Off-the-shelf iPSC-derived CAR-NK cells as monotherapy and in combination with antibodies

Abstract

Methods and compositions for use in cancer immunotherapy are provided. Exemplary compositions include functionally enhanced derived effector cells obtained from directed differentiation of genomically engineered iPSCs. In some embodiments, the derived cells provided herein have stable functional genome editing that results in improved or enhanced therapeutic effects. Therapeutic compositions and uses thereof are also provided that include functionally enhanced derived effector cells alone or in combination therapy with antibodies or checkpoint inhibitors.

Description

[Technical field]

[0001] (Related Applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 234,656, filed August 18, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] (Reference to electronically submitted sequence listing) The Sequence Listing entitled 184143-636601_SL.xml was created on Aug. 18, 2022, is 16,431 bytes in size, and is incorporated by reference in its entirety.

[0003] FIELD OF THEINVENTION The present disclosure relates generally to the field of off-the-shelf immune cell products. More specifically, the present disclosure relates to strategies for developing multifunctional effector cells capable of delivering therapeutically relevant properties in vivo. The cell products developed under the present disclosure address critical limitations of patient-derived cell therapy. [Background technology]

[0004] The field of adoptive cell therapy is currently focused on using patient- and donor-derived cells, making it particularly challenging to achieve consistent production of cancer immunotherapies and to provide therapy to all patients who may benefit. There is also a need to improve the efficacy and persistence of adoptively transferred lymphocytes to promote better patient outcomes. Lymphocytes, such as T cells and natural killer (NK) cells, are potent antitumor effectors that play a key role in innate and adaptive immunity. However, using these immune cells for adoptive cell therapy remains challenging and there is an unmet need for improvement. Thus, there remains a significant opportunity to fully exploit the potential of T cells and NK cells, or other lymphocytes, in adoptive immunotherapy. Summary of the Invention

[0005] There is a need for functionally improved effector cells that address a variety of issues, including response rate, cell exhaustion, loss of infused cells (viability and / or persistence), tumor escape due to target loss or lineage switching, precision of tumor targeting, off-target toxicity, off-tumor effects, efficacy against solid tumors, i.e., the tumor microenvironment and associated immune suppression, recruitment, trafficking, and invasion.

[0006] It is an object of embodiments of the present invention to provide methods and compositions for adoptive cell therapy, which comprises administering an adoptive cell therapy product generated from derived non-pluripotent cells differentiated from a single cell-derived iPSC (induced pluripotent stem cell) clonal line, which iPSC line contains one or several genetic modifications in its genome, in some embodiments, the one or several genetic modifications include one or more of DNA insertions, deletions, and substitutions, which remain retained and functional in the subsequent derived cells after differentiation, expansion, passaging, and / or transplantation.

[0007] The iPSC-derived non-pluripotent cells of the present application contain CD34 +These include, but are not limited to, cells, hemogenic endothelial cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic pluripotent progenitor cells, T cell precursors, NK cell precursors, T cells, NKT cells, NK cells, and B cells. The iPSC-derived non-pluripotent cells of the present application contain one or several genetic modifications in their genomes through differentiation from iPSCs containing the same genetic modifications. In some embodiments, the engineered clonal iPSC differentiation strategy to obtain genetically engineered derivative cells also benefits from the developmental potential of iPSCs in directed differentiation that is not significantly adversely affected by the engineered modalities of the iPSCs, and that the engineered modalities function as intended in the derivative cells. Furthermore, this strategy overcomes the current barriers in engineering primary lymphocytes such as T cells or NK cells obtained from peripheral blood, namely the difficulty in engineering such cells, resulting in cells that often lack reproducibility and uniformity and exhibit poor cell persistence with high cell death and low cell proliferation. Furthermore, this strategy avoids the generation of heterogeneous effector cell populations that are otherwise obtained using initially heterogeneous primary cell sources.

[0008] Thus, in one aspect, the invention provides a method of treating a subject suitable for adoptive cell therapy, (i) the subject has a hematological cancer, (ii) the method comprises administering to the subject at least a first cycle of an adoptive cell therapy product, the first cycle comprising one or more doses of the adoptive cell therapy product administered at a preselected frequency in a first effective amount, with the option of administering one or more additional cycles having one or more doses in a second effective amount during the course of treatment over a period of time, (iii) the first and second effective amounts are the same or different, and (iv) the product comprises engineered natural killer (NK) lineage cells comprising exogenous CD16 expression, IL15RF expression, CD38 knockout, and a BCMA-directed CAR (chimeric antigen receptor). In various embodiments, the hematological cancer comprises (i) multiple myeloma (MM) or (ii) relapsed or refractory MM (r / rMM). In some embodiments of the method, the course of treatment further comprises administering to the subject an effective amount of a tumor-targeting ADCC capable monoclonal antibody (mAb).

[0009] In some embodiments of the method, the course of treatment further comprises administering to the subject an initial dose of an effective amount of a monoclonal antibody at a starting time prior to the first cycle of administering the adoptive cell therapy product, the monoclonal antibody being an anti-CD38 monoclonal antibody. In various embodiments of the method of treatment, the starting time is about 8-12 days prior to the first cycle of administering the adoptive cell therapy product, and the initial dose of monoclonal antibody comprises 6-10 weekly (QW) doses, optionally followed by 6-10 biweekly (Q2W±1 day) doses. In some embodiments, the course of treatment further comprises administering to the subject an effective amount of the same anti-CD38 monoclonal antibody for 8 biweekly doses (Q2W±1 day) following administration of the initial dose of monoclonal antibody. In some embodiments, the course of treatment further comprises administering to the subject one dose of an effective amount of the same anti-CD38 monoclonal antibody every 4 weeks (Q4W±1 day) until the end of the doses. In some embodiments, the anti-CD38 monoclonal antibody comprises daratumumab. In some embodiments, the course of treatment comprises administering an anti-CD38 monoclonal antibody to a subject, and the method (i) does not require lymphatic conditioning or (ii) requires minimal lymphatic conditioning. In some embodiments, the lymphatic conditioning is CY / FLU-based.

