g., initiation, activation, and polyadenylation of gene transcripts) and viral replication. LTRs contain numerous regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences required for viral genome replication and integration. The U3 region contains enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region and contains polyadenylation sequences. The R (repetitive sequence) region is flanked by the U3 and U5 regions. In some embodiments, the R region contains a transactivation response (TAR) genetic element that interacts with a transactivator (tat) genetic element to enhance viral replication. Such elements are not required in embodiments where the U3 region of the 5' LTR is replaced by a heterogeneous promoter.

[0200] In some embodiments, the retroviral vector comprises a modified 5' LTR and / or 3' LTR. The 3' LTR is often modified to improve the safety of lentiviral or retroviral systems by making the virus replication-defective. In some embodiments, the retroviral vector is a self-inactivating (SIN) vector. As used herein, a SIN retroviral vector refers to a replication-defective retroviral vector in which the 3' LTR U3 region has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because the 3' LTR U3 region serves as a template for the 5' LTR U3 region during viral replication, thus preventing viral transcripts from being obtained without the U3 enhancer-promoter. In some embodiments, the 3' LTR is modified such that the U5 region is replaced by, for example, a desired polyadenylated sequence. It should be noted that modifications to the LTR, such as modifications to the 3' LTR, 5' LTR, or both 3' and 5' LTR, are also included in some embodiments of this disclosure.

[0201] In some embodiments, the U3 region of the 5' LTR is replaced by a heterologous promoter to drive transcription of the viral genome during viral particle production.

[0202] Examples of heterologous promoters that can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters.

[0203] Typical promoters can drive high levels of transcription in a Tat-independent manner. This substitution reduces the likelihood of recombination producing replication-competent viruses because the complete U3 sequence is absent in the virus-generating system.

[0204] Adjacent to the 5' LTR are the sequences required for genome reverse transcription (tRNA primer binding sites) and the sequences required for efficient packaging of viral RNA into particles (Ψ sites). As used herein, the terms "packaging signal" or "packaging sequence" refer to sequences located within the retroviral genome that are required for capsidation of the retroviral RNA strand during viral particle formation (see, for example, Clever et al., 1995 J. Virology, 69(4):2101-09). The packaging signal can be the minimal packaging signal (also known as the psi[Ψ] sequence) required for viral genome capsidation.

[0205] In some embodiments, the retroviral vector (e.g., a lentiviral vector) further comprises FLAP as used herein, the term "FLAP" referring to a nucleic acid whose sequence comprises a central polypurine segment and a central termination sequence (CPPT and CTS) of a retrovirus (e.g., HIV-1 or HIV-2). Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou et al. (2000) Cell 101:173. During reverse transcription, the central initiation of positive-strand DNA at the cPPT and the central termination at the CTS result in the formation of a triplet DNA structure: a central DNA lobe. While not wishing to be bound by any theory, the DNA lobe can act as a cis-activity determinant of lentiviral genome nuclear input and / or can increase viral titer. In some embodiments, the retroviral vector backbone comprises one or more FLAP elements upstream or downstream of the heterologous gene of interest in the vector. For example, in some embodiments, the transfer plasmid comprises FLAP elements. In some embodiments, the vectors of this disclosure comprise FLAP elements isolated from HIV-1.

[0206] In some embodiments, the retroviral vector (e.g., a lentiviral vector) further comprises an output element. In some embodiments, the retroviral vector comprises one or more output elements. The term "output element" refers to a cis-acting posttranscriptional regulatory element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm.

[0207] Examples of RNA output elements include (but are not limited to) human immunodeficiency virus (HIV) RREs (see, for example, Cullen et al., (1991) J. Virol. 65:1053; and Cullen et al., (1991) Cell 58: 423) and hepatitis B virus posttranscriptional regulatory elements (HPREs).

[0208] Generally, RNA export elements are located within the 3' UTR of a gene and can be inserted as one or more copies.

[0209] In some embodiments, the retroviral vector (e.g., a lentiviral vector) also contains posttranscriptional regulatory elements. Various posttranscriptional regulatory elements can increase the expression of heterologous nucleic acids, such as the marmot hepatitis virus posttranscriptional regulatory element (WPRE) (see Zufferey et al., (1999) J. Virol., 73:2886); the posttranscriptional regulatory element (HPRE) present in hepatitis B virus (Huang et al., Mol. Cell. Biol., 5:3864); the optimized posttranscriptional regulatory element (oPRE; see Schambach et al., (2006) Gene Therapy 13, 641-45); and so on (Liu et al., (1995), Genes Dev., 9:1766). Posttranscriptional regulatory elements are typically located at the 3' end of the heterologous nucleic acid sequence. This conformation synthesizes an mRNA transcript whose 5' portion contains the heterologous nucleic acid coding sequence and whose 3' portion contains the posttranscriptional regulatory element sequence. In some embodiments, the vectors of this disclosure lack or do not contain post-transcriptional regulatory elements (e.g., WPRE or HPRE) because, in some cases, these elements increase the risk of cell transformation and / or do not significantly or noticeably increase the amount of mRNA transcripts or increase mRNA stability. Therefore, in some embodiments, as an additional safety measure, the vectors of this disclosure lack or do not contain WPRE or HPRE.

[0210] Elements that guide efficient termination and polyadenylation of heterologous nucleic acid transcripts will increase heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. Therefore, in some embodiments, retroviral vectors (e.g., lentiviral vectors) also contain polyadenylation signals. As used herein, the term "polyadenylation signal" or "polyadenylation sequence" refers to a DNA sequence that guides the termination and polyadenylation of nascent RNA transcripts via RNA polymerase H. Efficient polyadenylation of recombinant transcripts is desirable because transcripts lacking polyadenylation signals are unstable and rapidly degrade. Illustrative examples of polyadenylation signals that can be used in vectors of this disclosure include desirable polyadenylation sequences (e.g., AATAAA, ATTAAA, AGTAAA), bovine growth hormone polyadenylation sequences (BGHpA), rabbit β-globulin polyadenylation sequences (rBgpA), or other suitable heterologous or endogenous polyadenylation sequences known in the art.

[0211] In some embodiments, the retroviral vector further comprises an insulator element.

[0212] Insulator elements help protect sequences expressed by retroviruses, such as therapeutic genes, from integration site effects, which can be mediated by cis-acting elements present in genomic DNA and lead to dysregulation of the expression of the transfected sequence (i.e., position effects; see, for example, Burgess-Beusse et al., (2002) Proc. Natl. Acad. Sci., USA, 99: 16433; and Zhan et al., 2001, Hum. Genet., 109:471). In some embodiments, the retroviral vector contains an insulator element in one or two LTRs or elsewhere in the vector region integrated into the cellular genome. Suitable insulators for use in this disclosure include, but are not limited to, chicken β-globulin insulators (see Chung et al., (1993). Cell 74: 505; Chung et al., (1997) Proc. Natl. Acad. Sci., USA 94:575; and Bell et al., 1999. Cell 98:387). Examples of insulator elements include, but are not limited to, insulators derived from B-globulin loci (e.g., chicken HS4).

[0213] Non-limiting examples of lentiviral vectors include pLVX-EF1α-AcGFP1-CI (Clontech catalog number 1984), pLVX-EF1α-IRES-mCherry (Clontech catalog number 1987), pLVX-Puro (Clontech catalog number 632159), pLVX-IRES-Puro (Clontech catalog number 632186), pLenti6 / V5-DEST™ (ThermoFisher), and pLenti6.2 / V5-DEST™ (ThermoFisher). Fisher), pLK0.1 (Addgene plasmid #10878), pLKO.3G (Addgene plasmid #14748), pSico (Addgene plasmid #11578), pLJMl-EGFP (Addgene plasmid #19319), FUGW (Addgene plasmid #14883), pLVTHM (Addgene plasmid #12247), pLVUT-tTR-KRAB (Addgene plasmid #11651). pLL3.7 (Addgene plasmid #11795), pLB (Addgene plasmid #11619), pWPXL (Addgene plasmid #12257), pWPI (Addgene plasmid #12254), EF.CMV.RFP (Addgene plasmid #17619), pLenti CMV Puro DEST (Addgene plasmid #17452), pLenti-puro (Addgene plasmid #39481), Addgene (Addgene plasmid #24129), pLX301 (Addgene plasmid #25895), pHIV-EGFP (Addgene plasmid #21373), pLV-mCherry (Addgene plasmid #36084), pLionII (Addgene plasmid #1730), pInducerl0-mir-RUP-PheS (Addgene plasmid #44011). These vectors can be modified to suit therapeutic uses. For example, selection markers (e.g., puro, EGFP, or mCherry) can be deleted or replaced with a second exogenous gene of interest.Other examples of lentiviral vectors are disclosed in U.S. Patent Nos. 7,629,153, 7,198,950, 8,329,462, 6,863,884, 6,682,907, 7,745,179, 7,250,299, 5,994,136, 6,287,814, 6,013,516, 6,797,512, 6,544,771, 5,834,256, 6,958,226, 6,207,455, 6,531,123, 5,352,694 and PCT Publication No. WO 2017 / 091786.

[0214] In some embodiments, a nucleic acid vector (e.g., a viral vector) encoding a heterologous protein (e.g., a CAR) disclosed herein is applied to a cell population (e.g., a T cell initiation population) at a multiplicity of infection (MOI) between 0 and 24 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24). In some embodiments, a nucleic acid vector encoding a heterologous protein (e.g., a CAR) is applied to a cell population (e.g., a T cell initiation population) at an MOI of about 4. In some embodiments, a nucleic acid vector encoding a heterologous protein (e.g., a CAR) is applied to a cell population (e.g., a T cell initiation population) at an MOI of about 5. In some embodiments, a nucleic acid vector encoding a heterologous protein (e.g., a CAR) is applied to a cell population (e.g., a T cell initiation population) at an MOI of about 6. In some embodiments, a nucleic acid vector encoding a heterologous protein (e.g., CAR) is administered to a cell population (e.g., a T cell initiation population) at an MOI of about 7. In some embodiments, a nucleic acid vector encoding a heterologous protein (e.g., CAR) is administered to a cell population (e.g., a T cell initiation population) at an MOI of about 8. In some embodiments, a nucleic acid vector encoding a heterologous protein (e.g., CAR) is administered to a cell population (e.g., a T cell initiation population) at an MOI of about 9. In some embodiments, a nucleic acid vector encoding a heterologous protein (e.g., CAR) is administered to a cell population (e.g., a T cell initiation population) at an MOI of about 10. In some embodiments, a nucleic acid vector encoding a heterologous protein (e.g., CAR) is administered to a cell population (e.g., a T cell initiation population) at an MOI of about 12. In some embodiments, a nucleic acid vector encoding a heterologous protein (e.g., CAR) is administered to a cell population (e.g., a T cell initiation population) at an MOI of about 14.

[0215] Nucleic acid transfer medium

[0216] Nucleic acid transfer mediators are another advantageous method for delivering polynucleotides encoding heterologous proteins disclosed herein to target cells (e.g., T cell initiation populations). In some embodiments, the nucleic acid transfer mediator is a nanoparticle, such as lipid nanoparticles (e.g., LNPs), non-lipid polymeric core-shell nanoparticles, or biodegradable nanoparticles. In some embodiments, the LNP comprises one or more of ionizable lipids, polyethylene glycol-modified lipids, structural lipids (e.g., cholesterol), and / or accessory lipids.

[0217] Not wanting to be bound by theory, we believe that the transfer medium described in this paper encapsulates nucleic acids encoding heterologous proteins, thus protecting them from degradation and providing efficient delivery of nucleic acids to target cells both in vivo and in vitro.

[0218] Cell culture conditions

[0219] In some embodiments, the contact between the activated T-cell population and the polynucleotide encoding a heterologous protein (e.g., CAR) is performed in a culture medium containing no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% serum (e.g., human serum). In some embodiments, the contact between the activated T-cell population and the polynucleotide encoding a heterologous protein (e.g., CAR) is performed in a culture medium containing 2% serum. In some embodiments, during the transfection step, the activated T-cell population is cultured in a medium containing basal medium and serum (e.g., 2% serum).

[0220] In some preferred aspects, the basal culture medium is serum-free. In some preferred methods, activated T cells are cultured in serum-free medium for a period of 24 to 60 hours after contact with a polynucleotide encoding a heterologous protein (e.g., CAR). In some methods of this disclosure, the culture step lasts for a period of 29 to 59 hours. In preferred methods of this disclosure, the culture step lasts for a period of 36 to 52 hours. In a more preferred aspect, the culture step lasts for a period of 46 to 50 hours. In some aspects, the culture step lasts for a period of approximately 48 hours.

[0221] The method disclosed herein may also include harvesting cultured T cells, wherein the harvested T cells express heterologous proteins.

[0222] In some embodiments, the culture medium does not contain any additional cytokines or growth factors other than proteins derived from serum.

[0223] In some embodiments, the contact between the activated T-cell population and the polynucleotide encoding a heterologous protein (e.g., CAR) is performed in a serum-free medium. In some embodiments, the contact between the activated T-cell population and the polynucleotide encoding a heterologous protein (e.g., CAR) is performed in a basal medium without serum supplementation. In some embodiments, the basal medium does not contain any cytokines or growth factors. In some embodiments, the basal medium does not contain any added proteins. It should be understood that cells in the medium may secrete proteins during cell culture (e.g., during the transfection step, or in the preceding activation step if there is no change in the medium between the activation and transfection steps). It should also be understood that reagents stimulating the CD3 / TCR complex and / or reagents stimulating co-stimulatory molecules may naturally degrade over time, releasing fragments into the medium. These proteins are not considered "added proteins".

[0224] In some embodiments, the contact between the activated T cell population and the polynucleotide encoding a heterologous protein (e.g., CAR) is performed in a culture medium containing one or more cytokines selected from the group consisting of IL-2, IL-7, IL-15, and IL-21. In some embodiments, the one or more cytokines are IL-2. In some embodiments, the one or more cytokines are IL-7 and IL-15. In some embodiments, the one or more cytokines are IL-2, IL-7, and IL-15. In some embodiments, the one or more cytokines are IL-21. In some embodiments, the one or more cytokines are IL-21, IL-7, and IL-15. In some embodiments, T cells are contacted with 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL of IL-2, alone or in combination with one or more other cytokines. In some embodiments, T cells are contacted with 100 ng / mL of IL-2. In some embodiments, T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL of IL-7, alone or in combination with one or more other cytokines. In some embodiments, T cells are contacted with 10 ng / mL IL-7. In some embodiments, T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL IL-15, alone or in combination with one or more other cytokines. In some embodiments, T cells are contacted with 10 ng / mL IL-15. In some embodiments, T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL IL-21, alone or in combination with one or more other cytokines. In some embodiments, T cells are contacted with 20 ng / mL IL-21, alone or in combination with one or more other cytokines. In some embodiments, T cells are cultured in the absence of any cytokines selected from IL-2, IL-7, IL-15, and IL-21. During the transfection step, serum-free medium (e.g., basal medium) or serum-supplemented medium may be provided in the presence or absence of the cytokines and combinations thereof disclosed herein.

[0225] In some embodiments, the contact between the activated T cell population and the polynucleotide encoding a foreign protein (e.g., CAR) is performed in a cytokine-free medium or without the addition of cytokines. In other embodiments, cytokines are added to the medium at the start of the preceding step (activation step). It should be understood that at the start of the transfection step, the effective concentration of cytokines may decrease due to degradation or increase due to secretion by cells (e.g., T cells) in the culture. In some embodiments, additional cytokines are added at the start of the transfection step (e.g., the same type and amount added as at the start of the activation step).

[0226] In some embodiments, the contact between the activated T cell population and the polynucleotide encoding a heterologous protein (e.g., CAR) occurs simultaneously with contact with one or more cytokines.

[0227] In some embodiments, the T-cell activated population is contacted with a polynucleotide encoding a heterologous protein (e.g., CAR) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 hours or longer after contact with one or more cytokines.

[0228] In some embodiments, the contact between the activated T cell population and the polynucleotide encoding a heterologous protein (e.g., CAR) is performed for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 hours or longer prior to contact with one or more cytokines.

[0229] In some respects, the culture step achieves an expansion of the harvested T cells between 1.0 and 4 times. In other respects, the culture step achieves an expansion of the harvested T cells between 1.2 and 4 times.

[0230] Culture duration

[0231] When T cells are contacted with polynucleotides encoding heterologous proteins (e.g., CARs) disclosed herein, cells can be cultured in cultures under conditions and time sufficient to promote the integration of nucleic acids into the genome of T cells to stably express heterologous proteins in T cells and / or obtain the desired T cell phenotype (e.g., TNSCM cell phenotype).

[0232] Surprisingly, the inventors have discovered that the two- to three-day manufacturing method of this disclosure yields extremely high CAR+ percentages only after a short culture period (typically about 48 hours). For example, T cells contacted with polynucleotides encoding heterologous proteins (e.g., CAR) of this disclosure are cultured in a culture for 24 to 72 hours (e.g., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours). In some embodiments, T cells contacted with polynucleotides encoding heterologous proteins (e.g., CARs) disclosed herein are cultured in a culture for approximately 48 hours (e.g., approximately 44, 45, 46, 47, 48, 49, 50, 51, or 52 hours).

[0233] Harvesting and Storage

[0234] After activation, introduction of heterologous nucleic acids, and culture, a population of T cells can be harvested for storage and subsequent therapeutic use. In some embodiments, no later than 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83 after the activation step begins. T cells are harvested at 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, or 36 hours for storage.

[0235] In some embodiments, T cells are harvested for storage no later than about 64 hours (e.g., 62, 63, 64, 65, or 66) after the activation step begins. In some embodiments, T cells are harvested for storage no later than 64 hours after the activation step begins. In some embodiments, T cells are harvested for storage no later than about 72 hours (e.g., 70, 71, 72, 73, or 74) after the activation step begins. In some embodiments, T cells are harvested for storage no later than 72 hours after the activation step begins.

[0236] In some embodiments, T cells are harvested for storage no later than 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 hours after the activation step (i.e., the start of the transfection step). In some embodiments, T cells are harvested for storage approximately 48 hours (e.g., 46, 47, 48, 49, or 50) hours after contacting the T cells with a polynucleotide encoding a heterologous protein (e.g., CAR).

[0237] In some embodiments, the harvesting step is accompanied by one or more assays designed to test one or more parameters, such as cell viability, cell count, purity (e.g., the fraction of T cells in the total cell population), the fraction of cells expressing heterologous proteins, T cell phenotype, T cell activation / exhaustion status, T cell proliferation relative to the starting population, T cell cytotoxicity, cytokine release, etc.

[0238] The harvesting step may be followed by a storage step, whereby the T cells generated according to the disclosed method are maintained under conditions suitable for preserving the cells (including their viability and functional and molecular characteristics) until subsequent therapeutic application or quality control testing. In some embodiments, the storage step includes one or more of the following: (1) reforming the cell population in a storage medium (e.g., a refrigerated medium, a frozen medium, or a cryopreservation medium); (2) transferring the cells to a suitable container for storage under appropriate storage conditions; and (3) maintaining the cells under appropriate conditions.

[0239] Characteristics of engineered T cells

[0240] In some embodiments, engineered T cells generated or available according to the methods of this disclosure are disclosed herein. In some embodiments, the T cells are engineered (e.g., genetically manipulated) to express a heterologous protein, such as CAR.

[0241] In some embodiments, engineered T cells or populations of engineered T cells stably express CAR, for example by integrating a heterologous nucleic acid sequence encoding CAR into the genome of the T cells.

[0242] Protein / CAR Expression

[0243] The brief two- to three-day methods used to manufacture the CAR T cells disclosed herein (including those using whole blood and / or PBMC samples as starting materials) produce engineered T cell populations expressing heterologous proteins (e.g., CAR) from the T cell starting population at a very high percentage, such that 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the cells in the T cell starting population express heterologous proteins (e.g., CAR).

[0244] In some embodiments, the expression of heterologous proteins (e.g., CARs) is measured in T cells at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more after the harvest step. In some embodiments, the expression of heterologous proteins (e.g., CARs) is measured in T cells at 3 days after the harvest step. In some embodiments, the expression of heterologous proteins (e.g., CARs) is measured in T cells at least 3, 4, 5, 6, 7, 8, 9, 10 days or more after the start of the transfection step, so that transient expression of the unintegrated vector is not significantly detected. Methods for quantifying heterologous protein expression at the genomic, transcriptomic, and proteomic levels in cells are well known in the art and include, but are not limited to, flow cytometry (e.g., fluorescence-assisted cell sorting, FACS), quantitative (q) PCR, digital (d) PCR, fluorescence imaging, integration site analysis, RNA sequencing, in situ hybridization, immunoprecipitation, and Topoganga assays.

[0245] Surprisingly, the inventors have discovered that the cells produced by the method of this disclosure for manufacturing CAR T cells exhibit an increased CAR+ percentage compared to cells produced by other existing methods. This increased CAR+ percentage (typically greater than at least 50-60%) can be obtained from the method of this disclosure using: (i) whole blood as the starting sample; (ii) T cells directly isolated from whole blood; (iii) simultaneous T cell isolation and activation; (iv) a transduction step approximately 10-25 hours (preferably approximately 18 hours) after T cell isolation; and (v) a brief culture step following transduction (typically approximately 40-60 hours, preferably approximately 48 hours).

