CN117915929A - Engineered Natural Killer (NK) cells and related methods - Google Patents

Engineered Natural Killer (NK) cells and related methods Download PDF

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CN117915929A
CN117915929A CN202280045649.8A CN202280045649A CN117915929A CN 117915929 A CN117915929 A CN 117915929A CN 202280045649 A CN202280045649 A CN 202280045649A CN 117915929 A CN117915929 A CN 117915929A
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cells
cell
enriched
engineered
composition
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G·迪皮埃罗
A·比格雷
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Indapta Therapeutics Inc
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Indapta Therapeutics Inc
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Priority claimed from PCT/US2022/035884 external-priority patent/WO2023278811A1/en
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Abstract

Provided herein are engineered Natural Killer (NK) cells (g-NK cells) deficient in FcR gamma chain expression, and further comprising recombinant Chimeric Antigen Receptors (CARs) and compositions thereof. Also provided herein are compositions containing these engineered NK cells and methods of making and using these engineered NK cells.

Description

Engineered Natural Killer (NK) cells and related methods
Cross Reference to Related Applications
The present application claims priority from U.S. provisional patent application No. 63/217,718, 2021, 7/1 entitled "ENGINEERED NATURAL KILLER (NK) CELLS AND RELATED METHODS", U.S. provisional patent application No. 63/217,722, 2021, 7/1 entitled "ENGINEERED NATURAL KILLER (NK) CELLS AND RELATED METHODS", and U.S. provisional patent application No. 63/217,726, 2021, 7/1 entitled "ENGINEERED NATURAL KILLER (NK) CELLS AND RELATED METHODS". The contents of these patent applications are incorporated herein by reference in their entirety.
Incorporated by reference into the sequence listing
The present application is presented with a sequence listing in electronic format. The sequence table is provided as a file named 77603_2001040_seqlising. Txt created at 6/30 of 2022, which has a size of 69,307 bytes. The information in the sequence listing in electronic format is incorporated herein by reference in its entirety.
Technical Field
The present disclosure provides engineered Natural Killer (NK) cells (g-NK cells) deficient in FcR gamma chain expression, and further includes recombinant Chimeric Antigen Receptors (CARs) and compositions thereof. The present disclosure also provides compositions containing engineered NK cells and methods of making and using engineered NK cells.
Background
Antibody-based therapies have been frequently used to treat cancer and other diseases. Responses to antibody therapies are typically focused on the direct inhibition of tumor cells by these antibodies (e.g., inhibition of growth factor receptors and subsequent induction of apoptosis), but the in vivo effects of these antibodies may be more complex and may involve the host immune system. Natural Killer (NK) cells are immune effector cells that mediate antibody-dependent cytotoxicity when Fc receptors (CD 16; fcgammaRIII) bind to the Fc portion of antibodies that bind antigen-bearing cells. NK cells, including specific specialized subpopulations thereof, are useful in therapeutic methods, including for improving response to antibody therapies. For therapeutic uses involving NK cells, improved methods are needed. Embodiments are provided herein that meet such needs.
Disclosure of Invention
Described herein are engineered Natural Killer (NK) cells (g-NK cells) deficient in FcR gamma chain expression, and also include recombinant Chimeric Antigen Receptors (CARs) and compositions thereof. Also described herein are compositions containing engineered NK cells and methods of making and using engineered NK cells.
In some aspects, provided herein are engineered Natural Killer (NK) cells (g-NK cells) that are deficient in FcR gamma chain expression, and the NK cells comprise a heterologous nucleic acid encoding a Chimeric Antigen Receptor (CAR).
In some aspects, provided herein are engineered Natural Killer (NK) cells (g-NK cells) deficient in FcR gamma chain expression, and the NK cells comprise a heterologous nucleic acid encoding an immunomodulatory agent.
In some aspects, provided herein are engineered Natural Killer (NK) cells (g-NK cells) deficient in FcR gamma chain expression, and the NK cells comprise a heterologous nucleic acid encoding a Chimeric Antigen Receptor (CAR) and a heterologous nucleic acid encoding an immunomodulatory agent.
In some embodiments, the CAR comprises 1) an antigen binding domain; 2) A flexible connector; 3) A transmembrane region; and 4) an intracellular signaling domain. In some embodiments, the antigen binding domain targets a tumor antigen. In some embodiments, the antigen binding domain is a single chain variable fragment (scFv). In some embodiments, the intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain. In some embodiments, the intracellular signaling domains comprise one or more of CD3 zeta, DAP10, DAP12, CD28, 4-1BB, or OX40 signaling domains. In some embodiments, the intracellular signaling domains comprise two or more signaling domains in CD3 zeta, DAP10, DAP12, CD28, 4-1BB, or OX 40. In some embodiments, the intracellular signaling domain comprises a primary signaling domain comprising a CD3 zeta signaling domain and a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is a CD28 or 4-1BB signaling domain.
In some embodiments, the heterologous nucleic acid encoding the CAR is stably integrated into the genome of the cell. In some embodiments, the heterologous nucleic acid encoding the CAR is transiently expressed.
In some embodiments, the immunomodulator is an immunosuppressant. In some embodiments, the immunomodulator is an immune activator. In some embodiments, the immunomodulatory agent is a cytokine. In some embodiments, the cytokine may be secreted from the engineered NK cell. In some embodiments, the secretable cytokine is IL-2 or a biological portion thereof; IL-15 or a biological portion thereof; or IL-21 or a biological part thereof; or a combination thereof. In other embodiments, the cytokine is membrane bound. In some embodiments, the membrane-bound cytokine is membrane-bound IL-2 (mbiL-2); membrane-bound IL-15 (mbIL-15); membrane-bound IL-21 (mbIL-21); or combinations thereof
In some embodiments, the heterologous nucleic acid encoding the immunomodulator is stably integrated into the genome of the cell. In some embodiments, a heterologous nucleic acid encoding an immunomodulatory agent is transiently expressed.
In some embodiments, the g-NK cells have the surface phenotype of CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of . In some embodiments, the g-NK cells also have a surface phenotype of NKG2a Negative of /CD161 Negative of . In some embodiments, the g-NK cell is also CD38 Negative of . In some embodiments, the g-NK cells have a surface phenotype further of CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative . In some embodiments, g-NK cells include CD16 158V/V (V158). In some embodiments, the g-NK cell is CD16 158V/F.
In some embodiments, there are compositions comprising a plurality of engineered g-NK cells from any of the above embodiments.
In some embodiments, greater than 50% or greater than about 50% of the NK cells or total cells in the composition are g-NK cells. In some embodiments, greater than 60% or greater than about 60% of the NK cells or total cells in the composition are g-NK cells. In some embodiments, greater than 70% or greater than about 70% of the NK cells or total cells in the composition are g-NK cells. In some embodiments, greater than 80% or greater than about 80% of the NK cells or total cells in the composition are g-NK cells. In some embodiments, greater than 90% or greater than about 90% of the NK cells or total cells in the composition are g-NK cells. In some embodiments, greater than 95% or greater than about 95% of the NK cells or total cells in the composition are g-NK cells.
In some embodiments, the plurality of engineered g-NK cells comprises greater than 20% or greater than about 20% g-NK cells comprising a heterologous nucleic acid encoding a CAR. In some embodiments, the plurality of engineered g-NK cells comprises greater than 30% or greater than about 30% g-NK cells comprising a heterologous nucleic acid encoding a CAR. In some embodiments, the plurality of engineered g-NK cells comprises greater than 40% or greater than about 40% g-NK cells comprising a heterologous nucleic acid encoding a CAR. In some embodiments, the plurality of engineered g-NK cells comprises greater than 50% or greater than about 50% g-NK cells comprising a heterologous nucleic acid encoding a CAR. In some embodiments, the plurality of engineered g-NK cells comprises greater than 60%, or greater than about 60%, g-NK cells comprising a heterologous nucleic acid encoding a CAR. In some embodiments, the plurality of engineered g-NK cells comprises greater than 70%, or greater than about 70%, g-NK cells comprising a heterologous nucleic acid encoding a CAR.
In some embodiments, the total composition comprises greater than 20% or greater than about 20% g-NK cells comprising the heterologous nucleic acid encoding the CAR. In some embodiments, the total composition comprises greater than 30% or greater than about 30% g-NK cells comprising the heterologous nucleic acid encoding the CAR. In some embodiments, the total composition comprises greater than 40% or greater than about 40% g-NK cells comprising the heterologous nucleic acid encoding the CAR. In some embodiments, the total composition comprises greater than 50% or greater than about 50% g-NK cells comprising the heterologous nucleic acid encoding the CAR. In some embodiments, the total composition comprises greater than 60% or greater than about 60% g-NK cells comprising the heterologous nucleic acid encoding the CAR. In some embodiments, the total composition comprises greater than 70%, or greater than about 70%, of g-NK cells comprising the heterologous nucleic acid encoding the CAR.
In some embodiments, the plurality of engineered g-NK cells comprises greater than 20% or greater than about 20% g-NK cells comprising a heterologous nucleic acid encoding an immunomodulatory agent. In some embodiments, the plurality of engineered g-NK cells comprises greater than 30% or greater than about 30% g-NK cells comprising a heterologous nucleic acid encoding an immunomodulatory agent. In some embodiments, the plurality of engineered g-NK cells comprises greater than 40% or greater than about 40% g-NK cells comprising a heterologous nucleic acid encoding an immunomodulatory agent. In some embodiments, the plurality of engineered g-NK cells comprises greater than 50% or greater than about 50% g-NK cells comprising a heterologous nucleic acid encoding an immunomodulatory agent. In some embodiments, the plurality of engineered g-NK cells comprises greater than 60% or greater than about 60% g-NK cells comprising a heterologous nucleic acid encoding an immunomodulatory agent. In some embodiments, the plurality of engineered g-NK cells comprises greater than 70% or greater than about 70% g-NK cells comprising a heterologous nucleic acid encoding an immunomodulatory agent.
In some embodiments, the total composition comprises greater than 20% or greater than about 20% g-NK cells comprising the heterologous nucleic acid encoding the immunomodulator. In some embodiments, the total composition comprises greater than 30% or greater than about 30% g-NK cells comprising the heterologous nucleic acid encoding the immunomodulator. In some embodiments, the total composition comprises greater than 40% or greater than about 40% g-NK cells comprising the heterologous nucleic acid encoding the immunomodulator. In some embodiments, the total composition comprises greater than 50% or greater than about 50% g-NK cells comprising the heterologous nucleic acid encoding the immunomodulator. In some embodiments, the total composition comprises greater than 60% or greater than about 60% g-NK cells comprising the heterologous nucleic acid encoding the immunomodulator. In some embodiments, the total composition comprises greater than 70% or greater than about 70% g-NK cells comprising the heterologous nucleic acid encoding the immunomodulator.
In some embodiments, the plurality of engineered g-NK cells comprises greater than 20% or greater than about 20% g-NK cells comprising a heterologous nucleic acid encoding a CAR and a heterologous nucleic acid encoding an immunomodulatory agent. In some embodiments, the plurality of engineered g-NK cells comprises greater than 30% or greater than about 30% g-NK cells comprising a heterologous nucleic acid encoding a CAR and a heterologous nucleic acid encoding an immunomodulatory agent. In some embodiments, the plurality of engineered g-NK cells comprises greater than 40% or greater than about 40% g-NK cells comprising a heterologous nucleic acid encoding a CAR and a heterologous nucleic acid encoding an immunomodulatory agent. In some embodiments, the plurality of engineered g-NK cells comprises greater than 50% or greater than about 50% g-NK cells comprising a heterologous nucleic acid encoding a CAR and a heterologous nucleic acid encoding an immunomodulatory agent. In some embodiments, the plurality of engineered g-NK cells comprises greater than 60%, or greater than about 60%, g-NK cells comprising a heterologous nucleic acid encoding a CAR and a heterologous nucleic acid encoding an immunomodulatory agent. In some embodiments, the plurality of engineered g-NK cells comprises greater than 70%, or greater than about 70%, g-NK cells comprising a heterologous nucleic acid encoding a CAR and a heterologous nucleic acid encoding an immunomodulatory agent.
In some embodiments, the total composition comprises greater than or greater than about 20% g-NK cells comprising a heterologous nucleic acid encoding a CAR and a heterologous nucleic acid encoding an immunomodulatory agent. In some embodiments, the total composition comprises greater than or greater than about 30% g-NK cells comprising a heterologous nucleic acid encoding a CAR and a heterologous nucleic acid encoding an immunomodulatory agent. In some embodiments, the total composition comprises greater than or greater than about 40% g-NK cells comprising a heterologous nucleic acid encoding a CAR and a heterologous nucleic acid encoding an immunomodulatory agent. In some embodiments, the total composition comprises greater than or greater than about 50% g-NK cells comprising a heterologous nucleic acid encoding a CAR and a heterologous nucleic acid encoding an immunomodulatory agent. In some embodiments, the total composition comprises greater than 60%, or greater than about 60%, of g-NK cells comprising the heterologous nucleic acid encoding the CAR and the heterologous nucleic acid encoding the immunomodulator. In some embodiments, the total composition comprises greater than 70%, or greater than about 70%, of g-NK cells comprising the heterologous nucleic acid encoding the CAR and the heterologous nucleic acid encoding the immunomodulator.
In some embodiments, greater than 70% or greater than about 70% of the g-NK cells are positive for perforin and greater than 70% or greater than about 70% of the g-NK cells are positive for granzyme B. In some embodiments, greater than 80% or greater than about 80% of the g-NK cells are positive for perforin and greater than 80% or greater than about 80% of the g-NK cells are positive for granzyme B. In some embodiments, greater than 90% or greater than about 90% of the g-NK cells are positive for perforin and greater than 90% or greater than about 90% of the g-NK cells are positive for granzyme B. In some embodiments, greater than 95% or greater than about 95% of the g-NK cells are positive for perforin and greater than 95% or greater than about 95% of the g-NK cells are positive for granzyme B.
In some embodiments, the average level of perforin expressed by the cells is at least twice or at least about twice the average level of perforin expressed by the cells of FcR gamma Positive and negative , based on the average fluorescence intensity (MFI), as measured by intracellular flow cytometry in cells positive for perforin; and/or in cells positive for granzyme B, the average level of granzyme B expressed by the cells is at least twice or at least about twice the average level of granzyme B expressed by the cells of FcR gamma Positive and negative , as measured by intracellular flow cytometry, based on the average fluorescence intensity (MFI).
In some embodiments, greater than 10% of the cells in the composition are capable of degranulation against tumor target cells, optionally as measured by CD107a expression, optionally wherein degranulation is measured in the absence of antibodies against tumor target cells. In some embodiments, greater than 10% of the cells in the composition are capable of producing interferon-gamma or TNF-alpha to the tumor target cells, optionally wherein the interferon-gamma or TNF-alpha is measured in the absence of antibodies to the tumor target cells.
In some embodiments, greater than 15% or greater than about 15% of the cells in the composition produce effector cytokines in the presence of cells expressing the target antigen (target cells) and antibodies to the target antigen (anti-target antibodies). In some embodiments, greater than 20% or greater than about 20% of the cells in the composition produce effector cytokines in the presence of cells expressing the target antigen (target cells) and antibodies to the target antigen (anti-target antibodies). In some embodiments, greater than 30% or greater than about 30% of the cells in the composition produce effector cytokines in the presence of cells expressing the target antigen (target cells) and antibodies directed against the target antigen (anti-target antibodies). In some embodiments, greater than 40% or greater than about 40% of the cells in the composition produce effector cytokines in the presence of cells expressing the target antigen (target cells) and antibodies to the target antigen (anti-target antibodies). In some embodiments, greater than 50% or greater than about 50% of the cells in the composition produce effector cytokines in the presence of cells expressing the target antigen (target cells) and antibodies directed against the target antigen (anti-target antibodies).
In some embodiments, greater than 30% or greater than about 30% of the total cells in the composition or g-NK cells in the composition are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 50% or greater than about 50% of the cells are negative or low level for NKG2A (NKG 2A Negative of ). In some embodiments, greater than 35% or greater than about 35% of the cells in the total cells in the composition or in g-NK cells in the composition are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 60% or greater than about 60% of the cells are negative or low level for NKG2A (NKG 2A Negative of ). Of the total cells in the composition or of the g-NK cells in the composition, greater than 40% or greater than about 40% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 70% or greater than about 70% of the cells are negative or low level for NKG2A (NKG 2A Negative of ). In some embodiments, greater than 45% or greater than about 45% of the total cells in the composition or g-NK cells in the composition are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 80% or greater than about 80% of the cells are negative or low level for NKG2A (NKG 2A Negative of ). In some embodiments, greater than 50% or greater than about 50% of the cells in the total cells in the composition or in g-NK cells in the composition are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 85% or greater than about 85% of the cells are negative or low level for NKG2A (NKG 2A Negative of ). In some embodiments, greater than 55% or greater than about 55% of the cells in the total cells in the composition or in g-NK cells in the composition are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 90% or greater than about 90% of the cells are negative or low level for NKG2A (NKG 2A Negative of ). In some embodiments, greater than 60% or greater than about 60% of the total cells in the composition or g-NK cells in the composition are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 95% or greater than about 95% of the cells are negative or low level for NKG2A (NKG 2A Negative of ).
In some embodiments, greater than 50% or greater than about 50% of the total cells in the composition or g-NK cells in the composition are CD38 Negative of . In some embodiments, greater than 60% or greater than about 60% of the total cells in the composition or g-NK cells in the composition are CD38 Negative of . In some embodiments, greater than 70% or greater than about 70% of the total cells in the composition or g-NK cells in the composition are CD38 Negative of . In some embodiments, greater than 80% or greater than about 80% of the total cells in the composition or g-NK cells in the composition are CD38 Negative of . In some embodiments, greater than 90% or greater than about 90% of the total cells in the composition or g-NK cells in the composition are CD38 Negative of .
In some embodiments, greater than 50% or greater than about 50% of the total cells in the composition or g-NK cells in the composition are CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of . In some embodiments, greater than 60% or greater than about 60% of the total cells in the composition or g-NK cells in the composition are CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of . In some embodiments, greater than 70% or greater than about 70% of the total cells in the composition or g-NK cells in the composition are CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of . In some embodiments, greater than 80% or greater than about 80% of the total cells in the composition or g-NK cells in the composition are CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of . In some embodiments, greater than 90% or greater than about 90% of the total cells in the composition or g-NK cells in the composition are CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of .
In some embodiments, greater than 50% or greater than about 50% of the total cells in the composition or g-NK cells in the composition are NKG2a Negative of /CD161 Negative of . In some embodiments, greater than 60% or greater than about 60% of the total cells in the composition or g-NK cells in the composition are NKG2a Negative of /CD161 Negative of . In some embodiments, greater than 70% or greater than about 70% of the total cells in the composition or g-NK cells in the composition are NKG2a Negative of /CD161 Negative of . In some embodiments, greater than 80% or greater than about 80% of the total cells in the composition or g-NK cells in the composition are NKG2a Negative of /CD161 Negative of . In some embodiments, greater than 90% or greater than about 90% of the total cells in the composition or g-NK cells in the composition are NKG2a Negative of /CD161 Negative of .
In some embodiments, the plurality of g-NK cells are CD16 (V/V) (V158). In some embodiments, the plurality of g-NK cells are CD16 158V/F.
In some embodiments, the composition comprises at least or at least about 10 8 cells. In some embodiments, the number of g-NK cells in the composition is from 10 8 cells to 10 12 cells or from about 10 8 cells to about 10 12 cells. In some embodiments, the number of g-NK cells in the composition is from 10 8 cells to 10 11 cells or from about 10 8 cells to about 10 11 cells. In some embodiments, the number of g-NK cells in the composition is from 10 8 cells to 10 10 cells or from about 10 8 cells to about 10 10 cells. In some embodiments, the number of g-NK cells in the composition is from 10 8 cells to 10 9 cells or from about 10 8 cells to about 10 9 cells. In some embodiments, the number of g-NK cells in the composition is from 10 9 cells to 10 12 cells or from about 10 9 cells to about 10 12 cells. In some embodiments, the number of g-NK cells in the composition is from 10 9 cells to 10 11 cells or from about 10 9 cells to about 10 11 cells. In some embodiments, the number of g-NK cells in the composition is from 10 9 cells to 10 10 cells or from about 10 9 cells to about 10 10 cells. In some embodiments, the number of g-NK cells in the composition is from 10 10 cells to 10 12 cells or from about 10 10 cells to about 10 12 cells. In some embodiments, the number of g-NK cells in the composition is from 10 10 cells to 10 11 cells or from about 10 10 cells to about 10 11 cells. In some embodiments, the number of g-NK cells in the composition is from 10 11 cells to 10 12 cells or from about 10 11 cells to about 10 12 cells.
In some embodiments, the number of g-NK cells in the composition is at or about 5X10 8 cells. In some embodiments, the number of g-NK cells in the composition is at or about 1X 10 9 cells. In some embodiments, the number of g-NK cells in the composition is at or about 5X10 10 cells. In some embodiments, the number of g-NK cells in the composition is at or about 1X 10 10 cells.
In some embodiments, the volume of the composition is between 50mL and 500mL or between about 50mL and about 500 mL. In some embodiments, the volume of the composition is optionally at or about 200mL.
In some embodiments, the cells in the composition are from a single donor subject, which has been amplified from the same biological sample.
In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a pharmaceutically acceptable excipient. In some embodiments, the composition is formulated in serum-free cryopreservation media that includes a cryoprotectant. In some embodiments, the cryoprotectant is DMSO and the cryopreservation medium is 5% to 10% DMSO (v/v). In some embodiments, the cryoprotectant is or is about 10% DMSO (v/v).
In some embodiments, the composition is sterile. In some embodiments, the composition comprises a sterile pouch. In some embodiments, the bag is a bag suitable for cryopreservation.
Also provided herein are methods of producing genetically engineered g-NK cells comprising introducing a heterologous nucleic acid encoding a Chimeric Antigen Receptor (CAR) into an FcR gamma chain expression deficient NK cell (g-NK cell).
In some aspects, provided herein are methods of producing genetically engineered g-NK cells comprising introducing a heterologous nucleic acid encoding an immunomodulatory agent into the g-NK cells, thereby producing genetically engineered g-NK cells.
In some aspects, provided herein are methods of producing genetically engineered g-NK cells, the methods comprising: (a) Introducing a heterologous nucleic acid encoding a Chimeric Antigen Receptor (CAR) into an FcR gamma chain expression deficient NK cell (g-NK cell), and (b) introducing a heterologous nucleic acid encoding an immunomodulatory agent into the g-NK cell, thereby producing a genetically engineered g-NK cell, wherein steps (a) and (b) are performed simultaneously or sequentially in any order.
In some embodiments, the CAR comprises 1) an antigen binding domain; 2) Flexible connectors (spacers); 3) A transmembrane region; and 4) an intracellular signaling domain. In some embodiments, the antigen binding domain targets a tumor antigen. In some embodiments, the antigen binding domain is a single chain variable fragment (scFv). In some embodiments, the intracellular signaling domain comprises one or more signaling domains from CD3 zeta, DAP10, DAP12, CD28, 4-1BB, or OX 40. In some embodiments, the intracellular signaling domains comprise two or more signaling domains from CD3 ζ, DAP10, DAP12, CD28, 4-1BB, or OX 40.
In some embodiments, the intracellular signaling domain comprises a primary signaling domain comprising a CD3 zeta signaling domain and a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is a CD28 or 4-1BB signaling domain.
In some embodiments, the heterologous nucleic acid encoding the CAR is introduced under conditions that stably integrate into the genome of the g-NK cell. In some embodiments, the heterologous nucleic acid encoding the CAR is included in a viral vector and introduced into g-NK cells by transduction. In some embodiments, the viral vector is a lentiviral vector.
In some embodiments, the nucleic acid encoding the CAR is introduced under conditions of transient expression in g-NK cells. In some embodiments, the nucleic acid encoding the CAR is introduced into the g-NK cell via a lipid nanoparticle. In some embodiments, the nucleic acid encoding the CAR is DNA. In some embodiments, the nucleic acid encoding the CAR is RNA. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is self-amplifying mRNA. In some embodiments, the nucleic acid is introduced into the g-NK cells via electroporation.
In some embodiments, the immunomodulator is an immunosuppressant. In some embodiments, the immunomodulator is an immune activator. In some embodiments, the immunomodulatory agent is a cytokine. In some embodiments, the cytokine may be secreted from the engineered NK cell. In some embodiments, the secretable cytokine is IL-2 or a biologically active portion thereof; IL-15 or a biologically active portion thereof; IL-21 or a biologically active portion thereof; or a combination thereof. In other embodiments, the cytokine is membrane bound. In some embodiments, the membrane-bound cytokine is membrane-bound IL-2 (mbiL-2); membrane-bound IL-15 (mbIL-15); or membrane-bound IL-21 (mbiL-21); or a combination thereof.
In some embodiments, the nucleic acid encoding the immunomodulator is introduced under conditions that stably integrate into the genome of the g-NK cell. In some embodiments, the nucleic acid encoding an immunomodulatory agent is included in a viral vector and introduced into g-NK cells by transduction. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the nucleic acid encoding the immunomodulator is introduced under conditions of transient expression in g-NK cells. In some embodiments, the nucleic acid encoding the immunomodulator is introduced by non-viral delivery. In some embodiments, the nucleic acid encoding the immunomodulator is introduced into the g-NK cell via a lipid nanoparticle. In some embodiments, the nucleic acid encoding the immunomodulator is DNA. In some embodiments, the nucleic acid encoding the immunomodulator is RNA. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is self-amplifying RNA. In some embodiments, the nucleic acid encoding the immunomodulator is introduced into the g-NK cell via electroporation.
In some embodiments, the nucleic acid encoding the CAR and the nucleic acid encoding the immunomodulator are encoded by the same polynucleotide and introduced together. In some embodiments, the nucleic acid encoding the CAR and the nucleic acid encoding the immunomodulator are separated by a polycistronic element of the polynucleotide sequence. In some embodiments, the polycistronic element is a self-cleaving peptide selected from the group consisting of T2A, P a and F2A. In some embodiments, the nucleic acid encoding the CAR and the nucleic acid encoding the immunomodulator are introduced simultaneously during an in vitro process for amplifying the population enriched in g-NK cells.
In some embodiments, the g-NK cell composition is produced by in vitro expansion of NK cells enriched from a biological sample from a subject, the NK cells being: (i) Negative or low level for CD3 and positive for CD57 (CD 3 Negative of CD57 Positive and negative ) or (ii) negative or low level for CD3 and positive for CD56 (CD 3 Negative of CD56 Positive and negative ), wherein the enriched NK cells are cultured with irradiated HLA-e+ feeder cells and one or more recombinant cytokines. In some embodiments, the one or more recombinant cytokines are selected from the group consisting of an effective amount of SCF, GSK3i, FLT3, IL-2, IL-6, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or a combination thereof. In some embodiments, the culturing is performed in the presence of two or more recombinant cytokines, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21. In some embodiments, one or more recombinant cytokines include IL-21.
In some embodiments, the introducing is performed during a method for expanding FcR gamma deficient NK (g-NK) cells, the method comprising: (A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and (B) culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21; wherein introducing a nucleic acid encoding a CAR is performed after step (a) and before, during or after step (B), thereby producing an engineered NK cell population; and wherein the method produces an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR.
In some embodiments, the introducing is performed during a method for amplifying FcR gamma deficient NK cells (g-NK), the method comprising: (A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; (B) Introducing a nucleic acid encoding a CAR into the enriched NK cell population, thereby producing an engineered NK cell population; and (C) culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and wherein the method produces an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR.
In some embodiments, the introducing is performed during a method for amplifying FcR gamma deficient NK cells (g-NK), the method comprising: (A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and (B) culturing the enriched NK cell population in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and (C) introducing a nucleic acid encoding the CAR into NK cells of an NK cell-expanded population, wherein the method produces an engineered NK cell-expanded population that is enriched for g-NK cells and comprises cells engineered with the CAR.
In some embodiments, the introducing is performed during a method for amplifying FcR gamma deficient NK cells (g-NK), the method comprising: (A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and (B) performing a first expansion comprising culturing the enriched NK cell population in a medium under conditions that expand NK cells to produce a first expanded population of NK cells; (C) Introducing a nucleic acid encoding a CAR into NK cells of the first amplified population of NK cells, thereby producing an engineered NK cell population; and (D) performing a second expansion comprising culturing the engineered NK cell population under conditions that further expand the NK cells, wherein the first expansion and/or the second expansion comprises culturing the enriched NK cell population in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21; and wherein the method produces an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR.
In some embodiments, the g-NK cell composition is produced by in vitro expansion of NK cells enriched from a biological sample from a subject, the NK cells being: (i) Negative or low level for CD3 and positive for CD57 (CD 3 Negative of CD57 Positive and negative ) or (ii) negative or low level for CD3 and positive for CD56 (CD 3 Negative of CD56 Positive and negative ), wherein the enriched NK cells are cultured with irradiated HLA-e+ feeder cells and one or more recombinant cytokines. In some embodiments, the one or more recombinant cytokines are selected from the group consisting of an effective amount of SCF, GSK3i, FLT3, IL-2, IL-6, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or a combination thereof. In some embodiments, the culturing is performed in the presence of two or more recombinant cytokines, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21.
In some embodiments, the introducing is performed during a method for expanding FcR gamma deficient NK (g-NK) cells, the method comprising: (A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and (B) culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21; wherein introducing a nucleic acid encoding an immunomodulatory agent is performed after step (a) and before, during, or after step (B), thereby producing an engineered NK cell population; and wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with an immunomodulatory agent.
In some embodiments, the introducing is performed during a method for amplifying FcR gamma deficient NK cells (g-NK), the method comprising: (A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; (B) Introducing a nucleic acid encoding an immunomodulatory agent into an enriched NK cell population, thereby producing an engineered NK cell population; and (C) culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with an immunomodulatory agent.
In some embodiments, the introducing is performed during a method for amplifying FcR gamma deficient NK cells (g-NK), the method comprising: (A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and (B) culturing the enriched NK cell population in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and (C) introducing a nucleic acid encoding an immunomodulator into NK cells of the NK cell-expanded population, wherein the method results in an engineered NK cell-expanded population that is enriched for g-NK cells and comprises cells engineered with the immunomodulator.
In some embodiments, the introducing is performed during a method for expanding FcR gamma deficient NK (g-NK) cells, the method comprising: (A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and (B) culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21; wherein introducing (i) a nucleic acid encoding a CAR and/or (ii) a nucleic acid encoding an immunomodulatory agent is performed after step (a) and before, during, or after step (B), wherein steps (i) and (ii) are performed simultaneously or sequentially in any order, thereby producing an engineered NK cell population; and wherein the method results in an engineered NK cell expansion population that is enriched for g-NK cells and comprises cells engineered with the CAR and the immunomodulator.
In some embodiments, the introducing is performed during a method for amplifying FcR gamma deficient NK cells (g-NK), the method comprising: (A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; (B) Introducing (i) a nucleic acid encoding a CAR and (ii) a nucleic acid encoding an immunomodulatory agent into an enriched NK cell population, wherein steps (i) and (ii) are performed simultaneously or sequentially in any order, thereby producing an engineered NK cell population; and (C) culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and wherein the method results in an engineered NK cell expansion population that is enriched for g-NK cells and comprises cells engineered with the CAR and the immunomodulator.
In some embodiments, the introducing is performed during a method for amplifying FcR gamma deficient NK cells (g-NK), the method comprising: (A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and (B) culturing the enriched NK cell population in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and (C) introducing (i) a nucleic acid encoding a CAR and (ii) a nucleic acid encoding an immunomodulator into NK cells of the NK cell-expanded population, wherein steps (i) and (ii) are performed simultaneously or sequentially in any order, wherein the method results in an engineered NK cell-expanded population that is enriched for g-NK cells and comprises cells engineered with the CAR and the immunomodulator.
In some embodiments, the introducing is performed during a method for amplifying FcR gamma deficient NK cells (g-NK), the method comprising: (A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and (B) performing a first expansion comprising culturing the enriched NK cell population in a medium under conditions that expand NK cells to produce a first expanded population of NK cells; (C) Introducing (i) a nucleic acid encoding a CAR and (ii) a nucleic acid encoding an immunomodulator into NK cells of a first amplified population of NK cells, wherein steps (i) and (ii) are performed simultaneously or sequentially in any order, thereby producing an engineered NK cell population; and (D) performing a second expansion comprising culturing the engineered NK cell population under conditions that further expand the NK cells, wherein the first expansion and/or the second expansion comprises culturing the enriched NK cell population in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21; and wherein the method results in an engineered NK cell expansion population that is enriched for g-NK cells and comprises cells engineered with the CAR and the immunomodulator.
In some embodiments, the NK cell enriched primary human cell population is obtained by selecting cells from a biological sample from a human subject, which are: (i) Negative or low level for CD3 and positive for CD57 (CD 3 Negative of CD57 Positive and negative ) or (ii) negative or low level for CD3 and positive for CD56 (CD 3 Negative of CD56 Positive and negative ).
In some embodiments, the population enriched for NK cells is a cell obtained by further selecting cells positive for NKG2C (NKG 2C Positive and negative ); the NK cell enriched population is a cell obtained by further selecting cells negative or low in level (NKG 2A Negative of ) for NKG 2A; alternatively, the NK cell enriched population is a cell obtained by further selecting cells positive for NKG2C and negative or low-level for NKG2A (NKG 2C Positive and negative NKG2A Negative of ).
In some embodiments of the present invention, in some embodiments, the human being is the subject: wherein at least or at least about 20% of Natural Killer (NK) cells in a peripheral blood sample from the subject are positive for NKG2C (NKG 2C Positive and negative ) and at least 70% of NK cells in a peripheral blood sample are negative for NKG2A or low levels (NKG 2A Negative of ). In some embodiments, the subject is CMV seropositive.
In some embodiments, the percentage of g-NK cells in a biological sample from the subject is greater than 5% or greater than about 5%. In some embodiments, the percentage of g-NK cells in a biological sample from the subject is greater than 10% or greater than about 10%. In some embodiments, the percentage of g-NK cells in a biological sample from the subject is greater than 30% or greater than about 30%.
In some embodiments, the percentage of g-NK cells in the enriched NK cell population is between 20% and 90% or between about 20% and about 90%. In some embodiments, the percentage of g-NK cells in the enriched NK cell population is between 40% and 90% or between about 40% and about 90%. In some embodiments, the percentage of g-NK cells in the enriched NK cell population is between 60% and 90% or between about 60% and about 90%.
In some embodiments, the NK cell enriched population is a cell that is negative or low in CD3 and positive for CD57 (CD 3 Negative of CD57 Positive and negative ) selected from a biological sample. In some embodiments, the NK cell enriched population is a cell that is negative or low in CD3 and positive for CD56 (CD 3 Negative of CD56 Positive and negative ) selected from a biological sample.
In some embodiments, the two or more recombinant cytokines further comprise an effective amount of SCF, GSK3i, FLT3, IL-6, IL-7, IL-15, IL-12, IL-18, IL-27, or a combination thereof. In some embodiments, the recombinant cytokine is IL-21 and IL-2. In some embodiments, the recombinant cytokine is IL-21, IL-2 and IL-15.
In some embodiments, recombinant IL-21 is added to the culture medium at a concentration of 10ng/mL to 100ng/mL or about 10ng/mL to about 100ng/mL during at least a portion of the culturing. In some embodiments, recombinant IL-21 is added to the culture medium during at least a portion of the culturing at a concentration of 25ng/mL or about 25 ng/mL. In some embodiments, recombinant IL-2 is added to the culture medium at a concentration of 10IU/mL to about 500IU/mL or about 10IU/mL to about 500IU/mL during at least a portion of the culture. In some embodiments, recombinant IL-2 is added to the culture medium at a concentration of 100IU/mL or about 100IU/mL during at least a portion of the culture. In some embodiments, recombinant IL-2 is added to the culture medium at a concentration of 500IU/mL or about 500IU/mL during at least a portion of the culture. In some embodiments, recombinant IL-15 is added to the culture medium at a concentration of 1ng/mL to 50ng/mL or about 1ng/mL to about 50ng/mL during at least a portion of the culturing. In some embodiments, recombinant IL-15 is added to the culture medium at a concentration of 10ng/mL or about 10ng/mL during at least a portion of the culture.
In some embodiments, the recombinant cytokine is added to the culture medium at or about the beginning of the culture. In some embodiments, the method further comprises changing the medium one or more times during the culturing, wherein fresh medium containing recombinant cytokines is added each time the medium is changed. In some embodiments, the medium exchange is performed every two or three days during the duration of the culture. In some embodiments, the medium exchange is performed up to 5 days after the first amplification, optionally up to 5 days after the first amplification.
In some embodiments, the human subject has a CD16 158V/V NK cell genotype or a CD16 158V/F NK cell genotype, optionally wherein the biological sample is from a human subject selected for a CD16 158V/VNK cell genotype or a CD16 158V/F NK cell genotype.
In some embodiments, the biological sample is or includes Peripheral Blood Mononuclear Cells (PBMCs), optionally a blood sample, an apheresis sample, or a leucocyte apheresis sample. In some embodiments, the HLA-E+ feeder cells are K562 cells (K562-HLA-E) transformed with HLA-E. In some embodiments, the HLA-E+ feeder cells are 221.AEH cells.
In some embodiments, the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is between 1:1 and 5:1, inclusive. In some embodiments, the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is between 1:1 and 3:1, inclusive. In some embodiments, the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is optionally at or about 2.5:1. In some embodiments, the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is optionally at or about 2:1. In some embodiments, the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is optionally about 1:1.
In some embodiments, the recombinant cytokine added to the culture medium during at least a portion of the culture is 500IU/mL IL-2, 10ng/mL IL-15, and 25ng/mL IL-21.
In some embodiments, the enriched NK cell population comprises enriched NK cells between 2.0×10 5 and 5.0×10 7 or between about 2.0×10 5 and about 5.0×10 7, inclusive. In some embodiments, the enriched NK cell population comprises enriched NK cells, inclusive, between 1.0×10 6 and 1.0×10 8 or between about 1.0×10 6 and about 1.0×10 8. In some embodiments, the enriched NK cell population comprises enriched NK cells between 1.0×10 7 and 5.0×10 8 or between about 1.0×10 7 and about 5.0×10 8, inclusive. In some embodiments, the enriched NK cell population comprises enriched NK cells, inclusive, between 1.0×10 7 and 1.0×10 9 or between about 1.0×10 7 and about 1.0×10 9. In some embodiments, the enriched NK cell population optionally comprises or comprises about 1.0 x10 6 enriched NK cells.
In some embodiments, the concentration of the enriched NK cell population at the beginning of the culture is between 0.05×10 6 enriched NK cells/mL and 1.0×10 6 enriched NK cells/mL or between about 0.05×10 6 enriched NK cells/mL and about 1.0×10 6 enriched NK cells/mL. In some embodiments, the concentration of the enriched NK cell population at the beginning of the culture is between 0.05×10 6 enriched NK cells/mL and 0.5×10 6 enriched NK cells/mL or between about 0.05×10 6 enriched NK cells/mL and about 0.5×10 6 enriched NK cells/mL. In some embodiments, the concentration of the enriched NK cell population at the beginning of the culture is optionally at or about 0.2 x 10 6 enriched NK cells/mL.
In some embodiments, the culturing is performed until such time as the method achieves expansion of at least or at least about 2.50X10 8 g-NK cells. In some embodiments, the culturing is performed until such time as the method achieves expansion of at least or at least about 5.00X 10 8 g-NK cells. In some embodiments, the culturing is performed until such time as the method achieves expansion of at least or at least about 1.0X10 9 g-NK cells. In some embodiments, the culturing is performed until such time as the method achieves expansion of at least or at least about 5.0X10 9 g-NK cells.
In some embodiments, the culturing is performed or performed for about or at least about 5 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 6 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 7 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 8 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 9 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 10 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 11 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 12 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 13 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 14 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 15 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 16 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 17 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 18 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 19 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 20 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 21 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 22 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 23 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 24 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 25 days.
In some embodiments, the method further comprises collecting the expanded population of engineered NK cells produced by the method. In some embodiments, the method further comprises formulating the engineered NK cell-expanded population in a pharmaceutically acceptable excipient. In some embodiments, the method further comprises formulating the engineered NK cell expansion population with a serum-free cryopreservation medium comprising a cryoprotectant. In some embodiments, the cryoprotectant is DMSO. In some embodiments, the cryoprotectant is optionally DMSO, and the cryopreservation medium is 5% to 10% DMSO (v/v), optionally at or about 10% DMSO (v/v).
In some embodiments, greater than 50% of the population of engineered NK cell expansion produced by the method is fcrγ Negative of . In some embodiments, greater than 60% of the population of engineered NK cell expansion produced by the method is fcrγ Negative of . In some embodiments, greater than 70% of the population of engineered NK cell expansion produced by the method is fcrγ Negative of . In some embodiments, greater than 80% of the population of engineered NK cell expansion produced by the method is fcrγ Negative of . In some embodiments, greater than 90% of the population of engineered NK cell expansion produced by the method is fcrγ Negative of . In some embodiments, greater than 95% of the population of engineered NK cell expansion produced by the method is fcrγ Negative of .
In some embodiments, a plurality of engineered NK cells are produced by a method according to any of the above embodiments.
Also provided herein are methods of treating a disease or disorder in a subject. In some embodiments, an effective amount of a cell of a composition according to any of the above embodiments is administered to an individual in need thereof.
In some embodiments, the disease or disorder is an inflammatory disorder. In some embodiments, the disease or disorder is an infection. In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is lymphoma. In some embodiments, the cancer is myeloma. In some embodiments, the disease or disorder is cancer, and the cancer comprises a solid tumor.
In some embodiments, the cancer is an adenocarcinoma of the stomach or gastroesophageal junction. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is brain cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is endocrine/neuroendocrine cancer. In some embodiments, the cancer is a head and neck cancer. In some embodiments, the cancer is a gastrointestinal stromal tumor. In some embodiments, the cancer is a bone giant cell tumor. In some embodiments, the cancer is renal cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is neuroblastoma. In some embodiments, the cancer is ovarian epithelial/fallopian tube/primary peritoneal cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is skin cancer. In some embodiments, the cancer is a soft tissue cancer.
In some embodiments, the composition is administered as a monotherapy. In some embodiments, the method further comprises administering an additional agent to the individual to treat the disease or disorder. In some embodiments, the additional agent is an antibody or Fc fusion protein. In some embodiments, the additional agent is an antibody that is a monoclonal antibody. In some embodiments, the antibody is a full length antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the disease or disorder is cancer, and the additional agent (optionally an antibody) recognizes a tumor antigen associated with the cancer. In some embodiments, the antibody recognizes a tumor antigen associated with cancer.
In some embodiments, the method comprises administering to the individual 1×10 5 cells to 50×10 9 cells or about 1×10 5 cells to about 50×10 9 cells of the g-NK cell composition. In some embodiments, the method comprises administering to the individual 1×10 8 cells to 50×10 9 NK cells or about 1×10 8 cells to about 50×10 9 NK cells of the g-NK cell composition. In some embodiments, the method optionally comprises administering to the individual 5×10 8 cells or 5×10 8 cells of the g-NK cell composition about 5×10 8 cells, 5×10 9 cells or about 5×10 9 cells of the g-NK cell composition or 10×10 9 cells or about 10×10 9 cells of the g-NK cell composition.
In some embodiments, the method further comprises administering an exogenous cytokine support to promote expansion or persistence of the administered NK cells in the subject. In some embodiments, the exogenous cytokine is optionally or optionally comprises IL-15.
In some embodiments, the subject has received lymphocyte removal therapy prior to administration of the dose of g-NK cells. In some embodiments, the therapy comprises fludarabine and/or cyclophosphamide. In some embodiments, lymphocyte depletion comprises administration of fludarabine at 20mg/m 2 to 40mg/m 2 or about 20mg/m 2 to about 40mg/m 2 of subject body surface area. In some embodiments, lymphocyte depletion optionally includes 30mg/m 2 or about 30mg/m 2 per day for 2 to 4 days, and/or administration of cyclophosphamide at 200mg/m 2 to 400mg/m 2 or about 200mg/m 2 to about 400mg/m 2 of subject body surface area, optionally at or about 300mg/m 2 per day for 2 to 4 days. In some embodiments, the lymphocyte removal therapy comprises fludarabine and cyclophosphamide. In some embodiments, the lymphoscavenging therapy comprises administering fludarabine at 30mg/m 2 or about 30mg/m 2 of subject body surface area per day, and cyclophosphamide at 300mg/m 2 or about 300mg/m 2 of subject body surface area per day, each for 2 to 4 days, optionally for 3 days.
In some embodiments, administration of a dose of g-NK cells begins within two weeks after initiation of lymphocyte depletion therapy. In some embodiments, administration of a dose of g-NK cells begins two weeks or about two weeks after initiation of lymphocyte depletion therapy.
In some embodiments, the individual is a human. In some embodiments, the NK cells in the composition are allogenic to the individual. In some embodiments, the NK cells in the composition are autologous to the subject.
Drawings
FIGS. 1A and 1B depict the expansion of g-NK cells expanded in the presence of 221.AEH or K562-mbiL15-41BBL feeder cells with or without IL-21 in NK cell culture medium. Figure 1A shows total NK cell count. FIG. 1B shows g-NK cell counts after 21 days of expansion.
FIGS. 2A and 2B depict up to Lei Tuoyou mab (daratumumab) and erlotinib (elotuzumab) -mediated cytotoxic activity 21 days after expansion of g-NK cells expanded in the presence of 221.AEH or K562-mbiL15-41BBL feeder cells with or without IL-21 in NK cell culture medium. FIG. 2A shows cytotoxicity of g-NK cells against LP1 cell line. FIG. 2B shows cytotoxicity of g-NK cells against MM.1S cell line.
Figures 3A-3D depict the levels of up Lei Tuoyou mab and erlotinib-mediated degranulation (CD 107a Positive and negative ) of g-NK cells expanded in the presence of 221.aeh or K562-mbIL15-41BBL feeder cells with or without IL-21 included in NK cell media. FIG. 3A shows the degranulation level of g-NK cells on LP1 cell line 13 days after expansion. FIG. 3B shows the degranulation level of g-NK cells against MM.1S cell line 13 days after expansion. FIG. 3C shows the degranulation levels of g-NK cells against the LP1 cell line 21 days after expansion. FIG. 3D shows the degranulation level of g-NK cells against MM.1S cell line 21 days after expansion.
FIGS. 4A-4D depict the levels of perforin and granzyme B expression in g-NK cells expanded in the presence of 221.AEH or K562-mbiL15-41BBL feeder cells with or without IL-21 in NK cell culture medium. FIG. 4A shows perforin and granzyme B expression at 13 days post-expansion as a percentage of g-NK cells. Fig. 4B shows total perforin and granzyme B expression at 13 days post amplification. FIG. 4C shows perforin and granzyme B expression at 21 days post-expansion as a percentage of g-NK cells. Fig. 4D shows total perforin and granzyme B expression at 21 days post amplification.
FIGS. 5A-5D depict the expression levels of up to Lei Tuoyou mab and erlotinib-mediated interferon-gamma of g-NK cells expanded in the presence of 221.AEH or K562-mbiL15-41BBL feeder cells with or without IL-21 in NK cell culture medium. FIG. 5A shows the expression levels of interferon-gamma by g-NK cells on the LP1 cell line at 13 days after expansion. FIG. 5B shows the expression levels of interferon-gamma by g-NK cells against MM.1S cell line 13 days after expansion. FIG. 5C shows the expression levels of interferon-gamma by g-NK cells on the LP1 cell line 21 days after expansion. FIG. 5D shows the expression levels of interferon-gamma by g-NK cells against MM.1S cell line at 21 days after expansion.
FIGS. 6A-6D depict the up-Lei Tuoyou mab and erlotinib-mediated TNF- α expression levels of expanded g-NK cells in the presence of 221.AEH or K562-mbiL15-41BBL feeder cells with or without IL-21 included in NK cell culture medium. FIG. 6A shows TNF- α expression levels of the LP1 cell line by g-NK cells 13 days after expansion. FIG. 6B shows TNF- α expression levels of g-NK cells on MM.1S cell line 13 days after expansion. FIG. 6C shows TNF- α expression levels of g-NK cells on the LP1 cell line 21 days after expansion. FIG. 6D shows TNF- α expression levels of g-NK cells against MM.1S cell line 21 days after expansion.
FIG. 7 depicts g-NK cell expansion of NK cells expanded for 15 days in the presence of various cytokine mixtures and concentrations.
FIGS. 8A to 8J show the cellular effector functions of g-NK cells expanded in the presence of various cytokine mixtures and concentrations.
FIGS. 8A and 8B depict the up Lei Tuoyou mab and erlotinib-mediated cytotoxic activity of g-NK cells expanded in the presence of various cytokine mixtures and concentrations. FIG. 8A shows cytotoxicity of g-NK cells against LP1 cell line. FIG. 8B shows cytotoxicity of g-NK cells against MM.1S cell line.
FIGS. 8C and 8D depict the levels of up to Lei Tuoyou mab and erlotinib-mediated degranulation (CD 107a Positive and negative ) of g-NK cells expanded in the presence of various cytokine mixtures and concentrations. FIG. 8C shows the degranulation level of g-NK cells against LP1 cell line. FIG. 8D shows degranulation levels of g-NK cells against MM.1S cell line.
FIGS. 8E and 8F depict the levels of perforin and granzyme B expression in g-NK cells expanded in the presence of various cytokine mixtures and concentrations. FIG. 8E shows perforin and granzyme B expression as a percentage of g-NK cells. Fig. 8F shows the expression of total perforin and granzyme B.
FIGS. 8G and 8H depict expression levels of up to Lei Tuoyou MAb and erlotinib-mediated interferon-gamma of G-NK cells expanded in the presence of various cytokine mixtures and concentrations. FIG. 8G shows the expression levels of interferon-gamma by G-NK cells on the LP1 cell line. FIG. 8H shows the expression level of interferon-gamma by g-NK cells against MM.1S cell line.
FIGS. 8I and 8J depict the levels of up Lei Tuoyou mAb and erlotinib-mediated TNF- α expression of g-NK cells expanded in the presence of various cytokine mixtures and concentrations. FIG. 8I shows TNF- α expression levels of the LP1 cell line by g-NK cells. FIG. 8J shows TNF- α expression levels of g-NK cells against the MM.1S cell line.
Fig. 9A to 9L show the expansion and cellular effector functions (n=6) of g-NK cells expanded in the presence of IL-21 for 14 days, compared to g-NK cells expanded in the absence of IL-21.
FIGS. 9A and 9B depict the expansion of g-NK cells expanded in the presence of IL-21 compared to g-NK cells expanded in the absence of IL-21. FIG. 9A shows the percentage of g-NK cells before and after expansion. FIG. 9B shows the number of amplified g-NK cells per 1 million NK cells. The values are mean.+ -. SE. For CD3 Negative of /CD57 Positive and negative +IL-21 amplification, as compared to CD3 Negative of /CD57 Positive and negative amplification in the absence of IL-21, #p <0.001. For comparison of CD3 Negative of /CD57 Positive and negative amplification with other CMV Positive and negative amplifications, p <0.05. For the comparison of CMV Positive and negative amplification with CMV Negative of CD3 Negative of amplification, p <0.001.
Fig. 9C depicts a comparison of the ratio of g-NK before and after amplification (percentage of total NK cells from cmv+ donor (n=8) and CMV-donor (n=6)). FIG. 9D depicts a comparison of n-fold amplification rates of g-NK from CMV+ and CMV-donors. FIG. 9E provides a representative flow diagram of Fc epsilon R1 gamma versus CD56 for CMV+ donors. FIG. 9F provides representative histograms of Fc εR1γ expression on CD3-/CD56+ NK cells of CMV+ donor and CMV-donor. The difference between cmv+ donor and CMV-donor before and after amplification was determined using independent sample t-test (fig. 9C and 9D). The values are mean.+ -. SE. * p <0.05, < p <0.01, and p <0.001.
FIGS. 9G and 9H depict up to Lei Tuoyou mab and erlotinib-mediated cytotoxic activity at 14 days post-expansion of G-NK cells expanded in the presence of IL-21 as compared to G-NK cells expanded in the absence of IL-21. FIG. 9G shows cytotoxicity of G-NK cells against LP1 cell line. FIG. 9H shows cytotoxicity of g-NK cells against MM.1S cell line. The values are mean.+ -. SE. For comparison of CD Negative of /CD57 Positive and negative +il-21 amplification with CD3 Negative of /CD57 Positive and negative amplification in the absence of IL-21, p <0.05, p <0.01, and p <0.001.
FIGS. 9I and 9J depict the levels of up to Lei Tuoyou mab and erlotinib-mediated degranulation of g-NK cells expanded in the presence of IL-21 (CD 107a Positive and negative ) compared to g-NK cells expanded in the absence of IL-21. FIG. 9I shows the degranulation levels of g-NK cells on the LP1 cell line at 14 days post-expansion. FIG. 9J shows the degranulation level of g-NK cells against MM.1S cell line at 14 days post-expansion. The values are mean.+ -. SE. For comparison of CD Negative of /CD57 Positive and negative +il-21 amplification with CD3 Negative of /CD57 Positive and negative amplification in the absence of IL-21, p <0.05, p <0.01, and p <0.001.
FIGS. 9K and 9L depict the levels of perforin and granzyme B expression in g-NK cells expanded in the presence of IL-21 as compared to g-NK cells expanded in the absence of IL-21. Fig. 9K shows perforin and granzyme B expression at 14 days post-expansion as a percentage of NK cells. Fig. 9L shows the expression of total perforin and granzyme B at 14 days post-amplification. The values are mean.+ -. SE. For comparison of CD Negative of /CD57 Positive and negative +il-21 amplification with CD3 Negative of /CD57 Positive and negative amplification in the absence of IL-21, p <0.05, p <0.01, and p <0.001.
Fig. 9M depicts baseline expression of perforin (left) and granzyme B (right) in expanded g-NK cells and cNK cells (n=5). To compare the expression of effector perforin and granzyme B between g-NK and cNK, a separate sample t-test was used. The values are mean.+ -. SE. Statistically significant differences from cNK cells are indicated with p < 0.001.
FIG. 9N depicts representative histograms of perforin and granzyme B expression of g-NK cells and cNK cells.
FIGS. 9O and 9P depict expression levels of up to Lei Tuoyou mab and erlotinib-mediated interferon-gamma in g-NK cells expanded in the presence of IL-21 as compared to g-NK cells expanded in the absence of IL-21. FIG. 9O shows the expression levels of interferon-gamma by g-NK cells on the LP1 cell line at 14 days after amplification. FIG. 9P shows the expression levels of interferon-gamma by g-NK cells against MM.1S cell line at 14 days after expansion. The values are mean.+ -. SE. For comparison of CD Negative of /CD57 Positive and negative +il-21 amplification with CD3 Negative of /CD57 Positive and negative amplification in the absence of IL-21, p <0.05, p <0.01, and p <0.001.
FIGS. 9Q and 9R depict the expression levels of TNF- α mediated by up to Lei Tuoyou mab and erlotinib in g-NK cells expanded in the presence of IL-21 as compared to g-NK cells expanded in the absence of IL-21. FIG. 9Q shows TNF- α expression levels of the LP1 cell line by g-NK cells at 14 days post-expansion. FIG. 9R shows TNF- α expression levels of g-NK cells on MM.1S cell line at 14 days post-expansion. The values are mean.+ -. SE. For comparison of CD Negative of /CD57 Positive and negative +il-21 amplification with CD3 Negative of /CD57 Positive and negative amplification in the absence of IL-21, p <0.05, p <0.01, and p <0.001.
Fig. 9S depicts the expression levels of up to Lei Tuoyou mab and erlotinib-mediated interferon-gamma by expanded g-NK cells versus cNK cells for the mm.1S cell line in different donors. FIG. 9T depicts the up Lei Tuoyou mab and erlotinib-mediated TNF- α expression levels of expanded g-NK cells versus MM.1S cell line in different donors compared to cNK cells.
Figure 10 depicts the amplification of g-NK amplified in the presence of IL-21/anti-IL-21 complex (n=4). The values are mean.+ -. SE. For amplification with IL-21 compared to amplification with IL-21/anti-IL-21 complex, #p <0.001.
Fig. 11A to 11H show NK cell effector functions of previously cryopreserved g-NK cells (n=4) compared to NK cell effector functions of freshly enriched g-NK cells. The values are mean.+ -. SE. For the fresh enriched g-NK cells and the previous cryopreserved g-NK cells compared, #p <0.05.
FIGS. 11A and 11B depict the levels of up Lei Tuoyou mAb and erlotinib-mediated degranulation (CD 107a Positive and negative ) of previously cryopreserved g-NK cells compared to freshly enriched g-NK cells. FIG. 11A shows degranulation levels of g-NK cells on LP1 cell lines. FIG. 11B shows degranulation levels of g-NK cells against MM.1S cell line.
FIGS. 11C and 11D depict the levels of perforin and granzyme B expression in previously cryopreserved g-NK cells compared to freshly enriched g-NK cells. FIG. 11C shows total perforin expression of g-NK cells. FIG. 11D shows total granzyme B expression of g-NK cells.
FIGS. 11E and 11F depict expression levels of up to Lei Tuoyou mAb and erlotinib-mediated interferon-gamma of previously cryopreserved g-NK cells compared to freshly enriched g-NK cells. FIG. 11E shows the expression levels of interferon-gamma by g-NK cells on the LP1 cell line. FIG. 11F shows the expression levels of interferon-gamma by g-NK cells against MM.1S cell line.
FIGS. 11G and 11H depict the expression levels of TNF- α mediated by up to Lei Tuoyou mAb and erlotinib of previously cryopreserved G-NK cells compared to freshly enriched G-NK cells. FIG. 37G shows TNF- α expression levels of the LP1 cell line by G-NK cells. FIG. 11H shows TNF- α expression levels of g-NK cells against MM.1S cell line.
Figures 12A-12C depict persistence of cNK (cryopreserved) and g-NK (cryopreserved or fresh) cells in NSG mice after infusion of a single dose of 1 x10 7 expanded cells. FIG. 12A shows the numbers of cNK cells and g-NK cells in peripheral blood collected on days 6, 16, 26 and 31 after infusion. Fig. 12B shows the number of NK cells present in the spleen at day 31 post infusion, i.e. at sacrifice. Fig. 12C shows the number of NK cells present in bone marrow at the time of sacrifice. For all 3 bars, n=3. The values are mean.+ -. SE. For comparison of cryopreserved cNK cells to fresh or cryopreserved g-NK cells, p <0.05, and p <0.001.
Fig. 13A-13D depict the expression of CD20 (rituximab target), CD38 (up to Lei Tuoyou mab target) and SLAMF7 (erlotinib target) on g-NK and cNK. FIG. 13A shows the percentage of amplified g-NK cells, non-amplified NK cells (CD 3 Negative of /CD56 Positive and negative ) and MM.1S cells expressing CD 20. FIG. 13B shows the percentage of amplified g-NK cells, non-amplified NK cells (CD 3 Negative of /CD56 Positive and negative ) and MM.1S cells expressing CD 38. FIG. 13C shows the percentage of expanded g-NK cells, unexpanded NK cells (CD 3 Negative of /CD56 Positive and negative ) and MM.1S cells expressing SLAMF 7. FIG. 13D shows the percentage of cNK and g-NK expressing CD38 before and after amplification. For all bars, n=3.
Fig. 13E depicts the Mean Fluorescence Intensity (MFI) of CD38 Positive and negative NK cells before and after expansion (n=4). FIG. 13F provides representative histograms depicting the reduction in CD38 expression of g-NK cells relative to cNK cells and MM.1S cells. The values are mean.+ -. SE. For the comparison of g-NK cells with all other cells, #p <0.001. Fig. 13G depicts a comparison of the co-phase killing induced by up to Lei Tuoyou mab by expanded G-NK cells and cNK cells (fratricide).
Fig. 14A to 14F show the effect of treatment with cNK and up to Lei Tuoyou mab (cNK +up to Lei Tuoyou mab) or g-NK and up to Lei Tuoyou mab (g-nk+up to Lei Tuoyou mab) on tumor burden and survival in a multiple myeloma mouse model. 5×10 5 luciferase-labeled mm.1s human myeloma cells were injected intravenously (i.v.) into the tail vein of female NSG mice. Expanded NK cells (6.0×10 6 cells/mouse) were administered weekly to NSG mice i.v. and injected i.p. up to Lei Tuoyou mab (10 μg/mouse) over a five week duration. Fig. 14A shows BLI imaging of mice twice weekly (left) on days 20, 27, 37, 41, 48 and 57 after tumor inoculation. The corresponding days after treatment are shown on the right side of the graph. The color indicates the intensity of the BLI (blue, lowest; red, highest). Fig. 14B shows the change over time of tumor BLI (photons/sec) in the g-nk+ Lei Tuoyou mab group relative to the control and cNK +up to Lei Tuoyou mab groups. For comparison of g-NK group with control or cNK groups, p <0.05. Fig. 14C shows the percent survival as a function of time, and the arrow indicates administration of therapy with cNK +up to Lei Tuoyou mab or g-nk+up to Lei Tuoyou mab. Fig. 14D presents the change in mouse body weight over time in the control group, cNK +up to Lei Tuoyou mab group and the g-nk+up to Lei Tuoyou mab group. Fig. 14E depicts the number of CD138 + tumor cells present in bone marrow at the time of sacrifice in mice treated with cNK +up to Lei Tuoyou mab and with g-nk+up to Lei Tuoyou mab. For the comparison of g-NK cells to cNK cells, p <0.001. The values are mean.+ -. SE. Fig. 14F shows a representative flowsheet using a gating strategy to resolve the presence of NK cells and tumor cells in control groups and mice treated with cNK +up to Lei Tuoyou mab or g-nk+up to Lei Tuoyou mab. N=8 for the control group and n=7 for the g-NK group or cNK group.
Fig. 14G presents all BLI images collected throughout the study for all control, cNK +up Lei Tuoyou mab and G-nk+up Lei Tuoyou mab-treated mice. The color indicates the intensity of the BLI (blue, lowest; red, highest). Fig. 14H depicts X-ray images obtained for all mice in the control group, cNK +up to Lei Tuoyou mab group, and g-nk+up to Lei Tuoyou mab group prior to sacrifice. Arrows indicate fractures and bone deformities. The date of sacrifice is indicated below each mouse.
Fig. 15A-15C present comparative data for NK cells that persisted in NSG mice after treatment with cNK + up to Lei Tuoyou mab or g-NK + up to Lei Tuoyou mab. All data presented the amount of cells detected at the time of sacrifice using flow cytometry. FIG. 15A shows the number of cNK cells and g-NK cells in blood. Fig. 15B shows the number of NK cells present in the spleen. Fig. 15C shows the number of NK cells present in bone marrow. The values are mean.+ -. SE. For the comparison of g-NK cells to cNK cells, p <0.001.
FIG. 16 depicts the percentage of g-NK (CD 45 Positive and negative /CD3 Negative of /CD56 Positive and negative /FcRγ Negative of ) within a subset of cells with an alternate extracellular surface phenotype of CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative /CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of or CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative /NKG2A Negative of /CD161 Negative of . Values are mean ± standard error.
FIG. 17 depicts post-transduction expression of GFP and CD20-CAR by g-NK cells in two separate experiments, each using a different donor.
FIG. 18 depicts the efficacy of g-NK cells on Raji lymphoma cells with or without CD20-CAR in the presence or absence of rituximab (anti-CD 20 monoclonal antibody).
Detailed Description
Provided herein are engineered Natural Killer (NK) cells (g-NK cells) deficient in FcR gamma chain expression, further comprising recombinant Chimeric Antigen Receptors (CARs) and compositions thereof. Fcrγ is also known as fcsr1γ, which is used interchangeably herein. Also provided herein are methods of producing genetically engineered g-NK cells, comprising introducing a nucleic acid encoding a CAR into a g-NK cell, thereby producing a genetically engineered g-NK cell. In some embodiments, the method of engineering the CAR results in transient expression of the CAR in the engineered NK cells. In some embodiments, the method of engineering the CAR results in stable expression of the CAR in the engineered NK cells.
Provided herein are engineered Natural Killer (NK) cells (g-NK cells) deficient in FcR gamma chain expression, further comprising a heterologous nucleic acid encoding an immunomodulatory agent, and compositions thereof. In some embodiments, the immune modulator may be any agent that may increase or enhance NK cell activity. In some embodiments, the immunomodulator is an exogenous cytokine, such as IL-15. Also provided herein are methods of producing genetically engineered g-NK cells comprising introducing a nucleic acid encoding an immunomodulatory agent into a g-NK cell, thereby producing genetically engineered g-NK cells. In some embodiments, the method of engineering an immunomodulatory agent (e.g., a cytokine such as IL-15) results in transient expression of the immunomodulatory agent in the engineered NK cells. In some embodiments, the method of engineering an immunomodulatory agent (e.g., a cytokine such as IL-15) results in stable expression of the immunomodulatory agent in the engineered NK cells.
Provided herein are engineered Natural Killer (NK) cells (g-NK cells) deficient in FcR gamma chain expression, further comprising a recombinant Chimeric Antigen Receptor (CAR) and a heterologous nucleic acid encoding an immunomodulatory agent, and compositions thereof. Also provided herein are methods of producing genetically engineered g-NK cells, the method comprising (a) introducing a nucleic acid encoding a CAR into a g-NK cell, and (b) introducing a nucleic acid encoding an immunomodulatory agent into a g-NK cell, thereby producing genetically engineered g-NK cells, wherein the introducing a nucleic acid encoding a CAR and the introducing a nucleic acid encoding an immunomodulatory agent can be performed simultaneously or sequentially in any order. In some embodiments, the method of engineering the CAR and the immunomodulatory agent (e.g., a cytokine such as IL-15) results in transient expression of the CAR and/or immunomodulatory agent in the engineered NK cells. In some embodiments, the method of engineering the CAR and the immunomodulatory agent (e.g., a cytokine such as IL-15) results in stable expression of the CAR and the immunomodulatory agent in the engineered NK cells. In some embodiments, one of the CAR or the immunomodulator is transiently expressed in the engineered cell, while the other of the CAR and the immunomodulator is stably expressed.
Natural Killer (NK) cells are congenital lymphocytes that are very important for mediating anti-viral and anti-Cancer immunity by cytokine and chemokine secretion and by release of cytotoxic particles (Vivier et al, science, volume 331, 6013: pages 44-49, 2011; caligiuri, blood, volume 112, 3: pages 461-469, 2008; roda et al, cancer res., volume 66, volume 1: pages 517-526, 2006). NK cells are effector cells that include the third largest group of lymphocytes and are important for host immune surveillance against tumor and pathogen infected cells. However, unlike T lymphocytes and B lymphocytes, NK cells use germline-encoded activation receptors and are considered to have only limited target recognition capacity (Bottino et al, curr Top Microbiol immunol., volume 298: pages 175-182, 2006; stewart et al, curr Top Microbiol immunol., volume 298: pages 1-21, 2006).
Activation of NK cells can occur through the following pathways: NK cell receptors bind directly to ligands on target cells, as seen by direct killing of tumor cells, or cross-linking of Fc receptors (CD 16; also known as CD16a or fcγriiia) occurs by binding to the Fc portion of antibodies that bind to antigen-bearing cells. Upon activation, NK cells produce large amounts of cytokines and chemokines while exhibiting potent cytolytic activity. NK cells are capable of killing tumor cells via antibody-dependent cell-mediated cytotoxicity (ADCC). In some cases, ADCC is triggered when a receptor on the NK cell surface (such as CD 16) recognizes IgGl or IgG3 antibodies that bind to the cell surface. This triggers the release of cytoplasmic granules containing perforin and granzyme, resulting in target cell death. Because NK cell expression activates Fc receptor CD16, which recognizes IgG coated target cells, target recognition was expanded (Ravetch and Bolland, annu Rev immunol., volume 19: pages 275-290, 2001; lanier, nat. Immunol., volume 9, 5: pages 495-502, 2008; bryceson and Long, curr Opin immunol., volume 20, 3: pages 344-352, 2008). ADCC and antibody-dependent cytokine/chemokine production are mediated primarily by NK cells.
CD16 also exists in a glycosyl phosphatidylinositol anchored form (also known as fcyriiib or CD 16B). It is understood that references herein to CD16 refer to the form of CD16a expressed on NK cells and involved in antibody dependent responses such as NK cell mediated ADCC, and not to the glycosyl phosphatidylinositol anchored form.
CD16 receptors are able to associate with the zeta chain (cd3ζ) and/or fcrγ chain of the adapter, TCR-CD3 complex, to transduce signals through an immune receptor tyrosine-based activation motif (ITAM). In some aspects, CD16 engagement (CD 16 cross-linking) initiates NK cell responses via intracellular signals generated by one or both of the CD16 associated adaptor chains fcrγ or cd3ζ. Triggering of CD16 results in phosphorylation of the gamma or zeta chain, which in turn recruits tyrosine kinases, syk and ZAP-70, initiating the signaling cascade, leading to rapid and efficient effector functions. The most well known effector function is to release cytoplasmic granules carrying toxic proteins to kill nearby target cells through the process of antibody-dependent cytotoxicity. CD16 cross-linking also results in the production of cytokines and chemokines which in turn activate and coordinate a range of immune responses.
This release of cytokines and chemokines can play a role in the anticancer activity of NK cells in vivo. NK cells also have small particles in their cytoplasm that contain perforin and protease (granzyme). Upon release from NK cells, perforins form pores in the cell membrane of the target cells through which granzymes and related molecules can enter, inducing apoptosis. The fact that NK cells induce apoptosis rather than necrosis of target cells is important-necrosis of virus-infected cells will release the virions, whereas apoptosis results in destruction of intracellular viruses.
A specialized subset of NK cells lacking FcR gamma adapter protein (also known as g-NK cells) is capable of mediating potent ADCC responses (see, e.g., published patent application No. US 2013/0295044). The mechanism of increased response may be due to changes in epigenetic modifications affecting fcrγ expression. g-NK cells express a large amount of signaling engagement zeta chain, but have a defect in expression of signaling engagement gamma chain. These gamma-deficient g-NK cells exhibit significantly enhanced activity when activated by antibodies compared to conventional NK cells. For example, g-NK cells can be activated by antibody-mediated CD16 cross-linking or by antibody-coated tumor cells. In some aspects, g-NK cells produce greater amounts of cytokines (e.g., IFN-gamma or TNF-alpha) and chemokines (e.g., MIP-1 alpha, MIP-1 beta, and RANTES) and/or exhibit a higher degranulation response than conventional NK cells expressing gamma chains. g-NK cells provide high expression of granzyme B, a component of the cytotoxic machinery of natural killer cells. Furthermore, g-NK cells have an extended lifetime compared to conventional NK cells, and their presence is maintained for a long period of time. In some embodiments, g-NK cells are stable in function and phenotype.
In some embodiments, g-NK cells are more effective in eliciting an ADCC response than conventional NK cells (e.g., NK cells without gamma chain defects). In some embodiments, g-NK cells are more potent than conventional NK cells in eliciting cell-mediated cytotoxicity, even in the absence of antibodies. In some cases, ADCC is the mechanism of action of therapeutic antibodies, including anti-cancer antibodies. In some aspects, cell therapies by administering NK cells can be used with antibodies for therapeutic and related purposes.
For example, certain therapeutic monoclonal antibodies, such as CD 38-targeting dashboards Lei Tuoyou mab and SLAMF 7-targeting erlotinib, are FDA approved for the treatment of diseases such as Multiple Myeloma (MM). While the clinical response of therapeutic antibodies is promising, they are generally not ideal. For example, while the initial clinical response is often encouraging, particularly for up to Lei Tuoyou mab, substantially all patients eventually develop progressive disease. Thus, new strategies are highly desirable to drive deeper relief or to overcome resistance to these agents. The embodiments (including compositions) provided address these needs.
Provided herein are engineered Natural Killer (NK) cells (g-NK cells) deficient in FcR gamma chain expression, further comprising recombinant Chimeric Antigen Receptors (CARs) and compositions containing the CARs. Provided herein are engineered Natural Killer (NK) cells (g-NK cells) deficient in FcR gamma chain expression, further comprising a heterologous nucleic acid encoding an immunomodulatory agent, and compositions thereof. Provided herein are engineered Natural Killer (NK) cells (g-NK cells) deficient in FcR gamma chain expression, further comprising a recombinant Chimeric Antigen Receptor (CAR) and a heterologous nucleic acid encoding an immunomodulator, and compositions containing the heterologous nucleic acid. Methods of engineering g-NK cells are also provided. In some embodiments, CAR-dependent antigen targeting by g-NK cells engineered or engineered g-NK cells can lead to improved results for patients due to improved affinity, cytotoxicity, and/or cytokine-mediated effector function of the g-NK cell subpopulation. In some embodiments, the engineered g-NK cells are administered to a subject in combination with a therapeutic antibody directed against a tumor or other pathogen or disease antigen. In some embodiments, antibody directed g-NK cell targeting can bring improved results to patients due to improved affinity, cytotoxicity, and/or cytokine mediated effector function of the g-NK cell subpopulation.
Although NK cells are typically activated when the Fc portion of an antibody binds to its Fc receptor (fcγriiia or CD16 a) and triggers activation and degranulation by processes involving the adaptor proteins cd3ζ and fcεr1γ, such methods involve administering an antibody directed against the desired target alone and exposing NK cells to the antibody. Furthermore, binding and cross-linking of the Fc receptor CD16 on conventional NK cells is involved in signaling via both cd3ζ and fcεr1γ, which can lead to variability in signaling depending on the expression of the signaling adapter in NK cells. Finally, NK cell activity often requires cytokine supports, such as through IL-15 support, to enhance cytotoxic activity; thus, the lack of sufficient supporting cytokines may limit the persistence of the response. Each of the above factors, alone or together, hamper the utility of certain NK cell therapies.
In several aspects, the engineered NK cells and compositions containing the engineered NK cells provided herein (such as produced by the provided methods) provide improved cell therapies. First, provided g-NK cells and compositions containing the g-NK cells (such as produced by the provided methods) are engineered to express a Chimeric Antigen Receptor (CAR), an immunomodulatory agent such as a co-stimulatory cytokine, or both. Expression of the CAR enables g-NK cells to be administered without the need for a separate monoclonal antibody to target the g-NK cells to target cells or tissues in the affected subject or individual. In addition, the ability of NK cells to produce and secrete exogenous immunomodulators such as cytokines means that NK cells have an intrinsic support of enhanced activity. Finally, the provided NK cell compositions are enriched for g-NK cells (i.e. Fc εR1γ deficient NK cells) which provide a number of advantages compared to conventional NK cells or NK cells enriched for other subpopulations. G-NK cells are a relatively rare subpopulation because G-NK cells are detectable only at levels of only about 3% -10% of total NK cells in 25% -30% of CMV seropositive individuals. The provided methods relate to methods that are particularly powerful in terms of the ability to amplify and enrich g-NK cells, thus allowing for the desired sufficient amplification for in vivo use, while also being suitable for engineering the enriched g-NK cells with a CAR, an immunomodulatory agent (e.g., a cytokine such as IL-15) or a CAR and an immunomodulatory agent (e.g., a cytokine such as IL-15) before, during, or after amplification of the enriched g-NK cells. In some embodiments, the engineered g-NK cells are administered to a subject in combination with a monoclonal antibody for targeting the g-NK cells to a target cell or tissue in the subject or individual affected. The provided cells and compositions produced by such methods are particularly powerful in their ability to target g-NK cells to an appropriate location in a subject or individual.
G-NK cells represent a relatively small percentage of NK cells in peripheral blood, limiting the ability to use these cells in therapeutic methods. In particular, in order to utilize g-NK cells in the clinic, a high preferential expansion rate is necessary, since g-NK cells are generally a rare population. Other methods for expanding NK cells are capable of achieving thousands of 14-day NK cell expansion rates, but they produce poorly differentiated, NKG2C Negative of FcεR1γ Positive and negative (FcRγ Positive and negative ) NK cells (Fujisaki et al 2009, cancer res., volume 69: pages 4010-4017; shah et al, 2013, PLoS One, 8:e76781). Furthermore, it was found herein that an amplification optimized for amplifying NK cells phenotypically overlapping with g-NK cells does not preferentially amplify g-NK cells to an amount supporting therapeutic use. In particular, it has been previously reported that NKG2C Positive and negative NK cells, which exhibit an overlap with the g-NK cell phenotype, can be preferentially expanded by using HLA-E transfected 221.AEH cells and including IL-15 in the medium (Bigley et al, 2016, clin. Exp. Immunol., volume 185:239-251). Culturing with such HLA-expressing cells that constitutively express HLA-E can push NK cells in the direction of the NKG2C Positive and negative /NKG2A Negative of phenotype (NKG 2C is an activating receptor for HLA-E and NKG2A is an inhibiting receptor for HLA-E). It is believed that such a method would be sufficient to amplify g-NK cells because such cells include g-NK cells within them. However, this method cannot achieve powerful expansion of g-NK cells.
The methods described herein are capable of producing a g-NK cell enriched NK cell composition that overcomes these limitations. The provided methods utilize a greater proportion of HLA-e+ feeder cells (e.g., 221.Aeh cells) to NK cells that are defective in HLA class I and HLA class II than previous methods. In particular, previous methods used a lower ratio of 221.Aeh cells, such as NK cells to 221.Aeh at a ratio of 10:1. It was found herein that a greater proportion of HLA-E expressing feeder cells (such as 221.AEH cells) resulted in greater overall expansion and a more biased g-NK phenotype. In some embodiments, a greater proportion of HLA-E+ feeder cells (e.g., 221.AEH cells) are possible by irradiating the feeder cells. In some aspects, the use of irradiated feeder cell lines is also advantageous because it provides a GMP-compatible approach. It was also found that any of the recombinant IL-2, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or combinations thereof, are supported for powerful amplification during amplification. In a specific embodiment of the provided method, the at least one recombinant cytokine is IL-2. In some embodiments, there are two or more recombinant cytokines, wherein at least one recombinant cytokine is IL-2 and at least one recombinant cytokine is IL-21.
The methods provided herein are based on the following findings: culturing NK cells for expansion in the presence of IL-21 can enhance NK cell function to produce cytokines or effector molecules such as perforin and granzyme B. Compositions containing NK cells produced by the expansion process herein are highly functional, exhibit potent proliferation, and work well even when they are not resuscitated after being cryogenically frozen. For example, NK cells produced by the provided process not only exhibit strong ADCC activity but they also exhibit antibody-independent cytotoxic activity when amplified in the presence of IL-21. Potent activity, including antibody-independent cytotoxic activity, is particularly suitable for the strategies described herein, wherein the cells are further engineered with CARs, immunomodulators, or CARs and immunomodulators, because after CAR is conjugated by target antigen or co-administered antibody whose Fc receptor CD16 is recognized by the target antigen, NK cells are primed and ready to exert effector activity. For example, effector molecules (e.g., perforins and granzymes) are spontaneously present in NK cells expanded by the provided methods, thereby providing cells that exhibit high cytotoxic potential. As shown herein, NK cell compositions produced by the provided processes including IL-21 (e.g., IL-2, IL-15, and IL-21) not only exhibit a higher percentage of NK cells positive for perforin or granzyme B, but also exhibit a higher average level or degree of expression of the molecule in the cell, as compared to NK cell compositions produced by processes including IL-2 alone without IL-21 addition. In addition, NK cell compositions produced by the methods provided herein including IL-21 (e.g., IL-2, IL-15, and IL-12) also produce g-NK cell compositions that exhibit significant effector activity (including degranulation and the ability to express more IFN-gamma and TNF-alpha) in response to target cells. This functional activity is highly retained even after cryopreservation and thawing of expanded NK cells. The marked increase in cytolytic enzymes and the more potent activation phenotype enhance the ability of the expanded g-NK cells to induce apoptosis in tumor targets. These activities are exemplified herein when binding to antibodies to a target antigen via CD16 cross-linking, although many of these activities are exemplified herein when binding to antibodies via CD16 cross-linking, similar activities occur when CAR binds to a target antigen, as signaling is also mediated via CD3 zeta. The potential utility of g-NK cells as monotherapy is also supported by the labeled antibody-independent effector phenotype, in addition to engineering the cells with CARs, immunomodulators (e.g., cytokines), or CARs and immunomodulators (e.g., cytokines).
Furthermore, in some embodiments, g - NK cells produce significantly greater amounts of cytokines than FcR gamma expressing natural killer cells. In another embodiment, the cytokine is interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), or a combination thereof. In one embodiment, g - NK cells produce significantly greater amounts of chemokines. In one embodiment, the chemokine is MIP-1α, MIP-1β, or a combination thereof. In another embodiment, g - NK cells produce cytokines or chemokines upon signaling via CD3 ζ, such as may occur via engagement of the CAR or in some cases stimulation via Fc receptor CD 16.
Furthermore, the findings herein demonstrate the potential provided for good persistence and proliferation of NK cells expanded in the presence of IL-21 over an extended period of time, which is greater than the potential of cells expanded, for example, in the presence of IL-2 alone without addition of IL-21. Furthermore, the results show that cryopreserved g-NK cells persist at levels comparable to fresh g-NK cells. This significantly improved persistence highlights the potential utility of fresh or cryopreserved g-NK as an off-the-shelf cell therapy to enhance target-directed cytotoxicity or antibody-mediated ADCC. This discovery of improved persistence is advantageous because the clinical utility of many NK cell therapies is hampered by limited NK cell persistence.
It was also found that enrichment of NK cells from a cell sample prior to the expansion method, such as by enrichment of CD16 or CD57 cells prior to expansion, further significantly increased the amount of g-NK cell expansion compared to the method of enriching NK cells based on CD3 depletion only initially. In another embodiment, another enrichment that can be performed prior to amplification is the enrichment of NK cells by positive selection of CD56 and negative selection or depletion of CD 38. In another embodiment, another enrichment that may be performed prior to amplification is the enrichment of NK cells by positive selection of CD56 followed by negative selection or depletion of NKG2a Negative of and negative selection or depletion of CD161 Negative of . In another embodiment, another enrichment that may be performed prior to amplification is the enrichment of NK cells by positive selection of CD57 followed by negative selection or depletion of NKG2A and/or positive selection of NKG 2C. In another embodiment, another enrichment that may be performed prior to amplification is the enrichment of NK cells by positive selection of CD56 followed by negative selection or depletion of NKG2A and/or positive selection of NKG 2C. In any of such embodiments, the enrichment of NKG2C Positive and negative and/or NKG2a Negative of NK cells may be performed after the expansion.
In any of such embodiments, the enriched NK cells may be enriched from a cell sample containing NK cells, such as from Peripheral Blood Mononuclear Cells (PBMCs). In some embodiments, T cells may be removed by negative selection or depletion of CD3 prior to enrichment of NK cells from the cell sample. In any of such embodiments, the enriched NK cells may be enriched from a biological sample (e.g., PBMCs) from a human subject containing NK cells (which have a relatively high proportion of g-NK cells), e.g., from a human subject selected for having a higher percentage of g-NK cells in NK cells. In any of such embodiments, the enriched NK cells can be enriched from a biological sample (e.g., PBMC) from a human subject containing NK cells, wherein the sample contains a relatively high proportion of NKG2C Positive and negative NK cells (e.g., about or greater than 20% NKG2C Positive and negative NK cells) and/or NKG2a Negative of NK cells (e.g., about or greater than 70% NKG2a Negative of NK cells). In any of such embodiments, the enriched NK cells can be enriched from a biological sample (e.g., PBMC) from a human subject containing NK cells, wherein the sample contains a relatively high proportion of NKG2C Positive and negative NK cells (e.g., about or greater than 20% NKG2C Positive and negative NK cells) and NKG2a Negative of NK cells (e.g., about or greater than 70% NKG2a Negative of NK cells). In particular embodiments, the subject from which the sample is taken is CMV seropositive, as such subject has more detectable g-NK cells in its peripheral blood.
In summary, from the first ten million enriched NK cells at the beginning of culture, the provided methods of expanding g-NK cells can achieve expansion of more than one billion cells, and in some cases up to one billion or more NK cells. In particular, the provided methods can produce high throughput (greater than 1000-fold) amplification rates while maintaining or in some cases increasing g-NK cell functionality after amplification. In some embodiments, the provided methods can produce g-NK cell populations expressing high levels of perforin and granzyme B. Furthermore, it was found that the provided methods are sufficient to expand previously frozen NK cells, which many existing methods involving resuscitating thawed NK cells are generally not achievable. In some embodiments, this is achieved by increasing the duration of the amplification schedule. In some embodiments, this is achieved by reducing the ratio of HLA-e+ feeder cells to NK cells, for example to about 1:1 ratio of 221.Aeh to NK cells. In some embodiments, this is accomplished by including any of recombinant IL-2, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or a combination thereof during amplification. In specific embodiments, at least one recombinant cytokine is IL-2. In some embodiments, the amplification is performed in the presence of two or more recombinant cytokines, wherein at least one recombinant cytokine is recombinant IL-21 and at least one recombinant cytokine is recombinant IL-2.
As shown herein, the provided engineered g-NK cells and compositions containing the engineered g-NK cells (such as produced by the provided methods) are useful for cancer therapy. In some embodiments, adoptive transfer of NK cells does not result in severe Graft Versus Host Disease (GVHD), and thus such cell therapies (including in combination with antibodies as antibody-directed NK cell therapies) can be used in an "off-the-shelf" manner for clinical use. In some aspects, NK cells can be further engineered to reduce or eliminate individual HLA molecules in NK cells, thereby increasing the allogeneic potential of the provided cell therapies.
All references, including patent applications, patent publications, and scientific literature and databases, cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual reference were specifically and individually indicated to be incorporated by reference.
For clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
I. Definition of the definition
Unless defined otherwise, all technical, symbolic, and other technical and scientific terms or specialized terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ease of reference, and the inclusion of such definitions herein is not necessarily to be construed as representing a substantial difference from what is commonly understood in the art.
As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules.
As used herein, the term "about" refers to a general range of error for the corresponding value as readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that refer to that value or parameter itself.
It should be understood that the aspects and embodiments of the invention described herein include, consist of, and consist essentially of the various aspects and embodiments.
As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally substituted group means that the group is unsubstituted or substituted.
As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or fully synthetic (such as recombinantly produced), including any fragment thereof that contains at least a portion of the variable heavy and/or light chain regions of an immunoglobulin molecule sufficient to form an antigen binding site and to specifically bind antigen when assembled. Thus, antibodies include any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen binding domain (antibody binding site). Typically, antibodies minimally comprise all or at least a portion of the variable heavy (V H) and/or variable light (V L) chains. Typically, the pairing of V H and V L together forms an antigen binding site, but in some cases a single V H or V L domain is sufficient for antigen binding. Antibodies may also include all or a portion of a constant region. References herein to antibodies include full length antibodies and antigen binding fragments. The term "immunoglobulin" (Ig) is used interchangeably herein with "antibody".
The terms "full length antibody", "whole antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Full length antibodies are antibodies that typically have two full length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH 4) and two full length light chains (VL-CL) and hinge regions, such as antibodies produced from mammalian species (e.g., human, mouse, rat, rabbit, non-human primate, etc.) by B cells that secrete the antibodies and synthetically produced antibodies with identical domains. Specifically, whole antibodies include antibodies having a heavy chain and a light chain comprising an Fc region. The constant domain may be a natural sequence constant domain (e.g., a human natural sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.
An "antibody fragment" includes a portion of an intact antibody, i.e., the antigen-binding and/or variable regions of an intact antibody. Antibody fragments include, but are not limited to, fab fragments, fab ' fragments, F (ab ') 2 fragments, fv fragments, disulfide-linked Fv (dsFv), fd fragments, fd ' fragments; a diabody; linear antibodies (see U.S. Pat. No. 5,641,870, example 2; zapata et al, protein Eng., vol. 8, 10: pages 1057-1062, 1995); single chain antibody molecules, including single chain Fv (scFv) or single chain Fab (scFab); an antigen binding fragment of any of the above and a multispecific antibody from an antibody fragment. For purposes herein, antibody fragments generally include antibody fragments sufficient to bind or crosslink with CD16 on the surface of NK cells.
The term "autologous" refers to cells or tissues derived from or taken from the individual's own tissues. For example, in the autologous transfer or transplantation of NK cells, the donor and recipient are the same person.