[0010] In various embodiments of the method of treatment, the method further comprises administering to the subject at least one daily dose of one or more chemotherapeutic agents prior to the first cycle of the adoptive cell therapy product, and the duration between the administration of the last daily dose of the one or more chemotherapeutic agents and the first cycle of the adoptive cell therapy product comprises a particular period of time. In some embodiments, the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU), optionally, CY and FLU are administered daily for three consecutive days, or the dose of CY is about 500 mg / m 2 and the dose of FLU is about 30 mg / m 2 In some embodiments, the duration of the period is (i) about 40 to 84 hours, or (ii) about 3 days.

[0011] In various embodiments of the method of treatment, the engineered NK lineage cells are derived from engineered induced pluripotent stem cells (iPSCs) that contain a polynucleotide encoding exogenous CD16, IL15RF, CD38 knockout, and a polynucleotide encoding a BCMA-directed CAR. In some embodiments, the effective amount of adoptive cell therapy product in a dose is about 5×10 7 cells ~ approx. 3 x 10 9 In some embodiments, the effective amount of adoptive cell therapy product in each dose is about 1×10 8 , 3×10 8 , 10×10 8 , or about 1.5 × 10 9 In some embodiments, (i) the daratumumab is in an amount of about 15 mg / kg to about 17 mg / kg, or (ii) the daratumumab is in an amount of about 16 mg / kg.

[0012] In various embodiments of the methods of treatment, subjects suitable for the adoptive cell therapy product have a diagnosis of MM, including (i) measurable disease, or (ii) not having a complete remission (CR) or having a relapse or having evidence of progressive disease (PD) after one or more prior therapies. In some embodiments, the MM, or previous therapy for relapsed or refractory MM, comprises (a) chemotherapy, immunochemotherapy, hematopoietic stem cell transplantation, chimeric antigen receptor (CAR) T-cell therapy, or any combination thereof, or (b) a proteasome inhibitor, an anti-CD38 antibody, an anti-SLAMF7 antibody, an immunomodulatory agent, stem-cell transplantation (SCT), or any combination thereof, or (c) bortezomib, carfilzomib, ixazomib, daratumumab, isatuximab, elotuzumab, thalidomide, lenalidomide, pomalidomide, or any combination thereof, or (d) carfilzomib.

[0013] In various embodiments of the method of treatment, the method includes administering (i) one cycle of an adoptive cell therapy product over about 29 days at one or two doses per cycle, (ii) two or more cycles of an adoptive cell therapy product, each cycle including one or two doses, (iii) a single dose per cycle over about 29 days, where one or more cycles of the adoptive cell therapy product are over an extended period based on clinical evaluation of disease response, or (iv) two doses per cycle over about 29 days, where one or more cycles of the adoptive cell therapy product are over an extended period based on clinical evaluation of disease response. In various embodiments of the method of treatment, administering the adoptive cell therapy product is (i) via intravenous infusion and / or (ii) at a location in an outpatient setting, and / or each dose of the adoptive cell therapy product is stored frozen and then thawed prior to administration.In various embodiments of the method of treatment, the BCMA-directed CAR comprises: (i) a variable heavy chain (VH) and a variable light chain (VL), wherein (a) the VH comprises a heavy chain complementary determining region 1 (H-CDR1) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO:8 (GFTFSRYW), a heavy chain complementary determining region 2 (H-CDR2) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO:9 (INPSSSTI), and a heavy chain complementary determining region 3 (H-CDR4) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO:10 (ASLYYDYGDAYDY). (b) a variable light chain (VL), comprising a light chain complementary determining region 1 (L-CDR1) having at least 80% sequence identity (e.g., 90% or 100% identity) with SEQ ID NO: 11 (QSVESN), a light chain complementary determining region 2 (L-CDR2) having at least 80% sequence identity (e.g., 90% or 100% identity) with SEQ ID NO: 12 (SAS), and a light chain complementary determining region 3 (L-CDR4) having at least 80% sequence identity (e.g., 90% or 100% identity) with SEQ ID NO: 13 (QQYNNYPLT); 3, L-CDR3), a variable heavy chain (VH) and a variable light chain (VL), or (ii) an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to SEQ ID NO:7.In some embodiments, the BCMA-directed CAR comprises the amino acid sequence of SEQ ID NO:7.