[0246] In some embodiments, the methods disclosed herein generate a population of engineered T cells expressing CAR that, compared to T cells that have not been contacted with target cells or have been contacted with control target cells that do not express the antigen, secrete an increased amount of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3) upon contact with target cells expressing an antigen bound to CAR (e.g., CD19). One or 32 proteins selected from the group consisting of the following: IFNg, granzyme B, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17A, IL-17F, IL-21, IL-22, IP-10, MCP1, MCP4, TNFα, TNFβ, TGFβ, GM-CSF, MIP1α, MIP1β, CCL11, perforin, RANTES, sCD137, and VEGF. In some embodiments, the secretion of one or more proteins after contact with target cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or higher, compared to the secretion of one or more proteins in the absence of target cells or in the presence of control target cells that do not express the antigen. In some embodiments, cytokine secretion in T cells is measured on days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more after the harvest step.

[0247] Methods for quantifying cytokine release from T cells are well known in the art and include, but are not limited to, ELISA, flow cytometry (e.g., cell counting bead array assay), and proteomics analysis (e.g., multiplex single-cell microarray analysis).

[0248] In certain preferred embodiments, the short manufacturing time provided by the methods of this disclosure produces CAR T cells that exhibit an improved multifunctional phenotype upon contact with a target (e.g., CD19). In certain preferred embodiments, the methods disclosed herein produce a population of engineered T cells expressing CAR, wherein at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20% or more are multifunctional CAR T cell populations that, compared to T cells that have not been contacted with target cells or have been contacted with control target cells that do not express an antigen, exhibit a multifunctional CAR... Upon contact with target cells expressing a CAR-binding antigen (e.g., CD19), the T cell population secretes increased amounts of one or more proteins (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32) selected from the group consisting of: IFNg, granzyme B, IL-1β. , IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17A, IL-17F, IL-21, IL -22, IP-10, MCP1, MCP4, TNFα, TNFβ, TGFβ, GM-CSF, MIP1α, MIP1β, CCL11, perforin, RANTES, sCD137 and VEGF. In some embodiments, the secretion of one or more proteins after contact with target cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or higher, compared to the secretion of one or more proteins in the absence of target cells or in the presence of control target cells that do not express the antigen. In some embodiments, cytokine secretion in T cells is measured on days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more after the harvest step.

[0249] In a preferred method of this disclosure, the harvested T cells comprise a population representing at least 6% of the harvested T cells and are pluripotent T cells upon activation based on a specific target. In some aspects, pluripotent T cells and / or a portion of their population simultaneously secrete granzyme B and TNFb. In some aspects, pluripotent T cells and / or a portion of their population simultaneously secrete granzyme B and IFNg. In a preferred aspect, at least 1% of the harvested T cells and / or a portion of their population simultaneously secrete granzyme B and IFNg. In some aspects, pluripotent T cells and / or a portion of their population simultaneously secrete MIP-1a and MIP-1b. In some methods, pluripotent T cells and / or a portion of their population simultaneously secrete IFNg and granzyme B.

[0250] In the preferred method of this disclosure, the pluripotent T cells and / or a portion of the population thereof comprise two or more of the following: cells that simultaneously secrete granzyme B and TNFb and / or a portion of the pluripotent T cell population; cells that simultaneously secrete granzyme B and IFNg and / or a portion of the pluripotent T cell population; cells that simultaneously secrete MIP-1a and MIP-1b and / or a portion of the pluripotent T cell population; and cells that simultaneously secrete IFNg and granzyme B and / or a portion of the pluripotent T cell population.

[0251] In some methods of this disclosure, a portion of a pluripotent T cell and / or a pluripotent T cell population includes: cells that simultaneously secrete granzyme B and TNFb and / or a portion of a pluripotent T cell population; cells that simultaneously secrete granzyme B and IFNg and / or a portion of a pluripotent T cell population; cells that simultaneously secrete MIP-1a and MIP-1b and / or a portion of a pluripotent T cell population; and cells that simultaneously secrete IFNg and granzyme B and / or a portion of a pluripotent T cell population.

[0252] In some embodiments, the methods disclosed herein generate an engineered T cell population expressing CAR that, upon contact with target cells expressing an antigen bound to CAR (e.g., CD19) in the absence of target cells or in the presence of control target cells that do not express the antigen, exhibits increased expression of one or more T cell activation markers, selected from the group consisting of CD69, CD25, and CD137.

[0253] In some embodiments, compared with the expression of one or more T cell activation markers selected from the group consisting of CD69, CD25, and CD137 in the absence of target cells or in the presence of control target cells that do not express antigens, the expression of one or more T cell activation markers is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more. In some embodiments, the expression of one or more T cell activation markers is measured at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more after the harvest step. Methods for quantifying the expression of T cell activation markers are well known in the art and include, but are not limited to, ELISA, flow cytometry, quantitative (q) PCR, digital (d) PCR, fluorescence imaging, in situ hybridization, and proteomics analysis.

[0254] In some embodiments, the methods disclosed herein generate a population of engineered T cells expressing CAR that exhibits increased cytotoxicity against target cells expressing an antigen bound to CAR, compared to cytotoxicity against cells not expressing an antigen (e.g., CD19). In some embodiments, cytotoxicity is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more compared to cytotoxicity against cells not expressing the antigen. In some embodiments, the cytotoxicity of engineered T cells against target cells expressing the antigen (e.g., CD19) is measured at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more after the harvest step. Methods for assessing the cytotoxicity of engineered T cells expressing heterologous proteins (e.g., CAR) to target cells are well known in the art and include, but are not limited to, chromium release assays, bioluminescence assays (e.g., luciferase-mediated bioluminescence imaging), real-time impedance-based analyses, flow cytometry (e.g., in combination with viability dyes such as CTV), and CFSE / PI assays.

[0255] In some embodiments, the methods disclosed herein generate a population of engineered T cells expressing CAR that exhibits increased proliferation upon contact with target cells expressing an antigen bound to CAR (e.g., CD19), compared to proliferation in the absence of target cells or in the presence of control target cells not expressing the antigen. In some embodiments, proliferation increases by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more compared to proliferation in the absence of target cells or in the presence of control target cells not expressing the antigen.

[0256] In some embodiments, T cell proliferation following contact with target cells expressing the antigen (e.g., CD19) is measured at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more after the harvest step. Methods for assessing the proliferation of engineered T cells expressing a heterologous protein (e.g., CAR) are well known in the art and include, but are not limited to, MTT assays, MTS assays, cell counting (e.g., by flow cytometry), CFSE / flow cytometry analysis, and [3H]thymidine incorporation.

[0257] In some embodiments, the methods disclosed herein generate engineered T cell populations with various phenotypes, such as naïve T cells characterized as CD45RO-CCR7+ and CD95-. N ) cells, characterized as CD45RO+ and CCR7+ central memory T (T) cells CM ) cells, characterized as CD45RO+ and CCR7- effector memory T cells (T cells) EM ) cells, characterized as CD45RO-, CCR7+ and CD95+ stem cell memory T (T) cells SCM ) cells, and effector memory T cells (T cells) characterized as CD45RO- and CCR7- that reexpress CD45RA. EMRA These T cell subsets are characterized by, but are not limited to, naive T cells characterized as CD45RA+, CCR7+, and CD95. N ) cells, characterized as CD45RA- and CCR7+ central memory T (T) cells CM ) cells, characterized as CD45RA- and CCR7- effector memory T cells (T cells) EM ) cells, characterized as CD45RA+, CCR7+ and CD95+ stem cell memory T (T) cells SCM ) cells, and effector memory T cells (T cells) characterized as CD45RA+ and CCR7- that reexpress CD45RA. EMRA )cell.

[0258] Surprisingly and advantageously, the method disclosed herein is capable of generating engineered T cell populations expressing heterologous proteins (e.g., CARs) that, compared to a T cell initiation population, include increased amounts of naïve and stem cell memory T cells (T cells). NSCM ) cells (identified by markers such as CD45RA+ / CD45RO- / CCR7+ / CD62L+; CD45RA+ / CCR7+; or CD45RO- / CCR7+). In some embodiments, the T cells generated according to the disclosed method contain T cells. NSCM The number of cells was higher than that of T cells in the T cell initiation population. NSCM The number of cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% of the T cell initiation population, or is a T cell initiation population. NSCM The number of cells is 2 times, 2.5 times, 3 times, 3.5 times, 4 times, or more. In some embodiments, the T cells generated according to the disclosed method contain stem cell memory T (T) cells. SCM The number of cells was higher than that of T cells in the initial T cell population. SCM The number of cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% in the T cell initiation population, or is a T cell initiation population. SCM The number of cells is 2, 2.5, 3, 3.5, 4 or more (identified by markers such as CD45RA+ / CD45RO- / CCR7+ / CD62L+ / CD95+; CD45RA+ / CCR7+ / CD95+; or CD45RO- / CCR7+ / CD95+).

[0259] In some preferred embodiments, the T cells produced according to the disclosed method contain at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more of the T cells harvested from all the T cells. NSCM Cells. In some embodiments, the T cells produced according to the disclosed method comprise at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more of the T cells harvested. SCM Cells. In some embodiments, T cells in the engineered T cell population are measured at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more after the harvest step. NSCM Cell count. Used to quantify T cells. NSCM Cellular methods are well known in the art and include, but are not limited to, flow cytometry (e.g., FACS) and fluorescence microscopy.

[0260] In some embodiments, the methods disclosed herein produce an engineered T cell population expressing a heterologous protein (e.g., CAR) that includes a reduced number of effector memory T (TEM) cells (identified by markers such as D45RA- / CD45RO+ / CCR7- / CD62L-; D45RA- / CCR7-; or CD45RO+ / CCR7-) compared to the initial T cell population. In some embodiments, the T cells produced according to the disclosed methods comprise an amount of TEM cells that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more lower than the number of TEM cells in the initial T cell population. In some embodiments, the number of TEM cells in the engineered T cell population is measured on days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more after the harvest step. Methods for quantifying TEM cells are well known in the art and include, but are not limited to, flow cytometry (e.g., FACS) and fluorescence microscopy.

[0261] Chimeric antigen receptor

[0262] In some embodiments, this document discloses T cells engineered (e.g., genetically modified) to express a heterologous protein. In some embodiments, the heterologous protein is a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises: (1) an extracellular domain containing an antigen-binding site that specifically binds to a target antigen; (2) a transmembrane domain; (3) an intracellular signal transduction domain; and optionally (4) a co-stimulatory domain. In some embodiments, the CAR disclosed herein also includes a hinge region.

[0263] In some embodiments, the CAR is a human CAR that comprises a whole-human sequence, such as a naturally occurring human sequence. In some embodiments, the extracellular domain is linked to one or more intracellular signaling domains, and in some embodiments, the intracellular signaling domains can mediate cellular activation via the antigen-receptor complex. In some embodiments, the transmembrane domain is linked to the extracellular domain. In some embodiments, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some embodiments, the transmembrane domain is selected or modified by amino acid substitution to prevent this domain from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0264] The CARs disclosed herein may comprise any number of amino acids, provided that the CAR retains its biological activity, such as the ability to specifically bind antigens, mediate cytotoxic activity, detect diseased cells in mammals, or treat or prevent diseases in mammals. In some embodiments, the CAR comprises 50 or more (e.g., 60 or more, 100 or more, or 500 or more) amino acids, but less than 1,000 (e.g., 900 or less, 800 or less, 700 or less, or 600 or less) amino acids. In some embodiments, the CAR is about 50 to about 700 amino acids (e.g., about 300 to about 1,000 amino acids (e.g., about 300 to about 800, about 300 to about 600, or about 400 to about 600 amino acids), or a range defined by any two of the foregoing values.

[0265] In some embodiments, the CAR contains additional amino acids at the amino or carboxyl terminus, or at both ends, of this moiety, which are not found in the amino acid sequence of the parent CAR. Desiredly, these additional amino acids do not interfere with the biological functions of the CAR, such as recognizing target cells, mediating cytotoxic activity, treating or preventing diseases or conditions. More ideally, the additional amino acids enhance the biological activity of the CAR compared to that of the parent CAR.

[0266] extracellular domain

[0267] In some embodiments, the CAR disclosed herein comprises an extracellular antigen-binding domain containing an antibody or an antigen-binding fragment thereof. Anticarrier proteins or other alternative scaffolds are also considered. The antigen-binding domain of the CAR can be a complete antibody or an antibody fragment (e.g., scFv). A complete antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains an N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains an N-terminal variable (VL) region and a C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The VH and VL regions have similar general structures, wherein each region includes three complementarity-determining regions (CDRs). The three CDRs, referred to as CDR1, CDR2, and CDR3, form the “hypervariable region” of the antibody responsible for antigen recognition and binding. The three CDR regions are connected by four frame regions whose sequences are relatively conserved.

[0268] Antigen-binding fragments of antibodies retain the antibody's ability to specifically bind to its antigens. Antibody fragments ideally include, for example, one or more CDRs or variable regions (or portions thereof). Examples of antibody fragments include, but are not limited to: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) Fab' fragments, which are monovalent fragments consisting of VL, VH, CL, CH1 domains and disulfide bridge thiol groups; (iii) F(ab')2 fragments, which are bivalent fragments comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iv) Fv fragments consisting of the VL and VH domains of an antibody single arm; (v) single-chain Fv (scFv), which is a monovalent molecule consisting of two domains (i.e., VL and VH) of an Fv fragment linked by a synthetic linker that allows the two domains to be synthesized into a single polypeptide chain; (vi) single-domain antibodies, such as V... H H or V NAR (vii) A bifunctional antibody, which is a dimer of polypeptide chains, wherein each polypeptide chain includes a VH linked to a VL via a peptide linker that is too short to allow pairing between VH and VL on the same polypeptide chain, thereby driving pairing between complementary domains on different VH-VL polypeptide chains to produce a dimer molecule with two functional antigen-binding sites. In some embodiments, the antigen-binding domain of the CAR includes an scFv that binds to the target antigen.

[0269] In some embodiments, the antibody or antigen-binding fragment thereof in the extracellular domain of the CAR may be obtained or derived from mammals, including but not limited to mice, rats, or humans. In some embodiments, the antigen-binding domain includes a variable region of a mouse or human monoclonal antibody or antigen-binding fragment thereof that binds to the antigen. In this respect, the antigen-binding domain includes a light chain variable region, a heavy chain variable region, or both a light chain variable region and a heavy chain variable region of a mouse or human monoclonal antibody or antigen-binding fragment thereof that binds to the antigen.

[0270] In some embodiments, the extracellular domain of the CAR disclosed herein includes a signal sequence. The signal sequence may be located at the N-terminus of an antigen recognition domain (e.g., a variable region of an antibody or its antigen-binding fragment). The signal sequence may include any suitable signal sequence. In one embodiment, the signal sequence is a human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor signal sequence or a CD8α signal sequence. For example, a CAR including a murine scFv may include a GM-CSF signal sequence, while a CAR including a human scFv may include a CD8α signal sequence. It should be understood that the N-terminal signal sequence is typically cleaved from the CAR protein after expression, but the nucleic acid encoding the CAR typically includes a sequence encoding the signal sequence.

[0271] In some embodiments, the antigen-binding domain binds to the target antigen (e.g., a peptide). In some embodiments, the antigen-binding domain specifically binds to the target antigen (e.g., a peptide).

[0272] Hinge domain

[0273] In some embodiments, the CAR further includes a hinge or spacer between the antigen-binding domain and the transmembrane domain. The hinge or spacer may be or include at least a portion of an immunoglobulin constant region or a variant or modification thereof, such as a hinge region, for example, a CD8α hinge, an IgG4 hinge region, and / or a CH1 / CL and / or FC region. In some embodiments, the constant region or portion is human IgG, such as IgG4 or IgG1. In some embodiments, a portion of the constant region serves as a spacer region between the antigen-recognizing component (e.g., scFv) and the transmembrane domain. The length of the spacer can provide increased cellular responsiveness after antigen binding compared to the absence of a spacer. In some embodiments, the length of the spacer is about 12 amino acids or no more than 12 amino acids. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and include any integer between the endpoints of any of the listed ranges. In some embodiments, the spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include CD8α hinges, separate IgG4 hinges, IgG4 hinges connected to CH2 and CH3 domains, or IgG4 hinges connected to a CH3 domain. In some embodiments, the CAR hinge comprises a CD8α, truncated CD8α, or CD28 hinge domain.

[0274] In some embodiments, the hinge region is a short amino acid sequence that can facilitate structural flexibility between polypeptide domains (e.g., between extracellular and transmembrane domains) (see, for example, Woof et al., Nal. Rev. Immunol. 4(2):89-99 (2004)). In some embodiments, the hinge region may comprise all or a portion of the extracellular region of any suitable transmembrane protein (e.g., CD8α).

[0275] In some embodiments, the hinge region is derived from the CD8α protein or the CD28 protein. In some embodiments, the hinge region is derived from the CD8α protein. In some embodiments, the hinge region is derived from the CD28 protein. In some embodiments, the hinge region is or comprises a hinge region from the CD28 protein or a functional segment thereof. In some embodiments, the hinge region is or comprises a hinge region from the CD8α protein or a functional segment thereof. In some embodiments, the hinge region is derived from the human CD8α protein or the human CD28 protein. In some embodiments, the hinge region is derived from the human CD8α protein. In some embodiments, the hinge region is or comprises a hinge region from the human CD28 protein or a functional segment thereof. In some embodiments, the hinge region is or comprises a hinge region from the human CD8α protein or a functional segment thereof.

[0276] In some embodiments, the hinge region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO: 28. In some embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID NO: 28. In some embodiments, the hinge region is derived from the same polypeptide as the transmembrane domain. In some embodiments, the hinge region and the transmembrane domain are derived from a CD8 polypeptide. In some embodiments, the hinge region and the transmembrane domain are derived from a CD8α polypeptide. In some embodiments, the hinge region and the transmembrane domain comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO: 19. In some embodiments, the hinge region and the transmembrane domain comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. In some embodiments, the hinge region and transmembrane domain comprise the amino acid sequence shown in SEQ ID NO: 19.

[0277] In some embodiments, the hinge region and transmembrane domain are encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO: 20. In some embodiments, the hinge region and transmembrane domain are encoded by a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 20. In some embodiments, the hinge region and transmembrane domain are encoded by a nucleic acid sequence as shown in SEQ ID NO: 20.

[0278] Transmembrane domain

[0279] In some embodiments, this document discloses modified T cells expressing a CAR comprising a transmembrane domain operatively linked to both an extracellular domain and an intracellular signaling domain of the CAR. The transmembrane domain may be any transmembrane domain derived from or obtained from any molecule known in the art (e.g., a type I transmembrane protein).

[0280] In some embodiments, the transmembrane domain of the CAR is derived from a natural source (e.g., a natural or wild-type peptide). In some embodiments, the transmembrane domain used according to this disclosure is derived from any suitable transmembrane protein or peptide known in the art. In some embodiments, the transmembrane domain is derived from CD3ε peptide, CD4 peptide, CD5 peptide, CD8 peptide, CD9 peptide, CD16 peptide, CD22 peptide, CD28 peptide, CD33 peptide, CD37 peptide, CD45 peptide, CD64 peptide, CD80 peptide, CD86 peptide, CD134 peptide, CD137 peptide, CD154 peptide, T cell receptor α chain peptide, T cell receptor β chain peptide, T cell receptor ζ chain peptide, or any derivative thereof and / or combinations thereof. In some embodiments, the transmembrane domain is or comprises a transmembrane domain or a functional fragment thereof derived from CD3ε polypeptide, CD4 polypeptide, CD5 polypeptide, CD8 polypeptide, CD9 polypeptide, CD16 polypeptide, CD22 polypeptide, CD28 polypeptide, CD33 polypeptide, CD37 polypeptide, CD45 polypeptide, CD64 polypeptide, CD80 polypeptide, CD86 polypeptide, CD134 polypeptide, CD137 polypeptide, CD154 polypeptide, T cell receptor α chain polypeptide, T cell receptor β chain polypeptide, T cell receptor ζ chain polypeptide, or any combination thereof. In some embodiments, the transmembrane domain is synthetically derived or engineered. In some embodiments, the synthetically derived or engineered transmembrane domain primarily comprises hydrophobic residues (e.g., leucine, valine, etc.). In some embodiments, the engineered transmembrane domain is or comprises any engineered transmembrane domain known in the art.

[0281] Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain primarily comprises hydrophobic residues such as leucine and valine. In some aspects, each end of the synthetic transmembrane domain has a triplet of phenylalanine, tryptophan, and valine. In some embodiments, linkages are achieved via linkers, spacers, and / or transmembrane domains.