The term "allogeneic" refers to cells or tissues that belong to or are obtained from the same species but are genetically different, and thus, in some cases, are immunocompatible. Generally, the term "allogeneic" is used to define cells transplanted from a donor to a recipient of the same species.
The term "enriched" with respect to a cell composition refers to a composition in which the number or percentage of cell types or populations is increased as compared to the number or percentage of cell types in the same volume of starting composition (such as starting composition obtained directly from or isolated from a subject). The term does not require that other cells, cell types or populations be completely removed from the composition nor that such enriched cells be present in the enriched composition at or even near 100%.
The term "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and/or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.
The term "heterologous" with respect to a protein or nucleic acid refers to a protein or nucleic acid that has been transformed or introduced into a cell. In some cases, the heterologous protein or nucleic acid is exogenous to the cell, for example, because it originates from another organism or individual, rather than a cell that expresses it. It will be appreciated that reference to "heterologous" does not exclude that the protein or nucleic acid may also be naturally expressed by the cell into which it is introduced. The heterologous nucleic acid or heterologous encoded protein can be introduced into NK cells, for example, by any of a variety of methods capable of introducing or transforming the nucleic acid (e.g., the nucleic acid encoding the heterologous protein) into the cell, including viral-based methods, such as by transduction or non-viral delivery methods, such as electroporation or lipid nanoparticle delivery. NK cells that have been introduced or transformed can carry exogenous or heterologous nucleic acids extrachromosomal or integrated into the chromosome. Integration into the genome of a cell and into self-replicating vectors generally results in transformed nucleic acid molecules having genetically stable inheritance. NK cells containing transformed nucleic acids are referred to as "genetically engineered" but are also interchangeably referred to as "recombinant" or "transformed".
As used herein, the term "introducing" encompasses a variety of methods of introducing DNA into a cell in vitro or in vivo, such methods including transformation, transduction, transfection (e.g., electroporation), lipid delivery, and infection. Vectors may be used to introduce DNA encoding a molecule into a cell. Possible vectors include plasmid vectors and viral vectors. Viral vectors include retroviral vectors, lentiviral vectors, or other vectors, such as adenoviral vectors or adeno-associated vectors. Lipid nanoparticles can also be used to introduce nucleic acids (DNA or mRNA) into cells.
The terms "polynucleotide", "nucleotide sequence", "nucleic acid molecule", "nucleic acid sequence" and "oligonucleotide" refer to a series of nucleotide bases (also referred to as "nucleotides") in DNA and RNA, and refer to any strand of two or more nucleotides. Polynucleotides, nucleotide sequences, nucleic acids, and the like may be single-stranded or double-stranded chimeric mixtures or derivatives or modified forms thereof. For example, they may be modified at the base moiety, sugar moiety or phosphate backbone to improve the stability of the molecule, its hybridization parameters, etc. Nucleotide sequences typically carry genetic information, including but not limited to information used by cellular machinery to make proteins and enzymes. These terms include double-or single-stranded genomic DNA, RNA, any synthetic and genetically manipulated polynucleotide, and sense and antisense polynucleotides. These terms also include nucleic acids containing modified bases.
The terms "protein," "peptide," and "polypeptide" are used interchangeably and refer to sequential chains of amino acids linked together via peptide bonds. These terms include individual proteins, groups or complexes of proteins that are associated together, as well as fragments or portions, variants, derivatives, and analogs of such proteins. Peptide sequences are presented herein using conventional symbols, starting from the amino or N-terminus on the left and proceeding to the carboxy or C-terminus on the right. Standard single-letter or three-letter abbreviations may be used.
As used herein in the context of a nucleic acid (e.g., gene, genomic region encoding a protein, promoter), the term "endogenous" refers to a native nucleic acid or protein in its natural location (e.g., within the genome of a cell). Conversely, as used herein in the context of nucleic acids (e.g., expression constructs, cdnas, indels, and nucleic acid vectors), the term "exogenous" refers to a nucleic acid that has been artificially introduced into the genome of a cell using, for example, genetic engineering techniques (such as transformation of heterologous nucleic acids or gene editing, e.g., CRISPR-based editing techniques).
The term "composition" refers to any mixture of two or more products, substances or compounds (including cells or antibodies). It may be a solution, suspension, liquid, powder, paste, aqueous solution, non-aqueous solution, or any combination thereof. The formulation is typically in a form that allows the biological activity of the active ingredient (e.g., antibody) to take effect.
By "pharmaceutically acceptable carrier" is meant an ingredient of the pharmaceutical formulation that is non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.
As used herein, a combination refers to any association between or among two or more items. The combination may be two or more separate items (such as two compositions or two collections), may be a mixture thereof (such as a single mixture of two or more items), or any variation thereof. The elements of a combination are typically functionally associated or related.
As used herein, a kit is a packaged combination that optionally includes other elements, such as additional medicaments and instructions for using the combination or elements thereof, for purposes including, but not limited to, therapeutic uses.
As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the individual or cell being treated during a clinical pathology. Desirable therapeutic effects include reducing the rate of disease progression, improving or ameliorating the disease state, and alleviating or improving prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with a disorder (e.g., eosinophil-mediated disease) are reduced or eliminated. For example, an individual is successfully "treated" if the treatment is such that it increases the quality of life of the person suffering from the disease, reduces the dosage of other drugs required to treat the disease, reduces the frequency of disease recurrence, reduces the severity of the disease, delays the progression or progression of the disease, and/or prolongs the survival of the individual.
An "effective amount" refers to an amount effective to achieve a desired or indicated effect (including therapeutic or prophylactic results) at least in the necessary dosages and for periods of time. An effective amount may be provided in one or more administrations. A "therapeutically effective amount" is at least the minimum cell dose required to achieve a measurable improvement in a particular condition. In some embodiments, a therapeutically effective amount is an amount of a composition that reduces the severity, duration, and/or symptoms associated with cancer, viral infection, microbial infection, or septic shock in an animal. The therapeutically effective amount herein may vary depending on factors such as the disease state, age, sex, and weight of the patient. A therapeutically effective amount may also be an amount in which the therapeutically beneficial effect exceeds any toxic or detrimental effect of the antibody. "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the necessary dosage and for the time period. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an early stage of the disease, the prophylactically effective amount may be less than the therapeutically effective amount.
As used herein, an "individual" or "subject" is a mammal. "mammal" for therapeutic purposes includes humans, domestic and farm animals, and zoo, sports or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, and the like. In some embodiments, the individual or subject is a human.
II engineering Fc receptor gamma deficient natural killer cells (g-Nk cells)
Provided herein are engineered Natural Killer (NK) cells (g-NK cells) deficient in fcrγ expression that express a heterologous agent, such as an antigen receptor, e.g., a Chimeric Antigen Receptor (CAR), that has been introduced into the g-NK cells.
Provided herein are engineered Natural Killer (NK) cells (g-NK cells) deficient in fcrγ expression that express heterologous agents, including immunomodulatory agents, such as cytokines, that have been introduced into the g-NK cells.
Provided herein are engineered Natural Killer (NK) cells (g-NK cells) deficient in fcrγ expression that express heterologous agents that have been introduced into the g-NK cells, including antigen receptors such as Chimeric Antigen Receptors (CARs) and immune modulators such as cytokines.
In some embodiments, the engineered NK cell is a g-NK cell deficient in fcrγ expression. In some embodiments, the g-NK cell subpopulation of NK cells can be detected by observing whether the NK cells or NK cell population express fcrγ, wherein the cells in which fcrγ is absent are g-NK cells. Fcrgamma protein is an intracellular protein. Thus, in some aspects, the presence or absence of fcrγ may be detected after treatment of the cells, e.g., by fixation and permeabilization, to allow detection of intracellular proteins.
In some cases, g-NK cells can also be identified by surface markers that are surrogate markers for g-NK cells. As described further below, certain combinations of cell surface markers have also been found to correlate with g-NK cell phenotype (i.e., cells lacking or defective in intracellular fcrγ expression), thereby providing a surrogate marker profile to identify or detect g-NK cells in a manner that does not damage the cells. In some embodiments, the surrogate marker profile of g-NK cells provided herein is based on positive surface expression of one or more markers CD16 (CD 16 Positive and negative )、NKG2C(NKG2C Positive and negative ) or CD57 (CD 57 positive) and/or based on low surface expression or negative surface expression of one or more markers CD7 (CD 7 Weak and weak / Negative of )、CD161(CD161 Negative of ) and/or NKG2A (NKG 2A Negative of ). In some embodiments, the cells are further evaluated for one or more surface markers of NK cells, such as CD45, CD3, and/or CD56. In some embodiments, the surrogate marker profile CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative /CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of can be used to identify, detect, enrich, and/or isolate g-NK cells. In some embodiments, the surrogate marker profile CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative /NKG2A Negative of /CD161 Negative of is used to identify, detect, enrich, and/or isolate g-NK cells. In some embodiments, g-NK cells of NKG2C Positive and negative and/or NKG2a Negative of are identified, detected, enriched, and/or isolated.
In some embodiments, the g-NK cells have the surface phenotype of CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of . In some embodiments, the g-NK cells also have a surface phenotype of NKG2a Negative of /CD161 Negative of . In some embodiments, the g-NK cells also have the surface phenotype of CD38 Negative of . In some embodiments, the g-NK cells have a surface phenotype further of CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative .
In some embodiments, the g-NK cells are engineered to express a CAR. In some embodiments, g-NK cells are engineered to express immunomodulators (e.g., exogenous cytokines). In some embodiments, the g-NK cells are engineered to express the CAR and an immunomodulatory agent (e.g., an exogenous cytokine). In some embodiments, the CAR is a fusion protein that generally includes an extracellular domain (the extracellular domain includes an antigen recognition region), a transmembrane domain, and an internal domain. The extracellular domain (i.e., antigen recognition region or antigen binding domain) and the transmembrane domain may be linked by a flexible linker. The internal domain may comprise an intracellular signaling domain that propagates an external cellular stimulus within the cell. In some embodiments, the CAR comprises 1) an antigen binding domain; 2) A flexible connector; 3) A transmembrane region; and 4) and intracellular signaling domains. In some embodiments, the CAR binds to a target antigen and induces cytotoxicity upon antigen binding. In some embodiments, the immunomodulator is an agent capable of modulating the immune function of NK cells. In some embodiments, the immunomodulator may be an immune activator. In other embodiments, the immunomodulator may be an immunosuppressant. In some embodiments, the immunomodulator is an exogenous cytokine, such as an interleukin or a functional portion thereof. Exemplary features of CARs and immunomodulators are described further in the following subsections.
In some embodiments, g-NK cells can be further engineered by gene editing as described in section III.
A. antigen receptors, e.g. chimeric antigen receptors
In the provided embodiments, g-NK cells are genetically engineered to express antigen receptors that bind to antigens of interest. In certain embodiments, the antigen receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the antigen receptor is a T Cell Receptor (TCR). The antigen receptor may bind to, for example, a tumor-specific or tumor-associated antigen or pathogen antigen. Thus, an engineered antigen receptor (e.g., CAR) is a recombinant antigen receptor that is intended to introduce some antigen specificity into NK cells. In some embodiments, an antigen receptor (such as a CAR) is stably integrated into g-NK cells. In other embodiments, the antigen receptor (e.g., CAR) is transiently expressed by g-NK cells. For example, g-NK cells include CARs with defined polypeptide sequences that are expressed by exogenous polynucleotides that have been introduced into immune effector cells (transiently or integrated into the genome). In provided embodiments, the engineered NK cells provided herein that include an antigen receptor (e.g., CAR) can be used in immunotherapy to target and destroy cells (e.g., cancer cells) associated with a disease or disorder that express a target antigen recognized by the antigen receptor (e.g., CAR).
In some embodiments, the antigen receptor is a Chimeric Antigen Receptor (CAR). CARs are typically encoded by a nucleic acid sequence (polynucleotide) that includes a leader sequence, an extracellular targeting domain (also referred to as an extracellular domain); for example, an antigen binding domain (such as an scFv), a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the CAR is a fusion protein comprising: an extracellular targeting domain (extracellular domain) comprising an antigen recognition or antigen binding domain; a transmembrane domain; and intracellular signaling domains. The extracellular domain and the transmembrane domain may be connected by a flexible linker (also referred to as a spacer). In some embodiments, the antigen binding domain, such as a single chain variable fragment (scFv) derived from a monoclonal antibody, recognizes a target antigen. In some embodiments, an antigen binding domain (e.g., scFv) is linked or fused to a transmembrane domain via a spacer. In some embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). Activation of the CAR fusion protein results in activation of the cell in response to recognition of its target by the scFv (or other antigen binding domain). When a cell expresses such a CAR, it can recognize and kill the target cell expressing the target antigen. This property makes CAR-expressing cells particularly attractive agents for specifically targeting cell activity to abnormal cells, including but not limited to cancer cells. Various CARs have been developed for target antigens, including tumor-associated antigens, for expression in various immune cells, including T lymphocytes and Natural Killer (NK) cells, to mediate cytotoxic activity against target cells expressing the antigen, and the immune cells may be engineered g-NK cells as disclosed herein.
In some embodiments, the leader sequence may be any of the signal peptide sequences described herein. An exemplary CD 8. Alpha. Signal peptide is shown in SEQ ID NO. 12. An exemplary GM-CSFRa signal peptide is shown in SEQ ID NO. 13. An exemplary IgK signal peptide is shown in SEQ ID NO. 14. An exemplary IgK signal peptide is shown in SEQ ID NO. 43.
Any kind of chimeric antigen receptor can be expressed in engineered NK cells, including those described in international PCT applications PCT/US 2018/024550, PCT/IB2019/000141, PCT/IB2019/000181 and/or PCT/US2020/020824, PCT/US2020,035752.
In certain embodiments, the extracellular antigen-binding domain specifically binds to an antigen. In some embodiments, the extracellular antigen-binding domain or targeting domain is derived from an antibody molecule and includes one or more Complementarity Determining Regions (CDRs) from the antibody molecule that confer antigen specificity to the CAR. In certain embodiments, the extracellular antigen-binding domain is a single chain variable fragment (scFv). In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the extracellular antigen-binding domain is a Fab (which is optionally crosslinked). In certain embodiments, the extracellular binding domain is F (ab') 2. In certain embodiments, any of the foregoing molecules may be included in a fusion protein having a heterologous sequence to form an extracellular antigen-binding domain. In certain embodiments, scfvs are identified by screening a scFv phage library with an antigen-Fc fusion protein.
In some embodiments, the scFv comprises a variable chain portion of an immunoglobulin light chain and an immunoglobulin heavy chain molecule separated by a flexible linker polypeptide. The order of the heavy and light chains is not limited and may be reversed. The flexible polypeptide linker allows the heavy and light chains to associate with each other and reconstruct the immunoglobulin antigen binding domain. In some embodiments, the flexible linker is a GS linker, such as shown in SEQ ID NO: 56. In some embodiments, the flexible linker is a Whitlow linker, such as shown in SEQ ID NO. 55. Suitably, the light chain variable region comprises three CDRs and the heavy chain variable region comprises three CDRs. Suitably, the CDRs for the antigen binding targeting domain are derived from an antibody molecule of any species (e.g., human, mouse, rat, rabbit, goat, sheep) and the framework regions between the CDRs are humanized or comprise sequences at least 85%, 90%, 95% or 99% identical to the human framework regions.
When the targeting domain of the CAR comprises an scFv, the immunoglobulin light chain and the immunoglobulin heavy chain are linked by polypeptide linkers of various lengths. Suitably, the polypeptide linker comprises a length of greater than or equal to 10 amino acids. Suitably, the polypeptide linker comprises a length of more than 10, 15, 20 or 25 amino acids. Suitably, the polypeptide linker comprises a length of less than or equal to 30 amino acids. Suitably, the polypeptide linker comprises a length of less than 15, 20, 25 or 30 amino acids. Suitably, the polypeptide linker comprises between 10 and 30 amino acids in length. Suitably, the polypeptide linker comprises between 10 and 25 amino acids in length. Suitably, the polypeptide linker comprises between 10 and 20 amino acids in length. Suitably, the polypeptide linker comprises between 10 and 15 amino acids in length. Suitably, the polypeptide linker comprises between 15 and 30 amino acids in length. Suitably, the polypeptide linker comprises between 20 and 30 amino acids in length. Suitably, the polypeptide linker comprises between 25 and 30 amino acids in length. Suitably, the polypeptide linker comprises a hydrophilic amino acid. Suitably, the polypeptide linker consists of hydrophilic amino acids. Suitably, the polypeptide linker comprises a G 4 S sequence (GGGGS). The G 4 S linker allows the linker to be flexible and protease resistant. Suitably, the G 4 S linker is repeated 1, 2, 3, 4, 5, 6, 7 or 8 times in succession in the polypeptide linker.
In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the antigen is a pathogen antigen, including, for example, a viral antigen or a bacterial antigen.
Binding of the extracellular antigen binding domain (e.g., scFv or analog thereof) of an antigen-targeted CAR can be confirmed by, for example, an enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or western blot assay. Each of these assays typically detects the presence of a protein-antibody complex of particular interest by using a labeling reagent (e.g., an antibody or scFv) that is specific for the complex of interest. For example, scFv may be radiolabeled and used in a Radioimmunoassay (RIA) (see, e.g., month 3 of Weintraub,B.,Principles of Radioimmunoassays,Seventh Training Course on Radioligand Assay Techniques,The Endocrine Society,1986, incorporated herein by reference). The radioisotope may be detected by means such as using a gamma counter or scintillation counter or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent label. Non-limiting examples of fluorescent markers include Green Fluorescent Protein (GFP), blue fluorescent protein (e.g., EBFP2, azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, cerulean and CyPet), and yellow fluorescent protein (e.g., YFP, citrine, venus and YPet).
In certain embodiments, the antigen recognizing receptor binds to a tumor-associated antigen or a tumor-specific antigen. Any suitable tumor-associated antigen or tumor-specific antigen (e.g., antigenic peptide) can be used in the embodiments described herein. The antigen may be, but is not limited to, a protein, a non-protein, a neoantigen, a post-translationally modified antigen, a peptide-MHC antigen, and/or an over-expressed antigen.
For example, tumor targets include, but are not limited to, CD38 (multiple myeloma); CD20 (lymphoma); epidermal growth factor receptor (EGFR; non-small cell lung cancer, epithelial cancer and glioma); type III variants of epidermal growth factor receptor (EGFRvIII; glioblastoma); human epidermal growth factor receptor 2 (HER 2; ovarian cancer, breast cancer, glioblastoma, colon cancer, osteosarcoma, and medulloblastoma); mesothelin (mesothelioma, ovarian cancer and pancreatic cancer); prostate specific membrane antigen (PSMA; prostate cancer); carcinoembryonic antigen (CEA; pancreatic, breast and colorectal cancers); disialoganglioside 2 (GD 2; neuroblastoma and melanoma); interleukin-13 Ra2 (glioma); glypican-3 (hepatocellular carcinoma); carbonic anhydrase IX (CAIX; renal cell carcinoma); l1 cell adhesion molecules (L1-CAM; neuroblastoma, melanoma and ovarian cancer); cancer antigen 125 (CA 125; epithelial ovarian cancer); CD133 (glioblastoma and cholangiocarcinoma); fibroblast activation protein (FAP; malignant pleural mesothelioma); cancer/testis antigen 1B (CTAG 1B; melanoma and ovarian cancer); mucin 1 (seminal vesicle cancer); and folate receptor-a (FR-a; ovarian cancer).
Other non-limiting examples of tumor antigens include, but are not limited to, carbonic Anhydrase IX (CAIX), carcinoembryonic antigen (CEA)、CD8、CD7、CD10、CD19、CD20、CD22、CD30、CD33、CLL1、CD34、CD38、CD41、CD44、CD49c、CD49f、CD56、CD66c、CD73、CD74、CD104、CD133、CD138、CD123、CD142、CD44V6、 Cytomegalovirus (CMV) infected cell antigen (e.g., cell surface antigen), skin lymphocyte-associated antigen (CLA; P-selectin glycoprotein ligand-1 (PSGL-1) one of the specified sugar type), epithelial glycoprotein-2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine protein kinase erb-B2,3,4 (erb-B2, 3, 4), folic acid binding protein (EBP), fetal acetylcholine receptor (AChR), folic acid receptor-alpha, ganglioside G2 (GD 2), ganglioside G3 (GD 3), human epidermal growth factor receptor 2 (HER 2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13 Ralpha 2), kappa-light chain, kinase insert domain receptor (KDR), lewis Y (LeY), LI cell adhesion molecule (L1 CAM), melanoma antigen family A,1 (MAGE-A1), mucin 16 (MUC 16), mucin 1 (MUC 1), mesothelin (MSLN), ERBB2, MAGE A3, P53, MARGI, GP100, protease 3 (PR 1), tyrosinase, survivin, hTERT, ephA2, NKG2D ligand, cancer-testis antigen NY-ESO-1, cancer embryo antigen (h 5T 4), cancer-testis antigen, prostate Stem Cell Antigen (PSCA), prostate Specific Membrane Antigen (PSMA), ROR1, four transmembrane protein 8 (TSPAN 8), tumor associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), wilms tumor protein (WT-1), cytokine receptor like factor 2 (CRLF 2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII and ERBB.
In some embodiments, the tumor antigen is CD19, ROR1, her2, PSMA, PSCA, mesothelin (MSLN), or CD20. In some embodiments, the tumor antigen is CD19, CD20, CD33, MSLN, or cytokine receptor like factor 2 (CRLF 2), which are expressed on leukemia or lymphoma. In some embodiments, the CAR binds to a target antigen selected from her2, EGFR, alpha folate receptor, CEA, cMET, MUC2, mesothelin, or ROR 1. In a certain embodiment, the target antigen is CD38, CD319/SLAMF-7, TNFRSF 17/BCMA, SYND1/CD138, CD229, CD47, her2/Neu, epidermal Growth Factor Receptor (EGFR), CD123/IL3-RA, CD19, CD20, CD22, mesothelin, epCAM, MUC1, MUC 16, tn antigen, NEU5GC, neuGcGM3, GD2, CLL-1 or HERV-K. In some embodiments, the target antigen is a blood cancer-associated antigen. For example, the target antigen may be CD38, CD319/SLAMF-7, TNFRSF 17/BCMA, SYND1/CD138, CD229, CD47, CD123/IL3-RA, CD19, CD20, CD22 or CLL-1.
A variety of antigen binding domains are known for binding into CARs. In one non-limiting example, g-NK cells are engineered with CD38 specific CARs (see, e.g., WO 2018/104562).
In some embodiments, g-NK cells are engineered with a bispecific CAR or multiple different CARs, wherein the affinity of the CAR is for two different ligands/antigens. Bispecific CAR-NK can be used to increase the number of potential binding sites on cancer cells, or alternatively to localize cancer cells to other immune effector cells that express ligands specific for NK-CARs. For use in cancer therapy, the bispecific CAR can bind to target tumor cells and effector cells, such as T cells, NK cells, or macrophages. Thus, for example, in the case of multiple myeloma, the bispecific CAR can bind to a T cell antigen (e.g., CD3, etc.) and a tumor cell marker (e.g., CD38, etc.). The bispecific CAR can alternatively bind to two different tumor cell markers, thereby increasing the overall binding affinity of NK cells to the target tumor cells. This can reduce the risk of developing resistance in cancer cells by down-regulating one of the target antigens. In this case, in multiple myeloma, one example is that the CAR binds to both CD38 and CS-1/SLAMF7 simultaneously. Another tumor cell marker that CARs are suitable to target is a "do-it-yourself" marker on the tumor, such as CD47.
In some embodiments, the engineered g-NK cells can include a bispecific CAR or multiple CARs expressed by the same NK cell. This allows NK cells to target two different antigens simultaneously. Suitably, the bispecific CAR is specific for any two of the following antigens: CD38, CD319/SLAMF-7, TNFRSF 17/BCMA, CD123/IL3-RA, SYND1/CD138, CD229, CD47, her2/Neu, epidermal Growth Factor Receptor (EGFR), CD19, CD20, CD22, mesothelin, epCAM, MUC1, MUC16, tn antigen, NEU5GC, neuGcGM3, GD2, CLL-1, CD123, HERV-K. Suitably, the bispecific nature of the CAR NK cells may allow binding to a tumor antigen and another immune cell, such as a T cell or dendritic cell. Suitably, the bispecific nature of the CAR NK cells may allow binding to a checkpoint inhibitor, such as PDL-1 or CD47. Suitably, the first CAR has CD38 specificity and the second CAR has specificity for any of SLAMF-7, BCMA, CD138, CD229, PDL-1 or CD47. Suitably, the first CAR is specific for CD38 and the second CAR is specific for SLAMF-7, BCMA, CD138, CD 229. Suitably, the first CAR is specific for CD38 and the second CAR is specific for SLAMF-7. Suitably, the first CAR is specific for CD38 and the second CAR is specific for BCMA. Suitably, the first CAR is specific for CD38 and the second CAR is specific for CD 138. Suitably, the first CAR is specific for CD38 and the second CAR is specific for CD 229.
In some embodiments, the transmembrane domain of the CAR comprises a hydrophobic amino acid residue and allows the CAR to anchor into the cell membrane of an engineered NK cell. Suitably, the transmembrane domain comprises an amino acid sequence derived from a transmembrane protein. Suitably, the transmembrane domain comprises the amino acid sequence of the transmembrane domain derived from the alpha, beta or zeta chain, CD27, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137 and CD 154 of the T cell receptor. Suitably, the CAR comprises a transmembrane domain having an amino acid sequence derived from the transmembrane domain of CD 8. Suitably, the CAR comprises a transmembrane domain having an amino acid sequence derived from a transmembrane domain of human CD8 a. In some embodiments, the CAR contains a transmembrane domain of CD8 a having the amino acid sequence shown in SEQ ID No. 61 or an amino acid sequence that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID No. 61. In some embodiments, the transmembrane domain is shown in SEQ ID NO. 61. In some embodiments, suitably, the CAR comprises a transmembrane domain having an amino acid sequence derived from a transmembrane domain of CD 28. Suitably, the CAR comprises a transmembrane domain having an amino acid sequence derived from the transmembrane domain of human CD 28. In some embodiments, the CAR contains a transmembrane domain of CD28 having the amino acid sequence set forth in SEQ ID No. 39 or an amino acid sequence that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID No. 39. In some embodiments, the transmembrane domain is shown in SEQ ID NO 39.
In some embodiments, the CAR may further comprise a spacer region between the antigen binding targeting domain and the transmembrane domain. In some embodiments, the spacer region comprises hydrophilic amino acids and allows flexibility of the targeting domain relative to the cell surface. Suitably, the spacer comprises more than 5, 10, 15, 20, 25 or 30 amino acids. Suitably, the spacer comprises less than 10, 15, 20, 25, 30 or 35 amino acids. In some embodiments, the spacer is a hinge region and includes the hinge sequence of a CD8 or immunoglobulin molecule.
In some embodiments, the spacer is or includes a CD8 hinge. In some embodiments, the spacer is the hinge region of human CD 8. In some embodiments, the CAR contains a CD8 hinge spacer sequence having the amino acid sequence set forth in SEQ ID No. 60 or an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID No. 60. In some embodiments, the sequence of the spacer is shown in SEQ ID NO. 60.
In some embodiments, the spacer comprises all or a portion of a hinge domain comprising IgG1 Fc or IgG4 Fc. In some embodiments, the spacer is an IgG4 Fc spacer. In some embodiments, the CAR contains an IgG4 Fc spacer having the amino acid sequence set forth in SEQ ID No. 38 or an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID No. 38. In some embodiments, the sequence of the spacer is shown in SEQ ID NO. 38. In some embodiments, the sequence of the spacer is the hinge portion of IgG1 Fc or IgG4 Fc. In some embodiments, the CAR contains an IgG4 hinge spacer. In some embodiments, the IgG4 hinge spacer has the amino acid sequence shown in SEQ ID NO:59 or an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO: 59. In some embodiments, the sequence of the spacer is shown in SEQ ID NO. 59.
In some embodiments, the intracellular signaling domain of the CAR increases the potency of the CAR and includes an intracellular signaling domain derived from a protein involved in immune cell signaling. Suitably, the one or more intracellular signaling domains comprise an intracellular signaling domain derived from cd3ζcd28, OX-40, 4-1BB, DAP10, DAP 12, 2B4 (CD 244), or any combination thereof. Suitably, the one or more intracellular signaling domains comprise an intracellular signaling domain derived from any two of cd3ζcd28, OX-40, 4-lBB, DAP10, DAP 12, 2B4 (CD 244), or any combination thereof.
In some embodiments, the inner domain of the CAR can include two additional signaling domains. For example, the CAR may include a primary intracellular signaling domain (such as a cd3ζ intracellular signaling domain), as well as an intracellular signaling domain from a co-stimulatory molecule, to provide additional signals to the cell, such as to further enhance the efficacy of the CAR-expressing immune cell. Thus, in some embodiments, a Chimeric Antigen Receptor (CAR) comprises: 1) An antigen binding domain; 2) A flexible connector; 3) A transmembrane region; and 4) an intracellular signaling region comprising a first primary intracellular signaling domain (such as a cd3ζ intracellular signaling domain) and a second co-stimulatory intracellular signaling domain. In some embodiments, the costimulatory domain may be a CD27, CD28, 4-1BB (CD 137), 0X40 (CD 134), CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and/or B7-H3 costimulatory domain. In some embodiments, the costimulatory domain can be CD27, CD28, 4-1BB (CD 137), 0X40 (CD 134), DAP10, DAP12, ICOS, and/or 2B4. In some embodiments, the costimulatory domain may be a CD27, CD28, 4-1BB, 2B4, DAP10, DAP12, 0X40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and/or B7-H3 costimulatory domain. In some embodiments, the costimulatory signaling domain is the signaling domain of CD 28. In some embodiments, the costimulatory signaling domain is a signaling domain of 4-1 BB.
In some embodiments, the CAR contains an intracellular signaling domain comprising a signaling domain of CD3 zeta having the amino acid sequence shown in SEQ ID No. 41 or an amino acid sequence that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID No. 41. In some embodiments, the CAR contains an intracellular signaling domain that contains a signaling domain of CD3 zeta having the amino acid sequence set forth in SEQ ID No. 41. In some embodiments, the CAR contains an intracellular signaling domain comprising a signaling domain of CD3 zeta having the amino acid sequence shown in SEQ ID No. 50 or an amino acid sequence exhibiting at least 85%, 90% or 95% sequence identity to SEQ ID No. 50. In some embodiments, the CAR contains an intracellular signaling domain that contains a signaling domain of CD3 zeta having the amino acid sequence set forth in SEQ ID No. 50.
In some embodiments, the CAR contains an intracellular signaling domain comprising a costimulatory signaling domain of CD28 having the amino acid sequence shown in SEQ ID No. 40 or an amino acid sequence that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID No. 40. In some embodiments, the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of CD28 having the amino acid sequence shown in SEQ ID No. 40. In some embodiments, the CAR contains an intracellular signaling domain comprising a costimulatory signaling domain of CD28 having the amino acid sequence shown in SEQ ID No. 52 or an amino acid sequence that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID No. 52. In some embodiments, the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of CD28 that has the amino acid sequence shown in SEQ ID No. 52.
In some embodiments, the CAR contains an intracellular signaling domain comprising a costimulatory signaling domain of 4-1BB having the amino acid sequence shown in SEQ ID No. 51 or an amino acid sequence that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID No. 51. In some embodiments, the CAR contains an intracellular signaling domain comprising a costimulatory signaling domain of 4-1BB having the amino acid sequence shown in SEQ ID NO. 51.
In some embodiments, the intracellular signaling domain may be a domain of CD3 zeta, CD28, and/or 4-1 BB.
Suitably, the CAR comprises at least two intracellular signaling domains derived from cd3ζ and 4-lBB. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequences shown in SEQ ID NO. 41 and SEQ ID NO. 51. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequences shown in SEQ ID NO. 50 and SEQ ID NO. 51.
In other embodiments, suitably, the CAR comprises at least two intracellular signaling domains derived from cd3ζ and CD 28. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequences shown in SEQ ID NO. 41 and SEQ ID NO. 40. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequences shown in SEQ ID NO. 41 and SEQ ID NO. 52. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequences shown in SEQ ID NO. 50 and SEQ ID NO. 40. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequences shown in SEQ ID NO. 50 and SEQ ID NO. 52.
In some embodiments, the antigen receptor (e.g., CAR) is encoded by a polynucleotide encoding a CAR having an NH 2 terminal leader sequence. The leader sequence (also known as a signal peptide) allows the expressed CAR construct to enter the Endoplasmic Reticulum (ER) and target the cell surface. The leader sequence is cleaved in the ER and the mature cell surface CAR does not have a leader sequence. Typically, the leader sequence will be in the range of 5 to 30 amino acids in length and include a stretch of hydrophobic amino acids. Suitably, the leader sequence comprises more than 5,10,15,20 or 25 amino acids in length. Suitably, the leader sequence comprises less than 10,15,20,25 or 30 amino acids in length. Suitably, the leader sequence comprises a sequence derived from any secreted protein. Suitably, the leader sequence comprises a sequence derived from a CD 8a leader sequence. In some embodiments, suitably, the leader sequence comprises a sequence derived from an IgK leader sequence. In some embodiments, the leader sequence is shown in SEQ ID NO. 43.
In some embodiments, the CAR is a CAR present in any of a variety of known engineered cell products. CARs may include, but are not limited to CARs engineered into cells:、JCARH125、CARVYKTITM(NJ-68284528;Janssen/Legend)、P-BCMA-101(Poseida)、PBCAR269A(Poseida)、P-BCMa-allo1(Poseida)、Allo-715(Pfizer/Allogene)、CT053(Carsgen)、Descartes-08(Cartesian)、PHE885(Novartis)、CTX120(CRISPR Therapeutics);/>、、/> Or/> 。
In some embodiments, the CAR comprises a CAR of a commercial CAR cell therapy. Non-limiting examples of CARs in commercial cell-based therapies include CARs engineered in cells: brexucabtagene autoleucel%)、axicabtagene ciloleucel(/>)、idecabtagene vicleucel(/>)、ciltacabtagene autoleucel(CARVYKTITM)、lisocabtagene maraleucel(/>)、tisagenlecleucel(/>)。
In some embodiments, g-NK cells are engineered with a CAR that binds to CD 19. Cluster of differentiation 19 (CD 19) is an epitope that can be detected on leukemia precursor cells. Human and murine amino acid and nucleic acid sequences can be found in public databases such as GenBank, uniProt and Swiss-prot. For example, the amino acid sequence of human CD19 can be found under UniProt/Swiss-Prot accession number P15391 and the nucleotide sequence encoding human CD19 can be found under accession number NM_ 001178098. CD19 is expressed in most B-lineage cancers, including, for example, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, and non-hodgkin's lymphoma. It is also an early marker for B cell progenitors. See, for example, nicholson et al mol.immun., volume 34, stages 16-17: pages 1157-1165, 1997. The antigen binding extracellular domain in the CAR polypeptides disclosed herein is specific for CD19 (e.g., human CD 19). In some examples, the antigen binding extracellular domain may comprise an scFv extracellular domain capable of binding to CD 19. In some embodiments, an anti-CD 19 CAR may include an anti-CD 19 single chain variable fragment (scFv) specific for CD19, followed by a spacer and transmembrane domain fused to an intracellular co-signaling domain (e.g., CD28 or 4-1 BB) and a CD3 zeta signaling domain.
In some embodiments, the extracellular binding domain of a CD19 CAR can include a heavy chain variable region (V H) set forth in SEQ ID NO:54 and a light chain variable region (V L) set forth in SEQ ID NO: 53. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as shown in SEQ ID NO: 56. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker shown in SEQ ID NO: 55. In some embodiments, the scFv has the amino acid sequence shown in SEQ ID NO. 57. In some embodiments, the scFv has the amino acid sequence shown in SEQ ID NO. 58. In some embodiments, the spacer is a CD8 hinge, such as shown in SEQ ID NO: 60. In some embodiments, the spacer is an IgG4 hinge, such as shown in SEQ ID NO: 59. In some embodiments, the intracellular signaling domain contains a 4-1BB costimulatory signaling domain and a CD3 zeta signaling domain, such as any of those described herein. In some embodiments, the intracellular signaling domain comprises a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any of those described herein. In some embodiments, it is understood that the CAR comprises any of the following sequences: it exhibits some sequence variation to any of the above or described SEQ ID NOs, such as at least 85%, 90%, 95% or more sequence identity thereto, and retains binding to CD19 as well as intracellular signaling and cytotoxic activity.
In some embodiments, the CAR comprises an anti-CD 19 CAR of a commercial CAR cell therapy. Non-limiting examples of anti-CD 19 CARs in commercial cell-based therapies include anti-CD 19 CARs engineered in cells:、/>、/> Or/> 。
CD20 has been demonstrated to be a therapeutic target for hematological malignancies (such as B-NHL) and is supported by approved and widely used monoclonal antibody therapies. Furthermore, the ubiquity of CD19, CD20 and CD22 antigens on malignant B cells makes them perfect targets for cell therapies. In some embodiments, the CAR contains an extracellular antigen-binding domain that binds to CD 20. In particular embodiments, the CD20 CAR comprises a CAR directed against CD20, wherein the CAR directed against CD20 comprises a single chain Fv antibody or antibody fragment (scFv). In some embodiments, an anti-CD 20 CAR may include an anti-CD 20 single chain variable fragment (scFv) specific for CD20, followed by a spacer and transmembrane domain fused to an intracellular co-signaling domain (e.g., CD28 or 4-1 BB) and a CD3 zeta signaling domain. In some embodiments, the CAR contains an anti-CD 20 scFv followed by an IgG4-Fc spacer, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR is a Leu16 CAR, such as Rufener et al, cancer immunol.res., volume 4, 2016: pages 509-519. See also GenBank accession # KX055828.
In some embodiments, the extracellular binding domain of a CD20 CAR can include a heavy chain variable region (V H) set forth in SEQ ID NO:36 and a light chain variable region (V L) set forth in SEQ ID NO: 35. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as shown in SEQ ID NO: 56. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker shown in SEQ ID NO: 55. In some embodiments, the anti-CD 20 scFv is shown in SEQ ID NO. 37. In some embodiments, the intracellular signaling domain contains a 4-1BB costimulatory signaling domain and a CD3 zeta signaling domain, such as any of those described herein. In some embodiments, the intracellular signaling domain comprises a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any of those described herein. In some embodiments, it is understood that the CAR comprises any of the following sequences: it exhibits some sequence variation to any of the above or described SEQ ID NOs, such as at least 85%, 90%, 95% or more sequence identity thereto, and retains binding to CD38 as well as intracellular signaling and cytotoxic activity. In some embodiments, the anti-CD 20 CAR comprises the scFv shown in SEQ ID NO:37 and an IgG4 Fc spacer (e.g., SEQ ID NO: 38), a CD28 transmembrane domain (e.g., SEQ ID NO: 39), a CD28 costimulatory signaling domain (e.g., SEQ ID NO: 40), and a CD3 zeta signaling domain (e.g., SEQ ID NO: 41). In some embodiments, the CD20 CAR has the amino acid sequence shown in SEQ ID NO. 42 or a sequence exhibiting at least 85%, at least 90% or at least 95% sequence identity with SEQ ID NO. 42. In some embodiments, the CD20 CAR has the sequence set forth in SEQ ID NO. 42. In some embodiments, the CAR is encoded by the polynucleotide shown in SEQ ID NO. 45 (e.g., mRNA).
In some embodiments, g-NK cells are engineered with a CAR that binds to BCMA. BCMA RNA has been commonly detected in multiple myeloma cells and other lymphomas, and several researchers have detected BCMA protein on the surface of plasma cells from multiple myeloma patients (see, e.g., novak et al, blood, volume 103, phase 2: pages 689-694, 2004; neri et al, CLINICAL CANCER RESEARCH, volume 73, phase 19: pages 5903-5909, 2007; bellucci et al, blood, volume 105, phase 10: pages 3945-3950, 2005; and Moreaux et al, blood, volume 703, phase 8: pages 3148-3157, 2004). CARs for targeting BCMA are known and include, but are not limited to, those described in U.S. patent No. 10,934,363 or WO 2018/028647. In some embodiments, the CAR contains an extracellular antigen-binding domain that binds to BCMA. In particular embodiments, BCMACAR comprises a BCMA-directed CAR, wherein the BCMA-directed CAR comprises a single chain Fv antibody or antibody fragment (scFv). In some embodiments, the anti-BCMACAR can include an anti-BCMA single chain variable fragment (scFv) specific for BCMA, followed by a spacer and transmembrane domain fused to an intracellular co-signaling domain (e.g., CD28 or 4-1 BB) and a CD3 zeta signaling domain.
In some embodiments, the extracellular binding domain of BCMACAR comprises an scFv derived from c11d5.3 (a murine monoclonal antibody), such as Carpenter et al, clin.cancer res., volume 19, phase 8: pages 2048-2060, 2013. See also PCT application publication No. WO2010/104949. scFv derived from c11d5.3 may include a heavy chain variable region (V H) and a light chain variable region (V L) of c11d5.3. In some embodiments, the VH has the amino acid sequence set forth in SEQ ID NO. 63 and the VL has the amino acid sequence set forth in SEQ ID NO. 62. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as shown in SEQ ID NO: 56. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker shown in SEQ ID NO: 55. In some embodiments, the scFv has the amino acid sequence shown in SEQ ID NO. 64. In some embodiments, the intracellular signaling domain contains a 4-1BB costimulatory signaling domain and a CD3 zeta signaling domain, such as any of those described herein. In some embodiments, the intracellular signaling domain comprises a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any of those described herein. In some embodiments, it is understood that the CAR comprises any of the following sequences: it exhibits some sequence variation to any of the above or described SEQ ID NOs, such as at least 85%, 90%, 95% or more sequence identity thereto, and retains binding to BCMA and intracellular signaling and cytotoxic activity.
In some embodiments, the extracellular binding domain of BCMACAR comprises an scFv derived from another murine monoclonal antibody c12a3.2, such as Carpenter et al, clin.cancer res., volume 19, phase 8: pages 2048-2060, 2013 and PCT application publication No. WO 2010/104949. In some embodiments, the VH has the amino acid sequence set forth in SEQ ID NO. 66 and the VL has the amino acid sequence set forth in SEQ ID NO. 65. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as shown in SEQ ID NO: 56. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker shown in SEQ ID NO: 55. In some embodiments, the scFv has the amino acid sequence shown in SEQ ID NO. 67. In some embodiments, the intracellular signaling domain contains a 4-1BB costimulatory signaling domain and a CD3 zeta signaling domain, such as any of those described herein. In some embodiments, the intracellular signaling domain comprises a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any of those described herein. In some embodiments, it is understood that the CAR comprises any of the following sequences: it exhibits some sequence variation to any of the above or described SEQ ID NOs, such as at least 85%, 90%, 95% or more sequence identity thereto, and retains binding to BCMA and intracellular signaling and cytotoxic activity.
In some embodiments, the extracellular binding domain of BCMACAR comprises a murine monoclonal antibody with high specificity for human BCMA, which is described in Friedman et al, hum.gene ter., volume 29, phase 5: pages 585-601, 2018, are called BB2121. See also PCT application publication No. WO2012163805.BB2121 is also known as an anti-BCMA 02 CAR. In some embodiments, the VH has the amino acid sequence set forth in SEQ ID NO. 68 and the VL has the amino acid sequence set forth in SEQ ID NO. 69. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as shown in SEQ ID NO: 56. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker shown in SEQ ID NO: 55. In some embodiments, the scFv has the amino acid sequence shown in SEQ ID NO. 70. In some embodiments, the intracellular signaling domain contains a 4-1BB costimulatory signaling domain and a CD3 zeta signaling domain, such as any of those described herein. In some embodiments, the intracellular signaling domain comprises a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any of those described herein. In some embodiments, it is understood that the CAR comprises any of the following sequences: it exhibits some sequence variation to any of the above or described SEQ ID NOs, such as at least 85%, 90%, 95% or more sequence identity thereto, and retains binding to BCMA and intracellular signaling and cytotoxic activity.
In some embodiments, the extracellular binding domain of BCMACAR comprises a single variable fragment of two heavy chains (VHH) that can bind to two epitopes of BCMA, such as Zhao et al, j.Hematol. Oncol., volume 11, phase 1: page 141, 2018, and is also referred to as LCAR-B38M. See also PCT application publication No. WO2018/028647. In some embodiments, the intracellular signaling domain contains a 4-1BB costimulatory signaling domain and a CD3 zeta signaling domain, such as any of those described herein. In some embodiments, the intracellular signaling domain comprises a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any of those described herein. In some embodiments, it is understood that the CAR comprises any of the following sequences: it exhibits some sequence variation to any of the above or described SEQ ID NOs, such as at least 85%, 90%, 95% or more sequence identity thereto, and retains binding to BCMA and intracellular signaling and cytotoxic activity.
In some embodiments, the extracellular binding domain of BCMACAR comprises a fully human heavy chain variable domain (FHVH), such as Lam et al, nat.Commun., volume 11, phase 1: on page 283, 2020, also known as FHVH. In some embodiments, the intracellular signaling domain contains a 4-1BB costimulatory signaling domain and a CD3 zeta signaling domain, such as any of those described herein. In some embodiments, the intracellular signaling domain comprises a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any of those described herein. In some embodiments, it is understood that the CAR comprises any of the following sequences: it exhibits some sequence variation to any of the above or described SEQ ID NOs, such as at least 85%, 90%, 95% or more sequence identity thereto, and retains binding to BCMA and intracellular signaling and cytotoxic activity.
In some embodiments, the CAR comprises an anti BCMACAR of a commercial CAR cell therapy. Non-limiting examples of antigens BCMACAR in commercial cell-based therapies include an engineered antigen BCMACAR in a cell: idecabtagene vicleucel%) Or ciltacabtagene autoleucel (CARVYKTI TM).
CD38 (cluster of differentiation 38), also known as cyclic ADP-ribose hydrolase, is a glycoprotein found on the surface of many immune cells (leukocytes), particularly T cells, including cd4+, cd8+, B lymphocytes and natural killer cells. CD38 also plays a role in cell adhesion, signaling and calcium signaling. Structural information about this protein can be found in the UniProtKB/Swiss-Prot database under reference P28907. In humans, the CD38 protein is encoded by the CD38 gene located on chromosome 4. CD38 is a multifunctional extracellular enzyme that catalyzes the synthesis and hydrolysis of cyclic ADP-ribose (cADPR) from NAD+ to ADP-ribose. These reaction products are thought to be necessary for intracellular ca2+ regulation. Moreover, loss of CD38 function is associated with impaired immune response and metabolic disorders (MALAVASI F et al, 2008, ,Evolution and function of the ADP ribosyl cyclase/CD38 gene family in physiology and pathology,Physiol.Rev.,, volume 88, 3: pages 841-886). CD38 protein is a marker for HIV infection, leukemia, myeloma, solid tumor, type II diabetes and bone metabolism. CD38 expression is an important prognostic factor Blood for B-cell chronic lymphocytic leukemia, volume 98: pages 181-186). In some embodiments, an anti-CD 38 CAR may include an anti-CD 38 single chain variable fragment (scFv) specific for CD38, followed by a spacer and transmembrane domain fused to an intracellular co-signaling domain (e.g., CD28 or 4-1 BB) and a CD3 zeta signaling domain.
In some embodiments, the extracellular binding domain of CD38 CAR can include a heavy chain variable region (V H) as set forth in SEQ ID NO. 46 or SEQ ID NO. 47 and a light chain variable region (V L) as set forth in SEQ ID NO. 48 or SEQ ID NO. 49. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as shown in SEQ ID NO: 56. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker shown in SEQ ID NO: 55. In some embodiments, the intracellular signaling domain contains a 4-1BB costimulatory signaling domain and a CD3 zeta signaling domain, such as any of those described herein. In some embodiments, the intracellular signaling domain comprises a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any of those described herein. In some embodiments, it is understood that the CAR comprises any of the following sequences: it exhibits some sequence variation to any of the above or described SEQ ID NOs, such as at least 85%, 90%, 95% or more sequence identity thereto, and retains binding to CD38 as well as intracellular signaling and cytotoxic activity.
B. Immunomodulators (e.g. cytokines)
In provided embodiments, an engineered g-NK cell or g-NK cells are engineered to express a heterologous immunomodulatory agent, such as an exogenous cytokine, e.g., an interleukin. In some embodiments, a heterologous nucleic acid encoding an immunomodulatory agent is stably integrated into the genome of a g-NK cell. In other embodiments, the heterologous nucleic acid encoding the immunomodulator is transiently expressed. In some embodiments, the immunomodulator is an immunosuppressant. In other embodiments, the immunomodulator is an immune activator. In some embodiments, the immune activator is a cytokine.
In provided embodiments, the engineered NK cells express a heterologous cytokine or functional portion thereof. According to the embodiments provided, in some embodiments, the NK cells are engineered to express a secreted form of the cytokine, while in some embodiments, the cytokine is membrane-bound. In some embodiments, the heterologous cytokine or functional portion thereof may be secreted from the cell. In some embodiments, the heterologous cytokine or functional portion thereof is expressed as a membrane-bound protein on the cell surface.
Cytokines are a large class of proteins that play an important role in cell signaling, particularly in the immune system environment. Cytokines have been shown to play a role as immunomodulators in autocrine, paracrine and endocrine signaling. Cytokines may act as immune activators, thereby stimulating an immune-mediated response, or as immunosuppressants, thereby attenuating an immune-mediated response. Cytokines include chemokines, interferons, interleukins, lymphokines and tumor necrosis factors, but generally do not include hormones or growth factors.
In some embodiments, the cytokine is an interleukin. Interleukins are a group of cytokines, typically secreted proteins and signaling molecules that mediate a broad immune response. For example, interleukin (IL) -2 plays a role in regulating the activity of leukocytes, whereas Interleukin (IL) -15 plays a major role in the development of inflammatory and protective immune responses to microbial invaders and parasites by regulating the cellular activities of the innate and adaptive immune systems. In some embodiments, NK cells (including the provided g-NK cells) activity or activities by IL-2, IL-21 and/or IL-15 or another cytokine described.
Because cytokines are required for NK cell activity, typical methods involve administering exogenous cytokines as exogenous cytokine supports to a subject in combination with NK cell therapy. However, in some aspects, the administration of exogenous cytokines may lead to the risk of systemic toxicity, which may occur particularly when certain cytokines are administered at high doses. In the embodiments provided, engineering NK cells with a secretable cytokine or membrane-bound cytokine provides a local source of the cytokine for NK cells while avoiding or reducing the risk of systemic toxicity.
In some embodiments of the engineered cells provided, the interleukin or functional portion thereof is introduced into g-NK cells or g-NK cell populations. In some embodiments, the interleukins include cytokines produced by immune cells such as lymphocytes, monocytes or macrophages. In some embodiments, the cytokine is an immune activating cytokine (also referred to as an immune activator) that can be used to induce NK cells, such as to promote NK cell survival, activation, and/or proliferation. For example, certain cytokines (such as IL-15 or IL-21) can prevent or reduce NK cells from undergoing senescence, such as by increasing their ability to expand in vitro or in vivo. In some embodiments, the interleukin or functional portion thereof is a partial peptide or an intact peptide of one or more of IL-2, IL-4, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-15, IL-18, or IL-21. In some embodiments, the cytokine is IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, flt3-L, SCF or IL-7. In some embodiments, the cytokine is IL-2 or a functional part thereof. In some embodiments, the cytokine is IL-12 or a functional part thereof. In some embodiments, the cytokine is IL-15 or a functional part thereof. In some embodiments, the cytokine is IL-21 or a functional part thereof. In some embodiments, the cytokine may be introduced with the corresponding receptor for the cytokine. In some embodiments, the step of engineering the heterologous cytokine into the engineered cell allows cytokine signaling, thereby maintaining or improving the cell growth, proliferation, expansion and/or effector function of NK cells, but reducing the risk of cytokine toxicity. In some embodiments, the introduced cytokine or in some cases its corresponding cytokine receptor is expressed on the cell surface. In some embodiments, cytokine signaling is constitutively active. In some embodiments, activation of cytokine signaling is inducible. In some embodiments, activation of cytokine signaling is transient or temporary.
Exemplary secreted and membrane-bound (mb) cytokines are known, as described, for example, in: patent publication No. US 2017/0073038; US2020/0199532, US 2021/0024959; and PCT patent publication nos. WO2015174928, WO 2019/126748, WO 2019/191495, WO2020056045, WO2021021907, WO 2021/01919, WO 2021/062281, any of which may be used in the engineered cells provided.
In some embodiments, the cytokine is IL-15 or a functional part thereof. IL-15 is a cytokine that regulates NK cell activation and proliferation. In some cases, IL-15 and IL-12 share similar biological activity. For example, IL-15 and IL-2 bind to a common receptor subunit and can compete for the same receptor. In some embodiments, IL-15 induces activation of JAK kinase and phosphorylation and activation of transcriptional activators STAT3, STAT5, and STAT 6. In some embodiments, IL-15 promotes or modulates one or more functional activities of NK cells, such as promoting NK cell survival, modulating activation and proliferation of NK cells and T cells, and supporting NK cell development from hematopoietic stem cells. In some embodiments, the functional moiety is a portion (e.g., comprising a truncated contiguous amino acid sequence of full-length IL-15) of IL-15 that retains one or more functions of full-length or mature IL-15 (such as promoting NK cell survival, modulating NK cell and T cell activation and proliferation, and supporting NK cell development from hematopoietic stem cells). All or a functional portion of IL-15 may be expressed as a membrane-bound polypeptide and/or a secreted polypeptide.
As will be appreciated by those skilled in the art, the sequences of a variety of IL-15 molecules are known in the art. In one aspect, IL-15 is wild-type IL-15. In some aspects, IL-15 is mammalian IL-15 (e.g., homo sapiens interleukin 15 (IL 15), transcriptional variant 3, mRNA, NCBI reference sequence: NM_000585.4, domestic dog interleukin 15 (IL 15), mRNA, NCBI reference sequence: NM_001197188.1, domestic cat interleukin 15 (IL 15), mRNA, NCBI reference sequence: NM_ 001009207.1). Examples of "mammals" or "mammals" include primates (e.g., humans), canines, felines, rodents, pigs, ruminants, and the like. Specific examples include humans, dogs, cats, horses, cattle, sheep, goats, rabbits, guinea pigs, rats and mice. In a specific aspect, mammalian IL-15 is human IL-15. Human IL-15 amino acid sequences include, for example, genbank accession number ：NR_751915.1、NP_000576.l、AAI00963.1、AAI00964.1、AAI00962.1、CAA71044.1、AAH18149.1、AAB97518.1、CAA63914.1 and CAA63913.1.
In some embodiments, the engineered NK cell comprises a heterologous nucleotide sequence encoding IL-15. In some embodiments, the IL-15 nucleotide sequence is shown in SEQ ID NO. 9, or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least about 98% sequence identity to SEQ ID NO. 9. In some embodiments, IL-15 is expressed by the cell in a mature form lacking the signal peptide sequence and in some cases also lacking the propeptide sequence. In some embodiments, IL-15 has the amino acid sequence shown in SEQ ID NO. 2 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least about 98% sequence identity to SEQ ID NO. 2.
In some embodiments, the IL-15 molecules are variants of human IL-5, e.g., having one or more amino acid changes, e.g., substitutions, to the amino acid sequence of human IL-15. In some embodiments, the IL-15 variant comprises or consists of a mutation at position 45, 51, 52 or 72, e.g., as described in US 2016/0184399. In some embodiments, the IL-15 variant comprises, or consists of, a substitution of N, S or one of L pair D, E, A, Y or P. In some embodiments, the mutation is selected from L45D, L45E, S51D, L D, N72D, N72E, N72A, N72S, N Y or N72P (with respect to the sequence of human IL-15, SEQ ID NO: 2).
In embodiments, IL-15 molecules include IL-15 variants, such as human IL-15 polypeptides having one or more amino acid substitutions. In some embodiments, the IL-15 molecule includes a substitution at position 72, e.g., a substitution of N for D. In one embodiment, the IL-15 molecule is an IL-15 polypeptide of SEQ ID NO. 2 (which contains the amino acid substitution N72D) or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto (which has IL-15Ra binding activity).
In some embodiments, the cytokine is IL-2 or a functional part thereof. In some embodiments, IL-2 is a member of a cytokine family, which also includes IL-4, IL-7, IL-9, IL-15 and IL-21.IL-2 is signal-transduced by a receptor complex consisting of three chains (called α, β and γ). All members of this cytokine receptor family share a gamma chain. Like IL-15, IL-2 promotes B-cell immunoglobulin production and induces NK cell differentiation and proliferation. Major differences between IL-2 and IL-15 were found in the adaptive immune response. For example, IL-2 is necessary for adaptive immunity to foreign pathogens, as it is the basis for the development of immunological memory. On the other hand, IL-15 is necessary to maintain a highly specific T cell response by supporting the survival of CD8 memory T cells. All or a functional portion of IL-2 may be expressed as a membrane-bound polypeptide and/or a secreted polypeptide. As will be appreciated by those skilled in the art, the sequences of a variety of IL-2 molecules are known in the art. In one aspect, IL-2 is wild-type IL-2. In some aspects, IL-2 is mammalian IL-2. In some embodiments, IL-2 is human IL-2.
In some embodiments, the engineered NK cell comprises a heterologous nucleotide sequence encoding IL-2. In some embodiments, IL-2 is expressed by cells in a mature form lacking the signal peptide sequence and in some cases also lacking the propeptide sequence. In some embodiments, IL-2 has the amino acid sequence set forth in SEQ ID NO. 1 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least about 98% sequence identity to SEQ ID NO. 1.
In some embodiments, the cytokine is IL-21 or a functional part thereof. IL-21 binds to the IL-21 receptor (IL-21R) and to the co-receptor (common gamma chain (CD 132)). IL-21 receptors have been identified on NK cells, T cells and B cells, indicating that IL-21 acts on cells of the hematopoietic lineage, in particular on lymphoprogenitors and lymphocytes. IL-21 has been shown to be a potent modulator of cytotoxic T cells and NK cells. (Parrish-Novak et al Nature, volume 408: pages 57-63, 2000; parrish-Novak et al J.Leuk.Bio., volume 72: pages 856-863, 202; collins et al Immunol.Res., volume 28: pages 131-140, 2003; brady et al J.Immunol., volume 172: pages 2048-2058, 2004). In murine studies, IL-21 enhanced NK cell maturation and effector function (Kasaian et al, immunity, 16:559-569, 2002).
As will be appreciated by those skilled in the art, the sequences of a variety of IL-21 molecules are known in the art. In one aspect, IL-21 is wild-type IL-21. In some aspects, IL-21 is mammalian IL-21. In one embodiment, the IL-21 sequence is a human IL-21 sequence. Human IL-21 amino acid sequences include, for example, genbank accession number ：AAU88182.1、EAX05226.1、CAI94500.1、CAJ47524.1、CAL81203.1、CAN87399.1、CAS03522.1、CAV33288.1、CBE74752.1、CBI70418.1、CBI85469.1、CBI85472.1、CBL93962.1、CCA63962.1、AAG29348.1、AAH66258.1、AAH66259.1、AAH66260.1、AAH66261.1、AAH66262.1、AAH69124.1 and ABG36529.1.
In some embodiments, the engineered NK cell comprises a heterologous nucleotide sequence encoding IL-21. In some embodiments, IL-21 is expressed by cells in a mature form lacking the signal peptide sequence and in some cases also lacking the propeptide sequence. In some embodiments, IL-21 has the amino acid sequence shown in SEQ ID NO. 3 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least about 98% sequence identity to SEQ ID NO. 3. In some embodiments, IL-21 has the sequence set forth in SEQ ID NO:4 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least about 98% sequence identity to SEQ ID No. 4.
The cytokine (e.g., IL-2, IL-15, or IL-21) amino acid sequence can include any functional portion of a mature cytokine, such as any functional portion of mature IL-2, mature IL-15, or mature IL-21. The functional moiety may be any moiety comprising consecutive amino acids of the interleukin to which it belongs, provided that the functional moiety specifically binds to the corresponding interleukin receptor. When used in reference to an interleukin, the term "functional moiety" refers to any portion or fragment of an interleukin that retains the biological activity of the interleukin to which it belongs (the parent interleukin). Functional moieties encompass, for example, interleukin moieties that retain the ability to specifically bind to a corresponding interleukin receptor, activate a downstream target of an interleukin, and/or induce one or more of differentiation, proliferation (or death) and activity of immune cells (e.g., NK cells), to the same extent as, or higher than, the parent interleukin. The biological activity of the functional portion of the interleukin may be measured using assays known in the art. With respect to the parent interleukin, the functional portion may include, for example, an amino acid sequence of about 60%, about 70%, about 80%, about 90%, about 95% or more of the parent mature interleukin.
Functional variants of the interleukins described herein are included within the scope of cytokines or functional moieties according to the provided embodiments. As used herein, the term "functional variant" refers to an interleukin that has substantial or significant sequence identity or similarity to a parent interleukin, which functional variant retains its original interleukin biological activity. Functional variants encompass, for example, those variants of an interleukin (parent interleukin) described herein that retain the ability to specifically bind to a corresponding interleukin receptor, activate a downstream target of the interleukin, and/or induce one or more of differentiation, proliferation (or death) and activity of immune cells (e.g., NK cells), to a similar, the same or higher degree than the parent interleukin. With respect to a parent interleukin, a functional variant may be, for example, at least about 80%, about 90%, about 95%, about 99% or more identical in amino acid sequence to the parent interleukin.
Functional variants may, for example, comprise the amino acid sequence of a parent interleukin with at least one conservative amino acid substitution. Alternatively or additionally, the functional variant may comprise an amino acid sequence of a parent interleukin having at least one non-conservative amino acid substitution. In some embodiments, amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions) do not interfere with or inhibit the biological activity of the functional variant as compared to the parent interleukin sequence. In some embodiments, amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions) may enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased compared to the parent interleukin.
In some embodiments, the amino acid substitution of an interleukin is a conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions may be: substitution of another acidic/negatively charged polar amino acid with an acidic/negatively charged polar amino acid (e.g., asp or glu), substitution of another amino acid with a nonpolar side chain with an amino acid with a nonpolar side chain (e.g., ala, gly, val, ile, leu, met, phe, pro, trp, cys, val, etc.), substitution of another basic/positively charged polar amino acid with a basic/positively charged polar amino acid (e.g., lys, his, arg, etc.), substitution of another uncharged amino acid with a polar side chain with an uncharged amino acid with a polar side chain (e.g., asn, gin, ser, thr, tyr, etc.), substitution of another amino acid with a beta-branched side chain with an amino acid with a beta-branched side chain (e.g., lie, thr, and val), substitution of another amino acid with an aromatic side chain with an amino acid with an aromatic side chain (e.g., his, phe, trp and tyr), etc.
In some embodiments, all or a functional portion of a cytokine (e.g., IL-2, IL-15, IL-21, or a functional portion of any of the foregoing) can be expressed by g-NK cells as secreted polypeptides in a variety of ways. For example, all or a functional portion of a cytokine may be expressed within and secreted from NK cells. In some embodiments, the secretable cytokine is free of transmembrane domains.
In some embodiments, cytokines can be secreted from the engineered g-NK cells. In some embodiments, the secretable cytokine is constitutively expressed. In other embodiments, the secretable cytokine is transiently expressed. In some embodiments, the secretable cytokine is under an inducible promoter. In some embodiments, the secretable cytokine is IL-2 or a functional portion thereof. In some embodiments, the amino acid sequence of IL-2 is or includes SEQ ID NO. 1. In some embodiments, the secretable cytokine is IL-15 or a functional portion thereof. In some embodiments, the amino acid sequence of IL-15 is or includes SEQ ID NO. 2. In some embodiments, the secretable cytokine is IL-21 or a functional portion thereof. In some embodiments, the amino acid sequence of IL-21 is or includes SEQ ID NO. 3. In some embodiments, the g-NK cells are engineered with two or more secretable cytokines, such as a combination of two or more of IL-2, IL-15, and IL-21.
While interleukins and other cytokines are usually secreted, they may also be membrane bound. When co-expressed with the CAR fusion protein, it is then possible to concentrate the immune cell activating cytokine and the CAR fusion protein in the vicinity of the target cell. When co-expressed with CAR fusion proteins in g-NK cells, g-NK cells exhibit improved targeting and killing capabilities and thus represent an attractive and effective therapeutic agent. In some cases, membrane-bound cytokines make it possible to concentrate immune cell activating cytokines in the vicinity of target cells, e.g., cells targeted by monoclonal antibodies administered in combination with g-NK cells as described herein.
In other embodiments, the cytokine is membrane bound (mb). In some embodiments, the membrane-bound cytokine is constitutively expressed. In other embodiments, the membrane-bound cytokine is transiently expressed. In some embodiments, the membrane-bound cytokine is under an inducible promoter. In some embodiments, the membrane-bound cytokine is membrane-bound IL-2 (mbiL-2). In some embodiments, the membrane-bound cytokine is membrane-bound IL-15 (mbiL-15). In some embodiments, the membrane-bound cytokine is membrane-bound IL-21 (mbiL-21). In some embodiments, g-NK cells are engineered with two or more membrane-bound cytokines (such as a combination of two or more of mbiL-2, mbiL-15, and mbiL-21). The membrane-bound cytokine may include any format of interleukin cytokine (e.g., IL-2, IL-15, or IL-21) formatted in a membrane-bound form, such as any of those described herein.
In some embodiments, all or a functional portion of a cytokine (e.g., IL-2, IL-15, IL-21, or a functional portion of any of the foregoing) can be expressed by g-NK cells as a membrane-bound cytokine in a variety of ways. In some embodiments, the cytokine or functional portion thereof can be directly or indirectly (e.g., ionically, nonionic, covalently) attached (e.g., conjugated or fused) to the surface of the g-NK cell (e.g., at the surface or within the membrane of the NK cell) using any of a variety of linkers known in the art (hermanson, g., bioconjugate Techniques, ACADEMIC PRESS, 1996). In some aspects, all or a functional portion of the cytokine is linked to all or a portion of the transmembrane protein. In one aspect, the NK cells express a fusion protein comprising all or a portion of a cytokine fused to all or a portion of a transmembrane protein. In some embodiments, the linker may be a peptide linker, such as a flexible linker. In some embodiments, the flexible linker comprises predominantly glycine and serine residues. For example, the flexible linker may include one or more repetitions of one or both of G4S and G3S (e.g., about 3 to about 15 or about 5 to about 12 repetitions of G4S and G3S). In some embodiments, the linker is a cleavable linker, such as a furin cleavable sequence. Exemplary furin cleavage sequences are described in Duckert et al, protein Engineering, design & selection, vol.17, phase 1: pages 107-112, 2004 and U.S. patent 8,871,906, each of which is incorporated herein by reference.
In particular aspects, the portion of the transmembrane protein comprises all or a portion of the transmembrane domain of the transmembrane protein. In some embodiments, the transmembrane protein may be any protein located on and/or within a membrane that is a phospholipid bilayer such as a biological membrane (e.g., a biological membrane such as a cell membrane). In some embodiments, the transmembrane domain is a domain of a transmembrane protein that is normally present within a membrane, particularly those that form channels and pores. In some embodiments, the transmembrane domain is a three-dimensional protein structure that is thermodynamically stable in a membrane (e.g., a membrane of a vesicle, such as a cell). Examples of transmembrane domains include a single alpha helix, a stable complex of several transmembrane alpha helices, a transmembrane beta barrel, a beta helix of gramicidin a, or any other structure. Transmembrane helices are typically about 20 amino acids long.
Examples of transmembrane proteins include receptors, ligands, immunoglobulins, glycophorins or combinations thereof. Specific examples of transmembrane proteins include, but are not limited to, CD 8a, CD4, CD3 epsilon, CD3 gamma, CD3 delta, CD3 zeta, CD28, CD137, fcerigamma, T cell receptors (TCRs such as tcra and/or tcrp), nicotinic acetylcholine receptors, GABA receptors, or combinations thereof. Specific examples of immunoglobulins include IgG, igA, igM, igE, igD or a combination thereof. Specific examples of glycophorins include glycophorin a, glycophorin D, or a combination thereof.
In some embodiments, the transmembrane domain is a CD28 transmembrane domain. An exemplary sequence of the CD28 transmembrane domain is shown in SEQ ID NO. 10.
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVG GVLACYSLLVTVAFIIFWVR(SEQ ID NO:10)
In some embodiments, the transmembrane domain is a CD8 transmembrane domain. An exemplary sequence of the CD8 transmembrane domain is shown in SEQ ID NO. 11.
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI WAPLAGTCGVLLLSLVITLYC(SEQ ID NO:11)
In some embodiments, the transmembrane domain is a CD4 transmembrane domain. An exemplary sequence of the CD4 transmembrane domain is shown in SEQ ID NO. 15.
MALIVLGGVAGLLLFIGLGIFF(SEQ ID NO:15)
In some embodiments, all or a functional portion of a cytokine (e.g., IL-2, IL-15, IL-21, or a functional portion of any of the foregoing) can be linked to other components, such as a signal peptide, a leader sequence, a secretion signal, a marker (e.g., a reporter gene), or any combination thereof.
In some embodiments, the nucleic acid sequence encoding all or a functional portion of a cytokine (e.g., IL-2, IL-15, IL-21, or a functional portion of any of the foregoing) is replaced with a nucleic acid sequence encoding a signal peptide from a heterologous protein. The heterologous protein may be, for example, CD8 alpha, CD28, tissue plasminogen activator (tPA), growth hormone, granulocyte-macrophage colony-stimulating factor (GM-CSF), GM-CSF receptor (GM-CSFRa), or an immunoglobulin (e.g., igE or IgK).
In some embodiments, all or a functional portion of a cytokine (e.g., IL-2, IL-15, IL-21, or a functional portion of any of the foregoing) is fused to a signal peptide of CD8 alpha. An exemplary CD 8. Alpha. Signal peptide is shown in SEQ ID NO. 12. In some embodiments, all or a functional portion of a cytokine (e.g., IL-15 or a functional portion thereof, IL-2 or a functional portion thereof, or IL-21 or a functional portion thereof) is fused to a signal peptide of GM-CSFRa (SEQ ID NO: 13). An exemplary GM-CSFRa signal peptide is shown in SEQ ID NO. 13. An exemplary IgK signal peptide is shown in SEQ ID NO. 14. An exemplary IgK signal peptide is shown in SEQ ID NO. 43.
In some embodiments, all or a functional portion of a cytokine (e.g., a functional portion of IL-2, IL-15, IL-21, or any of the foregoing) is fused to all or a portion of the signal peptide of CD 8a and the transmembrane domain of CD8 a. In some embodiments, the heterologous cytokine is the membrane bound IL-15 shown in SEQ ID NO. 7 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95% or at least about 98% sequence identity to SEQ ID NO. 7. In some embodiments, the heterologous cytokine is the membrane bound IL-15 shown in SEQ ID NO. 8 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95% or at least about 98% sequence identity to SEQ ID NO. 8.
In some embodiments, all or a functional portion of a cytokine (e.g., IL-2, IL-15, IL-21, or a functional portion of any of the foregoing) is fused to the Fc region of an immunoglobulin to produce a bivalent cytokine. In some embodiments, the cytokine-Fc fusion protein may be further linked to a transmembrane domain for expression as a membrane-bound cytokine.
In some embodiments, the heterologous cytokine is the membrane bound IL-15 shown in SEQ ID NO. 5 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95% or at least about 98% sequence identity to SEQ ID NO. 5.
In some embodiments, the heterologous cytokine is a membrane bound IL-21 shown in SEQ ID NO. 6 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95% or at least about 98% sequence identity to SEQ ID NO. 6.
In some embodiments, IL-15 and IL-15 receptor alpha (IL 15 RA) together engineered into cells. IL15RA specifically binds IL-15 with very high affinity and is able to bind IL-15 independently of other subunits. In some aspects, this property allows IL-15 to be produced by one cell, endocytosed by another cell, and then presented to a third cell. In some embodiments, g-NK cells express heterologous (e.g., exogenous) IL-15/IL-15Ra. In some embodiments, the g-NK cells are engineered with IL-15/IL-15R fusion proteins. In some embodiments, the g-NK cells are engineered with single chain IL-15/IL-15R fusion proteins. In some embodiments, IL-15/IL-15Ra is expressed as a membrane-bound IL-15.IL15Ra complex (e.g., imamura et al, blood,2014 124 (7): 108 and Hurton LV et al, PNAS, 2016). In some embodiments, the exogenous IL-15/IL-15Ra is secreted and expressed as a soluble il15ra.il15 complex (e.g., mortier E et al, JBC, 2006; bessard A, mol. Cancer ter., 2009; and Desbois M, j. Immunol., 2016). In some embodiments, provided engineered g-NK cells express a membrane-bound IL15/IL15Ra complex and a soluble (secreted) IL15Ra/IL15 complex. In some embodiments, the engineered g-NK cells express the il15.il15ra complex in membrane bound form with a cleavable linker.
C. Polynucleotide
In some embodiments, provided herein are polynucleotides having a nucleic acid sequence encoding an antigen receptor (such as a chimeric antigen receptor, including any of the chimeric antigen receptors provided). In some embodiments, provided herein are polynucleotides having a nucleic acid sequence encoding any one of the provided immunomodulatory agents (such as cytokines, including secretable cytokines or membrane-bound cytokines).
In some embodiments, the nucleic acid encoding an antigen receptor (such as a chimeric antigen receptor) and the nucleic acid encoding an immunomodulatory agent (such as a cytokine, including a secretable cytokine or a membrane-bound cytokine) are provided as separate polynucleotides.
In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding an antigen receptor (such as a chimeric antigen receptor) and a nucleic acid encoding an immunomodulator (such as a cytokine, including a secretable cytokine or a membrane-bound cytokine). Thus, in some aspects, the nucleic acid sequences are provided as part of the same polynucleotide. For example, provided embodiments include polynucleotides in which the engineered component is encoded by a polynucleotide comprising one or more protease cleavage sites, e.g., self-cleaving peptides, such as T2A, P2A, E a or F2A. Such sites are recognized and cleaved by proteases, which can lead to isolation (and separate expression) of the various component parts (e.g., cytokines and CARs) encoded by polynucleotides engineered into NK cells. Thus, according to embodiments, the various components of the engineered component may be delivered to the NK cells in a single vector or through multiple vectors.
Also provided herein are vectors encoding any one of the provided polynucleotides, such as for delivering the polynucleotides to a cell, e.g., a g-NK cell. In some embodiments, the vehicle is a vector, such as a viral vector or a non-viral vector. In some embodiments, the vector is a viral vector that is a lentiviral vector. In some embodiments, the vehicle is a liposome. In some embodiments, the vehicle is a lipid nanoparticle. Other vehicles (including carrier or non-carrier delivery vehicles) include those known to those of skill in the art, including any of the vehicles described below.
In some embodiments, according to the provided methods, polynucleotides are engineered into g-NK cells or compositions containing multiple g-NK cells. Exemplary methods of engineering NK cells are described below.
D. Methods of delivering heterologous agents
Disclosed herein are methods of producing genetically engineered g-NK cells comprising introducing a nucleic acid encoding a CAR into a g-NK cell, thereby producing a genetically engineered g-NK cell.
Disclosed herein are methods of producing genetically engineered g-NK cells comprising introducing a nucleic acid encoding an immunomodulatory agent (e.g., a secretable or soluble cytokine or membrane-bound cytokine) into a g-NK cell, thereby producing a genetically engineered g-NK cell.
Disclosed herein are methods of producing genetically engineered g-NK cells comprising (a) introducing a nucleic acid encoding a CAR into a g-NK cell, and (b) introducing a nucleic acid encoding an immunomodulatory agent (e.g., a secretable or soluble cytokine or membrane-bound cytokine) into a g-NK cell, thereby producing genetically engineered g-NK cells, wherein steps (a) and (b) are performed simultaneously or sequentially in any order.
The nucleic acid introduced into g-NK cells may be introduced for stable integration into the genome or for transient expression. Stable integration or transient expression may be selected based on a variety of factors including, but not limited to, the ability of a particular nucleic acid to efficiently integrate into the host genome or the amount of nucleic acid and its half-life.
In some embodiments, introducing the heterologous agent into the g-NK cells (e.g., an antigen receptor, such as a CAR and/or an immunomodulatory agent, such as the cytokines described) can be performed in a method of enriching a subpopulation of g-NK cells from a starting sample of NK cells. In some embodiments, the introduction of an antigen receptor (such as a CAR) can be performed in a method of enriching a subpopulation of g-NK cells from a starting sample of NK cells. In some embodiments, the introduction of an immunomodulatory agent (such as a cytokine) may be performed in a method of enriching a subpopulation of g-NK cells from a starting sample of NK cells. In some embodiments, the introduction of an antigen receptor (such as a CAR) and an immunomodulatory agent (such as a cytokine) can be performed in a method of enriching a subpopulation of g-NK cells from a starting sample of NK cells. Thus, it will be appreciated that the provided methods do not require engineering in particular only g-NK cells that have been selected for FcR gamma chain deficient NK cells (or only g-NK cells that have been selected or identified by g-NK surrogate marker profile), but may involve engineering cells that are to or have preferentially expanded g-NK cells or NK cell compositions enriched for g-NK cells. Thus, the final cell composition enriched in g-NK cells comprises g-NK cells that have incorporated a heterologous antigen receptor (e.g., a CAR), a heterologous immunomodulator (e.g., a secretable interleukin or a membrane-bound interleukin, such as IL-15 or IL-21), or a heterologous antigen receptor (e.g., a CAR) and a heterologous immunomodulator (e.g., a cytokine) (e.g., a secretable interleukin or a membrane-bound interleukin, such as IL-15 or IL-21). Exemplary methods of preparing and amplifying g-NK cell enriched compositions are provided in section V.
In some embodiments, the introduction of a heterologous agent (e.g., an antigen receptor, such as a CAR, and/or an immunomodulatory agent, such as the cytokines described) can be performed at any suitable time during the method of amplifying g-NK cells, such as described in section V. In some embodiments, the introduction of the antigen receptor (such as CAR) may be performed at any suitable time during the method of amplifying g-NK cells. In some embodiments, the introduction of an immunomodulatory agent (such as a cytokine) may be performed at any suitable time during the method of amplifying g-NK cells. In some embodiments, the introduction of the CAR and the immunomodulatory agent may be performed at any suitable time during the method of amplifying g-NK cells. In some embodiments, the introduction occurs after selecting cells from the subject (e.g., selecting or enriching cells for CD3 Negative of CD57 Positive and negative ) and prior to incubating or culturing the selected or enriched cells with feeder cells (e.g., HLA-E expressing feeder cells) for proliferation or expansion of NK cells. In some embodiments, introduction is performed after incubation or culture with feeder cells (e.g., HLA-E expressing feeder cells) and thus after the selected or enriched cells have proliferated or expanded. In some embodiments, the introduction is performed sequentially in any order with the methods for gene editing described herein.
In some embodiments, the expansion phase of the cells (such as described in section V) is divided into a first expansion and a second expansion. In some embodiments, prior to introduction (e.g., viral transduction), selected cells from the biological sample are cultured under expansion conditions for a first period of time, e.g., for the following period of time or for more than the following period of time: about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, or any time between these listed times (inclusive). In some embodiments, after the first expansion phase, an engineered construct encoding one or more heterologous agents, such as a chimeric antigen receptor and/or an immunomodulatory agent, such as the cytokines described, is introduced (e.g., transduced) into the expanded cells (e.g., NK cells). In some embodiments, after the first expansion phase, an engineered construct encoding one or more heterologous agents, such as chimeric antigen receptors, is introduced (e.g., transduced) into the expanded cells (e.g., NK cells). In some embodiments, after the first expansion period, an engineered construct encoding one or more heterologous agents (such as an immunomodulatory agent, such as a cytokine) is introduced (e.g., transduced) into the expanded cells (e.g., NK cells). In some embodiments, after the first expansion phase, an engineered construct encoding one or more heterologous agents (such as chimeric antigen receptors and immunomodulators, such as cytokines) is introduced (e.g., transduced) into the expanded cells (e.g., NK cells). Following introduction (e.g., viral transduction), the engineered cells are cultured for a second period of time, e.g., for or above the following period of time: about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, or any time between these listed times (inclusive).
The culture medium may be supplemented with HLA-E expressing feeder cells and/or one or more stimulatory agents (such as IL12 and/or IL 21) at any time during the culture. For example, one or more stimulators may be added at the beginning of the culture, such as at time point 0 (e.g., at the beginning of the culture). The one or more agents may be added a second, third, fourth, fifth, or more times. The subsequent addition may be the same or different from the concentration previously added. The interval between additions may vary, for example, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours or longer intervals, and any time therebetween (including endpoints). If the stimulus is added multiple times, the concentration of the first supplemental addition may be the same or different than the concentration of the second (and/or any supplemental addition). For example, in several embodiments, the concentration of the stimulus addition at multiple time points may be gradually increased, gradually decreased, remain constant, or vary between multiple non-equivalent concentrations.
In some embodiments, a nucleic acid encoding an antigen receptor (e.g., CAR) is introduced under conditions of transient expression in g-NK cells. In some embodiments, a nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine) is introduced under conditions of transient expression in g-NK cells. In some embodiments, a nucleic acid encoding an antigen receptor (e.g., CAR) and a nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine) are introduced under conditions of transient expression in g-NK cells. In some embodiments, the method for introducing a nucleic acid for transient expression includes any method that will produce a nucleic acid that can express its encoded contents shortly before degradation.
In some embodiments, a nucleic acid encoding an antigen receptor (e.g., CAR) is introduced under conditions of stable expression in g-NK cells. In some embodiments, a nucleic acid encoding an immunomodulatory agent (e.g., a cytokine such as a secretable or soluble cytokine or membrane-bound cytokine) is introduced under conditions of stable expression in g-NK cells. In some embodiments, a nucleic acid encoding an antigen receptor (e.g., CAR) and a nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine) are introduced under conditions of stable expression in g-NK cells. In some embodiments, the method for introducing nucleic acid for stable expression in a cell involves any such method: this method results in stable integration of the nucleic acid into the genome of the cell, such that if the cell into which the nucleic acid is integrated is divided, the nucleic acid can proliferate.
In some embodiments, one of the heterologous agents (e.g., one of a nucleic acid encoding an antigen receptor such as a CAR or a nucleic acid encoding an immunomodulatory agent such as a cytokine) is introduced into the cell by a method that results in transient expression in the cell, and the other of the heterologous agents (e.g., the other of a nucleic acid encoding an antigen receptor such as a CAR or a nucleic acid encoding an immunomodulatory agent such as a cytokine) is introduced into the cell by a method that results in stable expression in the cell. In some embodiments, two heterologous agents (e.g., both a nucleic acid encoding an antigen receptor such as a CAR and a nucleic acid encoding an immunomodulatory agent such as a cytokine) are introduced into a cell by a method that results in transient expression in the cell. In some embodiments, two heterologous agents (e.g., both a nucleic acid encoding an antigen receptor such as a CAR and a nucleic acid encoding an immunomodulatory agent such as a cytokine) are introduced into a cell by a method that results in stable expression in the cell.
Methods of delivering polynucleotides and compositions containing the polynucleotides are known to those of skill in the art. The person skilled in the art is able to select an appropriate method for transient or stable expression in a cell.
In some embodiments, the engineering of NK cells can be accomplished by transducing a cell composition with a polynucleotide encoding a heterologous agent (e.g., a CAR and/or an immunomodulatory agent, such as a cytokine) or a vector comprising the polynucleotide. In some embodiments, the engineering of NK cells can be accomplished by transducing a cell composition with a polynucleotide encoding an antigen receptor (such as a CAR) or a vector comprising the polynucleotide. In some embodiments, the engineering of NK cells can be accomplished by transducing a cell composition with a polynucleotide encoding an immunomodulatory agent (such as a cytokine) or a vector comprising the polynucleotide. In some embodiments, the engineering of NK cells can be accomplished by transducing a cell composition with a polynucleotide encoding an antigen receptor (such as a CAR) and immunomodulation (such as a cytokine) or a vector comprising the polynucleotide. The vector may be a viral vector, such as a lentiviral vector, a gamma retroviral vector, a recombinant AAV, an adenoviral vector, or an oncolytic viral vector. In other aspects, non-viral vectors, e.g., nanoparticles and liposomes, can also be used to introduce and deliver polynucleotides encoding heterologous agents (e.g., CARs and/or immunomodulators, such as cytokines) into NK cells. In other aspects, non-viral vectors such as nanoparticles and liposomes can also be used to introduce and deliver polynucleotides encoding antigen receptors (such as CARs) into NK cells. In other aspects, non-viral vectors such as nanoparticles and liposomes can also be used to introduce and deliver polynucleotides encoding immunomodulators (such as cytokines) into NK cells. In other aspects, non-viral vectors, e.g., nanoparticles and liposomes, can also be used to introduce and deliver polynucleotides encoding antigen receptors (such as CARs) and immune modulators (such as cytokines) into NK cells.
In some embodiments, vectors packaging polynucleotides encoding heterologous agents (e.g., CARs and/or immunomodulators, such as cytokines) can be used to deliver the packaged polynucleotides to g-NK cells or g-NK cell enriched cell compositions or populations. In some embodiments, vectors packaging polynucleotides encoding antigen receptors (such as CARs) can be used to deliver the packaged polynucleotides to g-NK cells or g-NK cell enriched cell compositions or populations. In some embodiments, vectors packaging polynucleotides encoding immunomodulators (such as cytokines) can be used to deliver the packaged polynucleotides to g-NK cells or g-NK cell enriched cell compositions or populations. In some embodiments, vectors packaging polynucleotides encoding antigen receptors (such as CARs) and immunomodulators (such as cytokines) can be used to deliver the packaged polynucleotides to g-NK cells or g-NK cell enriched cell compositions or populations. These vectors may be of any kind, including DNA vectors, RNA vectors, plasmids, viral vectors and particles. Viral vector technology is well known and described in Sambrook et al (2001, molecular Cloning: ALaboratory Manual, cold Spring Harbor Laboratory, new York). Viruses that may be used as vectors include, but are not limited to, lentiviral vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, herpes simplex virus vectors, retroviral vectors, oncolytic viruses, and the like.
Typically, the vector contains at least one origin of replication function in an organism, a promoter sequence and a suitable restriction endonuclease site, and one or more selectable markers, such as a drug resistance gene.
Promoters may include any DNA sequence recognized by the cellular transcription machinery that is required to initiate specific transcription of a polynucleotide sequence. The vector may include a native or non-native promoter operably linked to the polynucleotide. The promoters selected may be strong, weak, constitutive, inducible, tissue-specific, developmental stage-specific, and/or organism-specific. One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of the polynucleotide sequence to which it is operably linked. Another example of a promoter is the extended growth factor-1. Alpha (EF-1. Alpha). Other constitutive promoters may also be used, including but not limited to simian virus 40 (SV 40), mouse Mammary Tumor Virus (MMTV), human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoters, avian leukemia virus promoters, epstein barr virus immediate early promoters, rous sarcoma virus promoters, and human gene promoters including but not limited to phosphoglycerate kinase (PGK) promoters, actin promoters, myosin promoters, hemoglobin promoters, ubiquitin C (Ubc) promoters, human U6 small nucleoprotein promoters, and creatine kinase promoters. In some cases, inducible promoters such as, but not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters may be used.
Additional promoter elements (e.g., enhancers) may be used to regulate the frequency of transcription initiation. Such regions may be located 10 to 100 base pairs upstream or downstream of the start site. In some cases, two or more promoter elements may be used to activate transcription in concert or independently.
In some embodiments, the polynucleotide may be packaged into a viral vector or integrated into the viral genome, allowing for transient or stable expression of the polynucleotide. The viral vector may comprise a retroviral vector, including a lentiviral vector. To construct a retroviral vector, polynucleotide molecules encoding heterologous agents (e.g., CARs and/or immunomodulators, such as cytokines) are inserted into the viral genome at the location of certain viral sequences to produce replication defective viruses. To construct a retroviral vector, a polynucleotide molecule encoding an antigen receptor (such as a CAR) is inserted at the location of certain viral sequences in the viral genome to produce a replication defective virus. To construct retroviral vectors, polynucleotide molecules encoding immunomodulators (such as cytokines) are inserted into the viral genome at positions of certain viral sequences to produce replication defective viruses. To construct a retroviral vector, polynucleotide molecules encoding antigen receptors (such as CARs) and immune modulators (such as cytokines) are inserted at the location of certain viral sequences in the viral genome to produce replication defective viruses. The recombinant viral vector is then introduced into a packaging cell line containing the gag, pol and env genes but no LTR and packaging components. Recombinant retroviral particles are secreted into the culture medium and then collected, optionally concentrated, and used for gene transfer. Lentiviral vectors are particularly preferred because they are capable of infecting both dividing cells and non-dividing cells.