[0014] In another aspect, the invention provides a composition comprising engineered natural killer (NK) lineage cells for use in treating a hematological cancer in a subject, (i) the engineered NK lineage cells comprise exogenous CD16 expression, IL15RF expression, CD38 knockout, and a BCMA-directed CAR (chimeric antigen receptor), (ii) the use comprises a course of treatment comprising at least a first cycle of an adoptive cell therapy product comprising the engineered NK lineage cells, the first cycle comprising one or more doses of the adoptive cell therapy product at a preselected frequency of a first effective amount, with the option of one or more additional cycles having one or more doses of a second effective amount over a period of time, and (iii) the first and second effective amounts are the same or different. In various embodiments, the engineered NK lineage cells are derived from engineered induced pluripotent stem cells (iPSCs) comprising a polynucleotide encoding exogenous CD16, IL15RF, CD38 knockout, and a polynucleotide encoding a BCMA-directed CAR. In some embodiments, the course of treatment further comprises an effective amount of a tumor-targeting ADCC-capable monoclonal antibody (mAb). In some embodiments, the course of treatment further comprises an initial dose of a monoclonal antibody provided in an effective amount at a starting time prior to the first cycle of the adoptive cell therapy product, the monoclonal antibody being an anti-CD38 monoclonal antibody. In some embodiments, the anti-CD38 monoclonal antibody comprises daratumumab. In some embodiments, the course of treatment further comprises at least one daily dose of one or more chemotherapeutic agents prior to the first cycle of the adoptive cell therapy product, the duration between administration of the last daily dose of the one or more chemotherapeutic agents and the first cycle of the adoptive cell therapy product comprises a specific period of time. In some embodiments, the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU), optionally, CY and FLU are administered daily for three consecutive days, or the dose of CY is about 500 mg / m 2 and the dose of FLU is about 30 mg / m 2In some embodiments, the BCMA-directed CAR comprises (i) a variable heavy chain (VH) and a variable light chain (VL), (a) the VH comprising a heavy chain complementarity determining region 1 (H-CDR1) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 8 (GFTFSRYW), a heavy chain complementarity determining region 2 (H-CDR2) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 9 (INPSSSTI), and a heavy chain complementarity determining region 3 (H-CDR3) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 10 (ASLYYDYGDAYDY), and (b) a variable light chain (VL), wherein the VL is , a variable light chain (VL), a variable heavy chain (VH) and a variable light chain (VL) comprising a light chain complementarity determining region 1 (L-CDR1) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 11 (QSVESN), a light chain complementarity determining region 2 (L-CDR2) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 12 (SAS), and a light chain complementarity determining region 3 (L-CDR3) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 13 (QQYNNYPLT), or (ii) an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to SEQ ID NO: 7. In some embodiments, the BCMA-directed CAR comprises the amino acid sequence of SEQ ID NO: 7.

[0015] In another aspect, the invention provides for the use of engineered natural killer (NK) lineage cells in the manufacture of an adoptive cell therapy product for treating hematological cancer, (i) the engineered NK lineage cells comprising exogenous CD16 expression, IL15RF expression, CD38 knockout, and a BCMA-directed CAR (chimeric antigen receptor), (ii) the adoptive cell therapy product is for use in a course of treatment comprising at least a first cycle of an adoptive cell therapy product, the first cycle comprising one or more doses of the adoptive cell therapy product at a preselected frequency of a first effective amount, with the option of one or more additional cycles having one or more doses of a second effective amount over a period of time, and (iii) the first and second effective amounts are the same or different. In various embodiments, the engineered NK lineage cells are derived from engineered induced pluripotent stem cells (iPSCs) comprising a polynucleotide encoding exogenous CD16, IL15RF, CD38 knockout, and a polynucleotide encoding a BCMA-directed CAR. In some embodiments, the course of treatment further comprises an effective amount of a tumor-targeting ADCC-capable monoclonal antibody (mAb). In some embodiments, the course of treatment further comprises an initial dose of a monoclonal antibody provided in an effective amount at a starting time prior to the first cycle of the adoptive cell therapy product, the monoclonal antibody being an anti-CD38 monoclonal antibody. In some embodiments, the anti-CD38 monoclonal antibody comprises daratumumab. In some embodiments, the course of treatment further comprises at least one daily dose of one or more chemotherapeutic agents prior to the first cycle of the adoptive cell therapy product, the duration between administration of the last daily dose of the one or more chemotherapeutic agents and the first cycle of the adoptive cell therapy product comprises a specific period of time. In some embodiments, the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU), optionally, CY and FLU are administered daily for three consecutive days, or the dose of CY is about 500 mg / m 2 and the dose of FLU is about 30 mg / m 2In some embodiments, the BCMA-directed CAR comprises (i) a variable heavy chain (VH) and a variable light chain (VL), (a) the VH comprising a heavy chain complementarity determining region 1 (H-CDR1) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 8 (GFTFSRYW), a heavy chain complementarity determining region 2 (H-CDR2) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 9 (INPSSSTI), and a heavy chain complementarity determining region 3 (H-CDR3) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 10 (ASLYYDYGDAYDY), and (b) a variable light chain (VL), wherein the VL is , a variable light chain (VL), a variable heavy chain (VH) and a variable light chain (VL) comprising a light chain complementarity determining region 1 (L-CDR1) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 11 (QSVESN), a light chain complementarity determining region 2 (L-CDR2) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 12 (SAS), and a light chain complementarity determining region 3 (L-CDR3) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 13 (QQYNNYPLT), or (ii) an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to SEQ ID NO: 7. In some embodiments, the BCMA-directed CAR comprises the amino acid sequence of SEQ ID NO: 7.