[0282] This disclosure understands CD8 as a transmembrane glycoprotein that functions as a co-receptor for the T cell receptor (TCR) and is primarily expressed on the surface of T cells (e.g., cytotoxic T cells). The most common form of CD8 exists as a dimer composed of CD8α and CD8β chains. In some embodiments, the transmembrane domain is derived from the CD8α protein. In some embodiments, the transmembrane protein comprises an amino acid sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity with SEQ ID NO: 11. In some embodiments, the transmembrane protein comprises the amino acid sequence shown in SEQ ID NO: 11.

[0283] This disclosure further clarifies that CD28 is expressed on T cells and provides the co-stimulatory signal required for T cell activation. CD28 is a receptor for CD80 (B7.1) and CD86 (B7.2). In some embodiments, the CAR of this disclosure comprises a CD28 transmembrane domain. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least 770%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO: 12. In some embodiments, the transmembrane protein comprises the amino acid sequence shown in SEQ ID NO: 12.

[0284] Intracellular signal transduction domains

[0285] In some embodiments, upon binding of the CAR to the target antigen, a cytoplasmic domain or intracellular signaling domain of the receptor activates at least one normal effector function or response of an immune effector cell (e.g., a T cell engineered to express the receptor). For example, in some cases, the receptor induces T cell functions such as cytolytic activity or T helper activity, such as the secretion of cytokines or other factors. In some embodiments, for example, if the intracellular signaling domain of the antigen receptor component transduces effector signaling, the complete immune stimulation chain is replaced with a truncated portion of the intracellular signaling domain of the antigen receptor component or a co-stimulatory molecule. In some embodiments, one or more intracellular signaling domains include a cytoplasmic sequence of a T cell receptor (TCR) and, in some respects, those sequences of co-receptors that, in their natural environment, synergize with such a receptor to initiate signal transduction upon binding of the antigen receptor, and / or any derivatives or variants of such molecules, and / or any synthetic sequences having the same function.

[0286] In some respects, the receptor includes a primary cytoplasmic signaling sequence that regulates the primary activation of the TCR complex. Primary cytoplasmic signaling sequences that function in a stimulatory manner may contain signaling motifs known as immune receptor tyrosine-based activation motifs (ITAMs). Examples of primary cytoplasmic signaling sequences containing ITAMs include those derived from TCR or CD3ζ, FcRγ, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, FcεR1 (e.g., the FcεR1γ chain polypeptide), FcγRI, FcRβ, CD3γ, CD3δ, CD3ε, CD8, CD22, CD79a, CD79b, LIGHT, NKG2C, OX40, PD-1, CD66d, or any derivatives thereof, or any combination thereof.

[0287] In some embodiments, the receptor comprises an intracellular component of the TCR complex. It should be understood that the most common intracellular signaling domain used in CAR therapy is the intracellular signaling domain of CD3 zeta (CD3ζ). CD3ζ associates with T cell receptors to generate a signal and contains ITAM. In some embodiments, the intracellular signaling molecule in the CAR contains an intracellular signaling domain, a portion thereof, or a sequence derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises the CD3ζ domain. In some embodiments, the intracellular signaling domain comprises the human CD3ζ stimulatory signaling domain or a functional fragment thereof, such as the 112AA cytoplasmic domain of human CD3ζ isoform 3 (UniProt accession number: P20963.2).

[0288] In some embodiments, the intracellular signal transduction domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO: 23. In some embodiments, the intracellular signal transduction domain comprises the amino acid sequence shown in SEQ ID NO: 23.

[0289] In some embodiments, the intracellular signal transduction domain is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO: 24. In some embodiments, the intracellular signal transduction domain is encoded by a nucleic acid sequence as shown in SEQ ID NO: 24.

[0290] In some embodiments, the intracellular signal transduction domain comprises at least one intracellular signal transduction domain or a functional fragment thereof from 4-1BB peptide, B7H3 peptide, CD2 peptide, CD3γ peptide, CD3δ peptide, CD3ζ peptide, CD7 peptide, CD27 peptide, CD28 peptide, CD30 peptide, CD40 peptide, FcεRI peptide (e.g., FcεRIγ chain peptide), FcγRI peptide, LIGHT peptide, NKG2C peptide, OX40 peptide, PD-1 peptide, any derivative thereof, or any combination thereof. In some embodiments, the intracellular signal transduction domain comprises an intracellular signal transduction domain or a functional fragment thereof from CD3ζ peptide. In some embodiments, the intracellular signal transduction domain comprises an intracellular signal transduction domain or a functional fragment thereof from CD28 peptide. In some embodiments, the intracellular signal transduction domain comprises an intracellular signal transduction domain or a functional fragment thereof from CD28 peptide and an intracellular signal transduction domain or a functional fragment thereof from CD3ζ peptide. In some embodiments, the intracellular signal transduction domain comprises, from the N-terminus to the C-terminus, an intracellular signal transduction domain or a functional fragment thereof derived from the CD28 polypeptide and an intracellular signal transduction domain or a functional fragment thereof derived from the CD3ζ polypeptide.

[0291] In some embodiments, the intracellular signal transduction domain of this disclosure comprises at least one signal transduction sequence from 4-1BB peptide, B7-H3 peptide, CD2 peptide, CD3γ peptide, CD3δ peptide, CD3ζ peptide, CD7 peptide, CD27 peptide, CD28 peptide, CD30 peptide, CD40 peptide, FcεRI peptide (e.g., FcεRIγ chain peptide), FcγRI peptide, LIGHT peptide, NKG2C peptide, OX40 peptide, PD-1 peptide, or any combination thereof. In some embodiments, the intracellular signal transduction domain comprises at least one signal transduction sequence from a CD3ζ peptide. In some embodiments, the intracellular signal transduction domain comprises at least one signal transduction sequence from a CD28 peptide. In some embodiments, the intracellular signal transduction domain comprises at least one signal transduction sequence from a CD28 peptide and at least one signal transduction sequence from a CD3ζ peptide. In some embodiments, the intracellular signal transduction domain comprises at least one signal transduction sequence from a CD28 peptide and at least one signal transduction sequence from a CD3ζ peptide from the N-terminus to the C-terminus.

[0292] Co-stimulatory domain

[0293] In some embodiments, the CAR of this disclosure contains an intracellular domain of a T-cell costimulatory molecule, for example, located between a transmembrane domain and an intracellular signaling domain. In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of CD28 or 4-1BB, or a functional variant or portion thereof, such as the 41-amino acid cytoplasmic domain of human CD28 (UniProt accession number P10747.1) or the 42-amino acid cytoplasmic domain of human 4-1BB (UniProt accession number Q07011.1), or a functional variant or portion thereof.

[0294] In some embodiments, in addition to activation domains (e.g., primary activation domains) in the cytoplasmic portion, the receptor also encompasses one or more, such as two or more, co-stimulatory domains. Exemplary receptors include intracellular components of CD3-ζ and CD28, or intracellular components of CD3-ζ and 4-1BB.

[0295] In some embodiments, the receptor includes a co-stimulatory receptor comprising a signal transduction domain and / or transmembrane portion of a receptor such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same receptor contains both an activating component and a co-stimulatory component.

[0296] In some embodiments, the intracellular signal transduction domain includes a CD8a transmembrane and signal transduction domain connected to the CD3 (e.g., CD3-ζ) intracellular domain, and also includes a CD28 or 4-1BB (CD TNFRSF9) co-stimulatory domain connected to the CD3ζ intracellular domain.

[0297] In some embodiments, the intracellular signal transduction domain includes the CD28 intracellular signal transduction domain. In some embodiments, the intracellular signal transduction domain includes an intracellular signal transduction domain or a functional fragment thereof derived from the CD28 peptide. In some embodiments, the CD28 peptide intracellular signal transduction domain or a functional fragment thereof includes a co-stimulatory domain.

[0298] In some embodiments, the intracellular signal transduction domain disclosed herein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO: 21. In some embodiments, the intracellular signal transduction domain comprises the amino acid sequence shown in SEQ ID NO: 21.

[0299] In some embodiments, the intracellular signal transduction domain is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO: 22. In some embodiments, the intracellular signal transduction domain is encoded by a nucleic acid sequence as shown in SEQ ID NO: 22.

[0300] Chimeric antigen receptor - multiple domains

[0301] In some embodiments, the CAR disclosed herein comprises an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signal transduction domain. In some embodiments, the CAR disclosed herein comprises a signal peptide sequence (also referred to as a targeting signal, localization signal, localization sequence, leader sequence, or leader peptide), an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signal transduction domain. In some embodiments, the CAR disclosed herein comprises an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signal transduction domain from its N-terminus to its C-terminus. In some embodiments, the CAR disclosed herein comprises a signal peptide sequence, an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signal transduction domain from its N-terminus to its C-terminus. In some embodiments, the signal peptide sequence is cleaved from the CAR during or after its insertion into the membrane (e.g., the ER membrane) during CAR protein synthesis. In some embodiments, the domains or components of the CAR (e.g., extracellular domains, hinge regions, transmembrane domains, intracellular signal transduction domains, etc.) are directly connected or continuous. In some embodiments, the domains or components of the CAR are not directly connected or are discontinuous.

[0302] In some embodiments, the CAR as described herein comprises an intracellular signal transduction domain, wherein the intracellular signal transduction domain comprises: (a) a CD3ζ intracellular signal transduction domain or a functional fragment thereof; and (b) at least one of a 4-1BB, OX40, or CD28 intracellular signal transduction domain or a functional fragment thereof. In some embodiments, the 4-1BB intracellular signal transduction domain or a functional fragment thereof, the OX40 intracellular signal transduction domain, and / or the CD28 intracellular signal transduction domain or a functional fragment thereof are or comprise a co-stimulatory domain.

[0303] In some embodiments, the CAR of this disclosure comprises: (a) a CD28 transmembrane domain; and (b) an intracellular signal transduction domain comprising: (i) a CD3ζ intracellular signal transduction domain or a functional fragment thereof; and (ii) a CD28 intracellular signal transduction domain or a functional fragment thereof. In some embodiments, the CD28 intracellular signal transduction domain or a functional fragment thereof is or comprises a CD28 co-stimulatory domain.

[0304] In some embodiments, the CAR of this disclosure comprises: (a) a CD8α transmembrane domain; and (b) an intracellular signal transduction domain comprising: (i) a CD3ζ intracellular signal transduction domain or a functional fragment thereof; and (ii) a CD28, FcεRIγ chain, and / or 4-1BB intracellular signal transduction domain or a functional fragment thereof. In some embodiments, the CAR of this disclosure comprises: (a) a CD8α transmembrane domain; and (b) an intracellular signal transduction domain comprising: (i) a CD3ζ intracellular signal transduction domain or a functional fragment thereof; and (ii) a CD28, FcεRIγ chain, and 4-1BB intracellular signal transduction domain or a functional fragment thereof. In some embodiments, the CAR of this disclosure comprises: (a) a CD8α transmembrane domain; and (b) an intracellular signal transduction domain comprising: (i) a CD3ζ intracellular signal transduction domain or a functional fragment thereof; and (ii) a FcεRIγ chain intracellular signal transduction domain or a functional fragment thereof. In some embodiments, the CAR of this disclosure comprises: (a) a CD8α transmembrane domain; and (b) an intracellular signal transduction domain comprising: (i) a CD3ζ intracellular signal transduction domain or a functional fragment thereof; and (ii) a 4-1BB intracellular signal transduction domain or a functional fragment thereof. In some embodiments, the CD28 intracellular signal transduction domain or a functional fragment thereof is or includes a CD28 co-stimulatory domain. In some embodiments, the FcεRI intracellular signal transduction domain or a functional fragment thereof is or includes a FcεRI co-stimulatory domain. In some embodiments, the 4-1BB intracellular signal transduction domain or a functional fragment thereof is or includes a 4-1BB co-stimulatory domain.

[0305] In some embodiments, the CAR of this disclosure comprises: (a) a CD8α transmembrane domain; and (b) an intracellular signal transduction domain comprising: (i) a CD3ζ intracellular signal transduction domain or a functional fragment thereof; and (ii) a CD27 and / or CD28 intracellular signal transduction domain or a functional fragment thereof. In some embodiments, the CD27 intracellular signal transduction domain or a functional fragment thereof is or comprises a CD27 co-stimulatory domain. In some embodiments, the CD28 intracellular signal transduction domain or a functional fragment thereof is or comprises a CD28 co-stimulatory domain.

[0306] In some embodiments, the CAR of this disclosure comprises: (a) a CD28 transmembrane domain; and (b) an intracellular signal transduction domain comprising: (i) a CD3ζ intracellular signal transduction domain or a functional fragment thereof; and (ii) an intracellular signal transduction domain of the CD27, 4-1BB, and / or FcεRIγ chain or a functional fragment thereof. In some embodiments, the CAR of this disclosure comprises: (a) a CD28 transmembrane domain; and (b) an intracellular signal transduction domain comprising: (i) a CD3ζ intracellular signal transduction domain or a functional fragment thereof; and (ii) an intracellular signal transduction domain of the CD27, 4-1BB, and FcεRIγ chain or a functional fragment thereof. In some embodiments, the CAR of this disclosure comprises: (a) a CD28 transmembrane domain; and (b) an intracellular signal transduction domain comprising: (i) a CD3ζ intracellular signal transduction domain or a functional fragment thereof; and (ii) an intracellular signal transduction domain of the CD27 or a functional fragment thereof. In some embodiments, the CAR of this disclosure comprises: (a) a CD28 transmembrane domain; and (b) an intracellular signal transduction domain comprising: (i) a CD3ζ intracellular signal transduction domain or a functional fragment thereof; and (ii) a 4-1BB intracellular signal transduction domain or a functional fragment thereof. In some embodiments, the CAR of this disclosure comprises: (a) a CD28 transmembrane domain; and (b) an intracellular signal transduction domain comprising: (i) a CD3ζ intracellular signal transduction domain or a functional fragment thereof; and (ii) a FcεRIγ chain intracellular signal transduction domain or a functional fragment thereof. In some embodiments, the CD27 intracellular signal transduction domain or a functional fragment thereof is or includes a CD27 co-stimulatory domain. In some embodiments, the intracellular signal transduction domain or a functional fragment thereof is or includes an FcεRI co-stimulatory domain. In some embodiments, the 4-1BB intracellular signal transduction domain or a functional fragment thereof is or includes a 4-1BB co-stimulatory domain.

[0307] This disclosure also provides a CAR comprising an extracellular domain for any target molecule of interest (e.g., comprising any known antigen-binding domain, such as an antibody, scFv, etc.), and further comprising any transmembrane domains described herein (including any hinge domains described herein) and any intracellular signal transduction domains described herein (including any signaling sequence or motif described herein, any co-stimulatory domains, etc.) present in any combination thereof.

[0308] In some embodiments, the CAR comprises: (a) a hinge region, (b) a transmembrane domain derived from the human CD8α peptide, and (c) an intracellular signal transduction domain comprising: (i) a human CD3ζ intracellular signal transduction domain or a fragment thereof; and (ii) a human CD28 intracellular signal transduction domain or a fragment thereof, wherein the CD28 intracellular signal transduction domain or a fragment thereof is or comprises a co-stimulatory domain. In some embodiments, the CAR comprises: (a) a hinge region derived from the human CD8α peptide, (b) a transmembrane domain derived from the human CD8α peptide, and (c) an intracellular signal transduction domain comprising: (i) a human CD3ζ intracellular signal transduction domain; and (ii) a human CD28 intracellular signal transduction domain. In some embodiments, the CAR comprises the sequence shown in SEQ ID NO: 27.

[0309] In some embodiments, the CAR includes: (a) a hinge region, (b) a transmembrane domain derived from the human CD8α polypeptide, and (c) an intracellular signal transduction domain comprising: (i) a human CD3ζ intracellular signal transduction domain or a fragment thereof; and (ii) a human CD27 and / or CD28 intracellular signal transduction domain or a fragment thereof, wherein the CD27 and / or CD28 intracellular signal transduction domain or a fragment thereof is or contains a co-stimulatory domain.

[0310] In some embodiments, the CAR includes: (a) a hinge region, (b) a transmembrane domain derived from the human CD8α polypeptide, and (c) an intracellular signal transduction domain comprising: (i) an intracellular signal transduction domain of the human CD3ζ or a fragment thereof; and (ii) an intracellular signal transduction domain of the human CD28, human CD27, and / or FcεR1γ chain or a fragment thereof, wherein the intracellular signal transduction domain of the human CD28, human CD27, and / or FcεR1γ chain or a fragment thereof is or contains a co-stimulatory domain.

[0311] In some embodiments, the CAR comprises: (a) a hinge region, (b) a transmembrane domain derived from the human CD8α polypeptide, and (c) an intracellular signal transduction domain comprising: (i) an intracellular signal transduction domain of the human CD3ζ or a fragment thereof; and (ii) an intracellular signal transduction domain of the human CD28 and / or FcεR1γ chain or a fragment thereof, wherein the intracellular signal transduction domain of the CD28 and / or FcεR1γ chain or a fragment thereof is or contains a co-stimulatory domain.

[0312] In some embodiments, the CAR as described herein further comprises a signal peptide sequence. In some embodiments, the signal peptide is located at the N-terminus of an extracellular domain (e.g., at the N-terminus of an antigen-binding domain). The signal peptide used according to this disclosure may comprise any suitable signal peptide sequence. In some embodiments, the signal peptide sequence is a human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor signal peptide sequence or a CD8α signal peptide sequence. In some embodiments, the CAR provided herein comprises a human or humanized scFv comprising a CD8α signal peptide sequence. In some embodiments, the signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO: 15.

[0313] In some embodiments, the provided CAR comprises: (a) a CD8α hinge region comprising SEQ ID NO: 28, (b) a CD8α transmembrane domain comprising SEQ ID NO: 11, (c) a CD28 intracellular signal transduction domain comprising SEQ ID NO: 21, and (d) a CD3ζ intracellular signal transduction domain comprising SEQ ID NO: 23. In some embodiments, the provided CAR comprises, from the N-terminus to the C-terminus: (a) a CD8α hinge region comprising SEQ ID NO: 28, (b) a CD8α transmembrane domain comprising SEQ ID NO: 11, (c) a CD28 intracellular signal transduction domain comprising SEQ ID NO: 21, and (d) a CD3ζ intracellular signal transduction domain comprising SEQ ID NO: 23.

[0314] In some embodiments, the provided CAR comprises: (a) an antigen-binding domain comprising SEQ ID NO: 17, (b) a CD8α hinge region comprising SEQ ID NO: 28, (c) a CD8α transmembrane domain comprising SEQ ID NO: 11, (d) a CD28 intracellular signal transduction domain comprising SEQ ID NO: 21, and (e) a CD3ζ intracellular signal transduction domain comprising SEQ ID NO: 23. In some embodiments, the provided CAR comprises, from the N-terminus to the C-terminus: (a) an antigen-binding domain comprising SEQ ID NO: 17, (b) a CD8α hinge region comprising SEQ ID NO: 28, (c) a CD8α transmembrane domain comprising SEQ ID NO: 11, (d) a CD28 intracellular signal transduction domain comprising SEQ ID NO: 21, and (e) a CD3ζ intracellular signal transduction domain comprising SEQ ID NO: 23.

[0315] In some embodiments, the provided CAR comprises: (a) a CD8α signal peptide sequence comprising SEQ ID NO: 15, (b) an antigen-binding domain comprising SEQ ID NO: 17, (c) a CD8α hinge region as shown in SEQ ID NO: 28, (d) a CD8α transmembrane domain as shown in SEQ ID NO: 11, (e) a CD28 intracellular signal transduction domain as shown in SEQ ID NO: 21, and (f) a CD3ζ intracellular signal transduction domain as shown in SEQ ID NO: 23. In some embodiments, the provided CAR comprises, from the N-terminus to the C-terminus: (a) a CD8α signal peptide sequence comprising SEQ ID NO: 15, (b) an antigen-binding domain comprising SEQ ID NO: 17, (c) a CD8α hinge region as shown in SEQ ID NO: 28, (d) a CD8α transmembrane domain as shown in SEQ ID NO: 11, (e) a CD28 intracellular signal transduction domain as shown in SEQ ID NO: 21, and (f) a CD3ζ intracellular signal transduction domain as shown in SEQ ID NO: 23.

[0316] In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity with SEQ ID NO: 10. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 10. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 10. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 10. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 10. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 96% sequence identity with SEQ ID NO: 10. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 10. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 10. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 10. In some embodiments, the CAR of this disclosure comprises the amino acid sequence shown in SEQ ID NO: 10.

[0317] In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity with SEQ ID NO: 13. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 13. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 13. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 13. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 13. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 96% sequence identity with SEQ ID NO: 13. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 13. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 13. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 13. In some embodiments, the CAR of this disclosure comprises the amino acid sequence shown in SEQ ID NO: 13.