In some embodiments, a polynucleotide encoding a heterologous agent (e.g., a CAR and/or an immunomodulatory agent, such as a cytokine) is incorporated into a viral vector for delivery by transduction. In some embodiments, a polynucleotide encoding an antigen receptor (such as a CAR) is incorporated into a viral vector for delivery by transduction. In some embodiments, polynucleotides encoding immunomodulators (such as cytokines) are incorporated into viral vectors for delivery by transduction. In some embodiments, polynucleotides encoding antigen receptors (such as CARs) and immunomodulators (such as cytokines) are incorporated into viral vectors for delivery by transduction. Viral transduction is the process of deliberately introducing nucleic acids into eukaryotic cells by virus-mediated means.
In some embodiments, the viral vector is a lentiviral vector. Lentiviral vectors are particularly useful means for successful viral transduction, as they allow stable expression of genes contained within the nucleic acid transcript delivered. Lentiviral vectors express reverse transcriptase and integrase, both of which are required for stable expression of genes contained within the nucleic acid transcript being delivered. Reverse transcriptase converts RNA transcripts to DNA, and integrase inserts and integrates DNA into the genome of the target cell. Once the DNA has been stably integrated into the genome, it splits with the host. The gene of interest contained in the integrated DNA may be expressed constitutively or it may be inducible. As part of the host cell genome, it may be affected by cellular regulation, including activation or inhibition, depending on many factors in the target cell.
Lentiviruses are a subgroup of retroviridae viruses, because the viral RNA genome needs to be reverse transcribed into DNA before integration into the host genome. Thus, the most important feature of lentiviral vectors/particles is their integration of genetic material into the genome of the target cell/host cell. Some examples of lentiviruses include human immunodeficiency virus: HIV-1 and HIV-2, simian Immunodeficiency Virus (SIV), feline Immunodeficiency Virus (FIV), bovine Immunodeficiency Virus (BIV), Jem Blanana Virus (Jembrana Disease Virus) (JDV), equine Infectious Anemia Virus (EIAV), equine infectious anemia Virus, meydi-Weiner Virus (visna-maedi), and Caprine Arthritis Encephalitis Virus (CAEV).
Typically, the lentiviral particles that make up the gene delivery vehicle are themselves replication defective (also referred to as "self-inactivating"). Lentiviruses are capable of infecting both dividing and non-dividing cells due to the entry mechanism through the intact host nuclear envelope (Naldini L et al, curr. Opin. Bioiecknol,1998, volume 9: pages 457-463). Recombinant lentiviral vectors/particles have been generated by multiple attenuation of HIV virulence genes, e.g., gene Env, vif, vpr, vpu, nef and tat deletions, making the vector biologically safe. Correspondingly, lentiviral vectors, such as those derived from HIV-1/HIV-2, can mediate efficient delivery, integration and long-term expression of transgenes into non-dividing cells.
Lentiviral particles can be generated by co-expressing the viral packaging element and the vector genome itself in a producer cell, such as a human HEK293T cell. These elements are typically provided as three separate plasmids (in the second generation lentiviral system) or four separate plasmids (in the third generation lentiviral system). The producer cells are co-transfected with a plasmid encoding a lentiviral component, including the core (i.e., structural protein) and the enzyme component of the virus, as well as the envelope protein (referred to as the packaging system), and also with a plasmid encoding a genome, which includes the exogenous transgene to be transferred to the target cell, which is the vector itself (also referred to as the transfer vector). Typically, a plasmid or vector is included in the production cell line. The plasmid/vector is introduced into the producer cell line via transfection, transduction or infection. Methods of transfection, transduction or infection are well known to those skilled in the art. As non-limiting examples, packaging and transfer constructs can be introduced into production cell lines by calcium phosphate transfection, lipofection, or electroporation, typically with a dominant selection marker such as neomycin (neo), dihydrofolate reductase (DHFR), glutamine synthetase, or Adenosine Deaminase (ADA), and then selected and isolated in the presence of an appropriate drug.
The production cells produce recombinant viral particles containing exogenous genes, e.g., polynucleotides encoding heterologous agents (e.g., CARs and/or immunomodulators, such as cytokines). The producer cells produce recombinant viral particles containing exogenous genes, for example, polynucleotides encoding antigen receptors (such as CARs). The producer cells produce recombinant viral particles containing exogenous genes, for example, polynucleotides encoding immunomodulators (such as cytokines). The production cells produce recombinant viral particles containing exogenous genes, for example, polynucleotides encoding antigen receptors (such as CARs) and immunomodulators (such as cytokines). Recombinant viral particles are recovered from the culture medium and titrated by standard methods used by those skilled in the art. Recombinant lentiviral vectors can be used to infect target cells, such as g-NK cells or cell compositions or populations enriched for g-NK cells.
Cells that can be used to produce high titer lentiviral particles can include, but are not limited to, HEK293T cells, 293G cells, STAR cells (Relander et al, mol Ther.,2005, volume 11: pages 452-459), freeStyle TM 293 expression system (ThermoFisher, waltham, mass.), and other HEK 293T-based producer cell lines (e.g., stewart et al, hum Gene Ther.,2011, pages 2, volume 3: pages 357-369; lee et al, biotechnol Bioeng,2012, volume 10996: pages 1551-1560; throm et al, blood,2009, volume 113: pages 21: 5104-5110).
In some aspects, the envelope protein may be a heterologous envelope protein from another virus, such as the G protein of vesicular stomatitis virus (VSV G) or the baculovirus gp64 envelope protein. The VSV-G glycoprotein may be selected from the group consisting of, inter alia, species classified as vesicular virus: carajas virus (CJSV), chandizura virus (CHPV), cocal virus (COCV), isfahan virus (ISFV), maraba virus (MARAV), piry virus (PIRYV), vesicular Stomatitis Aiagoas Virus (VSAV), vesicular stomatitis Indiana virus (VSTV) and Vesicular Stomatitis New Jersey Virus (VSNJV), and/or provisionally classified as a species of the genus vesicular virus, such as the grass carp rhabdovirus, the Bean 157575 virus (Bean 157575), boteke virus (BTKV), calchaqui virus (CQFV), the American Eel Virus (EVA), the Gray Lodge virus (GLOV), the Jurona virus (JURY), the Klamath virus (KLAVj. Kwatta virus (KWAV), the La Joya Virus (LJV), the MALPAIS SPRING virus (MSPV), the Ehrlich Gong Shan bat virus (MEB V), the Ferine t virus (PERV), the shuttle fish fry rhabdovirus (PFRV), the Ferine t virus (Ferine t), the radii virus (Ferine t), the Spring Viremia of Carp Virus (SVCV), the Ferine t virus (Ferine t), the ulcerative rhabdovirus (Ferine t) and the Ferine t virus (Ferine t). Gp64 or other baculoviruses env proteins may be derived from the group of the Leucopia calis, the Apii graves, the silkworm nucleopolyhedrovirus, the Japanese moth nucleopolyhedrovirus, the hemlock virus, the hemlock (polyhedrosis virus), the hemlock (Ferine t) and the polyhedra virus (Ferine t) the polyhedrosis virus (Ferine t) the apple shell virus (Ferine t) Fall webworm (HYPHARITRIA CUNEA) nuclear polyhedrosis virus, chilo suppressalis nuclear polyhedrosis virus, dhori virus, thogoto virus, tussah (ANTHERAEA PEMYI) nuclear polyhedrosis virus or Batken virus.
Additional elements provided in the lentiviral particle may include a retroviral LTR (long terminal repeat) at the 5 'or 3' end, a retroviral export element, optionally a lentiviral Reverse Response Element (RRE), a promoter or active portion thereof, and a Locus Control Region (LCR) or active portion thereof. Other elements include a central polypurine tract (cPPT) sequence that increases transduction efficiency in non-dividing cells, a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) that enhances expression of the transgene and increases titer.
Methods of generating recombinant lentiviral particles are known to those skilled in the art, for example, as described in U.S. Pat. nos. 8,846,385, 7,745,179, 7,629,153, 7,575,924, 7,179,903 and 6,808,905. The lentiviral vector used may be selected from, but is not limited to pLVX、pLenti、pLenti6、pLJMl、FUGW、pWPXL、pWPI、pLenti CMV puro DEST、pLJMl-EGFP、pULTRA、pInducer2Q、pHIV-EGFP、pCW57.1、pTRPE、pELPS、pRRL and pLionII. Any known lentiviral vector may also be used (see U.S. Pat. Nos. 9,260,725, 9,068,199, 9,023,646, 8,900,858, 8,748,169, 8,709,799, 8,420,104, 8,329,462, 8,076,106, 6,013,516, and 5,994,136; international patent publication No. WO 2012079000).
Other retroviral vectors may also be used to package heterologous agents (e.g., CARs and/or immunomodulators, such as cytokines) for delivery into g-NK cells or g-NK cell enriched cell compositions or populations. Other retroviral vectors may also be used to package antigen receptors (such as CARs) for delivery into g-NK cells or g-NK cell enriched cell compositions or populations. Other retroviral vectors may also be used to package an immunomodulatory agent (such as a cytokine) for delivery to g-NK cells or to g-NK cell enriched cell compositions or populations. Other retroviral vectors may also be used to package antigen receptors (such as CARs) and immunomodulators (such as cytokines) for delivery into g-NK cells or g-NK cell enriched cell compositions or populations. Retroviral Vectors (RV) allow permanent integration of the transgene in the target cell. In addition to the complex HIV-1/2 based lentiviral vectors, simple gamma retrovirus based retroviral vectors have also been widely used to deliver therapeutic genes and have been clinically proven to be one of the most effective and powerful gene delivery systems capable of transducing a wide range of cell types. Exemplary classes of gamma retroviruses include Murine Leukemia Virus (MLV) and feline leukemia virus (FeLV).
In some embodiments, a gamma retroviral vector derived from a mammalian gamma retrovirus, such as Murine Leukemia Virus (MLV), is recombinant. The gamma retroviruses of the MLV family include the philic, amphotropic and polytropic subfamilies. The philic virus is only able to infect murine cells using the mCAT-1 receptor. Examples of philic viruses are moloney MLV and AKV. Amphotropic viruses infect mice, humans and other species through the Pit-2 receptor. One example of an amphotropic virus is the 4070A virus. The same (Xprl) receptor is used by both the tropic and the mutatropic viruses, but their species tropism is different. A xenotropic virus such as NZB-9-1 infects humans and other species but does not infect murine species, while a mutagen virus such as foci forming virus (MCF) infects mice, humans and other species.
Gamma retroviral vectors can be produced in packaging cells by co-transfecting the cells with several plasmids, including plasmids encoding retroviral structural polyproteins and enzymatic polyproteins (gag-pol), plasmids encoding envelope (env) proteins, and plasmids encoding vector mRNA comprising polynucleotides encoding heterologous agents (e.g., CARs and/or immunomodulators, such as cytokines) to be packaged in the newly formed viral particles. Gamma retroviral vectors can be produced in packaging cells by co-transfecting the cells with several plasmids, including plasmids encoding retroviral structural polyproteins and enzymatic polyproteins (gag-pol), plasmids encoding envelope (env) proteins, and plasmids encoding vector mRNA comprising polynucleotides encoding antigen receptors, such as CARs, to be packaged in newly formed viral particles. Gamma retroviral vectors can be produced in packaging cells by co-transfecting the cells with several plasmids, including plasmids encoding retroviral structural polyproteins and enzymatic polyproteins (gag-pol), plasmids encoding envelope (env) proteins, and plasmids encoding vector mRNA comprising polynucleotides encoding immunomodulators, such as cytokines, to be packaged in the newly formed viral particles. Gamma retroviral vectors can be produced in packaging cells by co-transfecting the cells with several plasmids, including plasmids encoding retroviral structural polyproteins and enzymatic polyproteins (gag-pol), plasmids encoding envelope (env) proteins, and plasmids encoding vector mRNA comprising polynucleotides encoding antigen receptors such as CARs and immunomodulators such as cytokines to be packaged in the newly formed viral particles.
In some aspects, the recombinant gamma retroviral vector is pseudotyped with envelope proteins from other viruses. The envelope glycoprotein is incorporated into the outer lipid layer of the viral particle, which may increase/alter cell tropism. Exemplary envelope proteins include the gibbon leukemia virus envelope protein (GALV) or vesicular stomatitis virus G protein (VSV-G), or the simian endogenous retrovirus envelope protein, or the measles virus H and F proteins, or the human immunodeficiency virus gp120 envelope protein, or the cocal vesicular virus envelope protein (see, e.g., U.S. patent application publication No. 2012/164118). In other aspects, the envelope glycoprotein may be genetically modified to incorporate targeting/binding ligands into the gamma retroviral vector, including but not limited to peptide ligands, single chain antibodies, and growth factors (Waehier et al, nat. Rev. Genet.,2007, volume 8, phase 8: pages 573-587). These engineered glycoproteins can re-target the vector to cells expressing their corresponding target moiety. In other aspects, a "molecular bridge" may be introduced to direct the vector to a particular cell. The molecular bridge has dual specificity: one end may recognize the viral glycoprotein and the other end may bind to a molecular determinant on the target cell. Such molecular bridges (e.g., ligand-receptors, avidin-biotin and chemical conjugates, monoclonal antibodies and engineered membrane fusion proteins) can direct the attachment of viral vectors to target cells for transduction (Yang et al, biotechnol bioeng.,2008, volume 101, phase 2: pages 357-368, and Maetzig et al, viruses,2011, volume 3: pages 677-713).
In some embodiments, the recombinant gamma retroviral vector is a self-inactivating (SIN) gamma retroviral vector. The vector may be replication-incompetent. The SIN vector may have a deletion in the 3' u3 region that originally includes enhancer/promoter activity. In addition, the 5' u3 region may be replaced with a strong promoter derived from cytomegalovirus or RSV (required in packaging cell lines) or selected internal promoter and/or enhancer elements. The choice of internal promoters may be made according to the specific requirements of the gene expression required for a specific purpose.
In some embodiments, a polynucleotide encoding a heterologous agent (e.g., a CAR and/or an immunomodulatory agent, such as a cytokine) is inserted into the recombinant viral genome. In some embodiments, a polynucleotide encoding an antigen receptor (such as a CAR) is inserted into the recombinant viral genome. In some embodiments, polynucleotides encoding immunomodulators (such as cytokines) are inserted into the recombinant viral genome. In some embodiments, polynucleotides encoding antigen receptors (such as CARs) and immune modulators (such as cytokines) are inserted into the recombinant viral genome. Other components of the viral mRNA of the recombinant gamma retroviral vector can be modified by insertion or removal of naturally occurring sequences (e.g., insertion of an IRES, insertion of a heterologous polynucleotide encoding a polypeptide or inhibitory nucleic acid of interest, replacement of a wild-type promoter with a more efficient promoter from a different retrovirus or virus shuffling, etc.). In some examples, the recombinant gamma retroviral vector may include a modified packaging signal, and/or a Primer Binding Site (PBS), and/or a 5' -enhancer/promoter element in the U3 region of the 5' -Long Terminal Repeat (LTR), and/or a modified 3' -SIN element in the US region of the 3-LTR. These modifications may increase titer and infectivity. A gamma retroviral vector suitable for delivering a heterologous agent (e.g., CAR and/or an immunomodulatory agent, such as a cytokine) may be selected from U.S. patent nos.: 8,828,718, 7,585,676, 7,351,585; U.S. patent application publication No.: US2007/048285; PCT application publication No.: WO2010/113037, WO2014/121005, WO2015/056014; and EP patent No.: vectors as disclosed in EP1757702, EP 1757703.
In some embodiments, polynucleotides encoding heterologous agents (e.g., CARs and/or immunomodulators, such as cytokines) can be packaged into recombinant adeno-associated virus (rAAV) vectors. In some embodiments, polynucleotides encoding antigen receptors, such as CARs, may be packaged into recombinant adeno-associated virus (rAAV) vectors. In some embodiments, polynucleotides encoding immunomodulators, such as cytokines, may be packaged into recombinant adeno-associated virus (rAAV) vectors. In some embodiments, polynucleotides encoding antigen receptors (such as CARs) and immunomodulators (such as cytokines) can be packaged into recombinant adeno-associated virus (rAAV) vectors. Such vectors or viral particles may be designed to utilize any known serotype capsid or combination of serotype capsids. Serotype capsids can include capsids from any identified AAV serotype and variants thereof, such as AAV1, AAV2G9, AAV3, AAV4-4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAVrh10. In some embodiments, the AAV serotype may be or have the sequences described in the following documents: U.S. publication No. US20030138772; pulicherla et al, molecular Therapy,2011, volume 19, phase 6: pages 1070-1078; U.S. patent No.: 6,156,303;7,198,951; U.S. patent publication No.: US2015/0159173 and US2014/0359799; and international patent publication No.: WO 1998/011024, WO2005/033321 and WO2014/14422.
AAV vectors include not only single stranded vectors, but also self-complementary AAV vectors (scAAV). scAAV vectors contain DNA that anneal together to form a double stranded vector genome. scAAV allows for rapid expression in cells by skipping second strand synthesis. rAAV vectors can be made by standard methods in the art, such as by triple transfection in sf9 insect cells or in suspension cell cultures of human cells (such as HEK293 cells).
In some embodiments, non-viral based methods may be used. For example, in some aspects, a vector comprising a polynucleotide can be transferred into a cell by a non-viral method by: physical methods such as needle, electroporation, acoustic perforation, water perforation; chemical carriers such as inorganic particles (e.g., calcium phosphate, silica, gold) and/or chemical methods. In other aspects, synthetic or natural biodegradable agents can be used for delivery, such as cationic lipids, lipid nanoemulsions, nanoparticles, peptide-based carriers, or polymer-based carriers.
In some embodiments, polynucleotides encoding heterologous agents (e.g., CARs and/or immunomodulators, such as cytokines) are designed as messenger RNAs (mrnas) for delivery. In some embodiments, polynucleotides encoding antigen receptors, such as CARs, are designed as messenger RNAs (mrnas) for delivery. In some embodiments, polynucleotides encoding immunomodulators, such as cytokines, are designed as messenger RNAs (mrnas) for delivery. In some embodiments, polynucleotides encoding antigen receptors (such as CARs) and immunomodulators (such as cytokines) are designed as messenger RNAs (mrnas) for delivery.
In some embodiments, a polynucleotide (such as mRNA) encoding a heterologous agent (e.g., a CAR and/or an immunomodulatory agent, such as a cytokine) is incorporated into the lipid nanoparticle. In some embodiments, a polynucleotide (such as mRNA) encoding an antigen receptor (such as a CAR) is incorporated into a lipid nanoparticle. In some embodiments, a polynucleotide (such as mRNA) encoding an immunomodulatory agent (such as a cytokine) is incorporated into a lipid nanoparticle. In some embodiments, a polynucleotide (such as mRNA) encoding an antigen receptor (such as a CAR) and an immunomodulatory agent (such as a cytokine) is incorporated into a lipid nanoparticle. In some embodiments, the formulation is a nanoparticle that may include at least one lipid. The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, and PEGylated lipids. In another aspect, the lipid may be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, and DODMA.
The lipid nanoparticles can be used to deliver encapsulated or associated (e.g., complexed) therapeutic agents, including mRNA. In particular, some nanoparticle compositions are particularly useful for delivering nucleic acids, including messenger RNAs (mrnas), antisense oligonucleotides, plasmid DNA, micrornas (mirnas), miRNA inhibitors (antagonists/enantiomers), complementary RNAs (micrornas) that interfere with messenger RNAs, DNA, multivalent RNAs, dicer substrate RNAs, complementary DNA (cdnas), and self-amplifying RNAs (sarnas). See, for example, U.S. patent No. 10,723,692B2.
Thus, the methods provided herein include methods for delivering nucleic acids (including DNA, RNA, mRNA and self-amplifying RNA (saRNA)) encoding heterologous agents (e.g., cars and/or immunomodulators, such as cytokines) into g-NK cells or g-NK cell-enriched cell compositions or populations. Also provided are methods for delivering nucleic acids encoding antigen receptors, such as cars, including DNA, RNA, mRNA and self-amplifying rnas (sarnas), into g-NK cells or g-NK cell-enriched cell compositions or populations. Methods for delivering nucleic acids encoding immunomodulators, such as cytokines, including DNA, RNA, mRNA and self-amplifying rnas (saRNA), into g-NK cells or g-NK cell-enriched cell compositions or populations are also provided. Also provided are methods for delivering nucleic acids (including DNA, RNA, mRNA and self-amplifying rnas (sarnas)) encoding antigen receptors (such as cars) and immune modulators (such as cytokines) into g-NK cells or g-NK cell-enriched cell compositions or populations. In some embodiments, a heterologous agent (e.g., a CAR and/or an immunomodulatory agent, such as a cytokine) is packaged or incorporated into the lipid nanoparticle to deliver nucleic acids, e.g., DNA, RNA, mRNA and self-amplifying RNA (saRNA). In some embodiments, antigen receptors (such as cars) are packaged or incorporated into lipid nanoparticles to deliver nucleic acids, e.g., DNA, RNA, mRNA and self-amplifying RNA (saRNA). In some embodiments, an immunomodulatory agent (such as a cytokine) is packaged or incorporated into the lipid nanoparticle to deliver nucleic acids, e.g., DNA, RNA, mRNA and self-amplifying RNA (saRNA). In some embodiments, antigen receptors (such as cars) and immunomodulators (such as cytokines) are packaged or incorporated into lipid nanoparticles to deliver nucleic acids, e.g., DNA, RNA, mRNA and self-amplifying RNA (saRNA). In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is self-amplifying RNA (saRNA).
In some embodiments, the mRNA is a self-amplifying mRNA. Self-amplifying RNA (saRNA) is capable of self-amplification by the presence of 5 'and 3' Conserved Sequence Elements (CSE) and the nsP1-4 gene, and subgenomic promoters. See, e.g., bloom, van den Berg and Arbuthnot, GENE THERAPY, 2021. After in situ translation, the nsP1-4 proteins form RdRP complexes that recognize the sheet-like CSE sequences and amplify the sequences contained within the RNA. Introduction of the saRNA into the target cell may be via lipid nanoparticle delivery. In some embodiments, such self-amplifying rnas may have the structural features or components of any of the teachings in international patent application publication No. WO201 1 05799.
In some embodiments, the provided methods involve the use of Lipid Nanoparticles (LNPs) comprising mRNA encoding a heterologous agent (e.g., a CAR and/or an immunomodulatory agent, such as a cytokine). In some embodiments, the provided methods involve the use of Lipid Nanoparticles (LNPs) comprising mRNA encoding an antigen receptor, such as a CAR. In some embodiments, the provided methods involve the use of Lipid Nanoparticles (LNPs) comprising mRNA encoding an immunomodulatory agent, such as a cytokine. In some embodiments, the provided methods involve the use of Lipid Nanoparticles (LNPs) comprising mRNA encoding an antigen receptor (such as a CAR) and an immunomodulatory agent (such as a cytokine). In some embodiments, mRNA encoding a heterologous agent (e.g., CAR and/or an immunomodulatory agent, such as a cytokine) can be produced using methods known in the art (such as in vitro transcription). In some embodiments, mRNA encoding an antigen receptor (such as a CAR) can be produced using methods known in the art (such as in vitro transcription). In some embodiments, mRNA encoding an immunomodulatory agent (such as a cytokine) may be produced using methods known in the art (such as in vitro transcription). In some embodiments, mRNA encoding an antigen receptor (such as a CAR) and an immunomodulatory agent (such as a cytokine) can be produced using methods known in the art (such as in vitro transcription). In some embodiments of the method, the mRNA includes a 5' cap. In some embodiments, the 5 'cap is an altered nucleotide on the 5' end of a primary transcript, such as a messenger RNA. In some aspects, the 5' cap of the mRNA improves one or more of RNA stability and processing, mRNA metabolism, processing and maturation of RNA transcripts in the nucleus, transport of the mRNA from the nucleus to the cytoplasm, mRNA stability, and efficient translation of the mRNA to the protein. In some embodiments, the 5 'cap may be a naturally occurring 5' cap or a cap different from the naturally occurring cap of the mRNA. The 5 'cap may be any 5' cap known to those skilled in the art. In certain embodiments, the 5' cap is selected from the group consisting of an anti-reverse cap analogue (ARCA) cap, a 7-methyl-guanosine (7 mG) cap, a,Analogues, vaccinia caps and analogues thereof. For example, the 5' cap may include, but is not limited to, an anti-reverse cap analogue (ARCA) (US 7074596), 7-methyl-guanosine,Analogs such as Cap 1 analogs (Trilink, san Diego, CA), or enzymatic capping using, for example, vaccinia capping enzymes, and the like. In some embodiments, the mRNA may be polyadenylation. The mRNA may contain various 5 'and 3' untranslated sequence elements to enhance expression of the encoded engineered heterologous agent (e.g., CAR and/or immunomodulator, such as a cytokine) and/or stability of the mRNA itself. The mRNA may contain various 5 'and 3' untranslated sequence elements to enhance expression of the encoded engineered antigen receptor (such as a CAR) and/or stability of the mRNA itself. The mRNA may contain various 5 'and 3' untranslated sequence elements to enhance expression of the encoded engineered immunomodulator (such as cytokines) and/or stability of the mRNA itself. The mRNA may contain various 5 'and 3' untranslated sequence elements to enhance expression of the encoded engineered antigen receptor (such as a CAR) and immune modulator (such as a cytokine) and/or stability of the mRNA itself. Such elements may include, for example, post-translational regulatory elements, such as woodchuck hepatitis virus post-translational regulatory elements.
In some embodiments, the mRNA includes at least one nucleoside modification. mRNA can contain modifications of naturally occurring nucleoside to nucleoside analogs. Any nucleoside analog known in the art is envisioned. Such nucleoside analogs can include, for example, those described in US 8,278,036. In certain embodiments of the method, the nucleoside modification is selected from the group consisting of a uridine to pseudouridine and a uridine to Nl-methyl pseudouridine modification. In a specific embodiment of this method, the nucleoside modification is a modification from uridine to pseudouridine.
LNPs particularly suitable for use in the methods herein include cationic lipids selected from the group consisting of: DLin-DMA (1, 2-dioleyloxy-3-dimethylaminopropane), DLin-MC3-DMA (dioleylmethylene-4-dimethylaminobutyrate), DLin-KC2-DMA (2, 2-dioleyl-4- (2-dimethylaminoethyl) - [1,3] -dioxolane), DODMA (1, 2-dioleyloxy-N, N-dimethyl-3-aminopropane), SS-OP (bis [2- (4- {2- [4- (cis-9-octadecenyloxy) phenylacetyloxy ] ethyl } piperidyl) ethyl ] disulfide) and derivatives thereof. DLin-MC3-DMA and its derivatives are described, for example, in WO 2010144740. DODMA and its derivatives are described, for example, in U.S. Pat. No. 3, 7,745,651 and Mok et al, 1999, biochimica et Biophysica Acta, volume 1419, phase 2: pages 137-150. DLin-DMA and its derivatives are described, for example, in US 7,799,565. DLin-KC2-DMA and its derivatives are described, for example, in US 9,139,554. SS-OP (NOF America Corporation, WHITE PLAINS, NY) is described, for example, in www.nofamerica.com/store/index.phpdispatch=products.view & product_id=962. Additional and non-limiting examples of cationic lipids include methylpyridinyl-dialkylic acid (MPDACA), palmitoyl-oleoyl-norarginine (PONA), guanidino-dialkylic acid (GUADCA), 1, 2-di-0-octadecenyl-3-trimethylammoniopropane (DOTMA), 1, 2-dioleoyl-3-trimethylammoniopropane (DOTAP), bis {2- [ N-methyl-N- (. Alpha. -D-tocopheryl hemisuccinate propyl) amino ] ethyl } disulfide (SS-33/3 AP 05), bis {2- [4- (. Alpha. -D-tocopheryl hemisuccinate ethyl) piperidinyl ] ethyl } disulfide (SS 33/4PE 15), bis {2- [4- (cis-9-octadecenyl ethyl) -1-piperidinyl ] ethyl } disulfide (SS 18/4PE 16), and bis {2- [4- (cis, cis-9, 12-octadecenyl ethyl) -1-piperidinyl ] ethyl } disulfide (SS 18/4PE 13). In other embodiments, the lipid nanoparticle further comprises one or more non-cationic lipids and lipid conjugates.
In some embodiments, the molar concentration of cationic lipid is from about 20% to about 80%, from about 30% to about 70%, from about 40% to about 60%, from about 45% to about 55% or about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or about 80% of the molar concentration of total lipid, wherein the molar concentration of total lipid is the sum of the molar concentrations of cationic lipid, non-cationic lipid and lipid conjugate. In certain embodiments, the molar ratio of cationic lipid to mRNA of the lipid nanoparticle is about 1 to about 20, about 2 to about 16, about 4 to about 12, about 6 to about 10, or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20.
In some embodiments, the lipid nanoparticle utilized in the methods disclosed herein can include at least one non-cationic lipid. In specific embodiments, the molar concentration of the non-cationic lipid is from about 20% to about 80%, from about 30% to about 70%, from about 40% to about 60%, from about 46% to about 50% or about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 48.5%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or about 80% of the molar concentration of the total lipid. In some embodiments, the non-cationic lipid comprises a phospholipid and a steroid.
In some embodiments, phospholipids useful in the lipid nanoparticles described herein include, but are not limited to, l, 2-distearoyl-sn-glycerol-3-phosphorylcholine (DSPC), l, 2-didecanoyl-sn-glycerol-3-phosphorylcholine (DDPC), l, 2-sinapis acyl-sn-glycerol-3-phosphate (sodium salt) (DEPA-NA), 1, 2-sinapis acyl-sn-glycerol-3-phosphorylcholine (DEPC), l, 2-sinapis acyl-sn-glycerol-3-phosphorylethanolamine (DEPE), 1, 2-sinapis acyl-sn-glycerol-3 [ phosphoric acid-racemic- (l-glycerol) (sodium salt) (DEPG-NA), 1, 2-di-oleoyl-sn-glycerol-3-phosphorylcholine (DLOPC), 1, 2-di-lauroyl-sn-glycerol-3-phosphoric acid (sodium salt) (DLPA-NA), 1, 2-di-lauroyl-sn-glycerol-3-phosphorylcholine (DLPA), 1, 2-di-sinapis-glycerol-3-phosphorylcholine (dlna), 1, 2-di-sinapis-glycerol-3-phosphorylcholine (dl-glycerol) (sodium salt) (DEPA-NA), 1, 2-di-stearoyl-sn-3-glycero-phosphate (dl-phosphorylcholine (dl-NA) 1, 2-dilauroyl-sn-glycero-3 [ rac- (l-glycero) (ammonium salt) (DLPG-NH 4), 1, 2-dilauroyl-sn-glycero-3-phosphoserine (sodium salt) (DLPS-NA), 1, 2-dimyristoyl-sn-glycero-3-phospho-choline (DMPA-NA), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1, 2-dimyristoyl-sn-glycero-3 [ phospho-rac- (l-glycero) (sodium salt) (DMPG-NA), 1, 2-dimyristoyl-sn-glycero-3 [ phospho-rac- (l-glycero) (ammonium salt) (DMPG-NH 4), 1, 2-dimyristoyl-sn-glycero-3 [ phospho-rac- (l-glycero-sodium salt) (DMPC), 1, 2-dimyristoyl-sn-glycero-3 [ phospho-3-phospho-ethanolamine (dmna), 1, 2-dioleoyl-sn-glycero-3-phosphoserine (sodium salt) (DOPA-NA), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DPPA-NA), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-dioleoyl-sn-glycero-3 [ phospho-rac- (l-glycero) (sodium salt) (DOPG-NA), 1, 2-dioleoyl-sn-glycero-3-phosphoserine (sodium salt) (DOPA-NA), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DPPA-NA), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-dioleoyl-sn-glycero-3 [ phospho-rac- (l-glycero) (sodium salt) (DPPG-NA), 1, 2-dioleoyl-sn-glycero-3-phosphoserine (sodium salt) (DPPA-NA), 1, 2-dioleoyl-sn-glycero-3-phospho-ethanolamine (DPPE), 1, 2-distearoyl-sn-glycero-3-phosphate (sodium salt) (DSPA-NA), 1, 2-distearoyl-sn-glycero-3-phosphate ethanolamine (DSPE), 1, 2-distearoyl-sn-glycero-3 [ phospho-rac- (l-glycero) (sodium salt) (DSPG-NA), 1, 2-distearoyl-sn-glycero-3 [ phospho-rac- (l-glycero) (ammonium salt) (DSPG-NH 4), 1, 2-distearoyl-sn-glycero-3-phosphate serine (sodium salt) (DSPS-NA), egg yolk PC (EPC), hydrogenated egg yolk PC (HEPC), hydrogenated Soybean PC (HSPC), l-myristoyl-sn-glycero-3-phosphate choline (LY S OPCM YRIS TIC), l-palmitoyl-sn-glycero-3-phosphate choline (LYSOPCPALMITIC), 1-stearoyl-sn-glycero-3-phosphate choline (LYSOPC STEARIC), l-myristoyl-2-palmitoyl-sn-glycero-3-phosphate choline (mpp-myristoyl-3-phospho-glycero-3-phosphate choline (mpp-c), and n-myristoyl-3-phosphate choline (msc), l-palmitoyl-2-myristoyl-sn-glycero-3-phosphorylcholine (PMPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), l-palmitoyl-2-oleoyl-sn-glycero-3 [ phospho-rac- (l-glycero) ] (sodium salt) (POPG-NA), l-palmitoyl-2-stearoyl-sn-glycero-3-phosphorylcholine (PS PC), l-stearoyl-2-myristoyl-sn-glycero-3-phosphorylcholine (SMPC), l-stearoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (SOPC), and l-stearoyl-2-palmitoyl-sn-glycero-3-phosphorylcholine (SPPC). In a specific embodiment, the phospholipid is DSPC. In a specific embodiment, the phospholipid is DOPE. In a specific embodiment, the phospholipid is DOPC.
In some embodiments, the lipid nanoparticle comprises a non-cationic lipid comprising one or more steroids. Steroids useful in the lipid nanoparticles described herein include, but are not limited to, cholestanes (such as cholesterol), cholanes (such as cholic acid), pregnanes (such as progesterone), androstanes (such as testosterone), and estranes (such as estradiol). Other steroids include, but are not limited to, cholesterol (sheep), cholesterol sulfate, chain sterol-d 6, cholesterol-d 7, cholestanol-d 7, chain sterols, stigmasterol, lanosterol, dehydrocholesterol, dihydrolanosterol, zymosterol, cholestanol, zymosterol-d 5, 14-desmethyl-lanosterol-d 6, 8 (9) -dehydrocholesterol, 8 (14) -dehydrocholesterol, diosgenin, DHEA sulfate, DHEA, lanosterol-d 6, dihydrolanosterol-d 7, campesterol-d 6, sitosterol, lanosterol-95, dihydroff-MAS-d 6, zymosterol-d 7, zymosterol, sitosterol, campestanol, campesterol, 7-dehydrodesmosterol, pregnenolone, sitosterol-d 7, dihydrot-MAS, delta 5-avenol, sterols, dihydroff-MAS, 24-methylenecholesterol, cholesterol, and cholesteryl derivatives and cholesterol. In particular embodiments, the lipid nanoparticle comprises cholesterol.
In some embodiments, the lipid nanoparticle comprises a lipid conjugate. Such lipid conjugates include, but are not limited to: ceramide PEG derivatives such as C8 PEG2000 ceramide, C16 PEG2000 ceramide, C8 PEG5000 ceramide, C16 PEG5000 ceramide, C8 PEG750 ceramide and C16 PEG750 ceramide, phosphoethanolamine PEG derivatives such as 16:0 PEG5000PE、14:0 PEG5000 PE、18:0 PEG5000 PE、18:1 PEG5000PE、16:0 PEG3000 PE、14:0 PEG3000 PE、18:0 PEG3000 PE、18:1 PEG3000 PE、16:0 PEG2000 PE、14:0 PEG2000 PE、18:0 PEG2000 PE、18:1 PEG2000 PE 16:0 PEG1000 PE、14:0 PEG1000 PE、18:0 PEG1000 PE、18:1 PEG 1000PE、16:0 PEG750 PE、14:0 PEG750 PE、18:0 PEG750 PE、18:1 PEG750 PE、16:0 PEG550 PE、14:0 PEG550 PE、18:0 PEG550 PE、18:1 PEG550 PE、16:0 PEG350 PE、14:0 PEG350 PE、18:0 PEG350 PE and 18:1 PEG350, sterol PEG derivatives such as Chol-PEG600, and glycerol PEG derivatives such as DMG-PEG5000、DSG-PEG5000、DPG-PEG5000、DMG-PEG3000、DSG-PEG3000、DPG-PEG3000、DMG-PEG2000、DSG-PEG2000、DPG-PEG2000、DMG-PEG1000、DSG-PEG1000、DPG-PEG1000、DMG-PEG750、DSG-PEG750、DPG-PEG750、DMG-PEG550、DSG-PEG550、DPG-PEG550、DMG-PEG350、DSG-PEG350 and DPG-PEG350. In some embodiments, the lipid conjugate is DMG-PEG. In some embodiments, the lipid conjugate is DMG-PEG2000. In some embodiments, the lipid conjugate is DMG-PEG5000.
The skilled person is able to select cationic lipids, non-cationic lipids and/or lipid conjugates comprising lipid nanoparticles and the relative molar ratio of these lipids to each other, such as based on the characteristics of the selected lipids, the nature of the delivery to the intended target cell (e.g. g-NK cell enriched composition) and the nature of the mRNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, the size, charge, pH, pKa, membrane fusion, and toxicity of the lipid selected. Thus, the molar ratio of each individual component can be adjusted accordingly.
The lipid nanoparticles used in the method may be prepared by various techniques known to those skilled in the art. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. patent publication nos. 20040142025 and 20070042031.
In some embodiments, the lipid nanoparticle will have a size in the range of about 25nm to about 500 nm. In some embodiments, the lipid nanoparticle has a size of about 50nm to about 300nm, or about 60nm to about 120 nm. The size of the lipid nanoparticle can be determined by quasi-electro-optical scattering (QELS), such as bloom field, ann.rev.biophys.bioeng., volume 10: 421a150, 1981. A variety of methods are known in the art for producing populations of lipid nanoparticles of a particular size range, such as sonication or homogenization. One such method is described in U.S. patent No. 4,737,323.
In some embodiments, the lipid nanoparticle includes an immune cell targeting molecule, such as a targeting ligand (e.g., an antibody, scFv protein, DART molecule, peptide, aptamer, etc.) anchored on the surface of the lipid nanoparticle that selectively binds the lipid nanoparticle to an NK cell, e.g., g-NK cell.
In some embodiments, the introduction of the nucleic acid may be performed by electroporation. In some embodiments, the nucleic acid is introduced into the g-NK cells via electroporation. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is saRNA. In some embodiments, nucleic acids (such as mRNA or saRNA) are incorporated into lipid nanoparticles for delivery by electroporation.
III Gene editing
In some embodiments, the g-NK cells can be genetically engineered by gene editing to alter, e.g., reduce, the expression of one or more genes by the g-NK cells, thereby altering one or more properties or activities of the NK cells. For example, policies for gene editing may include one or more policies that: reducing allophase killing (suicide) due to expression of the target antigen on g-NK cells; reducing undesirable immune reactivity that can lead to graft versus host disease (GvHD), particularly when infused into immunocompromised HLA-matched recipients, or in some cases also when infused into HLA-mismatched recipients; or to reduce immunosuppression of host factors, particularly in tumor microenvironments. In some embodiments, engineered g-NK cells (including those engineered by one or more gene editing strategies) exhibit enhanced NK cell response characteristics, such as enhanced target recognition, enhanced NK cell response level and/or duration, improved NK cell survival, delayed NK cell depletion, and/or enhanced target recognition, as compared to similar NK cells that have not undergone gene editing.
In some embodiments, the g-NK cells are generated by gene editing to disrupt or knock out the gene encoding the FcR gamma chain. In some embodiments, NK cells are genetically engineered to reduce or eliminate expression or activity of human FcR gamma chain proteins. Delta in some embodiments, the genetic disruption results in an insertion, deletion, or mutation in the gene, such as a frame shift mutation in the open reading frame and/or a premature stop codon. Methods for knocking out or disrupting FcR gamma chains in NK cells are described in PCT publication nos. WO2018/148462 and Liu et al, iScience,2020, volume 23: on page 101709.
Those of ordinary skill in the art will appreciate that there are many suitable methods for disrupting the FcR gamma chain gene. For example, the entire locus, such as the fcrγδ locus, may be deleted. In some cases, it may also be appropriate to delete a portion of the gene (e.g., an exon or domain). Specifically, the ITAM signaling domain of fcrγ may be deleted. Alternatively, the provided methods further comprise introducing one or more amino acid substitutions into a locus, such as an fcrγ locus, such as an inactivating mutation. In some embodiments, a stop codon may be introduced into an mRNA, such as fcrγ mRNA, to produce a truncated and/or inactivated form of an expressed gene, such as an fcrγ signaling adapter. In some embodiments, regulatory elements of a gene (such as the FcR gamma gene) may also be mutated or deleted in order to reduce expression, activity and/or signaling of the FcR gamma signaling adapter.
In some embodiments, gene disruption may be performed in mammalian cells using site-specific endonucleases. Endonucleases that allow for site-specific deletion of genes are well known in the art and may include TAL nucleases, meganucleases, zinc finger nucleases, CRISPR/Cas (e.g., cas 9) and Argonaute. Methods for producing engineered site-specific endonucleases are known in the art. The site-specific endonucleases can be engineered to recognize, delete, or modify specific genes, such as FcR gamma chain genes.
In some embodiments, the provided g-NK cells are engineered by editing the genome of the g-NK cells. In some embodiments, the editing of the genome may be performed in a method of enriching a subpopulation of g-NK cells from a starting sample of NK cells. Thus, it will be appreciated that the provided methods do not require the selection of only editing the genome of g-NK cells that have been selected for FcR gamma chain deficient NK cells (or only editing the genome of g-NK cells that have been selected or identified by g-NK surrogate marker profiling), but may involve gene editing of NK cell compositions that will or have preferentially expanded g-NK cells or enriched g-NK cells. Thus, the final cell composition enriched in g-NK cells includes g-NK cells that incorporate a heterologous antigen receptor (e.g., CAR), a heterologous cytokine (e.g., an interleukin or membrane-bound interleukin such as IL-15 or IL-21) or a heterologous antigen receptor and cytokine and that have been genetically edited. Exemplary methods of preparing and amplifying g-NK cell enriched compositions are provided in section V.
In some embodiments, editing of the genome may be performed at any suitable time during the method of amplifying g-NK cells, such as described in section V. In some embodiments, gene editing is performed after selecting cells from a subject (e.g., selecting or enriching cells of CD3 Negative of CD57 Positive and negative or CD3 Negative of CD56 Positive and negative ) and prior to incubating or culturing the selected or enriched cells with feeder cells (e.g., HLA-E expressing feeder cells) for proliferation or expansion of NK cells. In some embodiments, gene editing is performed after incubation or culture with feeder cells (e.g., HLA-E expressing feeder cells) and thus after the selected or enriched cells have proliferated or expanded. In some embodiments, gene editing is performed sequentially in any order with the method for introducing an antigen receptor (e.g., CAR) and/or an immunomodulatory agent (e.g., a cytokine, such as a secretable or membrane-bound interleukin, e.g., IL-15 or IL-21). In some embodiments, gene editing and the method for introducing an antigen receptor (such as a CAR) are performed sequentially in any order. In some embodiments, gene editing and methods for introducing an immunomodulatory agent (such as a cytokine) are performed sequentially in any order. In some embodiments, gene editing is performed sequentially in any order with the method for introducing an antigen receptor (such as a CAR) and an immunomodulatory agent (such as a cytokine).
Methods of knocking out target gene expression include, but are not limited to, zinc Finger Nucleases (ZFNs), tale effector domain nucleases (TALENs) and CRIPSR/Cas systems. Such methods generally comprise administering one or more polynucleotides encoding one or more nucleases to a cell such that the nucleases mediate modification of an endogenous gene (e.g., in the presence of one or more donor sequences) such that the donor is integrated into the endogenous gene targeted by the nucleases. Integration of one or more donor molecules occurs via Homology Directed Repair (HDR) or via non-homologous end joining (NHEJ) related repair. In certain embodiments, one or more pairs of nucleases are used, which may be encoded by the same or different nucleic acids.
In some embodiments, gene editing is performed using Zinc Finger Nucleases (ZFNs). ZFNs are fusion proteins comprising a non-specific cleavage domain (N) of a fokl endonuclease and a Zinc Finger Protein (ZFP). A pair of ZNFs are involved in identifying a specific locus in a target gene, one identifying an upstream sequence of a site to be modified, the other identifying a downstream sequence, and nuclease portions of ZFNs cleave at the specific locus, resulting in a knockout of the target gene. Methods for reducing gene expression using ZFNs are well known, for example, U.S. patent No. 9,045,763 and Durai et al ,Zinc Finger Nucleases:Custom-Designed Molecular Scissors for Genome Engineering of Plant and Mammalian cells,Nucleic Acid Research,, volume 33, 18: pages 5978-5990, 2005, the disclosures of which are incorporated herein by reference in their entirety.
In some embodiments, gene editing is performed using a transcription activator-like effector nuclease (TALEN). TALENs are similar to ZFNs in that they bind in pairs around a genomic locus and direct the same nonspecific nuclease FoKI to cleave the genome at a specific locus, but each domain recognizes a single nucleotide, rather than a DNA triplet. Methods for reducing gene expression using ZFNs are also well known, for example, U.S. Pat. No. 9,005,973 and Christian et al, TARGETING DNA Double-Strand Breaks with TAL Effector Nucleases, genetics, volume 186, phase 2: pages 757-761, the disclosures of which are incorporated herein by reference in their entirety.
In some embodiments, gene editing is performed using RNA-guided nucleases. In some embodiments, the RNA-guided nuclease is an RNA-guided DNA endonuclease. In some embodiments, the RNA-guided nuclease is a CRISPR nuclease. Non-limiting examples of RNA-directed nucleases include any of the nucleases described in PCT publication No. WO 2020/1684300 (e.g., table 2 therein). In some embodiments, the RNA-guided nuclease is a Cas9 or Cas12 nuclease. In some embodiments, the RNA-guided nuclease is Cpfl (Cas 12 a). In some embodiments Cpfl is an amino acid coccus Cpfl (AsCpfl).
In some embodiments, gene editing is performed with RNA-guided nucleases and guide RNAs (grnas) alone. The two components form a complex that is capable of associating with a particular nucleic acid sequence and editing DNA in or around the nucleic acid sequence, for example by performing one or more of a single strand break (SSB or split), a Double Strand Break (DSB), and/or a point mutation. In some embodiments, the gRNA includes crRNA and optionally tracrRNA. In some embodiments, the RNA-guided nuclease (e.g., cas9 or Cas 12) and the one or more grnas form a Ribonucleoprotein (RNP) complex that associates (i.e., targets) and cleaves a specific locus that is complementary to the targeting (or spacer) sequence of the gRNA (e.g., crRNA). In some embodiments, the Cas is a Cas9 nuclease, e.g., from streptococcus pyogenes. It is to be understood that endonucleases used herein are not limited to Cas9 (SpCas 9) of streptococcus pyogenes, which is commonly used to synthesize Cas 9. In one aspect, cas9 may be from a different bacterial source. Substitution of Cas9 can also be used to increase targeting specificity, requiring the use of fewer grnas. Thus, for example, cas can be derived from staphylococcus aureus (SaCas 9), amino acid coccus (AsCpf) genus, prevotella genus derived from spiralis (Lachnospiracase bacterium) genus and clustered regularly interspaced short palindromic repeats (Cpf 1) (LbCpf 1), neisseria meningitidis (NmCas 9), streptococcus thermophilus (StCas), campylobacter jejuni (CjCas 9), enhanced SpCas9 (eSpCas 9), spCas9-HF1, fokl fusion dCas9, or expanded Cas9 (xCas 9). Additionally, other cas endonucleases can be used instead of cas9 systems, such as CasX, casY, casl, cas4, csn2, cas13a, cas13b, cas13C, cas13d, C2C1, or C2C3, or any other type of engineered cas protein including primer editing.
In some embodiments, the genome editing system containing an RNA-guided nuclease (e.g., cas) and gRNA is implemented in certain embodiments as a protein/RNA complex (ribonucleoprotein or RNP) that is introduced into the cell to be edited. In some embodiments, the RNP complex is introduced into the cell in an encapsulating agent (such as a lipid or polymer microparticle or nanoparticle, micelle, or liposome). In certain embodiments, a genome editing system containing an RNA-guided nuclease (e.g., cas) and a gRNA is implemented as one or more nucleic acids encoding the RNA-guided nuclease and the guide RNA component. For example, in certain embodiments, the genome editing system is implemented as one or more vectors, e.g., viral vectors, such as adeno-associated viruses, that include such nucleic acids.
In functional terms, RNA-guided nucleases are defined as nucleases: (a) interacts with (e.g., complexes with) the gRNA; and (b) associating with and optionally cleaving or modifying a target region of DNA, which target region comprises (i) a sequence complementary to a targeting domain of the gRNA and optionally (ii) an additional sequence known as a "protospacer adjacent motif" or "PAM", together with the gRNA. PAM sequences are named for their sequential relationship to a "protospacer" sequence that is complementary to a gRNA targeting domain (or "spacer"). The PAM sequence and the protospacer sequence together define a target region or sequence for a particular RNA-guided nuclease/gRNA combination. Various RNA-guided nucleases may require a different sequential relationship between PAM and protospacer. For example, cas9 nucleases recognize PAM sequences that are 3 'of the protospacer, while Cpfl, on the other hand, typically recognize PAM sequences that are 5' of the protospacer. In addition to recognizing the specific sequential orientation of PAM and protospacer, RNA-guided nucleases can recognize specific PAM sequences. For example, staphylococcus aureus Cas9 recognizes PAM sequence NNGRRT or NNGRRV, where the N residue is immediately 3' of the region recognized by the gRNA targeting domain. Streptococcus pyogenes Cas9 recognizes the NGG PAM sequence. And new inland francisco (f.noviocada) Cpfl recognizes the TTN PAM sequence. PAM sequences for a variety of RNA-guided nucleases have been identified and Shmakov et al, 2015, molecular Cell, volume 60: pages 385-397, 5, 11, 2015 describe a strategy for identifying new PAM sequences.
It is to be understood and contemplated herein that the use of a particular Cas can alter the PAM sequence used by the Cas endonuclease (or alternative enzyme) for screening a target. Suitable PAM sequences, as used herein, include NGG(SpCas9 PAM)、NNGRRT(SaCas9 PAM)、NNNNGATT(NmCAs9 PAM)、NNNNRYAC(CjCas9 PAM)、NNAGAAW(St)、TTTV(LbCpf1 PAM and AsCpf1 PAM); TYCV (LbCpf 1 PAM variants and AsCpf1 PAM variants); wherein N can be any nucleotide; v= A, C or G; y=c or T; w=a or T; and r=a or G.
In some embodiments, the gRNA facilitates specific association (or "targeting") of an RNA-guided nuclease (e.g., cas, such as Cas9 or Cpfl) with a target sequence (such as a genomic sequence in a cell). The grnas may be single-molecular (including single RNA molecules, and alternatively referred to as chimeric), or modular (including more than one, typically two separate RNA molecules, such as CRISPR RNA (crRNA) and tracrRNA, which are typically associated with each other, e.g., by double-stranded binding). Guide RNAs, whether single-molecule or modular, include a "targeting domain" that is fully or partially complementary to a target domain within a target sequence, such as a DNA sequence in the genome of a cell that is desired to be edited. For example, with respect to Cas9, crRNA is a guide RNA that provides a targeting domain that is a nucleotide sequence complementary to a target DNA, and may also include tracr RNA that serves as a binding scaffold for Cas nucleases. With respect to Cpfl, which induces double-stranded DNA breaks under the direction of a single crRNA, there is no need for a tracrRNA, but rather the crRNA includes a 5' handle structure that is involved in Cpfl recognition and a guide fragment that interacts with the targeted DNA sequence through complementary binding. The targeting domain is typically 10-30 nucleotides in length, and in certain embodiments 16-24 nucleotides in length (e.g., 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length).
In some embodiments, a gRNA (in some cases crRNA) is any polynucleotide sequence that has sufficient complementarity to a target nucleic acid sequence to hybridize to the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid targeting complex to the target nucleic acid sequence. In some embodiments, the degree of complementarity is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or greater when aligned, optionally using a suitable alignment algorithm. The optimal alignment may be determined by using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, the Burrows-Wheeler transformation-based algorithm (e.g., burrows WHEELER ALIGNER), clustal 1W, clustal X, BLAT, and other algorithms known to those skilled in the art. The ability of the guide sequence (within the nucleic acid targeting guide RNA) to direct sequence specific binding of the nucleic acid targeting complex to the target nucleic acid sequence can be assessed by any suitable assay. For example, components of the nucleic acid-targeted CRISPR system (including the guide sequences to be tested) sufficient to form a nucleic acid-targeted complex can be provided to a host cell having a corresponding target nucleic acid sequence, such as by transfection with a vector encoding the components of the nucleic acid-targeted complex, followed by evaluation of preferential targeting (e.g., cleavage) within the target nucleic acid sequence. Similarly, cleavage of a target nucleic acid sequence can be assessed in a test tube by providing the target nucleic acid sequence, components of the nucleic acid targeting complex (including the guide sequence to be tested and a control guide sequence different from the test guide sequence), and comparing the binding or cleavage rate at the target sequence between the test guide sequence reaction and the control guide sequence reaction.
Methods for designing gRNAs are known to those skilled in the art (see, e.g., cui et al, 2018, INTERDISCIPLINARY SCIENCES: computational LIFE SCIENCES, volume 10:pages 455-465; PCT publication No. WO 2019/010384). Methods for selecting and validating target sequences and off-target assays have been previously described, for example, in Mali, hsu, fu et al, 2014, nat biotechnol, volume 32, phase 3: pages 279-84; heigwer et al, 2014, nat methods, volume 11, phase 2: pages 122-123; bae et al, 2014, bioenformats, volume 30, phase 10: pages 1473-1475; and Xiao a et al, 2014, bioinformatics, volume 30, phase 8: pages 1180-1182. As a non-limiting example, gRNA design may involve the use of software tools to optimize the selection of potential target sequences corresponding to a user target sequence, e.g., to minimize overall off-target activity of the entire genome. Although off-target activity is not limited to cleavage, cleavage efficiency at each off-target sequence can be predicted, for example, using an experimentally derived weighting scheme.
For example, a guide RNA comprising a targeting sequence of RNA nucleotides will comprise an RNA sequence corresponding to the targeting domain sequence provided as a DNA sequence, and this contains uracil instead of thymine nucleotides. For example, a guide RNA that includes a targeting domain sequence of RNA nucleotides and is described by a DNA sequence that includes a thymine molecule will have the same targeting domain of the corresponding RNA sequence, but includes uracil instead of thymine. It will be apparent to those skilled in the art that such targeting sequences will be linked to a suitable guide RNA scaffold, such as a crRNA scaffold sequence or a chimeric crRNA/tracerRNA scaffold sequence. Suitable gRNA scaffold sequences are known to those of ordinary skill in the art. For example, for Cpfl, suitable scaffold sequences include sequence U A AUUU CU ACUCUU GU AG AU (SEQ ID NO: 16) that is added to the 5' end of the targeting domain.
In some embodiments, the effort to enhance clinical ADCC response to antibodies (including MM antibodies) has been faced with difficulty, as NK cells also express certain antigens identical to tumor targets. Such antigens include, for example, CD38 and SLAMF7. Thus, when NK cell therapy is combined with antibodies directed against a target antigen (e.g., up to Lei Tuoyou mab and erlotinib for targeting CD38 and SLAMF7, respectively), or when NK cells express the CARs provided herein for the target antigen, the therapy can not only target cancer, but can also deplete the patient's NK cell population. For example, high CD38 expression results in rapid depletion of NK cells, particularly early in the course of treatment with Lei Tuoyou mab, thus largely eliminating the source of such innate immune cells, which could potentially drive more thorough tumor eradication.
In some embodiments, NK cells are edited to reduce expression of target antigens that are known or suspected to be also expressed by NK cells at certain levels. In some embodiments, gene editing is performed with a gRNA targeting a target antigen that is known or suspected to be expressed at a level by NK cells. In some embodiments, the NK cells express a CAR against CD38, and CD38 expression is reduced or eliminated in the NK cells. In some embodiments, the grnas used in the present disclosure are CD 38-targeted grnas (see, e.g., WO2019/222503, WO2021/087466, and WO2021/113853 for exemplary CD 38-targeted grnas).
In some embodiments, the gRNA targets molecules that are involved in the immunoreactivity of NK cells. In some embodiments, HLA class I expression is reduced on the surface of the engineered g-NK cells. The Human Leukocyte Antigen (HLA) system is a gene complex encoding Major Histocompatibility Complex (MHC) proteins in humans. HLA class I proteins have both long alpha and short beta B2M chains. Few HLA class I can be expressed without B2M, and expression of B2M is necessary for HLA class I proteins to present peptides from inside the cell. The present disclosure provides g-NK cells engineered to reduce B3M expression. Thus, these cells bypass immune surveillance and attach through cytotoxic T cells. In some embodiments, the gRNA used in the present disclosure is a β2 microglobulin (B2M) -targeted gRNA (see, e.g., WO 2020/1683300, WO2018/064694, WO2015/161276, or WO2017/152015 for exemplary B2M-targeted grnas).
In some embodiments, the gRNA targets molecules involved in immunosuppression of NK cell activity. Suitably, the engineered NK cell comprises reduced or no checkpoint inhibitory receptor function. Suitably, checkpoint inhibitory receptors with reduced or no function include one or more or all of CD96 (TACTILE), CD 152 (CTLA 4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC 7), SIGLEC9, TIGIT and/or TIM-3. Suitably, NK cells comprise reduced or no checkpoint inhibitory receptor function for two or more checkpoint inhibitory receptors. Suitably, the two or more checkpoint inhibitory receptors comprise CD96 (TACTILE), CD 152 (CTLA 4) or CD328 (SIGLEC 7) or CD279 (PD-1).
In some embodiments, the grnas used in the present disclosure are TIGIT-targeted grnas (see, e.g., WO 2020/1684300 for exemplary TIGIT-targeted grnas). In some embodiments, the grnas used in the present disclosure are PD-1-targeted grnas (see, e.g., WO2015/161276 or WO2017/152015 for exemplary PD-1-targeted grnas).
In some embodiments, the grnas used in the present disclosure are grnas that target adenosine receptors, such as the adenosine A2a receptor (ADORA 2 a) (see, e.g., WO 2020/1684300 for exemplary ADORA2 a-targeted grnas). In some embodiments, the grnas used in the present disclosure are grnas that target tgfβ receptors such as tgfβr2 (see, e.g., WO 2020/1684300 for exemplary tgfβr2-targeted grnas). In some embodiments, the grnas used in the present disclosure are grnas that target genes encoding cytokine-induced SH 2-containing proteins (CISH) (see, e.g., WO 2020/1684300 for exemplary CISH-targeted grnas).
In some embodiments, the RNA-guided nuclease-encoding and/or gRNA-encoding DNA can be delivered by, for example, a vector (e.g., a viral or non-viral vector), a non-vector-based method (e.g., using naked DNA or a DNA complex), or a combination thereof. In some embodiments, the nucleic acid encoding an RNA-guided nuclease (e.g., cas) and/or gRNA is delivered by AAV. Nucleic acids for gene editing may be delivered directly to cells as naked DNA or RNA (e.g., by transfection or electroporation), or may be conjugated to a molecule that facilitates uptake by target cells (e.g., N-acetylgalactosamine).
In some embodiments, the RNA-guided nuclease and the gRNA are delivered into the cell as Ribonucleoprotein (RNP) complexes. In some embodiments, the Cas and the gRNA are purified separately and then assembled to form the RNP. In some embodiments, one or more RNP complexes are delivered to the cell sequentially or simultaneously in any order. In some embodiments, the RNP complex is delivered into the cell by electroporation. In some embodiments, the RNP complex is delivered into the cell using a lipid nanoparticle.
In one non-limiting example, to make an RNP complex, the crRNA and tracrRNA may be mixed between about 50 μm and about 500 μm (e.g., 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 35, 375, 400, 425, 450, 475, or 500 μm), preferably between 100 μm and about 300 μm, most preferably about 200 μm at a concentration ratio of 1:1, 2:1, or 1:2, for about 5 minutes at 95 ℃ to form the crRNA: tracrRNA complex (i.e., guide RNA). The tracrRNA complex can then be mixed with a final dilution of Cas endonuclease (such as Cas 9) between about 20 μm and about 50 μm (e.g., 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, or 50 μm).
In specific embodiments, the RNP complex is introduced into NK cells (such as expanded NK cells enriched for g-NK cells as described in section V) by electroporation. Electroporation is a technique in which an electric field is applied to cells to increase cell membrane permeability. Application of an electric field causes a charge gradient across the membrane that attracts charged molecules (such as nucleic acids) across the cell membrane. Thus, in one aspect, disclosed herein is a method of genetically modifying NK cells, the method comprising: obtaining a guide RNA (gRNA) specific for a target DNA sequence in NK cells; b) A Ribonucleoprotein (RNP) complex is introduced into the target NK cell via electroporation, the complex comprising a Cas endonuclease (e.g., cas 9) complexed with a corresponding CRISPR/Cas guide RNA that hybridizes to a target sequence within the genomic DNA of the NK cell.
In some embodiments, following the introduction of NK cells (e.g., electroporation), the now modified NK cells may be proliferated in media comprising HLA-expressing feeder cells, generally irradiated feeder cells, and cytokines (e.g., IL-2 and IL-21), as described in section V, such as under conditions that induce stimulation, proliferation, or expansion of g-NK cell enriched NK cells. Genetically engineered cells thus retain viability and proliferative potential because they can be expanded using irradiated feeder cells after electroporation. It is to be understood and contemplated herein that the incubation period may be between 1 day and 14 days after introduction of the RNP complex, such as after electroporation (i.e., 1,2, 3,4,5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days), such as between 3 days and 7 days, for example, between 4 days and 6 days. In some aspects, the medium used to culture the engineered NK cells can also include cytokines, e.g., IL-2, IL-12, IL-15, IL-18, and/or IL-21, such as described in section V. In some embodiments, the medium contains IL-2 and IL-21.
IV composition and pharmaceutical formulation
Provided herein are compositions comprising engineered g-NK cells. In some embodiments, the engineered g-NK cells of the composition express the CAR. The composition may include a plurality of g-NK cells that express the CAR.
In some embodiments, the engineered g-NK cells of the composition express an immunomodulatory agent (e.g., a cytokine), which may be secreted or membrane-bound. The composition may include a plurality of g-NK cells expressing an immunomodulatory agent.
In some embodiments, the engineered g-NK cells of the composition express the CAR and an immunomodulatory agent (e.g., a cytokine), which may be secreted or membrane-bound. The composition can include a plurality of g-NK cells that express both the CAR and the immunomodulator. In some embodiments, the engineered NK cells comprise a plurality of engineered g-NK cells. In some embodiments, greater than 50% or greater than about 50% of the engineered NK cells are g-NK cells. In some embodiments, greater than 60% or greater than about 60% of the engineered NK cells are g-NK cells. In some embodiments, greater than 70% or greater than about 70% of the engineered NK cells are g-NK cells. In some embodiments, greater than 80% or greater than about 80% of the engineered NK cells are g-NK cells. In some embodiments, greater than 90% or greater than about 90% of the engineered NK cells are g-NK cells. In some embodiments, greater than 95% or greater than about 95% of the engineered NK cells are g-NK cells.
In some embodiments, the composition comprises greater than 50% or greater than about 50% g-NK cells. In some embodiments, the composition comprises greater than 60% or greater than about 60% g-NK cells. In some embodiments, the composition comprises greater than 70% or greater than about 70% g-NK cells. In some embodiments, the composition comprises greater than 80% or greater than about 80% g-NK cells. In some embodiments, the composition comprises greater than 90% or greater than about 90% g-NK cells. In some embodiments, the composition comprises greater than 95% or greater than about 95% g-NK cells.
In some embodiments, the plurality of NK cells of the composition comprises greater than 50% or greater than about 50% g-NK cells. In some embodiments, the plurality of NK cells of the composition comprises greater than 60% or greater than about 60% g-NK cells. In some embodiments, the plurality of NK cells of the composition comprises greater than 70% or greater than about 70% g-NK cells. In some embodiments, the plurality of NK cells of the composition comprises greater than 80% or greater than about 80% g-NK cells. In some embodiments, the plurality of NK cells of the composition comprises greater than 90% or greater than about 90% g-NK cells. In some embodiments, the plurality of NK cells of the composition comprises greater than 95% or greater than about 95% g-NK cells.
In some embodiments, greater than 20% or greater than about 20% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than 30% or greater than about 30% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than 40% or greater than about 40% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than 50% or greater than about 50% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than 60% or greater than about 60% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than 70% or greater than about 70% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than 80% or greater than about 80% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than 90% or greater than about 90% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than 95% or greater than about 95% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR.
In some embodiments, greater than 20% or greater than about 20% of the g-NK cells in the composition comprise the heterologous nucleic acid encoding the CAR. In some embodiments, greater than 30% or greater than about 30% of the g-NK cells in the composition comprise the heterologous nucleic acid encoding the CAR. In some embodiments, greater than 40% or greater than about 40% of the g-NK cells in the composition comprise the heterologous nucleic acid encoding the CAR. In some embodiments, greater than 50% or greater than about 50% of the g-NK cells in the composition comprise the heterologous nucleic acid encoding the CAR. In some embodiments, greater than 60% or greater than about 60% of the g-NK cells in the composition comprise the heterologous nucleic acid encoding the CAR. In some embodiments, greater than 70% or greater than about 70% of the g-NK cells in the composition comprise the heterologous nucleic acid encoding the CAR. In some embodiments, greater than 80% or greater than about 80% of the g-NK cells in the composition comprise the heterologous nucleic acid encoding the CAR. In some embodiments, greater than 90% or greater than about 90% of the g-NK cells in the composition comprise the heterologous nucleic acid encoding the CAR. In some embodiments, greater than 95% or greater than about 95% of the g-NK cells in the composition comprise the heterologous nucleic acid encoding the CAR.
In some embodiments, greater than 20% or greater than about 20% of the total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which may secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 30% or greater than about 30% of the total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which may secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 40% or greater than about 40% of the total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which may secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 50% or greater than about 50% of the total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which may secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 60% or greater than about 60% of the total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which may secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 70% or greater than about 70% of the total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which may secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 80% or greater than about 80% of the total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which may secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 90% or greater than about 90% of the total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which may secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 95% or greater than about 95% of the total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which may secrete a cytokine or a membrane-bound cytokine).
In some embodiments, greater than 20% or greater than about 20% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 30% or greater than about 30% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 40% or greater than about 40% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 50% or greater than about 50% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 60% or greater than about 60% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 70% or greater than about 70% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 80% or greater than about 80% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 90% or greater than about 90% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 95% or greater than about 95% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine).
In some embodiments, greater than 20% or greater than about 20% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 30% or greater than about 30% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 40% or greater than about 40% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 50% or greater than about 50% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 60% or greater than about 60% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 70% or greater than about 70% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 80% or greater than about 80% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 90% or greater than about 90% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 95% or greater than about 95% of the total cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine).
In some embodiments, greater than 20% or greater than about 20% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 30% or greater than about 30% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 40% or greater than about 40% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 50% or greater than about 50% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 60% or greater than about 60% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 70% or greater than about 70% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 80% or greater than about 80% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 90% or greater than about 90% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine). In some embodiments, greater than 95% or greater than about 95% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a cytokine, which can secrete a cytokine or a membrane-bound cytokine).
In particular, provided are compositions having g-NK cell enriched cell compositions. In some embodiments, the composition used in the provided methods contains g-NK cells that are expanded NK cells, such as produced by any one of the provided methods. In some embodiments, the composition contains NKG2C Positive and negative cells or a subpopulation thereof. In some embodiments, the composition comprises NKG2a Negative of cells or a subpopulation thereof. In some embodiments, the composition contains NKG2C Positive and negative /NKG2A Negative of cells or a subpopulation thereof.
In some embodiments, the composition comprises between about 5% and 99% NKG2C Positive and negative cells or a subpopulation thereof, or any percentage between 5% and 99% (inclusive) of NKG2C Positive and negative cells or subpopulations thereof. In some embodiments, the composition may include an increased or greater percentage of NKG2C Positive and negative cells or subpopulations thereof relative to total NK cells or total cells as compared to the percentage of naturally occurring NKG2C Positive and negative cells or subpopulations thereof relative to total NK cells or total cells in the subject from which the cells were isolated. In some embodiments, the percentage increase is at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, or more.
In some embodiments, the composition may comprise at least or at least about 20%, at least or at least about 30%, at least or at least about 40%, at least or at least about 50%, at least or at least about 60%, at least or at least about 65%, at least or at least about 70%, at least or at least about 75%, at least or at least about 80%, at least or at least about 81%, at least or at least about 82%, at least or at least about 83%, at least or at least about 84%, at least or at least about 85%, at least or at least about 86%, at least or at least about 87%, at least or at least about 88%, at least or at least about 89%, at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, or at least or substantially 100% NKG2C Positive and negative cells or subpopulations thereof. In some embodiments, the composition comprises more than 50% NKG2C Positive and negative cells or a subpopulation thereof. In another embodiment, the composition comprises more than 60% NKG2C Positive and negative cells or a subpopulation thereof. In another embodiment, the composition comprises more than 70% NKG2C Positive and negative cells or a subpopulation thereof. In another embodiment, the composition comprises more than 80% NKG2C Positive and negative cells or a subpopulation thereof. In some embodiments, provided compositions include compositions wherein the NKG2C Positive and negative cells or subpopulations thereof constitute at least or at least about 60%, at least or at least about 70%, at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95% or more of the cells in the composition or NK cells in the composition. In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than 60%, greater than 70%, or greater than 70%, greater than 80%, or greater than 80%, greater than 90%, or greater than 95% of the NKG2C Positive and negative cells in the composition comprise a heterologous nucleic acid encoding the CAR. In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the NKG2C Positive and negative cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than 60%, greater than 70%, or greater than 70%, greater than 80%, or greater than 80%, greater than 90%, or greater than 95% of the NKG2C Positive and negative cells in the composition comprise a heterologous nucleic acid encoding the CAR and an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine).
In some embodiments, the composition comprises between about 5% and 99% NKG2a Negative of cells or a subpopulation thereof, or any percentage between 5% and 99% (inclusive) of NKG2a Negative of cells or subpopulations thereof. In some embodiments, the composition may include an increased or greater percentage of NKG2a Negative of cells or subpopulations thereof relative to total NK cells or total cells as compared to the percentage of naturally occurring NKG2a Negative of cells or subpopulations thereof relative to total NK cells or total cells in the subject from which the cells were isolated. In some embodiments, the percentage increase is at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, or more.
In some embodiments, the composition may comprise at least or at least about 20%, at least or at least about 30%, at least or at least about 40%, at least or at least about 50%, at least or at least about 60%, at least or at least about 65%, at least or at least about 70%, at least or at least about 75%, at least or at least about 80%, at least or at least about 81%, at least or at least about 82%, at least or at least about 83%, at least or at least about 84%, at least or at least about 85%, at least or at least about 86%, at least or at least about 87%, at least or at least about 88%, at least or at least about 89%, at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, or at least or substantially 100% NKG2a Negative of cells or subpopulations thereof. In some embodiments, the composition comprises more than 50% NKG2a Negative of cells or a subpopulation thereof. In another embodiment, the composition comprises more than 60% NKG2a Negative of cells or a subpopulation thereof. In another embodiment, the composition comprises more than 70% NKG2a Negative of cells or a subpopulation thereof. In another embodiment, the composition comprises more than 80% NKG2a Negative of cells or a subpopulation thereof. In some embodiments, provided compositions include compositions wherein the NKG2a Negative of cells or subpopulations thereof constitute at least or at least about 60%, at least or at least about 70%, at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95% or more of the cells in the composition or NK cells in the composition. In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than 60%, greater than 70%, or greater than 70%, greater than 80%, or greater than 80%, greater than 90%, or greater than 95% of the NKG2a Negative of cells in the composition comprise a heterologous nucleic acid encoding the CAR. In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the NKG2a Negative of cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than 60%, greater than 70%, or greater than 70%, greater than 80%, or greater than 80%, greater than 90%, or greater than 95% of the NKG2a Negative of cells in the composition comprise a heterologous nucleic acid encoding the CAR and an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine).
In some embodiments, the composition comprises between about 5% and 99% NKG2C Positive and negative NKG2A Negative of cells or a subpopulation thereof, or any percentage between 5% and 99% (inclusive) of NKG2C Positive and negative NKG2A Negative of cells or subpopulations thereof. In some embodiments, the composition may include an increased or greater percentage of NKG2C Positive and negative NKG2A Negative of cells or subpopulations thereof relative to total NK cells or total cells as compared to the percentage of naturally occurring NKG2C Positive and negative NKG2A Negative of cells or subpopulations thereof relative to total NK cells or total cells in the subject from which the cells were isolated. In some embodiments, the percentage increase is at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, or more.
In some embodiments, the composition may comprise at least or at least about 20%, at least or at least about 30%, at least or at least about 40%, at least or at least about 50%, at least or at least about 60%, at least or at least about 65%, at least or at least about 70%, at least or at least about 75%, at least or at least about 80%, at least or at least about 81%, at least or at least about 82%, at least or at least about 83%, at least or at least about 84%, at least or at least about 85%, at least or at least about 86%, at least or at least about 87%, at least or at least about 88%, at least or at least about 89%, at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, or at least or substantially 100% NKG2C Positive and negative NKG2A Negative of cells or subpopulations thereof. In some embodiments, the composition comprises more than 50% NKG2C Positive and negative NKG2A Negative of cells or a subpopulation thereof. In another embodiment, the composition comprises more than 60% NKG2C Positive and negative NKG2A Negative of cells or a subpopulation thereof. In another embodiment, the composition comprises more than 70% NKG2C Positive and negative NKG2A Negative of cells or a subpopulation thereof. In another embodiment, the composition comprises more than 80% NKG2C Positive and negative NKG2A Negative of cells or a subpopulation thereof. In some embodiments, provided compositions include compositions wherein the NKG2C Positive and negative NKG2A Negative of cells or subpopulations thereof constitute at least or at least about 60%, at least or at least about 70%, at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95% or more of the cells in the composition or NK cells in the composition. In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than 60%, greater than 70%, or greater than 70%, greater than 80%, or greater than 80%, greater than 90%, or greater than 95% of the NKG2C Positive and negative NKG2A Negative of cells in the composition comprise a heterologous nucleic acid encoding the CAR. In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the NKG2C Positive and negative NKG2A Negative of cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than 60%, greater than 70%, or greater than 70%, greater than 80%, or greater than 80%, greater than 90%, or greater than 95% of the NKG2C Positive and negative NKG2A Negative of cells in the composition comprise a heterologous nucleic acid encoding the CAR and an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine).
In some embodiments, the composition comprises about 5% -99% g-NK cells, or any percentage between 5% and 99% (inclusive) g-NK cells. In some embodiments, the composition may include an increased or greater percentage of g-NK cells relative to total NK cells or total cells as compared to the percentage of naturally occurring g-NK cells relative to total NK cells or total cells in the subject from which the cells were isolated. In some embodiments, the percentage increase is at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, or more.
In some embodiments, the composition may comprise at least or at least about 20%, at least or at least about 30%, at least or at least about 40%, at least or at least about 50%, at least or at least about 60%, at least or at least about 65%, at least or at least about 70%, at least or at least about 75%, at least or at least about 80%, at least or at least about 81%, at least or at least about 82%, at least or at least about 83%, at least or at least about 84%, at least or at least about 85%, at least or at least about 86%, at least or at least about 87%, at least or at least about 88%, at least or at least about 89%, at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, or at least about 99%, or substantially 100% g-NK cells. In some embodiments, the composition comprises more than 50% g-NK cells. In another embodiment, the composition comprises more than 70% g-NK cells. In another embodiment, the composition comprises more than 80% g-NK cells. In some embodiments, provided compositions include compositions wherein the g-NK cells comprise at least or at least about 60%, at least or at least about 70%, at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95% or more of the cells in the composition or NK cells in the composition. In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the g-NK cells in the composition comprise the heterologous nucleic acid encoding the CAR. In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the g-NK cells in the composition comprise a heterologous nucleic acid encoding the CAR and an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine).
In some embodiments, the composition comprises a population of Natural Killer (NK) cell subpopulations, wherein at least or at least about 40%, at least or at least about 50%, at least or at least about 55%, at least or at least about 60%, at least or at least about 65%, at least or at least about 70%, at least or at least about 75%, at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, or at least about 95% of the cells in the composition have a g-NK cell surrogate marker profile of CD57 Positive and negative . In some embodiments, 70% to 90% or about 70% to about 90% of the cells in the composition have the phenotype CD57 Positive and negative . In some embodiments, at least or at least about 72%, at least or at least about 74%, at least or at least about 76%, at least or at least about 78%, at least or at least about 80%, at least or at least about 82%, at least or at least about 84%, at least or at least about 86%, at least or at least about 88%, at least or at least about 90%, at least or at least about 92%, at least or at least about 94%, at least or at least about 96%, or at least about 98% of the cells in the composition have a phenotype CD57 Positive and negative . In some embodiments of any of the provided embodiments, at least or at least about 60% of the cells in the composition comprise phenotype CD57 Positive and negative . In some embodiments of any of the provided embodiments, at least or at least about 70% of the cells in the composition comprise phenotype CD57 Positive and negative . In some embodiments, the phenotype further comprises the surface phenotype CD3 Negative of . In some embodiments, the phenotype further comprises the surface phenotype CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative . In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD57 Positive and negative cells in the composition comprise a heterologous nucleic acid that encodes a CAR. In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the CD57 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments, the phenotype further comprises the surface phenotype CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative . In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD57 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding the CAR and an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments, the phenotype further comprises the surface phenotype CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative . In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD3 Negative of CD57 Positive and negative cells in the composition comprise a heterologous nucleic acid that encodes a CAR. In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the CD3 Negative of CD57 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD3 Negative of CD57 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding the CAR and an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD57 Positive and negative CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative cells in the composition comprise a heterologous nucleic acid that encodes a CAR. In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the CD57 Positive and negative CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD57 Positive and negative CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding the CAR and an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any of the provided embodiments, of the cells having such a phenotype, greater than 50% of the cells are fcrγ Negative of , optionally between 50% and 90% or between about 50% and about 90% of the cells are fcrγ Negative of . In some embodiments of any of the provided embodiments, of the cells having such a phenotype, greater than 70% of the cells are fcrγ Negative of , optionally between 70% and 90% or between about 70% and about 90% of the cells are fcrγ Negative of .
In some embodiments, the composition comprises a population of Natural Killer (NK) cell subpopulations, wherein at least or at least about 40%, at least or at least about 50%, at least or at least about 55%, at least or at least about 60%, at least or at least about 65%, at least or at least about 70%, at least or at least about 75%, at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, or at least about 95% of the cells in the composition have a g-NK cell surrogate marker profile of CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of . In some embodiments, 70% to 90% or about 70% to about 90% of the cells in the composition have the phenotype CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of . In some embodiments, at least or at least about 72%, at least or at least about 74%, at least or at least about 76%, at least or at least about 78%, at least or at least about 80%, at least or at least about 82%, at least or at least about 84%, at least or at least about 86%, at least or at least about 88%, at least or at least about 90%, at least or at least about 92%, at least or at least about 94%, at least or at least about 96%, or at least about 98% of the cells in the composition have a phenotype CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of . In some embodiments of any of the provided embodiments, at least or at least about 60% of the cells in the composition comprise phenotype CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of . In some embodiments of any of the provided embodiments, at least or at least about 70% of the cells in the composition comprise phenotype CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of . In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of cells in the composition comprise a heterologous nucleic acid that encodes a CAR. In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of cells in the composition comprise a heterologous nucleic acid encoding the CAR and an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments, the phenotype further comprises the surface phenotype CD3 Negative of . In some embodiments, the phenotype further comprises the surface phenotype CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative . In some embodiments of any of the provided embodiments, of the cells having such a phenotype, greater than 50% of the cells are fcrγ Negative of , optionally between 50% and 90% or between about 50% and about 90% of the cells are fcrγ Negative of . In some embodiments of any of the provided embodiments, of the cells having such a phenotype, greater than 70% of the cells are fcrγ Negative of , optionally between 70% and 90% or between about 70% and about 90% of the cells are fcrγ Negative of .
In some embodiments, the composition comprises a population of Natural Killer (NK) cell subpopulations, wherein at least or at least about 40%, at least or at least about 50%, at least or at least about 55%, at least or at least about 60%, at least or at least about 65%, at least or at least about 70%, at least or at least about 75%, at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, or at least about 95% of the cells in the composition have a CD38 Negative of phenotype. In some embodiments, 70% to 90% or about 70% to about 90% of the cells in the composition have the phenotype CD38 Negative of . In some embodiments, at least or at least about 72%, at least or at least about 74%, at least or at least about 76%, at least or at least about 78%, at least or at least about 80%, at least or at least about 82%, at least or at least about 84%, at least or at least about 86%, at least or at least about 88%, at least or at least about 90%, at least or at least about 92%, at least or at least about 94%, at least or at least about 96%, or at least about 98% of the cells in the composition have a phenotype CD38 Negative of . In some embodiments of any of the provided embodiments, at least or at least about 60% of the cells in the composition comprise phenotype CD38 Negative of . In some embodiments of any of the provided embodiments, at least or at least about 70% of the cells in the composition comprise phenotype CD38 Negative of . In some embodiments, the phenotype further comprises the surface phenotype CD3 Negative of . In some embodiments, the phenotype further comprises the surface phenotype CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative . In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD38 Negative of cells in the composition comprise a heterologous nucleic acid encoding the CAR and an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD3 Negative of CD38 Negative of cells in the composition comprise a heterologous nucleic acid that encodes a CAR. In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the CD3 Negative of CD38 Negative of cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD38 Negative of cells in the composition comprise a heterologous nucleic acid encoding the CAR and an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD38 Negative of CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding the CAR. In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the CD38 Negative of CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD38 Negative of CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding the CAR and an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any of the provided embodiments, of the cells having such a phenotype, greater than 50% of the cells are fcrγ Negative of , optionally between 50% and 90% or between about 50% and about 90% of the cells are fcrγ Negative of . In some embodiments of any of the provided embodiments, of the cells having such a phenotype, greater than 70% of the cells are fcrγ Negative of , optionally between 70% and 90% or between about 70% and about 90% of the cells are fcrγ Negative of .
In some embodiments, the composition comprises a population of Natural Killer (NK) cell subpopulations, wherein at least or at least about 40%, at least or at least about 50%, at least or at least about 55%, at least or at least about 60%, at least or at least about 65%, at least or at least about 70%, at least or at least about 75%, at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, or at least about 95% of the cells in the composition have a CD16 Positive and negative phenotype. In some embodiments, 70% to 90% or about 70% to about 90% of the cells in the composition have the phenotype CD16 Positive and negative . In some embodiments, at least or at least about 72%, at least or at least about 74%, at least or at least about 76%, at least or at least about 78%, at least or at least about 80%, at least or at least about 82%, at least or at least about 84%, at least or at least about 86%, at least or at least about 88%, at least or at least about 90%, at least or at least about 92%, at least or at least about 94%, at least or at least about 96%, or at least about 98% of the cells in the composition have a phenotype CD16 Positive and negative . In some embodiments of any of the provided embodiments, at least or at least about 60% of the cells in the composition comprise phenotype CD16 Positive and negative . In some embodiments of any of the provided embodiments, at least or at least about 70% of the cells in the composition comprise phenotype CD16 Positive and negative . In some embodiments, the phenotype further comprises the surface phenotype CD3 Negative of . In some embodiments, the phenotype further comprises the surface phenotype CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative . In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD16 Positive and negative cells in the composition comprise a heterologous nucleic acid that encodes a CAR. In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the CD16 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD16 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding the CAR and an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD3 Negative of CD16 Positive and negative cells in the composition comprise a heterologous nucleic acid that encodes a CAR. In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the CD3 Negative of CD16 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD3 Negative of CD16 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding the CAR and an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95% greater than or greater than about 95% of the CD16 Positive and negative CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding the CAR. In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the CD16 Positive and negative CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD16 Positive and negative CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding the CAR and an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any of the provided embodiments, of the cells having such a phenotype, greater than 50% of the cells are fcrγ Negative of , optionally between 50% and 90% or between about 50% and about 90% of the cells are fcrγ Negative of . In some embodiments of any of the provided embodiments, of the cells having such a phenotype, greater than 70% of the cells are fcrγ Negative of , optionally between 70% and 90% or between about 70% and about 90% of the cells are fcrγ Negative of .
In some embodiments, the composition comprises a population of Natural Killer (NK) cell subpopulations, wherein at least or at least about 40%, at least or at least about 50%, at least or at least about 55%, at least or at least about 60%, at least or at least about 65%, at least or at least about 70%, at least or at least about 75%, at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, or at least about 95% of the cells in the composition have a g-NK cell surrogate marker profile of NKG2a Negative of /CD161 Negative of . In some embodiments, 70% to 90% or about 70% to about 90% of the cells in the composition have the phenotype NKG2a Negative of /CD161 Negative of . In some embodiments, at least or at least about 72%, at least or at least about 74%, at least or at least about 76%, at least or at least about 78%, at least or at least about 80%, at least or at least about 82%, at least or at least about 84%, at least or at least about 86%, at least or at least about 88%, at least or at least about 90%, at least or at least about 92%, at least or at least about 94%, at least or at least about 96%, or at least about 98% of the cells in the composition have a phenotype NKG2a Negative of /CD161 Negative of . In some embodiments of any of the provided embodiments, at least or at least about 60% of the cells in the composition comprise the phenotype NKG2a Negative of /CD161 Negative of . In some embodiments of any of the provided embodiments, at least or at least about 70% of the cells in the composition comprise the phenotype NKG2a Negative of /CD161 Negative of . In some embodiments, the phenotype further comprises the surface phenotype CD3 Negative of . In some embodiments, the phenotype further comprises the surface phenotype CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative . In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than 60%, greater than 70%, or greater than 70%, greater than 80%, or greater than 80%, greater than 90%, or greater than 95% of the NKG2a Negative of /CD161 Negative of cells in the composition comprise a heterologous nucleic acid encoding the CAR. In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the NKG2a Negative of /CD161 Negative of cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (such as a cytokine). In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the NKG2a Negative of /CD161 Negative of cells in the composition comprise a heterologous nucleic acid encoding an antigen receptor (such as a CAR) and an immunomodulatory agent (such as a cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the CD3 Negative of NKG2A Negative of /CD161 Negative of cells in the composition comprise a heterologous nucleic acid that encodes a CAR. In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the CD3 Negative of NKG2A Negative of /CD161 Negative of cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (such as a cytokine). In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the CD3 Negative of NKG2A Negative of /CD161 Negative of cells in the composition comprise a heterologous nucleic acid encoding an antigen receptor (such as a CAR) and an immunomodulatory agent (such as a cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than 60%, greater than 70%, or greater than 70%, greater than 80%, or greater than 80%, greater than 90%, or greater than 95% of the NKG2a Negative of /CD161 Negative of /CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding the CAR. In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the NKG2a Negative of /CD161 Negative of /CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (such as a cytokine). In some embodiments of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the NKG2a Negative of /CD161 Negative of /CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative cells in the composition comprise a heterologous nucleic acid encoding an antigen receptor (such as a CAR) and an immunomodulatory agent (such as a cytokine). In some embodiments of any of the provided embodiments, of the cells having such a phenotype, greater than 50% of the cells are fcrγ Negative of , optionally between 50% and 90% or between about 50% and about 90% of the cells are fcrγ Negative of . In some embodiments of any of the provided embodiments, of the cells having such a phenotype, greater than 70% of the cells are fcrγ Negative of , optionally between 70% and 90% or between about 70% and about 90% of the cells are fcrγ Negative of .