[0016] In another aspect, the invention provides a method of treating a subject diagnosed with multiple myeloma (MM), the method comprising: (i) administering to the subject at least a first cycle of an adoptive cell therapy product, the first cycle comprising at least a first dose at a first frequency of an adoptive cell therapy product administered in a first effective amount, with the option of administering one or more additional cycles having one or more additional doses at the same first frequency or at a different frequency in a second effective amount during the course of treatment over a period of time; and (ii) administering to the subject an effective amount of an anti-CD38 antibody for about 6-10 weeks. administering to the subject a weekly dose of a monoclonal antibody or an anti-SLAMF7 monoclonal antibody, wherein a first weekly dose of an anti-CD38 monoclonal antibody or an anti-SLAMF7 monoclonal antibody occurs about 8-12 days prior to step (i), wherein progression or recurrence of the MM tumor is prevented or reduced in the subject following treatment with an adoptive cell therapy product, the adoptive cell therapy comprising engineered natural killer (NK) lineage cells comprising a CD38 knockout and expressing exogenous CD16, IL15RF, and a BCMA-directed CAR. In various embodiments, the subject (i) has refractory or relapsed MM and / or (ii) has been treated with one or more therapies for MM, including (a) chemotherapy, immunochemotherapy, hematopoietic stem cell transplantation, chimeric antigen receptor (CAR) T-cell therapy, or any combination thereof; (b) a proteasome inhibitor, an anti-CD38 antibody, an anti-SLAMF7 antibody, an immunomodulatory agent, a stem cell transplant (SCT), or any combination thereof; or (c) bortezomib, carfilzomib, ixazomib, daratumumab, isatuximab, elotuzumab, thalidomide, lenalidomide, pomalidomide, or any combination thereof.

[0017] In various embodiments of the method of treatment, step (ii) further comprises administering biweekly doses of an effective amount of the same anti-CD38 monoclonal antibody or anti-SLAMF7 monoclonal antibody for an additional 6-10 weeks after completion of the weekly dose. In some embodiments, the anti-CD38 monoclonal antibody is daratumumab and / or the SLAMF7 monoclonal antibody is elotuzumab. In some embodiments of the method of treatment, the method further comprises administering to the subject daily doses of one or more chemotherapeutic agents for three consecutive days, wherein the duration between the administration of the last daily dose of the one or more chemotherapeutic agents and the first weekly dose of the adoptive cell therapy product is about 40-84 hours. In some embodiments, the one or more chemotherapeutic agents include cyclophosphamide (CY) and fludarabine (FLU), optionally wherein the daily dose of CY is about 500 mg / m 2 and the daily dose of FLU is about 30 mg / m 2 It is.

[0018] In various embodiments of the method of treatment, the method (a) does not require CY / FLU-based lymphatic conditioning or (b) requires minimal CY / FLU-based lymphatic conditioning, and step (ii) of the method comprises administering to the subject a weekly dose of an effective amount of an anti-CD38 monoclonal antibody. In some embodiments, the method does not require IL2 cytokine support to the subject during the course of treatment. In some embodiments, the engineered NK lineage cells are derived from engineered induced pluripotent stem cells (iPSCs) that include a CD38 knockout, a polynucleotide encoding exogenous CD16, and a polynucleotide encoding IL15RF. In some embodiments, the effective amount of adoptive cell therapy product in a dose is about 5×10 7 cells / dose~3×10 9 In some embodiments, the effective amount of adoptive cell therapy product in each dose is about 1×10 cells / dose. 8 , 3×10 8 , 10×10 8 , or about 1.5 × 10 9In some embodiments, (i) daratumumab is in an effective amount of about 15 mg / kg to about 17 mg / kg, and / or (ii) elotuzumab is in an effective amount of about 9 mg / kg to about 11 mg / kg. In some embodiments, (i) the effective amount of daratumumab is about 16 mg / kg, and (ii) the effective amount of elotuzumab is about 10 mg / kg.

[0019] In various embodiments of the method of treatment, the method further comprises assessing disease response after administration of the first cycle of the adoptive cell therapy product. In some embodiments, assessing disease response comprises assaying a bone marrow biopsy, peripheral blood sample, and urine sample from the subject for complete or partial response based on criteria including leukemic blast count, absolute neutrophil count, and / or platelet count, where (i) a complete response comprises (a) negative immunofixation to serum from the subject compared to serum immunofixation before the course of treatment, (b) negative immunofixation to urine from the subject compared to urine immunofixation before the course of treatment, (c) elimination of soft tissue plasmacytoma compared to plasmacytoma before the course of treatment, and / or (d) less than 5% plasma cells in the bone marrow compared to plasma cells in the bone marrow before the course of treatment, and / or (ii) a partial response comprises (a) a 25% or more but not more than 49% reduction in serum M protein levels compared to serum M protein before the course of treatment, and / or (b) about a 50-89% reduction in 24 hour urinary M protein levels compared to urinary M protein before the course of treatment. In some embodiments, administering the adoptive cell therapy product is (i) via intravenous infusion, and / or (ii) in an outpatient setting, and / or each dose of the adoptive cell therapy product is stored frozen and then thawed prior to administration.In various embodiments of the method of treatment, the BCMA-directed CAR comprises (i) a variable heavy chain (VH) and a variable light chain (VL), (a) the VH comprising a heavy chain complementarity determining region 1 (H-CDR1) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 8 (GFTFSRYW), a heavy chain complementarity determining region 2 (H-CDR2) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 9 (INPSSSTI), and a heavy chain complementarity determining region 3 (H-CDR3) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 10 (ASLYYDYGDAYDY), and (b) a variable light chain (VL), wherein the V wherein L comprises a variable light chain (VL), a variable heavy chain (VH) and a variable light chain (VL) comprising a light chain complementarity determining region 1 (L-CDR1) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 11 (QSVESN), a light chain complementarity determining region 2 (L-CDR2) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 12 (SAS), and a light chain complementarity determining region 3 (L-CDR3) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 13 (QQYNNYPLT), or (ii) an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to SEQ ID NO: 7. In some embodiments, the BCMA-directed CAR comprises the amino acid sequence of SEQ ID NO: 7.