[0318] In some embodiments, the CAR of this disclosure is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity with SEQ ID NO: 14. In some embodiments, the CAR of this disclosure is encoded by a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 14. In some embodiments, the CAR of this disclosure is encoded by a nucleic acid sequence having at least 85% sequence identity with SEQ ID NO: 14. In some embodiments, the CAR of this disclosure is encoded by a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 14. In some embodiments, the CAR of this disclosure is encoded by a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO: 14. In some embodiments, the CAR of this disclosure is encoded by a nucleic acid sequence having at least 96% sequence identity with SEQ ID NO: 14. In some embodiments, the CAR of this disclosure is encoded by a nucleic acid sequence having at least 97% sequence identity with SEQ ID NO: 14. In some embodiments, the CAR of this disclosure is encoded by a nucleic acid sequence having at least 98% sequence identity with SEQ ID NO: 14. In some embodiments, the CAR of this disclosure is encoded by a nucleic acid sequence having at least 99% sequence identity with SEQ ID NO: 14. In some embodiments, the CAR of this disclosure is encoded by the nucleic acid sequence shown in SEQ ID NO: 14.

[0319] Modification

[0320] A CAR may contain one or more modified synthetic amino acids in place of one or more naturally occurring amino acids. Exemplary modified amino acids include, but are not limited to: aminocyclohexanecarboxylic acid, leucine, α-aminodecanoic acid, homoserine, S-acetaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, 3-phenylserine, 3-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, and indoline-2-carboxylic acid. 1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornene)carboxylic acid, α,γ-diaminobutyric acid, α,γ-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0321] CARs (including their functional moieties and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized by, for example, disulfide bonds, or converted to acid addition salts and / or optionally dimerized or polymerized, or conjugated.

[0322] Pharmaceutical Composition

[0323] The engineered T cells disclosed herein can be incorporated into pharmaceutical compositions. In addition to the engineered T cells disclosed herein, these compositions may also contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other material may depend on the intended route of administration, such as intravenous, skin or subcutaneous, nasal, intramuscular, or intraperitoneal routes. Pharmaceutical compositions may contain one or more pharmaceutical excipients. Any suitable pharmaceutical excipient can be used, and those skilled in the art can select appropriate pharmaceutical excipients.

[0324] Therefore, the pharmaceutical excipients provided below are exemplary only and not limiting. Other pharmaceutical excipients include, for example, those described in Handbook of Pharmaceutical Excipients, Rowe et al. (eds.), 6th edition (2009), the entire contents of which are incorporated herein by reference.

[0325] Reagent test kit

[0326] In some embodiments, this document discloses a kit comprising one or more doses of engineered T cell (e.g., CAR-T cell) populations in a suitable container device, the cell populations being generated or obtained according to the methods disclosed herein.

[0327] In some embodiments, the kit includes a container component containing the engineered T cells described herein. In some embodiments, the container component is any suitable container that holds, for example, a liquid or lyophilized composition, including but not limited to vials, syringes, bottles, and intravenous (IV) bags or ampoules. The syringe holds any volume of liquid suitable for injection into a subject, including but not limited to 0.5 cc, 1 cc, 2 cc, 5 cc, 10 cc, or more. In some embodiments, the packaging and kit include a label that conveys information required by the US FDA or similar regulatory agency, such as a product description, the amount and mode of administration, and / or the indication for treatment. In some embodiments, the packaging provided herein includes any composition as described herein.

[0328] In some embodiments, the packaging and kit further include buffers, preservatives, and / or stabilizers in the pharmaceutical formulation. In some embodiments, each component of the kit is encapsulated in a separate container, and all various containers are contained in a single package. In some embodiments, the kit of this disclosure is designed for cryogenic or room temperature storage.

[0329] Additionally, in some embodiments, the formulation contains stabilizers to increase the shelf life of the kit and includes, for example, bovine serum albumin (BSA). In cases where the composition is lyophilized, in some embodiments, the kit contains an additional solution formulation to reconstitute the lyophilized formulation. Acceptable reconstitution solutions are well known in the art and include, for example, pharmaceutically acceptable phosphate-buffered saline (PBS).

[0330] In some embodiments, the kit may also include instructions for performing any of the methods described herein. The term "packaging material" refers to the physical structure that houses the components of the kit. In some embodiments, the packaging material keeps the components sterile and is made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, etc.). In some embodiments, a label or packaging insert includes appropriate written instructions (e.g., instructing the user of the kit to perform one or more of the methods disclosed herein). In some embodiments, the kit additionally includes a label or instructions for using the kit components in any of the methods disclosed herein. In some embodiments, the kit includes a compound in a package or dispenser and instructions for administering the compound as described herein.

[0331] In one aspect, this disclosure provides a method for preparing an engineered T cell population expressing a heterologous protein, the method comprising, in the following order: (a) contacting a T cell initial population with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide containing a nucleic acid sequence encoding a heterologous protein and culturing the T cells in a serum-free medium containing interleukin-2 (IL-2) protein for 29 to 71 hours; and (c) harvesting the T cells; optionally wherein step (b) is performed at least 1 hour after the start of step (a); and wherein the harvested T cells comprise one or more T cells engineered to express a heterologous protein.

[0332] In one aspect, this disclosure provides a method for preparing an engineered T cell population expressing a heterologous protein, the method comprising, in the following order: (a) contacting the T cell initiation population with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide containing a nucleic acid sequence encoding a heterologous protein and culturing the T cells in a culture medium containing interleukin-7 (IL-7) and interleukin-15 (IL-15) proteins for 31 to 71 hours; and (c) harvesting the T cells; wherein the harvested T cells comprise one or more T cells engineered to express a heterologous protein.

[0333] In one aspect, this disclosure provides a method for preparing an engineered T cell population expressing a heterologous protein, the method comprising, in the following order: (a) contacting the T cell initial population with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide containing a nucleic acid sequence encoding a heterologous protein; and (c) culturing the T cells in a serum-free medium containing interleukin-7 (IL-7) and / or interleukin-15 (IL-15) proteins for 30 to 60 hours; and (c) harvesting the T cells; wherein the harvested T cells comprise one or more T cells engineered to express a heterologous protein.

[0334] In one aspect, this disclosure provides a method for preparing an engineered T cell population expressing a heterologous protein, the method comprising, in the absence of cytokines, contacting a T cell starting population with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide containing a nucleic acid sequence encoding a heterologous protein and culturing the T cells in a culture medium containing at least one cytokine for 24 to 72 hours; and (c) harvesting the T cells; wherein step (b) is performed approximately 18 hours after step (a); and wherein the harvested T cells comprise one or more T cells engineered to express a heterologous protein. In some embodiments, the at least one cytokine in step (c) comprises one or more of IL-2, IL-7, IL-15, and / or IL-21 (e.g., 1, 2, 3, or 4). In some embodiments, the at least one cytokine in step (c) comprises IL-2. In some embodiments, the at least one cytokine in step (c) comprises IL-21. In some embodiments, the at least one cytokine in step (c) comprises IL-7 and IL-15. In some embodiments, at least one cytokine in step (c) comprises IL-2, IL-7, and IL-15. In some embodiments, at least one cytokine in step (c) comprises IL-21, IL-7, and IL-15.

[0335] In one aspect, this disclosure provides a method for preparing an engineered T cell population expressing a heterologous protein, the method comprising, in the following order: (a) contacting a T cell initiation population with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide containing a nucleic acid sequence encoding a heterologous protein and culturing the T cells in a medium containing interleukin-21 (IL-21) protein for 31 to 71 hours; and (c) harvesting the T cells; wherein the harvested T cells comprise one or more T cells engineered to express the heterologous protein. In some embodiments, the medium further comprises IL-7 and / or IL-15.

[0336] In one aspect, this disclosure provides a method for preparing an engineered T cell population expressing a heterologous protein, the method comprising, in the following order: (a) contacting a T cell starting population with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein and culturing the T cells in a culture medium for 24 to 72 hours; and (c) harvesting the T cells; wherein the culture medium does not contain IL-2, IL-7, IL-15, or IL-21; and wherein the harvested T cells comprise one or more T cells engineered to express a heterologous protein. In some embodiments, the culture medium is free of cytokines. In some embodiments, step (a) does not involve the use of IL-2, IL-7, IL-15, or IL-21. In some embodiments, step (a) is performed in the absence of cytokines. In some embodiments, step (c) does not involve the use of IL-2, IL-7, IL-15, or IL-21. In some embodiments, step (c) is performed in the absence of cytokines. In some embodiments, the method is performed in the absence of cytokines. In some embodiments, the culture medium is a basal medium.

[0337] In some embodiments, the culture medium contains serum. In some embodiments, the culture medium does not additionally contain added cytokines or growth factors. In some embodiments, the culture medium is serum-free.

[0338] In some embodiments, the T cell initiation population is approximately 1 × 10⁻⁶. 6 The cells were seeded into the culture at a concentration of approximately 100 cells / mL. In some embodiments, the T cell initiation population was seeded at approximately 2 × 10⁻⁶ cells / mL. 6 The cells were seeded into the culture at a concentration of approximately 5 × 10⁶ cells / mL. In some embodiments, the T cell initiation population was seeded at approximately 5 × 10⁶ cells / mL. 6 The cells were seeded in the culture at a concentration of approximately 1 × 10⁶ cells / mL. In some embodiments, (a) the T cell starting population was seeded at approximately 1 × 10⁶ cells / mL. 6 (a) T cells were seeded in the culture at a concentration of 1 × 10⁻⁶ cells / mL; and (b) the culture medium contained IL-2. In some embodiments, (a) the T cell initiation population was seeded at approximately 1 × 10⁻⁶ cells / mL.6 (a) T cells were seeded in the culture at a concentration of 1 × 10⁻⁶ cells / mL; and (b) the culture medium contained IL-7 and IL-15. In some embodiments, (a) the T cell initiation population was seeded at approximately 1 × 10⁻⁶ cells / mL. 6 (a) T cells were seeded in the culture at a concentration of 1 × 10⁻⁶ cells / mL; and (b) the culture medium contained IL-7, IL-15, and IL-21. In some embodiments, (a) the T cell initiation population was seeded at approximately 1 × 10⁻⁶ cells / mL. 6 (a) T cells were seeded in the culture at a concentration of 1 × 10⁻⁶ cells / mL; and (b) the culture medium contained IL-21. In some embodiments, (a) the T cell initiation population was seeded at approximately 1 × 10⁻⁶ cells / mL. 6 The cells were seeded in the culture at a concentration of 1 × 10⁶ cells / mL; and (b) the culture medium did not contain IL-2, IL-7, IL-15, or IL-21 (i.e., did not contain any of IL-2, IL-7, IL-15, and IL-21). In some embodiments, (a) the T cell initiation population was seeded at approximately 1 × 10⁶ cells / mL. 6 (a) T cells were seeded in the culture at a concentration of [number] cells / mL; and (b) the culture medium contained IL-2. In some embodiments, (a) the T cell initiation population was seeded at approximately 2 × 10 [cells / mL]. 6 (a) T cells were seeded in the culture at a concentration of [number] cells / mL; and (b) the culture medium contained IL-7 and IL-15. In some embodiments, (a) the T cell initiation population was seeded at approximately 2 × 10 [cells / mL]. 6 (a) T cells were seeded in the culture at a concentration of [number] cells / mL; and (b) the culture medium contained IL-7, IL-15, and IL-21. In some embodiments, (a) the T cell initiation population was seeded at approximately 2 × 10 [cells / mL]. 6 (a) T cells were seeded in the culture at a concentration of [number] cells / mL; and (b) the culture medium contained IL-21. In some embodiments, (a) the T cell initiation population was seeded at approximately 2 × 10 [cells / mL]. 6 The cells were seeded in the culture at a concentration of [number] cells / mL; and (b) the culture medium did not contain IL-2, IL-7, IL-15, or IL-21 (i.e., did not contain any of IL-2, IL-7, IL-15, and IL-21). In some embodiments, (a) the T cell initiation population was seeded at approximately 5 × 10 [cells / mL]. 6 (a) T cells were seeded in the culture at a concentration of [number] cells / mL; and (b) the culture medium contained IL-2. In some embodiments, (a) the T cell initiation population was seeded at approximately 5 × 10 [cells / mL]. 6 (a) T cells were seeded in the culture at a concentration of 10 cells / mL; and (b) the culture medium contained IL-7 and IL-15. In some embodiments, (a) the T cell initiation population was seeded at approximately 5 × 10⁻⁶ cells / mL. 6 (a) T cells were seeded in the culture at a concentration of 10 cells / mL; and (b) the culture medium contained IL-7, IL-15, and IL-21. In some embodiments, (a) the T cell initiation population was seeded at approximately 5 × 10⁻⁶ cells / mL. 6(a) T cells were seeded in the culture at a concentration of 10 cells / mL; and (b) the culture medium contained IL-21. In some embodiments, (a) the T cell initiation population was seeded at approximately 5 × 10⁻⁶ cells / mL. 6 (a) The culture medium is seeded at a concentration of cells / mL; and (b) the culture medium does not contain IL-2, IL-7, IL-15 or IL-21 (i.e., does not contain any of IL-2, IL-7, IL-15 and IL-21).

[0339] In some embodiments, the method further includes (d) maintaining the T cells harvested in step (c) at a temperature not higher than 38°C (e.g., not higher than 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9°C or lower). Temperatures between 2 and 8°C (e.g., 2, 3, 4, 5, 6, 7 or 8°C), or not lower than -80°C (e.g., not lower than -80, -81, -82, -83, -84, -85, -86, -87, -88, -89, -90, -91, -92, -93, -94, -95, -96, -97, -98, -99, -100°C or lower).

[0340] In some embodiments, one or more reagents activating CD3 and / or CD28 comprise an anti-CD3 antibody, an anti-CD28 antibody, or both. In some embodiments, one or more reagents activating CD3 and / or CD28 comprise a first reagent that binds CD3 and a second reagent that binds CD28. In some embodiments, the first and second reagents are the same reagent (e.g., a bispecific antibody that specifically binds CD3 and CD28).

[0341] In some embodiments, the method includes obtaining a sample containing a T-cell initiation population from the subject prior to step (a). In some embodiments, the method includes obtaining a sample containing a T-cell initiation population from the subject prior to step (a). In some embodiments, the sample is a whole blood sample obtained from the subject.

[0342] In some embodiments, the T cell initiation population includes helper T (Th) cells, cytotoxic T (Tc) cells, memory T (TM) cells, regulatory T (Treg) cells, and innate-like T cells. In some embodiments, Th cells include Th1 cells, Th2 cells, Th17 cells, Th9 cells, Tfh cells, and / or Th22 cells. In some embodiments, memory T cells include central memory T (TCM) cells, effector memory T (TEM) cells, tissue-resident memory T (TRM) cells, and virtual memory T (TVM) cells. In some embodiments, innate-like T cells are natural killer T (NKT) cells, mucosa-associated invariant T (MAIT) cells, and γδ T cells.

[0343] In some embodiments, polynucleotides are contained in the delivery medium. In some embodiments, the delivery medium is lipid nanoparticles. In some embodiments, the delivery medium is a nucleic acid carrier. In some embodiments, the nucleic acid carrier is a viral vector.

[0344] In some embodiments, the viral vector is a lentiviral vector.

[0345] In some embodiments, the method expands the T-cell initiation population after step (c) to no more than 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, 1.9-fold, 1.8-fold, 1.7-fold, 1.6-fold, 1.5-fold, 1.4-fold, 1.3-fold, 1.2-fold, 1.1-fold, or less. In some embodiments, after step (c), at least 20%, at least 30%, at least 40%, at least 50%, or at least 75% of the T-cell initiation population are engineered to express heterologous proteins.

[0346] In some embodiments, the heterologous protein comprises a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises: (a) an antigen-binding fragment of an anti-CD19 antibody; (b) a transmembrane domain; (c) an intracellular T cell signaling domain derived from human CD3ζ; and (d) an intracellular T cell signaling domain derived from human CD28.

[0347] In some embodiments, compared to T cells that do not come into contact with target cells, one or more of the T cells harvested in step (c) secrete an increased amount of one or more proteins (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32) selected from the group consisting of: IFN g, granzyme B, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17A, IL-17F, IL -21, IL-22, IP-IO, MCPI, MCP4, TNFα, TNFβ, TGFβ, GMCSF, MIP1α, MIP1β, CCL11, perforin, RANTES, sCD137 and VEGF. In some embodiments, the secretion of one or more proteins upon contact with target cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more higher than the secretion of one or more proteins in the absence of target cells.

[0348] In some embodiments, compared to the expression of one or more T cell activation markers in the absence of target cells, the one or more T cells harvested in step (c) exhibit increased expression of one or more T cell activation markers upon contact with target cells expressing the target antigen, wherein the one or more T cell activation markers are selected from the group consisting of CD69, CD25, and CD137. In some embodiments, compared to the expression of one or more T cell activation markers in the absence of target cells, the expression of one or more T cell activation markers selected from the group consisting of CD69, CD25, and CD137 is increased by at least 55%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more.

[0349] In some embodiments, the one or more T cells harvested in step (c) exhibit increased cytotoxicity against target cells expressing the target antigen (e.g., a disease-associated antigen, such as CD19) compared to cytotoxicity against cells not expressing the antigen. In some embodiments, the cytotoxicity is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more compared to cytotoxicity against cells not expressing the antigen.

[0350] In some embodiments, the one or more T cells harvested in step (c) exhibit increased proliferation upon contact with target cells expressing the target, compared to proliferation in the absence of target cells. In some embodiments, the proliferation increases by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more compared to proliferation in the absence of target cells.

[0351] In some embodiments, proliferation is measured from 0 to 240 hours after contact with target cells. In some embodiments, proliferation is measured at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36, 42, 48, 72, 96, 120, 144, 168, 192, 216, and / or 240 hours after contact with target cells. In some embodiments, proliferation is measured as the fold change in the number of CD3-positive (CD3+) cells in the T cells of step (c) compared to the number of CD3+ cells in the initial T cell population. In some embodiments, proliferation is measured as the fold change in the number of T cells expressing the heterologous protein in step (c) after contact with target cells expressing the target antigen, compared to the number of T cells expressing the heterologous protein in the absence of target cells.

[0352] In some embodiments, the T cells harvested in step (c) exhibit an increased amount of naïve and stem cell memory T cells (collectively, TNSCMs) (e.g., TSCMs) compared to the T cell starting population. In some embodiments, the T cells harvested in step (c) comprise an amount of TNSCMs (e.g., TSCMs) that is at least 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more higher than the T cell starting population. In some embodiments, the T cells harvested in step (c) exhibit substantially the same percentage of naïve and stem cell memory T (TNSCM) cells (e.g., TSCM cells) compared to the T cell starting population (e.g., the percentage of TNSCM cells in the T cells of step (c) increases or decreases by no more than 4%, 3%, 2%, or 1% or less (absolute difference between percentages of TNSCM cells in T cells) compared to the number of TNSCM cells in the T cell starting population). In some embodiments, the T cells harvested in step (c) contain a reduced amount of effector memory T (TEM) cells compared to the T cell starting population. In some embodiments, the T cells harvested in step (c) contain at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more of TEM cells compared to the T cell starting population. In some embodiments, the T cells harvested in step (c) exhibit substantially the same percentage of TEM cells as the T cell starting population (e.g., the percentage of TEM cells in the T cells in step (c) increases or decreases by no more than 4%, 3%, 2%, 1% or less (absolute difference between percentages of TEM cells in T cells) compared to the number of TEM cells in the T cell starting population).

[0353] In some embodiments, the method is performed outside the body.

[0354] Example

[0355] The following examples are provided to provide a description of how to use, prepare, and evaluate the compositions and methods described herein to those skilled in the art, and are intended purely as examples of this disclosure and not to limit the scope of what the inventors consider to be their invention.

[0356] Example 1: Preparation of T cells engineered to express chimeric antigen receptors using a 3-day process

[0357] A series of preliminary experiments were conducted to characterize the 2- to 3-day, 6-day, and 8-day methods for manufacturing CAR T cells from whole blood and / or PBMCs isolated from whole blood. For comparison, several experiments described below include a 3-day process requiring leukocyte separation and combined activation and transduction (“3-day alternative”). As such examples demonstrate, the currently disclosed method shows the production of CAR T cells using PBMCs extracted from small whole blood samples. This includes the successful production of CAR T cells using the two- to three-day process of this disclosure. Furthermore, as described below, the resulting CAR T cells exhibited favorable phenotypes, targeted cytotoxicity, and proliferation, and the yields showed feasible commercial production and scale-up. This series of preliminary experiments also validated that the preparation method of this disclosure is not only feasible but also capable of including preparation using PBMCs from whole blood samples in a rapid two- to three-day process without the need for leukocyte separation.

[0358] starting sample

[0359] T cells were extracted from peripheral blood mononuclear cells (PBMCs) isolated from donor 1 (a 27-year-old Asian male with a BMI of 25.4 and a smoker), or from donor 2 (a 31-year-old Caucasian male with a BMI of 42.0 and a non-smoker) or donor 3 (a 52-year-old mixed-race male with a BMI of 78.1 and a non-smoker) using leukocyte ablation. T cells were isolated from the samples using Miltenyi StraightFrom CD3 microbeads (Miltenyi Biotec: catalog number 130-090-874) and stored at 2–8°C until further processing.