In some embodiments, the composition comprises a population of NK cells, wherein greater than 50% or greater than about 50% of the NK cells in the composition are g-NK cells (fcrγ Negative of ) or NK cells expressing their surrogate marker profile. In some embodiments, the composition comprises a population of NK cells, wherein greater than 55% or greater than about 55% of the NK cells in the composition are g-NK cells (fcrγ Negative of ) or NK cells expressing their surrogate marker profile. In some embodiments, the composition comprises a population of NK cells, wherein greater than 60% or greater than about 60% of the NK cells in the composition are g-NK cells (fcrγ Negative of ) or NK cells expressing their surrogate marker profile. In some embodiments, the composition comprises a population of NK cells, wherein greater than 65% or greater than about 65% of the NK cells in the composition are g-NK cells (fcrγ Negative of ) or NK cells expressing their surrogate marker profile. In some embodiments, the composition comprises a population of NK cells, wherein greater than 70% or greater than about 70% of the NK cells in the composition are g-NK cells (fcrγ Negative of ) or NK cells expressing their surrogate marker profile. In some embodiments, the composition comprises a population of NK cells, wherein greater than 75% or greater than about 75% of the NK cells in the composition are g-NK cells (fcrγ Negative of ) or NK cells expressing their surrogate marker profile. In some embodiments, the composition comprises a population of NK cells, wherein greater than 80% or greater than about 80% of the NK cells in the composition are g-NK cells (fcrγ Negative of ) or NK cells expressing their surrogate marker profile. In some embodiments, the composition comprises a population of NK cells, wherein greater than 85% or greater than about 85% of the NK cells in the composition are g-NK cells (fcrγ Negative of ) or NK cells expressing their surrogate marker profile. In some embodiments, the composition comprises a population of NK cells, wherein greater than 90% or greater than about 90% of the NK cells in the composition are g-NK cells (fcrγ Negative of ) or NK cells expressing their surrogate marker profile. In some embodiments, the composition comprises a population of NK cells, wherein greater than 95% or greater than about 95% of the NK cells in the composition are g-NK cells (fcrγ Negative of ) or NK cells expressing their surrogate marker profile. The surrogate marker profile may be any surrogate marker profile described herein. For example, the surrogate marker profile may be CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of . In other examples, the surrogate marker profile may be NKG2a Negative of /CD161 Negative of . In other examples, the g-NK cell surrogate marker profile is CD38 Negative of . The surrogate surface marker profile may also include phenotype CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative .
In some embodiments, the g-NK cells of the composition, or a percentage thereof (e.g., greater than about 70%), are positive for perforin and/or granzyme B. In some embodiments, the natural killer cells in the composition are enriched for cells positive for perforin and granzyme B. In some cases, natural killer cells are positive for perforin and granzyme B. Perforin is a pore-forming cytolysin found in the granules of NK cells. After degranulation, perforin binds to the plasma membrane of the target cells and oligomerizes in a calcium-dependent manner to form pores on the target cells. Granzyme B is the most common serine protease in granules of natural killer cells and cytotoxic T cells. Granzyme B is secreted with perforin to mediate apoptosis of target cells. Methods for measuring the number of cells positive for perforin or granzyme B are known to those skilled in the art. Such methods include, for example, intracellular flow cytometry. In an example, the percentage or number of cells positive for perforin or granzyme B can be determined by the following procedure: permeabilization of the cells is performed, for example, using an intracellular staining kit (INSIDE STAIN KIT) from Miltenyi Biotec, followed by staining with antibodies to perforin and granzyme B. Cell staining can then be resolved, for example, using flow cytometry.
In some embodiments, greater than 70% or greater than about 70% of the g-NK cells of the composition are positive for perforin and greater than 70% or greater than about 70% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than 75% or greater than about 75% of the g-NK cells of the composition are positive for perforin and greater than 75% or greater than about 75% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than 80% or greater than about 80% of the g-NK cells of the composition are positive for perforin and greater than 80% or greater than about 80% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than 85% or greater than about 85% of the g-NK cells of the composition are positive for perforin and greater than 85% or greater than about 85% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than 90% or greater than about 90% of the g-NK cells of the composition are positive for perforin and greater than 90% or greater than about 90% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than 95% or greater than about 95% of the g-NK cells of the composition are positive for perforin and greater than 95% or greater than about 95% of the g-NK cells of the composition are positive for granzyme B. In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the cells positive for granzyme B and perforin in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than 60%, greater than 70%, or greater than 70%, greater than 80%, or greater than 80%, greater than 90%, or greater than 95%, of the cells positive for granzyme B and perforin in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than 60%, greater than 70%, or greater than 70%, greater than 80%, or greater than 80%, greater than 90%, or greater than 95%, of the cells positive for granzyme B and perforin in the composition comprise a heterologous nucleic acid encoding an antigen receptor (such as a CAR) and an immunomodulator (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine).
In some embodiments, the expression levels of perforin and granzyme B of NK cells (e.g., g-NK cells) can be measured by intracellular flow cytometry, and the expression levels are measured based on the level of Mean Fluorescence Intensity (MFI). In some embodiments, the expression levels of MFI-based perforin and granzyme B will differ between g-NK cells and FcR gamma Positive and negative cells. In some embodiments, the average level of perforin expressed by the perforin-positive g-NK cells of the composition is at least twice or at least about twice the average level of perforin expressed by FcR gamma Positive and negative NK cells, based on MFI levels. In some embodiments, the average level of perforin expressed by the perforin-positive g-NK cells of the composition is at least three times or at least about three times the average level of perforin expressed by FcR gamma Positive and negative NK cells, based on MFI levels. In some embodiments, the average level of perforin expressed by the perforin-positive g-NK cells of the composition is at least four times or at least about four times the average level of perforin expressed by FcR gamma Positive and negative NK cells, based on MFI levels. In some embodiments, the average level of granzyme B expressed by g-NK cells positive for granzyme B of the composition is at least twice or at least about twice the average level of granzyme B expressed by FcR gamma Positive and negative NK cells based on MFI levels. In some embodiments, the average level of granzyme B expressed by g-NK cells positive for granzyme B of the composition is at least three times or at least about three times the average level of granzyme B expressed by FcR gamma Positive and negative NK cells based on MFI levels. In some embodiments, the average level of granzyme B expressed by g-NK cells positive for granzyme B of the composition is at least four times or at least about four times the average level of granzyme B expressed by FcR gamma Positive and negative NK cells based on MFI levels.
In some embodiments, at least or at least about 50% of the cells in the composition are FcR gamma deficient NK cells (g-NK), wherein greater than 70% or greater than about 70% of the g-NK cells are positive for perforin and greater than 70% or greater than about 70% of the g-NK cells are positive for granzyme B. In some embodiments, greater than 80% or greater than about 80% of the g-NK cells are positive for perforin and greater than 80% or greater than about 80% of the g-NK cells are positive for granzyme B. In some embodiments, greater than 90% or greater than about 90% of the g-NK cells are positive for perforin and greater than 90% or greater than about 90% of the g-NK cells are positive for granzyme B. In some embodiments, greater than 95% or greater than about 95% of the g-NK cells are positive for perforin and greater than 95% or greater than about 95% of the g-NK cells are positive for granzyme B. In some embodiments, the g-NK cell is fcrγ Negative of . In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than 60%, greater than 70%, or greater than 70%, greater than 80%, or greater than 80%, greater than 90%, or greater than 95%, of the cells positive for perforin and granzyme B in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, the g-NK cell is fcrγ Negative of . In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than 60%, greater than 70%, or greater than 70%, greater than 80%, or greater than 80%, greater than 90%, or greater than 95%, of the cells positive for perforin and granzyme B in the composition comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine). In some embodiments, the g-NK cell is fcrγ Negative of . In some embodiments of any such embodiments, greater than 50%, greater than 60%, or greater than 60%, greater than 70%, or greater than 70%, greater than 80%, or greater than 80%, greater than 90%, or greater than 95%, of the cells positive for perforin and granzyme B in the composition comprise a heterologous nucleic acid encoding an antigen receptor (such as a CAR) and an immunomodulator (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine).
In some of any of the embodiments, the average level of perforin expressed by the cells is at least twice or at least about twice the average level of perforin expressed by the cells of FcR gamma Positive and negative , based on the average fluorescence intensity (MFI), as measured by intracellular flow cytometry in cells positive for perforin. In some of any of the embodiments, in cells positive for granzyme B, the mean level of granzyme B expressed by the cells is at least twice or at least about twice the mean level of granzyme B expressed by the cells of FcR gamma Positive and negative , based on Mean Fluorescence Intensity (MFI), as measured by intracellular flow cytometry.
In some embodiments, the natural killer cells in the composition are enriched for cells expressing or producing CD107A, IFN gamma and TNF-alpha. In some cases, expression or production or a degree of expression or production of such factors is inherent to the cells in the composition in the absence of the target antigen (i.e., in the absence of further stimulation). In some cases, the expression or production or a degree of expression or production is in the presence of cells expressing the target antigen (target cells) and antibodies directed against the target antigen (anti-target antibodies). For example, in some embodiments, the target cell may be a tumor cell line expressing CD38, and the antibody is an anti-CD 38 antibody (e.g., up to Lei Tuoyou mab). For example, in some embodiments, the target cell may be a tumor cell line that expresses CD20, and the antibody is an anti-CD 20 antibody (e.g., rituximab).
In some of any of the embodiments, optionally measured by CD107a expression, greater than 10% of the cells in the composition are capable of degranulation against tumor target cells, optionally wherein degranulation is measured in the absence of antibodies against tumor target cells. In some of any of the embodiments, greater than 15% or greater than about 15%, greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, or greater than 50% or greater than about 50% of the cells in the composition exhibit degranulation in the presence of cells expressing the target antigen (target cells) and antibodies to the target antigen (anti-target antibodies), optionally as measured by CD107a expression. In some of any of these embodiments, greater than 10% of the cells in the composition are also capable of producing interferon-gamma or TNF-alpha to the tumor target cells, optionally wherein the interferon-gamma or TNF-alpha is measured in the absence of antibodies to the tumor target cells. In some of any such embodiments, for example, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the cells positive for CD107a in the composition comprise a heterologous nucleic acid encoding a CAR, as measured in the presence or absence of cells expressing the target antigen and antibodies to the target antigen. In some of any of these embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the cells positive for CD107a comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine), e.g., measured in the presence or absence of the cell expressing the target antigen and the antibody to the target antigen in the composition. In some of any of these embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the cells positive for CD107a comprise a heterologous nucleic acid encoding an antigen receptor (such as a CAR) and an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine), e.g., measured in the presence or absence of the cell expressing the target antigen and the antibody to the target antigen. In some embodiments, in the cells in the composition, greater than 15% or greater than about 15%, greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, or greater than 50% or greater than about 50% of the cells expressing the target antigen (target cells) and the antibody directed against the target antigen (anti-target antibody) produce an effector cytokine. For example, in some embodiments, the target cell may be a tumor cell line expressing CD38, and the antibody is an anti-CD 38 antibody (e.g., up to Lei Tuoyou mab). For example, in some embodiments, the target cell may be a tumor cell line that expresses CD20, and the antibody is an anti-CD 20 antibody (e.g., rituximab).
In some embodiments, at least or at least about 50% of the cells in the composition are FcR gamma deficient (FcR gamma Negative of ) NK cells (g-NK), and wherein greater than 15% or greater than about 15% of the cells in the composition produce effector cytokines in the presence of cells expressing the target antigen (target cells) and antibodies to the target antigen (anti-target antibodies). In some embodiments, greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, or greater than 50% or greater than about 50% of the cells expressing the target antigen (target cells) and the antibody directed against the target antigen (anti-target antibody) produce the effector cytokine. For example, in some embodiments, the target cell may be a tumor cell line expressing CD38, and the antibody is an anti-CD 38 antibody (e.g., up to Lei Tuoyou mab). For example, in some embodiments, the target cell may be a tumor cell line that expresses CD20, and the antibody is an anti-CD 20 antibody (e.g., rituximab). In some of any of the embodiments, the effector cytokine is IFN-gamma or TNF-alpha. In some of any of the embodiments, the effector cytokines are IFN-gamma and TNF-alpha. In some of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the cells that produce an effector cytokine (e.g., IFN- γ or TNF- α) in the composition comprise a heterologous nucleic acid encoding the CAR, e.g., measured in the presence or absence of the cells that express the target antigen and the antibody to the target antigen. In some of any of the embodiments, the effector cytokines are IFN-gamma and TNF-alpha. In some of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the cells that produce an effector cytokine (e.g., IFN- γ or TNF- α) comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine), e.g., measured in the presence or absence of the cells that express the target antigen and antibodies to the target antigen. In some of any of the embodiments, the effector cytokines are IFN-gamma and TNF-alpha. In some of any such embodiments, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the cells that produce an effector cytokine (e.g., IFN- γ or TNF- α) comprise a heterologous nucleic acid encoding an antigen receptor (such as a CAR) and an immunomodulator (e.g., a cytokine, the secretable cytokine or membrane-bound cytokine), e.g., measured in the presence or absence of the cells that express the target antigen and antibodies to the target antigen in the composition.
In some of any of the embodiments, greater than 15% or greater than about 15%, greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, or greater than 50% or greater than about 50% of the cells in the composition exhibit degranulation in the presence of cells expressing the target antigen (target cells) and antibodies to the target antigen (anti-target antibodies), optionally as measured by CD107a expression. For example, in some embodiments, the target cell may be a tumor cell line expressing CD38, and the antibody is an anti-CD 38 antibody (e.g., up to Lei Tuoyou mab). For example, in some embodiments, the target cell may be a tumor cell line that expresses CD20, and the antibody is an anti-CD 20 antibody (e.g., rituximab).
In some embodiments, at least or at least about 50% of the cells in the composition are FcR gamma deficient (FcR gamma Negative of ) NK cells (g-NK), and wherein, optionally, greater than 15% or greater than about 15% of the cells in the composition exhibit degranulation in the presence of cells expressing the target antigen (target cells) and antibodies directed against the target antigen (anti-target antibodies), as measured by CD107a expression. In some embodiments, greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, or greater than 50% or greater than about 50% of the cells that express the target antigen (target cells) exhibit degranulation, optionally in the presence of cells that express the target antigen (target cells) and antibodies directed against the target antigen (anti-target antibodies), as measured by CD107a expression. For example, in some embodiments, the target cell may be a tumor cell line expressing CD38, and the antibody is an anti-CD 38 antibody (e.g., up to Lei Tuoyou mab). For example, in some embodiments, the target cell may be a tumor cell line that expresses CD20, and the antibody is an anti-CD 20 antibody (e.g., rituximab).
In some embodiments of any of the provided embodiments, greater than 60% or greater than about 60% of the cells in the composition are g-NK cells. In some embodiments of any of the provided embodiments, greater than 70% or greater than about 70% of the cells in the composition are g-NK cells. In some embodiments of any of the provided embodiments, greater than 80% or greater than about 80% of the cells in the composition are g-NK cells. In some embodiments of any of the provided embodiments, greater than 90% or greater than about 90% of the cells in the composition are g-NK cells. In some embodiments of any of the provided embodiments, greater than 95% or greater than about 95% of the cells in the composition are g-NK cells.
In some embodiments, g-NK cells exhibit g-NK cell surrogate marker profiles. In some embodiments, the g-NK cell surrogate marker profile is CD16 positive/CD 57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of . In some embodiments, the g-NK cell surrogate marker profile is NKG2a Negative of /CD161 Negative of . In some embodiments, the g-NK cell surrogate marker profile is CD38 Negative of . In some embodiments, the g-NK cell surrogate surface marker profile is also CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative .
In some embodiments of any of the preceding embodiments, greater than 60% or greater than about 60% of the cells are g-NK cells. In some embodiments of any of the preceding embodiments, greater than 70% or greater than about 70% of the cells are g-NK cells. In some embodiments of any of the preceding embodiments, greater than 80% or greater than about 80% of the cells are g-NK cells. In some embodiments of any of the preceding embodiments, greater than 90% or greater than about 90% of the cells are g-NK cells. In some embodiments of any of the preceding embodiments, greater than 95% or greater than about 95% of the cells are g-NK cells.
In some embodiments of any of the preceding embodiments, greater than 80% or about 80% of the cells are positive for perforin. In some embodiments of any of the preceding embodiments, greater than 90% or about 90% of the cells are positive for perforin. In some of any of the preceding embodiments, the average level of perforin expressed by the cells is at least twice or at least about twice the average level of perforin expressed by the cells of FcR gamma Positive and negative , based on the average fluorescence intensity (MFI), as measured by intracellular flow cytometry in cells positive for perforin.
In some embodiments of any of the preceding embodiments, greater than 80% or about 80% of the cells are positive for granzyme B. In some embodiments of any of the preceding embodiments, greater than 90% or about 90% of the cells are positive for granzyme B. In some of any of the preceding embodiments, in cells positive for granzyme B, the mean level of granzyme B expressed by the cells is at least twice or at least about twice the mean level of granzyme B expressed by the cells of FcR gamma Positive and negative , based on Mean Fluorescence Intensity (MFI), as measured by intracellular flow cytometry.
In some embodiments of any of the provided embodiments, the composition comprises from 10 6 cells to 10 12 cells or from about 10 6 cells to about 10 12 cells. In some of any of the provided embodiments, the composition comprises 10 to 10 cells or about 10 to about 10 cells, 10 to 10 cells or about 10 to about 10 cells 10 to 10 cells or about 10 to about 10 cells, 10 to 10 cells or about 10 to about 10 cells, 10 10 to 10 12 cells or about 10 10 to about 10 12 cells, 10 10 to 10 11 cells or about 10 10 to about 10 11 cells, or 10 11 to 10 12 cells or about 11 to about 10 12 cells.
In some embodiments of any of the provided embodiments, the composition comprises at least or at least about 10 6 cells. In some of any of the provided embodiments, the composition comprises 10 6 to 10 10 cells or about 10 6 to about 10 10 cells, 10 6 to 10 9 cells or about 10 6 to about 10 9 cells, 10 6 to 10 8 cells or about 10 6 to about 10 8 cells, 10 6 to 10 7 cells or about 10 6 to about 10 7 cells, 10 7 to 10 10 cells or about 10 7 to about 10 10 cells, 10 10 to 10 10 cells or about 10 10 cells, 10 10 to about 10 cells or about 10 cells 10 to about 10 10 cells, or about 10 to about 10 cells, or about 10 10 to about 10 cells.
In some embodiments of any of the provided embodiments, the composition comprises at least or at least about 10 8 cells. In some embodiments of any of the provided embodiments, the composition comprises at least or at least about 10 9 cells. In some embodiments of any of the provided embodiments, the composition comprises at least or at least about 10 10 cells. In some embodiments of any of the provided embodiments, the composition comprises at least or at least about 10 11 cells. In some embodiments of any of the provided embodiments, the composition comprises from 10 8 to 10 11 cells or from about 10 8 to about 10 11 cells. In some embodiments of any of the provided embodiments, the composition comprises from 10 8 to 10 10 cells or from about 10 8 to about 10 10 cells. In some embodiments of any of the provided embodiments, the composition comprises from 10 8 to 10 9 cells or from about 10 8 to about 10 9 cells. In some embodiments of any of the provided embodiments, the composition comprises from 10 9 to 10 11 cells or from about 10 9 to about 10 11 cells. In some embodiments of any of the provided embodiments, the composition comprises from 10 9 to 10 10 cells or from about 10 9 to about 10 10 cells. In some embodiments of any of the provided embodiments, the composition comprises from 10 10 to 10 11 cells or from about 10 10 to about 10 11 cells.
In some embodiments of any of the provided embodiments, the composition comprises at least or at least about 10 6 g-NK cells. In some of any of the provided embodiments, the composition comprises 10 6 to 10 10 g-NK cells or about 10 6 to about 10 10 g-NK cells, 10 6 to 10 9 g-NK cells or about 10 6 to about 10 9 g-NK cells, 10 6 to 10 8 g-NK cells or about 10 6 to about 10 8 g-NK cells, 10 6 to 10 7 g-NK cells or about 10 6 to about 10 7 g-NK cells, about 10 7 to 10 10 g-NK cells or about 10 7 to about 10 10 g-NK cells, 10 10 to 10 10 g-NK cells or about 10 10 g-NK cells, 10 10 to about 10 g-NK cells, 10 10 g- 10 or about 10 to about 10 NK cells, 10 10 to about 10 g-NK cells or about 10 10 g-NK cells, 10 to about 10 g-NK cells or about 10 10 g-NK cells, about 10 to about 10 g-NK cells, or about 10 10 g-NK cells. In some of any of the provided embodiments, the g-NK cell is fcrγ Negative of . In some of any of the provided embodiments, the g-NK cells are cells having a g-NK surrogate surface marker profile. In some embodiments, the g-NK cell surrogate surface marker profile is CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of . In some embodiments, the g-NK cell surrogate surface marker profile is NKG2a Negative of /CD161 Negative of . In some of any of the provided embodiments, the g-NK cell or cell having a g-NK surrogate marker profile further comprises a surface phenotype CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative . In some of any of the provided embodiments, the g-NK cell or cell having a g-NK surrogate marker profile further comprises a surface phenotype CD38 Negative of .
In any of the provided embodiments of the compositions, the cells in the composition are from the same donor. Thus, the composition does not include a mixed population of cells from one or more different donors. As provided herein, the amplification methods result in high-yield amplification of certain NK cell subsets, particularly g-NK cell subsets or NK cell subsets associated with or comprising surrogate markers for g-NK cells, such as any of the NK cell subsets described above, by 500-fold or more, 600-fold or more, 700-fold or more, 800-fold or more, 900-fold or more, 1000-fold or more. In some of any of the embodiments, the increase is at or above about 1000-fold. In some of any of the embodiments, the increase is at or above about 2000-fold. In some of any of the embodiments, the increase is at or above about 2500-fold. In some of any of the embodiments, the increase is at or above about 3000-fold. In some of any of the embodiments, the increase is at or above about 5000-fold. In some of any of the embodiments, the increase is or is about 10000 times greater. In some of any of the embodiments, the increase is at or above about 15000-fold. In some of any of the embodiments, the increase is at or above about 20000 times. In some of any of the embodiments, the increase is at or above about 25000 times. In some of any of the embodiments, the increase is at or above about 30000-fold. In some of any of the embodiments, the increase is or is about 35000-fold or greater. In specific embodiments, the expansion results in an increase in the number of certain NK cell subsets, particularly g-NK cell subsets or NK cell subsets (such as any of the above NK cell subsets) associated with or comprising a surrogate marker for g-NK cells, by a factor of or about 1,000. In specific embodiments, the expansion results in an increase or about 3,000-fold in the number of certain NK cell subsets, particularly g-NK cell subsets or NK cell subsets (such as any of the above NK cell subsets) associated with or comprising a surrogate marker for g-NK cells. In specific embodiments, the expansion results in an increase or about 35,000-fold in the number of certain NK cell subsets, particularly g-NK cell subsets or NK cell subsets (such as any of the above NK cell subsets) associated with or comprising a surrogate marker for g-NK cells.
In some cases, expansion achieved by the provided methods from an initial source of NK cells obtained from a single donor can produce a cellular composition, thereby providing multiple separate doses for administration to a subject in need thereof. Thus, the provided methods are particularly useful in allogeneic approaches. In some cases, a single expansion from an initial population of NK cells isolated from one donor according to the provided methods can result in greater than 20 or greater than about 20 individual doses for administration to a subject in need thereof, such as the following individual doses or about the following individual doses: 30 individual doses, 40 individual doses, 50 individual doses, 60 individual doses, 70 individual doses, 80 individual doses, 90 individual doses, 100 individual doses or individual doses that are values between any of the foregoing values. In some embodiments, the individual doses are from 1X 10 5 cells/kg to 1X 10 7 cells/kg or from about 1X 10 5 cells/kg to about 1X 10 7 cells/kg, such as from 1X 10 7 cells/kg to 7.5X 10 7 cells/kg or from about 1X 10 7 cells/kg to about 7.5X 10 7 cells/kg, from 1X 10 7 cells/kg to about 5X 10 7 cells/kg or from 1X 10 7 cells/kg to about 5X 10 7 cells/kg, from 1X 10 7 cells/kg to about 2.5X 10 7 cells/kg or from about 1X 10 7 cells/kg to about 2.5X 10 7 cells/kg, from 1X 10 7 cells/kg to about 1X 10 7 cells/kg or from about 1X 10 7 cells/kg to about 1X 10 7 cells/kg 1X 10 7 to 7.5X 10 7 cells/kg or about 1X 10 7 to about 7.5X 10 7 cells/kg, 1X 10 7 to 5X 10 7 cells/kg or about 1X 10 7 to about 5X 10 7 cells/kg, 1X 10 7 to 2.5X 10 7 cells/kg or about 1X 10 7 to about 2.5X 10 7 cells/kg, from 2.5X10 5 cells/kg to 1X 10 7 cells/kg or from about 2.5X10 5 cells/kg to about 1X 10 7 cells/kg, from 2.5X10 7 cells/kg to 7.5X10 7 cells/kg or from about 2.5X10 7 cells/kg to about 7.5X10 7 cells/kg, from 2.5X10 7 cells/kg to 5X 10 7 cells/kg or from about 2.5X10 7 cells/kg to about 5X 10 7 cells/kg, from 2.5X10 7 cells/kg to about 2.5X10 7 cells/kg or from about 2.5X10 7 cells/kg to about 2.5X10 7 cells/kg from 2.5X107 7 cells/kg to 1X 10 7 cells/kg or from about 2.5X107 7 cells/kg to about 1X 10 7 cells/kg, from 2.5X107 7 cells/kg to about 7.5X107 7 cells/kg or from about 2.5X107 7 cells/kg to about 7.5X107 7 cells/kg, from about 2.5X107 7 cells/kg to about 5X 10 7 cells/kg or from about 2.5X107. Sup. 7 cells/kg to about 5X 10 7 cells/kg, from about 5X 10 7 cells/kg to about 1X 10 7 cells/kg or from about 5X 10 7 cells/kg to about 1X 10 7 cells/kg, from 5X 10 5 cells/kg to 7.5X 10 6 cells/kg or from about 5X 10 5 cells/kg to about 7.5X 10 6 cells/kg, from 5X 10 6 cells/kg to about 5X 10 6 cells/kg or from about 5X 10 6 cells/kg to about 5X 10 6 cells/kg, from 5X 10 6 cells/kg to about 2.5X 10 6 cells/kg or from about 5X 10 6 cells/kg to about 2.5X 10 6 cells/kg, from 5X 10 6 cells/kg to about 1X 10 6 cells/kg or from about 5X 10 6 cells/kg to about 1X 10 6 cells/kg from 5X 10 6 cells/kg to 7.5X 10 6 cells/k or from about 5X 10 6 cells/kg to about 7.5X 10 6 cells/kg, from 1X 10 6 cells/kg to 1X 10 6 cells/kg or from about 1X 10 6 cells/kg to about 1X 10 6 cells/kg, from 1X 10 6 cells/kg to 7.5X 10 6 cells/kg or from about 1X 10 6 cells/kg to about 7.5X 10 6 cells/kg, from 1X 10 6 cells/kg to about 5X 10 6 cells/kg or from about 1X 10 6 cells/kg to about 5X 10 6 cells/kg, from about 1×10 6 cells/kg to 2.5×10 6 cells/kg or from about 1×10 6 cells/kg to about 2.5×10 6 cells/kg, from about 2.5×10 6 cells/kg to about 1×10 7 cells/kg or from about 2.5×10 6 cells/kg to about 1×10 7 cells/kg, from about 2.5×10 6 cells/kg to about 7.5×10 6 cells/kg or from about 2.5×10 6 cells/kg to about 7.5×10 6 cells/kg, from about 2.5×10 6 cells/kg to about 5×10 6 cells/kg or from about 2.5×10 6 cells/kg to about 5×10 6 cells/kg, from about 5×10 6 cells/kg to about 1×10 6 cells/kg or from about 5×10 6 cells/kg to about 1×10 6 cells/kg, from about 2.5×10 6 cells/kg to about 7.5×10 6 cells/kg or from about 2.5×10 6 cells/kg to about 7.5×10 6 cells/kg, from about 7.5×10 6 cells/kg or from about 2.5×10 to about 7.5×10 6 cells/kg. In some embodiments, the individual dose is 1×10 5 to 1×10 8 cells/kg or about 1×10 5 to about 1×10 8 cells/kg, such as about 2.5×10 8 to about 1×10 8 cells/kg or about 2.5×10 8 cells/kg to about 2.5×10 8 cells/kg, about 5×10 8 to about 5×10 8 cells/kg or about 5×10 8 cells/kg to about 1×10 8 cells/kg or about 7.5×10 8 cells/kg to about 1×10 8 cells/kg, about 1×10 8 cells/kg to about 1×10 8 cells/kg or about 1×10×2 to about 1×10 3.2 kg or about 5×10 to about 5×2 to about 3.5×10 8 cells/kg or about 7.5×10 to about 3×10 8 cells/kg or about 7.5×10 to about 3×2 cells/kg to about 1×10 8 cells/kg, from 1×10 7 cells/kg to 1×10 8 cells/kg or from about 1×10 7 cells/kg to about 1×10 8 cells/kg, from 2.5×10 7 cells/kg to 1×10 8 cells/kg or from about 2.5×10 7 cells/kg to about 1×10 8 cells/kg, from 5×10 7 cells/kg to 1×10 8 cells/kg or from about 5×10 7 cells/kg to about 1×10 8 cells/kg or from 7.5×10 7 cells/kg to 1×10 8 cells/kg or from about 7.5×10 7 cells/kg to about 1×10 8 cells/kg. In some embodiments, the individual doses are from 5×10 to 10×10 or from about 5×10 to about 10×10, such as from 5×10 to 5×10 or from about 5×10 to about 5×10, from 5×10 to 1×10 or from about 5×10 to about 1×10, from 5×10 to 5×10 or from about 5×10 to about 5×10, from 5×10 to 1×10 or from about 5×10 to about 1×10 or to about 10×10, from 1×10 to 5×10 or from about 1×10 to about 5×10, from 1×10 to 1×10 or from about 1×10 to about 1×10, from 1×10 to about 5×10, from 5×10 to about 1×10, from about 1×10 to about 1×10 or from about 1×10 to about 5×10, from about 1×10 to about 1×10. In some embodiments, the individual dose is or is about 5 x 10 8 cells. In some embodiments, the individual dose is or is about 1 x 10 9 cells. In some embodiments, the individual dose is or is about 5 x 10 9 cells. In some embodiments, the individual dose is or is about 1 x 10 10 cells. In any of the above embodiments, the dose is given in terms of the number of g-NK cells or NK cell subsets (such as any of the above NK cell subsets) (or the number of any of the aforementioned living cells) associated with or comprising the surrogate marker for g-NK cells. In any of the above embodiments, the dose is given as the number of cells in the composition of expanded cells produced by the method or the number of living cells of any of the foregoing.
Among these compositions are pharmaceutical compositions and formulations for administration (such as for adoptive cell therapy). In some embodiments, the engineered cells are formulated with a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers can include all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (gennaro, 2000, remington: THE SCIENCE AND PRACTICE of pharmacy, lippincott, williams and wilkins, philiadelphia, PA). Examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. Supplementary active compounds may also be incorporated into the compositions. The pharmaceutical carrier should be a carrier suitable for NK cells, such as saline solution, dextrose solution, or a solution comprising human serum albumin.
In some embodiments, the pharmaceutically acceptable carrier or vehicle for such compositions is any non-toxic aqueous solution in which NK cells can remain or remain viable for a time sufficient to allow administration of the viable NK cells. For example, the pharmaceutically acceptable carrier or vehicle may be a saline solution or a buffered saline solution. The pharmaceutically acceptable carrier or vehicle may also include various biological materials that may increase NK cell efficiency. Cell vehicles and carriers may include, for example, polysaccharides such as methylcellulose (m.c. tate, d.a. shear, s.w. hoffman, d.g. stein, m.c. laplace, biomaterials, volume 22: page 1113, 2001, which is incorporated herein by reference in its entirety), chitosan (suh J K F, MATTHEW H W T., biomaterials, volume 21: page 2589, 2000; lahiji A, sohrabi A, hungerford D S et al, JBiomed Mater Res, volume 51, page 586, 2000, each of which is incorporated herein by reference in its entirety), N-isopropylacrylamide copolymer P (NIPAM-co-AA) (Y.H.Bae, B.Vernon, C.K.Han, S.W.Kim, J.control.Release, volume 53, page 249, 1998; h.gappa, m.baudys, j.j.koh, s.w.kim, y.h.bae, tissue eng., volume 7, page 35, 2001, each of which is incorporated herein by reference in its entirety), and poly (oxyethylene)/poly (D, L-lactic-co-glycolic acid) (b.jeong, k.m.lee, a.gutowska, y.h.an, biomacromolecules, volume 3, page 865, 2002, which is incorporated herein by reference in its entirety), P (PF-co-EG) (Suggs L J, mikos ag, cell trans, volume 8, page 345, 1999, which is incorporated herein by reference in its entirety), PEO/PEG (mann B K, gobin A S, tsai A T, SCHMEDLEN R H, west J l., biomaterials, volume 22, page 3045, 2001; bryant S J, anseth KS., biomaterials, vol.22, page 619, 2001, each of which is incorporated herein by reference in its entirety), PVA (Chih-Ta Lee, po-Han Kung and Yu-Der Lee, carbohydrate Polymers, roll 61: page 348, 2005, which is incorporated herein by reference in its entirety), collagen (Lee C R, grodzinsky A J, spectrum m., biomaterials, volume 22: page 3145, 2001, which is incorporated herein by reference in its entirety), alginate (Bouhadir K H, lee K Y, alsberg E, damm K L, anderson K W, mooney D j., biotech Prog, volume 17: page 945, 2001; smidsrd O, skjak-Braek g., trends Biotech, volume 8: page 71, 1990, each of which is incorporated herein by reference in its entirety).
In some embodiments, NK cells (such as NKG2C Positive and negative cells or a subpopulation thereof) may be present in the composition in an effective amount. In some embodiments, the composition contains an effective amount of g-NK cells, such as FcR gamma Negative of cells or cells with their g-NK surrogate marker profile. The effective amount of cells can vary depending on the patient and the type, severity and extent of the disease. Thus, the physician can determine what the effective amount is after considering the health of the subject, the extent and severity of the disease, and other variables.
In certain embodiments, the amount of such cells in the composition is a therapeutically effective amount. In some embodiments, the amount is an amount that reduces the severity, duration, and/or symptoms associated with cancer, viral infection, microbial infection, or septic shock in an animal. In some embodiments, a therapeutically effective amount is a cell dose that results in at least 2.5%, at least 5%, at least 10%, at least 15%, at least 25%, at least 35%, at least 45%, at least 50%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% reduction in cancer growth or spread in a patient (or animal) or group of patients (or animals) to whom the composition described herein is administered relative to cancer growth or spread in the patient (or animal) or group of patients (or animals) to whom the composition is not administered. In some embodiments, a therapeutically effective amount is an amount that results in a cytotoxic activity that results in an activity that inhibits or reduces the growth of cancer, viruses, and microbial cells.
In some embodiments, the composition comprises an amount of NKG2C Positive and negative cells or a subpopulation thereof of from 10 5 to 10 12 cells or from about 10 5 to about 10 12 NKG2C Positive and negative cells or a subpopulation thereof, or from 10 5 to 10 8 cells or from about 10 5 to about 10 8 NKG2C Positive and negative cells or a subpopulation thereof, or from 10 6 to 10 12 cells or from about 10 6 to about 10 12 NKG2C Positive and negative cells or a subpopulation thereof, or from 10 8 to 10 11 cells or from about 10 8 to about 10 11 NKG2C Positive and negative cells or a subpopulation thereof, or from 10 9 to 10 10 cells or from about 10 9 to about 10 10 NKG2C Positive and negative cells or a subpopulation thereof. In some embodiments, the composition comprises greater than or greater than about 10 5 NKG2C Positive and negative cells or a subpopulation thereof, as or about 10 6 NKG2C Positive and negative cells or a subpopulation thereof, as or about 10 7 NKG2C Positive and negative cells or a subpopulation thereof, as or about 10 8 NKG2C Positive and negative cells or a subpopulation thereof, as or about 10 9 NKG2C Positive and negative cells or a subpopulation thereof, as or about 10 10 NKG2C Positive and negative cells or a subpopulation thereof, as or about 10 11 NKG2C Positive and negative cells or a subpopulation thereof, or as or about 10 12 NKG2C Positive and negative cells or a subpopulation thereof. In some embodiments, the amount may be administered to a subject suffering from a disease or disorder, such as to a cancer patient.
In some embodiments, the composition comprises an amount of g-NK cells of 10 5 to 10 12 or about 10 5 to about 10 12 g-NK cells, or of 10 5 to 10 8 or about 10 5 to about 10 8 g-NK cells, or of 10 6 to 10 12 or about 10 6 to about 10 12 g-NK cells, or of 10 8 to 10 11 or about 10 8 to about 10 11 g-NK cells, or of 10 9 to 10 10 or about 10 9 to about 10 10 g-NK cells. In some embodiments, the composition comprises greater than 10 5 or greater than about 10 5 g-NK cells, or about 10 6 g-NK cells, or about 10 7 g-NK cells, or about 10 8 g-NK cells, or about 10 9 g-NK cells, or about 10 10 g-NK cells, or about 10 11 g-NK cells, or about 10 12 g-NK cells. In some embodiments, the amount may be administered to a subject suffering from a disease or disorder, such as to a cancer patient.
In some embodiments, the volume of the composition is at least or at least about 10mL, at least or at least about 50mL, at least or at least about 100mL, at least or at least about 200mL, at least or at least about 300mL, at least or at least about 400mL, or at least about 500mL, such as at or about the following ranges: 10mL to 500mL, 10mL to 200mL, 10mL to 100mL, 10mL to 50mL, 50mL to 500mL, 50mL to 200mL, 50mL to 100mL, 100mL to 500mL, 100mL to 200mL, or 200mL to 500mL, each inclusive. In some embodiments, the cell density of the composition is at least or at least about 1 x 10 5 cells/mL, at least or at least about 5 x 10 5 cells/mL, at least or at least about 1 x 10 6 cells/mL, at least or at least about 5 x 10 6 cells/mL, at least or at least about 1 x 10 7 cells/mL, at least or at least about 5 x 10 7 cells/mL, or at least about 1 x 10 8 cells/mL. In some embodiments, the cell density of the composition is between or about the following densities: 1X 10 5 cells/mL and 1X 10 8 cells/mL, 1X 10 5 cells/mL and 1X 10 7 cells/mL, 1X 10 5 cells/mL and 1X 10 6 cells/mL, 1X 10 6 cells/mL and 1X 10 7 cells/mL, 1X 10 6 cells/mL and 1X 10 8 cells/mL, 1X 10 6 cells/mL and 1X 10 7 cells/mL, or 1X 10 7 cells/mL and 1X 10 8 cells/mL, each inclusive.
In some embodiments, the composition (including pharmaceutical compositions) is sterile. In some embodiments, the isolation, enrichment, or culture of the cells is performed in a closed or sterile environment, such as in a sterile culture bag, to minimize errors, user handling, and/or contamination. In some embodiments, sterility may be readily achieved, for example, by filtration through sterile filtration membranes. In some embodiments, the culturing is performed using a gas permeable culture vessel. In some embodiments, the culturing is performed using a bioreactor.
Also provided herein are compositions suitable for cryopreserving provided NK cells. In some embodiments, NK cells are cryopreserved in serum-free cryopreservation media. In some embodiments, the composition includes a cryoprotectant. In some embodiments, the cryoprotectant is or includes DMSO and/or glycerol. In some embodiments, the cryopreservation medium is DMSO (v/v) between 5% and 10% or between about 5% and about 10%. In some embodiments, the cryopreservation medium is or is about 5% DMSO (v/v). In some embodiments, the cryopreservation medium is or is about 6% DMSO (v/v). In some embodiments, the cryopreservation medium is or is about 7% DMSO (v/v). In some embodiments, the cryopreservation medium is or is about 8% dmso (v/v). In some embodiments, the cryopreservation medium is or is about 9% DMSO (v/v). In some embodiments, the cryopreservation medium is or is about 10% DMSO (v/v). In some embodiments, the cryopreservation media comprises a commercially available cryopreservation solution (CryoStor TMCS10).CryoStorTM CS10 is a cryopreservation media comprising 10% dimethyl sulfoxide (DMSO). In some embodiments, the compositions configured for cryopreservation may be stored at low temperatures (such as ultra-low temperatures), for example, in a temperature range from-40 ℃ to-150 ℃ (such as or about 80 ℃ + -6.0 ℃).
In some embodiments, the composition may be stored at ultra-low temperatures prior to administration to a patient. In some aspects, NK cell subsets (such as g-NK cells) can be isolated, processed, and expanded (such as according to the provided methods) and then stored at ultra-low temperatures prior to administration to a subject.
For example, a typical method for small-scale storage at ultra-low temperatures is described in U.S. patent No. 6,0168,991. For small scale, cells can be stored at ultra-low temperatures by low density suspension (e.g., at a concentration of about 200X 106/ml) in pre-cooled 5% Human Albumin Serum (HAS). An equivalent amount of 20% DMSO may be added to the HAS solution. An aliquot of the mixture may be placed in a vial and frozen overnight in an ultra-low temperature chamber at about-80 ℃.
In some embodiments, cryopreserved NK cells are prepared for administration by thawing. In some cases, NK cells may be administered to a subject immediately after thawing. In such embodiments, the composition is ready-to-use without any further treatment. In other cases, NK cells are further processed after thawing (such as by re-suspension with a pharmaceutically acceptable carrier, incubation with an activator or stimulator), or activated, washed, and re-suspended in a pharmaceutically acceptable buffer prior to administration to a subject.
V. method for expanding natural killer cell subpopulations
Also disclosed herein are methods of producing a composition comprising a population of genetically engineered g-NK cells or a plurality of genetically engineered g-NK cells comprising a heterologous nucleic acid encoding an antigen receptor (e.g., CAR). Also disclosed herein are methods of producing a composition comprising a genetically engineered g-NK cell population or a plurality of genetically engineered g-NK cells comprising a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or a membrane-bound cytokine). Also disclosed herein are methods of producing a composition comprising a population of genetically engineered g-NK cells or a plurality of genetically engineered g-NK cells comprising a heterologous nucleic acid encoding an antigen receptor (e.g., CAR) and an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine). In some embodiments, the methods comprise introducing a nucleic acid encoding a CAR into g-NK cells or into a cell composition or population enriched in g-NK cells or expanded g-NK cells.
Also disclosed herein are methods of producing a composition comprising a genetically engineered g-NK cell population or a plurality of genetically engineered g-NK cells comprising a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or a membrane-bound cytokine). In some embodiments, the methods comprise introducing a nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine) into g-NK cells or into a cell composition or population enriched in g-NK cells or expanded g-NK cells.
Also disclosed herein are methods of producing a composition comprising a population of genetically engineered g-NK cells or a plurality of genetically engineered g-NK cells comprising a heterologous nucleic acid encoding an antigen receptor (e.g., CAR) and an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine). In some embodiments, the methods comprise (a) introducing a nucleic acid encoding a CAR into g-NK cells or into a cell composition or population enriched in g-NK cells or expanded g-NK cells, and (b) introducing a nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine) into g-NK cells or into a cell composition or population enriched in g-NK cells or expanded g-NK cells, wherein steps (a) and (b) are performed simultaneously or sequentially in any order.
In some embodiments, one or more gene editing steps may also be performed to produce cells in which one or more genes have been edited (such as knocked out) in the genome of the engineered cell. In some embodiments, the method for gene editing (such as by introducing an RNA-guided nuclease (e.g., RNP complex) into g-NK cells or into a cell composition or population enriched in g-NK cells or expanded g-NK cells) can be performed simultaneously or sequentially in any order with the steps of introducing a heterologous nucleic acid encoding an antigen receptor (e.g., a CAR), a heterologous nucleic acid encoding an immune modulator (e.g., a cytokine such as a secretable or soluble cytokine or membrane-bound cytokine), or a heterologous nucleic acid encoding an antigen receptor (e.g., a CAR) and an immune modulator (e.g., a cytokine such as a secretable or soluble cytokine or membrane-bound cytokine).
In some embodiments, the step of engineering the cells may be performed in conjunction with a method for enriching and amplifying g-NK cells from a biological sample from a subject. Methods for enriching or amplifying g-NK cells may include those described in PCT publication No. WO2020/107002 or PCT patent application No. PCT/US 2021/028504. Exemplary methods for enriching g-NK cells and preferentially expanding such cells are described in further detail below.
In some aspects, nucleic acids encoding the CAR can be introduced into g-NK cells for stable integration into the genome or for transient expression. In some aspects, nucleic acids encoding immunomodulators may be introduced into g-NK cells for stable integration into the genome or for transient expression. In some aspects, nucleic acids encoding the CAR and the immunomodulator can be introduced into g-NK cells for stable integration into the genome or for transient expression. When the nucleic acid is introduced into g-NK cells for stable integration, it may be introduced prior to culturing the engineered NK cell population such that the nucleic acid is stably integrated and will proliferate in the engineered NK cell progeny. In some embodiments, the nucleic acid encoding the CAR is stably integrated into the genome. In some embodiments, the nucleic acid encoding the immunomodulator is stably integrated into the genome. In some embodiments, the nucleic acid encoding the CAR and the immunomodulatory agent is stably integrated into the genome. In some embodiments, the nucleic acid is introduced into the g-NK cell via a viral vector. In some embodiments, the viral vector is a lentiviral vector.
In some embodiments, the step of engineering the cells is performed prior to culturing or incubating the enriched g-NK cells under conditions of further expansion, such as for stable integration of the heterologous agent into the genome of the NK cells. In some embodiments, NK cells are isolated from a biological sample as described in section v.a below, then introduced into a heterologous nucleic acid encoding an antigen receptor (e.g., CAR), and then the cells are expanded using the methods described in section v.b. In some embodiments, NK cells are isolated from a biological sample as described in section v.a below, then introduced with a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine such as a secretable or soluble cytokine or membrane-bound cytokine), and then the cells are expanded using the methods described in section v.b. In some embodiments, NK cells are isolated from a biological sample as described in section v.a below, then introduced into a heterologous nucleic acid encoding an antigen receptor (e.g., CAR) and an immunomodulator (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine), and then the cells are expanded using the methods described in section v.b. In some embodiments, the cell is further engineered by a gene editing method simultaneously or sequentially with the introduction of the nucleic acid encoding the CAR. In some embodiments, the cells are further engineered by gene editing methods simultaneously or sequentially with the introduction of nucleic acids encoding an immunomodulatory agent (e.g., a cytokine such as a secretable or soluble cytokine or a membrane-bound cytokine). In some embodiments, the cells are further engineered by gene editing methods simultaneously or sequentially with the introduction of nucleic acids encoding the CAR and an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine). In some embodiments, the cells are engineered by gene editing prior to performing the amplification method described in section v.b.
In some embodiments, the step of engineering the cells is performed during the culturing or incubation of the enriched g-NK cells under conditions of further expansion, such as for stable integration of the heterologous agent into the genome of the NK cells. For example, NK cells are isolated from a biological sample as described in section v.a below, and then subjected to a first expansion phase according to the method described in section v.b. The first amplification stage is part of the total amplification stage described in section v.b, wherein the remainder of the amplification stage is achieved by performing a second amplification stage. For example, the first amplification period is at or about the following times: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days. In some embodiments, after the first expansion phase, the cells are collected, then a heterologous nucleic acid encoding an antigen receptor (e.g., CAR) is introduced, and then the cells are further expanded in a second expansion phase using the methods described in section v.b. In some embodiments, after the first expansion phase, the cells are harvested, then a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine) is introduced, and then the cells are further expanded in the second expansion phase using the methods described in section v.b. In some embodiments, after the first expansion phase, the cells are harvested, then a heterologous nucleic acid encoding an antigen receptor (e.g., CAR) and an immunomodulatory agent (e.g., a cytokine such as a secretable or soluble cytokine or membrane-bound cytokine) is introduced, and then the cells are further expanded in a second expansion phase using the methods described in section v.b. The second expansion phase is part of the total expansion phase described in section v.b, such as until a threshold number of g-NK cell-rich cells are expanded. For example, the second amplification period is at or about the following times: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days. In some embodiments, the cell is further engineered by a gene editing method simultaneously or sequentially with the introduction of the nucleic acid encoding the CAR. In some embodiments, the cells are further engineered by gene editing methods simultaneously or sequentially with the introduction of nucleic acids encoding an immunomodulatory agent (e.g., a cytokine such as a secretable or soluble cytokine or a membrane-bound cytokine). In some embodiments, the cells are further engineered by gene editing methods simultaneously or sequentially with the introduction of nucleic acids encoding the CAR and an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine). In some embodiments, the cells are engineered by gene editing after isolation or selection of the cells from the biological sample to enrich for g-NK cells as described in section v.a and prior to the first expansion.
In other aspects, nucleic acid encoding a CAR can be introduced into g-NK cells for transient expression. In other aspects, nucleic acids encoding immunomodulators may be introduced into g-NK cells for transient expression. In other aspects, nucleic acids encoding CARs and immune modulators can be introduced into g-NK cells for transient expression. When nucleic acid is introduced into g-NK cells for transient expression, it may be introduced after culturing the engineered NK cell population, as the transiently expressed nucleic acid may not persist long enough or proliferate sufficiently into all cells of the cultured population. In some embodiments, the nucleic acid encoding the CAR is transiently expressed in the engineered NK cells. In some embodiments, a nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine) is transiently expressed in an engineered NK cell. In some embodiments, nucleic acids encoding the CAR and an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine) are transiently expressed in the engineered NK cells. In some embodiments, the nucleic acid is introduced via nanoparticle delivery. In some embodiments, the nucleic acid is introduced via electroporation.
Thus, in some methods of amplifying g-NK cells, the enriched NK cell population should be cultured under amplification conditions prior to introducing the nucleic acid encoding the CAR into the NK cells of the amplified population. In some methods of amplifying g-NK cells, the enriched NK cell population should be cultured under amplification conditions prior to introducing the nucleic acid encoding the immunomodulator (e.g., cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine) into the NK cells of the amplified population. In some methods of amplifying g-NK cells, the enriched NK cell population should be cultured under amplification conditions prior to introducing nucleic acid encoding the CAR and an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine) into NK cells of the amplified population. This is particularly true in the case of CAR or immunomodulator transient expression (e.g., via mRNA).
Exemplary methods of expanding NK cells enriched in g-NK cells are described in the following sections V.A and V.B. In some embodiments, a method of amplifying g-NK cells comprises: (a) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; (b) Culturing the enriched NK cell population in a medium having: (i) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (ii) an effective amount of two or more recombinant cytokines for expanding NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; (c) Introducing a nucleic acid encoding a CAR into NK cells of an NK cell expansion population, wherein the method produces an engineered NK cell expansion population enriched in g-NK cells engineered with the CAR. In some embodiments, a method of amplifying g-NK cells comprises: (a) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; (b) Culturing the enriched NK cell population in a medium having: (i) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (ii) an effective amount of two or more recombinant cytokines for expanding NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; (c) Introducing a nucleic acid encoding an immunomodulator (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine) into NK cells of an expanded population of NK cells, wherein the method produces an expanded population of engineered NK cells enriched in g-NK cells engineered with the immunomodulator (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine). In some embodiments, a method of amplifying g-NK cells comprises: (a) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; (b) Culturing the enriched NK cell population in a medium having: (i) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (ii) an effective amount of two or more recombinant cytokines for expanding NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; (c) Introducing (i) a nucleic acid encoding a CAR, and (ii) a nucleic acid encoding an immunomodulator (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine) into NK cells of an NK cell expansion population, wherein steps (i) and (ii) are performed simultaneously or sequentially in any order, wherein the method produces an engineered NK cell expansion population enriched for g-NK cells engineered with the CAR and the immunomodulator (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine).
In some embodiments, the step of engineering the cells is performed after culturing or incubating the enriched g-NK cells under further expansion conditions, such as for transiently expressing the heterologous agent into the genome of the NK cells. In some embodiments, NK cells are isolated from a biological sample as described in section v.a below, amplified using the methods described in section v.b, and then introduced with a heterologous nucleic acid encoding an antigen receptor (e.g., CAR). In some embodiments, NK cells are isolated from a biological sample as described in section v.a below, amplified using the method described in section v.b, and then introduced with a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or a membrane-bound cytokine). In some embodiments, NK cells are isolated from a biological sample as described in section v.a below, amplified using the methods described in section v.b, and then introduced with a heterologous nucleic acid encoding an antigen receptor (e.g., CAR) and an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or a membrane-bound cytokine). In some embodiments, the cells are further engineered by gene editing methods simultaneously or sequentially with the introduction of nucleic acids encoding the CAR and/or an immunomodulatory agent (e.g., a cytokine, such as a secretable or soluble cytokine or membrane-bound cytokine). In some embodiments, the cells are engineered by gene editing prior to performing the amplification method described in section v.b.
A. method for selecting cells from biological sample to enrich g-NK cells
In some embodiments, g-NK cell compositions (including compositions comprising engineered g-NK cells) are produced by methods comprising a method for enriching g-NK cells by in vitro expansion of g-NK cells from a subpopulation of NK cells from a biological sample of a human subject. In some embodiments, a method for amplifying and producing a g-NK cell composition may comprise amplifying a cell subpopulation that is FcR gamma-deficient NK cells (g – NK) from a biological sample from a human subject. In some embodiments, the method may comprise amplifying NK cell subpopulations of NKG2C Positive and negative from a biological sample from a human subject. In some embodiments, the method may comprise amplifying a subpopulation of NK cells of NKG2a Negative of from a biological sample from a human subject. In some embodiments, the method comprises isolating a population of Natural Killer (NK) cell enriched cells from a biological sample from a human subject and culturing the cells under conditions such that the g-NK cell object and/or the NK cell subpopulation overlapping or sharing extracellular surface markers with the g-NK cell subpopulation preferentially grow and/or expand. For example, NK cells can be cultured using feeder cells or in the presence of cytokines to enhance the growth and/or expansion of g-NK cell objects and/or NK cell subsets that overlap with g-NK cell subsets or share extracellular surface markers. In some aspects, the provided methods can also amplify other NK cell subsets, such as any NK cells of NKG2C Positive and negative and/or NKG2a Negative of .
In some embodiments, the sample (e.g., a biological sample) is a sample containing a plurality of cell populations including NK cell populations. In some embodiments, the biological sample is or includes blood cells, such as peripheral blood mononuclear cells. In some aspects, the biological sample is a whole blood sample, an apheresis product, or a leucocyte apheresis product. In some embodiments, the sample is a sample of Peripheral Blood Mononuclear Cells (PBMCs). Thus, in some embodiments of the provided methods, a population of Peripheral Blood Mononuclear Cells (PBMCs) can be obtained. According to the provided methods, a sample containing a plurality of cell populations including NK cell populations can be used as cells for enriching or selecting NK cell subsets for expansion.
In some embodiments, the biological sample is from a healthy subject. In some embodiments, the biological sample is from a subject having a disease condition (e.g., cancer).
In some embodiments, the cells are isolated from or selected from a sample, such as a biological sample, e.g., a sample obtained from or derived from a subject, such as a subject having a particular disease or disorder or in need of or to which cell therapy is to be administered. In some aspects, the subject is a human, such as a subject that is a patient in need of a particular therapeutic intervention (such as adoptive cell therapy to isolate, treat, and/or engineer cells). Thus, in some embodiments, the cell is a primary cell, such as a primary human cell. Samples include tissues, body fluids, and other samples taken directly from a subject. The biological sample may be a sample obtained directly from a biological source or a processed sample. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebral spinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including treated samples derived therefrom. In some aspects, the sample is a blood or blood-derived sample, or is derived from an apheresis composition or a leucocyte apheresis composition product.
In some examples, the cells are obtained from circulating blood of the subject. In some aspects, the sample contains lymphocytes, including NK cells, T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and/or platelets, and in some aspects contains cells other than erythrocytes and platelets. In some embodiments, blood cells collected from the subject are washed, e.g., to remove plasma fractions and the cells are placed in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with Phosphate Buffered Saline (PBS). In some embodiments, the wash solution is free of calcium and/or magnesium and/or many or all divalent cations. In certain embodiments, components of the blood cell sample are removed and the cells are resuspended directly in culture medium. In some embodiments, the method includes a density-based cell separation method, such as by lysing erythrocytes and centrifuging by Percoll or Ficoll gradient (such as by usingDensity centrifugation) to prepare leukocytes from peripheral blood.
In some embodiments, the biological sample is from an enriched white blood cell apheresis product collected from normal peripheral blood. In some embodiments, the enriched white blood cell apheresis product may contain fresh cells. In some embodiments, the enriched white blood cell apheresis product is a cryopreserved sample that is thawed for use in the provided methods.
In some embodiments, the biological cell source contains 5 x 10 5 to 5 x 10 8 or about 5 x 10 5 to about 5 x 10 8 NK cells or g-NK cell subpopulations or NK cell subpopulations associated with or including a surrogate marker for g-NK cells. In some embodiments of the present invention, in some embodiments, the number of NK cells or g-NK cell subsets in the biological sample or NK cell subsets associated with or comprising surrogate markers for g-NK cells is from 5X 10 to 1X 10 or from about 5X 10 to about 1X 10, from 5X 10 to 5X 10 or from about 5X 10 to about 5X 10, from 5X 10 to 1X 10 or from about 5X 10 to about 1X 10, from 1X 10 to 1X 10 or from about 1X 10 to about 1X 10 from 1X 10 to 5X 10 or from about 1X 10 to about 5X 10, from 1X 10 to 1X 10 or from about 1X 10 to about 1X 10, from 1X 10 to 5X 10 or from about 1X 10 to about 5X 10, from 5X 10 to 1X 10 or from about 5X 10 to about 1X 10, from 5X 10 to 5X 10 or from about 5X 10 to about 5X 10, from 5X 10 to 1X 10 or from about 5X 10 to about 1X 10, from 1X 10 to 1X 10 or from about 1X 10 to about 1X 10, from 1X 10 to 5X 10 or from about 1X 10 to about 5X 10, or from 5 x 10 7 to 1 x 10 8 or from about 5 x 10 7 to about 1 x 10 8.
In some embodiments, the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 3% or greater than about 3%. In some embodiments, the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 5% or greater than about 5%. In some embodiments, the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 10% or greater than about 10%. In some embodiments, the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 12% or greater than about 12%. In some embodiments, the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 14% or greater than about 14%. In some embodiments, the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 16% or greater than about 16%. In some embodiments, the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 18% or greater than about 18%. In some embodiments, the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 20% or greater than about 20%. In some embodiments, the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 22% or greater than about 22%. In some embodiments, the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 24% or greater than about 24%. In some embodiments, the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 26% or greater than about 26%. In some embodiments, the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 28% or greater than about 28%. In some embodiments, the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 30% or greater than about 30%.
In some embodiments, the subject is selected if the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 3% or greater than about 3%. In some embodiments, the subject is selected if the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker for g-NK cells, is greater than 5% or greater than about 5%. In some embodiments, the subject is selected if the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 10% or greater than about 10%. In some embodiments, the subject is selected if the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 12% or greater than about 12%. In some embodiments, the subject is selected if the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 14% or greater than about 14%. In some embodiments, the subject is selected if the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 16% or greater than about 16%. In some embodiments, the subject is selected if the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 18% or greater than about 18%. In some embodiments, the subject is selected if the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 20% or greater than about 20%. In some embodiments, the subject is selected if the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 22% or greater than about 22%. In some embodiments, the subject is selected if the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 24% or greater than about 24%. In some embodiments, the subject is selected if the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 26% or greater than about 26%. In some embodiments, the subject is selected if the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 28% or greater than about 28%. In some embodiments, the subject is selected if the percentage of NK cells in the biological sample, g-NK cells or NK cell subpopulations associated with or including the surrogate marker of g-NK cells, is greater than 30% or greater than about 30%.
In some embodiments, the biological sample is from a CMV seropositive subject. CMV infection can result in phenotypic and functional differentiation of NK cells, including the development of high proportions of NKG2C expressing NK cells that exhibit enhanced antiviral activity. CMV-associated NK cells expressing NKG2C show altered DNA methylation patterns and reduced expression of signaling molecules such as FcRgamma (Schlums et al, immunity,2015, vol.42: pages 443-456). These NK cells are associated with more efficient antibody-dependent activation, expansion and function relative to conventional NK cell subsets. In some cases, the biological sample may be from a CMV seronegative subject, as NK cells with reduced fcrγ expression may also be detected in CMV seronegative individuals, albeit at lower levels in general. In some cases, the biological sample can be from a CMV seropositive individual.
In some embodiments, the subjects are selected based on the percentage of NK cells positive for NKG2C in the peripheral blood sample. In some embodiments, the subject is selected if at least or at least about 20% of NK cells in the peripheral blood sample are positive for NKG 2C. In some embodiments, the subject is selected if at least or at least about 25% of NK cells in the peripheral blood sample are positive for NKG 2C. In some embodiments, the subject is selected if at least or at least about 30% of NK cells in the peripheral blood sample are positive for NKG 2C. In some embodiments, the subject is selected if at least or at least about 35% of NK cells in the peripheral blood sample are positive for NKG 2C. In some embodiments, the subject is selected if at least or at least about 40% of NK cells in the peripheral blood sample are positive for NKG 2C. In some embodiments, the subject is selected if at least or at least about 45% of NK cells in the peripheral blood sample are positive for NKG 2C. In some embodiments, the subject is selected if at least or at least about 50% of NK cells in the peripheral blood sample are positive for NKG 2C. In some embodiments, the subject is selected if at least or at least about 55% of NK cells in the peripheral blood sample are positive for NKG 2C. In some embodiments, the subject is selected if at least or at least about 60% of NK cells in the peripheral blood sample are positive for NKG 2C.
In some embodiments, the subjects are selected based on the percentage of NK cells negative or low level for NKG2A in the peripheral blood sample. In some embodiments, the subject is selected if at least or at least about 70% of NK cells in the peripheral blood sample are negative or low in NKG 2A. In some embodiments, the subject is selected if at least or at least about 75% of NK cells in the peripheral blood sample are negative or low in NKG 2A. In some embodiments, the subject is selected if at least or at least about 80% of NK cells in the peripheral blood sample are negative or low in NKG 2A. In some embodiments, the subject is selected if at least or at least about 85% of NK cells in the peripheral blood sample are negative or low in NKG 2A. In some embodiments, the subject is selected if at least or at least about 90% of NK cells in the peripheral blood sample are negative or low in NKG 2A.
In some embodiments, the subjects are selected based on both the percentage of NK cells positive for NKG2C in the peripheral blood sample and the percentage of NK cells negative or low level for NKG2A in the peripheral blood sample. In some embodiments, the subject is selected if at least or at least about 20% of NK cells in the peripheral blood sample are positive for NKG2C and at least or at least about 70% of NK cells in the peripheral blood sample are negative or low in NKG 2A. In some embodiments, the subject is selected if at least or at least about 30% of NK cells in the peripheral blood sample are positive for NKG2C and at least or at least about 75% of NK cells in the peripheral blood sample are negative or low in NKG 2A. In some embodiments, the subject is selected if at least or at least about 40% of NK cells in the peripheral blood sample are positive for NKG2C and at least or at least about 80% of NK cells in the peripheral blood sample are negative or low level for NKG 2A. In some embodiments, the subject is selected if at least or at least about 50% of NK cells in the peripheral blood sample are positive for NKG2C and at least or at least about 85% of NK cells in the peripheral blood sample are negative or low in NKG 2A. In some embodiments, the subject is selected if at least or at least about 60% of NK cells in the peripheral blood sample are positive for NKG2C and at least or at least about 90% of NK cells in the peripheral blood sample are negative or low in NKG 2A. In some embodiments, the subject is selected if at least or at least about 60% of NK cells in the peripheral blood sample are positive for NKG2C and at least or at least about 95% of NK cells in the peripheral blood sample are negative or low in NKG 2A.
In some embodiments, if the subject is CMV seropositive and if the percentage of g-NK cells in a peripheral blood sample from the subject is greater than 30% or greater than about 30%, the percentage of NKG2C Positive and negative cells is greater than 20% or greater than about 20%, and the percentage of NKG2a Negative of cells is greater than 70% or greater than about 70%, then the subject is selected for cell expansion according to the provided methods.
In some embodiments, NK cells from the subject have a single nucleotide polymorphism in the CD16 gene, nucleotide 526[ thymine (T) → guanine (G) ] (SNP rs 396991), resulting in an amino acid (aa) substitution of valine (V) for phenylalanine (F) at position 158 in the mature (processed) form of the protein (F158V). In some embodiments, the NK cells have a CD16158V polymorphism (referred to herein as 158V/V) in both alleles. In some embodiments, the NK cells have a CD16158V polymorphism (referred to herein as 158V/F) in a single allele. It should be understood that references herein to the 158V+ genotype refer to both the 158V/V genotype and the 158V/F genotype. CD 16F 158V polymorphism has been found to be associated with significantly higher affinity for IgGl antibodies and has the ability to mount a more potent NK cell mediated ADCC response (Mellor et al, 2013, journal of Hematology & oncology, volume 6: page 1; musolino et al, 2008, journal of Clinical Oncology, volume 26: pages 1789-1796; and Hatjiharissi et al, 2007, blood, volume 110: pages 2561-2564). In some embodiments, antibody directed CD16 V+/g-NK cell targeting can bring improved results to patients due to improved affinity, cytotoxicity, and/or cytokine mediated effector functions of the CD169V+/g-NK cell subpopulation.
In some embodiments, provided methods comprise enriching or isolating NK cells or a subpopulation thereof from a biological sample of a subject identified as having a CD16158v+ NK cell genotype. In some embodiments, the method comprises screening the subject for the presence of a CD16158v+ nk cell genotype. In some embodiments, genomic DNA is extracted from a sample from a subject that is or includes NK cells, such as a blood sample or a bone marrow sample. In some embodiments, the sample is or includes blood cells, such as peripheral blood mononuclear cells. In some embodiments, the sample is or comprises an isolated NK cell. In some embodiments, the sample is a sample from a healthy donor subject. Any method for extracting DNA from a sample may be used. For example, nucleic acids can be readily isolated from a sample (e.g., cells) using standard techniques such as guanidinium thiocyanate-phenol-chloroform extraction (Chomocyznski et al, 1987, anal. Biochem., volume 162: page 156). Commercially available kits may also be conveniently used for extracting genomic DNA, such as Wizard genomic DNA purification kit (Promega, madison, wis.).
Any suitable sample may be genotyped. In any of the embodiments described herein, the genotyping reaction may be, for example, a pyrosequencing reaction, a DNA sequencing reaction, MASSARRAY MALDI-TOF, RFLP, allele-specific PCR, real-time allele identification, or a microarray. In some embodiments, PCR-based techniques of genomic DNA, such as RT-PCR, are performed using allele-specific primers for the polymorphism. PCR methods for amplifying target nucleic acid sequences in a sample are well known in the art and have been described, for example, in the following documents: innis et al, PCR Protocols (ACADEMIC PRESS, NY, 1990); taylor, polymerase chain reaction in 1991: basic PRINCIPLES AND automation, in PCR: A PRACTICAL Apprach, mcPherson et al, IRL Press, oxford; saiki et al, 1986, nature, volume 324: page 163; and U.S. Pat. nos. 4,683,195, 4,683,202 and 4,889,818, which are incorporated herein by reference in their entirety.
Primers for detecting the 158v+ polymorphism are known or can be readily designed by the skilled person, see for example, international published PCT application No. WO2012/061814; kim et al, 2006, blood, volume 108: pages 2720-2725; cartron et al, 2002, blood, volume 99: pages 754-758; koene et al, 1997, blood, volume 90: pages 1109-1114; hatijiharissi et al, 2007, blood, volume 110: stages 2561-2564; somboonyosdech et al, 2012, asian Biomedicine, volume 6: pages 883-889). In some embodiments, PCR may be performed using nested primers followed by allele-specific restriction enzyme digestion. In some embodiments, the first PCR primer comprises nucleic acid sequences 5'-ATA TTT ACA GAA TGG CAC AGG-3' (SEQ ID NO: 17) and 5'-GAC TTG GTA CCC AGG TTG AA-3' (SEQ ID NO: 18), while the second PCR primer is 5'-ATC AGA TTC GAT CCT ACT TCT GCA GGG GGC AT-3' (SEQ ID NO: 19) and 5'-ACG TGC TGA GCT TGA GTG ATG GTG ATG TTC AC-3' (SEQ ID NO: 20), which in some cases generates a 94bp fragment depending on the nature of the allele. In some embodiments, the primer pair comprises the nucleic acid sequences set forth in SEQ ID NO. 21 (CCCAACTCAA CTTCCCAGTG TGAT) and SEQ ID NO. 22 (GAAATCTACC TTTTCCTCTA ATAGGGCAAT). In some embodiments, the primer pair comprises the nucleic acid sequences set forth in SEQ ID NO. 21 (CCCAACTCAA CTTCCCAGTG TGAT) and SEQ ID NO. 23 (GAAATCTACC TTTTCCTCTA ATAGGGCAA). In some embodiments, the primer pair comprises the nucleic acid sequences set forth in SEQ ID NO. 21 (CCCAACTCAA CTTCCCAGTG TGAT) and SEQ ID NO. 24 (GAAATCTACC TTTTCCTCTA ATAGGGCA). In some embodiments, genotyping may be performed by quantitative real-time RT-PCR followed by RNA extraction using the primer sequences as follows: CD16 sense shown in SEQ ID NO. 25 (5'-CCAAAAGCCACACTCAAAGAC-3') and antisense shown in SEQ ID NO. 26 (5'-ACCCAGGTGGAAAGAATGATG-3') and TaqMan probe shown in SEQ ID NO. 27 (5'-AACATCACCATCACTCAAGGTTTGG-3').
To confirm genotyping, allele-specific primers may be used with a set of V allele-specific primers (e.g., the forward primer shown in SEQ ID NO:28,5'-CTG AAG ACA CAT TTT TAC TCC CAAA-3; and the reverse primer shown in SEQ ID NO:29,5' -TCC AAA AGC CAC ACT CAA AGA C-3 ') or a set of F allele-specific primers (e.g., the forward primer shown in SEQ ID NO:30,5' -CTG AAG ACA CAT TTT TAC TCC CAAC-3; and the reverse primer shown in SEQ ID NO:29,5'-TCC AAA AGC CAC ACT CAA AGA C-3').
The genomic sequence of CD16a is available in NCBI database as NG_ 009066.1. The gene ID of CD16A was 2214. Sequence information for CD16, including genetic polymorphisms, can be obtained from UniProt accession number P08637. The sequence of CD16 (F158) is shown in SEQ ID NO. 31 (residue F158 is bold and underlined). In some embodiments, CD16 (F158) further comprises a signal peptide as shown in MWQLLLPTALLLLVSA (SEQ ID NO: 32).
The sequence of CD16169V+ (resulting in the polymorphism of F169V) is designated as VAR_003960 and has the sequence shown in SEQ ID NO:33 (158 V+ polymorphism is shown in bold and underlined). In some embodiments, CD16 (158 V+) further comprises a signal peptide as shown in MWQLLLPTALLLLVSA (SEQ ID NO: 32).
In some embodiments, single Nucleotide Polymorphism (SNP) analysis is performed on genomic deoxyribonucleic acid (DNA) samples using allele-specific probes containing a fluorescent dye label (e.g., FAM or VIC) at the 5 'end and Minor Groove Binder (MGB) and non-fluorescent quencher (NFQ) at the 3' end, as well as unlabeled PCR primers to detect specific SNP targets. In some embodiments, the assay measures or detects the presence of a SNP by a change in fluorescence of a dye associated with the probe. In such embodiments, the probe hybridizes to the target DNA between the two unlabeled primers and the signal from the 5 'fluorescent dye is quenched by NFQ at its 3' end by Fluorescence Resonance Energy Transfer (FRET). During PCR, taq polymerase extends the unlabeled primer using the template as a guide and when the polymerase reaches the labeled probe, it cleaves the molecule separating the dye from the quencher. In some aspects, the qPCR instrument can detect fluorescence from the unquenched label. An exemplary reagent is a commercially available SNP assay, such as code c_25815666_10 of rs396991 (Applied Biosystems, catalog No. 4351379, for SNP genotyping of F158V in CD 16).
In some embodiments, subjects heterozygous or homozygous for the CD16 158V (F158V) polymorphism are identified. In some embodiments, subjects homozygous for the CD16 158V (F158V) polymorphism are identified. In some embodiments, NK cells or NK cell subsets are isolated or enriched from a biological sample of a subject identified as heterozygous or homozygous for the CD16 158V polymorphism. In some embodiments, NK cells or NK cell subsets are isolated or enriched from a biological sample of a subject identified as homozygous for the CD16 158V polymorphism.
In some embodiments, the method comprises enriching NK cells from the biological sample, such as from a population of PBMCs isolated or obtained from the subject. In some embodiments, the NK cell population enriched for NK cells is enriched by isolation or selection based on one or more cell-specific markers. The selection of a particular label or combination of surface labels is within the level of skill in the art. In some embodiments, the surface marker is any one or more ：CD11a、CD3、CD7、CD14、CD16、CD19、CD25、CD27、CD56、CD57、CD161、CD226、NKB1、CD62L;CD244、NKG2D、NKp30、NKp44、NKp46、NKG2A、NKG2C、KIR2DL1 and/or KIR2DL3 from the following surface antigens. In some embodiments, the surface marker is any one or more ：CD11a、CD3、CD7、CD14、CD16、CD19、CD25、CD27、CD38、CD56、CD57、CD161、CD226、NKB1、CD62L;CD244、NKG2D、NKp30、NKp44、NKp46、NKG2A、NKG2C、SLAMF7(CD319)、KIR2DL1 and/or KIR2DL3 from the following surface antigens. In particular embodiments, the one or more surface antigens include CD3 and one or more of the following surface antigens CD16, CD56, or CD57. In some embodiments, the one or more surface antigens are CD3 and CD57. In some embodiments, the one or more surface antigens are CD3, CD56, or CD16. In some embodiments, the one or more surface antigens are CD3, CD56, or CD38. In further embodiments, the one or more surface antigens are CD3, CD56, NKG2A, and CD161. In some embodiments, the one or more surface antigens are CD3, CD57, or NKG2C. In some embodiments, the one or more surface antigens are CD3, CD57, or NKG2A. In some embodiments, the one or more surface antigens are CD3, CD57, NKG2C, and NKG2A. In some embodiments, the one or more surface antigens are CD3 and CD56. In some embodiments, the one or more surface antigens are CD3, CD56, or NKG2C. In some embodiments, the one or more surface antigens are CD3, CD56, or NKG2A. In some embodiments, the one or more surface antigens are CD3, CD56, NKG2C, and NKG2A. Reagents for detecting such surface antigens, including fluorochrome conjugated antibodies, are well known and available to the skilled person.
In some embodiments, NK cell populations that are positive for (marker + or marker Positive and negative ) or express high levels (marker High height ) of one or more specific markers (such as surface markers) or cells that are negative for (marker or marker Positive and negative ) or express relatively low levels (marker or marker Positive and negative ) are enriched from a sample by the provided methods, such as by isolation or selection. Thus, it should be understood that the terms positive, pos or+) with respect to a marker or protein expressed on or in a cell are used interchangeably herein. Likewise, it should be understood that the term negative (negtive, neg or-) with respect to a marker or protein expressed on or in a cell is used interchangeably herein. Furthermore, it should be understood that references herein to cells of marker Negative of may refer to cells that are negative for the marker as well as cells that express relatively low levels of the marker, such as low levels that are not readily detectable as compared to control or background levels. In some cases, such markers are those that are not present or expressed at relatively low levels on certain NK cell populations but are present or expressed at relatively high levels on certain other lymphocyte populations (such as T cells). In some cases, such markers are those that are present on certain NK cell populations or expressed at relatively high levels but not on certain other lymphocyte populations (such as T cells or subpopulations thereof) or expressed at relatively low levels.
In some embodiments, any known separation method based on such markers may be used. In some embodiments, the separation is an affinity or immunoaffinity based separation. For example, in some aspects, isolation includes isolating cells and cell populations based on expression or expression levels of one or more markers (typically cell surface markers), e.g., by incubating with an antibody or binding partner that specifically binds such a marker, followed by a washing step, typically, and separating cells that have bound the antibody or binding partner from those cells that have not bound to the antibody or binding partner. In some embodiments, the incubation is static (without mixing). In some embodiments, the incubation is dynamic (mixing).
Such isolation steps may be based on positive selection in which cells to which the reagent has been bound are retained for further use and/or negative selection in which cells not bound to the antibody or binding partner are retained. In some examples, both fractions are reserved for further use. Isolation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell (such as those expressing a marker) refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Likewise, negative selection, removal, or depletion of a particular type of cell (such as those expressing a marker) refers to a reduction in the number or percentage of such cells, but does not necessarily result in complete removal of all such cells. For example, in some aspects, the negative selection of CD3 enriches the population of cells that are CD3 Negative of , but may also contain some residual or small percentage of other unselected cells, which in some cases may include a small percentage of cells that are CD3 Positive and negative that are still present in the enriched population. In some examples, positive selection of one of the CD57 Positive and negative or CD16 Positive and negative populations enriches the population, i.e., the CD57 Positive and negative or CD16 Positive and negative population, but may also contain some residual or small percentage of other non-selected cells, which in some cases may include the other of the CD57 or CD16 populations that are still present in the enriched population.
In some examples, multiple rounds of separation steps are performed, wherein fractions from positive or negative selection of one step are subjected to another separation step, such as subsequent positive or negative selection. In some examples, a single separation step may deplete cells expressing multiple markers simultaneously, such as by incubating the cells with multiple antibodies or binding partners, each antibody or binding partner specific for a marker targeted for negative selection. Likewise, multiple cell types can be positively selected simultaneously by incubating the cells with multiple antibodies or binding partners expressed on the multiple cell types.
In some aspects, the selection includes positive and/or negative selection steps based on expression of one or more of the surface antigens, such as in cells from a PBMC sample. In some embodiments, the isolation comprises positive selection of cells expressing CD56, cells expressing CD16 or cells expressing CD57 and/or negative selection of cells expressing CD38, and/or negative selection of cells expressing a non-NK cell marker, such as a T cell marker, for example, negative selection of cells expressing CD3 (CD 3 Negative of ). For example, in some embodiments, the isolation includes positive selection of cells expressing CD56, cells expressing CD16, or cells expressing CD57, and/or negative selection of cells expressing a non-NK cell marker, such as a T cell marker, e.g., negative selection of cells expressing CD3 (CD 3 Negative of ). In some embodiments, the isolation comprises positive selection of cells expressing CD56, cells expressing CD16, or cells expressing CD57, and/or negative selection of cells expressing CD38 (CD 38 Negative of ), negative selection of cells expressing CD161 (CD 161 Negative of ), negative selection of cells expressing NKG2A (NKG 2A Negative of ), and/or negative selection of cells expressing CD3 (CD 3 Negative of ). In some embodiments, selecting comprises isolating cells negative for CD3 (CD 3 Negative of ).
In some embodiments, the isolation includes negative selection of cells expressing CD3 (CD 3 Negative of ) and positive selection of cells expressing CD56 (CD 56 Positive and negative ). In some embodiments, the selection may also include negative selection of cells expressing CD38 (CD 38 Negative of ). In a specific embodiment, the isolated or selected cell is CD3 Negative of CD56 Positive and negative CD38 Negative of .
In some embodiments, the selection comprises negative selection of cells expressing CD3 (CD 3 Negative of ), positive selection of cells expressing CD56 (CD 56 Positive and negative ), followed by negative selection of cells expressing NKG2A (NKG 2A Negative of ) and negative selection of cells expressing CD161 (CD 161 Negative of ). In a specific embodiment, the isolated or selected cell is CD3 Negative of CD56 Positive and negative NKG2A Negative of CD161 Negative of .
In some embodiments, the selection includes negative selection of cells expressing CD3 (CD 3 Negative of ) and positive selection of cells expressing CD57 (CD 57 Positive and negative ). In a specific embodiment, the isolated or selected cell is CD3 Negative of CD57 Positive and negative .
In some embodiments, the selection includes negative selection of cells expressing CD3 (CD 3 Negative of ) and positive selection of cells expressing CD16 (CD 16 Positive and negative ). In a specific embodiment, the isolated or selected cell is CD3 Negative of CD16 Positive and negative .
In some embodiments, the selection includes negative selection of cells expressing CD3 (CD 3 Negative of ) and positive selection of cells expressing CD57 (CD 57 Positive and negative ). In a specific embodiment, the isolated or selected cell is CD3 Negative of CD57 Positive and negative . For example, CD57 Positive and negative NK cells can be isolated and enriched by depleting CD3 Positive and negative cells (negative selection for CD3 Positive and negative cells) followed by CD57 Positive and negative cell selection to enrich for NK cells. Isolation can be performed by immunoaffinity-based methods, such as the use of MACS TM microbeads. For example, in the negative selection of CD3 Negative of cells, CD3 microbeads can be used to deplete CD3 Positive and negative cells. Subsequently, CD57 microbeads can be used for CD57 enrichment of CD3 cell depleted PBMCs. NK cells enriched for CD3 Negative of /CD57 Positive and negative can then be used for expansion in the provided methods.
In some embodiments, the selection may also include positive selection of cells expressing NKG2C (NKG 2C Positive and negative ) and/or negative selection of cells NKG2A (NKG 2A Negative of ). In some embodiments, the selection comprises negative selection of cells expressing CD3 (CD 3 Negative of ), positive selection of cells expressing CD57 (CD 57 Positive and negative ), and positive selection of cells expressing NKG2C (NKG 2C Positive and negative ). In a specific embodiment, the isolated or selected cell is CD3 Negative of CD57 Positive and negative NKG2C Positive and negative . In some embodiments, the selection comprises negative selection of cells expressing CD3 (CD 3 Negative of ), positive selection of cells expressing CD57 (CD 57 Positive and negative ), and negative selection of cells expressing NKG2A (NKG 2A Negative of ). In a specific embodiment, the isolated or selected cell is CD3 Negative of CD57 Positive and negative NKG2A Negative of . In some embodiments, the selection comprises negative selection of cells expressing CD3 (CD 3 Negative of ), positive selection of cells expressing CD57 (CD 57 Positive and negative ), positive selection of cells expressing NKG2C (NKG 2C Positive and negative ), and negative selection of cells expressing NKG2A (NKG 2A Negative of ). In a specific embodiment, the isolated or selected cell is CD3 Negative of CD57 Positive and negative NKG2C Positive and negative NKG2A Negative of .
In some of any of the provided embodiments, the selecting may further comprise negative selection of cells expressing CD38 (CD 38 Negative of ). In a specific embodiment, the isolated or selected cell is CD3 Negative of CD57 Positive and negative CD38 Negative of . In a specific embodiment, the isolated or selected cell is CD3 Negative of CD57 Positive and negative CD38 Negative of NKG2C Positive and negative . In a specific embodiment, the isolated or selected cell is CD3 Negative of CD57 Positive and negative CD38 Negative of NKG2A Negative of . In a specific embodiment, the isolated or selected cell is CD3 Negative of CD57 Positive and negative CD38 Negative of NKG2C Positive and negative NKG2A Negative of .
In some embodiments, the selection includes negative selection of cells expressing CD3 (CD 3 Negative of ) and positive selection of cells expressing CD56 (CD 56 Positive and negative ). In a specific embodiment, the isolated or selected cell is CD3 Negative of CD56 Positive and negative . In some embodiments, the selection comprises negative selection of cells expressing CD3 (CD 3 Negative of ), positive selection of cells expressing CD56 (CD 56 Positive and negative ), and positive selection of cells expressing NKG2C (NKG 2C Positive and negative ). In a specific embodiment, the isolated or selected cell is CD3 Negative of CD56 Positive and negative NKG2C Positive and negative . In some embodiments, the selection comprises negative selection of cells expressing CD3 (CD 3 Negative of ), positive selection of cells expressing CD56 (CD 56 Positive and negative ), and negative selection of cells expressing NKG2A (NKG 2A Negative of ). In a specific embodiment, the isolated or selected cell is CD3 Negative of CD56 Positive and negative NKG2A Negative of . In some embodiments, the selection comprises negative selection of cells expressing CD3 (CD 3 Negative of ), positive selection of cells expressing CD56 (CD 56 Positive and negative ), positive selection of cells expressing NKG2C (NKG 2C Positive and negative ), and negative selection of cells expressing NKG2A (NKG 2A Negative of ). In a specific embodiment, the isolated or selected cell is CD3 Negative of CD56 Positive and negative NKG2C Positive and negative NKG2A Negative of .
In some of any of the provided embodiments, the selecting may further comprise negative selection of cells expressing CD38 (CD 38 Negative of ). In a specific embodiment, the isolated or selected cell is CD3 Negative of CD56 Positive and negative CD38 Negative of . In a specific embodiment, the isolated or selected cell is CD3 Negative of CD56 Positive and negative CD38 Negative of NKG2C Positive and negative . In a specific embodiment, the isolated or selected cell is CD3 Negative of CD56 Positive and negative CD38 Negative of NKG2A Negative of . In a specific embodiment, the isolated or selected cell is CD3 Negative of CD56 Positive and negative CD38 Negative of NKG2C Positive and negative NKG2A Negative of .
In some of any of the provided embodiments, the g-NK cells are cells having a g-NK surrogate surface marker profile. In some embodiments, the g-NK cell surrogate surface marker profile is CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of . In some embodiments, the g-NK cell surrogate surface marker profile is NKG2a Negative of /CD161 Negative of . In some of any of these embodiments, the g-NK cell surrogate surface marker profile is CD38 Negative of . In some of any such embodiments, CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative is used as an alternative surface marker profile for NK cells. In some of any of these embodiments, the g-NK cell surrogate surface marker profile further comprises an NK cell surrogate surface marker profile. In some of any of these embodiments, the g-NK cell surrogate surface marker profile further comprises CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative . In a specific embodiment, the g-NK cell replacement surface marker profile comprises CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative /CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of . In other specific embodiments, the g-NK cell replacement surface marker profile comprises CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative /NKG2A Negative of /CD161 Negative of . In other specific embodiments, the g-NK cell replacement surface marker profile comprises CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative /CD38 Negative of .
In some embodiments, methods of isolating, selecting, and/or enriching cells, such as by positive or negative selection based on expression of one or more cell surface markers, can include selection based on immunoaffinity. In some embodiments, immunoaffinity-based selection comprises contacting a sample containing cells (such as PBMCs) with an antibody or binding partner that specifically binds one or more cell surface markers. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a sphere or bead, e.g., microbeads, nanobeads (including agarose), magnetic beads, or paramagnetic beads, to allow separation of cells for positive and/or negative selection. In some embodiments, the ball or bead may be packed into a column for immunoaffinity chromatography, wherein a sample containing cells (such as PBMCs) is contacted with a matrix of the column and then eluted or released therefrom.
Incubation is typically performed under conditions in which an antibody or binding partner that specifically binds to an antibody or binding partner attached to a magnetic particle or bead specifically binds to a cell surface molecule (if present on cells within the sample).
In some aspects, the sample is placed in a magnetic field and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from unlabeled cells. For positive selection, cells attracted to the magnet were retained; for negative selection, cells that were not attracted (unlabeled cells) were retained. In some aspects, a combination of positive and negative selections is performed during the same selection step, wherein the positive and negative fractions are retained and further processed or subjected to further separation steps.
In some embodiments, the magnetically responsive particles remain attached to cells that are subsequently incubated and/or cultured. In some aspects, the particles remain attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods of removing magnetizable particles from cells are known and include, for example, the use of competitive non-labeled antibodies, magnetizable particles or antibodies conjugated with cleavable linkers, and the like. In some embodiments, the magnetizable particles are biodegradable.