[0020] In another aspect, the invention provides a method of multiple doses of targeted adoptive cell therapy in a subject in need of multiple doses of targeted adoptive cell therapy, comprising: (i) administering to the subject an effective amount of a targeted adoptive cell therapy product weekly over a course of treatment of about three weeks, the product comprising engineered immune cells expressing CD16, IL15RF, and a BCMA-directed CAR, wherein the engineered immune cells are CD38 negative; and (ii) detecting and comparing, at different given time points after administration of a first dose of adoptive cell therapy, one or more of the following: (a) presence of the engineered immune cells in the bone marrow of the subject; (b) presence of the engineered immune cells in the tumor of the subject; (c) protein markers of disease in the serum of the subject; (d) cytokines in a peripheral blood sample from the subject; and (e) circulating tumor DNA in a peripheral blood sample from the subject, wherein any of (a)-(e) is useful for assessing tumor burden, tumor immunobiology, and / or tumor therapy response, thereby determining the efficacy of multiple doses of targeted adoptive cell therapy. In some embodiments, the subject has multiple myeloma (MM). In some embodiments, an effective amount of an adoptive cell therapy product is about 5×10 7 cells / dose~1.5×10 9 cells / dose.

[0021] In various embodiments of the multiple dose targeted adoptive cell therapy method, the method further comprises administering to the subject an effective amount of an anti-CD38 monoclonal antibody or an anti-SLAMF7 monoclonal antibody weekly, wherein the first dose of the anti-CD38 monoclonal antibody or the anti-SLAMF7 monoclonal antibody occurs about 10 days prior to step (i). In some embodiments, the anti-CD38 monoclonal antibody is daratumumab, and / or the anti-SLAMF7 monoclonal antibody is elotuzumab, and / or the anti-CD20 monoclonal antibody is rituximab. In some embodiments, the BCMA-directed CAR comprises (i) a variable heavy chain (VH) and a variable light chain (VL), wherein (a) the VH comprises a heavy chain complementarity determining region 1 (H-CDR1) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 8 (GFTFSRYW), a heavy chain complementarity determining region 2 (H-CDR2) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 9 (INPSSSTI), and a heavy chain complementarity determining region 3 (H-CDR3) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 10 (ASLYYDYGDAYDY), and (b) a variable light chain (VL), wherein the VL comprises , a variable light chain (VL), a variable heavy chain (VH) and a variable light chain (VL) comprising a light chain complementarity determining region 1 (L-CDR1) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 11 (QSVESN), a light chain complementarity determining region 2 (L-CDR2) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 12 (SAS), and a light chain complementarity determining region 3 (L-CDR3) having at least 80% sequence identity (e.g., 90% or 100% identity) to SEQ ID NO: 13 (QQYNNYPLT), or (ii) an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to SEQ ID NO: 7. In some embodiments, the BCMA-directed CAR comprises the amino acid sequence of SEQ ID NO: 7.

[0022] In another aspect, the invention provides a method of re-treatment following one or more earlier therapies for MM according to the methods provided herein, wherein the one or more earlier therapies for MM comprise (a) chemotherapy, immunochemotherapy, hematopoietic stem cell transplantation, chimeric antigen receptor (CAR) T cell therapy, or any combination thereof; (b) a proteasome inhibitor, an anti-CD38 antibody, an anti-SLAMF7 antibody, an immunomodulatory agent, stem cell transplantation (SCT), or any combination thereof; (c) bortezomib, carfilzomib, ixazomib, daratumumab, isatuximab, elotuzumab, thalidomide, lenalidomide, pomalidomide, or any combination thereof; and / or (d) a BCMA-directed autologous CAR T cell therapy, idecabtagene vicleucel (bb2121, ide-cel).

[0023] Various objects and advantages of the compositions and methods provided herein will become apparent from the following description, taken in conjunction with the accompanying drawings, in which are set forth by way of illustration and example certain embodiments of the invention. [Brief description of the drawings]

[0024] [Figure 1] 1 is a graphical representation showing exemplary iNK cells for use in the immunotherapy methods described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Genome modification of iPSCs (induced pluripotent stem cells) includes polynucleotide insertion, deletion, and replacement. Exogenous gene expression in genome-engineered iPSCs often encounters problems such as gene silencing or reduced gene expression after long-term clonal expansion of the original genome-engineered iPSCs, after cell differentiation, and in dedifferentiated cell types from cells derived from genome-engineered iPSCs. Meanwhile, it is difficult to directly manipulate primary immune cells such as T cells or NK cells, which poses obstacles to the preparation and delivery of engineered immune cells for adoptive cell therapy. In various embodiments, the present invention provides an efficient and reliable targeted approach to stably integrate one or more exogenous genes, including suicide genes and other functional modalities, that confer improved therapeutic properties in terms of engraftment, trafficking, homing, migration, cytotoxicity, viability, maintenance, proliferation, longevity, self-renewal, persistence, and / or survival rate to iPSC-derived cells, including, but not limited to, HSCs (hematopoietic stem and progenitor cells), T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells.

[0026] definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0027] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0028] As used herein, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0029] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0030] The term "and / or" should be understood to mean either one or both of the alternatives.