[0360] The manufacturing process under study

[0361] Then use Figure 10 The various manufacturing methods outlined herein independently process isolated T cells, including the following protocols: A (“KYV 3-day alternative”), which relies on leukocyte isolation and combines activation and transduction; B (“KYV 3-day v1”), which can use PBMCs / whole blood; C (“KYV 3-day v2”), which can use PBMCs / whole blood; D (“KYV 6-day”), which can use PBMCs / whole blood; E (“KYV 8-day”), which can use PBMCs / whole blood; and F (“KYV 3-day v3”), which can use PBMCs / whole blood. For protocols AC and F, the initial starting cell population is 6 × 10⁶ cells / 10⁻⁶. 7 100 cells (a T cell population enriched with 85%-95% T cells), and for protocols D and E, 1 × 10⁻⁶ cells under conditions D and E. 7 Cells (a T cell population enriched with 85%-95% T cells). Cells were spaced at 3 × 10⁻⁶ cells per cell. 6 / cm2 (Conditions B, C, and F) or 1×10 6 / cm 2 (Conditions A, D, and E) were inoculated at densities in the G-Rex bioreactor.

[0362] TransAct (T Cell TransAct, human; Miltenyi Biotec; catalog number 130-111-160) activation reagent was added to cells under conditions A, B, C, D, and E. Cells were not activated under condition F. Cytokines were added along with the activation reagent under conditions B (100 ng / mL human IL-2), C (10 ng / mL human IL-7 and 10 ng / mL human IL-15), D (10 ng / mL human IL-7 and 10 ng / mL human IL-15), and E (10 ng / mL human IL-7 and 10 ng / mL human IL-15). Cells were cultured in a CO2 incubator in TexMACS medium supplemented with CTS immune cell SR.

[0363] T cells were transduced using KL-h198a28z, a third-generation lentiviral vector pseudotyped with self-inactivated (SIN) vesicular stomatitis virus (VSV)-G that encodes a chimeric antigen receptor binding to CD19. This CAR construct, named Hu19-CD828Z, has the amino acid sequence shown in SEQ ID NO: 13. The lentiviral vector contains an MSCV promoter and other regulatory factors, including a central polypurine segment / central termination sequence upstream of the promoter and a post-transcriptional regulatory element (PRE) downstream of the CAR expression sequence. The lentiviral vector KL-h198a28z was prepared using HEK 293T cell lines transiently transfected with a current-generation four-plasmid system. The plasmid encoding the envelope protein (pLTG1292) expressed the heterologous spike protein VSV-G under the control of the cytomegalovirus (CMV) promoter. The transduction step is initiated simultaneously with activation in condition A; 18 hours after the start of the activation step in conditions B and C; 18 hours after the inoculation step in condition F; and 24 hours after the start of the activation step in conditions D and E.

[0364] like Figure 10 As shown, cells were cultured with lentiviral vectors for different durations: 30 hours in condition A; 48 hours in conditions B, C, and E; 5 days in condition D; and 8 days in condition E. After harvest, conditions AC and F each produced approximately 3 × 10⁻⁶ cells. 7 Each cell, under condition D, produces approximately 1 × 102 cells. 8 10 cells, and condition E produces approximately 3 × 10 8Each cell.

[0365] Transduced T cells were frozen using a standard protocol. The cells were then thawed and characterized according to Example 2 below.

[0366] Example 2: Characterization of T cells transduced by lentiviral vector

[0367] vitality

[0368] Viability and cell count were measured using an automated cell counter, NucleoCounter NC-200™ (ChemoMetec A / S, Allerod, Denmark), which uses fluorescence detection to distinguish between live and non-live cells. Each test specimen was loaded into a proprietary cartridge containing two separate dyes for staining total nucleated cells and non-live cells, respectively. The software then calculated the viability percentage and cell count. Total cell counts were performed at 0 and 72 hours after thawing.

[0369] like Figure 2 As shown, each batch manufactured using the activated KYV 3-day process (“v1” and “v2”) exhibited better overall T-cell expansion during 72 hours post-thawing than the 8-day process, which is at least comparable to the KYV alternative 3-day process requiring leukocyte separation. Donor 1 showed minimal overall expansion, likely due to the low viability of the starting cell population.

[0370] CAR expression and T cell phenotype

[0371] Next, using a Cytoflex LX (Beckman Coulter) cytometer and flow cytometry, fluorescent antibodies were used to identify CD19 CAR and cell surface markers associated with Tn, Tscm, Tcm, Tem, and Temra memory T cell subsets to assess CAR expression and memory T cell phenotype. Specifically, Tn cells were identified as CD45RO- / CCR7+ / CD95-; Tscm cells as CD45RO- / CCR7+ / CD95+; Tcm cells as CD45RO+ / CCR7+; Tem cells as CD45RO+ / CCR7-; and Temra cells as CD45RO- / CCR7-. Fluorescence signals associated with each marker were acquired using CytExpert software (Beckman Coulter), and final data analysis was performed using FlowJo (BD Biosciences). Figure 3As shown, the KYV 3-day processes v1 and v2 exhibited extremely high CAR expression, generally exceeding 50% of CAR+ cells. This CAR+ percentage also surpasses that of the KYV alternative 3-day regimen. KYV 3-day processes v1 and v2 also showed higher CAR expression levels within CD4+ and CD8+ T cell subsets (data not shown). The KYV 6-day and KYV 8-day processes also demonstrated slightly better transduction efficiency than the KYV alternative 3-day regimen.

[0372] Regarding memory T cell subsets, such as Figure 4 As shown in A and 4B, cells produced via KYV Alternative 3 Days (which is dependent on leukocyte separation) and KYV 3 Days v3 methods maintained the highest percentage of Tnscm, exceeding 15%. Notably, Tnscm cells (CD45RO- / CCR7+) were observed to be almost entirely CD95+ (Tscm). The CAR+ percentage of cells from KYV Alternative 3 Days and KYV 3 Days v3 was 0 after thawing.

[0373] To assess additional memory phenotypes of CAR+ T cells. Thawed KYV replacement 3-day and KYV 3-day v3 cells were excluded from phenotyping due to low CAR expression, and in run 2, KYV 3-day v3 cells were completely excluded from phenotyping due to lack of transduction.

[0374] like Figures 5A-5D As shown, at 72 hours post-thawing, cells of KYV substitute day 3 and KYV day 3 v3 showed a similar percentage of Tnscm as cells of KYV day 3 v1 and KYV day 3 v2, reaching full CAR expression. The “enriched” samples were not transduced with lentivirus.

[0375] Overall, T cells processed using the 3-day process showed comparable or higher CAR expression compared to T cells produced using the KYV alternative 3-day method (dependent on leukocyte separation). Furthermore, considering donor-to-donor variability and CAR expression, the three 3-day processes showed similar Tnscm percentages to each other and higher Tnscm percentages compared to the longer KYV 8-day and KYV 6-day processes.

[0376] Cytotoxicity

[0377] The activity of CAR-T cells in killing CD19+ target cells was measured by flow cytometry using the Cytoflex LX (Beckman Coulter) cytometer.

[0378] In summary, as shown in Figure 5, CD19+ Raji or Nalm6 cells were labeled with cell traceviolet (CTV) and co-cultured with effector CAR-T cells at different effector-to-target (E:T) cell ratios for 18–20 hours. At each E:T ratio, the percentage of cell killing was determined by the ratio of live CTV-positive cells co-cultured with effector cells to live CTV-positive cells cultured in the absence of effector cells. Data analysis was performed using FlowJo (BD Biosciences).

[0379] Measure the percentage of cell lysis and plot it relative to the E:T ratio, such as Figure 6 The results are shown for Nalm6 cells. As provided in Table 1 below, the cytotoxic activity at a 1:1 E:T ratio is comparable between cells manufactured using process AE.

[0380] Table 1. Cytotoxicity of CAR-T cells produced according to the various protocols described herein.

[0381]

[0382] Single-cell cytokine secretion proteomic analysis

[0383] The single-cell cytokine secretion proteome was assessed using a human adaptive immune cytokine assay suite. CAR-T cells from condition C were compared with those from condition E by co-culturing them overnight with NALM-6 cells (E:T=3:1).

[0384] The human adaptive immune assay suite includes the following cytokines: CCL-I1, GM-CSF, granzyme B, IFNg, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17A, IL-17F, IL-21, IL-22, IP-10, MCP-I, MCP-4, MIP-1α, MIP-113, perforin, RANTES, sCD137, TNF-α, TNF-β, TGF-β, and VEGF.

[0385] Figures 7A-7D The percentage of cells secreting each of the 32 cytokines is shown, and higher levels of IFNg (IFNg) and TNF-β are observed in cells manufactured using the KYV 3-day v2 process compared to the KYV 9-day process.

[0386] Next, the pluripotency of CAR-T cells was assessed. Pluripotency is represented by the percentage of highly pluripotent cell subsets that simultaneously secrete multiple sets of cytokines after co-culturing with CD19+ target cells.

[0387] like Figure 8 As shown, CAR-T cells manufactured using the KYV 3-day v2 process exhibited higher pluripotency than CAR-T cells manufactured using the 9-day process. Furthermore, the percentage of pluripotent cells simultaneously secreting multiple sets of cytokines was assessed for each cytokine. Figure 9 The top 15 cytokines are shown, with cells manufactured using the KYV 3-day v2 process exhibiting the highest percentage of pluripotent cells capable of simultaneously secreting multiple sets of cytokines.

[0388] Example 3 - Evaluating methods for separating PBMCs

[0389] Given the positive results in Examples 1 and 2, where T cells were successfully isolated from PBMCs obtained from whole blood to generate CAR T cells, various PBMC isolation protocols were evaluated.

[0390] The donor provides a whole blood sample, from which PBMCs are then isolated using either the Rotea countercurrent centrifugation system (ThermoFisher Scientific) or the Corning X-LAB system (Corning Life Sciences). The Rotea system utilizes countercurrent centrifugation and is suitable for a wide range of cell processing applications, including PBMC isolation. The Corning X-LAB system is a sedimentation-based process that isolates monocytes (MNCs) in a closed, sterile, semi-automated manner without the need for density gradient media or manual transfer steps.

[0391] like Figure 11 As shown, the same donor study validated that the X-LAB system can achieve excellent PBMC enrichment from whole blood samples. Compared with the Rodea system, the X-LAB system provides higher average purity and less variability.

[0392] Example 4 - Assessing the stability of whole blood samples

[0393] Given the importance of obtaining T cells from whole blood samples for CAR T cell production, the stability of whole blood and its impact on T cell levels and viability were evaluated. In short, as... Figure 12 As shown, whole blood samples were obtained from the donor.

[0394] The composition of whole blood samples was evaluated over a three-day period using samples from two donors. During these three days, whole blood samples from both donors were enriched using clinical-grade Dynabeads, and the composition of the enriched samples was also evaluated.

[0395] like Figure 12 As shown, the percentages of CD3+, CD4+, and CD8+ cells (T cells) in whole blood samples remained stable over 72 hours, while the percentage of CD45+ cells decreased over time. For PBMC-enriched whole blood, samples obtained within 24 hours of sampling had higher yields than those obtained within 48 hours. Therefore, as shown, whole blood contains a sufficient number of T cells for CAR T cell production, with fresh whole blood providing the optimal starting product. Combined with the shortened CAR T cell production method of this disclosure, using fresh blood should yield high-quality, multifunctional final products.

[0396] Example 5 - Assessing the stability of whole blood samples

[0397] Based on the encouraging results regarding whole blood composition and stability in Example 4, this example conducted a series of experiments to evaluate the CAR-T cell preparation process disclosed herein that omits the PBMC step and instead isolates T cells directly from whole blood samples. Furthermore, this example not only evaluated the feasibility of using the step of directly isolating T cells from blood, but also the feasibility of combining this step with the simultaneous activation of the isolated T cells.

[0398] Figure 13 This document outlines the protocol used to evaluate the direct isolation of T cells from blood. In short, whole blood samples were obtained from three donors. These samples were used 24 or 48 hours after sampling. For the concurrent T cell isolation and activation protocol, whole blood samples were run using a ThermoFisher DynaCellect cell separator / washer system (ThermoFisher), and T cells from the whole blood were captured and activated using anti-CD3 / anti-CD28 Diano magnetic beads. T cells bound to the magnetic beads were collected using a G-Rex10M cell sorter. As a comparison, PBMCs were extracted from whole blood samples using either the X-LAB system or the Rodea system. T cells were extracted from PBMCs using the CliniMACS Plus cell separator (Miltenyi Biotec) and activated using TransACT. As a control, the DynaCellect cell isolation / activation protocol was also used for PBMCs extracted from the Rodea system to evaluate the relative efficacy of the comparative enrichment capabilities of the DynaCellect and CliniMACS systems and to determine whether concurrent activation / isolation affected enrichment.

[0399] like Figure 14 As shown, DynaCellect provides higher enrichment and final product yield compared to CliniMACS. This suggests that simultaneous activation and T cell isolation appear to yield higher isolated T cell yields from the sample, and consequently, higher yields of CAR-T cells can be generated using these T cells. As illustrated, DynaCellect provides an average fold increase of 1.2, compared to only 0.56 for CliniMACS.

[0400] like Figure 15 and Figure 16 As shown, regardless of whether whole blood or enriched PBMCs were used as starting materials, DynaCellect's simultaneous isolation / activation protocol produced high levels of CD3+ cells in all materials (whole blood, enriched PBMCs, and the final product), and made the CD4+ / CD8+ ratio more stable from the starting material to the final product in all donors. Therefore, the simultaneous activation / isolation method appears to be superior to separate isolation and activation protocols. Furthermore, this series of experiments clearly validates the method disclosed herein, which omits not only the leukocyte separation step but also the PBMC enrichment step when obtaining T cells from whole blood samples.

[0401] Example 6 - Phenotype of CAR T cells engineered from T cells isolated directly from whole blood.

[0402] Next, using a Cytoflex LX (Beckman Coulter) cytometer and flow cytometry, fluorescent antibodies were used to identify cell surface markers associated with Tn, Tscm, Tcm, Tem, and Temra memory T cell subsets to assess the memory T cell phenotype of T cells isolated directly from whole blood. Specifically, Tn cells were identified as CD45RO- / CCR7+ / CD95-; Tscm cells as CD45RO- / CCR7+ / CD95+; Tcm cells as CD45RO+ / CCR7+; Tem cells as CD45RO+ / CCR7-; and Temra cells as CD45RO- / CCR7-. Fluorescence signals associated with each marker were acquired using CytExpert software (Beckman Coulter), and final data analysis was performed using FlowJo (BD Biosciences). Figure 17As shown, compared with simulated (untransduced) control cells, cells generated using the KYV 3-day protocol and cells generated from T cells directly isolated from whole blood exhibited fewer differentiation phenotypes. Surprisingly, the products prepared from directly isolated T cells showed a significant increase in the expected Tcm and Tscm T cell types, consistent with the results of cells prepared using T cells isolated from PBMCs in Examples 1-2.

[0403] Example 7 - Functional characteristics of CAR T cells engineered from T cells isolated directly from whole blood.

[0404] The preparation of anti-CD19 CAR T cells used the KYV 3-day process as outlined in Examples 1 and 2, but T cells were isolated directly from whole blood from healthy donors instead of from PBMCs, and the TransACT activation step was omitted, with simultaneous activation and isolation using CD3 / CD28 Diano beads instead.

[0405] In short, T cells were transduced using KL-h198a28z, a third-generation lentiviral vector of self-inactivated (SIN) vesicular stomatitis virus (VSV)-G pseudotyped, which encodes a chimeric antigen receptor binding to CD19. This CAR construct, named Hu19-CD828Z, has the amino acid sequence shown in SEQ ID NO: 13. The lentiviral vector contains an MSCV promoter and other regulatory factors, including a central polypurine segment / central termination sequence upstream of the promoter and a post-transcriptional regulatory element (PRE) downstream of the CAR expression sequence. The lentiviral vector KL-h198a28z was prepared using HEK 293T cell lines transiently transfected with a current-generation four-plasmid system. The plasmid encoding the envelope protein (pLTG1292) expressed the heterologous spike protein VSV-G under the control of the cytomegalovirus (CMV) promoter. Figure 10 As shown in the KYV 3-day “v1” and “v2” processes, the isolation and activation steps begin simultaneously. 18 hours after the isolation / activation step, the cells are cultured with the lentiviral vector for 48 hours, and the transduced T cells are harvested, frozen using standard protocols, and thawed at a later time for characterization.

[0406] The short-term ability of CAR-T cells to specifically kill CD19+ target cells was measured by flow cytometry using a Cytoflex LX (Beckman Coulter) cytometer. Nalm6 cells were labeled with cell tracer purple (CTV) and co-cultured with effector CAR-T cells at different effector-to-target (E:T) cell ratios for 24 hours. At each E:T ratio, the percentage of cell killing was determined by the ratio of live CTV-positive cells co-cultured with effector cells to live CTV-positive cells cultured in the absence of effector cells. Data analysis was performed using FlowJo (BD Biosciences).

[0407] like Figure 18 As shown, after co-culturing CD19+ NALM6 target cells in vitro for 24 hours at an E:T (effectant:target) ratio of 0.1:1, the results showed that CAR-free simulated untransduced (UT) cells exhibited low non-specific killing activity compared to engineered anti-CD19 CAR T cells produced using the KYV 3-day process (using T cells simultaneously isolated and activated from whole blood), which showed significant target-dependent cytotoxicity.

[0408] The release of target-dependent cytokines from CAR T cells was also evaluated. Figure 19 Results of target-dependent cytokine release from CAR T cells derived from whole blood of healthy donors (HD) after 24 hours of in vitro co-culture with CD19+ NALM6 target cells at a specified E:T (effectant:target) ratio are provided. Supernatants were collected and analyzed by ELLA assay. Untransduced (UT) cells that do not express CAR showed low background levels of cytokine release. In contrast, CAR T cells exhibited well-defined target-dependent cytokine release, indicating target-dependent cytotoxic behavior.

[0409] Based on the positive results of the short-term killing assay, the exhaustion resistance of CAR T cells and the persistence of their target-dependent cytotoxicity were assessed using a long-term in vitro continuous re-excitation assay.

[0410] like Figure 20As shown, CD19-targeting CAR-T cells and CD19+ NALM6 target cells were co-cultured at a specified E:T ratio (CAR-T effector:NALM6 target), and the percentage of target cell killing was measured by flow cytometry at each specified time point. At each time point, a new round of target cells was added to the co-culture to assess the time-dependent, repeated cytotoxicity of CAR-T cells. Untransduced (“UT”) cells that did not express CAR did not show any cytotoxic activity. In contrast, CAR-T cells exhibited a durable immune response and provided repeated cytotoxic responses upon re-challenge at least 30 days after the first exposure.

[0411] Therefore, cells produced using the two- to three-day methods of this disclosure (which include methods for directly isolating and activating T cells from whole blood) provide an anti-depletion phenotype exhibiting the desired target-specific cytotoxic activity.

[0412] Advantageously, the resulting CAR T cells are not only effective, but have also been shown to expand at a higher rate upon contact with target cells than those using methods such as... Figure 10 The expansion rate of CAR T cells prepared using the “KYV 8-day” or “KYV 6-day” process outlined herein. As explained, the 8-day process: (i) uses T cells obtained from rich clusters (e.g., from leukocyte ablation samples or isolated PBMCs); (2) separate T cell isolation and activation steps; and (3) a longer culture and expansion period.

[0413] In short, anti-CD19 CAR-T cells were produced using either the conventional 8-day manufacturing process (“Conv”) or the KYV 3-day process outlined in this example. Both sets of CAR T cells were derived from whole blood starting material from healthy donors (HD). CAR-T cells were stimulated by co-culturing with mitomycin C-treated CD19+ NALM6 target cells added every 3–4 days at a 1:1 ratio. Vicell cell counting analysis was used to calculate the expansion of live T cells at each specified time point.

[0414] Figure 21 The results are provided in the figure. As shown, cells prepared using the 3-day process exhibited a higher target-specific expansion rate, indicating the desired in vivo immune profile. Therefore, the short-term CAR T cell manufacturing method of this disclosure (which includes those methods that use direct isolation of T cells from whole blood, simultaneous activation and isolation of T cells, and a short culture and expansion period) produces cells with favorable phenotypes, target-specific cytotoxicity, durable immune responses, and high expansion rates upon target stimulation.

[0415] As shown in the figure, CAR T cells produced using the method of this disclosure provide high CAR expression, including higher CAR expression within the CD4 and CD8 subsets, compared to other existing methods. Furthermore, the cells exhibit a high proportion of the desired Tcm and Tscm cell subsets. Single-cell cytokine analysis revealed higher levels of IFNg and TNF-β in cells produced using the method of this disclosure compared to conventional processes, which include other shortened processes, such as the KYV alternative 3-day process dependent on leukocyte ablation. Additionally, cells produced using the method of this disclosure exhibit a higher level of versatility compared to CAR T cells prepared using alternative methods, likely due to the shorter time interval between T cell ablation from whole blood and harvesting the final CAR T cell product. Moreover, the resulting CAR T cell product shows an increased level of antigen-induced proliferation compared to the other methods described.