In some embodiments, the affinity-based selection is via Magnetically Activated Cell Sorting (MACS) (Miltenyi Biotech, auburn, CA). Magnetically Activated Cell Sorting (MACS) systems are capable of selecting cells having magnetized particles attached thereto in high purity. In certain embodiments, MACS is operated in a mode in which non-target and target materials elute sequentially after application of an external magnetic field. That is, cells attached to the magnetized particles are held in place, while unattached material is eluted. Then, after this first elution step is completed, the substances that are trapped in the magnetic field and prevented from eluting are released in a way that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and removed from the heterogeneous cell population.
In some embodiments of any such embodiments, the method comprises administering to the subject IL-12, IL-15, IL-18, IL-2 and/or CCL5 prior to enriching (such as selecting and/or isolating) NK cells or a subpopulation thereof.
B. method for amplifying NK cells rich in g-NK cells
In embodiments of the provided methods, the enriched NK cells are incubated or cultured in the presence of feeder cells, such as under conditions that support proliferation and expansion of NK cell subsets, and in particular g-NK cell subsets.
In particular aspects, feeder cells include cells that stimulate or promote expansion of NKG2C Positive and negative and/or inhibit expansion of NKG2a Positive and negative cells. In some embodiments, the feeder cells are cells expressing or transfected with HLA-E or a hybrid HLA-E containing an HLA-A2 signal sequence. Examples of such heterozygosity are, for example, AEH heterozygous genes containing MHC class I (such as HLA-A 2) promoters and signal sequences, as well as HLA-E mature protein sequences, which in some cases can produce a mature protein that is identical to the mature protein encoded by the HLA-E gene but stably expressed on the cell surface (see, e.g., lee et al, 1998, journal of Immunology, volume 160: pages 4951-4960). In some embodiments, the cell is an LCL 721.221, K562 cell, or RMA-S cell transfected to express an MHC-E molecule stabilized in the presence of an MHC class I (such as HLA-A 2) leader sequence. Cell lines engineered to express cell surface HLA-E that are stable in the presence of MHC class I such as HLA-A2 leader peptide are known in the art (Lee et al, 1998, journal of Immunology, volume 160: pages 4951-4960; zhongguo et al, 2005, volume 13: pages 464-467; garcia et al, 2002, eur J.Immunol., volume 32: pages 936-944). In some embodiments, 221.aeh cells, such as irradiated 221.aeh cells, can be used as feeder cells, or any other HLA-E expressing cell line or otherwise HLA-negative irradiated HLA-E expressing cell line, such as K562. In some embodiments, the cell line may be transfected to express HLA-E. In some embodiments, K562 cells that express membrane-bound IL-15 (K562-mb 15) or membrane-bound IL-21 (K562-mb 21) can be used as feeder cells. Examples of such cell lines for use in the methods provided herein are 221-AEH cells.
In embodiments, HLA-expressing feeder cells are cryopreserved and thawed prior to use. In some embodiments, if the cells have been transfected to express HLA-E such as 221.aeh cells, the cells can be grown in the presence of a suitable nutrient (e.g., including serum or other suitable serum replacement) and a selection agent, and then used in the method. For example, in the case of 221.AEH cells, the cells can be cultured in cell culture medium supplemented with hygromycin B (e.g., 0.1% to 10%, such as at or about 1%) to maintain a selective pressure on the cells, thereby maintaining high levels of plasmid HLA-E. Cells can be maintained at a density of 1 x 10 5 cells/mL to 1 x 10 6 cells/mL until use.
In particular embodiments, HLA-E expressing feeder cells, e.g., 221.Aeh cells, added to the culture are non-dividing, such as by X-ray irradiation or gamma irradiation. HLA-E expressing feeder cells, such as 221.Aeh, can be irradiated on or before the day they are used in the provided methods. In some embodiments, the HLA-E expressing feeder cells are irradiated with gamma rays ranging from about 1000rad to 10000rad, such as 1000rad to 5000rad, to prevent cell division. In some embodiments, the feeder cells expressing HLA-E are irradiated with gamma rays ranging from about 10Gy to 100Gy, such as 10Gy to 50Gy, to prevent cell division. In some embodiments, the cells are irradiated to 100 Gy. In other embodiments, the irradiation is performed by x-ray irradiation. In some embodiments, the feeder cells expressing HLA-E are irradiated with x-rays ranging from about 10Gy to 100Gy, such as 10Gy to 50Gy, to prevent cell division. In some embodiments, an a Rad-Sure TM blood irradiation indicator may be used to provide positive visual verification of the irradiation. In aspects of the methods provided, feeder cells are never removed; as a result of the irradiation, NK cells will be directly cytotoxic to the feeder cells and the feeder cells will die during culture.
In some embodiments, the enriched, selected and/or isolated NK cells are incubated or cultured in the presence of HLA-E expressing feeder cells (e.g., 221.Aeh cells), such as an irradiated population thereof, the ratio of feeder cells to enriched NK cells being greater than or about 1:10 of the ratio of HLA-E feeder cells (e.g., 221.Aeh cells), such as an irradiated population thereof, to enriched NK cells, and the ratio of such feeder cells to enriched NK cells being or about 1:10 and being or about 10:1.
In some embodiments, the ratio of HLA-E expressing feeder cells (e.g., 221.AEH cells), such as an irradiated population thereof, is the ratio of such feeder cells to enriched NK cells, which is between 1:10 and 10:1 or between about 1:10 and about 10:1, between 1:10 and 5:1 or between about 1:10 and about 5:1, between 1:10 and 2.5:1 or between about 1:10 and about 2.5:1, between 1:10 and 1:1 or between about 1:10 and about 1:1, between 1:10 and 1:2.5 or between about 1:10 and about 1:2.5, between 1:10 and 1:5 or between about 1:10 and about 1:5, between about 1:5 and about 10:1, between about 1:5 and 5:1 or between about 1:5 and about 5:1, between 1:5 and 2.5:1 or between about 1:5 and about 2.5:1, between 1:5 and 1:1 or between about 1:5 and about 1:2.5, between 1:5 and about 1:1, between about 2.5 and about 1:1:1 or between about 1:5 and about 1:2.5.5.5 and about 1:1.1. Between 1:2.5 and 10:1 or between about 1:2.5 and about 10:1, between 1:2.5 and 5:1 or between about 1:2.5 and about 5:1, between 1:2.5 and 2.5:1 or between about 1:2.5 and about 2.5:1, between 1:2.5 and about 10:1 or between about 1:2.5 and about 1:1, between 1:1 and 10:1 or between about 1:1 and about 10:1, between 1:1 and 5:1 or between about 1:1 and about 5:1, between 1:1 and 3:1 or between about 1:1 and about 3:1, between 1:1 and 2.5:1 or between about 1:1 and about 2.5:1, between 2.5:1 and 10:1, between about 2.5:1 and about 10:1, between 1:1 and about 1, between about 1:1 and about 5:1 or between about 1:1 and about 5:1, between about 1:1 and about 10:1 or between about 1:1 and about 1:1:1 or between about 1:1:1 and about 5:1:1, between about 1 and about 1:1:1 and about 5:1 or between about 1:1:1 and about 1:1 and about 5:1:1 and about 5:1 and 5:1 or between 1 and between 1:1 and 1 and about 1:1:1 and 5:1 and between 1 and 5:1 and between 1 and about 1:1 and 5:1 and between 1 and between 1:1 and 1 and 5:5:5:5 and between 1 and between 5:5 and 1 and 5 and between 5:5 and between 5 and 1 and between 5 and 5:5 and between 5 and 1 and 5 and 5:5 and between 5 and between 5 each including an end value.
In some embodiments, the ratio of HLA-expressing feeder cells (e.g., 221.aeh cells) (such as an irradiated population thereof) is the ratio of such feeder cells to enriched NK cells, which is or is about 1.25:1, 1.5:1, 1.75:1, 2.0:1, 2.25:1, 2:5:1, 2.75:1, 3.0:1, 3.25:1, 3.5.:1, 3.75:1, 4.0:1, 4.25:1, 4.5:1, 4.75:1, or 5:1, or any value therebetween. In some embodiments, the ratio of HLA-expressing feeder cells (e.g., 221.Aeh cells), such as an irradiated population thereof, is the ratio of such feeder cells to enriched cells, which is less than or less than about 5:1. In some embodiments, the ratio of HLA-expressing feeder cells (e.g., 221.Aeh cells), such as irradiated populations thereof, is a ratio between 1:1 and 2.5:1 or between about 1:1 and about 2.5:1, inclusive. In some embodiments, the ratio of HLA-expressing feeder cells (e.g., 221.Aeh cells), such as an irradiated population thereof, is or is about 2.5:1 ratio. In some embodiments, the ratio of HLA-expressing feeder cells (e.g., 221.Aeh cells), such as an irradiated population thereof, is or is about 2:1 ratio.
In some cases, if the starting NK cell population has been cryopreserved, i.e., subjected to freezing/thawing, prior to expansion, a lower 221.Aeh to NK cell ratio may be employed than with the method using fresh NK cells. It was found here that a ratio of 221.Aeh to frozen/thawed NK cells of 1:1 resulted in comparable expansion in cultures containing a ratio of 221.Aeh to fresh NK cells of 2.5:1. In some aspects, the lower ratio ensures a higher number of NK cells in the culture to allow more cell-to-cell contact, which can play a role in promoting initial growth and expansion. In some embodiments, if an initially enriched NK cell population from the sample has been subjected to freezing/thawing, a ratio of 221.Aeh to frozen/thawed NK cells of about 2:1 to 1:2 is used or used. In a specific embodiment, the ratio is 1:1. It will be appreciated that higher ratios, such as 2.5:1 of 221.AEH to frozen/thawed NK cells, may be used, but this may require longer incubation times, for example, at or about 21 days, to reach the desired threshold density or number.
In some embodiments, NK cells are expanded by further adding non-dividing Peripheral Blood Mononuclear Cells (PBMCs) to the culture. In some aspects, the non-dividing feeder cells can comprise X-ray irradiated PBMC feeder cells. In some aspects, the non-dividing feeder cells may comprise gamma irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays ranging from about 1000rad to 10000rad, such as 1000rad to 5000rad, to prevent cell division. In some embodiments, the PBMCs are irradiated with gamma rays ranging from about 10Gy to 100Gy, such as 10Gy to 50Gy, to prevent cell division. In some aspects, during at least a portion of the incubation, the irradiated feeder cells are present in the medium concurrently with non-dividing (e.g., irradiated) HLA-E expressing feeder cells. In some aspects, non-dividing (e.g., irradiated) PBMC feeder cells, HLA-E expressing feeder cells, and enriched NK cells are added to the culture on the same day, such as on the day of culture initiation, e.g., at or about or near the same time.
In some embodiments, the incubating or culturing is further performed in the presence of irradiated PBMCs as feeder cells. In some embodiments, the irradiated PBMC feeder cells are autologous to or from the same subject as the isolated or selected NK cell enriched subject. In particular embodiments, the PBMCs are obtained from the same biological sample as used for enriching NK cells, e.g. whole blood or white blood cell apheresis or apheresis products. Once obtained, a portion of PBMCs was retained for irradiation prior to enrichment of NK cells as described above.
In some embodiments, the irradiated PBMCs are present as feeder cells in a ratio of such feeder cells to enriched NK cells of: from 1:10 to 10:1 or from about 1:10 to about 10:1, from 1:10 to 5:1 or from about 1:10 to about 5:1, from 1:10 to 2.5:1 or from about 1:10 to about 2.5:1, from 1:10 to 1:1 or from about 1:10 to about 1:1, from 1:10 to 1:2.5 or from about 1:10 to about 1:2.5 from 1:10 to 1:5 or from about 1:10 to about 1:5, from 1:5 to 10:1 or from about 1:5 to about 10:1, from 1:5 to 5:1 or from about 1:5 to about 5:1, from 1:5 to 2.5:1 or from about 1:5 to about 2.5:1, from 1:5 to 1:1 or from about 1:5 to about 1:1, from 1:5 to 1:2.5 or from about 1:5 to about 1:2.5 from 1:2.5 to 10:1 or from about 1:2.5 to about 10:1, from 1:2.5 to 5:1 or from about 1:2.5 to about 5:1, from 1:2.5 to 2.5:1 or from about 1:2.5 to about 2.5:1, from 1:2.5 to 1:1 or from about 1:2.5 to about 1:1, from 1:1 to 10:1 or from about 1:1 to about 10:1, from 1:1 to 5:1 or from about 1:1 to about 5:1, from 1:1 to 2.5:1 or from about 1:1 to about 2.5:1, from 2.5:1 to 10:1 or from about 2.5:1 to about 10:1, from 2.5:1 to about 5:1 or from about 2.5:1, or from about 5:1 to 10:1, or from about 5:1 to about 10:1.
In some embodiments, the irradiated PBMCs are present as feeder cells in a ratio of such feeder cells to enriched NK cells of: between 1:1 and 5:1 or between about 1:1 and about 5:1, such as at or about 1.25:1, 1.5:1, 1.75:1, 2.0:1, 2.25:1, 2:5:1, 2.75:1, 3.0:1, 3.25:1, 3.5:1, 3.75:1, 4.0:1, 4.25:1, 4.5:1, 4.75:1, or 5:1, or any value therebetween. In some embodiments, the irradiated PBMCs are present at a ratio of such feeder cells to enriched NK cells of at or about 5:1.
In particular embodiments, during at least a portion of the incubation or culture, one or more cells or cell types (such as T cells) of the irradiated PBMCs are activated and/or the incubation or culture is performed in the presence of at least one stimulatory agent capable of stimulating activation of one or more T cells of the PBMC feeder cells. In some embodiments, at least one stimulatory agent specifically binds to a member of the TCR complex. In some embodiments, the at least one stimulatory agent specifically binds CD3, optionally CD3 epsilon. In some aspects, the at least one stimulatory agent is an anti-CD 3 antibody or antigen binding fragment. Exemplary anti-CD 3 antibodies include mouse anti-human CD3 (OKT 3).
In some embodiments, the anti-CD 3 antibody or antigen binding fragment is present during at least a portion of the incubation that includes irradiated PBMC feeder cells. In some embodiments, the anti-CD 3 antibody or antigen binding fragment is added to the culture or the incubation at or about the same time as the irradiated PBMCs. For example, the anti-CD 3 antibody or antigen binding fragment is added at or about the beginning of the incubation or culture. In particular aspects, the anti-CD 3 antibody or antigen-binding fragment may be removed or its concentration reduced during the culture or incubation process, such as by exchanging or rinsing the medium. In particular embodiments, these methods do not include the addition back or replenishment of the medium with an anti-CD 3 antibody or antigen binding fragment after exchange or washing.
In some embodiments, the concentration of the anti-CD 3 antibody or antigen-binding fragment added or present during at least a portion of the culturing or incubation is between 10ng/mL and 5. Mu.g/mL or between about 10ng/mL and about 5. Mu.g/mL, such as between 10ng/mL and 2. Mu.g/mL or between about 10ng/mL and about 2. Mu.g/mL, between 10ng/mL and 1. Mu.g/mL or between about 10ng/mL and about 1. Mu.g/mL, between 10ng/mL and 500ng/mL or between about 10ng/mL and about 500ng/mL, between 10ng/mL and 100ng/mL or between about 10ng/mL, between 10ng/mL and 50ng/mL or between about 10ng/mL, between 50ng/mL and about 50ng/mL, between about 50ng/mL and about 5. Mu.g/mL, such as between 50ng/mL and 2 ng/mL, between about 50ng/mL and about 5 ng/mL, between about 50ng/mL, between 10ng and 100ng/mL, between about 50ng/mL and 100ng/mL, between 10ng/mL and 100ng/mL, between about 50ng/mL and about 50ng/mL, between about 50ng/mL and 50ng/mL, between about 50ng and 50ng/mL, between about 10ng and 100ng/mL and between about 5 mg/mL and between 10ng/mL Each including the end value between 100ng/mL and 1 μg/mL or between about 100ng/mL and about 1 μg/mL, between 100ng/mL and 500ng/mL or between about 100ng/mL and about 500ng/mL, between 500ng/mL and 5 μg/mL or between about 500ng/mL and about 5 μg/mL, between 500ng/mL and 2 μg/mL or between about 500ng/mL and about 2 μg/mL, between 500ng/mL and 1 μg/mL or between about 500ng/mL and about 1 μg/mL, between 1 μg/mL and 5 μg/mL or between about 1 μg/mL and about 5 μg/mL, between 1 μg/mL and 2 μg/mL or between about 1 μg/mL and about 2 μg/mL or between 2 μg/mL and 5 μg/mL or between about 2 μg/mL and about 5 μg/mL. In some embodiments, the concentration of the anti-CD 3 antibody or antigen-binding fragment is at or about 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, or 100ng/mL, or any value in between any of the foregoing. In some embodiments, the concentration of the anti-CD 3 antibody or antigen-binding fragment is at or about 50ng/mL.
In some embodiments, the term "antibody" refers to immunoglobulin molecules as well as antigen binding portions or fragments of immunoglobulin (Ig) molecules, i.e., molecules that comprise an antigen binding site that specifically binds to (immunoreacts with) an antigen. The term antibody includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof, such as dAb, fab, fab ', F (ab') 2, fv), single chain (scfv) or single domain antibodies (sdab). Typically, an "antigen binding fragment" comprises at least one CDR of an immunoglobulin heavy and/or light chain that binds to at least one epitope of an antigen of interest. In this regard, an antigen binding fragment may comprise 1,2, 3, 4, 5, or all 6 CDRs from the Variable Heavy (VH) and Variable Light (VL) sequences of an antibody that binds an antigen, such as typically six CDRs for an antibody containing VH and VL (for each of the heavy and light chains, "CDR1", "CDR2", and "CDR 3"), or three CDRs for an antibody containing a single variable domain.
An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') 2; a diabody; a linear antibody; variable heavy chain (V H) regions, single chain antibody molecules such as scFv and single domain V H monoclonal antibodies; and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibody is a single chain antibody fragment, such as an scFv, comprising a variable heavy chain region and/or a variable light chain region.
In some embodiments, incubation or culture is initiated in the presence of such enriched NK cells, such as selected and/or isolated NK cells, at a concentration of or about, or at least about, 0.05 x 10 6 enriched NK cells/mL, or about, 0.1 x 10 6 enriched NK cells/mL, or about, 0.2 x 10 6 enriched NK cells/mL, or about, 0.5 x 10 6 enriched NK cells/mL, or about, 1.0 x 10 6 enriched NK cells/mL. In embodiments of the provided methods, incubation or culture is initiated in the presence of such enriched NK cells, such as selected and/or isolated NK cells, at a concentration between 0.05X10 6 enriched NK cells/mL and 1.0X10 6 enriched NK cells/mL or between about 0.05X10 6 enriched NK cells/mL and about 1.0X10 6 enriched NK cells/mL, such as between 0.05X10 6 enriched NK cells/mL and 0.75X10 6 enriched NK cells/mL or between about 0.05X10 6 enriched NK cells/mL and about 0.75X10 6 enriched NK cells/mL or between about 0.0510 6 enriched NK cells/mL and 0.5X10 6 enriched NK cells/mL or between about 0.05X10 6 enriched NK cells/mL and about 0.5X10 NK cells/mL or between about 5 NK cells/NK 28 enriched NK cells/mL or between about 0.05X10.0510 enriched NK cells/mL and about 620510 enriched NK cells/mL or between about 20 NK cells/mL or between about 0.05X10.05X10.7X10 enriched NK cells/mL and about 20 enriched NK cells/mL or between about 20 NK cells/mL and about 20.05X10.7X10.7X10.enriched NK cells/mL is/mL Between 0.1X10 6 and 1.0X10 6 or between about 0.1X10 6 and about 1.0X10 6, between about 0.1X10 6 and 0.75X10 6 or between about 0.1X10 6 and about 0.75X10 6, between 0.1X10 6 and 0.5X10 6 or between about 0.1X10 6 and about 0.5X10 6, the cells are used for the preparation of a pharmaceutical composition between 0.1X10 6 enriched NK cells/mL and 0.20X10 6 enriched NK cells/mL or between about 0.1X10 6 enriched NK cells/mL and about 0.20X10 6 enriched NK cells/mL, between 0.20X10 6 enriched NK cells/mL and 1.0X10 6 enriched NK cells/mL or between about 0.20X10 6 enriched NK cells/mL and about 1.0X10 6 enriched NK cells/mL, between 0.20X10 6 enriched NK cells/mL and 0.75X10 6 enriched NK cells/mL or between about 0.20X10 6 enriched NK cells/mL and about 0.75X10 6 enriched NK cells/mL, included among the values of between 0.20X10 6 and 0.5X10 6 enriched NK cells/mL, between about 0.20X10 6 and about 0.5X10 6 enriched NK cells/mL, between 0.5X10 6 and 1.0X10 6 enriched NK cells/mL, or between about 0.5X10 6 and about 1.0X10 6 enriched NK cells/mL, between 0.5X10 6 and 0.75X10 6 enriched NK cells/mL, or between about 0.5X10 562 enriched NK cells/mL and about 0.75X10 6 enriched NK cells/mL, between 0.75X10 6 and 1.0X10 6 enriched NK cells/mL, between about 0.X10 and about 5 NK cells/mL, or between about 3410 NK cells/mL, are the respective values of between about 0.7X10.7X10 and about 1.7X10 NK cells/mL. In some embodiments, in the presence of such enriched NK cells, such as selected and/or isolated NK cells, incubation or culture is initiated at a concentration of at or about 0.2 x 10 6 enriched NK cells/mL.
In some embodiments of any such embodiments, the amount of enriched NK cells added or present at the beginning of the incubation or culture, such as the amount of enriched NK cells selected from or isolated from PBMCs as described above in section v.a, is at least or at least about 1×10 5 cells, at least or at least about 2×10 5 cells, at least or at least about 3×10 5 cells, at least or at least about 4×10 5 cells, at least or at least about 5×10 5 cells, at least or at least about 6×10 5 cells, at least or at least about 7×10 5 cells, at least or at least about 8×10 5 cells, at least or at least about 9×10 5 cells, at least or at least about 1×10 6 cells or more. In particular embodiments, the amount of enriched NK cells, such as the amount of enriched NK cells selected from or isolated from PBMCs as described above, is at least or about 1 x 10 6 cells.
In some embodiments, at the beginning of incubation or culture, an enriched NK cell population, such as the enriched NK cell population selected or isolated as described above in section v.a, comprises at least or at least about 2.0×10 6 enriched NK cells, at least or at least about 3.0×10 6 enriched NK cells, at least or at least about 4.0×10 6 enriched NK cells, at least or at least about 5.0×10 6 enriched NK cells, at least or at least about 6.0×10 6 enriched NK cells, at least or at least about 7.0×10 6 enriched NK cells, at least or at least about 8.0×10 6 enriched NK cells, at least or at least about 9.0×10 6 enriched NK cells, at least or at least about 1.0×10 7 enriched NK cells, at least or at least about 5.0×10 7 enriched NK cells, at least or at least about 1.0×10 8 enriched NK cells, at least or at least about 5.0×10 8 enriched NK cells, or at least about 1.0×10 NK cell 9 enriched. In some embodiments, the enriched NK cell population comprises at least or at least about 2.0 x 10 5 enriched NK cells. In some embodiments, the enriched NK cell population comprises at least or at least about 1.0 x 10 6 enriched NK cells. In some embodiments, the enriched NK cell population comprises at least or at least about 1.0 x 10 7 enriched NK cells.
In some embodiments, at the beginning of the incubation or culture, an enriched NK cell population, such as the enriched NK cell population selected or isolated as described above in section v.a, comprises the following enriched NK cells: between 2.0X10 5 enriched NK cells and 1.0X10 9 enriched NK cells or between about 2.0X10 5 enriched NK cells and about 1.0X10 9 enriched NK cells, between 2.0X10 9 enriched NK cells and 5.0X10 9 enriched NK cells or between about 2.0X10 9 enriched NK cells and about 5.0X10 9 enriched NK cells, between 2.0X10 9 enriched NK cells and 1.0X10 9 enriched NK cells or between about 2.0X10 9 enriched NK cells and about 1.0X10 9 enriched NK cells, between 2.0X10 9 enriched NK cells and 5.0X10 9 enriched NK cells or between about 2.0X10 9 enriched NK cells and about 5.0X10 9 enriched NK cells, between about 9 NK cells and about 5.0X10 enriched NK cells between 2.0X10 9 enriched NK cells and 1.0X10 9 enriched NK cells or between about 2.0X10 9 enriched NK cells and about 1.0X10 9 enriched NK cells, between 2.0X10 9 enriched NK cells and 5.0X10 9 enriched NK cells or between about 2.0X10 9 enriched NK cells and about 5.0X10 9 enriched NK cells, between 2.0X10 9 enriched NK cells and 1.0X10 9 enriched NK cells or between about 2.0X10 9 enriched NK cells and about 1.0X10 9 enriched NK cells, between 1.0X10 9 enriched NK cells and 1.0X10 9 enriched NK cells or between about 9 enriched NK cells and about 1.0X10.3932 enriched NK cells, the method may further comprise the step of enriching between 1.0×10 6 NK cells and 5.0×10 8 NK cells, or between about 1.0×10 372 NK cells and about 5.0×10 8 NK cells, between about 1.0×10 8 NK cells and 1.0×10 8 NK cells, or between about 1.0×10 8 NK cells and about 1.0×10 8 NK cells, between about 1.0×10 NK cells and 5.0×10 NK cells, or between about 1.0×10 NK cells and 5.0×3932 NK cells, or between about 1.0×10 NK cells and 5.0×10 NK cells, or between about 1.0×10 NK cells and 8 NK cells, or between about 1.0×10 NK cells and 1.0×10 NK cells, or between about 1.0×10 NK cells and 8 NK cells, or between about 1.0×10 NK cells and about 2 NK cells and 8 NK cells, or between about 1.0×10 NK cells and 8 NK cells or between about 1.0×10×10 NK cells or between about 2 NK cells and 8 NK cells or between about 1.0×10 NK cells and 8 NK cells The method may further comprise the step of enriching the cells with the cells between 5.0×10 6 NK cells and 5.0×10 7 NK cells, or between about 5.0×10 372 NK cells and about 5.0×10 7 NK cells, between 5.0×10 7 NK cells and 1.0×10 7 NK cells, or between about 5.0×10 NK cells and about 1.0×10 7 NK cells, between 1.0×10 NK cells and 1.0×10 NK cells, or between about 1.0×10 NK cells and 1.0×3932 NK cells, or between about 1.0×10 NK cells and about 1.0×10 NK cells, or between 1.0×10 NK cells and 5.0×3932 NK cells, or between about 1.0×10 NK cells and 7 NK cells, or between about 1.0×10 NK cells and about 1.10 NK cells, or between about 1.0×10 NK cells and 7 NK cells, or between about 1.0×10 NK cells and about 1.10 NK cells or between about 1.0×10 NK cells and 7 NK cells Between 5.0X10 7 enriched NK cells and 1.0X10 8 enriched NK cells or between about 5.0X10 7 enriched NK cells and about 1.0X10 8 enriched NK cells, between 1.0X10 8 enriched NK cells and 1.0X10 9 enriched NK cells or between about 1.0X10 8 enriched NK cells and about 1.0X10 9 enriched NK cells, between 1.0X10 8 enriched NK cells and 5.0X10 8 enriched NK cells or between about 1.0X10 8 enriched NK cells and about 5.0X10 8 enriched NK cells or between 5.0X10 8 enriched NK cells and 1.0X10 9 enriched NK cells or between about 5.0X10 8 enriched NK cells and about 1.0X10 9 enriched NK cells. In some embodiments, at the beginning of the culture or incubation, the enriched NK cell population comprises between 2.0×10 5 and 5.0×10 7 enriched NK cells or between about 2.0×10 5 and about 5.0×10 7 enriched NK cells. In some embodiments, at the beginning of the culturing or incubation, the enriched NK cell population comprises between 1.0×10 6 and 1.0×10 8 enriched NK cells or between about 1.0×10 6 and about 1.0×10 8 enriched NK cells. In some embodiments, at the beginning of the culture or incubation, the enriched NK cell population comprises between 1.0×10 7 and 5.0×10 8 enriched NK cells or between about 1.0×10 7 and about 5.0×10 8 enriched NK cells. In some embodiments, at the beginning of the culture or incubation, the enriched NK cell population comprises between 1.0×10 7 and 1.0×10 9 enriched NK cells or between about 1.0×10 7 and about 1.0×10 9 enriched NK cells.
In some embodiments of the present invention, in some embodiments, the percentage of g-NK cells in the enriched NK cell population present at the beginning of the culturing or incubation is between 20% and 90% or between about 20% and about 90%, between 20% and 80% or between about 20% and about 80%, between 20% and 70% or between about 20% and about 70%, between 20% and 60% or between about 20% and about 60%, between 20% and 50% or between about 20% and about 50%, between 20% and 40% or between about 20% and about 40%, between 20% and 30% or between about 20% and about 30%, between 30% and 90% or between about 30% and about 90%, between 30% and 80% or between about 30% and about 80%, between 30% and 70% or between about 30% and about 70%, between 30% and 60% or between about 30% and about 60%, or between about 30% and about 60%. Between 30% and 50%, or between about 30% and about 50%, between 30% and 40%, or between about 30% and about 40%, between 40% and 90%, or between about 40% and about 90%, between 40% and 80%, or between about 40% and about 80%, between 40% and 70%, or between about 40% and about 70%, between 40% and 60%, or between about 40% and about 60%, between 40% and 50%, or between about 40% and about 50%, the process of the present invention between 50% and 90% or between about 50% and about 90%, between 50% and 80% or between about 50% and about 80%, between 50% and 70% or between about 50% and about 70%, between 50% and 60% or between about 50% and about 60%, between 60% and 90% or between about 60% and about 90%, between 60% and 80% or between about 60% and about 80%, between 60% and 70%, or between about 60% and about 70%, between 70% and 90%, or between about 70% and about 90%, between 70% and 80%, or between about 70% and about 80%, or between 80% and 90%, or between about 80% and about 90%. In some embodiments, the percentage of g-NK cells in the enriched NK cell population at the beginning of the culture or incubation is between 20% and 90% or between about 20% and about 90%. In some embodiments, the percentage of g-NK cells in the enriched NK cell population at the beginning of the culture or incubation is between 40% and 90% or between about 40% and about 90%. In some embodiments, the percentage of g-NK cells in the enriched NK cell population at the beginning of the culture or incubation is between 60% and 90% or between about 60% and about 90%.
In some of these embodiments, the NK cells may be cultured with the growth factor. According to some embodiments, the at least one growth factor comprises a growth factor selected from the group consisting of SCF, GSK3i, FLT3, IL-2, IL-6, IL-7, IL-15, IL-12, IL-18, and IL-21. According to some embodiments, the at least one growth factor is IL-2 or IL-7 and IL-15. According to some embodiments, the at least one growth factor is IL-2, IL-21 or IL-7 and IL-15. In some embodiments, the growth factor is a recombinant cytokine, such as recombinant IL-2, recombinant IL-7, recombinant IL-21 or recombinant IL-15.
In some embodiments, NK cells are cultured in the presence of one or more recombinant cytokines. In some embodiments, the one or more recombinant cytokines include any one of SCF, GSK3i, FLT3, IL-2, IL-6, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or a combination thereof. In some embodiments, the one or more recombinant cytokines include any one of IL-2, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or a combination thereof. In some embodiments, at least one of the one or more recombinant cytokines is IL-21. In some embodiments, the one or more recombinant cytokines further include IL-2, IL-7, IL-15, IL-12, IL-18, or IL-27, or a combination thereof. In some embodiments, at least one of the one or more recombinant cytokines is IL-2. In some embodiments, the one or more recombinant cytokines are at least IL-2 and IL-21. In some embodiments, the one or more recombinant cytokines are IL-21 and IL-2. In some embodiments, the one or more recombinant cytokines are IL-21, IL-2 and IL-15. In some embodiments, the one or more recombinant cytokines are IL-21, IL-12, IL-15 and IL-18. In some embodiments, the one or more recombinant cytokines are IL-21, IL-2, IL-12, IL-15 and IL-18. In some embodiments, the one or more recombinant cytokines are IL-21, IL-15, IL-18 and IL-27. In some embodiments, the one or more recombinant cytokines are IL-21, IL-2, IL-15, IL-18 and IL-27. In some embodiments, the one or more recombinant cytokines are IL-2 and IL-15.
In particular embodiments, provided methods include incubating or culturing enriched NK cells and feeder cells in the presence of recombinant IL-2. In some embodiments, during at least a portion of the incubation, e.g., added at the beginning of the incubation and optionally added one or more times during the incubation, the recombinant IL-2 is present at a concentration between 1IU/mL and 500IU/mL or between about 1IU/mL and about 500IU/mL, such as between 1IU/mL and 250IU/mL or between about 1IU/mL and about 250IU/mL, between 1IU/mL and 100IU/mL or between about 1IU/mL and about 100IU/mL, between 1IU/mL and 50IU/mL or between about 1IU/mL and about 50IU/mL, between 50IU/mL and 500IU/mL or between about 50IU/mL and about 500IU/mL, between 50IU/mL and 250IU/mL or between about 50IU/mL and about 250IU/mL, between about 50IU/mL and about 100mL or between about 50IU/mL and about 100mL, between about 50IU/mL and about 100IU/mL, between about 500IU/mL and about 250IU/mL, between about 250IU/mL and about 500IU/mL or between about 250IU/mL and about 250 IU/mL. In some embodiments, the concentration of IL-2 is at or about 50IU/mL, 60IU/mL, 70IU/mL, 80IU/mL, 90IU/mL, 100IU/mL, 125IU/mL, 150IU/mL, 200IU/mL, or any value in between any of the foregoing, during at least a portion of the incubation, e.g., added at the beginning of the incubation, and optionally added one or more times during the incubation. In specific embodiments, the concentration of recombinant IL-2 added at the beginning of the culture and optionally added one or more times during the culture is at or about 100IU/mL. In specific embodiments, the concentration of recombinant IL-2 added at the beginning of the culture and optionally added one or more times during the culture is at or about 500IU/mL.
In particular embodiments, provided methods include incubating or culturing enriched NK cells and feeder cells in the presence of recombinant IL-21. In some embodiments, during at least a portion of the incubation, e.g., at the beginning of the incubation and optionally added one or more times during the incubation, recombinant IL-21 is present at a concentration between 1IU/mL and 500IU/mL or between about 1IU/mL and about 500IU/mL, such as between 1IU/mL and 250IU/mL or between about 1IU/mL and about 250IU/mL, between 1IU/mL and 100IU/mL or between about 1IU/mL and about 100IU/mL, between 1IU/mL and 50IU/mL or between about 1IU/mL and about 50IU/mL, between 50IU/mL and 500IU/mL or between about 50IU/mL and about 500IU/mL, between 50IU/mL and 250IU/mL or between about 50IU/mL and about 250IU/mL, between 50IU/mL and 100/mL or between about 50IU/mL and about 100/mL, between about 50IU/mL and about 100IU/mL, between about 500IU/mL and about 250IU/mL, between about 250IU/mL and about 500IU/mL or between about 250IU/mL and about 250 IU/mL. In some embodiments, the concentration of IL-21 is at or about 50IU/mL, 60IU/mL, 70IU/mL, 80IU/mL, 90IU/mL, 100IU/mL, 125IU/mL, 150IU/mL, 200IU/mL, or any value in between any of the foregoing, during at least a portion of the incubation, e.g., added at the beginning of the incubation, and optionally added one or more times during the incubation. In specific embodiments, the concentration of recombinant IL-21 added at the beginning of the culture and optionally added one or more times during the culture is at or about 100IU/mL.
In particular embodiments, provided methods include incubating or culturing enriched NK cells and feeder cells in the presence of recombinant IL-21. In particular embodiments, during at least a portion of the culturing, e.g., the concentration of recombinant IL-21 added at the beginning of the culturing and optionally added one or more times during the culturing, is between about 10ng/mL and about 100ng/mL, between about 10ng/mL and about 90ng/mL, between about 10ng/mL and about 80ng/mL, between about 10ng/mL and about 70ng/mL, between about 10ng/mL and about 60ng/mL, between about 10ng/mL and about 50ng/mL, between about 10ng/mL and about 40ng/mL, between about 10ng/mL and about 30ng/mL, between about 10ng/mL and about 20ng/mL, between about 20ng/mL and about 100ng/mL, between about 20ng/mL and about 90ng/mL, between about 20ng/mL and about 80ng/mL, between about 70ng/mL, between about 20ng/mL and about 60ng/mL, between about 20ng/mL, between about 40ng and about 40ng/mL, between about 30ng and about 30ng/mL, between about 20ng and about 40ng/mL, between about 30ng and about 90ng/mL, between about 20ng and about 40ng and about 30ng and about 90ng Values between about 40 and about 70, between about 40 and about 60, between about 60 and about 80, between about 60 and about 70, between about 70 and about 100, between about 70 and about 90, between about 70 and about 70, between about 80 and about 100, between about 80 and about 90, between about 90 and about 90, or between the inclusive of the values between about 40 and about 70, between about 40 and about 60, between about 60 and about 100. In particular embodiments, the concentration of recombinant IL-21 added during at least a portion of the culture, e.g., at the beginning of the culture, and optionally one or more times during the culture, is between about 10ng/mL and about 100ng/mL, inclusive. In specific embodiments, during at least a portion of the culture, e.g., at the beginning of the culture and optionally one or more times during the culture, the concentration of recombinant IL-21 is at or about 25ng/mL.
In particular embodiments, the concentration of recombinant IL-15 added at the beginning of the culture and optionally one or more times during the culture during at least a portion of the culture is about 1ng/mL to about 50ng/mL, about 1ng/mL to about 40ng/mL, about 1ng/mL to about 30ng/mL, about 1ng/mL to about 20ng/mL, about 1ng/mL to about 10ng/mL, about 1ng/mL to about 5ng/mL, about 5ng/mL to about 50ng/mL, about 5ng/mL to about 40ng/mL, about 5ng/mL to about 30ng/mL, about 5ng/mL to about 20ng/mL, about 5ng/mL to about 10ng/mL, about 10ng/mL to about 50mL, about 10ng/mL to about 40ng/mL, about 10ng/mL to about 30ng/mL, about 20ng to about 50ng/mL, about 20ng to about 40ng/mL, about 40ng to about 30ng/mL, about 30ng to about 30ng/mL, or about 50ng to about 30 ng/mL. In specific embodiments, the concentration of recombinant IL-15 added during at least a portion of the culture, e.g., at the beginning of the culture, and optionally one or more times during the culture, is from about 1ng/mL to about 50ng/mL. In specific embodiments, during at least a portion of the culture, for example, at the beginning of the culture and optionally during the culture to add one or more times recombinant IL-15 concentration is at or about 10ng/mL.
In specific embodiments, these methods comprise culturing in the presence of IL-2, IL-15, and IL-21. In embodiments of the provided methods, for example, the concentration of recombinant cytokine added to the culture at the beginning of the culture and optionally one or more times during the culture is between 50IU/mL and 500IU/mL IL-2 or between about 50IU/mL and about 500IU/mL IL-2, such as between 100IU/mL and 500IU/mL IL-2 or between about 100IU/mL and about 500IU/mL IL-2; between 1ng/mL and 50ng/mL IL-15 or between about 1ng/mL and about 50ng/mL IL-15, such as at or about 10ng/mL; and between 10ng/mL and 100ng/mL IL-21 or between about 10ng/mL and about 100ng/mL IL-21, such as at or about 25ng/mL. In particular embodiments, the addition is during at least a portion of the culture, such as at the beginning of the culture and optionally one or more 500IU/mL of IL-2, 10ng/mL of IL-15, and 25ng/mL of IL-21 during the culture. In particular embodiments, the addition is during at least a portion of the culture, such as at the beginning of the culture and optionally one or more times 100IU/mL of IL-2, 10ng/mL of IL-15, and 25ng/mL of IL-21 during the culture.
In some embodiments, the provided methods include incubating or culturing the enriched NK cells and feeder cells in the presence of recombinant IL-21 and adding the recombinant IL-21 as a complex with an anti-IL-21 antibody. In some embodiments, the anti-IL-21 antibody is contacted with recombinant IL-21 prior to culturing, thereby forming an IL-21/anti-IL-21 complex, and the IL-21/anti-IL-21 complex is added to the culture medium. In some embodiments, contacting recombinant IL-21 with an anti-IL-21 antibody to form an IL-21/anti-IL-21 complex is performed under conditions including a temperature and a time suitable for forming the complex. In some embodiments, the culturing is performed at 37 ℃ ± 2 for 30 minutes.
In some embodiments, the anti-IL is added at a concentration of between 100ng/mL and 500ng/mL or between about 100ng/mL and about 500ng/mL, between 100ng/mL and 400ng/mL or between about 100ng/mL and about 400ng/mL, between 100ng/mL and 300ng/mL or between about 100ng/mL and about 300ng/mL, between 100ng/mL and 200ng/mL or between about 100ng/mL and about 200ng/mL, between 200ng/mL and 500ng/mL or between about 200ng/mL and about 500ng/mL, between 200ng/mL and 400ng/mL or between about 200ng/mL and about 400ng/mL, between 200ng/mL and 300ng/mL or between about 200ng/mL and about 300ng/mL, between 300ng/mL and 500ng/mL or between about 300ng/mL, between 300ng/mL and 400ng/mL or between about 300ng/mL and about 400ng/mL, or between about 400ng and 400 ng/mL. In some embodiments, at 100ng/mL and 500ng/mL or between about 100ng/mL and about 500ng/mL concentration of anti IL-21 antibody. In some embodiments, the anti-IL-21 antibody is added at a concentration of 250 ng/mL.
In particular embodiments, the concentration of recombinant IL-21 used to form a complex with an anti-IL-21 antibody is between about 10ng/mL and about 100ng/mL, between about 10ng/mL and about 90ng/mL, between about 10ng/mL and about 80ng/mL, between about 10ng/mL and about 70ng/mL, between about 10ng/mL and about 60ng/mL, between about 10ng/mL and about 50ng/mL, between about 10ng/mL and about 40ng/mL, between about 10ng/mL and about 30ng/mL, between about 10ng/mL and about 20ng/mL, between about 20ng/mL and about 90ng/mL, between about 20ng/mL and about 80ng/mL, between about 20ng/mL and about 70ng/mL, between about 20ng/mL and about 60ng/mL, between about 20ng/mL and about 50ng/mL, between about 30ng/mL, between about 40ng/mL, between about 30ng/mL, between about 20ng and about 40ng/mL, between about 30ng/mL, between about 20ng/mL and about 90ng/mL, between about 40ng and about 80ng/mL, between about 30ng and about 40ng and about, between about 40 and about 60, between about 40 and about 50, between about 60 and about 70, between about 70 and about 100, between about 70 and about 90, between about 70 and about 80, between about 80 and about 100, between about 80 and about 90, or between about 90 and about 100, inclusive. In specific embodiments, the concentration of recombinant IL-21 for use in forming a complex with an anti-IL-21 antibody is between about 10ng/mL and about 100ng/mL, inclusive. In specific embodiments, the concentration of recombinant IL-21 used to form a complex with an anti-IL-21 antibody is at or about 25ng/mL.
In particular embodiments, during at least a portion of the culture, e.g., the concentration of recombinant IL-12 added at the beginning of the culture and optionally added one or more times during the culture is between about 1ng/mL and about 50ng/mL, between about 1ng/mL and about 40ng/mL, between about 1ng/mL and about 30ng/mL, between about 1ng/mL and about 20ng/mL, between about 1ng/mL and about 10ng/mL, between about 1ng/mL and about 5ng/mL, between about 5ng/mL and about 50ng/mL, between about 5ng/mL and about 40ng/mL, between about 5ng/mL and about 30ng/mL, between about 5ng/mL and about 20ng/mL, between about 5ng/mL and about 10ng/mL, between about 10ng/mL and about 50ng/mL, between about 10ng/mL and about 40ng/mL, between about 10ng/mL, about 30ng and about 20ng/mL, between about 40ng and about 40ng/mL, between about 20ng and about 40ng/mL, between about 30ng and about 40 ng/mL. In specific embodiments, during at least a portion of the culture, for example at the beginning of the culture and optionally during the culture to add one or more times recombinant IL-12 concentration between about 1ng/mL and about 50 ng/mL. In specific embodiments, during at least a portion of the culture, for example at the beginning of the culture and optionally during the culture to add one or more times recombinant IL-12 concentration is at or about 10ng/mL.
In particular embodiments, during at least a portion of the culturing, e.g., at the beginning of the culturing and optionally added one or more times during the culturing, the concentration of recombinant IL-18 is between about 1ng/mL and about 50ng/mL, between about 1ng/mL and about 40ng/mL, between about 1ng/mL and about 30ng/mL, between about 1ng/mL and about 20ng/mL, between about 1ng/mL and about 10ng/mL, between about 1ng/mL and about 5ng/mL, between about 5ng/mL and about 50ng/mL, between about 5ng/mL and about 40ng/mL, between about 5ng/mL and about 30ng/mL, between about 5ng/mL and about 20ng/mL, between about 5ng/mL and about 10ng/mL, between about 10ng/mL and about 50ng/mL, between about 10ng/mL and about 40ng/mL, between about 10ng/mL, about 30ng and about 20ng/mL, between about 40ng and about 40ng/mL, between about 20ng and about 40ng/mL, between about 30ng and about 40 ng/mL. In specific embodiments, during at least a portion of the culturing, e.g., at the beginning of the culturing, and optionally one or more times during the culturing, the concentration of recombinant IL-18 is between about 1ng/mL and about 50 ng/mL. In specific embodiments, during at least a portion of the culture, for example, at the beginning of the culture and optionally during the culture to add one or more times recombinant IL-18 concentration is at or about 10ng/mL.
In particular embodiments, the concentration of recombinant IL-27 is between about 1ng/mL and about 50ng/mL, between about 1ng/mL and about 40ng/mL, between about 1ng/mL and about 30ng/mL, between about 1ng/mL and about 20ng/mL, between about 1ng/mL and about 10ng/mL, between about 1ng/mL and about 5ng/mL, between about 5ng/mL and about 50ng/mL, between about 5ng/mL and about 40ng/mL, between about 5ng/mL and about 30ng/mL, between about 5ng/mL and about 20ng/mL, between about 5ng/mL and about 10ng/mL, between about 10ng/mL and about 50ng/mL, between about 10ng/mL and about 40ng/mL, between about 10ng/mL and about 30ng/mL, between about 20ng/mL, between about 40ng/mL and about 40ng/mL, between about 20ng/mL, between about 40ng/mL and about 30ng/mL, between about 20ng/mL, between about 40ng/mL and about 40ng/mL, between about 30ng/mL, between about 40ng and about 40ng/mL, between about 30ng and about 30 ng/mL. In specific embodiments, during at least a portion of the culturing, e.g., at the beginning of the culturing, and optionally one or more times during the culturing, the concentration of recombinant IL-27 is between about 1ng/mL and about 50 ng/mL. In specific embodiments, during at least a portion of the culture, e.g., at the beginning of the culture and optionally one or more times during the culture, the concentration of recombinant IL-27 is at or about 10ng/mL.
In some embodiments, the methods comprise exchanging a culture medium, which in some aspects comprises washing the cells. For example, the medium may be intermittently changed or rinsed during at least a portion of the culturing or incubation, such as daily, every other day, every third day, or weekly. In particular embodiments, the medium is changed or flushed beginning within 3 days to 7 days or within about 3 days to about 7 days after the start of the culture, such as at or about day 3, day 4, day 5, day 6, or day 7. In particular embodiments, the medium is replaced or flushed at or about day 5. For example, the medium is changed every 2 to 3 days on and after day 5.
Once the medium is removed or rinsed, the medium is replenished. In some embodiments, the supplemented media includes one or more growth factors or cytokines, such as any of the growth factors or cytokines described above. Thus, in some embodiments, the one or more growth factors or cytokines, such as recombinant IL-2, IL-15, and/or IL-21, are added intermittently during the incubation or culture. In some such aspects, the one or more growth factors or cytokines, such as recombinant IL-2, IL-15, and/or IL-21, are added at or about the beginning of the culture or incubation, and then added intermittently during the culture or incubation, such as each time the medium is replaced or flushed. In some embodiments, the one or more growth factors or cytokines, such as recombinant IL-2, IL-15, and/or IL-21, are added to the culture or the incubation beginning on day 0 (beginning of incubation), and each time the medium is replaced or flushed, are further added to supplement the culture or the incubation with the one or more growth factors or cytokines, such as recombinant IL-2, IL-15, and/or IL-21. In some embodiments, the methods comprise adding the one or more growth factors or cytokines, such as recombinant IL-2, IL-15, and/or IL-21, at the beginning of the culture (day 0), and every two or three days, such as at or about day 5, day 7, day 9, day 11, and day 14 of the culture or incubation, at each time the medium is washed or replaced during the culture.
In specific embodiments, in the presence of IL-2, IL-15 and IL-21 in the presence of at least one of the culture medium, and supplement the medium to include IL-2, IL-15 and IL-21 at least one. In some embodiments, in the presence of IL-2 and IL-21 culture, and supplement the medium to include IL-2 and IL-21. In some embodiments, in the presence of IL-2 and IL-15 culture, and supplement the medium to include IL-2 and IL-15. In some embodiments, in the presence of IL-15 and IL-21 culture, and supplement the medium to include IL-15 and IL21. In some embodiments, in the presence of IL-2, IL-15 and IL-21 in culture, and supplemented with medium to include IL-2, IL-15 and IL-21. In some embodiments, one or more additional cytokines can be used for NK cell expansion, including but not limited to recombinant IL-18, recombinant IL-7 and/or recombinant IL-12.
In some embodiments, the supplemented media includes one or more growth factors or cytokines, such as recombinant IL-2. Thus, in some embodiments, growth factors or cytokines (such as recombinant IL-2) are added intermittently during the incubation or culture. In some such aspects, the growth factor or cytokine, such as recombinant IL-2, is added at or about the beginning of the culture or incubation, and then intermittently during the culture or incubation, such as each time the medium is replaced or flushed. In some embodiments, a growth factor or cytokine, such as recombinant IL-2, is added to the culture or incubation beginning on day 0 (beginning of incubation) and further added to supplement the culture or incubation with a growth factor or cytokine (such as recombinant IL-2) each time the medium is replaced or flushed. In some embodiments, these methods include adding recombinant IL-2 at the beginning of the culture (day 0) and every two or three days during the culture at each wash or change of medium, e.g., at or about day 5, day 7, day 9, day 11, and day 14 of the culture or incubation. In any such embodiments, recombinant IL-2 is added to the culture or the incubator at a concentration between 1IU/mL and 500IU/mL or between about 1IU/mL and about 500IU/mL, such as at a value between 1IU/mL and 250IU/mL or between about 1IU/mL and about 250IU/mL, between 1IU/mL and 100IU/mL or between about 1IU/mL and about 100IU/mL, between 1IU/mL and 50IU/mL or between about 1IU/mL and about 50IU/mL, between 50IU/mL and 500IU/mL or between about 50IU/mL and about 500IU/mL, between about 50IU/mL and 250IU/mL or between about 50IU/mL and about 250IU/mL, between about 50IU/mL and about 100IU/mL, between 100IU/mL and 500IU/mL, between about 100IU/mL and about 100IU/mL, between about 500IU/mL and about 250IU/mL, between about 250IU/mL and about 250IU/mL, or between about 250IU/mL and about 250IU/mL, each. In some embodiments, recombinant IL-2 is present in the following amounts or about the amounts: 50IU/mL, 60IU/mL, 70IU/mL, 80IU/mL, 90IU/mL, 100IU/mL, 125IU/mL, 150IU/mL, 200IU/mL, or any value in between any of the foregoing is added to the culture or the incubator. In specific embodiments, the concentration of recombinant IL-2 is at or about 100IU/mL. In specific embodiments, the concentration of recombinant IL-2 is at or about 500IU/mL.
In some embodiments, the supplemented media includes one or more growth factors or cytokines, such as recombinant IL-21. Thus, in some embodiments, growth factors or cytokines (such as recombinant IL-21) are added intermittently during the incubation or culture. In some such aspects, a growth factor or cytokine, such as recombinant IL-21, is added at or about the beginning of the culture or incubation, and then intermittently during the culture or incubation, such as each time the medium is replaced or flushed. In some embodiments, a growth factor or cytokine, such as recombinant IL-21, is added to the culture or incubation beginning on day 0 (beginning of incubation) and further added to supplement the culture or incubation with a growth factor or cytokine (such as recombinant IL-21) each time the medium is replaced or flushed. In some embodiments, these methods include adding recombinant IL-21 at the beginning of the culture (day 0) and every two or three days during the culture at each wash or change of medium, e.g., at or about day 5, day 7, day 9, day 11, and day 14 of the culture or incubation. In any such embodiments, recombinant IL-21 is added to the culture or incubation at a concentration between, namely about 10ng/mL and about 100ng/mL, about 10ng/mL and about 90ng/mL, about 10ng/mL and about 80ng/mL, about 10ng/mL and about 70ng/mL, about 10ng/mL and about 60ng/mL, about 10ng/mL and about 50ng/mL, about 10ng/mL and about 40ng/mL, about 10ng/mL and about 30ng/mL, about 10ng/mL and about 20ng/mL, about 20ng/mL and about 100ng/mL, about 20ng/mL and about 90ng/mL, about 20ng/mL and about 80ng/mL, about 20ng/mL and about 70ng/mL, about 20ng/mL and about 60ng/mL, about 20ng/mL and about 50ng/mL, about 20ng/mL and about 40ng/mL, about 20ng/mL and about 30ng/mL, about 20ng/mL about 30 and about 100ng/mL, about 30 and about 90ng/mL, about 30 and about 80ng/mL, about 30 and about 70ng/mL, about 30 and about 60ng/mL, about 30 and about 50ng/mL, about 30 and about 40ng/mL, about 40 and about 100ng/mL, about 40 and about 90ng/mL, about 40 and about 80ng/mL, about 40 and about 70ng/mL, about 40 and about 60ng/mL, about 40 and about 50ng/mL, about 50 and about 100ng/mL, about 50 and about 90ng/mL, about 50 and about 80ng/mL, about 50 and about 70ng/mL, about, about 50ng/mL to about 60ng/mL, about 60ng/mL to about 100ng/mL, about 60ng/mL to about 90ng/mL, about 60ng/mL to about 80ng/mL, about 60ng/mL to about 70ng/mL, about 70ng/mL to about 100ng/mL, about 70ng/mL to about 90ng/mL, about 70ng/mL to about 80ng/mL, about 80ng/mL to about 100ng/mL, about 80ng/mL to about 90ng/mL, or about 90ng/mL to about 100ng/mL, inclusive. In specific embodiments, recombinant IL-21 is added to the culture or the incubation at a concentration between about 10ng/mL and about 100ng/mL, inclusive. Recombinant IL-21 is added to the culture or incubation at a concentration of 25ng/mL or about 25 ng/mL.
In some embodiments, the supplemented media includes one or more growth factors or cytokines, such as recombinant IL-21, which is added as a complex with an antibody (such as an anti-IL-21 antibody). Thus, in some embodiments, complexes, such as IL-21/anti-IL-21 antibody complexes, are added immediately during incubation or culture. In some such aspects, complexes, such as IL-21/anti-IL-21 antibody complexes, are added at or about the beginning of the culture or incubation, and then added intermittently during the culture or incubation, such as each time the medium is replaced or flushed. In some embodiments, a complex, such as an IL-21/anti-IL-21 antibody complex, is added to the culture or the incubation at the beginning of day 0 (beginning of incubation) and further added to supplement the culture or incubation with a complex, such as an IL-21/anti-IL-21 antibody complex, each time the medium is replaced or flushed. In some embodiments, the methods comprise adding a complex, such as an IL-21/anti-IL-21 antibody complex, at the beginning of the culture (day 0) and every two or three days at each wash or change of medium during the culture, for example, at or about day 5, day 7, day 9, day 11, and day 14 of the culture or incubation. In any such embodiments, the anti-IL-21 antibody is contacted with recombinant IL-21 to form an IL-21/anti-IL-21 complex and the IL-21/anti-IL-21 complex is added to the culture medium. In any such embodiments, contacting recombinant IL-21 with an anti-IL-21 antibody to form an IL-21/anti-IL-21 complex is performed under conditions including a temperature and a time suitable for forming the complex. In any such embodiment, the culturing is performed at 37 ℃ ± 2 for 30 minutes. In any such embodiments, the anti-IL is added at a concentration of between 100ng/mL and 500ng/mL or between about 100ng/mL and about 500ng/mL, between 100ng/mL and 400ng/mL or between about 100ng/mL and about 400ng/mL, between 100ng/mL and 300ng/mL or between about 100ng/mL and about 300ng/mL, between 100ng/mL and 200ng/mL or between about 100ng/mL and about 200ng/mL, between 200ng/mL and 500ng/mL or between about 200ng/mL and about 500ng/mL, between 200ng/mL and 400ng/mL or between about 200ng/mL and about 400ng/mL, between 200ng/mL and 300ng/mL or between about 200ng/mL and about 300ng/mL, between 300ng/mL and 500ng/mL or between about 300ng/mL and about 500ng/mL, between 300ng/mL and 400ng/mL or between about 400ng and 400ng/mL, or between about 400ng and 400 ng/mL. In some embodiments, at 100ng/mL and 500ng/mL or between about 100ng/mL and about 500ng/mL concentration of anti IL-21 antibody. In some embodiments, the anti-IL-21 antibody is added at a concentration of 250 ng/mL. In any such embodiment, the concentration of recombinant IL-21 used to form a complex with an anti-IL-21 antibody is between about 10ng/mL and about 100ng/mL, between about 10ng/mL and about 90ng/mL, between about 10ng/mL and about 80ng/mL, between about 10ng/mL and about 70ng/mL, between about 10ng/mL and about 60ng/mL, between about 10ng/mL and about 50ng/mL, between about 10ng/mL and about 40ng/mL, between about 10ng/mL and about 30ng/mL, between about 10ng/mL and about 20ng/mL, between about 20ng/mL and about 90ng/mL, between about 20ng/mL and about 80ng/mL, between about 20ng/mL and about 70ng/mL, between about 20ng/mL and about 60ng/mL, between about 20ng/mL and about 50ng/mL, between about 30ng/mL, between about 40ng/mL, between about 30ng/mL, between about 20ng and about 90ng/mL, between about 40ng/mL, between about 30ng and about 40ng/mL, between about 40ng and about 40ng/mL, between about 30ng and about 40ng and about 80ng/mL, between about 30ng and about 40ng and about, between about 40 and about 60, between about 40 and about 50, between about 60 and about 70, between about 70 and about 100, between about 70 and about 90, between about 70 and about 80, between about 80 and about 100, between about 80 and about 90, or between about 90 and about 100, inclusive. In specific embodiments, the concentration of recombinant IL-21 for use in forming a complex with an anti-IL-21 antibody is between about 10ng/mL and about 100ng/mL, inclusive. In specific embodiments, the concentration of recombinant IL-21 used to form a complex with an anti-IL-21 antibody is at or about 25ng/mL.
In some embodiments, the supplemented media includes one or more growth factors or cytokines, such as recombinant IL-15. Thus, in some embodiments, growth factors or cytokines (such as recombinant IL-15) are added intermittently during the incubation or culture. In some such aspects, a growth factor or cytokine, such as recombinant IL-15, is added at or about the beginning of the culture or incubation, and then intermittently during the culture or incubation, such as each time the medium is replaced or flushed. In some embodiments, a growth factor or cytokine, such as recombinant IL-15, is added to the culture or incubation beginning on day 0 (beginning of incubation) and further added to supplement the culture or incubation with a growth factor or cytokine (such as recombinant IL-15) each time the medium is replaced or flushed. In some embodiments, these methods include adding recombinant IL-15 at the beginning of the culture (day 0) and every two or three days during the culture at each wash or change of medium, e.g., at or about day 5, day 7, day 9, day 11, and day 14 of culture or incubation. In any such embodiments, recombinant IL-15 is added to the culture or the incubator at a concentration between about 1 and about 50ng/mL, about 1 and about 40ng/mL, about 1 and about 30ng/mL, about 1 and about 20ng/mL, about 1 and about 10ng/mL, about 1 and about 5ng/mL, about 5 and about 50ng/mL, about 5 and about 40ng/mL, about 5 and about 30ng/mL, about 5 and about 20ng/mL, about 5 and about 10ng/mL, about 10 and about 50ng/mL, about 10 and about 40ng/mL, about 10 and about 30ng/mL, about 20 and about 50ng/mL, about 20 and about 40ng/mL, about 40 and about 40ng/mL, or about 40ng and about 30 ng/mL. In any such embodiments, recombinant IL-15 is added to the culture or the incubation at a concentration of between about 1ng/mL and about 50ng/mL. In any such embodiments, recombinant IL-15 is added to the culture or incubation at a concentration of 10ng/mL or about 10 ng/mL. In a specific embodiment, 500IU/mL IL-2, 10ng/mL IL-15 and 25ng/mL IL-21 are added to the culture or the incubation.
In some embodiments, the medium to be supplemented includes one or more growth factors or cytokines, such as recombinant IL-12. Thus, in some embodiments, growth factors or cytokines (such as recombinant IL-12) are added intermittently during the incubation or culture. In some such aspects, a growth factor or cytokine, such as recombinant IL-12, is added at or about the beginning of the culture or incubation, and then intermittently added during the culture or incubation, such as each time the medium is replaced or flushed. In some embodiments, a growth factor or cytokine, such as recombinant IL-12, is added to the culture or incubation beginning on day 0 (beginning of incubation), and each time the medium is replaced or flushed, further added to supplement the culture or incubation with a growth factor or cytokine, such as recombinant IL-12. In some embodiments, these methods include at the beginning of the culture (day 0), and during the culture at every two or three days at each time the medium is washed or changed, for example at or about culture or incubation of day 5, day 7, day 9, day 11 and day 14, adding recombinant IL-12. In any such embodiments, recombinant IL-12 is added to the culture or the incubator at a concentration between about 1ng/mL and about 50ng/mL, about 1ng/mL and about 40ng/mL, about 1ng/mL and about 30ng/mL, about 1ng/mL and about 20ng/mL, about 1ng/mL and about 10ng/mL, about 1ng/mL and about 5ng/mL, about 5ng/mL and about 50ng/mL, about 5ng/mL and about 40ng/mL, about 5ng/mL and about 30ng/mL, about 5ng/mL and about 20ng/mL, about 5ng/mL and about 10ng/mL, about 10ng/mL and about 50ng/mL, about 10ng/mL and about 40ng/mL, about 10ng/mL and about 20ng/mL, about 20ng/mL and about 50ng/mL, about 20ng/mL and about 40ng/mL, about 20ng/mL and about 30ng/mL, about 30ng/mL and about 40ng/mL, about 40ng/mL and about 40 ng/mL. In any such embodiments, recombinant IL-12 is added to the culture or the incubation at a concentration of between about 1ng/mL and about 50ng/mL. In any such embodiments, recombinant IL-12 is added to the culture or incubation at a concentration of 10ng/mL or about 10 ng/mL.
In some embodiments, the supplemented media includes one or more growth factors or cytokines, such as recombinant IL-18. Thus, in some embodiments, growth factors or cytokines (such as recombinant IL-18) are added intermittently during the incubation or culture. In some such aspects, a growth factor or cytokine, such as recombinant IL-18, is added at or about the beginning of the culture or incubation, and then intermittently during the culture or incubation, such as each time the medium is replaced or flushed. In some embodiments, a growth factor or cytokine, such as recombinant IL-18, is added to the culture or incubation beginning on day 0 (beginning of incubation) and further added to supplement the culture or incubation with a growth factor or cytokine (such as recombinant IL-18) each time the medium is replaced or flushed. In some embodiments, these methods include adding recombinant IL-18 at the beginning of the culture (day 0) and every two or three days during the culture at each wash or change of medium, e.g., at or about day 5, day 7, day 9, day 11, and day 14 of culture or incubation. In any such embodiments, recombinant IL-18 is added to the culture or the incubator at a concentration between about 1 and about 50ng/mL, about 1 and about 40ng/mL, about 1 and about 30ng/mL, about 1 and about 20ng/mL, about 1 and about 10ng/mL, about 1 and about 5ng/mL, about 5 and about 50ng/mL, about 5 and about 40ng/mL, about 5 and about 30ng/mL, about 5 and about 20ng/mL, about 5 and about 10ng/mL, about 10 and about 50ng/mL, about 10 and about 40ng/mL, about 10 and about 30ng/mL, about 20 and about 50ng/mL, about 20 and about 40ng/mL, about 40 and about 40ng/mL, or about 40ng and about 30 ng/mL. In any such embodiments, recombinant IL-18 is added to the culture or the incubation at a concentration of between about 1ng/mL and about 50ng/mL. In any such embodiments, recombinant IL-18 is added to the culture or the incubation at a concentration of 10ng/mL or about 10 ng/mL.
In some embodiments, the supplemented media includes one or more growth factors or cytokines, such as recombinant IL-27. Thus, in some embodiments, growth factors or cytokines (such as recombinant IL-27) are added intermittently during the incubation or culture. In some such aspects, the growth factor or cytokine, such as recombinant IL-27, is added at or about the beginning of the culture or incubation, and then intermittently during the culture or incubation, such as each time the medium is replaced or flushed. In some embodiments, a growth factor or cytokine, such as recombinant IL-27, is added to the culture or incubation beginning on day 0 (beginning of incubation) and further added to supplement the culture or incubation with a growth factor or cytokine (such as recombinant IL-27) each time the medium is replaced or flushed. In some embodiments, these methods include adding recombinant IL-27 at the beginning of the culture (day 0) and every two or three days during the culture at each wash or change of medium, e.g., at or about day 5, day 7, day 9, day 11, and day 14 of the culture or incubation. In any such embodiments, recombinant IL-27 is added to the culture or the incubator at a concentration between about 1 and about 50ng/mL, about 1 and about 40ng/mL, about 1 and about 30ng/mL, about 1 and about 20ng/mL, about 1 and about 10ng/mL, about 1 and about 5ng/mL, about 5 and about 50ng/mL, about 5 and about 40ng/mL, about 5 and about 30ng/mL, about 5 and about 20ng/mL, about 5 and about 10ng/mL, about 10 and about 50ng/mL, about 10 and about 40ng/mL, about 10 and about 30ng/mL, about 20 and about 50ng/mL, about 20 and about 40ng/mL, about 40 and about 30ng/mL, about 20 and about 40ng/mL, about 30 and about 30ng/mL, or about 40ng and about 40 ng/mL. In any such embodiments, recombinant IL-27 is added to the culture or the incubation at a concentration of between about 1ng/mL and about 50ng/mL. In any such embodiments, recombinant IL-27 is added to the culture or incubation at a concentration of 10ng/mL or about 10 ng/mL.
In embodiments of the provided methods, culturing or incubating includes providing chemical and physical conditions (e.g., temperature, gas) that are required or useful for NK cell maintenance. Examples of chemical conditions that may support NK cell proliferation or expansion include, but are not limited to, buffers, nutrients, serum, vitamins, and antibiotics typically provided in growth (i.e., culture) media. In one embodiment, the NK medium comprises MEM alpha containing 10% FCS or MEM alpha containing 5% human serum +.CellGro SCGM (Cell Genix) of the FBS substitute (Lifeblood Products). Other media suitable for use in the present invention include, but are not limited to Glascow medium (Gibco Carlsbad Calif.), RPMI medium (Sigma-Aldrich, st Louis Mo.) or DMEM (Sigma-Aldrich, st Louis Mo.). It should be noted that many media contain nicotinamide as vitamin supplement, such as MEM alpha (8.19 μm nicotinamide), RPMI (8.19 μm nicotinamide), DMEM (32.78 μm nicotinamide) and Glascow media (16.39 μm nicotinamide).
In some embodiments, such as for applications in which cells are introduced (or reintroduced) into a human subject, culture is performed using a serum-free formulation, such as AIM V TM serum-free medium, MARROWMAX TM bone marrow medium, or serum-free Stem Cell Growth Medium (SCGM) for lymphocyte culture (e.g.GMP SCGM). Such media formulations and supplements are available from commercial sources. The culture may be supplemented with amino acids, antibiotics, and/or other growth factor cytokines as described to promote optimal viability, proliferation, functionality, and/or survival. In some embodiments, the serum-free medium may also be supplemented with a low percentage of human serum, such as 0.5% to 10% human serum, such as at or about 5% human serum. In such embodiments, the human serum may be human serum from human AB plasma (human AB serum) or autologous serum.
In some embodiments, the culturing with feeder cells and optionally cytokines (e.g., recombinant IL-2 or IL-21) is performed under conditions including a temperature suitable for growth or expansion of human NK cells, e.g., at least about 25 degrees celsius, typically at least about 30 degrees celsius, and typically at or about 37 degrees celsius. In some embodiments, the culturing is performed in 5% CO 2 at 37 ℃ ± 2.
In an embodiment of the provided method, the culturing comprises incubation under GMP conditions. In some embodiments, the incubation is performed in a closed system, which in some aspects may be a closed automated system. In some embodiments, the medium containing the one or more recombinant cytokines or growth factors is serum-free medium. In some embodiments, the incubation is performed with serum-free medium in a closed automated system.
In some embodiments, the expansion of NK cells is performed in a culture vessel suitable for cell expansion. In some embodiments, the culture vessel is a gas permeable culture vessel, such as a G-Rex system (e.g., G-Rex 10M, G-Rex 100M/100M-CS, or G-Rex 500M/500M-CS). In some embodiments, the culture vessel is a microplate, flask, bag, or other culture vessel suitable for expanding cells in a closed system. In some embodiments, the amplification may be performed in a bioreactor. In some embodiments, the cell expansion system is used to perform the expansion by transferring the cells to a gas permeable bag, such as in connection with a biological reaction chamber (e.g., xuri cell expansion system W25 (GE HEALTHCARE)). In embodiments, the cell expansion system includes a culture vessel, such as a bag, e.g., a gas-permeable cell bag, having a volume of about 50mL, about 100mL, about 200mL, about 300mL, about 400mL, about 500mL, about 600mL, about 700mL, about 800mL, about 900mL, about 1L, about 2L, about 3L, about 4L, about 5L, about 6L, about 7L, about 8L, about 9L, and about 10L, or any value between any of the foregoing. In some embodiments, the process is automated or semi-automated. In some aspects, the amplification culture is performed under static conditions. In some embodiments, the amplification culture is performed under shaking conditions. The medium may be added in bulk or may be added on a perfusion schedule. In some embodiments, the bioreactor maintains the temperature at or near 37 ℃, and maintains the CO2 level at or near 5%, wherein the steady air flow is, is about or at least 0.01L/min, 0.05L/min, 0.1L/min, 0.2L/min, 0.3L/min, 0.4L/min, 0.5L/min, 1.0L/min, 1.5L/min, or 2.0L/min, or greater than 2.0L/min. In certain embodiments, at least a portion of the culturing is performed with perfusion, such as at a rate of 290 ml/day, 580 ml/day, and/or 1160 ml/day.
In some aspects, the cells are expanded in a perfusion-capable, self-sealing expansion system. Perfusion can be accomplished by continuous addition of culture medium to the cells to ensure optimal growth rates are achieved.
Amplification methods can be performed under GMP conditions, including in a closed automated system and using serum-free medium. In some embodiments, any one or more steps of the method may be performed in a closed system or under GMP conditions. In certain embodiments, all process operations are performed in a GMP suite. In some embodiments, the closed system is used to perform one or more other processing steps of the method for manufacturing, generating, or producing a cell therapy. In some embodiments, one or more or all of the processing steps, e.g., isolation, selection and/or enrichment, processing, culturing steps (including incubations associated with cell expansion), and formulating steps, are performed using systems, devices or equipment integrated or self-contained in the system, and/or in an automated or programmable manner. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus that allows a user to program, control, evaluate, separate, engineer, and formulate the results of the steps and/or adjust various aspects thereof.
In some of any of the provided embodiments, the culturing is performed for a time until the method achieves expansion of at least or at least about 2.50X10 8 g-NK cells. In some of any of the provided embodiments, the culturing is performed for a time until the method achieves expansion of at least or at least about 5.0X10 8 g-NK cells. In some embodiments of any of the provided embodiments, the culturing is performed until the method achieves expansion of at least or at least about 1.0X10 9 g-NK cells. In some of any of the provided embodiments, the culturing is performed for a time until the method achieves expansion of at least or at least about 5.0X10 9 g-NK cells.
In some of any of the provided embodiments, the culturing is performed or performed about or at least about the following time: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 14 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 21 days.
In some embodiments of any of the provided embodiments, the culturing or incubating according to any of the provided methods is performed at or about or at least at or at least about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, or 25 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 14 days. In some embodiments, the culturing is performed or performed for about or at least or for at least about 21 days. In certain embodiments, longer culture durations are generally necessary if the enriched NK cells have been thawed after having been previously frozen or cryopreserved at the beginning of the culture. It is within the level of one skilled in the art to empirically determine the optimal number of days to culture cells based on factors such as the state of the cells at the beginning of the culture, the health or viability of the cells at the beginning or during the culture, and/or the desired threshold number of cells at the end of the culture (e.g., based on the desired cell application, such as the dose of cells administered to the subject for therapeutic purposes).
At the end of the culture, the cells were harvested. Cell collection or harvesting may be accomplished by centrifuging the cells from the culture vessel after the end of the culture. For example, cells are harvested by centrifugation after about 14 days of culture. After harvesting the cells, the cells are washed. Cell samples may be collected for functional or phenotypic testing. Any other cells not used for functional or phenotypic testing may be formulated alone. In some cases, the cells are formulated with a cryoprotectant to cryopreserve the cells.
In some embodiments, provided methods include the step of freezing (e.g., cryopreserving) the cells before or after isolation, selection, and/or enrichment. In some embodiments, provided methods include the step of freezing (e.g., cryopreserving) the cells prior to or after incubation and/or culture. In some embodiments, the method comprises cryopreserving the cells in the presence of a cryoprotectant, thereby producing a cryopreserved composition. In some aspects, the method comprises washing the cryopreserved composition under conditions that reduce or remove cryoprotectant prior to incubation and/or prior to administration to a subject. In some aspects, any of a variety of known freezing solutions and parameters may be used. In some embodiments, cells are frozen (e.g., cryogenically frozen or cryopreserved) in a medium and/or solution, wherein the final concentration is or is about 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or 5.0% DMSO, or between 1% and 15%, between 6% and 12%, between 5% and 10%, or between 6% and 8% DMSO. In particular embodiments, cells are frozen (e.g., cryogenically frozen or cryopreserved) in a medium and/or solution, wherein the final concentration is or is about 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5% or 0.25% HSA, or between 0.1% and-5%, between 0.25% and 4%, between 0.5% and 2% or between 1% and 2% HSA. One example involves the use of PBS or other suitable cell freezing medium containing 20% dmso and 8% Human Serum Albumin (HSA). Then diluted 1:1 with medium so that the final concentrations of DMSO and HSA were 10% and 4%, respectively. The cells are then typically frozen at a rate of about 1 ℃/minute to at or about-80 ℃ and stored in the gas phase of a liquid nitrogen storage tank. In some embodiments, the cells are frozen in a serum-free cryopreservation medium comprising a cryoprotectant. In some embodiments, the cryoprotectant is DMSO. In some embodiments, the cryopreservation medium is DMSO (v/v) between 5% and 10% or between about 5% and about 10%. In some embodiments, the cryopreservation medium is or is about 5% dmso (v/v). In some embodiments, the cryopreservation medium is or is about 6% dmso (v/v). In some embodiments, the cryopreservation medium is or is about 7% DMSO (v/v). In some embodiments, the cryopreservation medium is or is about 8% DMSO (v/v). In some embodiments, the cryopreservation medium is or is about 9% DMSO (v/v). In some embodiments, the cryopreservation medium is or is about 10% DMSO (v/v). In some embodiments, the cryopreservation media comprises a commercially available cryopreservation solution (CryoStor TM CS10 or CS 5). CryoStor TM CS10 is a cryopreservation medium containing 10% dimethyl sulfoxide (DMSO). CryoStor TM CS5 is a cryopreservation medium containing 5% dimethyl sulfoxide (DMSO). In some embodiments, the cryopreservation medium contains one or more additional excipients, such as plasma a or Human Serum Albumin (HSA).
In some embodiments, cells are cryopreserved at a density of 5×10 6 to×1×10 8 cells/mL. For example, cells may be stored at a density of between any of 5×10 6 cells/mL or about 5×10 6 cells/mL, 10×10 6 cells/mL or about 10×10 6 cells/mL, 15×10 6 cells/mL or about 15×10 6 cells/mL, 20×10 6 cells/mL or about 20×10 6 cells/mL, 25×10 6 cells/mL or about 25×10 6 cells/mL, 30×10 6 cells/mL or about 30×10 6 cells/mL, 40×10 6 cells/mL or about 40×10 6 cells/mL, 50×10 6 cells/mL or about 50×10 6 cells/mL, 60×10 6 cells/mL or about 60×10 6 cells/mL, 70×10 6 cells/mL or about 70×10 6 cells/mL, 80×10 6 cells/mL or about 80×10 6 cells/mL or about 80×10×48 cells/48 or about 40×10×57 cells/mL, or about 90×90 cells/mL, any of the preceding densities. Cells can be cryopreserved in any volume suitable for a cryopreservation vessel. In some embodiments, the cells are stored frozen in vials. The volume of cryopreservation medium may be between 1mL and 50mL or between about 1mL and about 50mL, such as at or about 1mL and 5mL. In some embodiments, the cells are stored frozen in a bag. The volume of cryopreservation medium may be between 10mL and 500mL or between about 10mL and about 500mL, such as at or about 100mL or at or about 200mL. The harvested and expanded cells may be cryopreserved in a low temperature environment, such as a temperature of-80 ℃ to-196 ℃. In some of any of the provided methods, the method produces an increased number of NKG2C Positive and negative cells at the end of the culture as compared to at the beginning of the culture. For example, the increase in NKG2C Positive and negative cells at the end of culture may be greater than or about 100-fold, greater than or about 200-fold, greater than or about 300-fold, greater than or about 400-fold, greater than or about 500-fold, greater than or about 600-fold, greater than or about 700-fold, or greater than or about 800-fold, as compared to at the beginning of culture. In some of any of the embodiments, the increase is at or above about 1000-fold. In some of any of the embodiments, the increase is at or above about 2000-fold. In some of any of the embodiments, the increase is at or above about 2500-fold. In some of any of the embodiments, the increase is at or above about 3000-fold. In some of any of the embodiments, the increase is at or above about 5000-fold. In some of any of the embodiments, the increase is or is about 10000 times greater. In some of any of the embodiments, the increase is at or above about 15000-fold. In some of any of the embodiments, the increase is at or above about 20000 times. In some of any of the embodiments, the increase is at or above about 25000 times. In some of any of the embodiments, the increase is at or above about 30000-fold. In some of any of the embodiments, the increase is or is about 35000-fold or greater. In some embodiments, culturing or incubating according to any of the provided methods is performed until the method achieves expansion of at least or about 2.50×10 NKG2C cells, at least or about 3.0×10 NKG2C cells, at least or about 4.0×10 NKG2C cells, at least or about 5.0×10 NKG2C cells, at least or about 6.0×10 NKG2C cells, at least or about 7.0×10 NKG2C cells, at least or about 8.0×10 NKG2C cells, at least or about 9.0×10 NKG2C cells, at least or about 1.0×10 NKG2C cells, at least or about 1.5×10 NKG2C cells, at least or about 2.0×10 NKG2C cells, at least or about 3.0×10 NKG2C cells, at least or about 4.0×10 NKG2C cells, at least or about 5×10 NKG2C cells, at least or about 5.0×10 NKG2C cells, at least or about 1.0×10 NKG2C cells, at least or more NKG2C cells, at least or about 1.0×10 NKG2C cells.
In some of any of the provided methods, the method produces an increased number of NKG2a Negative of cells at the end of the culture as compared to at the beginning of the culture. For example, the increase in NKG2a Negative of cells at the end of culture may be greater than or about 100-fold, greater than or about 200-fold, greater than or about 300-fold, greater than or about 400-fold, greater than or about 500-fold, greater than or about 600-fold, greater than or about 700-fold, or greater than or about 800-fold, as compared to at the beginning of culture. In some of any of the embodiments, the increase is at or above about 1000-fold. In some of any of the embodiments, the increase is at or above about 2000-fold. In some of any of the embodiments, the increase is at or above about 3000-fold. In some of any of the embodiments, the increase is at or above about 2500-fold. In some of any of the embodiments, the increase is at or above about 5000-fold. In some of any of the embodiments, the increase is or is about 10000 times greater. In some of any of the embodiments, the increase is at or above about 15000-fold. In some of any of the embodiments, the increase is at or above about 20000 times. In some of any of the embodiments, the increase is at or above about 25000 times. In some of any of the embodiments, the increase is at or above about 30000-fold. In some of any of the embodiments, the increase is or is about 35000-fold or greater. In some embodiments, culturing or incubating according to any of the provided methods is performed until the method achieves expansion of at least or about 2.50×10 NKG2A cells, at least or about 3.0×10 NKG2A cells, at least or about 4.0×10 NKG2A cells, at least or about 5.0×10 NKG2A cells, at least or about 6.0×10 NKG2A cells, at least or about 7.0×10 NKG2A cells, at least or about 8.0×10 NKG2A cells, at least or about 9.0×10 NKG2A cells, at least or about 1.0×10 NKG2A cells, at least or about 1.5×10 NKG2A cells, at least or about 2.0×10 NKG2A cells, at least or about 3.0×10 NKG2A cells, at least or about 4.0×10 NKG2A cells, at least or about 5.0×10 NKG2A cells, at least or about 5×10 NKG2A cells, at least or about 1.0×10 NKG2A cells, at least or more than about 1.0×10 NKG2A cells.
In some of any of the provided methods, the method produces an increased number of NKG2C Positive and negative NKG2A Negative of cells at the end of the culture as compared to at the beginning of the culture. For example, the increase in NKG2C Positive and negative NKG2A Negative of cells at the end of culture may be greater than or about 100-fold, greater than or about 200-fold, greater than or about 300-fold, greater than or about 400-fold, greater than or about 500-fold, greater than or about 600-fold, greater than or about 700-fold, or greater than or about 800-fold, as compared to at the beginning of culture. In some of any of the embodiments, the increase is at or above about 1000-fold. In some of any of the embodiments, the increase is at or above about 2000-fold. In some of any of the embodiments, the increase is at or above about 2500-fold. In some of any of the embodiments, the increase is at or above about 3000-fold. In some of any of the embodiments, the increase is at or above about 5000-fold. In some of any of the embodiments, the increase is or is about 10000 times greater. In some of any of the embodiments, the increase is at or above about 15000-fold. In some of any of the embodiments, the increase is at or above about 20000 times. In some of any of the embodiments, the increase is at or above about 25000 times. In some of any of the embodiments, the increase is at or above about 30000-fold. In some of any of the embodiments, the increase is or is about 35000-fold or greater. In some embodiments, culturing or incubating according to any of the provided methods is performed until the method achieves expansion of at least or about 2.50×10 NKG2C cells, at least or about 3.0×10 NKG2C cells, at least or about 4.0×10 NKG2C cells, at least or about 5.0×10 NKG2C cells, at least or about 6.0×10 NKG2C cells, at least or about 7.0×10 NKG2C cells, at least or about 8.0×10 NKG2C cells, at least or about 9.0×10 NKG2C cells, at least or about 1.0×10 NKG2C cells, at least or about 1.5×10 NKG2C cells, at least or about 2.0×10 NKG2C cells, at least or about 3.0×10 NKG2C cells, at least or about 4.0×10 NKG2C cells, at least or about 5×10 NKG2C cells, at least or about 5.0×10 NKG2C cells, at least or about 1.0×10 NKG2C cells, at least or more NKG2C cells, at least or about 1.0×10 NKG2C cells.
In some of any of the provided methods, the method produces an increased number of g-NK cells at the end of the culture compared to at the beginning of the culture. For example, the increase in g-NK cells at the end of culture can be greater than or about 100-fold, greater than or about 200-fold, greater than or about 300-fold, greater than or about 400-fold, greater than or about 500-fold, greater than or about 600-fold, greater than or about 700-fold, or greater than or about 800-fold as compared to at the beginning of culture. In some of any of the embodiments, the increase is at or above about 1000-fold. In some of any of the embodiments, the increase is at or above about 2000-fold. In some of any of the embodiments, the increase is at or above about 2500-fold. In some of any of the embodiments, the increase is at or above about 3000-fold. In some of any of the embodiments, the increase is at or above about 5000-fold. In some of any of the embodiments, the increase is or is about 10000 times greater. In some of any of the embodiments, the increase is at or above about 15000-fold. In some of any of the embodiments, the increase is at or above about 20000 times. In some of any of the embodiments, the increase is at or above about 25000 times. In some of any of the embodiments, the increase is at or above about 30000-fold. In some of any of the embodiments, the increase is or is about 35000-fold or greater. In some embodiments, culturing or incubating according to any of the provided methods is performed until the method achieves expansion of at least or at least about 2.50×10 8 g-NK cells, at least or at least about 3.0×10 8 g-NK cells, at least or at least about 4.0×10 8 g-NK cells, at least or at least about 5.0×10 8 g-NK cells, at least or at least about 6.0×10 8 g-NK cells, at least or at least about 7.0×10 8 g-NK cells, at least or at least about 8.0×10 8 g-NK cells, at least or at least about 9.0×10 8 g-NK cells, at least or at least about 1.0×10 9 g-NK cells, at least or at least about 1.5×10 9 g-NK cells, at least or at least about 2.0×10 9 g-NK cells, at least or at least about 3.0×10 9 g-NK cells, at least or at least about 4×10×10 8 g-NK cells, at least or at least about 7.0×10×9696 g-NK cells, at least or at least about 8.0×10×10 8 g-NK cells, at least or at least about 9.0×10×10 8 g-NK cells, at least or at least about 1.0×10×10 9 g-NK cells, at least about 1.5×10×35 g-NK cells, at least about 2.0×35 g-NK cells, at least about 2×35 g-t.
In some embodiments, the provided methods result in preferential expansion of g-NK cells. In some aspects, g-NK cells are identified by distinguishing NK cells from other lymphocytes or immune cells, and distinguishing the presence, absence, or level of surface expression of one or more different markers of g-NK cells from conventional NK cells. In embodiments, surface expression can be determined by flow cytometry, for example, by staining with an antibody that specifically binds to a label and detecting binding of the antibody to the label. Similar methods can be performed to assess the expression of intracellular markers, except that such methods typically include methods for immobilization and permeabilization prior to staining to detect intracellular proteins by flow cytometry. In some embodiments, immobilization is achieved using formaldehyde (e.g., 0.01%), followed by disruption of the membrane using a detergent (e.g., 0.1% to 1% detergent, e.g., at or about 0.5%), such as Triton, NP-50, tween 20, saponin, digitonin, or Leucoperm.
Antibodies and other binding entities can be used to detect the expression level of a marker protein to identify, detect, enrich and/or isolate g - NK cells. Suitable antibodies may include polyclonal, monoclonal, fragments (such as Fab fragments), single chain antibodies, and other forms of specific binding molecules.
In some embodiments, a cell (e.g., NK cell subpopulation) is positive for a particular marker if a particular marker (which may be an intracellular marker or a surface marker) is present on or in the cell that is detectable. In embodiments, surface expression is positive if staining is detected at a level substantially higher than that detected by the same procedure with an isotype-matched control under otherwise identical conditions and/or at a level substantially similar to or in some cases higher than that of cells known to be positive for the marker and/or higher than that of cells known to be negative for the marker.
In some embodiments, a cell (e.g., NK cell subpopulation) is negative for a particular marker if no detectable presence of the particular marker (which may be an intracellular marker or a surface marker) is present on or in the cell. In embodiments, surface expression is negative if staining is detected at a level substantially higher than that detected by the same procedure with an isotype-matched control under otherwise identical conditions and/or staining is undetectable at a level substantially lower than that of cells known to be positive for the marker and/or at a level substantially similar to cells known to be negative for the marker.