[0031] As used herein, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a level, value, number, frequency, percentage, dimension, size, amount, weight, or length range of approximately ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0032] As used herein, the term "substantially" or "essentially" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more compared to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "substantially the same" or "essentially the same" refers to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range that is about the same as the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0033] As used herein, the terms "substantially free" and "essentially free" are used interchangeably and, when used to describe a composition such as a cell population or culture medium, refer to a composition that is free of a particular substance or source thereof, such as, for example, 95% free, 96% free, 97% free, 98% free, 99% free, etc., of a particular substance or source thereof, or is undetectable as measured by conventional means. The term "free" or "essentially free" of a particular component or substance in a composition also means that such component or substance is (1) not included in the composition at any concentration, or (2) included in the composition but at a low density and functionally inactive. A similar meaning may be applied to the term "absent," which refers to the absence of a particular substance or source thereof in a composition.

[0034] Throughout this specification, unless the context requires otherwise, "comprise", "comprises", and "comprising" will be understood to mean the inclusion of a stated step or element or group of steps or elements, but not to the exclusion of any other step or element or group of steps or elements. In certain embodiments, the terms "include", "having", "contain", and "comprise" are used interchangeably.

[0035] "Consisting of" means inclusive of and limited to everything that follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0036] "Consisting essentially of" means the inclusion of any elements listed after the phrase, limited to other elements that do not interfere with or contribute to the activity or operation specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that other elements are not optional and may or may not be present depending on whether they affect the activity or operation of the listed elements.

[0037] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0038] The term "ex vivo" generally refers to activities carried out outside of a living organism, such as experiments or measurements carried out in or on living tissue in an artificial environment outside of the body, preferably with minimal alteration of natural conditions. In certain embodiments, "ex vivo" procedures include live cells or tissues taken from a living organism and cultured in a laboratory setting, usually under sterile conditions, typically for a few hours or up to about 24 hours, but up to 48 hours or 72 hours or more depending on the circumstances. In certain embodiments, such tissues or cells may be collected and frozen, and later thawed for ex vivo processing. Tissue culture experiments or procedures lasting longer than a few days using live cells or tissues are typically considered to be "in vitro", although in certain embodiments, the term may be used interchangeably with ex vivo.

[0039] The term "in vivo" generally refers to activities that take place inside a living organism.

[0040] As used herein, the term "reprogramming" or "dedifferentiation" or "increasing cell potential" or "increasing developmental potential" refers to a method of increasing the potential of a cell or dedifferentiating a cell into a less differentiated state. For example, a cell with increased cell potential has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in a non-reprogrammed state. In other words, a reprogrammed cell is a cell that is in a less differentiated state than the same cell in a non-reprogrammed state.

[0041] As used herein, the term "differentiation" refers to the process by which an unspecialized ("uncommitted") or less specialized cell acquires the characteristics of a specialized cell, such as, for example, a blood cell or a muscle cell. A differentiated or differentiation-induced cell is a cell that assumes a more specialized ("committed") position within the lineage of a cell. The term "committed" as applied to the process of differentiation refers to a cell that has progressed down a differentiation pathway to a position where it will continue to differentiate into a particular cell type or subset of cell types under normal circumstances, and cannot, under normal circumstances, differentiate into a different cell type and revert to a less differentiated cell type. As used herein, the term "pluripotency" refers to the ability of a cell (i.e., the embryo itself) to form all lineages of an organism or somatic cells. For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers, ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential ranging from incompletely or partially pluripotent cells (e.g., epiblast stem cells or EpiSCs), which are unable to give rise to an entire organism, to more primitive, more pluripotent cells (e.g., embryonic stem cells), which are able to give rise to an entire organism.

[0042] As used herein, the term "induced pluripotent stem cells" or iPSCs refers to stem cells produced in vitro from differentiated adult, neonatal, or fetal cells that have been induced or altered, i.e., reprogrammed, using reprogramming factors and / or small molecule chemical drive methods into cells capable of differentiating into tissues of all three germ layers or dermal layers: mesoderm, endoderm, and ectoderm. Generated iPSCs do not refer to naturally occurring cells.

[0043] As used herein, the term "embryonic stem cell" refers to the naturally occurring pluripotent stem cells of the inner cell mass of blastocyst. Embryonic stem cells are pluripotent and generate all derivatives of the three primary germ layers during development, i.e., ectoderm, endoderm, and mesoderm. They do not contribute to extraembryonic membranes or placenta, i.e., they are not totipotent.

[0044] As used herein, the term "pluripotent stem cell" refers to a cell that has the developmental potential to differentiate into cells of one or more germ layers (ectoderm, mesoderm, and endoderm), but not all three. Thus, pluripotent cells can also be referred to as "partially differentiated cells." Pluripotent cells are well known in the art, and examples of pluripotent cells include adult stem cells, such as hematopoietic stem cells and neural stem cells. "Pluripotency" indicates that a cell can form many types of cells in a given lineage, but not cells of other lineages. For example, pluripotent hematopoietic cells can form many different types of blood cells (red, white, platelets, etc.), but cannot form neurons. Thus, the term "multipotency" refers to a state of a cell that has a degree of developmental potential that is less than totipotency and pluripotency.

[0045] Pluripotency can be determined, in part, by assessing the pluripotency characteristics of the cells, including, but not limited to, (i) pluripotent stem cell morphology, (ii) the potential for unlimited self-renewal, (iii) expression of pluripotent stem cell markers, including, but not limited to, SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30, and / or CD50, (iv) the ability to differentiate into all three somatic lineages (ectoderm, mesoderm, and endoderm), (v) teratoma formation composed of the three somatic lineages, and (vi) formation of embryoid bodies composed of cells from the three somatic lineages.