[0416] Therefore, as shown in the figure, the method disclosed herein provides the shortest path from the sample to the CAR T cell product, while providing cells of higher quality than existing CAR T cell manufacturing processes.

[0417] Example 8 - T cell subsets from whole blood samples

[0418] To evaluate the subsets of CAR T cells obtained from whole blood, three groups of CAR T cells were generated using whole blood starting samples: “AR037”, which used T cells isolated from whole blood (1 week old, at 2–8°C) and downstream processed according to the conventional 9-day process in Examples 1–2; “AR039”, which did not perform any downstream processing on the fresh whole blood material; and “AR050”, which applied the 3-day v2 (CPD-23-007) process to isolate and engineer T cells from fresh whole blood.

[0419] Figure 22-23 The TBNK / memory phenotypes of pre- and post-enrichment materials, as well as the TBNK / memory phenotypes of the final CAR T cell products of AR037 (e.g., post-expansion), are provided. Figure 24 The paper provides the TBNK / memory phenotypes of AR037 in materials before and after enrichment. Figure 25 The paper provides the TBNK / memory phenotypes of AR050 in materials before and after enrichment.

[0420] As shown in the figure, live CAR T cells were generated using whole blood as the starting material, with Tnscm accounting for a high percentage. Figure 23 As shown, the number of Tnscm increases during cell expansion. Surprisingly, this is achieved using a shortened 3-day process ( Figure 25The initial number of Tnscm cells exceeds that of the 8-day process. Given the increased number shown in the final product of the 8-day process, the 3-day process should produce a final product containing a very high percentage of Tnscm cells.

[0421] Example 9 - Ingenui-T Platform

[0422] Traditionally, ablation has been the source of cell starting material for T-cell therapy products because conventional manufacturing processes require large numbers of T cells to generate sufficient modified T cells to achieve therapeutic doses for cancer patients. The time-consuming and invasive nature of ablation cell collection procedures, coupled with logistical limitations in transporting ablation products to manufacturing sites, imposes a burden on patients. These are current challenges facing CAR T-cell products, limiting their accessibility and thus necessitating a new approach. Challenges in obtaining optimal leukocyte ablation products for CAR T-cell manufacturing include operational barriers such as access to qualified ablation centers with adequate resources and trained personnel. 1 ), as well as technical issues (such as vascular access, contamination from other cell types, and effective management of adverse events during the collection process). 2 ).

[0423] Furthermore, conventional CAR T-cell fabrication involves long-term culturing of patient-derived material for 7–10 days, a design intended to maximize expansion and produce a final product with a highly differentiated T-cell phenotype. However, oncology studies have shown that T cells with a younger, more stem cell-like phenotype are associated with better clinical benefit compared to T cells with differentiation memory, effector function, or an exhausted phenotype (signature markers of more differentiated T-cell types). Shorter fabrication processes could mitigate these issues and produce improved CAR T-cell products. 3 .

[0424] Starting with whole blood extraction instead of apheresis and shortening the time required for CAR T-cell production has the potential to revolutionize the patient experience of receiving CAR T-cell therapy by addressing key challenges faced by conventional methods. This optimization could reduce production costs. 4 This increases treatment accessibility and the overall feasibility of CAR T-cell therapy.

[0425] Ingenui-T is a next-generation CAR T-cell manufacturing platform originally developed for the treatment of autoimmune diseases, utilizing the same all-human anti-CD19 CAR construct as KYV-101. KYV-101 is an investigational autologous anti-CD19 CAR T-cell therapy (manufactured using conventional methods) currently being studied in patients with B-cell-driven autoimmune diseases, including lupus nephritis, systemic sclerosis, myasthenia gravis, multiple sclerosis, and other diseases for which there is strong evidence of B-cell involvement in the pathological processes. This example provides an exemplary use of the novel Ingenui-T manufacturing platform disclosed herein, highlighting its ability to produce highly pure and functional CAR T-cells.

[0426] Importantly, the Ingenui-T platform produces CAR T cells with potent functional characteristics and a lower differentiation phenotype compared to CAR T cells generated in conventional manufacturing processes using cell-derived starting materials derived from the isolation process. By circumventing the challenges associated with isolation, the Ingenui-T platform paves a promising path to improving the efficiency and accessibility of CAR T cell therapy, reducing costs, and ultimately advancing its application in the field of autoimmune diseases.

[0427] method

[0428] Patient whole blood and white blood cell apheresis

[0429] Peripheral whole blood (up to 200 mL) was collected from healthy donors (n=9, AllCells or Bloodworks Northwest, USA) and transported fresh for immediate processing. Cryopreservation was intentionally avoided to maximize cell viability. Cell counting was performed to quantify the infused blood cell population. Cryopreserved leukocyte separation material matched to the donors was also obtained (n=4, AllCells, USA) to generate CAR T cells according to standard manufacturing processes.

[0430] Ingenui-T Manufacturing Platform

[0431] Figure 26 An overview of exemplary uses of the Ingenui-T manufacturing platform disclosed herein for generating engineered immune cells using whole blood as a starting material is provided.

[0432] like Figure 26In summary, up to 200 mL of collected whole blood was added directly to a Gibco™ CTS™ DynaCellect™ magnetic separation system (Thermo Fisher Scientific, Waltham, MA), and T cells were enriched and activated using anti-CD3 / CD28 Dynabeads (CTS™ Removable Dynabeads™ CD3 / CD28 Kit; Thermo Fisher Scientific, Waltham, MA) at a defined ratio. The isolated and activated T cells were counted, CD3+ T cell purity was analyzed by flow cytometry, and they were seeded into containers containing a medium rich in cytokines (human interleukin-2 [IL-2], IL-21, IL-15, IL-7, or combinations thereof). Less than 24 hours post-seedling, T cells were transduced at a fixed multiple of infection (MOI) using a lentiviral vector encoding the Hu19-CD828Z anti-CD19 CAR construct. This is the same construct used in KYV-101, a first-in-class, all-human autologous anti-CD19 CAR T-cell therapy (Kyverna Therapeutics, Emeryville, CA). After a targeted in vitro cell culture period of less than 72 hours post-inoculation, magnetic beads were removed from the culture, cells were harvested, formulated into final product containers, and cryopreserved. Simultaneously, untransduced cells were generated using the same manufacturing process in the absence of lentiviral transduction, serving as control cells.

[0433] Conventional research-grade CAR T cell manufacturing

[0434] For routine research-only (RUO) production of anti-CD19 CAR T cells, the produced cell products are intended to represent the KYV-101 product. Frozen leukocyte separation material was thawed, washed, and antibody-driven T cell separation was performed using magnetic beads (Miltenyi Biotec). The separated T cells were counted, and CD3+ was analyzed by flow cytometry. + T cells were purified and activated using T CellTransAct (Miltenyi Biotec) in the presence of supporting cytokines (human IL-2, IL-21, IL-15, IL-7, or combinations thereof). Similar to the Ingenui-T platform, T cells were transduced at a fixed MOI using the same lentiviral vector (Kyvema Therapeutics, Emeryville, CA) incorporating the Hu19-CD828Z anti-CD19CAR construct. Cells were then cultured for 8–10 days, followed by harvesting, formulation, and cryopreservation of the final product.

[0435] In vitro CAR T cell phenotyping

[0436] Flow cytometry is used to analyze CD3. + T cell purity, CD4 + and CD8 + T cell populations and anti-CD19 CAR expression. Based on the expression of CD45RO, CCR7, and CD95 surface markers, the CD4 and CD8 T cell memory phenotypes of the Ingenui-T final product were compared to the T cell memory population in whole blood starting materials. Similarly, the T cell memory phenotypes in conventional CAR T cells were compared to the T cell memory population in the separation-starting materials (matched to whole blood Ingenui-T cell donors). CAR expression was analyzed at 0 and 72 hours after thawing of the final drug product to ensure accurate measurement of expression after stable integration.

[0437] In vitro CAR T cell functional activity

[0438] To assess the functional activity of CAR T cells in a short-term, single-challenge cytotoxicity assay, donor-matched Ingenui-T cells or conventional CAR T cells were co-cultured with CD19+ NALM6 target cells expressing mCherry fluorescent reporter protein at a specified effector-to-target (E:T) ratio for 120 hours. Target-specific cytotoxic activity was assessed by imaging the co-cultures using an Incucyte Sx5 (Sartorius) instrument and calculating the survival or growth of fluorescent target cells over time (normalized relative to the signal intensity at the start of co-culture).

[0439] To assess the long-term functionality of CAR T cells, donor-matched Ingenui-T cells or conventional CAR T cells were sequentially re-elected every 2–3 days with CD19+ NALM6 target cells at a specified E:T ratio. At each time point, the sample was split in two: one part was used to assess the percentage of cytotoxicity, and the other part was used to replate with fresh target cells. The percentage of cytotoxicity at each time point was calculated by measuring target cell viability using flow cytometry and normalized to the target cell viability in the absence of CAR T effector cells.

[0440] To evaluate the cytolytic activity of CAR T cells against autologous primary B cells, Ingenui-T cells or control untransduced T cells were co-cultured with peripheral blood mononuclear cells (PBMCs) obtained from donor-matched leukocyte ablation material. Based on CAR... +The number of Ingenui-T cells (effectants) and total PBMCs (targets) defined the effector-to-target (E:T) ratio. Target-specific cytolytic activity against B cells was measured by flow cytometry after 48 hours. B cells utilize gated CD3... - The expression of intracellular CD19 or CD20 is defined to ensure proper detection of B cells even in the presence of interaction with anti-CD19 CAR T cells. The percentage of cell lysis against B cells is calculated by normalizing the survival rate of B cells relative to cultures containing only PBMCs.

[0441] result

[0442] Ingenui-T cell manufacturing platform

[0443] The aim is to demonstrate the technical feasibility of generating anti-CD19 CAR T cells using fresh whole blood as the starting material in a shortened manufacturing process. Figure 26 As shown, fresh whole blood from healthy donors was loaded onto the DynaCellect platform, and T cells were simultaneously isolated and activated using anti-CD3 / CD28 Dino magnetic beads at a defined bead-to-cell ratio. Isolated / activated T cells were sampled to confirm isolation purity (>95% CD3+) by flow cytometry and then seeded into cultures containing cytokines IL-2, IL-7, IL-15, IL-21, or combinations thereof. During the first 24 hours of culture, transduction was performed at a fixed MOI using a lentiviral vector encoding an anti-CD19 CAR construct, followed by a brief culture period to allow cell recovery and transgene integration (<72 hours post-inoculation). After this brief culture period, CAR T cells were collected to remove beads and then reconstituted in cryopreservation medium. T cell purity analysis of the final Ingenui-T cell product showed an initial T cell frequency of 42.3 ± 6.8% derived from whole blood and a final T cell percentage of 93.9 ± 1.6%. These results are comparable to T cell enrichment obtained using donor-matched cells via conventional CAR T cell manufacturing processes (in conventional separation, the initial T cell frequency was 46.3 ± 7.2%, and the final T cell purity was 94.0 ± 3.3%; Table 1).

[0444] Given the short culture time, Ingenui-T cells exhibited minimal expansion during the manufacturing process, resulting in a change in the total T cell count of 0.68 ± 0.09 fold from culture seeding to final formulation (including any losses due to washing and bead removal procedures). However, the final yield of the Ingenui-T cell product was 38.5 ± 6.6 × 10⁻⁶. 61 T cells / 100 mL of starting whole blood. Product properties were tested at harvest and 72 hours after thawing to mimic product performance in patients. 72 hours after thawing, CAR+ expression in Ingenui-T cells ranged from 45.1% to 54.5%, which was statistically similar to the 37.4%–56.3% CAR+ expression obtained from conventional CAR T cell manufacturing processes derived from ablation (Table 1). This indicates that using the Ingenui-T platform, anti-CD19 CAR T cells can be successfully manufactured directly from whole blood in a shortened manufacturing process at a scale sufficient to meet the therapeutic dose requirements of patients with B-cell-driven autoimmune diseases.

[0445] Phenotypic and functional comparison of Ingenui-T cells and conventional CAR T cells

[0446] To demonstrate the pharmacological activity of anti-CD19 CAR T cells generated in the Ingenui-T platform, we performed a series of phenotypic and functional in vitro characterization experiments, comparing Ingenui-T cells with CAR T cells expressing the same anti-CD19 CAR construct generated using a conventional manufacturing process. Whole blood used for the Ingenui-T process and absorptions used for the conventional process were derived from the same donor and were part of the same collection. As expected, due to the shortened culture period in the Ingenui-T platform, Ingenui-T cells contained a lower-differentiated T cell memory phenotype than CAR T cells generated in the conventional manufacturing process. Whole blood-derived Ingenui-T cells retained a T cell memory phenotype very similar to that observed in the starting material, and the overall effector / memory population (combined T cells) exhibited a similar phenotype. CM T EM and T E The number of people in the group has increased slightly.

[0447] From starting materials to the final Ingenui-T product, the effector / memory population increased from a mean of 48.8 ± 5.6% to 69.4 ± 4.8% in the CD4+ T cell fraction and from 42.9 ± 3.9% to 46.6 ± 5.5% in the CD8+ T cell fraction, while also increasing in the T cells... N +T SCM Maintaining a fairly large proportion of cells within the population ( Figure 27A ).

[0448] In contrast, most CAR T cells obtained from conventional manufacturing processes (cultured for about 9 days) converted into effector / memory populations, with the percentages of CD4+ and CD8+ T cells increasing from an average of 58.4±3.5% to 94.0±2.8% and from 40.1±5.2% to 86.2±4.9%, respectively.

[0449] Importantly, minimizing the in vitro differentiation of Ingenui-T cells is expected to preserve their activation potential for expansion and activity in patients. This, in turn, enables the administration of significantly lower doses to achieve equivalent therapeutic efficacy in clinical applications, considering the inherently smaller initial number of T cells obtainable from whole blood compared to ablation, a key characteristic of Ingenui-T.

[0450] The functional activity of Ingenui-T cells was evaluated in short-term and long-term in vitro preclinical assays to demonstrate target-specific cytotoxicity against CD19-expressing cells. In a short-term cytotoxicity assay against CD19+ NALM6 tumor cells (as a representative target cell line), the cytotoxic activity of Ingenui-T cells was compared with that of donor-matched, dissected conventional CAR T cells (from conventional 9-day cultures expressing the same CAR construct).

[0451] like Figure 27B As shown, in Incucyte-based imaging assays, Ingenui-T cells controlled the growth of NALM6 target cells with a lower E:T ratio than conventional CAR T cells over a 120-hour period. This reflects the expected increase in CAR T cell potency and target-mediated CAR T cell proliferation due to the less differentiated memory phenotype of Ingenui-T cells. Minimal cytotoxicity was observed against the control CD19-negative target cell line (CEM / C1), and no cytotoxic activity was observed in untransduced Ingenui-T cells (i.e., those not expressing CAR) (data for CD19-positive targets are not shown). These results confirm the anti-CD19 target-specific activity of Ingenui-T cells and their increased functional potency compared to conventional CAR T cells produced in conventional manufacturing processes.

[0452] To further evaluate the functional activity of Ingenui-T cells, we performed a long-term continuous re-challenge assay in vitro. In this assay, CAR T cells and CD19+ NALM6 target cells were co-cultured at a specified E:T ratio starting from day 0, and then the same number of NALM6 target cells were added every 2 or 3 days to assess the potency and durability of target-specific continuous cytotoxicity over extended periods.

[0453] like Figure 27CAs shown, Ingenui-T cells sustained target cell killing for a significantly longer time at a given E:T ratio, and the E:T ratio required to maintain the same killing duration was less than one-quarter that of donor-matched CAR T cells generated by a 9-day culture process. This finding further confirms our expectation that the younger differentiation phenotype of Ingenui-T cells produces higher functional potency, stronger proliferation (data not shown), and longer-lasting cytolytic activity compared to conventional CAR T cells, which are more prone to exhaustion and loss of function over time.

[0454] Finally, the in vitro cytolytic activity of Ingenui-T cells was evaluated against autologous primary B cells, which are target cells that need to be eliminated in the treatment of patients with B-cell-driven autoimmune diseases. When Ingenui-T cells were co-cultured with autologous total peripheral blood mononuclear cells (PBMCs) for 48 hours, B cells were eliminated in a specific and dose-dependent manner. Figure 27D As expected, when tested at a dose-limiting E:T ratio (e.g., 0.011:1), Ingenui-T cells exhibited stronger B-cell killing efficacy than conventional CAR T cells. Figure 27D (And data not displayed).

[0455] These preclinical assays provide proof-of-concept data demonstrating the generation of highly functional anti-CD19 CAR T cells using fresh autologous whole blood as a starting material using our Ingenui-T platform. These results pave the way for the clinical development of Ingenui-T cells as a therapy for autoimmune diseases, which will improve patient experience, increase treatment accessibility, and reduce costs by eliminating the need for patients to undergo ablation procedures.

[0456]

[0457] Example 10 - Ingenui-T Platform - Leukocyte Separation and Whole Blood (WB) Starting Material SM

[0458] This example provides additional data relating to cells manufactured using the Ingenui-T platform with leukocyte ablation or WB SM using the methods disclosed herein. The aim was to characterize anti-CD19 CAR T cells prepared in a shortened manufacturing process using fresh whole blood material as the starting material and to compare them with those prepared using a longer 9-day process and / or leukocyte ablation SM as the starting material.

[0459] In short, use Figure 26 The method outlined in the text is used to prepare and manufacture CAR T cells, in which... Figure 26The starting material obtained from the subjects was derived from leukocyte separation samples.

[0460] Peripheral whole blood and leukocyte separation (SM) is obtained from healthy donors, collected, and transported in fresh condition for immediate processing.

[0461] like Figure 26 In summary, 100 mL of whole blood or leukocyte ablation sample was collected and aliquots were added directly to a Gibco™ CTS™ DynaCellect™ magnetic separation system (Thermo Fisher Scientific, Waltham, MA). T cells were enriched and activated using anti-CD3 / CD28 dyno beads (CTS™ Removable Dynabeads™ CD3 / CD28 Kit; Thermo Fisher Scientific, Waltham, MA) at a defined ratio. The isolated and activated T cells were counted, CD3+ T cell purity was analyzed by flow cytometry, and they were seeded into containers containing a medium rich in cytokines (human interleukin-2 [IL-2], IL-21, IL-15, IL-7, or combinations thereof). Less than 24 hours post-seedling, T cells were transduced at a fixed multiple of infection (MOI) using a lentiviral vector encoding the Hu19-CD828Z anti-CD19 CAR construct. This is the same construct used in KYV-101 described above. After targeted in vitro cell culture, magnetic beads were removed from the culture, cells were harvested, formulated into final product containers, and cryopreserved. Simultaneously, untransduced cells were generated using the same manufacturing process in the absence of lentiviral transduction, serving as control cells.

[0462] like Figure 28A As shown, flow cytometry was used to analyze the overall T cell expansion, T cell viability, and T cell purity of CAR-T cells generated from samples prepared using the 3-day process on the Ingenui-T platform with initial leukocyte separation. Across various cytokine cultures, the 3-day process consistently produced T cells with an expansion greater than 1-fold, viability exceeding 90%, and purity exceeding 95%. Importantly, these results remained consistent regardless of whether the T cells were transduced with exogenous immune receptors.

[0463] Similarly, such as Figure 28B As shown, when whole blood samples are used as starting material, the 3-day process disclosed herein produces a high final concentration of expanded T cells with extremely high T cell purity.

[0464] These results are comparable to T cell enrichment obtained using donor-matched cells via conventional CAR T cell manufacturing processes. Particularly for cells prepared from WB SM, Ingenui-T cells exhibited minimal expansion during the manufacturing process despite shorter culture times, while simultaneously producing a high final yield. Therefore, not only was a sufficient quantity of the final product produced, but it was also generated with limited expansion, which should enable the production of T cells with the favorable phenotype described above.

[0465] Figure 29A The percentage of CAR+ expression, analyzed by flow cytometry, is shown in CAR-T cells manufactured using a KYV 3-day process with leukocyte ablation material as the starting material. At harvest, CAR expression was analyzed in total CD3+ T cells, or in CD4+ or CD8+ T cells. KYV 3-day conditions “A”, “B”, “C”, and “D” indicate the different culture cytokines used. N=4 healthy donors were used under each condition. As explained in Example 8, CAR+ expression in Ingenui-T cells (leukocyte ablation SM) was statistically similar to that obtained from conventional CAR T cell manufacturing processes 72 hours after thawing of the drug product.

[0466] Similarly, such as Figure 29B As shown, when whole blood samples were used as the starting material, the CAR+ expression percentage unexpectedly appeared to exceed the results obtained using a 3-day process with leukocyte separation samples as the starting material.