In some embodiments, if a lower level of a particular marker is present detectably on or in a cell than a cell known to be positive for the marker, the cell (e.g., NK cell subpopulation) is low level (lo or min) for the particular marker. In embodiments, surface expression can be determined by flow cytometry, e.g., by staining with an antibody that specifically binds to the marker and detecting binding of the antibody to the marker, wherein if the level of staining is lower than for cells known to be positive for the marker, expression on the surface or intracellular (depending on the method used) is at a low level.
In some embodiments, the g-NK cells are cells having an NK cell phenotype (e.g., CD45 Positive and negative 、CD3 Negative of and/or CD56 Positive and negative ) and express one or more markers that identify or are associated with a subpopulation of g-NK cells.
In some embodiments, as disclosed in patent application No. US2013/0295044 or Zhang et al, 2013, j.immunol., volume 190: the identification of g-NK cells is described in pages 1402-1406.
In some embodiments, the g-NK cell is a cell that does not express a substantial amount of fcrγ but expresses at least one marker of natural killer cells. The amino acid sequence of the FcRgamma chain (Homo sapiens, also known as high affinity immunoglobulin gamma Fc receptor I) is available in the NCBI database under accession number NP-004097.1 (GI: 4758344) and is reproduced below under SEQ ID NO: 34.
In some embodiments, the g-NK cell subpopulation of NK cells can be detected by observing whether fcrγ is expressed by the NK cell population or NK cell subpopulation. In some cases, g-NK cells are identified as cells that do not express FcRgamma. Fcrgamma protein is an intracellular protein. Thus, in some aspects, the presence or absence of fcrγ may be detected after treatment of the cells, e.g., by fixation and permeabilization, to allow detection of intracellular proteins. In some embodiments, the cells are further assessed for one or more surface markers (CD 45, CD3, and/or CD 56) prior to intracellular detection, such as prior to fixing the cells. In some embodiments, g-NK cells are identified, detected, enriched, and/or isolated as cells of CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative /FcRγ Negative of .
In some embodiments, greater than 50% or greater than about 50% of NK cells in the expanded population are fcrγ Negative of . In some embodiments, greater than 60% or greater than about 60% of NK cells in the expanded population are fcrγ Negative of . In some embodiments, greater than 70% or greater than about 70% of NK cells in the expanded population are fcrγ Negative of . In some embodiments, greater than 80% or greater than about 80% of NK cells in the expanded population are fcrγ Negative of . In some embodiments, greater than 90% or greater than about 90% of NK cells in the expanded population are fcrγ Negative of . In some embodiments, greater than 95% or greater than about 95% of NK cells in the expanded population are fcrγ Negative of . For example, the methods herein generally produce g-NK cell products of high purity (e.g., 70% to 90%).
In some embodiments, it may be useful to detect the expression of g-NK cells without employing intracellular staining, such as, for example, if the cells of the sample are to be subjected to cell sorting or functional assays. Although treatments that allow intracellular staining of FcR gamma (e.g., immobilization and permeabilization) can be used to confirm the identity of a substantially pure cell population, in many cases cell surface markers can be employed that can be detected without damaging the cells when identifying, detecting or isolating g - NK cells. Thus, in some embodiments, g-NK cells are identified using a surrogate marker profile that correlates with the lack of fcrγ in the NK cell subpopulation. In some embodiments, surrogate marker profiling is particularly useful when it is difficult or impossible to assess the presence or absence of an intracellular protein (such as fcrγ) depending on the particular application of the cell.
It is found herein that certain combinations of cell surface markers are associated with the g-NK cell phenotype, i.e. cell deficiency or lack of intracellular expression of fcrγ, thereby providing a surrogate marker profile to identify or detect g-NK cells in a manner that does not damage the cells. In some embodiments, the surrogate marker profile of g-NK cells provided herein is based on positive surface expression of one or more markers CD16 (CD 16 Positive and negative )、NKG2C(NKG2C Positive and negative ) or CD57 (CD 57 positive) and/or based on low surface expression or negative surface expression of one or more markers CD7 (CD 7 Weak and weak / Negative of )、CD161(CD161 Negative of ) and/or NKG2A (NKG 2A Negative of ). In some embodiments, the cells are further evaluated for one or more surface markers of NK cells, such as CD45, CD3, and/or CD56. In some embodiments, the surrogate marker profile CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative /CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of can be used to identify, detect, enrich, and/or isolate g-NK cells. In some embodiments, the surrogate marker profile CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative /NKG2A Negative of /CD161 Negative of is used to identify, detect, enrich, and/or isolate g-NK cells. In some embodiments, g-NK cells of NKG2C Positive and negative and/or NKG2a Negative of are identified, detected, enriched, and/or isolated.
In some embodiments, greater than 30% or greater than about 30% of NK cells in the expanded population are positive for NKG2C and/or greater than 50% or greater than about 50% of NK cells in the expanded population are negative or low level for NKG 2A. In some embodiments, greater than 35% or greater than about 35% of NK cells in the expanded population are positive for NKG2C and/or greater than 60% or greater than about 60% of NK cells in the expanded population are negative or low level for NKG 2A. In some embodiments, greater than 40% or greater than about 40% of NK cells in the expanded population are positive for NKG2C and/or greater than 70% or greater than about 70% of NK cells in the expanded population are negative or low level for NKG 2A. In some embodiments, greater than 45% or greater than about 45% of NK cells in the expanded population are positive for NKG2C and/or greater than 80% or greater than about 80% of NK cells in the expanded population are negative or low level for NKG 2A. In some embodiments, greater than 50% or greater than about 50% of NK cells in the expanded population are positive for NKG2C and/or greater than 85% or greater than about 85% of NK cells in the expanded population are negative or low level for NKG 2A. In some embodiments, greater than 55% or greater than about 55% of NK cells in the expanded population are positive for NKG2C and/or greater than 90% or greater than about 90% of NK cells in the expanded population are negative or low level for NKG 2A. In some embodiments, greater than 60% or greater than about 60% of NK cells in the expanded population are positive for NKG2C and/or greater than 95% or greater than about 95% of NK cells in the expanded population are negative or low level for NKG 2A.
In some embodiments, greater than 70% or greater than about 70% of the g-NK cells in the expanded population are positive for perforin and greater than 70% or greater than about 70% of the g-NK cells in the expanded population are positive for granzyme B. In some embodiments, greater than 75% or greater than about 75% of the g-NK cells in the expanded population are positive for perforin and greater than 75% or greater than about 75% of the g-NK cells in the expanded population are positive for granzyme B. In some embodiments, greater than 80% or greater than about 80% of the g-NK cells in the expanded population are positive for perforin and greater than 80% or greater than about 80% of the g-NK cells in the expanded population are positive for granzyme B. In some embodiments, greater than 85% or greater than about 85% of the g-NK cells in the expanded population are positive for perforin and greater than 85% or greater than about 85% of the g-NK cells in the expanded population are positive for granzyme B. In some embodiments, greater than 90% or greater than about 90% of the g-NK cells in the expanded population are positive for perforin and greater than 90% or greater than about 90% of the g-NK cells in the expanded population are positive for granzyme B. In some embodiments, greater than 95% or greater than about 95% of the g-NK cells in the expanded population are positive for perforin and greater than 95% or greater than about 95% of the g-NK cells in the expanded population are positive for granzyme B.
In some embodiments of any such embodiments, greater than 20%, greater than 30%, or greater than about 30%, greater than 40%, or greater than about 40%, greater than 50%, or greater than about 50%, greater than 60%, or greater than about 60%, greater than 70%, or greater than about 70%, greater than 80%, or greater than about 80%, greater than 90%, or greater than about 90%, or greater than 95%, or greater than about 95% of the total cells in the amplified population comprise a heterologous nucleic acid encoding the CAR. In some embodiments of any such embodiments, greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the total cells in the amplified population comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a secretable or membrane-bound cytokine as described). In some embodiments of any such embodiments, greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the total cells in the amplified population comprise a heterologous nucleic acid encoding the CAR and an immunomodulatory agent (e.g., a secretable or membrane-bound cytokine as described). In some embodiments of any such embodiment, greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the g-NK cells in the expanded population or subpopulations of cells having an surrogate marker profile of g-NK cells as described herein comprise a heterologous nucleic acid encoding a CAR. In some embodiments of any such embodiments, greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the g-NK cells or cell subpopulations having a surrogate marker profile of g-NK cells as described herein comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a secretable or membrane-bound cytokine as described). In some of any such embodiments, greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, greater than 90% or greater than about 90%, or greater than 95% or greater than about 95% of the g-NK cells or cell subpopulations having a surrogate marker profile of g-NK cells as described herein comprise a heterologous nucleic acid encoding a CAR and an immunomodulatory agent (e.g., a secretable or membrane-bound cytokine as described).
Any of a number of functional or phenotypic activities of cells expanded by the provided methods can be assessed, including but not limited to cytotoxic activity, degranulation, the ability to produce or secrete cytokines, and expression of one or more intracellular or surface phenotypic markers. Methods of assessing such activity are known and are exemplified herein and in the working examples.
In some embodiments, antibody-dependent cellular cytotoxicity (ADCC) cytotoxic activity against target cells may be used as a measure of functionality. For ADCC cytotoxicity assays, cells from expansion may be co-cultured with appropriate target cells in the presence or absence of antibodies specific for target antigens on the target cells. For example, for anti-myeloma cytotoxicity, any of a number of Multiple Myeloma (MM) target cells (e.g., AM01, KMS11, KMS18, KMS34, LP1, or mm.1 s) can be used and assayed with an anti-CD 38 (e.g., up to Lei Tuoyou mab) or an anti-CD 319 antibody (e.g., erlotinib). Cell killing can be measured by a number of methods. For example, cells can be stained with Propidium Iodide (PI), and the number of NK cells, live target cells, and dead target cells can be resolved, such as by flow cytometry.
In some embodiments, greater than 10% or greater than about 10% of the g-NK cells in the expanded population are capable of degranulation against tumor cells. Degranulation can be measured by assessing the expression of CD 107A. For example, in some embodiments, greater than 20% or greater than about 20% of the g-NK cells in the expanded population are capable of degranulation against tumor cells. In some embodiments, greater than 30% or greater than about 30% of the g-NK cells in the expanded population are capable of degranulation against tumor cells. In some embodiments, greater than 40% or greater than about 40% of the g-NK cells in the expanded population are capable of degranulation against tumor cells. In some embodiments, the ability to degranulate is measured in the absence of antibodies to tumor cells.
In some embodiments, greater than 10% or greater than about 10% of the g-NK cells in the expanded population are capable of producing an effector cytokine, such as interferon-gamma or TNF-alpha, against tumor cells. In some embodiments, greater than 20% or greater than about 20% of the g-NK cells in the expanded population are capable of producing an effector cytokine, such as interferon-gamma or TNF-alpha, against the tumor cells. In some embodiments, greater than 30% or greater than about 30% of the g-NK cells in the expanded population are capable of producing an effector cytokine, such as interferon-gamma or TNF-alpha, against tumor cells. In some embodiments, greater than 40% or greater than about 40% of the g-NK cells in the expanded population are capable of producing an effector cytokine, such as interferon-gamma or TNF-alpha, against tumor cells. In some embodiments, the ability to produce interferon-gamma or TNF-alpha is measured in the absence of antibodies to tumor cells.
Provided herein are methods for identifying or detecting g-NK cells in a sample containing a population of cells by employing a surrogate marker profile of the g-NK cells. In some embodiments, the methods comprise contacting the cell sample with a binding molecule, such as an antibody or antigen binding fragment specific for one or more markers CD16, CD57, CD7, CD161, NKG2C and/or NKG 2A. In some embodiments, the methods further comprise contacting the cell sample with a binding molecule, such as an antibody or antigen binding fragment specific for CD45, CD3, and/or CD 56. In some embodiments of these methods, the one or more binding molecules may be contacted with the sample simultaneously. In some embodiments of these methods, the one or more binding molecules may be contacted with the sample sequentially. In some embodiments, after contacting, the methods may include one or more washes under conditions that preserve cells that have bound to the one or more binding molecules and/or separate unbound binding molecules from the sample.
In some embodiments, each of the one or more binding molecules (e.g., antibodies) can be directly or indirectly attached to a label for detecting cells positive or negative for the label. For example, a binding molecule (e.g., an antibody) may be conjugated, coupled, or linked to a label. Labels are well known to those skilled in the art. Labels contemplated herein include, but are not limited to, fluorescent dyes, fluorescent proteins, radioisotopes, chromophores, metal ions, gold particles (e.g., colloidal gold particles), silver particles, particles having strong light scattering properties, magnetic particles (e.g., magnetic bead particles, such asMagnetic beads), polypeptides (e.g., FLAG TM tags, human influenza Hemagglutinin (HA) tags, etc.), enzymes such as peroxidases (e.g., horseradish peroxidase) or phosphatases (e.g., alkaline phosphatase), streptavidin, biotin, luminescent compounds (e.g., chemiluminescent substrates), oligonucleotides, members of specific binding pairs (e.g., ligands and their receptors), and other labels well known in the art for visualizing or detecting binding molecules (e.g., antibodies) when directly or indirectly attached to the antibodies.
Many well known methods for assessing the expression level of a surface marker or protein may be used, such as detection by affinity-based methods, e.g. immunoaffinity-based methods, e.g. in the context of a surface marker, such as by flow cytometry. In some embodiments, the label is a fluorophore and the method for detecting or identifying g-NK cells is by flow cytometry. In some embodiments, different labels are used for each of the different labels by polychromatic flow cytometry.
In some embodiments, the methods comprise contacting the sample with binding molecules specific for CD45, CD3, CD56, CD57, CD7, and CD 161. In some such embodiments, g-NK cells are identified or detected as cells having the g-NK cell substitution marker profile CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative /CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of .
In some embodiments, the methods comprise contacting the sample with binding molecules specific for CD45, CD3, CD56, NKG2A, and CD 161. In some such embodiments, g-NK cells are identified or detected as cells having the g-NK cell substitution marker profile CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative /NKG2A Negative of /CD161 Negative of .
In some embodiments, the provided methods can further comprise isolating or enriching g-NK cells, such as g-NK cells that preferentially expand according to any of the provided methods. In some such embodiments, a substantially pure g-NK cell population, such as a cell population containing greater than or greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more g-NK cells, such as determined using a combination of any of the groups or markers, can be obtained. Antibodies and other binding molecules can be used to detect the presence or absence of the expression level of the marker protein for isolating or enriching g - NK cells. In some embodiments, the separation or enrichment is performed by fluorescence activated cell sorting (FAC). In an example of such a method, g-NK cells are identified or detected by flow cytometry using the method described above for staining cells for a variety of cell surface markers, and the stained cells are carried in a fluid stream for collecting cells positive or negative for the markers associated with g-NK cells.
VI kits and articles of manufacture
Provided herein are articles of manufacture and kits comprising provided compositions containing NK cells enriched for a particular subpopulation (such as g-NK cells). In some embodiments, g-NK cells are engineered, such as with a nucleic acid encoding an antigen receptor (such as a CAR). In some embodiments, g-NK cells are engineered, such as with nucleic acids encoding immunomodulators (such as cytokines). In some embodiments, g-NK cells are engineered, such as with nucleic acids encoding antigen receptors (such as CARs) and immune modulators (such as cytokines). The cells in these compositions are engineered g-NK cells that comprise a heterologous nucleic acid encoding an antigen receptor (e.g., CAR). The cells in these compositions are engineered g-NK cells that comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine). The cells in these compositions are engineered g-NK cells comprising a heterologous nucleic acid encoding an antigen receptor (e.g., CAR) and an immunomodulatory agent (e.g., cytokine). In some embodiments, the composition is produced by any of the provided methods. In some embodiments, the kit comprises any one of the provided compositions and instructions for administering the compositions as monotherapy.
In some embodiments, provided herein are kits comprising any one of the provided compositions and additional agents. Exemplary additional agents include any of the agents described herein. In some embodiments, the additional agent comprises an Fc domain. In some embodiments, the additional agent is an Fc fusion protein or antibody. In some embodiments, the additional agent is an antibody. In some embodiments, the additional agent is a human antibody, a humanized antibody, or a chimeric antibody. In some of these embodiments, the additional agent is a full length antibody. Exemplary antibodies include any of the antibodies described.
The kit may optionally include one or more components, such as instructions for use, devices, and additional reagents (e.g., sterile water or saline solution for diluting the composition and/or reconstituting the lyophilized protein), as well as components for performing such methods, such as tubing, containers, and syringes. In some embodiments, the kit may further comprise reagents for collecting a sample, preparing and processing a sample, and/or reagents for quantifying the amount of one or more surface markers in a sample, such as but not limited to detection reagents, such as antibodies, buffers, substrates for enzyme staining, chromophores, or other materials, such as slides, containers, microtiter plates, and optionally instructions for performing these methods. Those of skill in the art will recognize many other possible containers and plates and reagents that may be used according to the provided methods.
In some embodiments, the kit may be provided as articles of manufacture comprising packaging materials for packaging cells, antibodies or reagents, or combinations thereof, or one or more other components. For example, the kit may include containers, bottles, tubes, vials, and any packaging material suitable for separating or organizing the kit components. The one or more containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the one or more containers hold a composition comprising cells or antibodies or other reagents for use in these methods. The articles or kits herein may include cells, antibodies, or reagents in separate containers or in the same container.
In some embodiments, the one or more containers containing the composition may be disposable vials or multiple use vials, which may allow for reuse of the composition in some cases. In some embodiments, the article of manufacture or kit may further comprise a second container comprising a suitable diluent. The article of manufacture or kit may also include other materials desirable from a commercial, therapeutic, and user standpoint, including other buffers, diluents, filters, needles, syringes, therapeutic agents, and/or package insert with instructions for use.
In some embodiments, the kit may optionally include instructions. The instructions generally include descriptions of the tangible expression of cell compositions, reagents, and/or antibodies, and optionally other components included in the kit, and methods of using these. In some embodiments, the instructions indicate methods of using a cell composition and an antibody to administer to a subject to treat a disease or disorder, such as according to any of the provided embodiments. In some embodiments, the instructions are provided as indicia or package insert on or associated with the container. In some embodiments, the instructions may indicate instructions for reconstitution and/or use of the composition.
VII therapeutic methods
Provided herein are compositions for treating a disease or disorder in a subject and methods involving provided g-NK cell compositions comprising engineered g-NK cells (also referred to as engineered g-NK cell compositions), including any of the g-NK cells described herein. Such methods and uses may include the use of any of the compositions as described herein, including those described in section IV or those produced by the methods herein as described in section II or V. In some embodiments, provided herein are methods of treating a disorder in an individual, the method comprising administering to an individual in need thereof any one of the provided g-NK compositions comprising engineered g-NK cells. The cells in these compositions are engineered g-NK cells that comprise a heterologous nucleic acid encoding an antigen receptor (e.g., CAR). The cells in these compositions are engineered g-NK cells that comprise a heterologous nucleic acid encoding an immunomodulatory agent (e.g., a cytokine). The cells in these compositions are engineered g-NK cells comprising a heterologous nucleic acid encoding an antigen receptor (e.g., CAR) and an immunomodulatory agent (e.g., cytokine). In particular embodiments, the compositions are produced by the methods provided herein. Such methods and uses include therapeutic methods and uses, for example, involving administering a therapeutic cell or composition comprising the same to a subject suffering from a disease, condition, or disorder. In some cases, the disease or disorder is a tumor or cancer. In some embodiments, the disease or disorder is a viral infection. In some embodiments, the cells or pharmaceutical compositions thereof are administered in an amount effective to treat the disease or disorder. Uses include the use of cells or pharmaceutical compositions thereof in such methods and treatments, and in the manufacture of medicaments for performing such methods of treatment. In some embodiments, the methods thereby treat a disease or condition or disorder in a subject.
In some embodiments, any of the provided methods and uses may be provided NK cell compositions comprising engineered g-NK cells, which may include methods and uses as described in PCT publication No. WO2020/107002 or PCT application No. PCT/US 2021/028504.
In some embodiments, the methods of treatment or uses involve administering to an individual an effective amount of cells of a g-NK cell composition provided herein, such as a composition comprising engineered g-NK cells as provided herein, including any such composition comprising expanded NK cells produced by the provided methods. In some embodiments, 10 5 to 10 12 or about 10 5 to about 10 12, or 10 5 to 10 8 or about 10 5 to about 10 8, or 10 6 to 10 12 or about 10 6 to about 10 12, or 10 8 to 10 11 or about 10 8 to about 10 11, or 10 9 to 10 10, or about 10 9 to about 10 10 such g-NK cell compositions provided herein (such as compositions comprising engineered NK cells as provided herein, including any compositions produced by the provided methods) are administered to an individual subject. In some embodiments, a dose of cells comprising 10 5 or greater than 10 5 or greater than about 10 5、106 or greater than 10 6 or greater than about 10 6、107 or greater than 10 7 or greater than about 10 7、108 or greater than 10 8 or greater than about 10 8、109 or greater than 10 9 or greater than about 10 9、1010 or greater than 10 10 or greater than about 10 10、1011 or greater than 10 11 or greater than about 10 11, or 10 12 or greater than 10 12 or greater than about 10 12 cells of such g-NK cell compositions provided herein (such as compositions comprising engineered NK cells as provided herein, including any compositions produced by the provided methods) is administered to an individual.
In some embodiments, the method of treatment or use involves administering to an individual an effective amount of cells of any provided NK cell composition (including any engineered g-NK cell composition as described herein). In some embodiments, from 10 5 to 10 12 or from about 10 5 to about 10 12, or from 10 5 to 10 8 or from about 10 5 to about 10 8, or from 10 6 to 10 12 or from about 10 6 to about 10 12, or from 10 8 to 10 11 or from about 10 8 to about 10 11, or from 10 9 to 10 10, or from about 10 9 to about 10 10 cells from any of the provided compositions containing engineered g-NK cells are administered to an individual subject. In some embodiments, a dose of cells comprising 10 5 or greater than 10 5 or greater than about 10 5、106 or greater than 10 6 or greater than about 10 6、107 or greater than 10 7 or greater than about 10 7、108 or greater than 10 8 or greater than about 10 8、109 or greater than 10 9 or greater than about 10 9、1010 or greater than 10 10 or greater than about 10 10、1011 or greater than 10 11 or greater than about 10 11, or 10 12 or greater than 10 12 or greater than about 10 12 cells from any of the provided compositions containing engineered g-NK cells is administered to an individual. In some embodiments, any provided composition comprising engineered g-NK cells of from about 10 6 to 10 10 such cells per kg is administered to a subject.
In some embodiments, the composition containing engineered g-NK cells may be administered at a predetermined number of doses once a week.
In some embodiments, the predetermined number of weekly doses is one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine doses, ten doses, eleven doses, or twelve doses. In some embodiments, once weekly dose administration lasts for 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, or longer. In some embodiments, six (6) weekly doses of g-NK cell composition are administered. In some embodiments, the weekly dose is administered over a continuous number of weeks.
In some embodiments, the weekly dose is administered in a cyclic regimen. In some embodiments, the cycling regimen is a 14 day period. In some embodiments, the once weekly dose is administered twice in a 14 day cycle. In some embodiments, the 14 day cycle is repeated twice. In some embodiments, the 14 day cycle is repeated three times.
In some embodiments, an effective amount of each of the disclosed cells or compositions containing the engineered g-NK cells disclosed herein is administered to the subject once a week for 5 weeks.
In some embodiments, each dose of cells of the g-NK cell composition containing engineered g-NK cells may be from 1 x 10 8 cells to 50 x 10 9 cells or from about 1 x 10 8 cells to about 50 x 10 9 cells of the g-NK cell composition. In some embodiments, each dose of cells of the g-NK cell composition containing engineered g-NK cells may be or may be about 5 x 10 8 cells of the g-NK cell composition. In some embodiments, each dose of cells of the g-NK cell composition containing engineered g-NK cells may be or may be about 5 x 10 9 cells of the g-NK cell composition. In some embodiments, each dose of cells of the g-NK cell composition containing engineered g-NK cells may be or may be about 10 x 10 9 cells of the g-NK cell composition.
In some embodiments, the dosage administered is from 1X 10 5 cells/kg to 1X 10 7 cells/kg or from about 1X 10 5 cells/kg to about 1X 10 7 cells/kg, depending on any of the provided methods of treatment or uses, such as from 1X 10 7 cells/kg to 7.5X 10 7 cells/kg or from about 1X 10 7 cells/kg to about 7.5X 10 7 cells/kg, from 1X 10 7 cells/kg to about 5X 10 7 cells/kg or from 1X 10 7 cells/kg to about 5X 10 7 cells/kg, from 1X 10 7 cells/kg to about 2.5X 10 7 cells/kg or from about 1X 10 7 cells/kg to about 2.5X 10 7 cells/kg, from 1X 10 7 cells/kg to about 1X 10 7 cells/kg or from about 1X 10 7 cells/kg to about 1X 10 7 cells/kg 1X 10 7 to 7.5X 10 7 cells/kg or about 1X 10 7 to about 7.5X 10 7 cells/kg, 1X 10 7 to 5X 10 7 cells/kg or about 1X 10 7 to about 5X 10 7 cells/kg, 1X 10 7 to 2.5X 10 7 cells/kg or about 1X 10 7 to about 2.5X 10 7 cells/kg, from 2.5X10 5 cells/kg to 1X 10 7 cells/kg or from about 2.5X10 5 cells/kg to about 1X 10 7 cells/kg, from 2.5X10 7 cells/kg to 7.5X10 7 cells/kg or from about 2.5X10 7 cells/kg to about 7.5X10 7 cells/kg, from 2.5X10 7 cells/kg to 5X 10 7 cells/kg or from about 2.5X10 7 cells/kg to about 5X 10 7 cells/kg, from 2.5X10 7 cells/kg to about 2.5X10 7 cells/kg or from about 2.5X10 7 cells/kg to about 2.5X10 7 cells/kg from 2.5X107 7 cells/kg to 1X 10 7 cells/kg or from about 2.5X107 7 cells/kg to about 1X 10 7 cells/kg, from 2.5X107 7 cells/kg to about 7.5X107 7 cells/kg or from about 2.5X107 7 cells/kg to about 7.5X107 7 cells/kg, from about 2.5X107 7 cells/kg to about 5X 10 7 cells/kg or from about 2.5X107. Sup. 7 cells/kg to about 5X 10 7 cells/kg, from about 5X 10 7 cells/kg to about 1X 10 7 cells/kg or from about 5X 10 7 cells/kg to about 1X 10 7 cells/kg, from 5X 10 5 cells/kg to 7.5X 10 6 cells/kg or from about 5X 10 5 cells/kg to about 7.5X 10 6 cells/kg, from 5X 10 6 cells/kg to about 5X 10 6 cells/kg or from about 5X 10 6 cells/kg to about 5X 10 6 cells/kg, from 5X 10 6 cells/kg to about 2.5X 10 6 cells/kg or from about 5X 10 6 cells/kg to about 2.5X 10 6 cells/kg, from 5X 10 6 cells/kg to about 1X 10 6 cells/kg or from about 5X 10 6 cells/kg to about 1X 10 6 cells/kg from 5X 10 6 cells/kg to 7.5X 10 6 cells/kg or from about 5X 10 6 cells/kg to about 7.5X 10 6 cells/kg, from 1X 10 6 cells/kg to 1X 10 6 cells/kg or from about 1X 10 6 cells/kg to about 1X 10 6 cells/kg, from 1X 10 6 cells/kg to about 7.5X 10 6 cells/kg or from about 1X 10 6 cells/kg to about 7.5X 10 6 cells/kg, from 1X 10 6 cells/kg to about 5X 10 6 cells/kg or from about 1X 10 6 cells/kg to about 5X 10 6 cells/kg, from about 1×10 6 cells/kg to 2.5×10 6 cells/kg or from about 1×10 6 cells/kg to about 2.5×10 6 cells/kg, from about 2.5×10 6 cells/kg to about 1×10 7 cells/kg or from about 2.5×10 6 cells/kg to about 1×10 7 cells/kg, from about 2.5×10 6 cells/kg to about 7.5×10 6 cells/kg or from about 2.5×10 6 cells/kg to about 7.5×10 6 cells/kg, from about 2.5×10 6 cells/kg to about 5×10 6 cells/kg or from about 2.5×10 6 cells/kg to about 5×10 6 cells/kg, from about 5×10 6 cells/kg to about 1×10 6 cells/kg or from about 5×10 6 cells/kg to about 1×10 6 cells/kg, from about 2.5×10 6 cells/kg to about 7.5×10 6 cells/kg or from about 2.5×10 6 cells/kg to about 7.5×10 6 cells/kg, from about 7.5×10 6 cells/kg or from about 2.5×10 to about 7.5×10 6 cells/kg. In some embodiments, the dose administered is 1×10 to 1×10 8 cells/kg or about 1×10 5 cells/kg to about 1×10 8 cells/kg, such as about 2.5×10 8 cells/kg to about 1×10 8 cells/kg or about 2.5×10 8 cells/kg to about 5×10 8 cells/kg, about 5×10 8 cells/kg to about 1×10 8 cells/kg or about 5×10 8 cells/kg to about 1×10 8 cells/kg, about 7.5×10 8 cells/kg to about 1×10 8 cells/kg or about 7.5×10 8 cells/kg to about 1×10 8 cells/kg, about 1×10 8 cells/kg to about 1×10 8 cells/kg or about 1×10×2 cells/kg to about 1×10×3932 cells/kg to about 5×10 kg to about 1×10 cells/kg, about 5×10×2 cells/kg to about 2.2×2 cells/8 kg to about 1×10×2 cells/kg or about 5×10×2 cells/kg to about 7.3×10×2 cells/kg to about 1×10×2 cells/kg to about 1×10.3932 cells/kg or about 7×2×2 cells/kg to about 1×10.3932 cells/kg to about 1×2×2 cells/3932.3932 cells/kg, from 1×10 7 cells/kg to 1×10 8 cells/kg or from about 1×10 7 cells/kg to about 1×10 8 cells/kg, from 2.5×10 7 cells/kg to 1×10 8 cells/kg or from about 2.5×10 7 cells/kg to about 1×10 8 cells/kg, from 5×10 7 cells/kg to 1×10 8 cells/kg or from about 5×10 7 cells/kg to about 1×10 8 cells/kg or from 7.5×10 7 cells/kg to 1×10 8 cells/kg or from about 7.5×10 7 cells/kg to about 1×10 8 cells/kg.
In some embodiments, the dose is given in terms of the number of g-NK cells or NK cell subsets (such as any NK cell subset described herein) in the composition (or the number of any of the aforementioned living cells) associated with or comprising the surrogate marker for g-NK cells. In any of the above embodiments, the dose is given as the number of cells or the number of any of the aforementioned living cells in the provided composition of engineered cells (such as produced by the provided methods).
In some embodiments, according to any therapeutic methods or uses of the dosage of 5X 10 7 to 10X 10 9 or about 5X 10 7 to about 10X 10 9 containing engineered g-NK cell composition, such as from 5 x 10 7 to 5 x 10 9 or from about 5 x 10 9 to 1 x 10 9 or from about 5 x 10 9 to about 1 x 10 9 to 5 x 10 9 or from about 5 x 10 9 to about 1 x 10 9 or from about 5 x 10 9 to about 1 x 10 9 to about 10 x 10 9 or to about 10 x 10 9 to about 5 x 10 9 or from about 1 x 10 9 to about 5 x 10 9 to about 1 x 10 9 or about 1 x 2 x. 10 9 to about 1×10 9 to 5×10 9 or about 1×10 9 to about 5×10 9 to about 10×10 9 or about 5×10 9 to about 10×10 9 to about 5×10 9 or about 5×10 9 to about 10×10 9 or about 5×10 9 to about 1×10 9 to about 10×10 9 or about 1×10 9 to about 10×10 9 to about 5×10 9 or about 1×10 9 to about 5×10 9 or from 5 x 10 9 to 10 x 10 9 or from about 5 x 10 9 to about 10 x 10 9 cells. In some embodiments, the dose administered is 5×10 8 cells or about 5×10 8 cells of the g-NK cell composition containing engineered g-NK cells. In some embodiments, the dose administered is 1X 10 9 cells or about 1X 10 9 cells of a g-NK cell composition containing engineered g-NK cells. In some embodiments, the dose administered is 5×10 9 cells or about 5×10 9 cells of the g-NK cell composition containing engineered g-NK cells. In some embodiments, the dose administered is 1X 10 10 cells or about 1X 10 10 cells of a g-NK cell composition containing engineered g-NK cells. In any of the above embodiments, the dose is given as the number of cells or the number of living cells of any of the foregoing in the composition of engineered cells produced by the provided methods. In some embodiments, the dose is given in terms of the number of g-NK cells or NK cell subsets (such as any of the NK cell subsets described herein) (or the number of any of the aforementioned living cells) associated with or comprising the surrogate marker of g-NK cells.
In some embodiments, a dose of cells of a composition containing engineered g-NK cells is administered to an individual shortly after expansion and/or engineering according to the provided methods. In other embodiments, the g-NK cell composition containing engineered g-NK cells is stored prior to administration, such as by the methods described above. For example, NK cells may be stored for more than 6, 12, 18, or 24 months prior to administration to an individual.
In some embodiments, the provided compositions containing engineered NK cells and their subpopulations (such as g-NK cells) can be administered to a subject by any convenient route, including parenteral routes, such as subcutaneous, intramuscular, intravenous, and/or epidural routes of administration.
In particular embodiments, the provided compositions are administered by intravenous infusion. In some embodiments, about 10 x 10 6 cells to 10 x 10 9 cells are administered or administered by intravenous infusion in a volume of 1mL to 100 mL. In some embodiments, about 50 x 10 6 cells are administered or administered. In some embodiments, about 1×10 9 cells are administered or administered. In some embodiments, about 5 x 10 9 cells are administered or administered. In some embodiments, about 10 x 10 9 cells are administered or administered. Determining the cell volume for infusion to administer the cell number is within the level of skill in the art. In one example, 0.5×10 9 cells are administered by intravenous infusion of a volume of about 20mL of a composition, such as a thawed cryopreserved composition, formulated at a concentration of 2.5×10 7 cells/mL or about 2.5×10 7 cells/mL (e.g., or about 5×10 9 cells in 200 mL).
The provided engineered g-NK cell compositions are useful in methods of treating individuals suffering from a tumor or hyperproliferative disorder or microbial infection, such as viral infection, yeast infection, fungal infection, protozoal infection, and/or bacterial infection. The provided engineered g-NK cell compositions can be administered for the treatment of animals, such as mammals, e.g., human subjects.
In some examples, the methods include treating a hyperproliferative disorder, such as a hematological malignancy or a solid tumor. Examples of the types of cancers and proliferative disorders that can be treated with the compositions described herein include, but are not limited to: multiple myeloma, leukemia (e.g., myelogenous, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic myelogenous (granulocytic) leukemia, and chronic lymphocytic leukemia), lymphoma (e.g., hodgkin's and non-hodgkin's disease), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, angiosarcoma, endothelial sarcoma, ewing's tumor, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, hepatoma, wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytic tumor, oligodendroglioma, melanoma, neuroblastoma, retinoblastoma, dysplasia and hyperplasia. Treatment and/or prevention of cancer includes, but is not limited to, alleviation of one or more symptoms associated with cancer, inhibition or reduction of cancer progression, promotion of cancer regression, and/or promotion of an immune response.
In some examples, the methods include treating a viral infection, such as an infection caused by the presence of a virus in vivo. Viral infections may be caused by DNA or RNA viruses and include chronic or persistent viral infections, which are viral infections that are capable of infecting a host and propagating within the host cell for a long period of time (typically weeks, months or years) before proving fatal. Chronically infectious viruses that can be treated according to the present invention include, for example, human Papillomaviruses (HPV), herpes simplex viruses and other herpesviruses, hepatitis c, hepatitis b, and other hepatitis viruses, human immunodeficiency viruses and measles viruses, all of which can produce significant clinical disease. Long-term infection can ultimately lead to induction of a disease, such as in the case of hepatitis c virus liver cancer, which can be fatal to the patient. Other chronic viral infections that can be treated according to the present invention include Epstein Barr Virus (EBV) and other viruses, such as those that can be associated with tumors.
Examples of viral infections that may be treated or prevented with the compositions and methods described herein include, but are not limited to, viral infections caused by: coronaviruses (e.g., SARS-CoV-2, where infection is COVID-19), retroviruses (e.g., human T cell lymphotropic virus (HTLV) type I and type II, and Human Immunodeficiency Virus (HIV)), herpesviruses (e.g., herpes Simplex Virus (HSV) type I and type II, EB virus, and cytomegalovirus), arenaviruses (e.g., lassa-fever virus), paramyxoviruses (e.g., measles virus, human respiratory syncytial virus, and pneumovirus), adenoviruses, bunyaviruses (e.g., hantavirus), cornaviruses, filoviruses (e.g., ebola virus), flaviviruses (e.g., hepatitis C Virus (HCV), yellow fever virus, and japanese encephalitis virus), hepadnaviruses (e.g., hepatitis B Virus (HBV)), orthomyxoviruses (sendai virus and influenza virus A, B, and C), papovaviruses (e.g., papillomaviruses), picornaviruses (e.g., rhinoviruses, enteroviruses, and hepatitis a virus), poxviruses, reoviruses (e.g., rotaviruses), membrane viruses (e.g., rotaviruses), such as togaviruses, and rabies viruses (e.g., rabies). Treatment and/or prevention of a viral infection includes, but is not limited to, alleviation of one or more symptoms associated with the infection, inhibition, reduction or suppression of viral replication, and/or enhancement of an immune response.
In some embodiments, the engineered g-NK cells and compositions containing the engineered g-NK cells are used in methods of treating yeast or bacterial infections. Examples of yeast or bacterial infections include infections related to: streptococcus pyogenes, streptococcus pneumoniae, neisseria gonorrhoeae, neisseria meningitidis, corynebacterium diphtheriae, clostridium botulinum, clostridium perfringens, clostridium tetani, haemophilus influenzae, klebsiella pneumoniae, klebsiella, sclerostin Lei Baiba, staphylococcus aureus, vibrio cholerae, escherichia coli, pseudomonas aeruginosa, campylobacter fetus (vibrio), campylobacter jejuni, aeromonas hydrophila, bacillus cereus, edwardsiella tarda, yersinia enterocolitica, plague bacillus pseudotuberculosis, shigella dysenteriae, shigella flexneri, shigella sonnei, salmonella typhimurium, treponema pallidum, leptospira maculosa, treponema pallidum, borrelia, bordetella icterus, mycobacterium tuberculosis, toxoplasmosis, pneumocystis carinii, brucella tularensis, brucella hyos, brucella suis, helicobacter pylori, or combinations thereof.
In some embodiments, the provided engineered g-NK cells and compositions thereof are useful as monotherapy in the treatment of diseases or disorders.
A. Combination therapy
In some embodiments, a composition comprising engineered g-NK cells as provided herein may be administered in combination therapy with one or more other agents for treating a disease or disorder in a subject. In such embodiments, the compositions provided herein containing engineered g-NK cells can be administered prior to, concurrently with, or subsequent to (after) administration of one or more other agents.
In some embodiments, the disclosed methods of treating a subject with the provided engineered g-NK cells and compositions can be combined with a therapeutic monoclonal antibody, such as an anti-tumor antigen or anti-cancer antibody, an anti-viral antibody, or an anti-bacterial antibody.
In some embodiments, a dose of engineered g-NK cells can be administered simultaneously or sequentially with antimicrobial, antiviral, and other therapeutic agents. In some embodiments, these methods are performed in combination with administering a chemotherapeutic agent, a cytotoxic agent, or an immunomodulatory agent to a subject.
In some embodiments provided herein, an engineered g-NK cell or composition containing the same may be administered to an individual in combination with a cytokine and/or a growth factor. Because cytokines are required for NK cell activity, typical methods involve administering exogenous cytokines as exogenous cytokine supports to a subject in combination with NK cell therapy.
Exemplary combination therapies are described in the following subsections.
1. Antibody combinations
In some embodiments, compositions containing the engineered g-NK cells provided herein exhibit enhanced activity when activated by or contacted with antibodies or Fc-containing proteins, such as compared to conventional NK cells. For example, g-NK cells can be activated by antibody-mediated CD16 cross-linking or by antibody-coated tumor cells. In some embodiments, provided herein are methods of treating a disorder in an individual, the method comprising administering to a subject an engineered g-NK cell or composition thereof and an antibody.
One of ordinary skill in the art can select an appropriate therapeutic (e.g., anti-cancer) monoclonal antibody to administer to a subject with the provided engineered g-NK cells and compositions described herein, such as depending on the particular disease or disorder of the individual. Suitable antibodies may include polyclonal, monoclonal, fragments (such as Fab fragments), single chain antibodies, and other forms of specific binding molecules.
In some embodiments, the antibody may also comprise a humanized antibody or a human antibody. Humanized forms of non-human antibodies are chimeric igs, ig chains or fragments (such as Fv, fab, fab ', F (ab') 2, or other antigen-binding subsequences of antibodies) that contain minimal sequences derived from non-human igs. In some embodiments, the antibody comprises an Fc domain.
Typically, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization was accomplished by replacing the corresponding sequences of the human antibodies with rodent CDR or CDR sequences (Jones et al, 1986; riechmann et al, 1988; verhoeyen et al, 1988). Such "humanized" antibodies are chimeric antibodies (1989) in which significantly less than the complete human variable domain has been replaced by the corresponding sequence from a non-human species. Indeed, humanized antibodies are typically human antibodies in which some CDR residues and possibly some Fc residues are substituted by residues from similar sites in rodent antibodies. Humanized antibodies include human antibodies (recipient antibodies) in which residues from the Complementarity Determining Regions (CDRs) of the recipient are replaced by residues from CDRs of a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired specificity, affinity and capacity. In some cases, the Fv framework residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies may comprise residues that are present in neither the recipient antibody nor the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which most, if not all, of the CDR regions correspond to those of a non-human Ig and most, if all, of the FR regions are those of a human antibody consensus sequence. Preferably, the humanized antibody further comprises at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al, 1986; presta, 1992; riechmann et al, 1988).
Human antibodies can also be produced using a variety of techniques, including phage display libraries (Hoogenboom et al, 1991; marks et al, 1991) and the preparation of human mAbs (Boerner et al, 1991; reisfeld and Sell, 1985). Similarly, the introduction of human Ig genes into transgenic animals in which endogenous antibody genes have been partially or fully inactivated can be used to synthesize human abs. After challenge, human antibody production was observed, which was highly similar in all respects to that observed in humans, including gene rearrangement, assembly and antibody repertoire (1997 a;1997b;1997c;1997d;1997; fishwild et al, 1996;1997; 2001;1996;1997; lonberg and Huszar,1995; lonberg et al, 1994; marks et al, 1992; 1997).
In particular, the cells of the invention can be targeted to tumors by administering antibodies that recognize tumor-associated antigens. One of ordinary skill in the art will appreciate that the g-NK cells of the present invention are suitable for use with a variety of antibodies that recognize tumor-associated antigens. Non-limiting examples of tumor-associated antigens include: CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40, CD52, CD56, CD70, CD74, CD140, epCAM, CEA, gpA, mesothelin, alpha-fetoprotein, mucin, PDGFR-alpha, TAG-72, CAIX, PSMA, folate binding protein, disperse factor receptor kinase, ganglioside, cytokeratin, frizzled receptor, VEGF, VEGFR, integrin αvβ3, integrin α5β1, EGFR, EGFL7, ERBB2 (HER 2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGLF2, EPHA3, FR-alpha, phosphatidylserine, adhesive proteoglycan 1, SLAMF7 (CD 319), TRAILR1, TRAILR2, RANKL, FAP, vimentin, or tenascin. In some cases, the antibody is an anti-CD 20 antibody (e.g., rituximab), an anti-HER 2 antibody (e.g., cetuximab), an anti-CD 52 antibody, an anti-EGFR antibody, and an anti-CD 38 antibody (e.g., up to Lei Tuoyou mab), an anti-SLAMF 7 antibody (e.g., erltuzumab).
Non-limiting antibodies that may be used in the methods provided in combination therapies with cell compositions comprising g-NK cells include trastuzumab @) Ramucirumab (/ >)) Alemtuzumab (TECENTRIQ TM), nivolumab (/ >)) Rivarox You Shan antibody (Imfinzi TM) and avermectin antibody) Pembrolizumab (/ >)) Bevacizumab (/ >)) Everolimus (/ >)) Pertuzumab (/ >)) Enmetrastuzumab (/ >)) Cetuximab (/ >)) Dinozumab @) Rituximab (/ >)) Alemtuzumab (/ >)) Offalo mab (/ >) Ababine You Tuozhu mab (/ >) Cetuximab (Portrazza TM), temozolomide (/ >)) Vibutuximab (/ >)) Setuximab (/ >)) Bortezomib (/ >)) The drug comprises the components of Tadalton (R) Lei Tuoyou monoclonal antibody (Darzalex TM), erltuzumab (EMPLICITI TM), denotuzumab (Unituxin TM), olympic monoclonal antibody (Lartruvo TM), oryzizumab, ai Satuo Ximab 、Truxima、Blitzima、Ritemvia、Rituzena、Herzuma、Ruxience、ABP 798、Kanjinti、Ogivry、BI 695500、Novex(RTXM83)、 toximod or Ontruzant, or analogues thereof. Exemplary antibodies include rituximab, trastuzumab, aletuzumab, cetuximab, up to Lei Tuoyou mab, valtuzumab, up to Lei Tuoyou mab, ubbelo-tuximab, oxcarbazeb or erltuzumab.
In some embodiments, the antibody may be an anti-PD-1 or anti-PD-L1 antibody. Antibodies targeting PD-1 or PD-L1 include, but are not limited to, nivolumab, pembrolizumab, or atumumab.
Antibodies specific for the selected cancer type may be selected and include any antibody approved for the treatment of cancer. Examples include trastuzumab (Herceptin) for breast cancer, rituximab for lymphoma @ for) And cetuximab (Erbitux) for head and neck squamous cell carcinoma. Those skilled in the art are familiar with FDA-approved monoclonal antibodies capable of binding to a particular tumor or disease antigen, any of which may be used in accordance with the provided methods of treating a tumor or disease.
In some embodiments, the methods are used to treat adenocarcinoma of the stomach or gastroesophageal junction, and the antibody is trastuzumab @) Or ramucirumab (/ >))。
In some embodiments, the methods are used to treat bladder cancer, and the antibody is alemtuzumab (TECENTRIQ TM), nivolumab @) Rivarox You Shan antibody (Imfinzi TM) and avermectin antibody) Or pembrolizumab (/ >))。
In some embodiments, the methods are used to treat brain cancer and the antibody is bevacizumab @)。
In some embodiments, the methods are used to treat breast cancer and the antibody is trastuzumab @)。
In some embodiments, these methods are used to treat cervical cancer and the antibody is bevacizumab @)。
In some embodiments, these methods are used to treat colorectal cancer and the antibody is cetuximab @) Panitumumab (/ >)) Bevacizumab (/ >)) Or ramucirumab (/ >))。
In some embodiments, these methods are used to treat endocrine/neuroendocrine tumors, and the antibody is avermectin @)。
In some embodiments, these methods are used to treat head and neck cancer, and the antibody is cetuximab @) Pembrolizumab (/ >)) Nawuzumab (/ >)) Trastuzumab or ramucirumab.
In some embodiments, these methods are used to treat bone cancer, and the antibody is denomab)。
In some embodiments, these methods are used to treat kidney cancer and the antibody is bevacizumab @) Or Nawuzumab (/ >))。
In some embodiments, these methods are used to treat leukemia, and the antibody is rituximab @) Alemtuzumab (/ >)) Offalo mab (/ >) Ababine You Tuozhu mab (/ >) Or Bonauzumab (/ >))。
In some embodiments, the methods are used to treat lung cancer and the antibody is bevacizumab @) Ramucirumab (/ >)) Nawuzumab (/ >)) Xitumumab (Portrazza TM), pembrolizumab @) Or alemtuzumab (TECENTRIQ TM).
In some embodiments, these methods are used to treat lymphomas and the antibody is temozolomide @) Vibutuximab (/ >)) Rituximab (/ >)) Setuximab (/ >)) Ababine You Tuozhu mab (/ >) Nawuzumab (/ >)) Or pembrolizumab (/ >))。/>
In some embodiments, the methods are used to treat multiple myeloma and the antibody is bortezomib @) Either up Lei Tuoyou mab (Darzalex TM) or erlotinib (EMPLICITI TM).
In some embodiments, the methods are for treating neuroblastoma, and the antibody is denotuzumab (Unituxin TM).
In some embodiments, these methods are used to treat ovarian epithelial/fallopian tube/primary peritoneal cancer, and the antibody is bevacizumab @)。
In some embodiments, the methods are used to treat pancreatic cancer and the antibody is cetuximab @) Or bevacizumab (/ >))。
In some embodiments, the method is for treating skin cancer and the antibody is ipilimumab @) Pembrolizumab (/ >)) Avermectin (/ >)) Or Nawuzumab (/ >))。
In some embodiments, the method is for treating soft tissue sarcoma, and the antibody is olamumab (Lartruvo TM).
In some embodiments, a population of g-NK cells described herein and an effective dose of a bispecific antibody are administered to a subject. In some embodiments, the bispecific antibody comprises a first binding domain that specifically binds to a surface antigen on an immune cell (e.g., NK cell or macrophage) and a second binding domain. In some embodiments, the first binding domain specifically binds to an activating receptor, such as CD16 (CD 16 a), on NK cells or macrophages. In some embodiments, the second binding domain specifically binds a tumor-associated antigen. The tumor-associated antigen of the target may be selected based on the type of cancer, including, but not limited to, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40, CD52, CD56, CD70, CD74, CD140, epCAM, CEA, gpA33, mesothelin, alpha-fetoprotein, mucin, PDGFR-alpha, TAG-72, CAIX, PSMA, folate binding protein, disperse factor receptor kinase, ganglioside, cytokeratin, frizzled receptor, VEGF, VEGFR, integrin αvβ3, integrin α5β1, EGFR, EGFL7, ERBB2 (HER 2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGLF2, EPHA3, FR-alpha, phosphatidylserine, adhesive proteoglycan 1, SLAMF7 (CD 319), TRAILR, TRAILR, kl, FAP, vimentin, or tenascin. In some embodiments, the first binding domain specifically binds CD16 and the second binding domain specifically binds CD30.
The engineered g-NK cells and the additional agent can be administered sequentially or simultaneously. In some embodiments, additional agents may be administered prior to administration of g-NK cells. In some embodiments, additional agents may be administered after the administration of the engineered g-NK cells. For example, the engineered g-NK cells can be administered concurrently with antibodies specific for the selected cancer type. Alternatively, the engineered g-NK cells may be administered at a selected time different from the time at which the antibody specific for the selected cancer type is administered.
In specific examples, an effective dose of the antibody is administered to the subject before, after, or substantially simultaneously with the population containing the engineered g-NK cells. In some examples, about 0.1mg/kg to about 100mg/kg of antibody (such as about 0.5mg/kg to about 10mg/kg, about 1mg/kg to about 20mg/kg, about 10mg/kg to about 50mg/kg, about 20mg/kg to about 100mg/kg, e.g., about 0.5mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 8mg/kg, about 10mg/kg, about 16mg/kg, about 20mg/kg, about 24mg/kg, about 36mg/kg, about 48mg/kg, about 60mg/kg, about 75mg/kg, or about 100 mg/kg) is administered to the subject. An effective amount of the antibody may be selected by a skilled clinician in view of the particular antibody, the particular disease or disorder (e.g., tumor or other disorder), the general disorder of the subject, any additional treatment the subject is receiving or has previously received, and other relevant factors. A population comprising engineered g-NK cells described herein is also administered to a subject. Both antibodies and engineered g-NK cell populations are typically administered parenterally (e.g., intravenously); however, injection or infusion to a tumor or near tumor (local administration) or administration to the abdominal cavity may also be used. The skilled person can determine a suitable route of administration.
In some embodiments, an effective dose of one or more antibodies to a virus and a g-NK cell population described herein is administered to a subject for treatment of the virus. In some embodiments, the one or more antibodies are antibodies that bind to a spike glycoprotein, such as the spike glycoprotein of SARS-Cov-2. In some embodiments, a population comprising engineered g-NK cells described herein is administered to a subject along with an effective dose of an Fc fusion protein, e.g., a recombinant ACE2-Fc fusion protein. In some embodiments, a population of g-NK cells described herein and an antibody-containing serum against the virus, e.g., an antibody against SARS-Cov-2, are administered to a subject. In some embodiments, the serum is convalescence serum collected from a patient recovering from an infection caused by the same virus. In some embodiments, convalescence serum from multiple patients recovered from infection by the same virus is collected, combined and administered to a subject in need thereof along with a population containing engineered g-NK cells described herein.
2. Cytokines and growth factors
In some embodiments provided herein, an engineered g-NK cell or composition containing the same may be administered to an individual in combination with a cytokine and/or a growth factor. In some embodiments provided herein, the engineered g-NK cells or compositions containing the same can be administered to an individual in combination with additional exogenously administered cytokines and/or growth factors. Because cytokines are required for NK cell activity, typical methods involve administering exogenous cytokines as exogenous cytokine supports to a subject in combination with NK cell therapy.
According to some embodiments, the at least one growth factor or cytokine comprises a growth factor selected from the group consisting of SCF, FLT3, IL-2, IL-7, IL-15, IL-12, IL-21 and IL-27.
In some embodiments, at least one cytokine is administered to the subject in combination with administration of engineered g-NK cells or a composition thereof.
Cytokines are a large class of proteins that play an important role in cell signaling, particularly in the immune system environment. Cytokines have been shown to play a role as immunomodulators in autocrine, paracrine and endocrine signaling. Cytokines may act as immune activators, thereby stimulating an immune-mediated response, or as immunosuppressants, thereby attenuating an immune-mediated response. Cytokines include chemokines, interferons, interleukins, lymphokines and tumor necrosis factors, but generally do not include hormones or growth factors.
In some embodiments, the cytokine is an interleukin. Interleukins are a group of cytokines, typically secreted proteins and signaling molecules that mediate a broad immune response. For example, interleukin (IL) -2 plays a role in regulating the activity of leukocytes, whereas Interleukin (IL) -15 plays a major role in the development of inflammatory and protective immune responses to microbial invaders and parasites by regulating the cellular activities of the innate and adaptive immune systems. In some embodiments, NK cells (including the provided g-NK cells) activity or activities by IL-2, IL-21 and/or IL-15 or another cytokine described.
In some embodiments, the interleukins include cytokines produced by immune cells such as lymphocytes, monocytes or macrophages. In some embodiments, the cytokine is an immunocompetent cytokine that can be used to induce NK cells, such as to promote NK cell survival, activation, and/or proliferation. For example, certain cytokines (such as IL-15 or IL-21) can prevent or reduce NK cells from undergoing senescence, such as by increasing their ability to expand in vitro or in vivo. In some embodiments, the interleukin or functional portion thereof is a partial peptide or an intact peptide of one or more of IL-2, IL-4, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-15, IL-18, or IL-21. In some embodiments, the cytokine is IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, flt3-L, SCF or IL-7. In some embodiments, the cytokine is IL-2. In some embodiments, the cytokine is IL-12. In some embodiments, the cytokine is IL-15. In some embodiments, the cytokine is IL-21. In some embodiments, the cytokine may be administered with the corresponding receptor for the cytokine. In some embodiments, the step of administering the cytokine with the engineered g-NK cell allows cytokine signaling, thereby maintaining or improving the cell growth, proliferation, expansion, and/or effector function of the NK cell.
In specific embodiments, recombinant IL-2 is administered to a subject. In other specific embodiments, recombinant IL-15 is administered to a subject. In other specific embodiments, recombinant IL-21 is administered to a subject.
In some embodiments, the cytokine is IL-15 or a functional part thereof. IL-15 is a cytokine that regulates NK cell activation and proliferation. In some cases, IL-15 and IL-12 share similar biological activity. For example, IL-15 and IL-2 bind to a common receptor subunit and can compete for the same receptor. In some embodiments, IL-15 induces activation of JAK kinase and phosphorylation and activation of transcriptional activators STAT3, STAT5, and STAT 6. In some embodiments, IL-15 promotes or modulates one or more functional activities of NK cells, such as promoting NK cell survival, modulating activation and proliferation of NK cells and T cells, and supporting NK cell development from hematopoietic stem cells. In some embodiments, the functional moiety is a portion (e.g., comprising a truncated contiguous amino acid sequence of full-length IL-15) of IL-15 that retains one or more functions of full-length or mature IL-15 (such as promoting NK cell survival, modulating NK cell and T cell activation and proliferation, and supporting NK cell development from hematopoietic stem cells). All or a functional portion of IL-15 may be administered to a subject.
As will be appreciated by those skilled in the art, the sequences of a variety of IL-15 molecules are known in the art. In one aspect, IL-15 is wild-type IL-15. In some aspects, IL-15 is mammalian IL-15 (e.g., homo sapiens interleukin 15 (IL 15), transcriptional variant 3, mRNA, NCBI reference sequence: NM_000585.4, domestic dog interleukin 15 (IL 15), mRNA, NCBI reference sequence: NM_001197188.1, domestic cat interleukin 15 (IL 15), mRNA, NCBI reference sequence: NM_ 001009207.1). Examples of "mammals" or "mammals" include primates (e.g., humans), canines, felines, rodents, pigs, ruminants, and the like. Specific examples include humans, dogs, cats, horses, cattle, sheep, goats, rabbits, guinea pigs, rats and mice. In a specific aspect, mammalian IL-15 is human IL-15. Human IL-15 amino acid sequences include, for example, genbank accession number ：NR_751915.1、NP_000576.l、AAI00963.1、AAI00964.1、AAI00962.1、CAA71044.1、AAH18149.1、AAB97518.1、CAA63914.1 and CAA63913.1.
In some embodiments, the IL-15 nucleotide sequence is shown in SEQ ID NO. 9, or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least about 98% sequence identity to SEQ ID NO. 9. In some embodiments, IL-15 is a mature form lacking the signal peptide sequence and in some cases also lacking the propeptide sequence. In some embodiments, IL-15 has the amino acid sequence shown in SEQ ID NO. 2 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least about 98% sequence identity to SEQ ID NO. 2.
In some embodiments, the IL-15 molecules are variants of human IL-5, e.g., having one or more amino acid changes, e.g., substitutions, to the amino acid sequence of human IL-15. In some embodiments, the IL-15 variant comprises or consists of a mutation at position 45, 51, 52 or 72, e.g., as described in US 2016/0184399. In some embodiments, the IL-15 variant comprises, or consists of, a substitution of N, S or one of L pair D, E, A, Y or P. In some embodiments, the mutation is selected from L45D, L45E, S51D, L D, N72D, N72E, N72A, N72S, N Y or N72P (with respect to the sequence of human IL-15, SEQ ID NO: 2).
In embodiments, IL-15 molecules include IL-15 variants, such as human IL-15 polypeptides having one or more amino acid substitutions. In some embodiments, the IL-15 molecule includes a substitution at position 72, e.g., a substitution of N for D. In one embodiment, the IL-15 molecule is the IL-15N72D polypeptide of SEQ ID NO. 2 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto (which has IL-15Ra binding activity).
In some embodiments, IL-15 is administered with IL-15 receptor alpha (IL 15 RA), such as in a complex therewith or as a fusion therewith. IL15RA specifically binds IL-15 with very high affinity and is able to bind IL1-5 independently of other subunits. In some aspects, this property allows IL-15 to be produced by one cell, endocytosed by another cell, and then presented to a third cell. In some embodiments, IL-15/IL-15Ra is administered to a subject. In some embodiments, the IL-15/IL-15R fusion protein is administered to a subject. In some embodiments, a single chain IL-15/IL-15R fusion protein is administered to a subject. In some embodiments, the IL-15/IL-15Ra is a soluble il15ra.il15 complex (e.g., mortier E et al, JBC, 2006; bessard A, mol. Cancer ter., 2009; and Desbois M, j. Immunol., 2016).
In some embodiments, the cytokine is IL-2 or a functional part thereof. In some embodiments, IL-2 is a member of a cytokine family, which also includes IL-4, IL-7, IL-9, IL-15 and IL-21.IL-2 is signal-transduced by a receptor complex consisting of three chains (called α, β and γ). All members of this cytokine receptor family share a gamma chain. Like IL-15, IL-2 promotes B-cell immunoglobulin production and induces NK cell differentiation and proliferation. Major differences between IL-2 and IL-15 were found in the adaptive immune response. For example, IL-2 is necessary for adaptive immunity to foreign pathogens, as it is the basis for the development of immunological memory. On the other hand, IL-15 is necessary to maintain a highly specific T cell response by supporting the survival of CD8 memory T cells. All or a functional portion of IL-2 may be expressed as a membrane-bound polypeptide and/or a secreted polypeptide. As will be appreciated by those skilled in the art, the sequences of a variety of IL-2 molecules are known in the art. In one aspect, IL-2 is wild-type IL-2. In some aspects, IL-2 is mammalian IL-2. In some embodiments, IL-2 is human IL-2.
In some embodiments, IL-2 is a mature form lacking the signal peptide sequence and in some cases also lacking the propeptide sequence. In some embodiments, IL-2 has the amino acid sequence set forth in SEQ ID NO. 1 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least about 98% sequence identity to SEQ ID NO. 1.
In some embodiments, the cytokine is IL-21 or a functional part thereof. IL-21 binds to the IL-21 receptor (IL-21R) and to the co-receptor (common gamma chain (CD 132)). IL-21 receptors have been identified on NK cells, T cells and B cells, indicating that IL-21 acts on cells of the hematopoietic lineage, in particular on lymphoprogenitors and lymphocytes. IL-21 has been shown to be a potent modulator of cytotoxic T cells and NK cells. (Parrish-Novak et al Nature, volume 408: pages 57-63, 2000; parrish-Novak et al J.Leuk.Bio., volume 72: pages 856-863, 202; collins et al Immunol.Res., volume 28: pages 131-140, 2003; brady et al J.Immunol., volume 172: pages 2048-2058, 2004). In murine studies, IL-21 enhanced NK cell maturation and effector function (Kasaian et al, immunity, 16:559-569, 2002).
As will be appreciated by those skilled in the art, the sequences of a variety of IL-21 molecules are known in the art. In one aspect, IL-21 is wild-type IL-21. In some aspects, IL-21 is mammalian IL-21. In one embodiment, the IL-21 sequence is a human IL-21 sequence. Human IL-21 amino acid sequences include, for example, genbank accession number ：AAU88182.1、EAX05226.1、CAI94500.1、CAJ47524.1、CAL81203.1、CAN87399.1、CAS03522.1、CAV33288.1、CBE74752.1、CBI70418.1、CBI85469.1、CBI85472.1、CBL93962.1、CCA63962.1、AAG29348.1、AAH66258.1、AAH66259.1、AAH66260.1、AAH66261.1、AAH66262.1、AAH69124.1 and ABG36529.1.
In some embodiments, IL-21 is a mature form lacking the signal peptide sequence and in some cases also lacking the propeptide sequence. In some embodiments, IL-21 has the amino acid sequence shown in SEQ ID NO. 3 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least about 98% sequence identity to SEQ ID NO. 3. In some embodiments, IL-21 has the amino acid sequence shown in SEQ ID NO. 4 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least about 98% sequence identity to SEQ ID NO. 4.
The cytokine (e.g., IL-2, IL-15, or IL-21) amino acid sequence can include any functional portion of a mature cytokine, such as mature IL-2, mature IL-15, or any functional portion of mature IL-15. The functional moiety may be any moiety comprising consecutive amino acids of the interleukin to which it belongs, provided that the functional moiety specifically binds to the corresponding interleukin receptor. When used in reference to an interleukin, the term "functional moiety" refers to any portion or fragment of an interleukin that retains the biological activity of the interleukin to which it belongs (the parent interleukin). Functional moieties encompass, for example, interleukin moieties that retain the ability to specifically bind to a corresponding interleukin receptor, activate a downstream target of an interleukin, and/or induce one or more of differentiation, proliferation (or death) and activity of immune cells (e.g., NK cells), to the same extent as, or higher than, the parent interleukin. The biological activity of the functional portion of the interleukin may be measured using assays known in the art. With respect to the parent interleukin, the functional portion may include, for example, an amino acid sequence of about 60%, about 70%, about 80%, about 90%, about 95% or more of the parent mature interleukin.
Functional variants of the interleukins described herein are included within the scope of cytokines or functional moieties according to the provided embodiments. As used herein, the term "functional variant" refers to an interleukin that has substantial or significant sequence identity or similarity to a parent interleukin, which functional variant retains its original interleukin biological activity. Functional variants encompass, for example, those variants of an interleukin (parent interleukin) described herein that retain the ability to specifically bind to a corresponding interleukin receptor, activate a downstream target of the interleukin, and/or induce one or more of differentiation, proliferation (or death) and activity of immune cells (e.g., NK cells), to a similar, the same or higher degree than the parent interleukin. With respect to a parent interleukin, a functional variant may be, for example, at least about 80%, about 90%, about 95%, about 99% or more identical in amino acid sequence to the parent interleukin.
Functional variants may, for example, comprise the amino acid sequence of a parent interleukin with at least one conservative amino acid substitution. Alternatively or additionally, the functional variant may comprise an amino acid sequence of a parent interleukin having at least one non-conservative amino acid substitution. In some embodiments, amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions) do not interfere with or inhibit the biological activity of the functional variant as compared to the parent interleukin sequence. In some embodiments, amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions) may enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased compared to the parent interleukin.
In some embodiments, the amino acid substitution of an interleukin is a conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions may be: substitution of another acidic/negatively charged polar amino acid with an acidic/negatively charged polar amino acid (e.g., asp or glu), substitution of another amino acid with a nonpolar side chain with an amino acid with a nonpolar side chain (e.g., ala, gly, val, ile, leu, met, phe, pro, trp, cys, val, etc.), substitution of another basic/positively charged polar amino acid with a basic/positively charged polar amino acid (e.g., lys, his, arg, etc.), substitution of another uncharged amino acid with a polar side chain with an uncharged amino acid with a polar side chain (e.g., asn, gin, ser, thr, tyr, etc.), substitution of another amino acid with a beta-branched side chain with an amino acid with a beta-branched side chain (e.g., lie, thr, and val), substitution of another amino acid with an aromatic side chain with an amino acid with an aromatic side chain (e.g., his, phe, trp and tyr), etc.
In some embodiments, a dose of the provided engineered g-NK cell composition and the cytokine or growth factor are administered sequentially. For example, g-NK cells may be administered first, followed by cytokines and/or growth factors. In some embodiments, a dose of cells containing engineered g-NK cells is administered concurrently with cytokines or growth factors.
In some embodiments, one or more cytokines (such as IL-2, IL-15, IL-21, IL-27 and/or IL-12) are administered to a subject to support the survival and/or growth of NK cells. Cytokines may be administered before, after, or substantially simultaneously with NK cells. In some examples, the cytokine may be administered after the NK cells. In a specific example, the cytokine is administered to the subject within about 1 hour to 8 hours of administration of the NK cells (such as within about 1 hour to 4 hours, within about 2 hours to 6 hours, within about 4 hours to 6 hours, or within about 5 hours to 8 hours).
3. Cytotoxic agents for lymphocyte removal therapy
In some embodiments, the provided methods can further comprise administering a dose of cells containing engineered g-NK cells with another treatment, such as with a chemotherapeutic or cytotoxic agent or other treatment.
In some aspects, provided methods can further comprise administering one or more lymphocyte removal therapies, such as prior to or concurrent with the beginning of administration of the g-NK cell composition comprising engineered g-NK cells. In some embodiments, the lymphoscavenging therapy comprises administration of a phosphoramide, such as cyclophosphamide. In some embodiments, the lymphoscavenging therapy may include administration of fludarabine.
In some aspects, pre-treating a subject with an immune depleting (e.g., lymphocyte depleting) therapy can improve the efficacy of Adoptive Cell Therapy (ACT). In some embodiments, the lymphoscavenging therapy comprises a combination of cyclosporine and fludarabine.
Such pretreatment may be targeted to reduce the risk of one or more of the various outcomes that may inhibit the efficacy of the therapy. These phenomena include what are known as "cytokine pooling" by which T cells, B cells, NK cells compete with TIL for homeostasis and activate cytokines such as IL-2, IL-7 and/or IL-15; modulating T cells, NK cells, or other cells of the immune system to inhibit TIL; negative regulators in the tumor microenvironment. Muranski et al, NAT CLIN PRACT oncol, 12 months; volume 3, phase 12: pages 668-681, 2006.
Thus, in some embodiments, the provided methods further involve administering to the subject a lymphoproliferative therapy. In some embodiments, the method comprises administering lymphocyte removal therapy to the subject prior to administering the dose of cells. In some embodiments, the lymphoscavenging therapy comprises a chemotherapeutic agent, such as fludarabine and/or cyclophosphamide. In some embodiments, administration of the cell and/or lymph clearing therapy is via outpatient delivery.
In some embodiments, the methods comprise administering a pretreatment agent, such as a lymphocyte depleting agent or a chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, to the subject prior to administering the dose of cells. For example, a pretreatment agent, such as a lymphocyte depleting agent or a chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, may be administered to the subject at least 2 days prior to the first or subsequent dose, such as at least 3,4,5, 6, or 7 days prior. In some embodiments, a pretreatment agent, such as a lymphocyte depleting agent or a chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, is administered to the subject no more than 7 days, such as no more than 6, 5, 4, 3, or 2 days prior to administration of the dose of cells. In some embodiments, a pretreatment agent, such as a lymphocyte depleting agent or a chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, is administered to the subject no more than 14 days, such as no more than 13, 12, 11, 10, 9, or 8 days before administration of the dose of cells.
In some embodiments, the subject is pretreated with a dose of cyclophosphamide between 20mg/kg and 100mg/kg or between about 20mg/kg and about 100mg/kg, such as between 40mg/kg and 80mg/kg or between about 40mg/kg and about 80 mg/kg. In some aspects, the subject is pretreated with or with about 60mg/kg cyclophosphamide. In some embodiments, fludarabine may be administered in a single dose or may be administered in multiple doses, such as daily, every other day, or every third day. In some embodiments, cyclophosphamide is administered once daily for one or two days.
In some embodiments, when the lymphatic depleting agent comprises fludarabine, between 1mg/m 2 and 100mg/m 2 or between about 1mg/m 2 and about 100mg/m 2, such as between the following doses or between about the following doses: the fludarabine is administered to the subject at a dose of 10mg/m 2 and 75mg/m 2、15mg/m2 and 50mg/m 2、20mg/m2 and 30mg/m 2, or 24mg/m 2 and 26mg/m 2. In some cases, 25mg/m 2 of fludarabine is administered to the subject. In some embodiments, fludarabine may be administered in a single dose or may be administered in multiple doses, such as daily, every other day, or every third day. In some embodiments, fludarabine is administered daily, such as for 1 to 5 days, for example for 3 to 5 days.
In some embodiments, the lymphatic depleting agent comprises a combination of agents, such as a combination of cyclophosphamide and fludarabine. Thus, combinations of agents may include cyclophosphamide at any dose or dosing regimen, such as those described above, and fludarabine at any dose or dosing regimen, such as those described above. For example, in some aspects, 60mg/kg (about 2g/m 2) of cyclophosphamide and 3 to 5 doses of 25mg/m 2 fludarabine are administered to the subject prior to administration of the dose of cells.
In some embodiments, the subject has received lymphocyte removal therapy prior to administration of the dose of g-NK cells. In some embodiments, the lymphoscavenging therapy comprises fludarabine and/or cyclophosphamide. In some embodiments, lymphodepletion comprises administering fludarabine at 20mg/m 2 to 40mg/m 2 or about 20mg/m 2 to about 40mg/m 2 of subject body surface area, optionally at or about 30mg/m 2, daily for 2 to 4 days, and/or administering cyclophosphamide at 200mg/m 2 to 400mg/m 2 or about 200mg/m 2 to about 400mg/m 2 of subject body surface area, optionally at or about 300mg/m 2, daily for 2 to 4 days.
In some embodiments, the lymphoscavenging therapy comprises fludarabine and cyclophosphamide. In some embodiments, the lymphoscavenging therapy comprises administering fludarabine at 30mg/m 2 or about 30mg/m 2 of subject body surface area per day, and cyclophosphamide at 300mg/m 2 or about 300mg/m 2 of subject body surface area per day, each for 2 to 4 days, optionally for 3 days.
In some embodiments, administration of the pretreatment agent prior to infusion of the dose of cells improves the outcome of the treatment. For example, in some aspects, a pretreatment, such as a lymphocyte depleting agent or a chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, improves the efficacy of treatment with the dose or increases the persistence of NK cells in the subject. In some embodiments, the pretreatment treatment increases disease-free survival, such as the percentage of subjects that survive a given period of time after administration of the dose of cells and that do not exhibit minimal residual or molecularly detectable disease. In some embodiments, the time to reach median disease-free survival is increased.
Once the cells are administered to a subject (e.g., a human), the biological activity of the engineered cell population is measured in some aspects by any of a number of known methods. The evaluation parameters include specific binding of engineered or natural T cells or other immune cells to the antigen, either in vivo, e.g., by imaging, or in vitro, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of NK cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described, for example, in the following: kochenderfer et al, J.Immunothepy, volume 32, phase 7: pages 689-702, 2009; and Herman et al, j.immunological Methods, volume 285, phase 1: pages 25-40, 2004. In certain embodiments, the biological activity of the cells may also be measured by assaying the expression and/or secretion of certain cytokines or other effector molecules (such as CD107a, ifnγ, and TNF). In some aspects, biological activity is measured by assessing clinical outcome (such as a decrease in tumor burden or burden). In some aspects, the toxicity results, the duration and/or expansion of cells, and/or the presence or absence of a host immune response are assessed.
Exemplary embodiments
The provided embodiments include:
1. An engineered Natural Killer (NK) cell (g-NK cell) deficient in FcR gamma chain expression, and the NK cell comprising a heterologous nucleic acid encoding a Chimeric Antigen Receptor (CAR).
2. The engineered NK cell of embodiment 1, wherein said CAR comprises 1) an antigen binding domain; 2) A flexible connector; 3) A transmembrane region; and 4) an intracellular signaling domain.
3. The engineered NK cell of embodiment 2, wherein said antigen binding domain targets a tumor antigen.
4. The engineered NK cell of embodiment 2 or 3, wherein said antigen binding domain is a single chain variable fragment (scFv).
5. The engineered NK cell of any one of embodiments 2-4, wherein the intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain.
6. The engineered NK cell of any one of embodiments 2-5, wherein the intracellular signaling domain comprises one or more signaling domains of cd3ζ, DAP10, DAP12, CD28, 4-1BB, or OX 40.
7. The engineered NK cell of any one of embodiments 2 to 5, wherein said intracellular signaling domain comprises two or more signaling domains of cd3ζ, DAP10, DAP12, CD28, 4-1BB, or OX 40.
8. The engineered NK cell of any one of embodiments 1-7, wherein the heterologous nucleic acid encoding the CAR is stably integrated into the genome of the cell.
9. The engineered NK cell of any one of embodiments 1-7, wherein the heterologous nucleic acid encoding the CAR is transiently expressed.
10. The engineered NK cell of any one of embodiments 1to 9, wherein said g-NK cell has the surface phenotype of CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of .
11. The engineered NK cell of any one of embodiments 1 to 10, wherein said g-NK cell further has the surface phenotype of NKG2a Negative of /CD161 Negative of .
12. The engineered NK cell of any one of embodiments 1 to 11, wherein said g-NK cell is also CD38 Negative of .
13. The engineered NK cell of any one of embodiments 1 to 12, wherein said g-NK cell further has the surface phenotype of CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative .
14. The engineered NK cell of any one of embodiments 1 to 13, wherein said g-NK cell comprises CD 16V/V (V158)
15. The engineered NK cell of any one of embodiments 1 to 13, wherein said g-NK cell is CD16 158V/F.
16. A composition comprising a plurality of engineered g-NK cells according to any one of embodiments 1 to 15.
17. The composition of embodiment 16, wherein greater than 50% or greater than about 50% of the NK cells or total cells in the composition are g-NK cells.
18. The composition of embodiment 16, wherein greater than 60% or greater than about 60% of the NK cells or total cells in the composition are g-NK cells.
19. The composition of embodiment 16, wherein greater than 70% or greater than about 70% of the NK cells or total cells in the composition are g-NK cells.
20. The composition of embodiment 16, wherein greater than 80% or greater than about 80% of the NK cells or total cells in the composition are g-NK cells.
21. The composition of embodiment 16, wherein greater than 90% or greater than about 90% of the NK cells or total cells in the composition are g-NK cells.
22. The composition of embodiment 16, wherein greater than 95% or greater than about 95% of the NK cells or total cells in the composition are g-NK cells.
23. The composition of any one of embodiments 16-22, wherein the plurality of engineered g-NK cells comprises greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, or greater than 70% or greater than about 70% g-NK cells comprising a heterologous nucleic acid encoding the CAR.
24. The composition of any one of embodiments 16-23, wherein the total composition comprises greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, or greater than 70% or greater than about 70% g-NK cells comprising a heterologous nucleic acid encoding the CAR.
25. The composition of any one of embodiments 16 to 24, wherein greater than 70% or greater than about 70% of the g-NK cells are positive for perforin and greater than 70% or greater than about 70% of the g-NK cells are positive for granzyme B.
26. The composition of any one of embodiments 16 to 25, wherein greater than 80% or greater than about 80% of the g-NK cells are positive for perforin and greater than 80% or greater than about 80% of the g-NK cells are positive for granzyme B.
27. The composition of any one of embodiments 16 to 26, wherein greater than 90% or greater than about 90% of the g-NK cells are positive for perforin and greater than 90% or greater than about 90% of the g-NK cells are positive for granzyme B.
28. The composition of any one of embodiments 16-27, wherein greater than 95% or greater than about 95% of the g-NK cells are positive for perforin and greater than 95% or greater than about 95% of the g-NK cells are positive for granzyme B.
29. The composition according to any one of embodiments 25 to 28, wherein:
In the cells positive for perforin, the cells express perforin at an average level of at least twice or at least about twice that of FcR gamma Positive and negative based on Mean Fluorescence Intensity (MFI) as measured by intracellular flow cytometry; and/or
In the cells positive for granzyme B, the cells express granzyme B at an average level of at least twice or at least about twice as high as that of FcR gamma Positive and negative based on average fluorescence intensity (MFI) as measured by intracellular flow cytometry.
30. The composition of any one of embodiments 16 to 29, wherein optionally greater than 10% of the cells in the composition are capable of degranulation against tumor target cells as measured by CD107a expression, optionally wherein the degranulation is measured in the absence of antibodies against the tumor target cells.
31. The composition of any one of embodiments 16-30, wherein greater than 10% of the cells in the composition are further capable of producing interferon-gamma or TNF-alpha to a tumor target cell, optionally wherein the interferon-gamma or TNF-alpha is measured in the absence of antibodies to the tumor target cell.
32. The composition of any one of embodiments 16 to 31, wherein in the cells in the composition, greater than 15% or greater than about 15%, greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, or greater than 50% or greater than about 50% of the cells expressing a target antigen (target cells) and antibodies to the target antigen (anti-target antibodies) produce an effector cytokine.
33. The composition of any one of embodiments 16 to 32, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 30% or greater than about 30% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 50% or greater than about 50% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
34. The composition of any one of embodiments 16 to 33, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 35% or greater than about 35% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 60% or greater than about 60% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
35. The composition of any one of embodiments 16 to 34, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 40% or greater than about 40% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 70% or greater than about 70% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
36. The composition of any one of embodiments 16 to 34, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 45% or greater than about 45% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 80% or greater than about 80% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
37. The composition of any one of embodiments 16 to 34, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 50% or greater than about 50% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 85% or greater than about 85% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
38. The composition of any one of embodiments 16 to 34, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 55% or greater than about 55% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 90% or greater than about 90% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
39. The composition of any one of embodiments 16 to 34, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 60% or greater than about 60% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 95% or greater than about 95% of the cells are negative or low level for NKG2A (NKG 2A Negative of ).
40. The composition of any one of embodiments 16 to 39, wherein greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, or greater than 90% or greater than about 90% of the cells in the total cells in the composition or the g-NK cells in the composition are CD38 Negative of .
41. The composition of any one of embodiments 16 to 39, wherein greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, or greater than about 90% of the cells in the total cells in the composition or the g-NK cells in the composition are CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of .
42. The composition of any one of embodiments 16 to 39, wherein greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, or greater than 90% or greater than about 90% of the cells in the total cells in the composition or the g-NK cells in the composition are NKG2a Negative of /CD161 Negative of .
43. The composition of any one of embodiments 16 to 42, wherein the plurality of g-NK cells is CD16 158V/V (V158).
44. The composition of any one of embodiments 16 to 42, wherein the plurality of g-NK cells is CD16 158V/F.
45. The composition of any one of embodiments 16 to 44, wherein the composition comprises at least or at least about 10 8 cells.
46. The composition according to any one of embodiments 16 to 45, wherein the number of g-NK cells in the composition is from 10 8 to 10 12 cells or from about 10 8 to about 10 12 cells, from 10 8 to 10 11 cells or from about 10 8 to about 10 11 cells, from 10 8 to 10 10 cells or from about 10 8 to about 10 10 cells, from 10 8 to 10 9 cells or from about 10 8 to about 10 9 cells, from 10 9 cells to 10 12 cells or from about 10 9 cells to about 10 12 cells, from 10 12 to 10 12 cells or from about 10 12 to about 10 cells, from about 10 12 to about 12 cells or from 10 12 to 12 cells or from 10 to 12 cells.
47. The composition of any one of embodiments 16 to 46, wherein the number of g-NK cells in the composition is or is about 5 x 10 8 cells, or is about 1 x 10 9 cells, or is about 5 x 10 10 cells, or is about 1 x 10 10 cells.
48. The composition of any of embodiments 16-47, wherein the volume of the composition is between 50mL and 500mL or between about 50mL and about 500mL, optionally at or about 200mL.
49. The composition of any one of embodiments 16-48, wherein the cells in the composition are from a single donor subject, the cells having been expanded from the same biological sample.
50. The composition according to any one of embodiments 16 to 49, wherein the composition is a pharmaceutical composition.
51. The composition according to any one of embodiments 16 to 50, comprising a pharmaceutically acceptable excipient.
52. The composition of any one of embodiments 16 to 51, wherein the composition is formulated in a serum-free cryopreservation medium comprising a cryoprotectant.
53. The composition of embodiment 52, wherein the cryoprotectant is DMSO and the cryopreservation medium is 5% to 10% DMSO (v/v).
54. The composition of embodiment 53, wherein the cryoprotectant is or is about 10% DMSO (v/v).
55. The composition of any one of embodiments 16 to 54, which is sterile.
56. A sterile bag comprising the composition according to any one of embodiments 16 to 55.
57. The sterile pouch of embodiment 56, wherein the pouch is a cryopreservation compatible pouch.
58. A method of producing a genetically engineered g-NK cell, the method comprising introducing a heterologous nucleic acid encoding a Chimeric Antigen Receptor (CAR) into an FcR gamma chain expression deficient NK cell (g-NK cell).
59. The method of embodiment 58, wherein the CAR comprises 1) an antigen binding domain; 2) Flexible connectors (spacers); 3) A transmembrane region; and 4) an intracellular signaling domain.
60. The method of embodiment 59, wherein the antigen binding domain targets a tumor antigen.
61. The method of embodiment 59 or 60, wherein the antigen binding domain is a single chain variable fragment (scFv).
62. The method of any one of embodiments 59-61, wherein the intracellular signaling domain comprises one or more signaling domains from cd3ζ, DAP10, DAP12, CD28, 4-1BB, or OX 40.
63. The method of any one of embodiments 59 to 62, wherein the intracellular signaling domain comprises two or more signaling domains from CD28, 4-1BB, or OX 40.
64. The method of any one of embodiments 58 to 63, wherein the heterologous nucleic acid encoding the CAR is introduced under conditions that stably integrate into the genome of the g-NK cell.
65. The method of any one of embodiments 58 to 64, wherein the heterologous nucleic acid encoding the CAR is contained in a viral vector and introduced into the g-NK cell by transduction.
66. The method of embodiment 65, wherein the viral vector is a lentiviral vector.
67. The method of any one of embodiments 58 to 63, wherein the nucleic acid encoding the CAR is introduced under conditions of transient expression in the g-NK cells.
68. The method of any one of embodiments 58 to 63 and 67, wherein the nucleic acid encoding the CAR is introduced by non-viral delivery.
69. The method of any one of embodiments 58 to 63, 67, and 68, wherein said nucleic acid encoding said CAR is introduced into said g-NK cells via a lipid nanoparticle.
70. The method of any one of embodiments 58 to 69, wherein the nucleic acid encoding the CAR is DNA.
71. The method of any one of embodiments 58-63 and 67-79, wherein the nucleic acid encoding the CAR is RNA.
72. The method of embodiment 71, wherein the RNA is mRNA.
73. The method of embodiment 72, wherein the RNA is self-amplifying mRNA.
74. The method of any one of embodiments 58-63 and 67-73, wherein said nucleic acid is introduced into said g-NK cells via electroporation.
75. The method of any one of embodiments 58 to 74, wherein the g-NK cell composition is produced by in vitro expansion of NK cells enriched from a biological sample from a subject, the NK cells being: (i) Negative or low level for CD3 and positive for CD57 (CD 3 Negative of CD57 Positive and negative ) or (ii) negative or low level for CD3 and positive for CD56 (CD 3 Negative of CD56 Positive and negative ), wherein the enriched NK cells are cultured with irradiated HLA-e+ feeder cells and one or more recombinant cytokines.
76. The method of embodiment 75, wherein the one or more recombinant cytokines are selected from the group consisting of an effective amount of SCF, GSK3i, FLT3, IL-2, IL-6, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or a combination thereof.
77. The method of embodiment 75 or embodiment 76, wherein the culturing is performed in the presence of two or more recombinant cytokines, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21.
78. The method of any one of embodiments 58 to 77, wherein the introducing is performed during a method for expanding FcR γ -deficient NK (g-NK) cells, the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21;
Wherein introducing the nucleic acid encoding the CAR is performed after step (a) and before, during or after step (B), thereby producing an engineered NK cell population; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR.
79. The method of any one of embodiments 58 to 77, wherein the introducing is performed during a method for amplifying FcR γ -deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject;
(B) Introducing the nucleic acid encoding a CAR into the enriched NK cell population, thereby producing an engineered NK cell population; and
(C) Culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR.
80. The method of any one of embodiments 58 to 77, wherein the introducing is performed during a method for amplifying FcR γ -deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Culturing the enriched NK cell population in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and
(C) Introducing said nucleic acid encoding a CAR into NK cells of said NK cell expansion population,
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR.
81. The method of any one of embodiments 58 to 77, wherein the introducing is performed during a method for amplifying FcR γ -deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Performing a first expansion comprising culturing the enriched NK cell population in a medium under conditions to expand the NK cells to produce a first expanded population of NK cells;
(C) Introducing the nucleic acid encoding a CAR into NK cells of the first amplified population of NK cells, thereby producing an engineered NK cell population; and
(D) Performing a second expansion comprising culturing said population of engineered NK cells under conditions that further expand said NK cells,
Wherein the first expansion and/or second expansion comprises culturing the enriched NK cell population in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR.
82. The method according to any one of embodiments 75 to 81, wherein the population of NK cell enriched primary human cells is obtained by selecting cells from a biological sample from a human subject, the cells being: (i) Negative or low level for CD3 and positive for CD57 (CD 3 Negative of CD57 Positive and negative ) or (ii) negative or low level for CD3 and positive for CD56 (CD 3 Negative of CD56 Positive and negative ).
83. The method of embodiment 82, wherein:
The population enriched for NK cells is a cell obtained by further selecting cells positive for NKG2C (NKG 2C Positive and negative );
The population enriched for NK cells is a cell obtained by further selecting cells negative for NKG2A or low in level (NKG 2A Negative of ); or alternatively
The population enriched for NK cells is a cell obtained by further selecting cells positive for NKG2C and negative or low level for NKG2A (NKG 2C Positive and negative NKG2A Negative of ).