[0046] Two types of pluripotency have been described so far: a "primed" or "metastable" state of pluripotency similar to the epiblast stem cells (EpiSCs) of late blastocysts, and a "naive" or "ground" state similar to the inner cell mass of pluripotent early / preimplantation blastocysts. While both pluripotent states exhibit the above-mentioned characteristics, the naive or ground state further exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and survival in single cell culture, (iii) global reduction in DNA methylation, (iv) reduced deposition of H3K27me3 repressive chromatin marks on developmental regulatory gene promoters, and (v) reduced expression of differentiation markers compared to primed state pluripotent cells. Standard methodologies of cell reprogramming, in which exogenous pluripotency genes are introduced into somatic cells, expressed, and then either silenced or removed from the resulting pluripotent cells, appear to generally have characteristics of a primed state for pluripotency. Under standard pluripotent cell culture conditions, such cells remain in a primed state and characteristics of the ground state are observed unless exogenous transgene expression is maintained.

[0047] As used herein, the term "pluripotent stem cell morphology" refers to the classical morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a round and small shape with a high nuclear to cytoplasmic ratio, prominent presence of nucleoli, and typical intercellular spacing.

[0048] As used herein, the term "subject" refers to any animal, preferably a human patient, livestock, or other domesticated animals.

[0049] "Pluripotency factor" or "reprogramming factor" refers to an agent that can increase the developmental potential of a cell, alone or in combination with other agents. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of a cell. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0050] "Culture" or "cell culture" refers to the maintenance, growth and / or differentiation of cells in an in vitro environment. "Cell culture medium", "culture medium" (in each case singular "medium"), "supplement" and "medium supplement" refer to a nutritional composition in which a cell culture is cultivated.

[0051] "Culturing" or "maintaining" refers to sustaining, propagating (growing), and / or differentiating a tissue or cells outside the body, for example, in a sterile plastic (or coated plastic) cell culture dish or flask. "Culturing" or "maintaining" can utilize culture medium as a source of nutrients, hormones, and / or other factors that aid in the growth and / or maintenance of the cells.

[0052] As used herein, the term "mesoderm" refers to one of three germ layers that emerge during early embryonic development and give rise to a variety of specialized cell types, including blood cells of the circulatory system, muscle, heart, dermis, skeleton, and other supportive and connective tissues.

[0053] As used herein, the term "definitive hemogenic endothelium" (HE) or "pluripotent stem cell-derived definitive hemogenic endothelium" (iHE) refers to a subset of endothelial cells that give rise to hematopoietic stem and progenitor cells in a process called endothelial-hematopoietic conversion. Hematopoietic cell development in the embryo progresses sequentially from lateral plate mesoderm through hemangioblasts to definitive hemogenic endothelial cells and hematopoietic precursors.

[0054] The terms "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells", or "hematopoietic precursor cells" refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation, including multipotent hematopoietic stem cells (blood cells), myeloid precursors, megakaryocyte precursors, erythroid precursors, and lymphoid precursors. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid (T cells, B cells, NK cells). As used herein, the term "secondary hematopoietic stem cells" refers to CD34+ hematopoietic cells that can give rise to both mature myeloid and lymphoid cell types, including T cells, NK cells, and B cells. Hematopoietic cells also include various subsets of primitive hematopoietic cells that give rise to primitive erythrocytes, megakaryocytes, and macrophages.

[0055] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to a major type of white blood cell that has completed maturation in the thymus and has a variety of roles in the immune system, including the recognition of specific foreign antigens in the body, and the activation and inactivation of other immune cells. A T cell can be any T cell, e.g., a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. T cells are classified as CD3 + T cells can be CD4 + / CD8 +Double positive T cells, CD4 + Helper T cells (e.g., Th1 and Th2 cells), CD8 + The T cells can be of any type and at any stage of development, including, but not limited to, T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (γδ T cells), and the like. Additional types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells). The term "T cells" can also refer to genetically engineered T cells, such as T cells modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells or T cell-like effector cells can also be differentiated from stem or progenitor cells ("derived T cells" or "derived T cell-like effector cells", or collectively "derived T lineage cells"). Derived T cell-like effector cells may have T cell lineage in some respects, but at the same time have one or more functional attributes not present in primary T cells. In this application, T cells, T cell-like effector cells, derived T cells, derived T cell-like effector cells, or derived T lineage cells are collectively referred to as "T lineage cells".

[0056] "CD4 +"CD4 cells" refers to a subset of T cells that express CD4 on their surface and are associated with cellular immune responses. They are characterized by their secretion profile after stimulation, which may include secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. The "CD4" molecule is a 55 kD glycoprotein originally defined as a differentiation antigen for T lymphocytes, but is also found on other cells, including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is involved as the relevant recognition element of the MHC (major histocompatibility complex) class II-restricted immune response. In T lymphocytes, it defines the helper / inducer subsets.

[0057] "CD8 + "CD8 cells" refers to a subset of T cells that express CD8 on their surface, are MHC class I restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is the associated recognition element of major histocompatibility complex class I restricted interactions.