[0467] Based on the expression of CD45RO, CCR7, and CD95 surface markers, the CD4 and CD8 T cell memory phenotypes of the Ingenui-T final product generated using leukocyte separation starting material (SM) or whole blood (WB) SM were compared with the T cell memory population in whole blood / leukocyte separation starting material. Similarly, the T cell memory phenotypes in conventional CAR T cells were compared with the T cell memory population in leukocyte separation starting material (matched to whole blood Ingenui-T cell donors). CAR expression was analyzed at 0 and 72 hours after thawing of the final drug product to ensure accurate measurement of expression after stable integration.

[0468] Figure 30A The image shows the CD4+ to CD8+ ratio of CAR-T cells produced using the leukocyte separation technique (SM) and a 3-day process, compared to similar cells produced using a conventional 9-day process. As shown in the figure, the CD4:CD8 ratio of CAR-T cells produced using the 3-day process is similar to that of the longer 9-day process.

[0469] Figure 30B Similar results were shown for cells manufactured using a 3-day process but with whole blood (WB) as the starting material.

[0470] To demonstrate the pharmacological activity of anti-CD19 CAR T cells generated in the Ingenui-T platform, a series of phenotypic and functional in vitro characterization experiments were performed, comparing Ingenui-T cells (using WB or leukocyte separation SM) with CAR T cells expressing the same anti-CD19 CAR construct generated using a conventional (9-day) manufacturing process.

[0471] like Figures 31A-31B As shown, compared to the 9-day process, the 3-day process of this disclosure using leukocyte separation SM produces T cells with fewer effector T cells and a larger proportion of TNSCM cells (especially Tscm cells). Surprisingly, as... Figure 31C As shown, when using WB SM, the 3-day method of this disclosure produced T cells with an even higher proportion of TNSCM cells. Surprisingly, these TNSCM cells included a larger proportion of naïve T cells compared to methods using leukoablation SM as the starting material and the 9-day method using WB SM as the starting material.

[0472] Whole blood used in the Ingenui-T process and leukocyte-sparing smears used in the 3-day and 9-day processes were derived from the same donor and were part of the same collection. As expected, due to the shortened culture period in the Ingenui-T platform, Ingenui-T cells contained a less differentiated T cell memory phenotype than CAR T cells produced in conventional manufacturing processes. Whole blood-derived Ingenui-T cells retained a T cell memory phenotype very similar to that observed in the starting material, and the overall effector / memory population (combined T cells) exhibited a similar phenotype. CM T EM and T E The population has increased slightly. These are significant improvements over the existing 9-day process. Therefore, although the cells are transduced with the same exogenous CAR, they are still fundamentally different end products.

[0473] The functional activity of Ingenui-T cells was assessed using Western blot analysis (WB SM) and leukocyte ablation SM to demonstrate target-specific cytotoxicity against CD19-expressing cells. In a short-term cytotoxicity assay against CD19+ NALM6 tumor cells (as a representative target cell line), the cytotoxic activity of Ingenui-T cells was compared with that of donor-matched ablation-derived conventional CAR T cells (from conventional 9-day cultures expressing the same CAR construct).

[0474] Figure 32The percentage of cell lysis in CD19+ NALM6 target cells or CD19-CEM / C1 control cells after co-culturing with anti-CD19 CAR-T cells prepared by the KYV 3-day process at a specified E:T (effectant:target) ratio is shown. N=2 donors are illustrated. Cell lysis activity was measured by chemiluminescence assay and normalized relative to target cells alone (0:1). As shown, the 3-day process cells produced a clear target-dependent cytotoxic response against CD19+ expressing cells.

[0475] Figure 33A The results of killing or growth of CD19+ NALM6 target cells during 120 hours of co-culture with anti-CD19 CAR-T cells manufactured using leukocyte separation material in a KYV 3-day process and co-cultured at a specified E:T (effectant:target) ratio of 0.3:1 or 1:1. NALM6 growth was measured by fluorescence in an Incucyte-based imaging assay and normalized relative to time=0. KYV 3-day conditions “C1”, “C2”, “C3”, “C4” indicate different culture cytokines used. “NT” = untransduced control T cells. A representative donor from n=4 is illustrated. Similarly, Figure 33B Similar results were provided regarding the killing or growth of CD19+ NALM6 target cells during 120 hours of co-culture with anti-CD19 CAR-T cells manufactured from freshly collected whole blood in a KYV 3-day process, co-cultured at a specified E:T (effectant:target) ratio of 0.3:1 or 1:1. NALM6 growth was measured by fluorescence in an Incucyte-based imaging assay and normalized relative to time=0. “NT” = untransduced control T cells. “Conv 9 days” refers to donor-matched CAR-T cells manufactured from leukocyte separation starting material in a standard 9-day culture process. A representative donor from n=4 is illustrated.

[0476] like Figure 33A As shown, in an Incucyte-based imaging assay, Ingenui-T cells (leukocyte separation SM) controlled the growth of NALM6 target cells over a 120-hour period with a lower E:T ratio than conventional CAR T cells. Similar results were observed in cells generated using WBSM. Figure 33BT cells generated using a three-day process (whether starting with WB SM or leukoablation samples) produced a durable, target-specific cytotoxic response, exceeding the efficacy of comparable cells generated using a longer process (e.g., 9 days). Even compared to cells generated using leukoablation samples, cells generated using WB SM exhibited a more potent and durable cytotoxic response.

[0477] This reflects the expected CAR T cell potency and target-mediated CAR T cell proliferation. Furthermore, minimal cytotoxicity was observed against the control CD19-negative target cell line (CEM / C1), and no cytotoxic activity was observed in untransduced Ingenui-T cells. These results confirm the anti-CD19 target-specific activity of Ingenui-T cells prepared by WB SM.

[0478] The in vitro cytolytic activity of Ingenui-T cells was evaluated against autologous primary B cells, which are target cells that need to be eliminated in the treatment of patients with B-cell-driven autoimmune diseases. To assess the cytolytic activity of CAR T cells against autologous primary B cells, Ingenui-T cells or control untransduced T cells were co-cultured with peripheral blood mononuclear cells (PBMCs) obtained from donor-matched leukocyte ablation material. + The number of Ingenui-T cells (effectants) and total PBMCs (targets) defined the effector-to-target (E:T) ratio. Target-specific cytolytic activity against B cells was measured by flow cytometry after 48 hours. B cells utilize gated CD3... - The expression of intracellular CD19 or CD20 is defined to ensure proper detection of B cells even in the presence of interaction with anti-CD19 CAR T cells. The percentage of cell lysis against B cells is calculated by normalizing the survival rate of B cells relative to cultures containing only PBMCs.

[0479] When Ingenui-T cells prepared by WB SM were co-cultured with autologous total peripheral blood mononuclear cells (PBMCs) for 48 hours, B cells were eliminated in a specific and dose-dependent manner. Figure 34 When tested at a dose-limiting E:T ratio, Ingenui-T cells exhibited stronger B-cell killing efficacy than conventional CAR T cells.

[0480] Figure 35The study demonstrates IFN-γ production in anti-CD19 CAR-T cells manufactured from freshly collected whole blood using a KYV 3-day process, co-cultured with CD19+ NALM6 target cells or CD19- CEMC1 control cells at a specified E:T (effectant:target) ratio. Culture supernatants were collected and analyzed by ELLA. The N=2 donors are illustrated. These results demonstrate that anti-CD19 CAR-T cells prepared by the KYV 3-day process respond to target-dependent cytokine release from CD19+ expressing target cells.

[0481] Figure 36A Cytokine release was demonstrated from anti-CD19 CAR-T cells manufactured from leukocyte separation starting material in a KYV 3-day process and co-cultured with CD19+NALM6 target cells at a specified E:T (effectant:target) ratio of 0.3:1 or 1:1. Figures 36B-36C The cytokine release from anti-CD19 CAR-T cells, manufactured from freshly collected whole blood in a KYV 3-day process, is shown. These cells were co-cultured with CD19+ NALM6 target cells at a specified E:T (effectant:target) ratio of 0.3:1 or 1:1. Culture supernatants were collected and analyzed by MSD for the specified cytokines. KYV 3-day conditions “C1”, “C2”, “C3”, and “C4” indicate different culture cytokines used in the manufacturing process. “Conv 9 days” refers to donor-matched CAR-T cells manufactured from leukocyte separation starting material in a standard 9-day culture process. “NT” = untransduced control T cells. N = 4 healthy donors for each condition. As shown, cells generated using the 3-day method of this disclosure provide effector-dose-dependent CAR-mediated cytokine release in response to CD19+ target cells.

[0482] The long-term continuous cytotoxic activity of anti-CD19 CAR-T cells manufactured by the KYV 3-day process in response to CD19+ target cells was compared with that of the conventional 9-day process. Figure 37This figure shows the duration of in vitro cytotoxicity of KYV 3-day or Conv 9-day anti-CD19 CAR T cells derived from healthy donors in a series of rechallenge assays against CD19+ NALM6 tumor cells. KYV 3-day CAR T cells were derived from leukocyte ablation starting material (“APH”, top panel) or freshly collected whole blood (“WB”, bottom panel). CAR T cells were co-cultured in triplicate with NALM6 target cells at a specified effector:target (E:T) ratio, and NALM6 cell viability was analyzed by flow cytometry every 2–3 days. For each individual replicate, the time (in days) required for loss of CAR-mediated cytotoxic activity was measured, defined as the assay time point at which >95% target cell viability was detected. Data represent n=4 donors. As shown, WB cells produced a more durable long-term cytotoxic response.

[0483] Figure 38 This study presents data on the in vitro expansion of anti-CD19 CAR-T cells manufactured using the KYV 3-day process in response to CD19+ target cells compared to the conventional 9-day process. The data provide the responses of KYV 3-day or conventional (“Conv”) 9-day anti-CD19 CAR T cells to repeated stimulation in co-culture with CD19+ REH target cells. KYV 3-day CAR T cells were derived from leukocyte isolation starting material (“APH”, n=4) or other freshly collected starting material (“WB”, n=3) and compared to donor-matched Conv 9-day CAR T cells derived from leukocyte isolation material. CAR T cells were co-cultured with mitomycin C-treated REH target cells at a 1:1 ratio, and cells were replated with new target cells every 3–4 days. The total fold increase of CAR+ T cells (gated by flow cytometry analysis) was measured on day 16. As shown in the figure, cells exhibit expansion potential upon contact with appropriate targets.

[0484] Based on these encouraging results, the in vivo activity of cells processed using freshly harvested starting materials for 3 days was evaluated in mice.

[0485] Figures 39A-39B Data on the in vivo activity of anti-CD19 CAR-T cells manufactured by the KYV 3-day process compared to the conventional 9-day process are presented in CD19+ NALM6 tumor-bearing NSG mice.

[0486] Figure 39AThe average NALM6 tumor growth in NSG mice treated with a specified dose of donor-matched anti-CD19 CAR T cells manufactured using either a KYV 3-day process or a conventional (“Conv”) 9-day process, both starting with leukocyte ablation (“APH”) as the starting material, is shown. NALM6-luciferase-containing tumor cells were intravenously injected into mice on day 7 prior to T cell transfer. A specified dose of CAR T cells was injected intravenously into mice on day 0. Tumor burden in each animal was measured twice weekly using IVIS bioluminescence imaging and is shown as total flux (photons / second). Data are presented as mean ± SEM for all animals in each group. Data represent two studies using n=2 independent donors.

[0487] Figure 39B Individual NALM6 tumor growth curves are shown in NSG mice treated with donor-matched anti-CD19 CAR T cells at a dose of 1e6 CAR+ T cells. The CAR T cells were manufactured using a KYV 3-day process derived from freshly collected whole blood (“WB”) or a conventional (“Conv”) 9-day process derived from leukocyte ablation starting material. Tumor cells were inoculated and, as... Figure 39A The mice were treated and analyzed. N=5 animals per group.

[0488] To further evaluate the success rate of the method disclosed herein in producing CAR T cells using freshly collected whole blood as the starting material, a 9-day process was developed based on the KYV 3-day process and an alternative 3-day process. Figure 10 ). Figure 40 This outlines the 9-day whole blood process.

[0489] In short, using one of two 9-day culture processes, T cells directly isolated from healthy donor (HD) whole blood (WB) are engineered into anti-CD19 CAR-T drug products (DP). For example... Figure 40As shown, T cells were isolated from whole blood using anti-CD3 microbeads followed by TransAct activation (n=1), or simultaneously isolated and activated using anti-CD3 / CD28 dinobeads (n=2). Less than 24 hours post-inoculation, T cells were transduced at a fixed multiple of infection (MOI) using a lentiviral vector encoding the Hu19-CD828Z anti-CD19 CAR construct. The transduced cells were then cultured for a total of 9 days (from inoculation). Figures 41A-41E provide data characterizing the products prepared using these 9-day whole blood processes based on the 3-day whole blood process of this disclosure. As shown in Figure 41A, for both whole blood 9-day methods, whether using anti-CD3 microbeads to isolate T cells from WB followed by TransAct activation (solid triangles, n=1) or using anti-CD3 / CD28 dinobeads for simultaneous isolation and activation (solid circles, n=2), the final yields of the drug products were high and appear to continue the trend established by the 3-day whole blood method described herein (e.g., Figure 28B Figure 41B provides flow cytometry data showing the CAR+ cell percentage within the DP. Figure 41C provides flow cytometry data showing the T cell purity percentage within the DP compared to the WB starting material (SM). As shown, both variants of the 9-day whole blood method achieved successful CAR expression and >90% T cell purity in the final product. This is comparable to the 3-day whole blood method (e.g., Figures 29A-29B ).

[0490] Figure 41D provides flow cytometry data showing the CD4 / 8 fraction in the drug product, while Figure 41E provides flow cytometry data showing the T cell memory phenotype within DP compared to WB SM at the end of CAR T cell production (n=3, combined from the two isolation methods described in Figures 41A-41C). The CD4 / 8 fraction is similar to the 3-day method. However, the T cell memory phenotype shows different final drug products compared to the aforementioned 3-day whole blood method and the conventional 9-day process (not using whole blood as the starting material). In the final drug products produced by these 9-day methods based on the whole blood 3-day method, the T cell memory or effector phenotype (e.g., primarily Tem cells) is similar to that seen in the "conventional" 9-day process using isolation material as the starting material, compared to the "conventional" 9-day process (e.g., ...). Figure 31C Therefore, compared to the 3-day process (which has a higher proportion of Tscm cells and naive T cells), the 9-day whole blood process also produces a lower TNSCM component (e.g., Figure 31C The 9-day whole blood method of this disclosure provides an alternative to the method, although it requires 9 days in manufacturing steps and still depends on obtaining the starting material for the ablation. Therefore, the method of this disclosure can alleviate the manufacturing bottleneck associated with obtaining the ablation sample, while providing cell products with comparable or better therapeutic efficacy.

[0491] To evaluate the therapeutic potential of T cells generated using this 9-day process, the CD19-dependent CAR-mediated cytotoxicity of the cells was assessed by co-culturing CAR T cells derived from WB starting material with CD19+ NALM6 target cells or CD19- CEMC1 control cells, or by co-culturing untransduced control T cells with CD19+ NALM6 target cells. Cell lysis of target cells was assessed by chemiluminescence after 24 hours of co-culture. Figure 42 The cell lysis % result is provided. Figure 43 Corresponding cytokine secretion data were provided. As shown in the figure, cells generated from whole blood using the 9-day method described in this paper provided a target-dependent cytotoxic response.

[0492] As shown in the figure, the Ingenui-T process using whole blood as starting material for 3 days and 9 days yielded a final product with therapeutic potential. To confirm certain aspects of the Ingenui-T process, the drug product produced by the 3-day method (as expected) contained a higher effector / memory population of T cells due to the shorter culture period compared to the whole blood 9-day process, which itself had an effector / memory population comparable to the "conventional" 9-day process. Whole blood-derived Ingenui-T cells retained a T cell memory phenotype very similar to that observed in the starting material, and as the culture process lengthened, the overall effector / memory population (combined T cells) increased. CM T EM and T E The population increased slightly. Both the 3-day and 9-day processes are superior to the existing conventional 9-day process. Therefore, although the cells are transduced using the same exogenous CAR, they are still fundamentally different end products.

[0493] These preclinical assays provided proof-of-concept data (including in vivo data from a three-day process) demonstrating that the Ingenui-T platform can generate highly functional anti-CD19 CAR T cells using fresh autologous whole blood as a starting material. These results pave the way for the clinical development of Ingenui-T cells as a therapy for autoimmune diseases, which will improve patient experience, increase treatment accessibility, and reduce costs by eliminating the need for patients to undergo ablation procedures.

[0494] discuss

[0495] The Ingenui-T platform focuses on improving patient experience and reducing the manufacturing costs of CAR T-cell therapy. This next-generation manufacturing process uses autologous whole blood as a starting material and employs a rapid (<3 days) manufacturing process to produce potent CAR T-cell products with proven target-specific cytotoxic activity. This manufacturing process represents a significant departure from traditional methods that require isotope separation (a laborious and resource-intensive process) and lengthy cell culture.

[0496] The use of whole blood eliminates the need for ablation, alleviating the burden on patients by reducing the need for lengthy and invasive collection processes, ultimately improving the overall patient experience. Furthermore, the limited number of beds available for ablation restricts the number of patients who can be treated, and this problem is becoming increasingly acute as CAR T-cell and CAR T-therapies expand to non-oncology indications (such as autoimmune diseases), where the number of patients is significantly higher (millions compared to tens of thousands). The Ingenui-T platform shortens the process and produces a potent product by minimizing in vitro cell differentiation through minimal culture time, potentially providing equivalent therapeutic benefits at lower doses, while enabling the use of a limited volume of whole blood (instead of ablation) as the starting material. Simplifying the process by collecting up to 300 mL of whole blood and minimizing culture time not only reduces and optimizes resource utilization but also reduces time spent in specialized facilities and the involvement of highly skilled personnel, thereby improving the cost-effectiveness of CAR T-cell therapy. This reduction in the total cost of manufacturing the product and the less burden on patients promises to make the therapy more accessible and affordable. This optimization also aligns with the scalability goals of CAR T-cell therapy, addressing a key need in the field.

[0497] The results of Ingenui-T cell fabrication demonstrate the ability of this process to enrich T cells from the blood and successfully generate potent anti-CD19 CAR T cells with activity comparable to or better than anti-CD19 CAR T cells derived from isolated materials in conventional fabrication processes. Phenotypic characterization revealed a less differentiated phenotype in Ingenui-T cells compared to conventionally fabricated CAR T cells. This characteristic may have clinical significance, as less differentiated T cells are associated with enhanced in vivo expansion and efficacy.

[0498] The Ingenui-T anti-CD19 CAR T-cell product, currently under development for the treatment of B-cell-driven autoimmune diseases, pioneers a novel therapeutic paradigm, addressing key challenges associated with traditional ablation-based manufacturing methods. This platform, which reduces patient burden, is cost-effective, and employs a unique approach, underscores its potential to significantly impact the feasibility and accessibility of CAR T-cell therapy for autoimmune diseases.

[0499] Other embodiments

[0500] Various modifications and variations of this disclosure will be apparent to those skilled in the art without departing from its scope and spirit. Although this disclosure has been described in conjunction with specific embodiments, it should be understood that the claimed disclosure should not be unduly limited to such specific embodiments. In fact, various modifications to the described modes of implementation that will be apparent to those skilled in the art are intended to be within the scope of this disclosure. Other embodiments are described in the claims.

[0501] While preferred embodiments of the invention 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. Those skilled in the art will now recognize numerous variations, modifications, and substitutions without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. The following claims are intended to define the scope of the invention and therefore cover the methods and structures within the scope of these claims and their equivalents.

[0502] By incorporating via reference

[0503] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same degree, as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference.

[0504] References

[0505] (1)Mikhael, J., Fowler, J. & Shah, N. Chimeric Antigen Receptor T-Cell Therapies: Barriers and Solutions to Access. JCO Oncology Practice 18,800-807 (2022).

[0506] (2) Qayed, M. et al. Leukapheresis guidance and best practices for optimal chimeric antigen receptor T-cell manufacturing. Cytotherapy 24, 869-878 (2022).

[0507] (3) Bulliard, Y., Andersson, B. S., Baysal, M. A., Damiano, J. & Tsimberidou, A. M. Reprogramming T cell differentiation and exhaustion in CAR-T cell therapy. J Hematol Oncol 16, 108 (2023).

[0508] (4) Ghassemi, S. et al. Rapid manufacturing of non-activated potent CAR T cells. Nat Biomed Eng 6, 118-128 (2022).

[0509] Sequence Appendix

[0510]

[0511]

[0512]

[0513]

Claims

1. A method for generating an engineered T cell population expressing a heterologous protein, the method comprising: Obtain whole blood samples from the donor; T cells from the whole blood sample are separated and activated by binding to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a carrier. Between 10 and 25 hours after binding to the T cells, the activated T cells are brought into contact with nucleic acids encoding a foreign protein; The T cells in contact with the nucleic acid were cultured in serum-free medium for a period of time between 24 and 60 hours. as well as Harvest the cultured T cells, wherein the harvested T cells express the heterologous protein.