84. The method according to any one of embodiments 75 to 83, wherein the human subject is a subject: wherein at least or at least about 20% of Natural Killer (NK) cells in a peripheral blood sample from the subject are positive for NKG2C (NKG 2C Positive and negative ) and at least 70% of NK cells in the peripheral blood sample are negative for NKG2A or low level (NKG 2A Negative of ).
85. The method of any one of embodiments 75 to 84, wherein the subject is CMV seropositive.
86. The method of any one of embodiments 75 to 85, wherein the percentage of g-NK cells in the biological sample from the subject is greater than 5% or greater than about 5%, greater than 10% or greater than about 10%, or greater than 30% or greater than about 30%.
87. The method of any one of embodiments 75 to 86, wherein the percentage of g-NK cells in the enriched NK cell population is between 20% and 90%, or between about 20% and about 90%, or between 40% and 90%, or between about 40% and about 90%, or between 60% and 90%, or between about 60% and about 90%.
88. The method of any one of embodiments 75 to 87, wherein the population enriched for NK cells is a cell negative or low level for CD3 and positive for CD57 (CD 3 Negative of CD57 Positive and negative ) selected from the biological sample.
89. The method of any one of embodiments 75 to 87, wherein the population enriched for NK cells is a cell negative or low level for CD3 and positive for CD56 (CD 3 Negative of CD56 Positive and negative ) selected from the biological sample.
90. The method of any one of embodiments 77-89, wherein said two or more recombinant cytokines further comprise an effective amount of SCF, GSK3i, FLT3, IL-6, IL-7, IL-15, IL-12, IL-18, IL-27, or a combination thereof.
91. The method of any one of embodiments 75 to 89, wherein the recombinant cytokine is IL-21 and IL-2.
92. The method according to any one of embodiments 75 to 90, wherein the recombinant cytokine is IL-21, IL-2, and IL-15.
93. The method of any one of embodiments 75 to 92, wherein recombinant IL-21 is added to the culture medium at a concentration of 10ng/mL to 100ng/mL or about 10ng/mL to about 100ng/mL during at least a portion of the culturing.
94. The method of any one of embodiments 75 to 93, wherein recombinant IL-21 is added to the culture medium at a concentration of 25ng/mL or about 25ng/mL during at least a portion of the culturing.
95. The method of any one of embodiments 75 to 94, wherein recombinant IL-2 is added to the culture medium at a concentration of 10IU/mL to 500IU/mL or about 10IU/mL to about 500IU/mL during at least a portion of the culturing.
96. The method of any one of embodiments 75 to 95, wherein recombinant IL-2 is added to the culture medium at a concentration of 100IU/mL or about 100IU/mL during at least a portion of the culturing.
97. The method of any one of embodiments 75 to 96, wherein recombinant IL-2 is added to the culture medium at a concentration of 500IU/mL or about 500IU/mL during at least a portion of the culturing.
98. The method of any one of embodiments 75 to 90 and 92 to 97, wherein recombinant IL-15 is added to the culture medium at a concentration of 1ng/mL to 50ng/mL or about 1ng/mL to about 50ng/mL during at least a portion of the culturing.
99. The method of any one of embodiments 75 to 90 and 92 to 97, wherein recombinant IL-15 is added to the culture medium at a concentration of 10ng/mL or about 10ng/mL during at least a portion of the culturing.
100. The method according to any one of embodiments 75 to 99, wherein the addition of the recombinant cytokine to the culture medium is initiated at or about the beginning of the culturing.
101. The method according to any one of embodiments 75 to 100, wherein the method further comprises replacing the medium one or more times during the culturing, wherein fresh medium containing the recombinant cytokine is added each time the medium is replaced.
102. The method of embodiment 101, wherein the replacement of the medium is performed every two or three days during the duration of the culturing.
103. The method of embodiment 101 or embodiment 102, wherein the medium change is performed up to 5 days after the first amplification without a medium change, optionally up to 5 days after the first amplification without a medium change.
104. The method according to any one of embodiments 75 to 103, wherein the human subject has a CD16 158V/V NK cell genotype or a CD16 158V/F NK cell genotype, optionally wherein the biological sample is from a human subject selected for the CD16 158V/V NK cell genotype or the CD16 158V/F NK cell genotype.
105. The method according to any one of embodiments 75 to 104, wherein the biological sample is or comprises Peripheral Blood Mononuclear Cells (PBMCs), optionally a blood sample, an apheresis sample or a leucocyte apheresis sample.
106. The method of any one of embodiments 75-105, wherein the HLA-e+ feeder cells are K562 cells (K562-HLA-E) transformed with HLA-E.
107. The method of any one of embodiments 75 to 106, wherein the HLA-e+ feeder cells are 221.Aeh cells.
108. The method of any one of embodiments 75 to 107, wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is between 1:1 and 5:1, inclusive, between 1:1 and 3:1, inclusive, optionally at or about 2.5:1 or at or about 2:1 or about 1:1.
109. The method of any one of embodiments 75 to 108, wherein the recombinant cytokine added to the medium during at least a portion of the culturing is 500IU/mL IL-2, 10ng/mL IL-15, and 25ng/mL IL-21.
110. The method of any one of embodiments 75 to 109, wherein the enriched NK cell population comprises the following enriched NK cells: the population of cells optionally comprises enriched cells at the end of each of between 2.0×10 5 enriched NK cells and 5.0×10 7 enriched NK cells or between about 2.0×10 5 enriched NK cells and about 5.0×10 7 enriched NK cells, between 1.0×10 6 enriched NK cells and 1.0×10 8 enriched NK cells or between about 1.0×10 6 enriched NK cells and about 1.0×10 8 enriched NK cells, between 1.0×10 7 enriched NK cells and 5.0×10 8 enriched NK cells or between about 1.0×10 7 enriched NK cells and about 5.0×10 8 enriched NK cells or between 1.0×10 7 enriched NK cells and 1.0×10 9 enriched NK cells or between about 1.0×10 7 enriched NK cells and about 1.0×10 9 enriched NK cells, including enriched cells at the end of each of said population optionally comprising enriched cells of about 1×25 NK cells.
111. The method of any one of embodiments 75 to 110, wherein the enriched NK cell population has a concentration at the beginning of the culturing of between 0.05 x 10 6 enriched NK cells/mL and 1.0 x 10 6 enriched NK cells/mL or between about 0.05 x 10 6 enriched NK cells/mL and about 1.0 x 10 6 enriched NK cells/mL or between or about 0.05 x 10 6 enriched NK cells/mL and 0.5 x 10 6 enriched NK cells/mL, optionally wherein the enriched NK cell population has or has a concentration of about 0.2 x 10 6 enriched NK cells/mL at the beginning of the culturing.
112. The method of any one of embodiments 75 to 111, wherein said culturing is performed until said method achieves a time to expand at least or at least about 2.50X10 8 g-NK cells, at least or at least about 5.00X10 8 g-NK cells, at least or at least about 1.0X10 9 g-NK cells, or at least about 5.0X10 9 g-NK cells.
113. The method according to any one of embodiments 75 to 112, wherein the culturing is performed or performed about or at least or for at least the following time: about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 days.
114. The method of any one of embodiments 75 to 113, further comprising collecting the expanded population of engineered NK cells produced by the method.
115. The method of any one of embodiments 75 to 114, further comprising formulating the expanded population of engineered NK cells in a pharmaceutically acceptable excipient.
116. The method of embodiment 115, further comprising formulating the expanded population of engineered NK cells with a serum-free cryopreservation medium comprising a cryoprotectant.
117. The method of embodiment 116, wherein the cryoprotectant is DMSO, optionally wherein the cryoprotectant is DMSO and the cryopreservation medium is 5% to 10% DMSO (v/v), optionally at or about 10% DMSO (v/v).
118. The method of any one of embodiments 75 to 117, wherein in the engineered NK cell expanded population produced by the method, greater than 50% of the population is FcR gamma Negative of , greater than 60% of the population is FcR gamma Negative of , greater than 70% of the population is FcR gamma Negative of ,, greater than 80% of the population is FcR gamma Negative of , greater than 90% of the population is FcR gamma Negative of , or greater than 95% of the population is FcR gamma Negative of .
119. A composition comprising a plurality of engineered NK cells produced by the method according to any one of embodiments 58 to 118.
120. A method of treating a disease or disorder in a subject, the method comprising administering to an individual in need thereof an effective amount of cells of the composition of any one of embodiments 16-55 and 119.
121. The method of embodiment 120, wherein the disease or disorder is selected from an inflammatory disorder, an infection, or a cancer.
122. The method of embodiment 120 or embodiment 121, wherein the disease or disorder is cancer and the cancer is leukemia, lymphoma, or myeloma.
123. The method of embodiment 120 or embodiment 121, wherein the disease or disorder is cancer and the cancer comprises a solid tumor.
124. The method of embodiment 123, wherein the cancer is selected from the group consisting of adenocarcinoma of the stomach or gastroesophageal junction, bladder cancer, breast cancer, brain cancer, cervical cancer, colorectal cancer, endocrine/neuroendocrine cancer, head and neck cancer, gastrointestinal stromal cancer, bone giant cell tumor, kidney cancer, liver cancer, lung cancer, neuroblastoma, ovarian epithelial/fallopian tube/primary peritoneal cancer, pancreatic cancer, prostate cancer, skin cancer, and soft tissue cancer.
125. The method according to any one of embodiments 120 to 124, wherein the composition is administered as monotherapy.
126. The method of any one of embodiments 120 to 125, comprising administering to the individual 1 x 10 5 to 50 x 10 9 or about 1 x 10 5 to about 50 x 10 9 cells of the g-NK cell composition.
127. The method of any one of embodiments 120 to 126, comprising administering from 1 x 10 8 cells to 50 x 10 9 NK cells or from about 1 x 10 8 cells to about 50 x 10 9 NK cells of the g-NK cell composition, optionally from 5 x 10 8 cells or about 5 x 10 8 cells of the g-NK cell composition, from 5 x 10 9 cells or about 5 x 10 9 cells of the g-NK cell composition, or from 10 x 10 9 cells or about 10 x 10 9 cells of the g-NK cell composition.
128. The method of any one of embodiments 120 to 127, further comprising administering exogenous cytokine support to promote expansion or persistence of the administered NK cells in the subject, optionally wherein the exogenous cytokine is or comprises IL-15.
129. The method of any one of embodiments 120 to 128, wherein the subject has received lymphocyte removal therapy prior to said administering said dose of g-NK cells.
130. The method of embodiment 129, wherein the lymphocyte depletion therapy comprises fludarabine and/or cyclophosphamide.
131. The method of embodiment 129 or embodiment 130, wherein the lymphoremoval comprises the administration of fludarabine at 20mg/m 2 to 40mg/m 2 or about 20mg/m 2 to about 40mg/m 2 of body surface area of the subject, optionally at or about 30mg/m 2, for 2 to 4 days per day, and/or the administration of cyclophosphamide at 200mg/m 2 to 400mg/m 2 or about 200mg/m 2 to about 400mg/m 2 of body surface area of the subject, optionally at or about 300mg/m 2, per day for 2 to 4 days.
132. The method of any one of embodiments 129 to 131, wherein the lymphocyte scavenging therapy comprises fludarabine and cyclophosphamide.
133. The method of any one of embodiments 129 to 132, wherein the lymphoremoval therapy comprises the administration of fludarabine at 30mg/m 2 or about 30mg/m 2 of the subject body surface area per day and the administration of cyclophosphamide at 300mg/m 2 or about 300mg/m 2 of the subject body surface area per day, each for 2 to 4 days, optionally for 3 days.
134. The method of any of embodiments 129 to 133, wherein administration of the cells begins within two weeks or at or about two weeks after initiation of the lymphocyte removal therapy.
135. The method of any one of embodiments 120 to 169, wherein the individual is a human.
136. The method of any one of embodiments 120 to 135, wherein the NK cells in the composition are allogeneic to the individual.
137. The method of any one of embodiments 120 to 136, wherein the NK cells in the composition are autologous to the subject.
The provided embodiments include:
1. an engineered Natural Killer (NK) cell (g-NK cell) deficient in FcR gamma chain expression, and the NK cell comprising a heterologous nucleic acid encoding an immunomodulatory agent.
2. The engineered NK cell of embodiment 1, wherein said immunomodulator is an immunosuppressant.
3. The engineered NK cell of embodiment 1, wherein the immunomodulator is an immune activator.
4. The engineered NK cell of embodiment 1 or embodiment 3, wherein said immunomodulatory agent is a cytokine.
5. The engineered NK cell of any one of embodiments 1-4, wherein said cytokine is secreted from said engineered NK cell.
6. The engineered NK cell according to embodiment 5, wherein said secretable cytokine is IL-2 or a biological portion thereof; IL-15 or a biological portion thereof; or IL-21 or a biological part thereof; or a combination thereof.
7. The engineered NK cell of any one of embodiments 1-5, wherein said cytokine is membrane-bound.
8. The engineered NK cell of embodiment 7, wherein said membrane-bound cytokine is membrane-bound IL-2 (mbIL-2); membrane-bound IL-15 (mbIL-15); membrane-bound IL-21 (mbIL-21); or a combination thereof.
9. The engineered NK cell of any one of embodiments 1-8, wherein the heterologous nucleic acid encoding the immunomodulator is stably integrated into the genome of the cell.
10. The engineered NK cell of any one of embodiments 1-8, wherein the heterologous nucleic acid encoding the immunomodulator is transiently expressed.
11. The engineered NK cell of any one of embodiments 1 to 10, wherein said g-NK cell has the surface phenotype of CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of .
12. The engineered NK cell of any one of embodiments 1 to 11, wherein said g-NK cell further has the surface phenotype of NKG2a Negative of /CD161 Negative of .
13. The engineered NK cell of any one of embodiments 1 to 12, wherein said g-NK cell is also CD38 Negative of .
14. The engineered NK cell of any one of embodiments 1 to 13, wherein said g-NK cell further has the surface phenotype of CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative .
15. The engineered NK cell of any one of embodiments 1 to 14, wherein said g-NK cell comprises CD 16V/V (V158)
16. The engineered NK cell of any one of embodiments 1 to 14, wherein said g-NK cell is CD16 158V/F.
17. A composition comprising a plurality of engineered g-NK cells according to any one of embodiments 1 to 16.
18. The composition of embodiment 17, wherein greater than 50% or greater than about 50% of the NK cells or total cells in the composition are g-NK cells.
19. The composition of embodiment 17, wherein greater than 60 or greater than about 60% of the NK cells or total cells in the composition are g-NK cells.
20. The composition of embodiment 17, wherein greater than 70% or greater than about 70% of the NK cells or total cells in the composition are g-NK cells.
21. The composition of embodiment 17, wherein greater than 80% or greater than about 80% of the NK cells or total cells in the composition are g-NK cells.
22. The composition of embodiment 17, wherein greater than 90% or greater than about 90% of the NK cells or total cells in the composition are g-NK cells.
23. The composition of embodiment 17, wherein greater than 95% or greater than about 95% of the NK cells or total cells in the composition are g-NK cells.
24. The composition of any one of embodiments 17 to 23, wherein the plurality of engineered g-NK cells comprises greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, or greater than 70% or greater than about 70% g-NK cells comprising a heterologous nucleic acid encoding the immunomodulatory agent.
25. The composition of any one of embodiments 17 to 24, wherein the total composition comprises greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, or greater than 70% or greater than about 70% g-NK cells comprising the heterologous nucleic acid encoding the immunomodulator.
26. The composition of any one of embodiments 17 to 25, wherein greater than 70% or greater than about 70% of the g-NK cells are positive for perforin and greater than 70% or greater than about 70% of the g-NK cells are positive for granzyme B.
27. The composition of any one of embodiments 17 to 26, wherein greater than 80% or greater than about 80% of the g-NK cells are positive for perforin and greater than 80% or greater than about 80% of the g-NK cells are positive for granzyme B.
28. The composition of any one of embodiments 17 to 27, wherein greater than 90% or greater than about 90% of the g-NK cells are positive for perforin and greater than 90% or greater than about 90% of the g-NK cells are positive for granzyme B.
29. The composition of any one of embodiments 17 to 28, wherein greater than 95% or greater than about 95% of the g-NK cells are positive for perforin and greater than 95% or greater than about 95% of the g-NK cells are positive for granzyme B.
30. The composition according to any one of embodiments 26 to 29, wherein:
In the cells positive for perforin, the cells express perforin at an average level of at least twice or at least about twice that of FcR gamma Positive and negative based on Mean Fluorescence Intensity (MFI) as measured by intracellular flow cytometry; and/or
In the cells positive for granzyme B, the cells express granzyme B at an average level of at least twice or at least about twice as high as that of FcR gamma Positive and negative based on average fluorescence intensity (MFI) as measured by intracellular flow cytometry.
31. The composition of any one of embodiments 17 to 30, wherein optionally greater than 10% of the cells in the composition are capable of degranulation against tumor target cells as measured by CD107a expression, optionally wherein the degranulation is measured in the absence of antibodies against the tumor target cells.
32. The composition of any one of embodiments 17-31, wherein greater than 10% of the cells in the composition are further capable of producing interferon-gamma or TNF-alpha to a tumor target cell, optionally wherein the interferon-gamma or TNF-alpha is measured in the absence of antibodies to the tumor target cell.
33. The composition of any one of embodiments 17 to 32, wherein in the cells in the composition, greater than 15% or greater than about 15%, greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, or greater than 50% or greater than about 50% of the cells expressing a target antigen (target cells) and antibodies to the target antigen (anti-target antibodies) produce an effector cytokine.
34. The composition of any one of embodiments 17 to 33, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 30% or greater than about 30% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 50% or greater than about 50% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
35. The composition of any one of embodiments 17 to 33, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 35% or greater than about 35% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 60% or greater than about 60% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
36. The composition of any one of embodiments 17 to 33, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 40% or greater than about 40% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 70% or greater than about 70% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
37. The composition of any one of embodiments 17 to 33, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 45% or greater than about 45% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 80% or greater than about 80% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
38. The composition of any one of embodiments 17 to 33, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 50% or greater than about 50% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 85% or greater than about 85% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
39. The composition of any one of embodiments 17 to 33, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 55% or greater than about 55% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 90% or greater than about 90% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
40. The composition of any one of embodiments 17 to 33, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 60% or greater than about 60% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 95% or greater than about 95% of the cells are negative or low level for NKG2A (NKG 2A Negative of ).
41. The composition of any one of embodiments 17 to 40, wherein greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, or greater than 90% or greater than about 90% of the cells in the total cells in the composition or the g-NK cells in the composition are CD38 Negative of .
42. The composition of any one of embodiments 17 to 40, wherein greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, or greater than 90% or greater than about 90% of the cells in the total cells in the composition or the g-NK cells in the composition are CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of .
43. The composition of any one of embodiments 17 to 40, wherein greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, or greater than 90% or greater than about 90% of the cells in the total cells in the composition or the g-NK cells in the composition are NKG2a Negative of /CD161 Negative of .
44. The composition of any one of embodiments 17 to 43, wherein the plurality of g-NK cells is CD16 158V/V (V158).
45. The composition of any one of embodiments 17 to 44, wherein the plurality of g-NK cells is CD16 158V/F.
46. The composition of any one of embodiments 17 to 45, wherein the composition comprises at least or at least about 10 8 cells.
47. The composition according to any one of embodiments 17 to 46, wherein the number of g-NK cells in the composition is from 10 8 to 10 12 cells or from about 10 8 to about 10 12 cells, from 10 8 to 10 11 cells or from about 10 8 to about 10 11 cells, from 10 8 to 10 10 cells or from about 10 8 to about 10 10 cells, from 10 8 to 10 9 cells or from about 10 8 to about 10 9 cells, from 10 9 cells to 10 12 cells or from about 10 9 cells to about 10 12 cells, from 10 12 to 10 12 cells or from about 10 12 to about 10 cells, from about 10 12 to about 12 cells or from 10 12 to 12 cells or from 10 to 12 cells.
48. The composition of any one of embodiments 17 to 47, wherein the number of g-NK cells in the composition is or is about 5 x 10 8 cells, or is about 1x 10 9 cells, or is about 5 x 10 10 cells, or is about 1x 10 10 cells.
49. The composition of any of embodiments 17-48, wherein the volume of the composition is between 50mL and 500mL or between about 50mL and about 500mL, optionally at or about 200mL.
50. The composition of any one of embodiments 17 to 49, wherein the cells in the composition are from a single donor subject, the cells having been expanded from the same biological sample.
51. The composition according to any one of embodiments 17 to 50, wherein the composition is a pharmaceutical composition.
52. The composition according to any one of embodiments 17 to 51, comprising a pharmaceutically acceptable excipient.
53. The composition of any one of embodiments 17 to 52, wherein the composition is formulated in a serum-free cryopreservation medium comprising a cryoprotectant.
54. The composition of embodiment 53, wherein the cryoprotectant is DMSO and the cryopreservation medium is 5% to 10% DMSO (v/v).
55. The composition of embodiment 54, wherein the cryoprotectant is or is about 10% DMSO (v/v).
56. The composition of any one of embodiments 17 to 55, which is sterile.
57. A sterile bag comprising the composition according to any one of embodiments 17 to 56.
58. The sterile bag of embodiment 57, wherein the bag is a cryopreservation compatible bag.
59. A method of producing a genetically engineered g-NK cell, the method comprising introducing into the g-NK cell a heterologous nucleic acid encoding an immunomodulatory agent, thereby producing a genetically engineered g-NK cell.
60. The method of embodiment 59, wherein the immunomodulator is an immunosuppressant.
61. The method of embodiment 59, wherein the immunomodulator is an immune activator.
62. The method of embodiment 59 or embodiment 61, wherein the immunomodulatory agent is a cytokine.
63. The method of embodiment 62, wherein said cytokine is secreted from said engineered NK cell.
64. The method of embodiment 63, wherein the secretable cytokine is IL-2 or a biologically active portion thereof; IL-15 or a biologically active portion thereof; IL-21 or a biologically active portion thereof; or a combination thereof.
65. The method of embodiment 62, wherein the immune activator is membrane-bound.
66. The method of embodiment 65, wherein the membrane-bound cytokine is membrane-bound IL-2 (mbIL-2); membrane-bound IL-15 (mbIL-15); or membrane-bound IL-21 (mbiL-21); or a combination thereof.
67. The method of any one of embodiments 59 to 66, wherein the nucleic acid encoding the immunomodulator is introduced under conditions that stably integrate into the genome of the g-NK cell.
68. The method of any one of embodiments 59 to 67, wherein the nucleic acid encoding an immunomodulatory agent is contained in a viral vector and introduced into the g-NK cell by transduction.
69. The method of embodiment 68, wherein the viral vector is a lentiviral vector.
70. The method of any one of embodiments 59 to 66, wherein the nucleic acid encoding the immunomodulator is introduced under conditions of transient expression in the g-NK cell.
71. The method of any one of embodiments 59 to 66 and 70, wherein the nucleic acid encoding the immunomodulatory agent is introduced by non-viral delivery.
72. The method of any one of embodiments 59 to 66, 70 and 71, wherein the nucleic acid encoding the immunomodulatory agent is introduced into the g-NK cell via a lipid nanoparticle.
73. The method of any one of embodiments 67 to 72, wherein the nucleic acid encoding the immunomodulatory agent is DNA.
74. The method of any one of embodiments 70-73, wherein the nucleic acid encoding the immunomodulatory agent is RNA.
75. The method of embodiment 74, wherein the RNA is mRNA.
76. The method of embodiment 75, wherein the RNA is self-amplifying mRNA.
77. The method of any one of embodiments 59 to 66 and 70 to 75, wherein the nucleic acid encoding the immunomodulatory agent is introduced into the g-NK cells via electroporation.
78. The method of any one of embodiments 59 to 77, wherein the g-NK cell composition is produced by in vitro expansion of NK cells enriched from a biological sample from a subject, the NK cells being: (i) Negative or low level for CD3 and positive for CD57 (CD 3 Negative of CD57 Positive and negative ) or (ii) negative or low level for CD3 and positive for CD56 (CD 3 Negative of CD56 Positive and negative ), wherein the enriched NK cells are cultured with irradiated HLA-e+ feeder cells and one or more recombinant cytokines.
79. The method of embodiment 78, wherein the one or more recombinant cytokines are selected from the group consisting of an effective amount of SCF, GSK3i, FLT3, IL-2, IL-6, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or a combination thereof.
80. The method of embodiment 78 or embodiment 79, wherein the culturing is performed in the presence of two or more recombinant cytokines, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21.
81. The method of any one of embodiments 59 to 80, wherein the introducing is performed during a method for expanding FcR γ -deficient NK (g-NK) cells, the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21;
Wherein introducing the nucleic acid encoding the immunomodulator is performed after step (a) and before, during or after step (B), thereby producing an engineered NK cell population; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with an immunomodulatory agent.
82. The method of any one of embodiments 59 to 80, wherein the introducing is performed during a method for amplifying FcR γ -deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject;
(B) Introducing a nucleic acid encoding an immunomodulatory agent into the enriched NK cell population, thereby producing an engineered NK cell population; and
(C) Culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with an immunomodulatory agent.
83. The method of any one of embodiments 59 to 80, wherein the introducing is performed during a method for amplifying FcR γ -deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Culturing the enriched NK cell population in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and
(C) Introducing said nucleic acid encoding an immunomodulator into NK cells of said amplified population of NK cells,
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with an immunomodulatory agent.
84. The method of any one of embodiments 59 to 80, wherein the introducing is performed during a method for amplifying FcR γ -deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Performing a first expansion comprising culturing the enriched NK cell population in a medium under conditions to expand the NK cells to produce a first expanded population of NK cells;
(C) Introducing said nucleic acid encoding an immunomodulatory agent into NK cells of said first expanded population of NK cells, thereby producing an engineered NK cell population; and
(D) Performing a second expansion comprising culturing said population of engineered NK cells under conditions that further expand said NK cells,
Wherein the first expansion and/or second expansion comprises culturing the enriched NK cell population in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with an immunomodulatory agent.
85. The method according to any one of embodiments 78 to 84, wherein the population of NK cell enriched primary human cells is obtained by selecting cells from a biological sample from a human subject, the cells being: (i) Negative or low level for CD3 and positive for CD57 (CD 3 Negative of CD57 Positive and negative ) or (ii) negative or low level for CD3 and positive for CD56 (CD 3 Negative of CD56 Positive and negative ).
86. The method of embodiment 85, wherein:
The population enriched for NK cells is a cell obtained by further selecting cells positive for NKG2C (NKG 2C Positive and negative );
The population enriched for NK cells is a cell obtained by further selecting cells negative for NKG2A or low in level (NKG 2A Negative of ); or alternatively
The population enriched for NK cells is a cell obtained by further selecting cells positive for NKG2C and negative or low level for NKG2A (NKG 2C Positive and negative NKG2A Negative of ).
87. The method according to any one of embodiments 78 to 86, wherein the human subject is a subject: wherein at least or at least about 20% of Natural Killer (NK) cells in a peripheral blood sample from the subject are positive for NKG2C (NKG 2C Positive and negative ) and at least 70% of NK cells in the peripheral blood sample are negative for NKG2A or low level (NKG 2A Negative of ).
88. The method of any one of embodiments 78 to 87, wherein the subject is CMV seropositive.
89. The method of any one of embodiments 78 to 88, wherein the percentage of g-NK cells in the biological sample from the subject is greater than 5% or greater than about 5%, greater than 10% or greater than about 10%, or greater than 30% or greater than about 30%.
90. The method of any one of embodiments 78 to 89, wherein the percentage of g-NK cells in the enriched NK cell population is between 20% and 90%, or between about 20% and about 90%, or between 40% and 90%, or between about 40% and about 90%, or between 60% and 90%, or between about 60% and about 90%.
91. The method of any one of embodiments 78 to 90, wherein the population enriched for NK cells is a cell that is negative or low in level for CD3 and positive for CD57 (CD 3 Negative of CD57 Positive and negative ) selected from the biological sample.
92. The method of any one of embodiments 78 to 90, wherein the population enriched for NK cells is a cell that is negative or low in level for CD3 and positive for CD56 (CD 3 Negative of CD56 Positive and negative ) selected from the biological sample.
93. The method of any one of embodiments 80-92, wherein the two or more recombinant cytokines further comprise an effective amount of SCF, GSK3i, FLT3, IL-6, IL-7, IL-15, IL-12, IL-18, IL-27, or a combination thereof.
94. The method of any one of embodiments 78 to 92, wherein the recombinant cytokine is IL-21 and IL-2.
95. The method of any one of embodiments 78 to 93, wherein the recombinant cytokine is IL-21, IL-2, and IL-15.
96. The method of any one of embodiments 78 to 95, wherein recombinant IL-21 is added to the culture medium at a concentration of 10ng/mL or about 10ng/mL to about 100ng/mL during at least a portion of the culturing.
97. The method of any one of embodiments 78 to 96, wherein recombinant IL-21 is added to the culture medium at a concentration of 25ng/mL or about 25ng/mL during at least a portion of the culturing.
98. The method of any one of embodiments 78 to 97, wherein recombinant IL-2 is added to the culture medium at a concentration of 10IU/mL to 500IU/mL or about 10IU/mL to about 500IU/mL during at least a portion of the culturing.
99. The method of any one of embodiments 78 to 98, wherein recombinant IL-2 is added to the culture medium at a concentration of 100IU/mL or about 100IU/mL during at least a portion of the culturing.
100. The method of any one of embodiments 78 to 99, wherein recombinant IL-2 is added to the culture medium at a concentration of 500IU/mL or about 500IU/mL during at least a portion of the culturing.
101. The method of any one of embodiments 78 to 93 and 95 to 100, wherein recombinant IL-15 is added to the medium at a concentration of 1ng/mL to 50ng/mL or about 1ng/mL to about 50ng/mL during at least a portion of the culturing.
102. The method of any one of embodiments 78 to 93 and 95 to 101, wherein recombinant IL-15 is added to the culture medium at a concentration of 10ng/mL or about 10ng/mL during at least a portion of the culturing.
103. The method of any one of embodiments 78 to 102, wherein the adding of the recombinant cytokine to the culture medium is initiated at or about the beginning of the culturing.
104. The method according to any one of embodiments 78 to 103, wherein the method further comprises changing the medium one or more times during the culturing, wherein fresh medium containing the recombinant cytokine is added each time the medium is changed.
105. The method of embodiment 104, wherein the replacement of the medium is performed every two or three days during the duration of the culturing.
106. The method of embodiment 104 or embodiment 105, wherein the medium change is performed up to 5 days after the first amplification without a medium change, optionally up to 5 days after the first amplification without a medium change.
107. The method of any one of embodiments 78 to 106, wherein said human subject has a CD16 158V/V NK cell genotype or a CD16 158V/F NK cell genotype, optionally wherein said biological sample is from a human subject selected for said CD16 158V/V NK cell genotype or said CD16 158V/F NK cell genotype.
108. The method of any one of embodiments 78 to 107, wherein the biological sample is or comprises Peripheral Blood Mononuclear Cells (PBMCs), optionally a blood sample, an apheresis sample, or a leucocyte apheresis sample.
109. The method of any one of embodiments 78 to 108, wherein the HLA-e+ feeder cells are K562 cells (K562-HLA-E) transformed with HLA-E.
110. The method of any one of embodiments 78 to 109, wherein the HLA-e+ feeder cells are 221.Aeh cells.
111. The method of any one of embodiments 78 to 110, wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is between 1:1 and 5:1, inclusive, between 1:1 and 3:1, inclusive, optionally at or about 2.5:1 or at or about 2:1 or about 1:1.
112. The method of any one of embodiments 78 to 111, wherein the recombinant cytokine added to the medium during at least a portion of the culturing is 500IU/mL IL-2, 10ng/mL IL-15, and 25ng/mL IL-21.
113. The method of any one of embodiments 78 to 112, wherein the enriched NK cell population comprises the following enriched NK cells: the population of cells optionally comprises enriched cells at the end of each of between 2.0×10 5 enriched NK cells and 5.0×10 7 enriched NK cells or between about 2.0×10 5 enriched NK cells and about 5.0×10 7 enriched NK cells, between 1.0×10 6 enriched NK cells and 1.0×10 8 enriched NK cells or between about 1.0×10 6 enriched NK cells and about 1.0×10 8 enriched NK cells, between 1.0×10 7 enriched NK cells and 5.0×10 8 enriched NK cells or between about 1.0×10 7 enriched NK cells and about 5.0×10 8 enriched NK cells or between 1.0×10 7 enriched NK cells and 1.0×10 9 enriched NK cells or between about 1.0×10 7 enriched NK cells and about 1.0×10 9 enriched NK cells, including enriched cells at the end of each of said population optionally comprising enriched cells of about 1×25 NK cells.
114. The method of any one of embodiments 78 to 113, wherein the enriched NK cell population has a concentration at the beginning of the culturing of between 0.05 x 10 6 enriched NK cells/mL and 1.0 x 10 6 enriched NK cells/mL or between about 0.05 x 10 6 enriched NK cells/mL and about 1.0 x 10 6 enriched NK cells/mL or between about 0.05 x 10 6 enriched NK cells/mL and 0.5 x 10 6 enriched NK cells/mL, optionally wherein the enriched NK cell population has or has a concentration of about 0.2 x 10 6 enriched NK cells/mL at the beginning of the culturing.
115. The method of any one of embodiments 78 to 114, wherein said culturing is performed until said method achieves a time of expansion of at least or at least about 2.50X10 8 g-NK cells, at least or at least about 5.00X10 8 g-NK cells, at least or at least about 1.0X10 9 g-NK cells, or at least about 5.0X10 9 g-NK cells.
116. The method according to any one of embodiments 78 to 115, wherein the culturing is performed or performed for about or at least about the following time: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 days.
117. The method of any one of embodiments 78 to 116, further comprising collecting the expanded population of engineered NK cells produced by the method.
118. The method of any one of embodiments 78 to 117, further comprising formulating the expanded population of engineered NK cells in a pharmaceutically acceptable excipient.
119. The method of embodiment 118, further comprising formulating the expanded population of engineered NK cells with a serum-free cryopreservation medium comprising a cryoprotectant.
120. The method of embodiment 119, wherein the cryoprotectant is DMSO, optionally wherein the cryoprotectant is DMSO and the cryopreservation medium is 5% to 10% DMSO (v/v), optionally at or about 10% DMSO (v/v).
121. The method of any one of embodiments 78 to 120, wherein in the engineered NK cell expansion population produced by the method, greater than 50% of the population is FcR gamma Negative of , greater than 60% of the population is FcR gamma Negative of , greater than 70% of the population is FcR gamma Negative of ,, greater than 80% of the population is FcR gamma Negative of , greater than 90% of the population is FcR gamma Negative of , or greater than 95% of the population is FcR gamma Negative of .
122. A composition comprising a plurality of engineered NK cells produced by the method according to any one of embodiments 59 to 121.
123. A method of treating a disease or disorder in a subject, the method comprising administering to an individual in need thereof an effective amount of cells of the composition of any one of embodiments 17-56 and 122.
124. The method according to embodiment 123, wherein the disease or disorder is selected from an inflammatory disorder, an infection, or a cancer.
125. The method of embodiment 123 or embodiment 124, wherein the disease or disorder is cancer and the cancer is leukemia, lymphoma, or myeloma.
126. The method of embodiment 123 or embodiment 124, wherein the disease or disorder is cancer and the cancer comprises a solid tumor.
127. The method of embodiment 126, wherein the cancer is selected from the group consisting of adenocarcinoma of the stomach or gastroesophageal junction, bladder cancer, breast cancer, brain cancer, cervical cancer, colorectal cancer, endocrine/neuroendocrine cancer, head and neck cancer, gastrointestinal stromal cancer, bone giant cell tumor, kidney cancer, liver cancer, lung cancer, neuroblastoma, ovarian epithelial/fallopian tube/primary peritoneal cancer, pancreatic cancer, prostate cancer, skin cancer, and soft tissue cancer.
128. The method according to any one of embodiments 123 to 127, wherein the composition is administered as monotherapy.
129. The method of any one of embodiments 123-127, further comprising administering to the individual an additional agent to treat the disease or disorder.
130. The method of embodiment 129, wherein the additional agent is an antibody or Fc fusion protein.
131. The method of embodiment 130, wherein the disease or disorder is cancer and the additional agent, optionally an antibody, recognizes a tumor antigen associated with the cancer.
132. The method of any one of embodiments 123 to 131, comprising administering to the individual 1 x 10 5 to 50 x 10 9 or about 1 x 10 5 to about 50 x 10 9 cells of the g-NK cell composition.
133. The method of any one of embodiments 123 to 132, comprising administering from 1 x 10 8 cells to 50 x 10 9 NK cells or from about 1 x 10 8 cells to about 50 x 10 9 NK cells of the g-NK cell composition, optionally from 5 x 10 8 cells or about 5 x 10 8 cells of the g-NK cell composition, from 5 x 10 9 cells or about 5 x 10 9 cells of the g-NK cell composition, or from 10 x 10 9 cells or about 10 x 10 9 cells of the g-NK cell composition.
134. The method of any one of embodiments 123 to 133, wherein the subject has received lymphocyte removal therapy prior to said administering said dose of g-NK cells.
135. The method of embodiment 134, wherein the lymphocyte depletion therapy comprises fludarabine and/or cyclophosphamide.
136 The method of embodiment 134 or embodiment 135, wherein said lymphoremoval comprises said administration of fludarabine at 20mg/m 2 to 40mg/m 2 or about 20mg/m 2 to about 40mg/m 2, optionally at or about 30mg/m 2, for 2 to 4 days per day, and/or said administration of cyclophosphamide at 200mg/m 2 to 400mg/m 2 or about 200mg/m 2 to about 400mg/m 2, optionally at or about 300mg/m 2, per day, for 2 to 4 days.
137. The method of any of embodiments 134-136, wherein the lymphocyte depletion therapy comprises fludarabine and cyclophosphamide
138. The method of any of embodiments 134-137, wherein the lymphoremoval therapy comprises the administration of fludarabine at 30mg/m 2 or about 30mg/m 2 of the subject body surface area per day, and cyclophosphamide at 300mg/m 2 or about 300mg/m 2 of the subject body surface area per day, each for 2 to 4 days, optionally for 3 days.
139. The method of any one of embodiments 134 to 138, wherein administering a dose of g-NK cells is initiated within two weeks or at or about two weeks after initiation of the lymphocyte depletion therapy.
140. The method of any one of embodiments 123 to 139, wherein the individual is a human.
141. The method of any one of embodiments 123 to 140, wherein the NK cells in the composition are allogeneic to the individual.
142. The method of any one of embodiments 123 to 140, wherein the NK cells in the composition are autologous to the subject.
The provided embodiments include:
1. an engineered Natural Killer (NK) cell (g-NK cell) deficient in FcR gamma chain expression, and the NK cell comprising a heterologous nucleic acid encoding a Chimeric Antigen Receptor (CAR) and a heterologous nucleic acid encoding an immunomodulator.
2. The engineered NK cell of embodiment 1, wherein said immunomodulator is an immunosuppressant.
3. The engineered NK cell of embodiment 1, wherein the immunomodulator is an immune activator.
4. The engineered NK cell of embodiment 1 or embodiment 3, wherein said immunomodulatory agent is a cytokine.
5. The engineered NK cell of any one of embodiments 1-4, wherein said cytokine is secreted from said engineered NK cell.
6. The engineered NK cell according to embodiment 5, wherein said secretable cytokine is IL-2 or a biological portion thereof; IL-15 or a biological portion thereof; or IL-21 or a biological part thereof; or a combination thereof.
7. The engineered NK cell of any one of embodiments 1-5, wherein said cytokine is membrane-bound.
8. The engineered NK cell of embodiment 7, wherein said membrane-bound cytokine is membrane-bound IL-2 (mbIL-2); membrane-bound IL-15 (mbIL-15); membrane-bound IL-21 (mbIL-21); or a combination thereof.
9. The engineered NK cell of any one of embodiments 1 to 8, wherein said CAR comprises 1) an antigen binding domain; 2) A flexible connector; 3) A transmembrane region; and 4) an intracellular signaling domain.
10. The engineered NK cell of embodiment 9, wherein said antigen binding domain targets a tumor antigen.
11. The engineered NK cell of embodiment 9 or 10, wherein said antigen binding domain is a single chain variable fragment (scFv).
12. The engineered NK cell of any one of embodiments 9 to 11, wherein said intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain.
13. The engineered NK cell of any one of embodiments 9 to 12, wherein said intracellular signaling domain comprises one or more signaling domains of cd3ζ, DAP10, DAP12, CD28, 4-1BB, or OX 40.
14. The engineered NK cell of any one of embodiments 9 to 12, wherein said intracellular signaling domain comprises two or more signaling domains of cd3ζ, DAP10, DAP12, CD28, 4-1BB, or OX 40.
15. The engineered NK cell of any one of embodiments 1-14, wherein the heterologous nucleic acid encoding the CAR is stably integrated into the genome of the cell.
16. The engineered NK cell of any one of embodiments 1-14, wherein the heterologous nucleic acid encoding the CAR is transiently expressed.
17. The engineered NK cell of any one of embodiments 1-16, wherein the heterologous nucleic acid encoding the immunomodulator is stably integrated into the genome of the cell.
18. The engineered NK cell of any one of embodiments 1-16, wherein the heterologous nucleic acid encoding the immunomodulator is transiently expressed.
19. The engineered NK cell of any one of embodiments 1 to 18, wherein said g-NK cell has the surface phenotype of CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of .
20. The engineered NK cell of any one of embodiments 1 to 19, wherein said g-NK cell further has the surface phenotype of NKG2a Negative of /CD161 Negative of .
21. The engineered NK cell of any one of embodiments 1 to 20, wherein said g-NK cell is also CD38 Negative of .
22. The engineered NK cell of any one of embodiments 1 to 21, wherein said g-NK cell further has the surface phenotype of CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative .
23. The engineered NK cell of any one of embodiments 1 to 22, wherein said g-NK cell comprises CD 16V/V (V158)
24. The engineered NK cell of any one of embodiments 1 to 22, wherein said g-NK cell is CD16 158V/F.
25. A composition comprising a plurality of engineered g-NK cells according to any one of embodiments 1 to 24.
26. The composition of embodiment 25, wherein greater than 50% or greater than about 50% of the NK cells or total cells in the composition are g-NK cells.
27. The composition of embodiment 25, wherein greater than 60% or greater than about 60% of the NK cells or total cells in the composition are g-NK cells.
28. The composition of embodiment 25, wherein greater than 70% or greater than about 70% of the NK cells or total cells in the composition are g-NK cells.
29. The composition of embodiment 25, wherein greater than 80% or greater than about 80% of the NK cells or total cells in the composition are g-NK cells.
30. The composition of embodiment 25, wherein greater than 90% or greater than about 90% of the NK cells or total cells in the composition are g-NK cells.
31. The composition of embodiment 25, wherein greater than 95% or greater than about 95% of the NK cells or total cells in the composition are g-NK cells.
32. The composition of any one of embodiments 25-31, wherein the plurality of engineered g-NK cells comprises greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, or greater than 70% or greater than about 70% g-NK cells comprising a heterologous nucleic acid encoding the CAR and a heterologous nucleic acid encoding the immunomodulatory agent.
33. The composition of any one of embodiments 25-32, wherein the total composition comprises greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, or greater than 70% or greater than about 70% g-NK cells comprising a heterologous nucleic acid encoding the CAR and a heterologous nucleic acid encoding the immunomodulatory agent.
34. The composition of any one of embodiments 25-33, wherein the plurality of engineered g-NK cells comprises greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, or greater than 70% or greater than about 70% g-NK cells comprising a heterologous nucleic acid encoding the CAR.
35. The composition of any one of embodiments 25 to 34, wherein the plurality of engineered g-NK cells comprises greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, or greater than 70% or greater than about 70% g-NK cells comprising a heterologous nucleic acid encoding the immunomodulatory agent.
36. The composition of any one of embodiments 25-35, wherein the total composition comprises greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, or greater than 70% or greater than about 70% g-NK cells comprising a heterologous nucleic acid encoding the CAR.
37. The composition of any one of embodiments 25 to 36, wherein the total composition comprises greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, or greater than 70% or greater than about 70% g-NK cells comprising the heterologous nucleic acid encoding the immunomodulator.
38. The composition of any one of embodiments 25 to 37, wherein greater than 70% or greater than about 70% of the g-NK cells are positive for perforin and greater than 70% or greater than about 70% of the g-NK cells are positive for granzyme B.
39. The composition of any one of embodiments 25-38, wherein greater than 80% or greater than about 80% of the g-NK cells are positive for perforin and greater than 80% or greater than about 80% of the g-NK cells are positive for granzyme B.
40. The composition of any one of embodiments 25-39, wherein greater than 90% or greater than about 90% of the g-NK cells are positive for perforin and greater than 90% or greater than about 90% of the g-NK cells are positive for granzyme B.
41. The composition of any one of embodiments 25-39, wherein greater than 95% or greater than about 95% of the g-NK cells are positive for perforin and greater than 95% or greater than about 95% of the g-NK cells are positive for granzyme B.
42. The composition according to any one of embodiments 38 to 41, wherein:
In the cells positive for perforin, the cells express perforin at an average level of at least twice or at least about twice that of FcR gamma Positive and negative based on Mean Fluorescence Intensity (MFI) as measured by intracellular flow cytometry; and/or
In the cells positive for granzyme B, the cells express granzyme B at an average level of at least twice or at least about twice as high as that of FcR gamma Positive and negative based on average fluorescence intensity (MFI) as measured by intracellular flow cytometry.
43. The composition of any one of embodiments 25 to 42, wherein optionally greater than 10% of the cells in the composition are capable of degranulation against tumor target cells as measured by CD107a expression, optionally wherein the degranulation is measured in the absence of antibodies against the tumor target cells.
44. The composition of any one of embodiments 25-43, wherein greater than 10% of the cells in the composition are further capable of producing interferon-gamma or TNF-alpha to a tumor target cell, optionally wherein the interferon-gamma or TNF-alpha is measured in the absence of antibodies to the tumor target cell.
45. The composition of any one of embodiments 25-44, wherein in the cells in the composition, greater than 15% or greater than about 15%, greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, or greater than 50% or greater than about 50% of the cells expressing a target antigen (target cells) and antibodies to the target antigen (anti-target antibodies) produce an effector cytokine.
46. The composition of any one of embodiments 25 to 45, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 30% or greater than about 30% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 50% or greater than about 50% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
47. The composition of any one of embodiments 25 to 45, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 35% or greater than about 35% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 60% or greater than about 60% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
48. The composition of any one of embodiments 25 to 45, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 40% or greater than about 40% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 70% or greater than about 70% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
49. The composition of any one of embodiments 25 to 45, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 45% or greater than about 45% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 80% or greater than about 80% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
50. The composition of any one of embodiments 25 to 45, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 50% or greater than about 50% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 85% or greater than about 85% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
51. The composition of any one of embodiments 25 to 45, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 55% or greater than about 55% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 90% or greater than about 90% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
52. The composition of any one of embodiments 25 to 45, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 60% or greater than about 60% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 95% or greater than about 95% of the cells are negative or low level for NKG2A (NKG 2A Negative of ).
53. The composition of any one of embodiments 25 to 52, wherein greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, or greater than 90% or greater than about 90% of the cells in the total cells in the composition or the g-NK cells in the composition are CD38 Negative of .
54. The composition of any one of embodiments 25 to 52, wherein greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, or greater than 90% or greater than about 90% of the cells in the total cells in the composition or the g-NK cells in the composition are CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of .
55. The composition of any one of embodiments 25 to 52, wherein greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, or greater than 90% or greater than about 90% of the cells in the total cells in the composition or the g-NK cells in the composition are NKG2a Negative of /CD161 Negative of .
56. The composition of any one of embodiments 25 to 55, wherein the plurality of g-NK cells is CD16 158V/V (V158).
57. The composition of any one of embodiments 25 to 56, wherein the plurality of g-NK cells is CD16 158V/F.
58. The composition of any one of embodiments 25-57, wherein the immunomodulatory agent is a cytokine, optionally comprising IL-2 or a biologically active portion thereof, IL-15 or a biologically active portion thereof, or IL-21 or a biologically active portion thereof.
59. The composition of embodiment 58, wherein the cytokine is secreted from an engineered cell of the composition or is membrane-bound.
60. The composition of any one of embodiments 25 to 59, wherein the composition comprises at least or at least about 10 8 cells.
61. The composition according to any one of embodiments 25 to 60, wherein the number of g-NK cells in the composition is from 10 8 to 10 12 cells or from about 10 8 to about 10 12 cells, from 10 8 to 10 11 cells or from about 10 8 to about 10 11 cells, from 10 8 to 10 10 cells or from about 10 8 to about 10 10 cells, from 10 8 to 10 9 cells or from about 10 8 to about 10 9 cells, from 10 9 cells to 10 12 cells or from about 10 9 cells to about 10 12 cells, from 10 12 to 10 12 cells or from about 10 12 to about 10 cells, from about 10 12 to about 12 cells or from 10 12 to 12 cells or from 10 to 12 cells.
62. The composition of any one of embodiments 25 to 61, wherein the number of g-NK cells in the composition is or is about 5 x 10 8 cells, or is about 1 x 10 9 cells, or is about 5 x 10 10 cells, or is about 1 x 10 10 cells.
63. The composition of any of embodiments 25-62, wherein the volume of the composition is between 50mL and 500mL or between about 50mL and about 500mL, optionally at or about 200mL.
64. The composition of any one of embodiments 25-63, wherein the cells in the composition are from a single donor subject, the cells having been expanded from the same biological sample.
65. The composition according to any one of embodiments 25 to 64, wherein the composition is a pharmaceutical composition.
66. The composition according to any one of embodiments 25 to 65, comprising a pharmaceutically acceptable excipient.
67. The composition of any one of embodiments 25 to 66, wherein the composition is formulated in a serum-free cryopreservation medium comprising a cryoprotectant.
68. The composition of embodiment 67, wherein the cryoprotectant is DMSO and the cryopreservation medium is 5% to 10% DMSO (v/v).
69. The composition of embodiment 68, wherein the cryoprotectant is or is about 10% DMSO (v/v).
70. The composition of any one of embodiments 25 to 69, which is sterile.
71. A sterile bag comprising the composition according to any one of embodiments 25 to 70.
72. The sterile bag of embodiment 71, wherein the bag is a cryopreservation compatible bag.
73. A method of producing genetically engineered g-NK cells, the method comprising:
(a) Introducing a heterologous nucleic acid encoding a Chimeric Antigen Receptor (CAR) into an FcR gamma chain expression deficient NK cell (g-NK cell), and
(B) Introducing a heterologous nucleic acid encoding an immunomodulator into said g-NK cell,
Thereby producing genetically engineered g-NK cells, wherein steps (a) and (b) are performed simultaneously or sequentially in any order.
74. The method of embodiment 73, wherein the immunomodulator is an immunosuppressant.
75. The method of embodiment 73, wherein the immunomodulator is an immune activator.
76. The method of embodiment 73 or embodiment 75, wherein the immunomodulatory agent is a cytokine.
77. The method of embodiment 76, wherein said cytokine is secreted from said engineered NK cell.
78. The method of embodiment 77, wherein the secretable cytokine is IL-2 or a biologically active portion thereof; IL-15 or a biologically active portion thereof; IL-21 or a biologically active portion thereof; or a combination thereof.
79. The method of embodiment 76, wherein the immune activator is membrane-bound.
80. The method of embodiment 79, wherein the membrane-bound cytokine is membrane-bound IL-2 (mbIL-2); membrane-bound IL-15 (mbIL-15); or membrane-bound IL-21 (mbiL-21); or a combination thereof.
81. The method of any one of embodiments 73 to 80, wherein the CAR comprises 1) an antigen binding domain; 2) Flexible connectors (spacers); 3) A transmembrane region; and 4) an intracellular signaling domain.
82. The method of embodiment 81, wherein the antigen binding domain targets a tumor antigen.
83. The method of embodiment 81 or 82, wherein the antigen binding domain is a single chain variable fragment (scFv).
84. The method of any one of embodiments 81-83, wherein the intracellular signaling domain comprises one or more signaling domains from cd3ζ, DAP10, DAP12, CD28, 4-1BB, or OX 40.
85. The method of any one of embodiments 81-84, wherein the intracellular signaling domain comprises two or more signaling domains from CD28, 4-1BB or OX 40.
86. The method of any one of embodiments 73-85, wherein the heterologous nucleic acid encoding the CAR is introduced under conditions that stably integrate into the genome of the g-NK cell.
87. The method of any one of embodiments 73-86, wherein the heterologous nucleic acid encoding the CAR is contained in a viral vector and introduced into the g-NK cell by transduction
88. The method of embodiment 87, wherein the viral vector is a lentiviral vector.
89. The method of any one of embodiments 73-85, wherein the nucleic acid encoding the CAR is introduced under conditions of transient expression in the g-NK cells.
90. The method of any one of embodiments 73-85 and 89, wherein the nucleic acid encoding the CAR is introduced by non-viral delivery.
91. The method of any one of embodiments 73 to 85, 89, and 90, wherein the nucleic acid encoding the CAR is introduced into the g-NK cell via a lipid nanoparticle.
92. The method of any one of embodiments 73-91, wherein the nucleic acid encoding the CAR is DNA.
93. The method of any one of embodiments 73-85 and 89-91, wherein the nucleic acid encoding the CAR is RNA.
94. The method of embodiment 93, wherein the RNA is mRNA.
95. The method of any one of embodiments 73 to 85 and 89 to 94, wherein the nucleic acid is introduced into the g-NK cells via electroporation.
96. The method of any one of embodiments 73 to 95, wherein the nucleic acid encoding the immunomodulator is introduced under conditions that stably integrate into the genome of the g-NK cell.
97. The method according to any one of embodiments 73 to 96, wherein the nucleic acid encoding an immunomodulatory agent is contained in a viral vector and introduced into the g-NK cell by transduction.
98. The method of embodiment 97, wherein the viral vector is a lentiviral vector.
99. The method of any one of embodiments 73 to 95, wherein the nucleic acid encoding the immunomodulator is introduced under conditions of transient expression in the g-NK cells.
100. The method of any one of embodiments 73 to 95 and 99, wherein the nucleic acid encoding the immunomodulatory agent is introduced by non-viral delivery.
101. The method of any one of embodiments 73 to 95, 99 and 100, wherein the nucleic acid encoding the immunomodulatory agent is introduced into the g-NK cells via a lipid nanoparticle.
102. The method according to any one of embodiments 96-101, wherein the nucleic acid encoding the immunomodulator is DNA.
103. The method according to any one of embodiments 99 to 101, wherein the nucleic acid encoding the immunomodulator is RNA.
104. The method of embodiment 103, wherein the RNA is mRNA.
105. The method of any one of embodiments 73 to 85 and 99 to 104, wherein the nucleic acid encoding the immunomodulatory agent is introduced into the g-NK cells via electroporation.
106. The method of embodiments 94 and 104, wherein the RNA is self-amplified mRNA.
107. The method of any one of embodiments 73-106, wherein the nucleic acid encoding the CAR and the nucleic acid encoding the immunomodulatory agent are encoded by the same polynucleotide and introduced together.
108. The method of embodiment 107, wherein the nucleic acid encoding the CAR and the nucleic acid encoding the immunomodulator are separated by a polycistronic element of a polynucleotide sequence.
109. The method of embodiment 108, wherein the polycistronic element is a self-cleaving peptide selected from the group consisting of T2A, P a and F2A.
110. The method of any one of embodiments 73-109, wherein the nucleic acid encoding the CAR and the nucleic acid encoding the immunomodulator are introduced simultaneously during an in vitro process for amplifying a population enriched in g-NK cells.
111. The method of any one of embodiments 73 to 110, wherein the g-NK cell composition is produced by in vitro expansion of NK cells enriched from a biological sample from a subject, the NK cells being: (i) Negative or low level for CD3 and positive for CD57 (CD 3 Negative of CD57 Positive and negative ) or (ii) negative or low level for CD3 and positive for CD56 (CD 3 Negative of CD56 Positive and negative ), wherein the enriched NK cells are cultured with irradiated HLA-e+ feeder cells and one or more recombinant cytokines.
112. The method of embodiment 111, wherein the one or more recombinant cytokines are selected from the group consisting of an effective amount of SCF, GSK3i, FLT3, IL-2, IL-6, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or a combination thereof.
113. The method of embodiment 111 or embodiment 112, wherein the culturing is performed in the presence of two or more recombinant cytokines, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21.
114. A method according to any one of embodiments 73 to 113, wherein the introducing is performed during a method for expanding FcR γ -deficient NK (g-NK) cells, the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21;
Wherein introducing (i) the nucleic acid encoding the CAR and/or (ii) the nucleic acid encoding the immunomodulator is performed after step (a) and before, during or after step (B), wherein steps (i) and (ii) are performed simultaneously or sequentially in any order, thereby producing an engineered NK cell population; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR and an immunomodulatory agent.
115. A method according to any one of embodiments 73 to 113, wherein the introducing is performed during a method for amplifying FcR γ -deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject;
(B) Introducing (i) a nucleic acid encoding a CAR and (ii) a nucleic acid encoding an immunomodulatory agent into the enriched NK cell population, wherein steps (i) and (ii) are performed simultaneously or sequentially in any order, thereby producing an engineered NK cell population; and
(C) Culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR and an immunomodulatory agent.
116. A method according to any one of embodiments 73 to 113, wherein the introducing is performed during a method for amplifying FcR γ -deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Culturing the enriched NK cell population in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and
(C) Introducing (i) said nucleic acid encoding a CAR and (ii) said nucleic acid encoding an immunomodulator into NK cells of said amplified population of NK cells, wherein steps (i) and (ii) are performed simultaneously or sequentially in any order,
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR and an immunomodulatory agent.
117. A method according to any one of embodiments 73 to 113, wherein the introducing is performed during a method for amplifying FcR γ -deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Performing a first expansion comprising culturing the enriched NK cell population in a medium under conditions to expand the NK cells to produce a first expanded population of NK cells;
(C) Introducing (i) the nucleic acid encoding a CAR and (ii) the nucleic acid encoding an immunomodulatory agent into NK cells of the first amplified population of NK cells, wherein steps (i) and (ii) are performed simultaneously or sequentially in any order, thereby producing an engineered NK cell population; and
(D) Performing a second expansion comprising culturing said population of engineered NK cells under conditions that further expand said NK cells,
Wherein the first expansion and/or second expansion comprises culturing the enriched NK cell population in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR and an immunomodulatory agent.
118. The method of any one of embodiments 111 to 117, wherein the population of NK cell enriched primary human cells is obtained by selecting cells from a biological sample from a human subject, the cells being: (i) Negative or low level for CD3 and positive for CD57 (CD 3 Negative of CD57 Positive and negative ) or (ii) negative or low level for CD3 and positive for CD56 (CD 3 Negative of CD56 Positive and negative ).
119. The method of embodiment 118, wherein:
The population enriched for NK cells is a cell obtained by further selecting cells positive for NKG2C (NKG 2C Positive and negative );
The population enriched for NK cells is a cell obtained by further selecting cells negative for NKG2A or low in level (NKG 2A Negative of ); or alternatively
The population enriched for NK cells is a cell obtained by further selecting cells positive for NKG2C and negative or low level for NKG2A (NKG 2C Positive and negative NKG2A Negative of ).
120. The method of any one of embodiments 111 to 118, wherein the human subject is a subject: wherein at least or at least about 20% of Natural Killer (NK) cells in a peripheral blood sample from the subject are positive for NKG2C (NKG 2C Positive and negative ) and at least 70% of NK cells in the peripheral blood sample are negative for NKG2A or low level (NKG 2A Negative of ).
121. The method of any one of embodiments 111 to 120, wherein the subject is CMV seropositive.
122. The method of any one of embodiments 111 to 121, wherein the percentage of g-NK cells in the biological sample from the subject is greater than 5% or greater than about 5%, greater than 10% or greater than about 10%, or greater than 30% or greater than about 30%.
123. The method of any one of embodiments 111 to 122, wherein the percentage of g-NK cells in the enriched NK cell population is between 20% and 90%, or between about 20% and about 90%, or between 40% and 90%, or between about 40% and about 90%, or between 60% and 90%, or between about 60% and about 90%.
124. The method of any one of embodiments 111 to 123, wherein the population enriched for NK cells is a cell that is negative or low level for CD3 and positive for CD57 (CD 3 Negative of CD57 Positive and negative ) selected from the biological sample.
125. The method of any one of embodiments 111 to 123, wherein the population enriched for NK cells is a cell that is negative or low level for CD3 and positive for CD56 (CD 3 Negative of CD56 Positive and negative ) selected from the biological sample.
126. The method of any one of embodiments 113-125, wherein the two or more recombinant cytokines further comprise an effective amount of SCF, GSK3i, FLT3, IL-6, IL-7, IL-15, IL-12, IL-18, IL-27, or a combination thereof.
127. The method of any one of embodiments 111 to 125, wherein the recombinant cytokine is IL-21 and IL-2.
128. The method of any one of embodiments 111 to 126, wherein the recombinant cytokine is IL-21, IL-2, and IL-15.
129. The method of any one of embodiments 111 to 128, wherein recombinant IL-21 is added to the culture medium at a concentration of 10ng/mL or about 10ng/mL to about 100ng/mL during at least a portion of the culturing.
130. The method of any one of embodiments 111-129, wherein recombinant IL-21 is added to the culture medium at a concentration of 25ng/mL or about 25ng/mL during at least a portion of the culturing.
131. The method of any one of embodiments 111 to 130, wherein recombinant IL-2 is added to the culture medium at a concentration of 10IU/mL to about 500IU/mL or about 10IU/mL to about 500IU/mL during at least a portion of the culturing.
132. The method of any one of embodiments 111 to 131, wherein recombinant IL-2 is added to the culture medium at a concentration of 100IU/mL or about 100IU/mL during at least a portion of the culturing.
133. The method of any one of embodiments 111 to 132, wherein recombinant IL-2 is added to the culture medium at a concentration of 500IU/mL or about 500IU/mL during at least a portion of the culturing.
134. The method of any one of embodiments 111 to 126 and 128 to 134, wherein recombinant IL-15 is added to the medium at a concentration of 1ng/mL to 50ng/mL or about 1ng/mL to about 50ng/mL during at least a portion of the culturing.
135. The method of any one of embodiments 111-126 and 128-134, wherein recombinant IL-15 is added to the culture medium at a concentration of 10ng/mL or about 10ng/mL during at least a portion of the culturing.
136. The method of any one of embodiments 111 to 135, wherein the adding of the recombinant cytokine to the culture medium is initiated at or about the beginning of the culturing.
137. The method of any one of embodiments 111 to 136, wherein the method further comprises replacing the medium one or more times during the culturing, wherein fresh medium containing the recombinant cytokine is added each time the medium is replaced.
138. The method of embodiment 137, wherein said replacing of said medium is performed every two or three days during the duration of said culturing.
139. The method of embodiment 137 or embodiment 138, wherein the medium exchange is performed up to 5 days after the first amplification without a medium exchange, optionally up to 5 days after the first amplification without a medium exchange.
140. The method of any one of embodiments 111 to 139, wherein the human subject has a CD16 158V/V NK cell genotype or a CD16 158V/F NK cell genotype, optionally wherein the biological sample is from a human subject selected for the CD16 158V/V NK cell genotype or the CD16 158V/F NK cell genotype.
141. The method of any one of embodiments 111 to 140, wherein the biological sample is or comprises Peripheral Blood Mononuclear Cells (PBMCs), optionally a blood sample, an apheresis sample, or a leucocyte apheresis sample.
142. The method of any one of embodiments 111 to 141, wherein the HLA-e+ feeder cells are K562 cells (K562-HLA-E) transformed with HLA-E.
143. The method of any one of embodiments 111 to 145, wherein the HLA-e+ feeder cells are 221.Aeh cells.
144. The method of any one of embodiments 111 to 143, wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is between 1:1 and 5:1, inclusive, between 1:1 and 3:1, inclusive, optionally at or about 2.5:1 or at or about 2:1 or about 1:1.
145. The method of any one of embodiments 111 to 144, wherein the recombinant cytokine added to the medium during at least a portion of the culturing is 500IU/mL IL-2, 10ng/mL IL-15, and 25ng/mL IL-21.
146. The method of any one of embodiments 111 to 145, wherein the enriched NK cell population comprises the following enriched NK cells: the population of cells optionally comprises enriched cells at the end of each of between 2.0×10 5 enriched NK cells and 5.0×10 7 enriched NK cells or between about 2.0×10 5 enriched NK cells and about 5.0×10 7 enriched NK cells, between 1.0×10 6 enriched NK cells and 1.0×10 8 enriched NK cells or between about 1.0×10 6 enriched NK cells and about 1.0×10 8 enriched NK cells, between 1.0×10 7 enriched NK cells and 5.0×10 8 enriched NK cells or between about 1.0×10 7 enriched NK cells and about 5.0×10 8 enriched NK cells or between 1.0×10 7 enriched NK cells and 1.0×10 9 enriched NK cells or between about 1.0×10 7 enriched NK cells and about 1.0×10 9 enriched NK cells, including enriched cells at the end of each of said population optionally comprising enriched cells of about 1×25 NK cells.
147. The method of any one of embodiments 111 to 146, wherein the enriched NK cell population has a concentration at the beginning of the culturing of between 0.05 x 10 6 enriched NK cells/mL and 1.0 x 10 6 enriched NK cells/mL or between about 0.05 x 10 6 enriched NK cells/mL and about 1.0 x 10 6 enriched NK cells/mL or between about 0.05 x 10 6 enriched NK cells/mL and 0.5 x 10 6 enriched NK cells/mL, optionally wherein the enriched NK cell population has or has a concentration of about 0.2 x 10 6 enriched NK cells/mL at the beginning of the culturing.
148. The method of any one of embodiments 111 to 146, wherein the culturing is performed until the method achieves a time to expand at least or at least about 2.50 x 10 8 g-NK cells, at least or at least about 5.00 x 10 8 g-NK cells, at least or at least about 1.0 x 10 9 g-NK cells, or at least about 5.0 x 10 9 g-NK cells.
149. The method of any one of embodiments 111 to 148, wherein the culturing is performed or performed about or at least about the following time: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 days.
150. The method of any one of embodiments 111 to 149, further comprising collecting the expanded population of engineered NK cells produced by the method.
151. The method of any one of embodiments 111 to 150, further comprising formulating the expanded population of engineered NK cells in a pharmaceutically acceptable excipient.
152. The method of embodiment 151, further comprising formulating the expanded population of engineered NK cells with a serum-free cryopreservation medium comprising a cryoprotectant.
153. The method of embodiment 152, wherein the cryoprotectant is DMSO, optionally wherein the cryoprotectant is DMSO and the cryopreservation medium is 5% to 10% DMSO (v/v), optionally at or about 10% DMSO (v/v).
154. The method of any one of embodiments 111 to 153, wherein in the engineered NK cell-expanded population produced by the method, greater than 50% of the population is FcR gamma Negative of , greater than 60% of the population is FcR gamma Negative of , greater than 70% of the population is FcR gamma Negative of ,, greater than 80% of the population is FcR gamma Negative of , greater than 90% of the population is FcR gamma Negative of , or greater than 95% of the population is FcR gamma Negative of .
155. A composition comprising a plurality of engineered NK cells produced by the method according to any one of embodiments 73 to 154.
156. A method of treating a disease or disorder in a subject, the method comprising administering to an individual in need thereof an effective amount of cells of the composition of any one of embodiments 25-72 and 155.
157. The method of embodiment 156, wherein the disease or disorder is selected from an inflammatory disorder, an infection, or a cancer.
158. The method of embodiment 156 or embodiment 157, wherein the disease or disorder is cancer and the cancer is leukemia, lymphoma, or myeloma.
159. The method of embodiment 156 or embodiment 157, wherein the disease or disorder is cancer and the cancer comprises a solid tumor.
160. The method of embodiment 159, wherein the cancer is selected from the group consisting of adenocarcinoma of the stomach or gastroesophageal junction, bladder cancer, breast cancer, brain cancer, cervical cancer, colorectal cancer, endocrine/neuroendocrine cancer, head and neck cancer, gastrointestinal stromal cancer, bone giant cell tumor, kidney cancer, liver cancer, lung cancer, neuroblastoma, ovarian epithelial/fallopian tube/primary peritoneal cancer, pancreatic cancer, prostate cancer, skin cancer, and soft tissue cancer.
161. The method according to any one of embodiments 156-160, wherein the composition is administered as monotherapy.
162. The method of any one of embodiments 156 to 161, comprising administering to the individual 1 x 10 5 to 50 x 10 9 or about 1 x 10 5 to about 50 x 10 9 cells of the g-NK cell composition.
163. The method of any one of embodiments 156 to 162, comprising administering 1 x 10 8 cells to 50 x 10 9 NK cells or about 1 x 10 8 cells to about 50 x 10 9 NK cells of said g-NK cell composition, optionally 5 x 10 8 cells or about 5 x 10 8 cells of said g-NK cell composition, 5 x 10 9 cells or about 5 x 10 9 cells of said g-NK cell composition, or 10 x 10 9 cells or about 10 x 10 9 cells of said g-NK cell composition.
164. The method of any one of embodiments 156 to 163, wherein said subject has received lymphocyte removal therapy prior to said administering said dose of g-NK cells.
165. The method of embodiment 164, wherein the lymphocyte depletion therapy comprises fludarabine and/or cyclophosphamide.
166. The method of embodiment 164 or embodiment 165, wherein the lymphoremoval comprises the administration of fludarabine at 20mg/m 2 to 40mg/m 2 or about 20mg/m 2 to about 40mg/m 2 of body surface area of the subject, optionally at or about 30mg/m 2, for 2 to 4 days per day, and/or the administration of cyclophosphamide at 200mg/m 2 to 400mg/m 2 or about 200mg/m 2 to about 400mg/m 2 of body surface area of the subject, optionally at or about 300mg/m 2, per day for 2 to 4 days.
167. The method of any one of embodiments 164 to 166, wherein the lymphocyte depletion therapy comprises fludarabine and cyclophosphamide.
168. The method of any one of embodiments 164 to 167, wherein the lymphoremoval therapy comprises the administration of fludarabine at 30mg/m 2 or about 30mg/m 2 of the subject body surface area per day, and cyclophosphamide at 300mg/m 2 or about 300mg/m 2 of the subject body surface area per day, each for 2 to 4 days, optionally for 3 days.
169. The method of any one of embodiments 164 to 168, wherein administering a dose of g-NK cells is initiated within two weeks or at or about two weeks after initiation of the lymphocyte depletion therapy.
170. The method of any one of embodiments 156-169, wherein the subject is a human.
171. The method of any one of embodiments 156 to 170 wherein the NK cells in the composition are allogeneic to the individual.
172. The method of any one of embodiments 156 to 170 wherein the NK cells in the composition are autologous to the subject.
IX. example
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1: amplification of g-NK cells in the Presence of different cytokines
50ML of fresh whole blood from CMV seropositive donors (NKG 2C Positive and negative and NKG2A Negative of NK-cell percentages of 56.24% and 11.68%, respectively) was collected into ACD vacuum blood collection tubes and diluted with PBS 1:1. According to the instruction of the manufacturer byPBMC were isolated by density centrifugation. After harvesting the PBMC-containing buffy coats, PBMCs were washed with PBS and counted. After cell counting, magnetic bead separation was performed to increase the frequency of g-NK cells. Magnetic bead separation is CD3 depletion followed by CD57 enrichment to isolate CD57 Positive and negative NK cells.
Transgenic lymphoma cell lines 221.AEH (Lee et al, 1998, journal of Immunology, vol.160: pages 4951-4960) and transgenic leukemia cell lines K562-mb15-41BBL (Fujisaki et al, 2009, CANCER RESEARCH, vol.69, 9: pages 4010-4017) were prepared as feeder cells for NK cell expansion. Feeder cells were taken from fresh cultures (i.e., rather than cryopreserved stock) and irradiated prior to use. AEH and K562-mb15-41BBL cells were expanded, with an seeding density of 5X 10 5 cells/mL and a subculturing density of 2X 10 5 cells/mL. The medium used to grow 221.AEH feeder cells was RPMI-1640 with 10% FBS and 200. Mu.g/mL hygromycin B. The medium used to grow K562-mb15-41BBL feeder cells was RPMI-1640 with 10% FBS.
Non-cryopreserved NK cells were expanded under four different conditions: IL-2 at a ratio of 2:1AEH: NK cells to 500 IU/mL; IL-2 was added at a ratio of 2:1K562-mb15-41BBL to NK cells of 500 IU/mL; IL-2 was added at a ratio of 1:1:1AEH: K562-mb15-41BBL: NK cells to 500 IU/mL; IL-2 at a ratio of 2:1AEH to NK cells of 500IU/mL, IL-15 at 10ng/mL and IL-21 at 25 ng/mL. All amplifications were performed in CellGenix GMP SCGM medium supplemented with 5% human AB serum and the corresponding cytokines. Co-cultured cells were cultured at 37℃in 5% CO 2 for 21 days. Cells were counted each time media was changed or replenished (day 5, day 7, day 10, day 13, day 16, day 19 and day 21) and the percentage of g-NK was assessed by flow cytometry on day 0, day 13 and day 21.
As shown in fig. 1A-1B, the addition of IL-21 to the expansion medium resulted in a significant increase in g-NK cell expansion. Total g-NK cell count (fcer1γ -deficient cells, also interchangeably referred to herein as fcrγ) is highest for g-NK cells expanded in the presence of IL-21 (fig. 1A). For the IL-21 in the presence of amplified g-NK cells, by 21 days g-NK cell fold expansion is also highest (figure 1B).
Taken together, these results indicate that the presence of IL-21 improves the expansion of g-NK cells.
Example 2: cellular effector function of g-NK cells amplified in the presence of different cytokines
In this study, NK cell effector function was measured in the presence of different feeder cells and cytokines, including in g-NK cells expanded in the presence of IL-21 as described in example 1. Target cell lines LP1 and MM.1S at a ratio of 0.5:1NK:MM cells were used and assayed using antibodies to Lei Tuoyou mAb and erlotinib as described below.
A. Cell-mediated cytotoxicity
After thawing the expanded NK cells, 10 4 NK cells were co-cultured with MM target cells in a 1:1NK cell to MM cell ratio in the presence of 1. Mu.g/mL up to Lei Tuoyou mAb (anti-CD 38) or 1. Mu.g/mL erlotinib (anti-CD 319). After 4 hours of incubation in a CO 2 incubator at 37 ℃, the cells were washed and stained with anti-CD 3 and CD56 antibodies to quantify the number of NK cells. After the last wash, propidium Iodide (PI) was added and the number of NK cells, live and dead target cells was resolved using 4-color flow cytometry (Bigley et al, 2016, clin. Exp. Immunol., volume 185: pages 239-251).
As shown in fig. 2A-2B, g-NK cells expanded in the presence of IL-21 for 21 days were stronger against cell-mediated cytotoxicity of CD38 High height MM cell line LP1 (fig. 2A) and SLAMF7 High height MM cell line mm.1s (fig. 2B) than the g-NK cells expanded in the absence of IL-21. In the absence of antibodies and in the presence of up to Lei Tuoyou mab or erlotinib, greater cell-mediated cytotoxicity of IL-21 expanded g-NK cells was observed.
Taken together, these results indicate that g-NK cells expanded in the presence of IL-21 have enhanced cell-mediated cytotoxicity against tumor cells compared to g-NK cells expanded in the absence of IL-21.
B. degranulation process
After thawing the expanded NK cells, 2.0X10. 10 5 NK cells were co-cultured with MM target cells in a ratio of 1:1NK cells to MM cells in the presence of 1. Mu.g/mL up to Lei Tuoyou mab or 1. Mu.g/mL of erlotinib. For the degranulation assay, 2 μl of VioGreen conjugated anti-CD 107a was added to the CO-culture, incubated in a CO 2 incubator at 37 ℃ for one hour, followed by 4 μl of BD GolgiStop containing monensin. For cytokine expression assays, 6 μl of BD GolgiStop containing briaferin a was added. The cells were then cultured at 37℃for a further five hours in a CO2 incubator. After incubation, cells were harvested, washed and stained with 0.5 μl of anti-CD 45 antibody, 0.5 μl of anti-CD 3 antibody and 1 μl of anti-CD 56 antibody (all antibodies were purchased from Miltenyi Biotec). Cells were then fixed and permeabilized using an internal staining kit from Miltenyi Biotec according to the manufacturer's instructions. Cells were then stained with 1 μl of anti-fcrγ,2 μl of anti-perforin, 2 μl of anti-granzyme B, 2 μl of interferon- γ, and 2 μl of TNF- α antibodies, as described in table E1. After the last wash, cells were resolved using eight-color flow cytometry.
Table e1. Antibody panel for functional assays.
As shown in fig. 3A-3D, g-NK cells expanded in the presence of IL-21 were degranulated more against CD38 High height MM cell line LP1 (fig. 3A and 3C) and SLAMF7 High height MM cell line mm.1s (fig. 3B and 3D) than the g-NK cells expanded without IL-21 after 13 days of expansion (fig. 3A-3B) and 21 days (fig. 3C-3D). In the absence of antibodies and in the presence of up to Lei Tuoyou mab or erlotinib, greater degranulation of IL-21 expanded g-NK cells was observed.
Taken together, these results demonstrate that g-NK cells expanded in the presence of IL-21 have enhanced degranulation against tumor cells compared to g-NK cells expanded in the absence of IL-21.
C. expression of perforin and granzyme B
As shown in fig. 4A-4D, after 13 days of expansion (fig. 4A-4B) and 21 days (fig. 4C-4D), g-NK cells expanded in the presence of IL-21 expressed more cytolytic perforin than g-NK cells expanded in the absence of IL-21 as measured by the percentage of perforin-positive cells (fig. 4A and 4C) and total perforin expression (MFI) (fig. 4B and 4D). Furthermore, after 13 and 21 days of expansion, g-NK cells expanded in the presence of IL-21 expressed more pro-apoptotic protease granzyme B than g-NK cells expanded in the absence of IL-21 as measured by the percentage of granzyme B positive cells (fig. 4A and 4C) and total granzyme B expression (MFI) (fig. 4B and 4D).
Taken together, these results indicate that g-NK cells expanded in the presence of IL-21 have enhanced expression of perforin and granzyme B compared to g-NK cells expanded in the absence of IL-21.
D. Expression of interferon-gamma
As shown in fig. 5A-5D, g-NK cells expanded in the presence of IL-21 expressed more interferon- γ against CD38 High height MM cell line LP1 (fig. 5A and 5C) and SLAMF7 High height MM cell line mm.1s (fig. 5B and 5D) after 13 days of expansion (fig. 5A-5B) and 21 days (fig. 5C-5D) than in the absence of IL-21. In the absence of antibodies and in the presence of up to Lei Tuoyou mab or erlotinib, greater expression of interferon-gamma was observed in IL-21 expanded g-NK cells.
Taken together, these results indicate that g-NK cells expanded in the presence of IL-21 have enhanced expression of interferon-gamma against tumor cells compared to g-NK cells expanded in the absence of IL-21.
Expression of TNF-alpha
As shown in fig. 6A-6D, g-NK cells expanded in the presence of IL-21 expressed more TNF-a against CD38 High height MM cell line LP1 (fig. 6A and 6C) and SLAMF7 High height MM cell line mm.1s (fig. 6B and 6D) after 13 days of expansion (fig. 6A-6B) and 21 days (fig. 6C-6D) than in the absence of IL-21. In the absence of antibodies and in the presence of up to Lei Tuoyou mab or erlotinib, greater expression of TNF- α was observed in IL-21 expanded g-NK cells.
Taken together, these results indicate that g-NK cells expanded in the presence of IL-21 have enhanced expression of TNF- α against tumor cells compared to g-NK cells expanded in the absence of IL-21.
Example 3: amplification of g-NK cells in the Presence of additional cytokines
In another study, the rate of expansion of NK cells expanded in the presence of various combinations of cytokine mixtures and concentrations was compared. NK cells were harvested from the same donor as in example 1 as described above. NK cells were seeded at a density of 2X 10 5 cells/mL and subcultured density and co-cultured with irradiated 221.AEH feeder cells at a ratio of 2:1. AEH: NK cells. For NK cell expansion, the following concentrations of cytokines were added: IL-2 at 100IU/mL (low IL-2) or 500IU/mL (IL-2); IL-15 at 10 ng/mL; IL-21 at 25 ng/mL; IL-12 at 10 ng/mL; IL-18 at 10 ng/mL; and/or 10ng/mL IL-27. All amplifications were performed in CellGenix GMP SCGM medium supplemented with 5% human AB serum and the corresponding cytokines.
As shown in FIG. 7, NK cells expanded in the presence of IL-21 have higher g-NK cell expansion rate than NK cells expanded in the presence of IL-2 and IL-15, IL-12, IL-15 and IL-18, and IL-15, IL-18 and IL-27 themselves. The combination of cytokines that results in the highest g-NK cell expansion rate in the presence or absence of IL-15 is IL-2 and IL-21.
Taken together, these results indicate that the presence of IL-21 improves the rate of expansion of g-NK cells more than other cytokine mixtures.
Example 4: cellular effector function of g-NK cells amplified in the presence of additional cytokines
NK cell effector function was measured in g-NK cells expanded for 15 days in the presence of cytokines including in the presence of IL-21 as described in example 3. Target cell lines LP1 and MM.1S at a ratio of 0.5:1NK:MM cells were used and antibody up to Lei Tuoyou mab and erlotinib were used for assays as described in example 2.
A. Cell-mediated cytotoxicity
As shown in FIGS. 8A and 8B, g-NK cells expanded in the presence of IL-2, IL-15 and IL-21 have greater cell-mediated cytotoxicity against CD38 High height MM cell line LP1 (FIG. 8A) and SLAMF7 High height MM cell line MM.1S (FIG. 8B) than g-NK cells expanded in the presence of IL-2 and IL-15. In the absence of antibodies and in the presence of up to Lei Tuoyou mab or erlotinib, greater cell-mediated cytotoxicity was observed for g-NK cells expanded in the presence of IL-2, IL-15 and IL-21.
Taken together, these results indicate that g-NK cells expanded in the presence of IL-2, IL-15 and IL-21 have enhanced cell-mediated cytotoxicity against tumor cells compared to g-NK cells expanded in the presence of IL-2 and IL-15.
B. degranulation process
As shown in FIGS. 8C and 8D, g-NK cells expanded in the presence of IL-2, IL-15 and IL-21 were degranulated more against CD38 High height MM cell line LP1 (FIG. 8C) and SLAMF7 High height MM cell line MM.1S (FIG. 8D) than g-NK cells expanded in the presence of IL-2 and IL-15. For g-NK cells expanded in the presence of IL-2, IL-15 and IL-21, greater degranulation was observed under all conditions, including in the absence of antibodies.
Taken together, these results demonstrate that g-NK cells expanded in the presence of IL-2, IL-15 and IL-21 have enhanced degranulation against tumor cells compared to g-NK cells expanded in the presence of IL-2 and IL-15.
C. expression of perforin and granzyme B
As shown in fig. 8E and 8F, g-NK cells expanded in the presence of IL-2, IL-15 and IL-21 expressed more of the cytolytic protein perforin than the g-NK cells expanded in the presence of IL-2 and IL-15 as measured by the percentage of perforin positive cells (fig. 8E) and total perforin expression (MFI) (fig. 8F). Furthermore, g-NK cells expanded in the presence of IL-2, IL-15 and IL-21 expressed more pro-apoptotic protease granzyme B than g-NK cells expanded in the presence of IL-2 and IL-15 as measured by the percentage of granzyme B positive cells (FIG. 8E) and total granzyme B expression (MFI) (FIG. 8F). Addition of IL-2, IL-15, IL-18, IL-21 and IL-27 to the amplification medium enhanced the expression of granzyme B of g-NK cells.
Taken together, these results indicate that g-NK cells expanded in the presence of IL-2, IL-15 and IL-21 have enhanced expression of perforin and granzyme B compared to g-NK cells expanded in the presence of IL-2 and IL-15.
D. Expression of interferon-gamma
As shown in FIGS. 8G-8H, G-NK cells expanded in the presence of IL-2, IL-15 and IL-21 expressed more interferon-gamma against CD38 High height MM cell line LP1 (FIG. 8G) and SLAMF7 High height MM cell line MM.1S (FIG. 8H) than G-NK cells expanded in the presence of IL-2 and IL-15. For g-NK cells expanded in the presence of IL-2, IL-15 and IL-21, greater expression of interferon-gamma was observed under all conditions, including in the absence of antibodies. The addition of IL-2, IL-12, IL-15, IL-18 and IL-21 to the amplification medium enhanced the expression of interferon-gamma by g-NK cells under all conditions, including in the absence of antibodies. The addition of IL-2, IL-15, IL-18, IL-21 and IL-27 to the amplification medium enhanced the expression of interferon-gamma by g-NK cells under all conditions, including in the absence of antibodies.
Taken together, these results indicate that g-NK cells expanded in the presence of IL-2, IL-15 and IL-21 have enhanced expression of interferon-gamma against tumor cells compared to g-NK cells expanded in the presence of IL-2 and IL-15.
Expression of TNF-alpha
As shown in FIGS. 8I-8J, g-NK cells expanded in the presence of IL-2, IL-15 and IL-21 expressed more TNF- α against CD38 High height MM cell line LP1 (FIG. 8I) and SLAMF7 High height MM cell line MM.1S (FIG. 8J) than g-NK cells expanded in the presence of IL-2 and IL-15. For g-NK cells expanded in the presence of IL-2, IL-15 and IL-21, greater expression of TNF- α was observed under all conditions, including in the absence of antibodies. The addition of IL-2, IL-15, IL-18, IL-21 and IL-27 to the amplification medium enhanced antibody-induced expression of TNF- α by g-NK cells under all conditions, including in the absence of antibodies.
Taken together, these results indicate that g-NK cells expanded in the presence of IL-2, IL-15 and IL-21 have enhanced expression of TNF- α against tumor cells compared to g-NK cells expanded in the presence of IL-2 and IL-15.
Example 5: amplification and cellular effector function of g-NK cells amplified in the Presence of IL-21
In this study, the rate of expansion and NK cell effector function of NK cells expanded in the presence of IL-21 were compared with those of NK cells expanded in the absence of IL-21. According to the instruction of the manufacturer byDensity centrifugation human Peripheral Blood Mononuclear Cells (PBMC) were isolated from whole blood from CMV positive human donors or CMV seronegative donors for comparison. The donors were CMV seropositive (n=8) and CMV seronegative (n=6) (age 37.8±10.6 years; 8 men and 6 women).
PBMCs were harvested from the buffy coat, washed, and surviving CD45 Positive and negative cells were assessed by flow cytometry. NK cells were enriched by immunoaffinity-based magnetic bead isolation using MILTENYI MACS TM microbeads, i.e. by depletion of CD3 Positive and negative cells to remove T cells (CD 3 depletion, CD3 Negative of ) or by depletion of CD3 followed by positive selection of CD57 to enrich for CD57 Positive and negative NK cells (CD 3 Negative of CD57 Positive and negative ). The latter method of initially enriching for CD3 Negative of /CD57 Positive and negative cells prior to expansion was used in subsequent experiments to expand g-NK cells. As a further comparison, NK cells were enriched by CD3 depletion followed by positive selection of CD16 (enriched CD16 Positive and negative NK cells and monocytes (CD 3 negative CD57 positive). If PBMC had been cryopreserved prior to NK cell enrichment, NK cells were seeded at a density of 2X 10 5 cells/mL and NK cells were seeded at a subculture density of 2X 10 5 cells/mL. NK cells were co-cultured with gamma irradiated (100 Gy) 221.AEH feeder cells at a ratio of 2:1. AEH: NK cells and expanded in the presence of IL-2 (500 IU/mL), IL-15 (10 ng/mL) and IL-21 (25 ng/mL), or IL-2 alone (500 IU/mL). If PBMC had been cryopreserved prior to NK cell enrichment, 1:1 irradiated 221.AEH cells: NK cells were used, as further described in example 6. All expanded medium was replaced with 5% human AB serum and corresponding cytokines in CellGenix GMP SCGM medium and every 2 days and the expanded medium was used for the subsequent function of cryopreservation of 90% FBS cells.
Expansion and cellular effector function were assessed 14 days after expansion. Target cell lines LP1 and MM.1S at a ratio of 0.5:1NK:MM cells were used and antibody up to Lei Tuoyou mab and erlotinib were used for assays as described in example 2.