2. The method of claim 1, wherein the T cells are isolated directly from the whole blood sample without performing an intermediate T cell separation step and / or leukocyte separation.

3. The method of claim 1, wherein prior to the binding step, the method comprises isolating peripheral blood mononuclear cells (PBMCs) containing the T cells from the whole blood sample without a leukocyte separation step.

4. The method according to any one of claims 1 to 3, wherein the harvested T cells comprise between about 10% and 60% CD45RO- / CCR7+ T cells (Tnscm).

5. The method of claim 4, wherein the harvested T cells contain at least 18% Tnscm.

6. The method of claim 4, wherein the harvested T cells contain at least 22% Tnscm.

7. The method of claim 4, wherein the harvested T cells contain at least 25% Tnscm.

8. The method according to any one of claims 4 to 7, wherein the Tnscm comprises naive T cells (Tn) and stem cell memory T cells (Tscm).

9. The method of claim 8, wherein the Tnscm contains more Tscm than Tn.

10. The method of claim 9, wherein the Tscm contained in the Tnscm is at least 1.5 times the Tn.

11. The method of claim 10, wherein the Tnscm contains at least twice the amount of Tscm.

12. The method of claim 11, wherein the Tscm contained in the Tnscm is at least 3 times the amount of Tn.

13. The method of claim 12, wherein the Tscm contained in the Tnscm is at least 5 times the size of Tn.

14. The method of claim 13, wherein the Tscm contained in the Tnscm is at least 10 times the size of Tn.

15. The method of claim 14, wherein the Tscm contained in the Tnscm is at least 50 times the size of Tn.

16. The method according to any one of claims 1 to 15, wherein the contact step occurs between 15 and 19 hours after the bonding step.

17. The method according to any one of claims 1 to 16, wherein the contact step occurs between 17 and 19 hours after the bonding step.

18. The method according to any one of claims 1 to 17, wherein the contact step occurs about 18 hours after the bonding step.

19. The method according to any one of claims 1 to 18, wherein the cultivation step lasts for a period of time between 36 hours and 52 hours.

20. The method according to any one of claims 1 to 19, wherein the culture step lasts for a period of time between 46 hours and 50 hours.

21. The method according to any one of claims 1 to 20, wherein the culture step lasts for a period of about 48 hours.

22. The method according to any one of claims 1 to 21, wherein the one or more anti-CD3 antibodies and the one or more anti-CD28 antibodies are attached to the same carrier.

23. The method according to any one of claims 1 to 22, wherein the one or more anti-CD3 antibodies and / or the one or more anti-CD28 antibodies are attached to the vector via a cleavable linker.

24. The method of claim 22, wherein the adapter is an enzymatically cleavable, hydrolyzable, redox-cleavable, phosphate-based, acid-based, ester-based, peptide-based, disulfide-based, nitrophenylmethyl, methoxymethyl-based, and / or photocleavable adapter.

25. The method of claim 23 or claim 24, further comprising contacting the activated T cells with a stimulant that lyses the connector, thereby releasing the T cells from the surface.

26. The method according to any one of claims 1 to 25, wherein the surface is a solid surface.

27. The method of claim 26, wherein the solid surface is a bead, a pore, a chip, or a microfluidic channel.

28. The method of claim 28, wherein the solid surface is a bead.

29. The method according to any one of claims 1 to 25, wherein the surface comprises a polymer.

30. The method of claim 29, wherein the polymer is a hydrogel.

31. The method of claim 29, wherein the surface comprises a polymer scaffold.

32. The method according to any one of claims 1 to 31, wherein the harvested T cells comprise a population comprising at least 6% of the harvested T cells and are pluripotent T cells upon activation based on a specific target.

33. The method of claim 32, wherein the multifunctional T cells and / or a portion of their population simultaneously secrete granzyme B and TNFb.

34. The method of claim 32, wherein the multifunctional T cells and / or a portion of their population simultaneously secrete granzyme B and IFNg.

35. The method of claim 34, wherein at least 1% of the harvested T cells and / or a portion of their population simultaneously secrete granzyme B and IFNg.

36. The method of claim 32, wherein the pluripotent T cells and / or a portion of their population simultaneously secrete MIP-1a and MIP-1b.

37. The method of claim 32, wherein the multifunctional T cells and / or a portion of their population simultaneously secrete IFNg and granzyme B.

38. The method of claim 32, wherein the pluripotent T cells and / or a portion of their population comprise two or more of the following: Cells that simultaneously secrete granzyme B and TNFb and / or a portion of the multifunctional T cell population; Cells that simultaneously secrete granzyme B and IFNg and / or a portion of the aforementioned multifunctional T cell population; Cells that simultaneously secrete MIP-1a and MIP-1b and / or a portion of the aforementioned multifunctional T cell population; and Cells that simultaneously secrete IFNg and granzyme B and / or a portion of the aforementioned multifunctional T cell population.

39. The method of claim 38, wherein the pluripotent T cells and / or a portion of the pluripotent T cell population comprises: Cells that simultaneously secrete granzyme B and TNFb and / or a portion of the multifunctional T cell population; Cells that simultaneously secrete granzyme B and IFNg and / or a portion of the aforementioned multifunctional T cell population; Cells that simultaneously secrete MIP-1a and MIP-1b and / or a portion of the aforementioned multifunctional T cell population; and Cells that simultaneously secrete IFNg and granzyme B and / or a portion of the aforementioned multifunctional T cell population.

40. The method according to any one of claims 1 to 39, wherein the serum-free culture medium comprises at least one cytokine.

41. The method of claim 40, wherein the at least one cytokine comprises one or more of IL-2, IL-21, IL-7 and IL-15.

42. The method of claim 40, wherein the at least one cytokine comprises one or more of IL-21, IL-7 and IL-15 and does not contain IL-2.

43. The method according to any one of claims 1 to 42, wherein the number of isolated T cells from the whole blood sample is about 1 × 10⁻⁶. 6 With approximately 1×10 8 Between 100 total T cells, and the number of T cells harvested is approximately 1 × 10⁻⁶. 8 With approximately 5×10 8 between.

44. The method of claim 43, wherein the number of isolated T cells from the whole blood sample is approximately 5 × 10⁻⁶. 7 With approximately 7.5 × 10 7 Between 100 total T cells, and the number of T cells harvested was approximately 7.5 × 10⁻⁶. 7 With approximately 1.2 × 10 8 between.

45. The method of claim 43 or claim 44, wherein the culture step achieves an expansion of the harvested T cells between 1.0 and 4 times.

46. ​​A method for generating an engineered T cell population expressing a heterologous protein, the method comprising: Obtain whole blood samples from the donor; T cells from the whole blood sample are bound to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a carrier, thereby isolating and activating the T cells from the whole blood sample without the need for intermediate PBMC separation or leukocyte separation steps. This allows the activated T cells to come into contact with nucleic acids encoding foreign proteins; The T cells in contact with the nucleic acid were cultured in serum-free medium. as well as Harvest the cultured T cells, wherein the harvested T cells express the heterologous protein.

47. The method of claim 46, wherein the contact step occurs between 10 and 25 hours after binding to the T cells.

48. The method of claim 46 or claim 47, wherein the T cells in contact with the nucleic acid are cultured for a period of 28 hours to 60 hours.

49. The method according to any one of claims 46 to 48, wherein the harvested T cells comprise between about 10% and 60% CD45RO- / CCR7+ T cells (Tnscm).

50. The method of claim 49, wherein the harvested T cells contain at least 18% Tnscm.

51. The method of claim 49, wherein the harvested T cells contain at least 22% Tnscm.

52. The method of claim 49, wherein the harvested T cells contain at least 25% Tnscm.

53. The method according to any one of claims 49 to 52, wherein the Tnscm comprises naive T cells (Tn) and stem cell memory T cells (Tscm).

54. The method of claim 53, wherein the Tnscm contains more Tscm than Tn.

55. The method of claim 54, wherein the Tscm contained in the Tnscm is at least 1.5 times the Tn.

56. The method of claim 55, wherein the Tnscm comprises at least twice the amount of Tn.

57. The method of claim 56, wherein the Tscm contained in the Tnscm is at least 3 times the size of Tn.

58. The method of claim 57, wherein the Tscm contained in the Tnscm is at least 5 times the size of Tn.

59. The method of claim 58, wherein the Tscm contained in the Tnscm is at least 10 times the size of Tn.

60. The method of claim 59, wherein the Tscm contained in the Tnscm is at least 50 times the size of Tn.

61. The method according to any one of claims 46 to 60, wherein the contact step occurs between 15 and 19 hours after the bonding step.

62. The method according to any one of claims 46 to 61, wherein the contact step occurs between 17 and 19 hours after the bonding step.

63. The method according to any one of claims 46 to 62, wherein the contact step occurs about 18 hours after the bonding step.

64. The method according to any one of claims 46 to 63, wherein the cultivation step lasts for a period of time between 36 hours and 52 hours.

65. The method according to any one of claims 46 to 64, wherein the culture step lasts for a period of time between 46 hours and 50 hours.

66. The method according to any one of claims 46 to 65, wherein the culture step lasts for a period of about 48 hours.

67. The method according to any one of claims 46 to 66, wherein the one or more anti-CD3 antibodies and the one or more anti-CD28 antibodies are attached to the same carrier.

68. The method according to any one of claims 46 to 67, wherein the one or more anti-CD3 antibodies and / or the one or more anti-CD28 antibodies are attached to the vector via a cleavable linker.

69. The method of claim 68, wherein the adapter is an enzymatically cleavable, hydrolyzable, redox-cleavable, phosphate-based, acid-cleavable, ester-based, peptide-based, disulfide-based, nitrobenzyl-based, methoxymethyl-based, and / or photocleavable adapter.

70. The method of claim 68 or claim 69, further comprising contacting the activated T cells with a stimulant that lyses the connector, thereby releasing the T cells from the surface.

71. The method according to any one of claims 46 to 70, wherein the surface is a solid surface.

72. The method of claim 71, wherein the solid surface is a bead, a pore, a chip, or a microfluidic channel.

73. The method of claim 72, wherein the solid surface is a bead.

74. The method according to any one of claims 46 to 70, wherein the surface comprises a polymer.

75. The method of claim 74, wherein the polymer is a hydrogel.

76. The method of claim 74, wherein the surface comprises a polymer.

77. The method of claim 74, wherein the polymer forms a polymer scaffold.

78. The method of any one of claims 46 to 77, wherein the harvested T cells comprise a population comprising at least 6% of the harvested T cells and are pluripotent T cells upon activation based on a specific target.

79. The method of claim 78, wherein the multifunctional T cells and / or a portion of their population simultaneously secrete granzyme B and TNFb.

80. The method of claim 78, wherein the multifunctional T cells and / or a portion of their population simultaneously secrete granzyme B and IFNg.

81. The method of claim 80, wherein at least 1% of the harvested T cells and / or a portion of their population simultaneously secrete granzyme B and IFNg.

82. The method of claim 78, wherein the pluripotent T cells and / or a portion of their population simultaneously secrete MIP-1a and MIP-1b.

83. The method of claim 78, wherein the multifunctional T cells and / or a portion of their population simultaneously secrete IFNg and granzyme B.

84. The method of claim 78, wherein the pluripotent T cells and / or a portion of the pluripotent T cell population comprises two or more of the following: Cells that simultaneously secrete granzyme B and TNFb and / or a portion of the multifunctional T cell population; Cells that simultaneously secrete granzyme B and IFNg and / or a portion of the aforementioned multifunctional T cell population; Cells that simultaneously secrete MIP-1a and MIP-1b and / or a portion of the aforementioned multifunctional T cell population; and Cells that simultaneously secrete IFNg and granzyme B and / or a portion of the aforementioned multifunctional T cell population.

85. The method of claim 78, wherein the pluripotent T cell comprises: Cells that simultaneously secrete granzyme B and TNFb and / or a portion of the multifunctional T cell population; Cells that simultaneously secrete granzyme B and IFNg and / or a portion of the aforementioned multifunctional T cell population; Cells that simultaneously secrete MIP-1a and MIP-1b and / or a portion of the aforementioned multifunctional T cell population; and Cells that simultaneously secrete IFNg and granzyme B and / or a portion of the aforementioned multifunctional T cell population.

86. The method according to any one of claims 46 to 85, wherein the serum-free culture medium comprises at least one cytokine.

87. The method of claim 86, wherein the at least one cytokine comprises one or more of IL-2, IL-21, IL-7 and IL-15.

88. The method of claim 86, wherein the at least one cytokine comprises one or more of IL-21, IL-7 and IL-15 and does not comprise IL-2.

89. The method according to any one of claims 46 to 88, wherein the number of isolated T cells from said whole blood sample is about 1 × 10⁻⁶. 6 With approximately 1×10 8 Between 100 total T cells, and the number of T cells harvested is approximately 1 × 10⁻⁶. 6 With approximately 1.5 × 10 8 between.

90. The method of claim 89, wherein the number of isolated T cells from the whole blood sample is approximately 5 × 10⁻⁶. 7 With approximately 7.5 × 10 7 Between 100 total T cells, and the number of T cells harvested was approximately 7.5 × 10⁻⁶. 7 With approximately 1.2 × 10 8 between.

91. The method of claim 89 or claim 90, wherein the culture step achieves an expansion of the harvested T cells between 1.0 and 4.0 times.

92. A method for generating an engineered T cell population expressing a heterologous protein, the method comprising: Obtain whole blood samples from the donor; T cells from the whole blood sample are separated from the whole blood sample by binding one or more anti-CD4 antibodies and one or more anti-CD8 antibodies attached to a carrier. Between 10 and 25 hours after binding to the T cells, the activated T cells are brought into contact with nucleic acids encoding a foreign protein; The T cells in contact with the nucleic acid were cultured in serum-free medium for a period of time between 24 and 60 hours. as well as Harvest the cultured T cells, wherein the harvested T cells express the heterologous protein.

93. The method of claim 92, wherein the T cells are isolated directly from the whole blood sample without the need for an intermediate T cell separation step and / or leukocyte separation.

94. The method of claim 92, wherein prior to the binding step, the method comprises isolating peripheral blood mononuclear cells (PBMCs) containing the T cells from the whole blood sample without a leukocyte separation step.

95. The method according to any one of claims 92 to 94, further comprising the step of activating the isolated T cells.

96. The method according to any one of claims 92 to 95, wherein the harvested T cells comprise between about 10% and 60% CD45RO- / CCR7+ T cells (Tnscm).

97. The method of claim 96, wherein the Tnscm comprises naive T cells (Tn) and stem cell memory T cells (Tscm).

98. The method of claim 97, wherein the Tnscm contains more Tscm than Tn.

99. The method according to any one of claims 92 to 98, wherein the contact step occurs between 15 and 19 hours after the bonding step.

100. The method according to any one of claims 92 to 99, wherein the cultivation step lasts for a period of time between 36 hours and 52 hours.

101. The method according to any one of claims 92 to 100, wherein the one or more anti-CD4 antibodies and the one or more anti-CD8 antibodies are attached to the same carrier.

102. The method according to any one of claims 92 to 100, wherein the one or more anti-CD4 antibodies and / or the one or more anti-CD8 antibodies are attached to the vector via a cleavable linker.

103. The method of claim 102, wherein the adapter is an enzymatically cleavable, hydrolyzable, redox-cleavable, phosphate-based, acid-cleavable, ester-based, peptide-based, disulfide-based, nitrobenzyl-based, methoxymethyl-based, and / or photocleavable adapter.

104. The method of claim 102 or claim 103, further comprising contacting the activated T cells with a stimulant that lyses the connector, thereby releasing the T cells from the surface.

105. The method according to any one of claims 92 to 104, wherein the surface is a solid surface.

106. The method of claim 105, wherein the solid surface is a bead, a pore, a chip, or a microfluidic channel.

107. The method according to any one of claims 92 to 104, wherein the surface comprises a polymer.

108. The method of claim 107, wherein the polymer is a hydrogel.

109. The method of claim 107, wherein the surface comprises a polymer scaffold.

110. The method of any one of claims 92 to 109, wherein the harvested T cells comprise a population comprising at least 6% of the harvested T cells and are pluripotent T cells upon activation based on a specific target.

111. The method of claim 110, wherein the multifunctional T cells and / or a portion of their population simultaneously secrete granzyme B and TNFb.

112. The method of claim 110, wherein the multifunctional T cells and / or a portion of their population simultaneously secrete granzyme B and IFNg.

113. The method of claim 110, wherein the pluripotent T cells and / or a portion of their population simultaneously secrete MIP-1a and MIP-1b.

114. The method of claim 110, wherein the multifunctional T cells and / or a portion of their population simultaneously secrete IFNg and granzyme B.

115. The method according to any one of claims 92 to 114, wherein the serum-free culture medium comprises at least one cytokine.

116. The method of claim 115, wherein the at least one cytokine comprises one or more of IL-2, IL-21, IL-7, and IL-15.

117. The method of claim 115, wherein the at least one cytokine comprises one or more of IL-21, IL-7 and IL-15 and does not contain IL-2.

118. The method according to any one of claims 92 to 117, wherein the number of isolated T cells from said whole blood sample is about 1 × 10⁻⁶. 6 With approximately 1×10 8 Between 100 total T cells, and the number of T cells harvested is approximately 1 × 10⁻⁶. 8 With approximately 5×10 8 between.

119. The method of claim 118, wherein the number of isolated T cells from the whole blood sample is approximately 5 × 10⁻⁶. 7 With approximately 7.5 × 10 7 Between 100 total T cells, and the number of T cells harvested was approximately 7.5 × 10⁻⁶. 7 With approximately 1.2 × 10 8 between.

120. The method of claim 118 or claim 119, wherein the culture step achieves an expansion of the harvested T cells between 1.0 and 4 times.

121. A method for generating an engineered T cell population expressing a heterologous protein, comprising: Obtain whole blood samples from the donor; T cells from the whole blood sample are bound to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a carrier, thereby isolating and activating the T cells from the whole blood sample, wherein the T cells are isolated directly from the whole blood sample without the need for intermediate T cell isolation steps and / or leukocyte separation. Between 20 and 28 hours after binding to the T cells, the activated T cells are brought into contact with nucleic acids encoding a foreign protein; The T cells in contact with the nucleic acid were cultured in serum-free medium for a period of 4 to 9 days; and Harvest the cultured T cells, wherein the harvested T cells express the heterologous protein.

122. A method for generating an engineered T cell population expressing a heterologous protein, comprising: Obtain whole blood samples from the donor; T cells from the whole blood sample are bound to one or more anti-CD4 antibodies and one or more anti-CD8 antibodies attached to a carrier, thereby isolating and activating the T cells from the whole blood sample, wherein the T cells are isolated directly from the whole blood sample without the need for intermediate T cell separation steps and / or leukocyte separation. Between 20 and 28 hours after binding to the T cells, the activated T cells are brought into contact with nucleic acids encoding a foreign protein; The T cells in contact with the nucleic acid were cultured in serum-free medium for a period of 4 to 9 days; and Harvest the cultured T cells, wherein the harvested T cells express the heterologous protein.

123. The method of claim 122, further comprising the step of activating the isolated T cells.

124. The method according to any one of claims 121 to 123, wherein the contact step occurs between about 22 and 26 hours after the bonding step.

125. The method of claim 124, wherein the contacting step occurs between approximately 23 and 25 hours after the bonding step.

126. The method of claim 124, wherein the contacting step occurs approximately 24 hours after the bonding step.

127. The method according to any one of claims 121 to 126, wherein the cultivation step lasts for a period of about 5 to about 7 days.

128. The method according to any one of claims 121 to 126, wherein the culture step lasts for a period of about 6 days.

129. The method according to any one of claims 121 to 126, wherein the culture step lasts for a period of about 8 days.

130. The method according to any one of claims 121 to 129, wherein the harvested T cells comprise between about 10% and 60% CD45RO- / CCR7+ T cells (Tnscm).

130. The method of claim 129, wherein the Tnscm comprises naive T cells (Tn) and stem cell memory T cells (Tscm).

131. The method of claim 130, wherein the Tnscm contains more Tscm than Tn.

132. The method of any one of claims 121 to 131, wherein the harvested T cells comprise a population comprising at least 6% of the harvested T cells and are pluripotent T cells upon activation based on a specific target.

133. The method of claim 132, wherein the multifunctional T cells and / or a portion of their population simultaneously secrete granzyme B and TNFb.

134. The method of claim 132, wherein the multifunctional T cells and / or a portion of their population simultaneously secrete granzyme B and IFNg.

135. The method of claim 132, wherein the pluripotent T cells and / or a portion of their population simultaneously secrete MIP-1a and MIP-1b.

136. The method of claim 132, wherein the multifunctional T cells and / or a portion of their population simultaneously secrete IFNg and granzyme B.

137. The method according to any one of claims 121 to 136, wherein the serum-free culture medium comprises at least one cytokine.

138. The method of claim 137, wherein the at least one cytokine comprises one or more of IL-2, IL-21, IL-7 and IL-15.

139. The method of claim 137, wherein the at least one cytokine comprises one or more of IL-21, IL-7 and IL-15 and does not contain IL-2.

Images