In some of the studies described in the examples that follow, the phenotypic and functional activity of g-NK cells was compared with cNK cells. Since the yield of cNK cells from CMV seronegative donors was insufficient and preferential expansion of g-NK cells from CMV seropositive donors using the methods described above (results are described in section a below), cNK cells were expanded using alternative methods for in vitro function and in vivo studies. The expansion method uses K652-mbiL15-41BBL feeder cells and 500IU/mL IL-2 to expand cNK cells 180+ -89-fold (n=5CMV Negative of ) over 2 weeks (Fujisaki et al 2009, cancer Res., volume 68, 9: pages 4010-4017). In 5CMV negative donors (age 38.9.+ -. 9.8 years; 3 men and 2 women), the proportion of g-NK cells was 1.5.+ -. 0.5% before expansion and 1.6.+ -. 0.4% after expansion.
A.g-NK cell expansion rate
Cells were counted at medium change and the percentage of g-NK cells was assessed by flow cytometry on day 0 and day 14. As shown in FIGS. 9A and 9B, NK cells initially enriched for CD3 Negative of /CD57 Positive and negative cells prior to expansion and then expanded in the presence of IL-21 had higher expansion rates of g-NK cells than those under similar conditions but without IL-21. As measured using intracellular staining and flow cytometry of FcR gamma, higher rates of expansion of g-NK cells were observed when both the percentage of g-NK cells (fig. 9A) and the count (fig. 9B) were measured.
Prior to expansion, the proportion of g-NK cells in CMV seropositive donors was 30.8.+ -. 3.1% (total NK cells%) whereas the proportion of g-NK cells in CMV seronegative donors was only 1.8.+ -. 0.3% (total NK cells%). After expansion after initial enrichment of CD3 Negative of /CD57 Positive and negative cells, the proportion of g-NK cells increased to 84.0±1.4% for CMV seropositive donors, but not for CMV seronegative donors (1.5±0.4%) (fig. 9C). Representative flow cytometry plots and histograms depicting the proportion of g-NK cells in CMV seropositive and seronegative donors are shown in fig. 9E and 9F. The percentage of NKG2C positive/NKG 2A negative NK cells in the g-NK subpopulation ranged from 1.7% to 51% (26.8±13.9%). Thus, there is a phenotypic overlap between g-NK and NKG2C Positive and negative /NKG2C Negative of NK cells, but they are not identical.
Representative expansion of g-NK cells is shown in FIG. 9D, where the expansion method is shown to increase the proportion of g-NK cells from CMV seropositive donors with detectable g-NK populations, where the total NK cell number is increased by at least 400-fold.
Taken together, these results indicate that the presence of IL-21 improves the expansion of g-NK cells.
B. Cell-mediated cytotoxicity
As shown in fig. 9G and 9H, NK cells expanded in the presence of IL-21 had greater cell-mediated cytotoxicity against CD38 High height MM cell line LP1 (fig. 9G) and SLAMF7 High height MM cell line mm.1s (fig. 9H) than G-NK cells expanded in the absence of IL-21. In the absence of antibodies and in the presence of up to Lei Tuoyou mab or erlotinib, greater cell-mediated cytotoxicity of IL-21 expanded g-NK cells was observed.
Taken together, these results indicate that g-NK cells expanded in the presence of IL-21 have enhanced cell-mediated cytotoxicity against tumor cells compared to g-NK cells expanded in the absence of IL-21.
C. degranulation process
As shown in FIGS. 9I and 9J, g-NK cells expanded in the presence of IL-21 were degranulated more against CD38 High height MM cell line LP1 (FIG. 9I) and SLAMF7 High height MM cell line MM.1S (FIG. 9J) than g-NK cells expanded in the absence of IL-21. In the absence of antibodies and in the presence of up to Lei Tuoyou mab or erlotinib, greater degranulation of IL-21 expanded g-NK cells was observed.
Taken together, these results demonstrate that g-NK cells expanded in the presence of IL-21 have enhanced degranulation against tumor cells compared to g-NK cells expanded in the absence of IL-21.
D. expression of perforin and granzyme B
As shown in fig. 9K and 9L, g-NK cells expanded in the presence of IL-21 expressed more lysin perforin than g-NK cells expanded without IL-21 as measured by total perforin expression (GMFI) (fig. 9L) rather than the percentage of perforin-positive cells (fig. 9K). Furthermore, g-NK cells expanded in the presence of IL-21 expressed more pro-apoptotic proteinase B than g-NK cells expanded in the absence of IL-21 as measured by the percentage of granzyme B positive cells (FIG. 9K) and total granzyme B expression (GMFI) (FIG. 9L).
Baseline expression of perforin (fig. 9M, left) and granzyme B (fig. 9M, right) was also significantly higher in expanded g-NK cells than cNK cells (n=5). Representative histogram of perforin and granzyme B expression of NK and cNK cells is shown in fig. 9N.
Taken together, these results indicate that g-NK cells expanded in the presence of IL-21 have enhanced expression of perforin and granzyme B against tumor cells compared to g-NK cells expanded in the absence of IL-21.
E. Expression of interferon-gamma
As shown in FIGS. 9O and 9P, g-NK cells expanded in the presence of IL-21 expressed more interferon-gamma against CD38 High height MM cell line LP1 (FIG. 9O) and SLAMF7 High height MM cell line MM.1S (FIG. 9P) than g-NK cells expanded in the absence of IL-21. In the absence of antibodies and in the presence of up to Lei Tuoyou mab or erlotinib, greater expression of interferon-gamma was observed in IL-21 expanded g-NK cells.
Taken together, these results indicate that g-NK cells expanded in the presence of IL-21 have enhanced expression of interferon-gamma against tumor cells compared to g-NK cells expanded in the absence of IL-21.
Expression of TNF-alpha
As shown in FIGS. 9Q and 9R, g-NK cells expanded in the presence of IL-21 expressed more TNF- α against CD38 High height MM cell line LP1 (FIG. 9Q) and SLAMF7 High height MM cell line MM.1S (FIG. 9R) than g-NK cells expanded in the absence of IL-21. In the absence of antibodies and in the presence of up to Lei Tuoyou mab or erlotinib, greater expression of TNF- α was observed in IL-21 expanded g-NK cells.
Taken together, these results indicate that g-NK cells expanded in the presence of IL-21 have enhanced expression of TNF- α against tumor cells compared to g-NK cells expanded in the absence of IL-21.
Comparison of effector functions in G.g-NK donors
G-NK cells and cNK cells were expanded as described and effector activity was compared between different donors. The assay was performed as described in example 2 using a target cell line mm.1s at a ratio of 0.5:1nk: mm cells and using antibodies to Lei Tuoyou mab and erlotinib. After co-culture, cells were fixed and permeabilized, and analyzed for interferon-gamma (ifnγ) and TNF-alpha (tnfα) by intracellular cytokine staining. The results depicted in fig. 9S (ifnγ) and fig. 9T (tnfα) show low donor variability between g-NK donors, with standard error of mAb-dependent ifnγ and tnfα responses less than 5. Similar results were also observed for other effector functions. The results indicate that the effector functions of all g-NK donors are superior to those of all cNK donors tested.
Example 6: amplification of g-NK cells in the Presence of IL-21/anti-IL-21 Complex
Cryopreserved PBMCs were thawed and enriched for CD3 Negative of CD57 Positive and negative NK cells via magnetic sorting. Prior to expansion of these NK cells, IL-21/anti-IL-21 complexes are formed by combining IL-21 with anti-IL-21 antibodies. IL-21 and anti-IL-21 antibodies were incubated at 37℃for 30 minutes at concentrations of 25ng/mL and 250ng/mL, respectively. The complex was then added to NK cell expansion medium with 500IU/mL IL-2 and 10ng/mL IL-15. NK cells were co-cultured with irradiated 221.AEH feeder cells at a ratio of 1:1NK:221.AEH feeder cells. For comparison, NK cells were also expanded in the presence of IL-2, IL-15 and IL-21 at concentrations of 500IU/mL, 10ng/mL and 25ng/mL, respectively.
As shown in FIG. 10, the g-NK cells expanded in the presence of IL-2, IL-15 and IL-21/anti-IL-21 complex had a higher expansion rate than the g-NK cells expanded in the presence of IL-2, IL-15 and IL-21.
Example 7: maintenance of g-NK cell effector function after cryopreservation
The NK cell effector function of previously cryopreserved g-NK cells was compared to that of freshly enriched (i.e., not cryopreserved) g-NK cells (n=4). CD3 Negative of /CD57 Positive and negative enriched NK cells were co-cultured with irradiated 221.AEH feeder cells at a 2:1 221.AEH: NK cell ratio and in the presence of 500IU/mL IL-2, 10ng/mL IL-15 and 25ng/mL IL-21. After expansion, NK cells were freshly evaluated for function or frozen in 90% FBS containing 10% DMSO at a concentration of 2000 ten thousand cells per 1.8ml of cryopreservation medium. NK cell effector function was assessed against LP1 and MM.1S cell lines in the absence of antibody and 1. Mu.g/mL up to Lei Tuoyou mab or 1. Mu.g/mL erlotinib.
A. Degranulation process
As shown in fig. 11A and 11B, previously cryopreserved g-NK cells had comparable degranulation levels to fresh g-NK cells against CD38 High height MM cell line LP1 (fig. 11A) and SLAMF7 High height MM cell line mm.1s (fig. 11B). Comparable degranulation levels were observed in the absence of antibodies and up to Lei Tuoyou mab or erlotinib.
Taken together, these results demonstrate that g-NK cell degranulation in response to multiple myeloma target cells is maintained after cryopreservation.
B. expression of perforin and granzyme B
As shown in FIGS. 11C and 11D, previously cryopreserved g-NK cells had perforin (FIG. 11C) and granzyme B expression comparable to fresh g-NK cells (FIG. 11D). Taken together, these results indicate that the expression of g-NK cell perforin and granzyme B is maintained after cryopreservation.
C. Expression of interferon-gamma
As shown in fig. 11E and 11F, previously cryopreserved g-NK cells had comparable expression levels of interferon- γ with fresh g-NK cells against CD38 High height MM cell line LP1 (fig. 11E) and SLAMF7 High height MM cell line mm.1s (fig. 11F). Comparable interferon-gamma expression was observed in the absence of antibodies and in the presence of up Lei Tuoyou mab or erlotinib.
Taken together, these results demonstrate that g-NK cell interferon-gamma expression in response to multiple myeloma target cells was maintained after cryopreservation.
TNF-alpha expression
As shown in fig. 11G and 11H, previously cryopreserved G-NK cells had reduced TNF- α expression levels compared to fresh G-NK cells against CD38 High height MM cell line LP1 (fig. 11G) and SLAMF7 High height MM cell line mm.1s (fig. 11H). Reduced TNF- α expression was observed in the absence of antibodies and in the presence of up Lei Tuoyou mab or erlotinib.
Taken together, these results demonstrate that g-NK cell TNF- α expression in response to multiple myeloma target cells is reduced after cryopreservation.
Example 8: evaluation of persistence of g-NK cells in vivo compared to cNK cells
NK cells expanded substantially as described in example 5 were injected into mice and biological samples were analyzed using flow cytometry to assess their persistence.
G-NK cells were expanded after initial enrichment of CD3 Negative of /CD57 Positive and negative cells from cryopreserved PBMC as described in example 5, followed by expansion with irradiated 221.AEH feeder cells at a ratio of 1:1. AEH: NK cells in the presence of IL-2 (500 IU/mL), IL-15 (10 ng/mL) and IL-21 (25 ng/mL) stimulatory cytokines. Due to insufficient yield of cNK cells from CMV seronegative donors, cNK cells were expanded using the alternative method described in example 5. cNK cells were expanded for 2 weeks using the transgenic leukemia cell line K562-mb15-41BBL and IL-2. All cells were expanded from cryopreserved PBMCs and cryopreserved feeder cells. The cryopreservation medium for the cells was CS-10 (Biolife Solutions, bothel, WA, USA). Cryopreserved cell products were thawed quickly in a hot water bath (37 ℃) prior to administration to mice.
A single dose of 1 x 10 7 expanded NK cells (fresh g-NK, cryopreserved g-NK or cryopreserved cNK cells) were injected intravenously via the tail vein into female nod. Cg-PrkDc scidIL2rgtm1Wjl/SzJ (NSG) mice (n=9, 3 per group). To provide NK cell support, about 2 μg/mouse human recombinant IL-15 was administered via the i.p. route every three days (see table 2). Blood collected on day 6, day 16, day 26 and day 31 after infusion was analyzed immediately by flow cytometry. Mice were sacrificed on day 31 and bone marrow and spleen were collected for immediate flow cytometry analysis.
TABLE 2 durability study design
Figures 12A-C show enhanced persistence of fresh and cryopreserved g-NK cells relative to cNK cells in peripheral blood (figure 12A), spleen (figure 12B) and bone marrow (figure 12C). The persistence of cryopreserved g-NK cells was 90% higher than that observed in peripheral blood for cryopreserved cNK cells at multiple time points (p < 0.001) (fig. 12A) and at day 31 at the time of sacrifice (p < 0.001) spleen (p < 0.001) (fig. 12B) and bone marrow (p < 0.05) (fig. 12C). Fig. 12A also shows that the levels of fresh and cryopreserved g-NK cells continued at comparable levels until at least day 26 of the study.
The results are consistent with the observation that g-NK cells exhibit significantly improved persistence. These results demonstrate the utility of fresh or cryopreserved g-NK as a viable off-the-shelf cell therapy to enhance mAb ADCC.
Example 9: evaluation of the influence of CD38 and SLAMF7 on g-NK cells and the homophase insecticidal Activity of g-NK cells
This example demonstrates in part that g-NK cells are protected from antibodies due to the lack of target surface markers.
G-NK cells were substantially expanded by the method described in example 5 with certain exceptions: 1) the ratio of AEH target cells to NK cells was 2.5:1 (compared to 2:1 ratio in example 5), 2) NK cells were exposed to lower levels of IL-2 (100 IU/ml compared to 500IU/ml in example 5), and 3) IL-21 was absent during expansion. Approximately 2.0X10 5 NK cells and/or MM.1S or Raji cells were aliquoted into flow tubes and stained with 2. Mu.L of 7-AAD viability dye and 2. Mu.L of anti-CD 45, 2. Mu.L of anti-CD 20, 2. Mu.L of anti-CD 38, 2. Mu.L of anti-CD 3, 10. Mu.L of anti-SLAMF 7 and 2. Mu.L of anti-CD 56 antibodies as described in Table E3. After incubation at 4 ℃ for 10 min, the cells were washed and stained intracellular with anti-FceRI antibody (Millipore). After completion of the staining procedure, the percentage of CD20, CD38 and SLAMF7 expressing g-NK, cNK and mm.1s or Raji cells was assessed by 8-color flow cytometry (Miltenyi MACSQuant Analyzer, 10).
Table E3. flow cytometry group assayed CD20, CD38 and SLAMF7 expression on NK, MM and Raji cells.
* FcRg is an intracellular epitope
The expression of CD20, CD38 and SLAMF7 on g-NK, cNK and mm.1s cells is shown in fig. 13A-13D. Both g-NK and cNK lack expression of CD20 highly expressed on Raji lymphoma cells (fig. 13A). g-NK expressed much lower than cNK and MM.1S cells on CD38 (see FIG. 13B; both p < 0.001). SLAMF7 expression was not different between g-NK and cNK (p=0.9), but both g-NK and cNK showed significantly lower expression of SLAMF7 than mm.1s cells (see fig. 13C; both p < 0.001). The percentage decrease in CD38 Positive and negative NK cells was also observed on amplified g-NK when compared to amplified cNK (see fig. 13d, p < 0.001). Furthermore, the intensity of CD38 expression (MFI) on CD38 positive g-NK cells was reduced relative to CD38 positive cNK and MM1/S cells (fig. 13e, p < 0.001). Representative bar graphs depicting reduced CD38 expression of g-NK cells relative to cNK and mm.1s cells are shown in fig. 13F.
The lack of g-NK expression of CD20, CD38 or SLAMF7 provides protection against mAb-induced isotype killing by rituximab (anti-CD 20), up Lei Tuoyou mAb (anti-CD 38) or erlotinib (anti-SLAMF 7). In summary, the data further demonstrate how g-NK has a persistence advantage when compared to cNK, particularly in the presence of therapeutic antibodies such as up to Lei Tuoyou mab.
Similar results were observed by the amplification method described in example 5 in the presence of IL-21, indicating that there was no difference in CD38 or SLAMF7 expression between the amplified g-NK cells with or without IL-21. In a further evaluation, the same-phase killing rate of the expanded g-NK cells was compared with the same-phase killing rate of the expanded cNK cells. CD38 expression on g-NK cells was significantly lower than cNK cells as shown in FIGS. 13B and 13D-13F, and as shown in FIG. 13C, same low levels of SLAMF7 were present on g-NK and cNK cells. These results indicate that g-NK cells lack the potential for allo-killing against these targets, as if NK cells express mAb targets, ADCC activity can result in NK cells other than tumors being eliminated by allo-killing. The findings that cNK cells expressed high levels of CD38 are consistent with previous results, indicating that >90% of CD38 High height NK cells in patients were rapidly depleted after reaching Lei Tuoyou mab therapy (Casneuf et al, 2017, blood Adv, volume 1, 23: pages 2105-2114).
Using the method substantially as described in example 5, six (6) unique donors were used to generate amplified g-NK (6 CMV+,3 men and 3 women, age 39.+ -. 7 years) and 8 unique donors were used to amplify cNK (8 CMV-,4 men and 4 women, age 38.+ -. 9 years). The proportion of g-NK is 85.+ -. 4% for the g-NK donor and 2.+ -. 1% for the cNK donor.
To evaluate the homophase killing, about 1X 10 4 expanded NK cells (g-NK or cNK) were cultured in the presence of 1. Mu.g/mL up to Lei Tuoyou monoclonal antibody (anti-CD 38). After 4 hours of incubation at 37 ℃ in a 5% CO 2 incubator, the cells were washed and stained with anti-CD 3 and anti-CD 56 antibodies to quantify the number of NK cells. After the last wash, propidium Iodide (PI) was added and the number of live and dead NK cells was resolved using 3-color flow cytometry (Bigley et al, 2016, clin. Exp. Immunol., volume 185: pages 239-251). As shown in FIG. 13G, G-NK cells had a homophase killing as low as 1/13 of that of cNK. Similar experiments with erlotinib showed that no homogeneous killing was detected with either g-NK or cNK treated with erlotinib.
Together with the results of g-NK cells expanded in the absence of IL-21, these results are consistent with the ability of g-NK cells to confer enhanced mAb antitumor activity in MM without suffering from allo-killing-related depletion.
Example 10: in vivo efficacy in diffuse in situ xenograft mm.1s model of multiple myeloma
The in vivo efficacy of NK cells (expanded g-NK cells or cNK cells) in combination with up to Lei Tuoyou mab was assessed by measuring tumor suppression and survival in a murine model of multiple myeloma. g-NK cells were expanded as described in example 5 after initial enrichment of CD3 Negative of /CD57 Positive and negative cells from cryopreserved PBMC followed by expansion with irradiated 221.AEH feeder cells at a ratio of 1:1. AEH: NK cells in the presence of IL-2 (500 IU/mL), IL-15 (10 ng/mL) and IL-21 (25 ng/mL) stimulatory cytokines. Due to insufficient yield of cNK cells from CMV seronegative donors, cNK cells were expanded using the alternative method described in example 5. cNK cells were expanded for 2 weeks using the transgenic leukemia cell line K562-mb15-41BBL and IL-2. All cells were expanded from cryopreserved PBMCs and cryopreserved feeder cells.
Approximately 5×10 5 luciferase-labeled mm.1s human myeloma cells were injected intravenously into the tail vein of female NSG mice and allowed to grow for 14 days. Monoclonal antibodies were administered via the i.p. route for Lei Tuoyou mab and 6.0x10 6 expanded g-NK or cNK cells were administered intravenously weekly for 5 weeks. Two weeks after tumor administration, 2 μg/mouse human recombinant IL-15 was administered via the i.p. route every three days to provide NK cell support. Table 4 summarizes the mice groups treated in this study.
Bioluminescence imaging (BLI) was performed twice weekly to monitor tumor burden. Mice were checked daily for signs of discomfort and tolerance, and body weight was measured twice weekly starting one week after tumor inoculation. Mice were imaged 15 minutes after subcutaneous injection of 150mg/kg D-luciferin. Total flux (photons/sec) was quantified for whole mice using LIVING IMAGE software (PerkinElmer). Tumor-bearing mice are sacrificed when symptomatic myeloma such as hindlimb paralysis, combing, and/or sleepiness occurs. The time of sacrifice was used as representative of survival. All surviving mice were sacrificed 43 days after initial NK cell administration for tissue collection. At the completion of the study, g-NK, cNK and mm.1s (CD 138 positive/CD 45 negative) cells from the biological samples were quantified using flow cytometry to determine tumor burden and NK cell survival.
Table e4.Mm efficacy study design
Co-administration of g-NK and up to Lei Tuoyou mab resulted in significant tumor suppression and improved survival compared to treatment with cNK and up to Lei Tuoyou mab. As shown in fig. 14A, g-NK cell up to Lei Tuoyou mab abrogated myeloma tumor burden in 5 out of 7 mice, as demonstrated by BLI imaging 5 weeks after treatment. Quantitative BLI analysis showed that g-NK plus Lei Tuoyou mab induced sustained and statistically significant tumor regression (fig. 14B). Kaplan-Meier survival analysis showed that the total survival probability of g-NK plus Lei Tuoyou mab treated mice was significantly better than those treated with vehicle or with cNK and up to Lei Tuoyou mab (p < 0.0001) (fig. 14C). All mice given g-NK cells were energetic, no weight loss or toxicity was observed at the end of the study, while all control mice or mice treated with cNK cells and up to Lei Tuoyou mab had severe weight loss and died of myeloma before the end of the study (fig. 14D). Interestingly, one of the mice treated with g-NK cells was not dosed until day 21 after tumor inoculation, due to anesthesia-induced asphyxiation of one of the mice therein, and the mice had no detectable tumor BLI at the end of the study, although the g-NK mice had the highest peak BLI (fig. 14A, mice labeled #). Of the 7 mice dosed with g-NK cells, only 2 mice had the lowest detectable amount of residual tumor BLI.
Flow cytometry analysis of bone marrow confirmed that 5 g-NK treated mice with no detectable tumor BLI were virtually tumor-free (no CD138 positive cells in bone marrow). The average tumor burden was reduced by more than 99% for all 7 g-NK treated mice relative to mice treated with cNK and up to Lei Tuoyou mab (p <0.001; FIG. 14E). Representative flow cytometry spots depicting tumor burden and sustained NK cells in bone marrow are shown in fig. 14F. All BLI images taken during the study are shown in fig. 14G. X-ray images were obtained from all mice prior to sacrifice and it was determined that control mice or mice treated with cNK cells and up to Lei Tuoyou mab had fractures and deformities of hind limb bones, while one of the mice treated with g-NK cells and up to Lei Tuoyou mab had any skeletal deformity (fig. 14H).
NK cell analysis in blood, spleen and bone marrow showed a large increase in persistence of g-NK cells relative to cNK cells in mice treated with up to Lei Tuoyou mAb (FIGS. 15A-15C). Notably, the g-NK cell numbers in blood were >90% higher than cNK cells (fig. 15A), >95% higher in the spleen (fig. 15B), and >99% higher in the bone marrow (fig. 15C).
Taken together, these results further support the superiority of g-NK cells (including compared to cNK cells) in enhancing mAb efficacy in vivo, and demonstrate that g-NK cells administered in combination with up to Lei Tuoyou mAb can potentially cure MM. Furthermore, these results support that increased survival and resistance to homokilling resulted in excellent anti-tumor effects and persistence of g-NK cells.
Example 11: identification of g-NK surrogate surface markers
Studies were performed to identify combinations of extracellular surface markers that could be used as surrogate surface markers to identify g-NK cells that were negative for the intracellular marker FcεR1γ (FcRγ Negative of ). The percentage of g-NK cells in human peripheral blood samples was determined by flow cytometry, by intracellular staining of fceri 1 gamma and by extracellular staining of CD45, CD3 and CD56 to identify the g-NK cell subpopulation CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative /FcRγ Negative of . As shown in FIG. 16, among the g-NK cells in the sample, cells having NK cell phenotype CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative and cells having CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of or NKG2A Negative of /CD161 Negative of extracellular surface phenotype are highly correlated with the presence of g-NK cells in the sample. Specifically, the percentage of g-NK cells in either CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of or NKG2a Negative of /CD161 Negative of NK cell subpopulation is greater than 80%.
Example 12: feasibility and cytotoxic efficacy of transduction of g-NK cells with CD20 CAR
Studies were performed to assess expression and efficacy of Chimeric Antigen Receptors (CARs) engineered in g-NK cells. G-NK cells were expanded from enriched CD3 Negative of /CD57 Positive and negative NK cells from peripheral blood by co-culturing with irradiated 221.AEH feeder cells at a ratio of 2:1. AEH: NK cells in the presence of 500IU/mL IL-2, 10ng/mL IL-15 and 25ng/mL IL-21, substantially as described in example 5. Cells were electroporated using the Neon TM transfection system. Cells were washed with PBS prior to electroporation and resuspended in Opti-MEM TM medium at a cell density of 4.8X107/mL. mu.L of cells were mixed with 14.4. Mu.g of 1mg/ML GFP MRNA or CAR-CD20 mRNA. CAR-CD20 the amino acid sequence of a CAR consisting of a murine anti-CD 20 (Leu 16) scFv that binds to the CD20 polypeptide (SEQ ID NO: 37), the IgG4 Fc spacer (SEQ ID NO: 38), the CD28 transmembrane domain (SEQ ID NO: 39), the CD28 intracellular co-stimulatory signaling domain (SEQ ID NO: 39) and the CD3 zeta primary signaling domain (SEQ ID NO: 41) is shown in SEQ ID NO:42 (GenBank number KX 055829.1) and also generates an HA tag at the 5' end. The anti-CD 20 mRNA is shown in SEQ ID NO. 45.
The mixture of g-NK cells and mRNA was shocked with a first pulse of 1820 (20 ms) followed by a second pulse of 500V (100 ms). Then in 500IU/mL IL-2, 10ng/mL IL-15 and 25ng/mL IL-21 2mL NKCells are cultured in a medium. Cells were incubated at 37℃for 24 hours with 5% CO 2.
After incubation, cells were analyzed by flow cytometry to evaluate the levels of GFP and CAR-CD20 expression. Post-transduction expression of GFP and CD20-CAR from g-NK cells from two separate experiments using different donors is shown in figure 17. The percentage of GFP or CAR-CD20 positive cells is reported as the percentage of surviving cells expressing GFP or CAR-CD 20.
By using rituximab @ a) In the presence or absence of cells were incubated with 60,000 Raji lymphoma cells (targets) at different effector to target (E: T) ratios to analyze the cytotoxicity of g-NK cells (effectors) with or without CAR-CD 20. Rituximab (/ >) used) Is 5. Mu.g/ml. Raji cells were pre-stained with APC-conjugated mouse anti-human CD19 mAb. Residual dye was removed by washing with RPMI 1640 medium (assay medium) containing 10% FBS and 1% penicillin-streptomycin. Pre-stained Raji cells were validated by flow cytometry to confirm that all target cells were successfully labeled.
The g-NK cells expanded on day 17 or CAR-CD20 g-NK cells 36 hours after electroporation were mixed with pre-stained Raji target cells in E:T ratios of 0.5:1, 2.5:1 and 5:1 in 1mL assay medium. The mixture was precipitated at 300g for 5 minutes and then incubated with 5% CO 2 for 4 hours at 37 ℃. Cytotoxicity was assessed using 4-color flow cytometry. To identify NK cells (CD 3 Negative of /CD56 Positive and negative ), PE conjugated mouse anti-human CD56 mAb and FITC conjugated mouse anti-human CD3 mAb were used. To distinguish live target cells (CD 19 Positive and negative /PI Negative of ) from dead target cells (CD 19 Positive and negative /PI Positive and negative ), propidium Iodide (PI) was used.
FIG. 18 demonstrates that at rituximab @) With or without the efficacy of g-NK cells of CD20-CAR against Raji lymphoma cells. The addition of CD20-CAR enhanced the efficacy of g-NK cells as monotherapy. Whether or not CD20-CAR is expressed, rituximab (/ >) The addition of (2) also enhances the efficacy of g-NK cells to similar levels.
This result demonstrates that CARs are functional in g-NK cells lacking FcR gamma chain expression. This result further demonstrates the surprising finding that the addition of CAR does not disrupt the ADCC mechanism of g-NK cells conjugated via CD16 by IgG1 mAb. Thus, in addition to the activity of CAR-engineered g-NK cell monotherapy, these results support combination therapies of g-NK cells engineered with a CAR and an Fc targeting agent (such as an IgG1 mAb), including methods of using CAR targeting antigens other than mAb. Such dual targeting strategies can potentially be additive or compensatory functionally, depending on antigen expression and possible antigen loss of the tumor.
The present invention is not intended to be limited in scope to the specifically disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to these compositions and methods will be apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure, and are intended to fall within the scope of the disclosure.
Sequence listing
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Sequence listing
<110> Indapta treatment Co
<120> Engineered Natural Killer (NK) cells and related methods
<130> 77603-20010.40
<140> Not yet assigned
<141> Filed concurrently with the present application
<150> US 63/217,718
<151> 2021-07-01
<150> US 63/217,722
<151> 2021-07-01
<150> US 63/217,726
<151> 2021-07-01
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Gly Asn Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg
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Lys Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr
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His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser
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Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu
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Gln Met Phe Ile Asn Thr Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro
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Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu
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Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
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Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly
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His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser
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Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu
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gaagatctta ttcaatctat gcatattgat gctactttat atacggaaag tgatgttcac 240
cccagttgca aagtaacagc aatgaagtgc tttctcttgg agttacaagt tatttcactt 300
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1 5 10 15
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<210> 11
<211> 69
<212> PRT
<213> Artificial sequence
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Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
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Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
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Ile Thr Leu Tyr Cys
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<210> 12
<211> 21
<212> PRT
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1 5 10 15
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<210> 13
<211> 22
<212> PRT
<213> Artificial sequence
<220>
<223> GM-CSFRa Signal peptide
<400> 13
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro
20
<210> 14
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> IgK Signal peptide
<400> 14
Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Pro
1 5 10 15
Gly Ser Ser Gly
20
<210> 15
<211> 22
<212> PRT
<213> Artificial sequence
<220>
<223> CD4 transmembrane Domain
<400> 15
Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile
1 5 10 15
Gly Leu Gly Ile Phe Phe
20
<210> 16
<211> 20
<212> RNA
<213> Artificial sequence
<220>
<223> CPFL CRRNA scaffold sequences
<400> 16
uaauuucuac ucuuguagau 20
<210> 17
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Primer
<400> 17
atatttacag aatggcacag g 21
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> Primer
<400> 18
gacttggtac ccaggttgaa 20
<210> 19
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223> Primer
<400> 19
atcagattcg atcctacttc tgcagggggc at 32
<210> 20
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223> Primer
<400> 20
acgtgctgag cttgagtgat ggtgatgttc ac 32
<210> 21
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> Primer
<400> 21
cccaactcaa cttcccagtg tgat 24
<210> 22
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223> Primer
<400> 22
gaaatctacc ttttcctcta atagggcaat 30
<210> 23
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223> Primer
<400> 23
gaaatctacc ttttcctcta atagggcaa 29
<210> 24
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<223> Primer
<400> 24
gaaatctacc ttttcctcta atagggca 28
<210> 25
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Primer
<400> 25
ccaaaagcca cactcaaaga c 21
<210> 26
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Primer
<400> 26
acccaggtgg aaagaatgat g 21
<210> 27
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223> Primer
<400> 27
aacatcacca tcactcaagg tttgg 25
<210> 28
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223> Primer
<400> 28
ctgaagacac atttttactc ccaaa 25
<210> 29
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> Primer
<400> 29
tccaaaagcc acactcaaag ac 22
<210> 30
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223> Primer
<400> 30
ctgaagacac atttttactc ccaac 25
<210> 31
<211> 238
<212> PRT
<213> Artificial sequence
<220>
<223> CD16（F158）
<400> 31
Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro
1 5 10 15
Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln
20 25 30
Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu
35 40 45
Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr
50 55 60
Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu
65 70 75 80
Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln
85 90 95
Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys
100 105 110
His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn
115 120 125
Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro
130 135 140
Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe
145 150 155 160
Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln
165 170 175
Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln
180 185 190
Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly
195 200 205
Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp
210 215 220
Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys
225 230 235
<210> 32
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> Signal peptide
<400> 32
Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala
1 5 10 15
<210> 33
<211> 238
<212> PRT
<213> Artificial sequence
<220>
<223> CD16 (V158 or 158 V+)
<400> 33
Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro
1 5 10 15
Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln
20 25 30
Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu
35 40 45
Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr
50 55 60
Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu
65 70 75 80
Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln
85 90 95
Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys
100 105 110
His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn
115 120 125
Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro
130 135 140
Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val
145 150 155 160
Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln
165 170 175
Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln
180 185 190
Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly
195 200 205
Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp
210 215 220
Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys
225 230 235
<210> 34
<211> 86
<212> PRT
<213> Artificial sequence
<220>
<223> FcRγ
<400> 34
Met Ile Pro Ala Val Val Leu Leu Leu Leu Leu Leu Val Glu Gln Ala
1 5 10 15
Ala Ala Leu Gly Glu Pro Gln Leu Cys Tyr Ile Leu Asp Ala Ile Leu
20 25 30
Phe Leu Tyr Gly Ile Val Leu Thr Leu Leu Tyr Cys Arg Leu Lys Ile
35 40 45
Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val
50 55 60
Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys
65 70 75 80
His Glu Lys Pro Pro Gln
85
<210> 35
<211> 105
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-CD 20 (Leu 16) VL
<400> 35
Ile Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu
1 5 10 15
Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Asp
20 25 30
Trp Tyr Gln Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala
35 40 45
Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly
50 55 60
Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp
65 70 75 80
Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe
85 90 95
Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 36
<211> 117
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-CD 20 (Leu 16) VH
<400> 36
Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys
1 5 10 15
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln
20 25 30
Thr Pro Gly Gln Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn
35 40 45
Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr
50 55 60
Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr
65 70 75 80
Ser Glu Asp Ser Ala Asp Tyr Tyr Cys Ala Arg Ser Asn Tyr Tyr Gly
85 90 95
Ser Ser Tyr Trp Phe Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr
100 105 110
Val Ser Ser Leu Asp
115
<210> 37
<211> 247
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-CD 20 (Leu 16) scFv
<400> 37
Ile Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu
1 5 10 15
Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Asp
20 25 30
Trp Tyr Gln Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala
35 40 45
Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly
50 55 60
Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp
65 70 75 80
Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe
85 90 95
Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly Gly Gly
100 105 110
Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Val Gln Leu Gln
115 120 125
Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser
130 135 140
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val
145 150 155 160
Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro
165 170 175
Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr
180 185 190
Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser
195 200 205
Leu Thr Ser Glu Asp Ser Ala Asp Tyr Tyr Cys Ala Arg Ser Asn Tyr
210 215 220
Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp Gly Ala Gly Thr Thr
225 230 235 240
Val Thr Val Ser Ser Leu Asp
245
<210> 38
<211> 230
<212> PRT
<213> Artificial sequence
<220>
<223> IgG4 Fc spacer
<400> 38
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
1 5 10 15
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly Lys Met
225 230
<210> 39
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> CD28 transmembrane Domain
<400> 39
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210> 40
<211> 41
<212> PRT
<213> Artificial sequence
<220>
<223> CD28 costimulatory signaling domain
<400> 40
Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 41
<211> 112
<212> PRT
<213> Artificial sequence
<220>
<223> CD3 zeta signaling domain
<400> 41
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 42
<211> 657
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-CD 20 CAR
<400> 42
Ile Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu
1 5 10 15
Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Asp
20 25 30
Trp Tyr Gln Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala
35 40 45
Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly
50 55 60
Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp
65 70 75 80
Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe
85 90 95
Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly Gly Gly
100 105 110
Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Val Gln Leu Gln
115 120 125
Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser
130 135 140
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val
145 150 155 160
Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro
165 170 175
Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr
180 185 190
Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser
195 200 205
Leu Thr Ser Glu Asp Ser Ala Asp Tyr Tyr Cys Ala Arg Ser Asn Tyr
210 215 220
Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp Gly Ala Gly Thr Thr
225 230 235 240
Val Thr Val Ser Ser Leu Asp Glu Ser Lys Tyr Gly Pro Pro Cys Pro
245 250 255
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
260 265 270
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
275 280 285
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
290 295 300
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
305 310 315 320
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
325 330 335
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
340 345 350
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
355 360 365
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
370 375 380
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
385 390 395 400
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
405 410 415
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
420 425 430
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
435 440 445
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
450 455 460
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Met Phe Trp Val
465 470 475 480
Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr
485 490 495
Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly
500 505 510
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
515 520 525
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
530 535 540
Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
545 550 555 560
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
565 570 575
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
580 585 590
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
595 600 605
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
610 615 620
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
625 630 635 640
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
645 650 655
Arg
<210> 43
<211> 21
<212> PRT
<213> Artificial sequence
<220>
<223> IgK Signal peptide
<400> 43
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp
20
<210> 44
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> HA tag
<400> 44
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
1 5
<210> 45
<211> 2064
<212> DNA
<213> Artificial sequence
<220>
<223> Anti-CD 20 CAR
<400> 45
atggagacag acacactcct gctatgggtg ctgctgctct gggttccagg ttccacaggt 60
gactacccat acgatgttcc agattacgct attgtgctga cccaatctcc agctatcctg 120
tctgcatctc caggggagaa ggtcacaatg acttgcaggg ccagctcaag tgtaaattac 180
atggactggt accagaagaa gccaggatcc tcccccaaac cctggattta tgccacatcc 240
aacctggctt ctggagtccc tgctcgcttc agtggcagtg ggtctgggac ctcttactct 300
ctcacaatca gcagagtgga ggctgaagat gctgccactt attactgcca gcagtggagt 360
tttaatccac ccacgttcgg aggggggacc aagctggaaa taaaaggcag tactagcggt 420
ggtggctccg ggggcggttc cggtgggggc ggcagcagcg aggtgcagct gcagcagtct 480
ggggctgagc tggtgaagcc tggggcctca gtgaagatgt cctgcaaggc ttctggctac 540
acatttacca gttacaatat gcactgggta aagcagacac ctggacaggg cctggaatgg 600
attggagcta tttatccagg aaatggtgat acttcctaca atcagaagtt caaaggcaag 660
gccacattga ctgcagacaa atcctccagc acagcctaca tgcagctcag cagcctgaca 720
tctgaggact ctgcggacta ttactgtgca agatctaatt attacggtag tagctactgg 780
ttcttcgatg tctggggcgc agggaccacg gtcaccgtct cctcactcga cgaatctaag 840
tacggaccgc cctgcccccc ttgccctgcc cccgagttcc tgggcggacc cagcgtgttc 900
ctgttccccc ccaagcccaa ggacaccctg atgatcagcc ggacccccga ggtgacctgc 960
gtggtggtgg acgtgagcca ggaagatccc gaggtccagt tcaattggta cgtggacggc 1020
gtggaagtgc acaacgccaa gaccaagccc agagaggaac agttcaacag cacctaccgg 1080
gtggtgtctg tgctgaccgt gctgcaccag gactggctga acggcaaaga atacaagtgc 1140
aaggtgtcca acaagggcct gcccagcagc atcgaaaaga ccatcagcaa ggccaagggc 1200
cagcctcgcg agccccaggt gtacaccctg cctccctccc aggaagagat gaccaagaac 1260
caggtgtccc tgacctgcct ggtgaagggc ttctacccca gcgacatcgc cgtggagtgg 1320
gagagcaacg gccagcctga gaacaactac aagaccaccc ctcccgtgct ggacagcgac 1380
ggcagcttct tcctgtacag ccggctgacc gtggacaaga gccggtggca ggaaggcaac 1440
gtctttagct gcagcgtgat gcacgaggcc ctgcacaacc actacaccca gaagagcctg 1500
agcctgtccc tgggcaagat gttctgggtg ctggtggtgg tgggcggggt gctggcctgc 1560
tacagcctgc tggtgacagt ggccttcatc atcttttggg tgcggagcaa gcggagcaga 1620
ggcggccaca gcgactacat gaacatgacc cccagacggc ctggccccac ccggaagcac 1680
taccagccct acgccccacc cagggacttt gccgcctaca gaagccgggt gaagttcagc 1740
agaagcgccg acgcccctgc ctaccagcag ggccagaatc agctgtacaa cgagctgaac 1800
ctgggcagaa gggaagagta cgacgtcctg gataagcgga gaggccggga ccctgagatg 1860
ggcggcaagc ctcggcggaa gaacccccag gaaggcctgt ataacgaact gcagaaagac 1920
aagatggccg aggcctacag cgagatcggc atgaagggcg agcggaggcg gggcaagggc 1980
cacgacggcc tgtatcaggg cctgtccacc gccaccaagg atacctacga cgccctgcac 2040
atgcaggccc tgcccccaag gtga 2064
<210> 46
<211> 121
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-CD 38 VH
<400> 46
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Phe Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Ile Arg Phe Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Leu Ile Ala Asp Lys Ser Thr Asn Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Gly Glu Pro Gly Glu Arg Asp Pro Asp Ala Val Asp Ile Trp Gly
100 105 110
Gln Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 47
<211> 122
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-CD 38 VH
<400> 47
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Asn Tyr
20 25 30
Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Tyr Pro His Asp Ser Asp Ala Arg Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Phe Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg His Val Gly Trp Gly Ser Arg Tyr Trp Tyr Phe Asp Leu Trp
100 105 110
Gly Arg Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 48
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-CD 38 VL
<400> 48
Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Ser
20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Ser Ser Ser Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 49
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-CD 38 VL
<400> 49
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr
20 25 30
Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 50
<211> 112
<212> PRT
<213> Artificial sequence
<220>
<223> CD3ζ
<400> 50
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 51
<211> 42
<212> PRT
<213> Artificial sequence
<220>
<223> 4-1BB costimulatory Domain
<400> 51
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 52
<211> 41
<212> PRT
<213> Artificial sequence
<220>
<223> CD28 Co-stimulatory Domain
<400> 52
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 53
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-CD 19 VL
<400> 53
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr
100 105
<210> 54
<211> 120
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-CD 19 VH
<400> 54
Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 55
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> Whitlow linker
<400> 55
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
1 5 10 15
Lys Gly
<210> 56
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> GS linker
<400> 56
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 57
<211> 245
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-CD 19 scFv
<400> 57
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys
115 120 125
Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser
130 135 140
Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser
145 150 155 160
Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile
165 170 175
Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu
180 185 190
Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn
195 200 205
Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr
210 215 220
Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
225 230 235 240
Val Thr Val Ser Ser
245
<210> 58
<211> 242
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-CD 19 scFv
<400> 58
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu
115 120 125
Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys
130 135 140
Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg
145 150 155 160
Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser
165 170 175
Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile
180 185 190
Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln
195 200 205
Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly
210 215 220
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
225 230 235 240
Ser Ser
<210> 59
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> IgG4 hinge
<400> 59
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
1 5 10
<210> 60
<211> 25
<212> PRT
<213> Artificial sequence
<220>
<223> CD8 hinge
<400> 60
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu
20 25
<210> 61
<211> 44
<212> PRT
<213> Artificial sequence
<220>
<223> CD8 transmembrane
<400> 61
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
1 5 10 15
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly
20 25 30
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
35 40
<210> 62
<211> 111
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-BCMA C11D5.3 VL
<400> 62
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Met Ser Leu Gly
1 5 10 15
Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Val Ile
20 25 30
Gly Ala His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Thr Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp
65 70 75 80
Pro Val Glu Glu Asp Asp Val Ala Ile Tyr Ser Cys Leu Gln Ser Arg
85 90 95
Ile Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 63
<211> 117
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-BCMA C11D5.3 VH
<400> 63
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ser Ile Asn Trp Val Lys Arg Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp Phe
50 55 60
Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Tyr Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser
115
<210> 64
<211> 246
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-BCMA C11D5.3 scFv
<400> 64
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Met Ser Leu Gly
1 5 10 15
Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Val Ile
20 25 30
Gly Ala His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Thr Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp
65 70 75 80
Pro Val Glu Glu Asp Asp Val Ala Ile Tyr Ser Cys Leu Gln Ser Arg
85 90 95
Ile Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
100 105 110
Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys
115 120 125
Gly Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly
130 135 140
Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
145 150 155 160
Tyr Ser Ile Asn Trp Val Lys Arg Ala Pro Gly Lys Gly Leu Lys Trp
165 170 175
Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp
180 185 190
Phe Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala
195 200 205
Tyr Leu Gln Ile Asn Asn Leu Lys Tyr Glu Asp Thr Ala Thr Tyr Phe
210 215 220
Cys Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
225 230 235 240
Ser Val Thr Val Ser Ser
245
<210> 65
<211> 111
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-BCMA C12A3.2VL
<400> 65
Asp Ile Val Leu Thr Gln Ser Pro Pro Ser Leu Ala Met Ser Leu Gly
1 5 10 15
Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Thr Ile Leu
20 25 30
Gly Ser His Leu Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Thr Leu Leu Ile Gln Leu Ala Ser Asn Val Gln Thr Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp
65 70 75 80
Pro Val Glu Glu Asp Asp Val Ala Val Tyr Tyr Cys Leu Gln Ser Arg
85 90 95
Thr Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 66
<211> 117
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-BCMA C12A3.2 VH
<400> 66
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Arg His Tyr
20 25 30
Ser Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Arg Ile Asn Thr Glu Ser Gly Val Pro Ile Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Val Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Val Ile Asn Asn Leu Lys Asp Glu Asp Thr Ala Ser Tyr Phe Cys
85 90 95
Ser Asn Asp Tyr Leu Tyr Ser Leu Asp Phe Trp Gly Gln Gly Thr Ala
100 105 110
Leu Thr Val Ser Ser
115
<210> 67
<211> 246
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-BCMA C12A3.2 scFv
<400> 67
Asp Ile Val Leu Thr Gln Ser Pro Pro Ser Leu Ala Met Ser Leu Gly
1 5 10 15
Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Thr Ile Leu
20 25 30
Gly Ser His Leu Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Thr Leu Leu Ile Gln Leu Ala Ser Asn Val Gln Thr Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp
65 70 75 80
Pro Val Glu Glu Asp Asp Val Ala Val Tyr Tyr Cys Leu Gln Ser Arg
85 90 95
Thr Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
100 105 110
Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys
115 120 125
Gly Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly
130 135 140
Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Arg His
145 150 155 160
Tyr Ser Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp
165 170 175
Met Gly Arg Ile Asn Thr Glu Ser Gly Val Pro Ile Tyr Ala Asp Asp
180 185 190
Phe Lys Gly Arg Phe Ala Phe Ser Val Glu Thr Ser Ala Ser Thr Ala
195 200 205
Tyr Leu Val Ile Asn Asn Leu Lys Asp Glu Asp Thr Ala Ser Tyr Phe
210 215 220
Cys Ser Asn Asp Tyr Leu Tyr Ser Leu Asp Phe Trp Gly Gln Gly Thr
225 230 235 240
Ala Leu Thr Val Ser Ser
245
<210> 68
<211> 110
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-BCMA BCMA02 VH
<400> 68
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ser Ile Asn Trp Val Lys Arg Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp Phe
50 55 60
Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Tyr Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
<210> 69
<211> 110
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-BCMA BCMA02 VL
<400> 69
Asp Ile Val Leu Thr Gln Ser Pro Pro Ser Leu Ala Met Ser Leu Gly
1 5 10 15
Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Thr Ile Leu
20 25 30
Gly Ser His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Thr Leu Leu Ile Gln Leu Ala Ser Asn Val Gln Thr Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp
65 70 75 80
Pro Val Glu Glu Asp Asp Val Ala Val Tyr Tyr Cys Leu Gln Ser Arg
85 90 95
Thr Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110
<210> 70
<211> 246
<212> PRT
<213> Artificial sequence
<220>
<223> Anti-BCMA 02 scFv
<400> 70
Asp Ile Val Leu Thr Gln Ser Pro Pro Ser Leu Ala Met Ser Leu Gly
1 5 10 15
Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Thr Ile Leu
20 25 30
Gly Ser His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Thr Leu Leu Ile Gln Leu Ala Ser Asn Val Gln Thr Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp
65 70 75 80
Pro Val Glu Glu Asp Asp Val Ala Val Tyr Tyr Cys Leu Gln Ser Arg
85 90 95
Thr Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
100 105 110
Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys
115 120 125
Gly Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly
130 135 140
Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
145 150 155 160
Tyr Ser Ile Asn Trp Val Lys Arg Ala Pro Gly Lys Gly Leu Lys Trp
165 170 175
Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp
180 185 190
Phe Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala
195 200 205
Tyr Leu Gln Ile Asn Asn Leu Lys Tyr Glu Asp Thr Ala Thr Tyr Phe
210 215 220
Cys Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
225 230 235 240
Ser Val Thr Val Ser Ser
245

Claims (197)

1. An engineered Natural Killer (NK) cell (g-NK cell) deficient in FcR gamma chain expression, and the engineered NK cell comprising a heterologous nucleic acid encoding a Chimeric Antigen Receptor (CAR).
2. An engineered Natural Killer (NK) cell (g-NK cell) deficient in FcR gamma chain expression, and the engineered NK cell comprising a heterologous nucleic acid encoding an immunomodulatory agent.
3. An engineered Natural Killer (NK) cell (g-NK cell) deficient in FcR gamma chain expression, and the engineered NK cell comprising a heterologous nucleic acid encoding a Chimeric Antigen Receptor (CAR) and a heterologous nucleic acid encoding an immunomodulator.
4. The engineered NK cell of claim 1 or claim 3, wherein said CAR comprises 1) an antigen binding domain; 2) A flexible connector; 3) A transmembrane region; and 4) an intracellular signaling domain.
5. The engineered NK cell of claim 4, wherein said antigen binding domain targets a tumor antigen.
6. The engineered NK cell of claim 4 or claim 5, wherein said antigen binding domain is a single chain variable fragment (scFv).
7. The engineered NK cell of any one of claims 4 to 6, wherein said intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain.
8. The engineered NK cell of any one of claims 4 to 7, wherein said intracellular signaling domain comprises one or more signaling domains of cd3ζ, DAP10, DAP12, CD28, 4-1BB, or OX 40.
9. The engineered NK cell of any one of claims 4 to 7, wherein said intracellular signaling domain comprises two or more signaling domains of cd3ζ, DAP10, DAP12, CD28, 4-1BB, or OX 40.
10. The engineered NK cell of any one of claims 4 to 9, wherein said intracellular signaling domain comprises a primary signaling domain comprising a signaling domain of cd3ζ and a costimulatory signaling domain.
11. The engineered NK cell of claim 10, wherein the costimulatory signaling domain is the signaling domain of CD28 or 4-1 BB.
12. The engineered NK cell of any one of claims 1 or 3 to 11, wherein the heterologous nucleic acid encoding the CAR is stably integrated into the genome of the cell.
13. The engineered NK cell of any one of claims 1 or 3 to 11, wherein the heterologous nucleic acid encoding the CAR is transiently expressed.
14. The engineered NK cell of claim 2 or claim 3, wherein said immunomodulator is an immunosuppressant.
15. The engineered NK cell of claim 2 or claim 3, wherein said immunomodulator is an immune activator.
16. The engineered NK cell of any one of claims 2, 3 or 15, wherein said immunomodulatory agent is a cytokine.
17. The engineered NK cell of claim 16, wherein said cytokine is secreted from said engineered NK cell.
18. The engineered NK cell of claim 17, wherein said secretable cytokine is IL-2 or a biological portion thereof; IL-15 or a biological portion thereof; or IL-21 or a biological part thereof; or a combination thereof.
19. The engineered NK cell of claim 16, wherein said cytokine is membrane-bound.
20. The engineered NK cell of claim 19, wherein said membrane-bound cytokine is membrane-bound IL-2 (mbIL-2); membrane-bound IL-15 (mbIL-15); membrane-bound IL-21 (mbIL-21); or a combination thereof.
21. The engineered NK cell of any one of claims 2 to 20, wherein said heterologous nucleic acid encoding said immunomodulator is stably integrated into the genome of said cell.
22. The engineered NK cell of any one of claims 2 to 21, wherein said heterologous nucleic acid encoding said immunomodulator is transiently expressed.
23. The engineered NK cell of any one of claims 1 to 22, wherein said g-NK cell has the surface phenotype of CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of .
24. The engineered NK cell of any one of claims 1 to 23, wherein the g-NK cell further has the surface phenotype of NKG2a Negative of /CD161 Negative of .
25. The engineered NK cell of any one of claims 1 to 24, wherein said g-NK cell is also CD38 Negative of .
26. The engineered NK cell of any one of claims 1 to 25, wherein said g-NK cell further has the surface phenotype of CD45 Positive and negative /CD3 Negative of /CD56 Positive and negative .
27. The engineered NK cell of any one of claims 1 to 26, wherein said g-NK cell comprises CD 16V/V (V158)
28. The engineered NK cell of any one of claims 1 to 26, wherein said g-NK cell is CD16 158V/F.
29. A composition comprising a plurality of engineered g-NK cells of any one of claims 1, 3 to 13 or 23 to 28.
30. A composition comprising a plurality of engineered g-NK cells of any one of claims 2 or 14 to 28.
31. A composition comprising a plurality of engineered g-NK cells of any one of claims 3 to 28.
32. The composition of any one of claims 29 to 31, wherein greater than 50% or greater than about 50% of the NK cells or total cells or greater than 60% or greater than about 60% of the NK cells or total cells in the composition are g-NK cells.
33. The composition of any one of claims 29 to 31, wherein greater than 70% or greater than about 70% of the NK cells or total cells in the composition are g-NK cells.
34. The composition of any one of claims 29 to 31, wherein greater than 80% or greater than about 80% of the NK cells or total cells in the composition are g-NK cells.
35. The composition of any one of claims 29 to 31, wherein greater than 90% or greater than about 90% of the NK cells or total cells in the composition are g-NK cells.
36. The composition of any one of claims 29 to 31, wherein greater than 95% or greater than about 95% of the NK cells or total cells in the composition are g-NK cells.
37. The composition of any one of claims 29 or 31-36, wherein the plurality of engineered g-NK cells comprises greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, or greater than 70% or greater than about 70% g-NK cells comprising a heterologous nucleic acid encoding the CAR.
38. The composition of any one of claims 29 or 31-37, wherein the total composition comprises greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, or greater than 70% or greater than about 70% g-NK cells comprising a heterologous nucleic acid encoding the CAR.
39. The composition of any one of claims 30-36, wherein the plurality of engineered g-NK cells comprises greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, or greater than 70% or greater than about 70% g-NK cells comprising a heterologous nucleic acid encoding the immunomodulatory agent.
40. The composition of any one of claims 30-36 or 39, wherein the total composition comprises greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, or greater than 70% or greater than about 70% g-NK cells comprising a heterologous nucleic acid encoding the immunomodulatory agent.
41. The composition of any one of claims 31-36, wherein the plurality of engineered g-NK cells comprises greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, or greater than 70% or greater than about 70% g-NK cells comprising a heterologous nucleic acid encoding the CAR and a heterologous nucleic acid encoding the immunomodulatory agent.
42. The composition of any one of claims 31-36 or 41, wherein the total composition comprises greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, or greater than 70% or greater than about 70% g-NK cells comprising a heterologous nucleic acid encoding the CAR and the heterologous nucleic acid encoding the immunomodulatory agent.
43. The composition of any one of claims 29 to 42, wherein greater than 70% or greater than about 70% of the g-NK cells are positive for perforin and greater than 70% or greater than about 70% of the g-NK cells are positive for granzyme B.
44. The composition of any one of claims 29 to 43, wherein greater than 80% or greater than about 80% of the g-NK cells are positive for perforin and greater than 80% or greater than about 80% of the g-NK cells are positive for granzyme B.
45. The composition of any one of claims 29 to 44, wherein greater than 90% or greater than about 90% of the g-NK cells are positive for perforin and greater than 90% or greater than about 90% of the g-NK cells are positive for granzyme B.
46. The composition of any one of claims 29-45, wherein greater than 95% or greater than about 95% of the g-NK cells are positive for perforin and greater than 95% or greater than about 95% of the g-NK cells are positive for granzyme B.
47. The composition of any one of claims 43 to 46, wherein:
In the cells positive for perforin, the cells express perforin at an average level of at least twice or at least about twice that of FcR gamma Positive and negative based on Mean Fluorescence Intensity (MFI) as measured by intracellular flow cytometry; and/or
In the cells positive for granzyme B, the cells express granzyme B at an average level of at least twice or at least about twice as high as that of FcR gamma Positive and negative based on average fluorescence intensity (MFI) as measured by intracellular flow cytometry.
48. The composition of any one of claims 29 to 47, wherein optionally greater than 10% of the cells in the composition are capable of degranulation against tumor target cells as measured by CD107a expression, optionally wherein the degranulation is measured in the absence of antibodies against the tumor target cells.
49. The composition of any one of claims 29 to 48, wherein greater than 10% of the cells in the composition are further capable of producing interferon-gamma or TNF-alpha to a tumor target cell, optionally wherein the interferon-gamma or TNF-alpha is measured in the absence of antibodies to the tumor target cell.
50. The composition of any one of claims 29 to 49, wherein in the cells in the composition, greater than 15% or greater than about 15%, greater than 20% or greater than about 20%, greater than 30% or greater than about 30%, greater than 40% or greater than about 40%, or greater than 50% or greater than about 50% of the cells expressing a target antigen (target cells) and antibodies to the target antigen (anti-target antibodies) produce an effector cytokine.
51. The composition of any one of claims 29 to 50, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 30% or greater than about 30% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 50% or greater than about 50% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
52. The composition of any one of claims 29 to 51, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 35% or greater than about 35% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 60% or greater than about 60% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
53. The composition of any one of claims 29 to 52, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 40% or greater than about 40% of cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 70% or greater than about 70% of cells are negative for NKG2A or low level (NKG 2A Negative of ).
54. The composition of any one of claims 29 to 52, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 45% or greater than about 45% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 80% or greater than about 80% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
55. The composition of any one of claims 29 to 52, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 50% or greater than about 50% of cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 85% or greater than about 85% of cells are negative for NKG2A or low level (NKG 2A Negative of ).
56. The composition of any one of claims 29 to 52, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 55% or greater than about 55% of the cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 90% or greater than about 90% of the cells are negative for NKG2A or low level (NKG 2A Negative of ).
57. The composition of any one of claims 29 to 52, wherein in the total cells in the composition or in the g-NK cells in the composition, greater than 60% or greater than about 60% of cells are positive for NKG2C (NKG 2C Positive and negative ) and/or greater than 95% or greater than about 95% of cells are negative or low level for NKG2A (NKG 2A Negative of ).
58. The composition of any one of claims 29 to 57, wherein greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, or greater than 90% or greater than about 90% of the cells in the total cells in the composition or the g-NK cells in the composition are CD38 Negative of .
59. The composition of any one of claims 29 to 57, wherein greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, or greater than 90% or greater than about 90% of the cells in the total cells in the composition or the g-NK cells in the composition are CD16 Positive and negative /CD57 Positive and negative /CD7 Weak and weak / Negative of /CD161 Negative of .
60. The composition of any one of claims 29 to 57, wherein greater than 50% or greater than about 50%, greater than 60% or greater than about 60%, greater than 70% or greater than about 70%, greater than 80% or greater than about 80%, or greater than 90% or greater than about 90% of the cells in the total cells in the composition or the g-NK cells in the composition are NKG2a Negative of /CD161 Negative of .
61. The composition of any one of claims 29 to 60, wherein the plurality of g-NK cells are CD16 158V/V (V158).
62. The composition of any one of claims 29 to 60, wherein the plurality of g-NK cells are CD16 158V/F.
63. The composition of any one of claims 29 to 62, wherein the composition comprises at least 10 8 cells or at least about 10 8 cells.
64. The composition of any one of claims 29 to 63, wherein the number of g-NK cells in the composition is 10 8 to 10 12 cells or about 10 8 to about 10 12 cells, 10 8 to 10 11 cells or about 10 8 to about 10 11 cells, 10 8 to 10 10 cells or about 10 8 to about 10 10 cells, 10 8 to 10 9 cells or about 10 8 to about 10 9 cells, 10 9 cells to 10 12 cells or about 10 9 cells to about 10 12 cells, 10 12 to 10 12 cells or about 10 12 cells, 10 12 to 12 cells, or about 10 12 cells, 12 to 12 cells, 12 cells or about 10 12 cells is 10 12 to 12 cells, 12 cells or about 10 12 cells is 10 12 cells, 12 to 12 cells, 12 cells or about 10 12 cells.
65. The composition of any one of claims 29 to 64, wherein the number of g-NK cells in the composition is or is about 5 x 10 8 cells, or is about 1x 10 9 cells, or is about 5 x 10 10 cells, or is about 1x 10 10 cells.
66. The composition of any one of claims 29 to 65, wherein the volume of the composition is between 50mL and 500mL or between about 50mL and about 500mL, optionally 200mL or about 200mL.
67. The composition of any one of claims 29 to 66, wherein the cells in the composition are from a single donor subject, the cells having been expanded from the same biological sample.
68. The composition of any one of claims 29 to 67, wherein the composition is a pharmaceutical composition.
69. The composition of any one of claims 29 to 68, comprising a pharmaceutically acceptable excipient.
70. The composition of any one of claims 29 to 69, wherein the composition is formulated in a serum-free cryopreservation medium comprising a cryoprotectant.
71. The composition of claim 70, wherein the cryoprotectant is DMSO and the cryopreservation medium is 5% to 10% DMSO (v/v).
72. The composition of claim 71, wherein the cryoprotectant is or is about 10% dmso (v/v).
73. The composition of any one of claims 29 to 72, which is sterile.
74. A sterile bag comprising the composition of any one of claims 29 to 73.
75. The sterile bag of claim 74, wherein the bag is a cryopreservation compatible bag.
76. A method of producing a genetically engineered g-NK cell, the method comprising introducing a heterologous nucleic acid encoding a Chimeric Antigen Receptor (CAR) into an FcR gamma chain expression deficient NK cell (g-NK cell).
77. A method of producing a genetically engineered g-NK cell, the method comprising introducing into the g-NK cell a heterologous nucleic acid encoding an immunomodulatory agent, thereby producing a genetically engineered g-NK cell.
78. A method of producing genetically engineered g-NK cells, the method comprising:
(a) Introducing a heterologous nucleic acid encoding a Chimeric Antigen Receptor (CAR) into an FcR gamma chain expression deficient NK cell (g-NK cell), and
(B) Introducing a heterologous nucleic acid encoding an immunomodulator into said g-NK cell,
Thereby producing genetically engineered g-NK cells, wherein steps (a) and (b) are performed simultaneously or sequentially in any order.
79. The method of claim 76 or claim 78, wherein the CAR comprises 1) an antigen binding domain; 2) Flexible connectors (spacers); 3) A transmembrane region; and 4) an intracellular signaling domain.
80. The method of claim 79, wherein the antigen binding domain targets a tumor antigen.
81. The method of claim 79 or 80, wherein the antigen binding domain is a single chain variable fragment (scFv).
82. The method of any one of claims 79 to 81, wherein the intracellular signaling domain comprises one or more signaling domains from cd3ζ, DAP10, DAP12, CD28, 4-1BB, or OX 40.
83. The method of any one of claims 79 to 82, wherein the intracellular signaling domain comprises two or more signaling domains from cd3ζ, DAP10, DAP12, CD28, 4-1BB, or OX 40.
84. The method of any one of claims 79 to 83, wherein the intracellular signaling domain comprises a primary signaling domain comprising a signaling domain of cd3ζ and a costimulatory signaling domain.
85. The method of claim 84, wherein the co-stimulatory signaling domain is a signaling domain of CD28 or 4-1 BB.
86. The method of any one of claims 76 or 78 to 85, wherein the heterologous nucleic acid encoding the CAR is introduced under conditions that stably integrate into the genome of the g-NK cell.
87. The method of any one of claims 76 or 78 to 86, wherein the heterologous nucleic acid encoding the CAR is comprised in a viral vector and is introduced into the g-NK cell by transduction.
88. The method of claim 87, wherein the viral vector is a lentiviral vector.
89. The method of any one of claims 76 or 78 to 85, wherein the nucleic acid encoding the CAR is introduced under conditions of transient expression in the g-NK cells.
90. The method of any one of claims 76, 78 to 85 or 89, wherein the nucleic acid encoding the CAR is introduced by non-viral delivery.
91. The method of any one of claims 76, 78 to 85 or 89 to 90, wherein the nucleic acid encoding the CAR is introduced into the g-NK cell via a lipid nanoparticle.
92. The method of any one of claims 76 or 78 to 91, wherein the nucleic acid encoding the CAR is DNA.
93. The method of any one of claims 76, 78 to 85, or 89 to 91, wherein the nucleic acid encoding the CAR is RNA.
94. The method of claim 93, wherein the RNA is mRNA.
95. The method of claim 94, wherein the RNA is self-amplifying mRNA.
96. The method of any one of claims 76, 78 to 85 or 89 to 95, wherein the nucleic acid is introduced into the g-NK cell via electroporation.
97. The method of claim 77 or claim 78, wherein said immunomodulatory agent is an immunosuppressant.
98. The method of any one of claims 77 or 78, wherein the immunomodulator is an immune activator.
99. The method of claim 77, 78 or claim 98, wherein the immunomodulatory agent is a cytokine.
100. The method of claim 99, wherein the cytokine is secreted from the engineered NK cell.
101. The method of claim 100, wherein the secretable cytokine is IL-2 or a biologically active portion thereof; IL-15 or a biologically active portion thereof; IL-21 or a biologically active portion thereof; or a combination thereof.
102. The method of claim 99, wherein the cytokine is membrane-bound.
103. The method of claim 102, wherein the membrane-bound cytokine is membrane-bound IL-2 (mbIL-2); membrane-bound IL-15 (mbIL-15); or membrane-bound IL-21 (mbiL-21); or a combination thereof.
104. The method of any one of claims 77-103, wherein said nucleic acid encoding said immunomodulator is introduced under conditions that stably integrate into the genome of said g-NK cells.
105. The method of any one of claims 77-104, wherein said nucleic acid encoding an immunomodulatory agent is contained in a viral vector and introduced into said g-NK cell by transduction.
106. The method of claim 105, wherein the viral vector is a lentiviral vector.
107. The method of any one of claims 77-103, wherein said nucleic acid encoding said immunomodulator is introduced under conditions of transient expression in said g-NK cells.
108. The method of any one of claims 77-85, 89-90, or 97-107, wherein the nucleic acid encoding the immunomodulator is introduced by non-viral delivery.
109. The method of any one of claims 77-85, 89-91, or 97-108, wherein the nucleic acid encoding the immunomodulatory agent is introduced into the g-NK cell via a lipid nanoparticle.
110. The method of any one of claims 77-109, wherein the nucleic acid encoding the immunomodulatory agent is DNA.
111. The method of any one of claims 77-85, 89-91, or 97-109, wherein the nucleic acid encoding the immunomodulatory agent is RNA.
112. The method of claim 111, wherein the RNA is mRNA.
113. The method of claim 112, wherein the RNA is self-amplifying mRNA.
114. The method of any one of claims 77-85, 89-103 or 107-113, wherein the nucleic acid encoding the immunomodulatory agent is introduced into the g-NK cell via electroporation.
115. The method of any one of claims 78 to 114, wherein the nucleic acid encoding the CAR and the nucleic acid encoding the immunomodulatory agent are encoded by the same polynucleotide and introduced together.
116. The method of claim 115, wherein the nucleic acid encoding the CAR and the nucleic acid encoding the immunomodulator are separated by a polycistronic element of a polynucleotide sequence.
117. The method of claim 116, wherein the polycistronic element is a self-cleaving peptide selected from the group consisting of T2A, P a and F2A.
118. The method of any one of claims 78 to 114, wherein the nucleic acid encoding the CAR and the nucleic acid encoding the immunomodulatory agent are introduced simultaneously during an in vitro process for amplifying a population enriched in g-NK cells.
119. The method of any one of claims 76 to 118, wherein the g-NK cell composition is produced by in vitro expansion of NK cells enriched from a biological sample from a subject, the NK cells being: (i) Negative or low level for CD3 and positive for CD57 (CD 3 Negative of CD57 Positive and negative ) or (ii) negative or low level for CD3 and positive for CD56 (CD 3 Negative of CD56 Positive and negative ), wherein the enriched NK cells are cultured with irradiated HLA-e+ feeder cells and one or more recombinant cytokines. 76. The method of claim 75, wherein the one or more recombinant cytokines are selected from the group consisting of SCF, GSK3i, FLT3, IL-2, IL-6, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or a combination thereof in an effective amount.
120. The method of claim 119, wherein the one or more recombinant cytokines comprise IL-21.
121. The method of claim 119 or claim 120, wherein culturing is performed in the presence of two or more recombinant cytokines, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21.
122. The method of any one of claims 76, 79 to 96, or 119 to 121, wherein the introducing is performed during a method for expanding FcR gamma-deficient NK (g-NK) cells, the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21;
Wherein introducing the nucleic acid encoding the CAR is performed after step (a) and before, during or after step (B), thereby producing an engineered NK cell population; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR.
123. The method of any one of claims 76, 79 to 96 or 119 to 121, wherein the introducing is performed during a method for amplifying FcR gamma-deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject;
(B) Introducing the nucleic acid encoding a CAR into the enriched NK cell population, thereby producing an engineered NK cell population; and
(C) Culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR.
124. The method of any one of claims 76, 79 to 96 or 119 to 121, wherein the introducing is performed during a method for amplifying FcR gamma-deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Culturing the enriched NK cell population in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and
(C) Introducing said nucleic acid encoding a CAR into NK cells of said NK cell expansion population,
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR.
125. The method of any one of claims 76, 79 to 96 or 119 to 121, wherein the introducing is performed during a method for amplifying FcR gamma-deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Performing a first expansion comprising culturing the enriched NK cell population in a medium under conditions to expand the NK cells to produce a first expanded population of NK cells;
(C) Introducing the nucleic acid encoding a CAR into NK cells of the first amplified population of NK cells, thereby producing an engineered NK cell population; and
(D) Performing a second expansion comprising culturing said population of engineered NK cells under conditions that further expand said NK cells,
Wherein the first expansion and/or second expansion comprises culturing the enriched NK cell population in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR.
126. The method of any one of claims 77, 97-114, or 119-121, wherein the introducing is performed during a method for expanding FcR gamma-deficient NK (g-NK) cells, the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21;
Wherein introducing the nucleic acid encoding the immunomodulator is performed after step (a) and before, during or after step (B), thereby producing an engineered NK cell population; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with an immunomodulatory agent.
127. The method of any one of claims 77, 97-114, or 119-121, wherein the introducing is performed during a method for amplifying FcR gamma-deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject;
(B) Introducing the nucleic acid encoding an immunomodulatory agent into the enriched NK cell population, thereby producing an engineered NK cell population; and
(C) Culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with an immunomodulatory agent.
128. The method of any one of claims 77, 97-114, or 119-121, wherein the introducing is performed during a method for amplifying FcR gamma-deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Culturing the enriched NK cell population in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and
(C) Introducing said nucleic acid encoding an immunomodulator into NK cells of said amplified population of NK cells,
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with an immunomodulatory agent.
129. The method of any one of claims 77, 97-114, or 119-121, wherein the introducing is performed during a method for amplifying FcR gamma-deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Performing a first expansion comprising culturing the enriched NK cell population in a medium under conditions to expand the NK cells to produce a first expanded population of NK cells;
(C) Introducing said nucleic acid encoding an immunomodulatory agent into NK cells of said first expanded population of NK cells, thereby producing an engineered NK cell population; and
(D) Performing a second expansion comprising culturing said population of engineered NK cells under conditions that further expand said NK cells,
Wherein the first expansion and/or second expansion comprises culturing the enriched NK cell population in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with an immunomodulatory agent.
130. The method of any one of claims 78 to 121, wherein the introducing is performed during a method for expanding FcR γ -deficient NK (g-NK) cells, the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21;
Wherein introducing (i) the nucleic acid encoding the CAR and/or (ii) the nucleic acid encoding the immunomodulator is performed after step (a) and before, during or after step (B), wherein steps (i) and (ii) are performed simultaneously or sequentially in any order, thereby producing an engineered NK cell population; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR and an immunomodulatory agent.
131. The method of any one of claims 78 to 121, wherein the introducing is performed during a method for amplifying FcR γ -deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject;
(B) Introducing (i) a nucleic acid encoding a CAR and (ii) a nucleic acid encoding an immunomodulatory agent into the enriched NK cell population, wherein steps (i) and (ii) are performed simultaneously or sequentially in any order, thereby producing an engineered NK cell population; and
(C) Culturing the population of engineered NK cells in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR and an immunomodulatory agent.
132. The method of any one of claims 78 to 121, wherein the introducing is performed during a method for amplifying FcR γ -deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Culturing the enriched NK cell population in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21, thereby producing an NK cell expanded population; and
(C) Introducing (i) said nucleic acid encoding a CAR and (ii) said nucleic acid encoding an immunomodulator into NK cells of said amplified population of NK cells, wherein steps (i) and (ii) are performed simultaneously or sequentially in any order,
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR and an immunomodulatory agent.
133. The method of any one of claims 78 to 121, wherein the introducing is performed during a method for amplifying FcR γ -deficient NK cells (g-NK), the method comprising:
(A) Obtaining a population of primary human cells enriched for Natural Killer (NK) cells, wherein the population enriched for NK cells is selected from a biological sample from a human subject; and
(B) Performing a first expansion comprising culturing the enriched NK cell population in a medium under conditions to expand the NK cells to produce a first expanded population of NK cells;
(C) Introducing (i) the nucleic acid encoding a CAR and (ii) the nucleic acid encoding an immunomodulatory agent into NK cells of the first amplified population of NK cells, wherein steps (i) and (ii) are performed simultaneously or sequentially in any order, thereby producing an engineered NK cell population; and
(D) Performing a second expansion comprising culturing said population of engineered NK cells under conditions that further expand said NK cells,
Wherein the first expansion and/or second expansion comprises culturing the enriched NK cell population in a medium having: (1) An irradiated HLA-e+ feeder cell, wherein the feeder cell is deficient in HLA class I and HLA class II, and wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is from 1:10 to 10:1; and (2) an effective amount of two or more recombinant cytokines for expanding the NK cells, wherein at least one recombinant cytokine is Interleukin (IL) -2 and at least one recombinant cytokine is IL-21; and
Wherein the method results in an expanded population of engineered NK cells that is enriched for g-NK cells and comprises cells engineered with a CAR and an immunomodulatory agent.
134. The method of any one of claims 119-133, wherein the population of NK cell-enriched primary human cells is obtained by selecting cells from a biological sample from a human subject, the cells being: (i) Negative or low level for CD3 and positive for CD57 (CD 3 Negative of CD57 Positive and negative ) or (ii) negative or low level for CD3 and positive for CD56 (CD 3 Negative of CD56 Positive and negative ).
135. The method of claim 134, wherein:
The population enriched for NK cells is a cell obtained by further selecting cells positive for NKG2C (NKG 2C Positive and negative );
The population enriched for NK cells is a cell obtained by further selecting cells negative for NKG2A or low in level (NKG 2A Negative of ); or alternatively
The population enriched for NK cells is a cell obtained by further selecting cells positive for NKG2C and negative or low level for NKG2A (NKG 2C Positive and negative NKG2A Negative of ).
136. The method of any one of claims 119-135, wherein the human subject is a subject: wherein at least or at least about 20% of Natural Killer (NK) cells in a peripheral blood sample from the subject are positive for NKG2C (NKG 2C Positive and negative ) and at least 70% of NK cells in the peripheral blood sample are negative for NKG2A or low level (NKG 2A Negative of ).
137. The method of any one of claims 119-136, wherein the subject is CMV seropositive.
138. The method of any one of claims 119-137, wherein the percentage of g-NK cells in the biological sample from the subject is greater than 5% or greater than about 5%, greater than 10% or greater than about 10%, or greater than 30% or greater than about 30%.
139. The method of any one of claims 119-138, wherein the percentage of g-NK cells in the enriched NK cell population is between 20% and 90%, or between about 20% and about 90%, or between 40% and 90%, or between about 40% and about 90%, or between 60% and 90%, or between about 60% and about 90%.
140. The method of any one of claims 119-139, wherein the population enriched for NK cells is a cell that is negative or low level for CD3 and positive for CD57 (CD 3 Negative of CD57 Positive and negative ) selected from the biological sample.
141. The method of any one of claims 119-140, wherein the population enriched for NK cells is a cell that is negative or low in level for CD3 and positive for CD56 (CD 3 Negative of CD56 Positive and negative ) selected from the biological sample.
142. The method of any one of claims 119-141, wherein the two or more recombinant cytokines further comprise an effective amount of SCF, GSK3i, FLT3, IL-6, IL-7, IL-15, IL-12, IL-18, IL-27, or a combination thereof.
143. The method of any one of claims 114-142, wherein the recombinant cytokine is IL-21 and IL-2.
144. The method of any one of claims 114-142, wherein the recombinant cytokine is IL-21, IL-2, and IL-15.
145. The method of any one of claims 114-144, wherein recombinant IL-21 is added to the culture medium at a concentration of 10ng/mL to 100ng/mL or about 10ng/mL to about 100ng/mL during at least a portion of the culturing.
146. The method of any one of claims 114-145, wherein recombinant IL-21 is added to the culture medium at a concentration of 25ng/mL or about 25ng/mL during at least a portion of the culturing.
147. The method of any one of claims 114-146, wherein recombinant IL-2 is added to the culture medium at a concentration of 10IU/mL to 500IU/mL, or about 10IU/mL to about 500IU/mL, during at least a portion of the culturing.
148. The method of any one of claims 114-147, wherein recombinant IL-2 is added to the culture medium at a concentration of 100IU/mL, or about 100IU/mL, during at least a portion of the culturing.
149. The method of any one of claims 114-148, wherein recombinant IL-2 is added to the culture medium at a concentration of 500IU/mL or about 500IU/mL during at least a portion of the culturing.
150. The method of any one of claims 114-142 and 144-149, wherein recombinant IL-15 is added to the medium at a concentration of 1ng/mL to 50ng/mL or about 1ng/mL to about 50ng/mL during at least a portion of the culturing.
151. The method of any one of claims 114-142 and 144-148, wherein recombinant IL-15 is added to the culture medium at a concentration of 10ng/mL or about 10ng/mL during at least a portion of the culturing.
152. The method of any one of claims 114-151, wherein the addition of the recombinant cytokine to the culture medium is initiated at or about the beginning of the culture.
153. The method of any one of claims 114-152, wherein the method further comprises replacing the medium one or more times during the culturing, wherein fresh medium containing the recombinant cytokine is added each time the medium is replaced.
154. The method of claim 153, wherein the replacement of the medium is performed every two or three days during the duration of the culturing.
155. The method of claim 153 or claim 154, wherein the replacement of medium is performed after no medium replacement for up to 5 days after the first amplification, optionally after no medium replacement for up to 5 days after the first amplification.
156. The method of any one of claims 114-155, wherein the human subject has a CD16 158V/V NK cell genotype or a CD16 158V/F NK cell genotype, optionally wherein the biological sample is from a human subject selected for the CD16 158V/V NK cell genotype or the CD16 158V/F NK cell genotype.
157. The method of any one of claims 114 to 156, wherein the biological sample is or comprises Peripheral Blood Mononuclear Cells (PBMCs), optionally a blood sample, an apheresis sample, or a leucocyte apheresis sample.
158. The method of any one of claims 114-157, wherein the HLA-e+ feeder cells are K562 cells (K562-HLA-E) transformed with HLA-E.
159. The method of any one of claims 114 to 158, wherein the HLA-e+ feeder cells are 221.Aeh cells.
160. The method of any one of claims 114 to 159, wherein the ratio of irradiated HLA-e+ feeder cells to enriched NK cells is between 1:1 and 5:1, inclusive, between 1:1 and 3:1, inclusive, optionally 2.5:1 or about 2:1 or about 1:1.
161. The method of any one of claims 114-160, wherein the recombinant cytokine added to the culture medium during at least a portion of the culturing is 500IU/mL IL-2, 10ng/mL IL-15, and 25ng/mL IL-21.
162. The method of any one of claims 114 to 161, wherein the enriched NK cell population comprises enriched NK cells of: the population of cells optionally comprises enriched cells at the end of each of between 2.0×10 5 enriched NK cells and 5.0×10 7 enriched NK cells or between about 2.0×10 5 enriched NK cells and about 5.0×10 7 enriched NK cells, between 1.0×10 6 enriched NK cells and 1.0×10 8 enriched NK cells or between about 1.0×10 6 enriched NK cells and about 1.0×10 8 enriched NK cells, between 1.0×10 7 enriched NK cells and 5.0×10 8 enriched NK cells or between about 1.0×10 7 enriched NK cells and about 5.0×10 8 enriched NK cells or between 1.0×10 7 enriched NK cells and 1.0×10 9 enriched NK cells or between about 1.0×10 7 enriched NK cells and about 1.0×10 9 enriched NK cells, including enriched cells at the end of each of said population optionally comprising enriched cells of about 1×25 NK cells.
163. The method of any one of claims 114 to 162, wherein the enriched NK cell population has a concentration at the beginning of the culturing of between 0.05 x 10 6 enriched NK cells/mL and 1.0 x 10 6 enriched NK cells/mL or between about 0.05 x 10 6 enriched NK cells/mL and about 1.0 x 10 6 enriched NK cells/mL or between 0.05 x 10 6 enriched NK cells/mL and 0.5 x 10 6 enriched NK cells/mL or between about 0.05 x 10 6 enriched NK cells/mL and about 0.5 x 10 6 enriched NK cells/mL, optionally wherein the enriched NK cell population has or has a concentration of about 0.2 x 10 6 enriched NK cells/mL at the beginning of the culturing.
164. The method of any one of claims 114 to 163, wherein the culturing is performed until the method achieves a time to expand at least or at least about 2.50 x 10 8 g-NK cells, at least or at least about 5.00 x 10 8 g-NK cells, at least or at least about 1.0 x 10 9 g-NK cells, or at least about 5.0 x 10 9 g-NK cells.
165. The method of any one of claims 114 to 164, wherein the culturing is performed or performed for about or at least about the following time: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 days.
166. The method of any one of claims 114 to 165, further comprising collecting the expanded population of engineered NK cells produced by the method.
167. The method of any one of claims 114 to 166, further comprising formulating the expanded population of engineered NK cells in a pharmaceutically acceptable excipient.
168. The method of claim 167, further comprising formulating the expanded population of engineered NK cells with a serum-free cryopreservation medium comprising a cryoprotectant.
169. The method of claim 168, wherein the cryoprotectant is DMSO, optionally wherein the cryoprotectant is DMSO and the cryopreservation medium is 5% to 10% DMSO (v/v), optionally at or about 10% DMSO (v/v).
170. The method of any one of claims 114 to 169, wherein in the population of engineered NK cell expansion produced by the method, greater than 50% of the population is
Fcrgamma Negative of , greater than 60% of the population is fcrgamma Negative of , greater than 70% of the population is fcrgamma Negative of ,,, greater than 80% of the population is fcrgamma Negative of , greater than 90% of the population is fcrgamma Negative of , or greater than 95% of the population is fcrgamma Negative of .
171. A composition comprising a plurality of engineered g-NK cells produced by the method of any one of claims 76, 78 to 96, 119 to 125, or 134 to 170.
172. A composition comprising a plurality of engineered g-NK cells produced by the method of any one of claims 77, 97 to 114, 119 to 121, or 126 to 129, or 134 to 170.
173. A composition comprising a plurality of engineered g-NK cells produced by the method of any one of claims 77 to 121 or 130 to 170.
174. A method of treating a disease or disorder in a subject, the method comprising administering to an individual in need thereof an effective amount of cells of the composition of any one of claims 29-73 or 171-173.
175. The method of claim 174, wherein the disease or disorder is selected from an inflammatory disorder, an infection, or a cancer.
176. The method of claim 174 or claim 175, wherein the disease or disorder is cancer and the cancer is leukemia, lymphoma, or myeloma.
177. The method of claim 174 or claim 175, wherein the disease or disorder is cancer and the cancer comprises a solid tumor.
178. The method of claim 177, wherein the cancer is selected from the group consisting of adenocarcinoma of the stomach or gastroesophageal junction, bladder cancer, breast cancer, brain cancer, cervical cancer, colorectal cancer, endocrine/neuroendocrine cancer, head and neck cancer, gastrointestinal stromal cancer, bone giant cell tumor, renal cancer, liver cancer, lung cancer, neuroblastoma, ovarian epithelial/fallopian tube/primary peritoneal cancer, pancreatic cancer, prostate cancer, skin cancer, and soft tissue cancer.
179. The method of any one of claims 174 to 178, wherein the composition is administered as monotherapy.
180. The method of any one of claims 174-179, further comprising administering to the individual an additional agent to treat the disease or disorder.
181. The method of claim 180, wherein the additional agent is an antibody or Fc fusion protein.
182. The method of claim 180 or claim 181, wherein the additional agent is an antibody that is a monoclonal antibody.
183. The method of claim 181 or claim 182, wherein the antibody is a full-length antibody.
184. The method of any one of claims 181 to 183 wherein the antibody is an IgG1 antibody.
185. The method of claims 180-184, wherein the disease or disorder is cancer and the additional agent, optionally the antibody, recognizes a tumor antigen associated with the cancer.
186. The method of any one of claims 174 to 185, comprising administering to the individual 1 x 10 5 to 50 x 10 9 cells or about 1 x 10 5 to about 50 x 10 9 cells of the g-NK cell composition.
187. The method of any one of claims 174 to 186, comprising administering from 1x 10 8 cells to 50 x 10 9 NK cells or from about 1x 10 8 cells to about 50 x 10 9 NK cells of the g-NK cell composition, optionally from 5x 10 8 cells or about 5x 10 8 cells of the g-NK cell composition, from 5x 10 9 cells or about 5x 10 9 cells of the g-NK cell composition, or from 10 x 10 9 cells or about 10 x 10 9 cells of the g-NK cell composition.
188. The method of any one of claims 174 to 187, further comprising administering exogenous cytokine support to promote expansion or persistence of the administered NK cells in the subject, optionally wherein the exogenous cytokine is or comprises IL-15.
189. The method of any one of claims 174 to 188, wherein the subject has received lymphocyte removal therapy prior to the administration of the dose of g-NK cells.
190. The method of claim 129, wherein the lymphocyte removal therapy comprises fludarabine and/or cyclophosphamide.
191. The method of claim 189 or claim 190, wherein the lymphocyte depletion comprises administering fludarabine daily for 2 to 4 days at 20mg/m 2 to 40mg/m 2 or about 20mg/m 2 to about 40mg/m 2 of the subject body surface area, optionally 30mg/m 2 or about 30mg/m 2, and/or administering cyclophosphamide daily for 2 to 4 days at 200mg/m 2 to 400mg/m 2 or about 200mg/m 2 to about 400mg/m 2 of the subject body surface area, optionally 300mg/m 2 or about 300mg/m 2.
192. The method of any one of claims 189 to 191 wherein the lymphocyte depletion therapy comprises fludarabine and cyclophosphamide.
193. The method of any one of claims 189 to 192, wherein the lymphoremoval therapy comprises administering fludarabine at 30mg/m 2 or about 30mg/m 2 of the subject body surface area per day, and cyclophosphamide at 300mg/m 2 or about 300mg/m 2 of the subject body surface area per day, each for 2 to 4 days, optionally for 3 days.
194. The method of any one of claims 189 to 193, wherein administering the cells is initiated within two weeks or at or about two weeks after initiation of the lymphocyte removal therapy.
195. The method of any one of claims 174 to 194, wherein the subject is a human.
196. The method of any one of claims 174 to 195, wherein the NK cells in the composition are allogeneic to the individual.
197. The method of any one of claims 189 to 195, wherein the NK cells in the composition are autologous to the subject.
CN202280045649.8A 2021-07-01 2022-06-30 Engineered Natural Killer (NK) cells and related methods Pending CN117915929A (en)

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