[0058] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by expression of CD56 or CD16 and the absence of the T cell receptor (CD3). The NK cells can be any NK cell, e.g., cultured NK cells (e.g., primary NK cells), or NK cells derived from cultured or expanded NK cells, or cell line NK cells (e.g., NK-92), or NK cells obtained from a mammal that is healthy or has a disease state. As used herein, the terms "adaptive NK cells" and "memory NK cells" are interchangeable and are phenotypically CD3+ and CD4+ expression. - and CD56 + and refers to a subset of NK cells that express at least one of NKG2C and CD57, and optionally CD16, but lack expression of one or more of PLZF, SYK, FceRγ, and EAT-2. In some embodiments, CD56 +The isolated subpopulation of NK cells includes expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and / or DNAM-1. + may be dim or bright expression. NK cells or NK cell-like effector cells may be differentiated from stem or progenitor cells ("derived NK cells" or "derived NK cell-like effector cells", or collectively "derived NK lineage cells"). Derived NK cell-like effector cells may have NK cell lineage in some respects, but at the same time have one or more functional attributes not present in primary NK cells. In this application, NK cells, NK cell-like effector cells, derived NK cells, derived NK cell-like effector cells, or derived NK lineage cells are collectively referred to as "NK lineage cells".

[0059] As used herein, the term "NKT cells" or "natural killer T cells" refers to CD1d-restricted T cells that express the T cell receptor (TCR). Unlike conventional T cells, which detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by the non-classical MHC molecule, CD1d. Two types of NKT cells are recognized. Invariant or type I NKT cells express a very limited TCR repertoire - a canonical α chain (Vα24-Jα18 in humans) associated with a limited range of β chains (Vβ11 in humans). A second population of NKT cells, called non-classical or non-invariant type II NKT cells, presents a more heterogeneous use of TCRαβ. Type I NKT cells are considered to be suitable for immunotherapy. Adaptive or invariant (Type I) NKT cells can be identified by expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0060] The term "effector cell" generally refers to a specific cell in the immune system that performs a specific activity in response to stimulation and / or activation, or that provides a specific function upon activation. As used herein, the term "effector cell" includes, and in some contexts is interchangeable with, immune cells, "differentiated immune cells," and primary or differentiated cells that have been edited and / or regulated to perform a specific activity in response to stimulation and / or activation. Non-limiting examples of effector cells include primary or iPSC-derived T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0061] As used herein, terms such as "isolated" refer to a cell or population of cells that has been separated from its original environment, i.e., the environment of the isolated cell is substantially free of at least one component found in the environment in which the "non-isolated" reference cell resides. The term includes cells that have been removed from some or all components as found in their natural environment, e.g., isolated from a tissue or biopsy sample. The term also includes cells that have been removed from at least one, some, or all components as they are found in a non-native environment, e.g., isolated from a cell culture or cell suspension. Thus, an "isolated cell" is partially or completely separated from at least one component, including other substances, cells, or cell populations, as found in nature, or as grown, stored, or persisted in a non-native environment. Specific examples of isolated cells include partially pure cell compositions, substantially pure cell compositions, and cells cultured in a medium that does not occur in nature. Isolated cells can be obtained from isolating a desired cell or population thereof from other substances or cells in the environment, or from removing one or more other cell populations or subpopulations from the environment.

[0062] As used herein, terms such as "purify" refer to increasing purity. For example, purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0063] As used herein, the term "encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually listed in the sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, may be referred to as encoding the protein or other product of that gene or cDNA.

[0064] A "construct" refers to a complex of polymers or molecules, including polynucleotides, that are delivered to a host cell, either in vitro or in vivo. As used herein, a "vector" refers to any nucleic acid construct that can direct the delivery or transfer of foreign genetic material to a target cell and can replicate and / or express in the target cell. As used herein, the term "vector" includes the construct that is delivered. A vector can be a linear or circular molecule. A vector can be integrating or non-integrating. The main types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, and the like.

[0065] "Integration" means that one or more nucleotides of the construct are stably inserted into the cell genome, i.e., covalently linked to a nucleic acid sequence in the chromosomal DNA of the cell. "Targeted integration" means that the nucleotides of the construct are inserted into the chromosomal or mitochondrial DNA of the cell at a preselected site or "integration site". As used herein, the term "integration" further refers to a process that includes the insertion of one or more exogenous sequences or nucleotides of the construct, with or without deletion of the endogenous sequence or nucleotide at the integration site. If there is a deletion at the insertion site, "integration" may further include replacement of the deleted nucleotide with the endogenous sequence or one or more inserted nucleotides.

[0066] As used herein, the term "exogenous" is intended to mean that the referenced molecule or activity is introduced into the host cell or is non-native to the host cell. The molecule can be introduced, for example, by introducing an encoding nucleic acid into the genetic material of the host, for example, by integration into a chromosome of the host, or as non-chromosomal genetic material, for example, a plasmid. Thus, the term used in reference to expression of an encoding nucleic acid refers to introducing the encoding nucleic acid into the cell in an expressible form. The term "endogenous" refers to a referenced molecule or activity present in the host cell. Similarly, when used in reference to expression of an encoding nucleic acid, the term refers to expression of an encoding nucleic acid that is contained within the cell and not exogenously introduced.

[0067] As used herein, a "gene of interest" or a "polynucleotide sequence of interest" is a DNA sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into RNA and, in some cases, translated into a polypeptide in vivo. A gene or polynucleotide of interest may include, but is not limited to, a prokaryotic sequence, a cDNA from eukaryotic mRNA, a genomic DNA sequence from eukaryotic (e.g., mammalian) DNA, and a synthetic DNA sequence. For example, a gene of interest may code for an miRNA, an shRNA, a natural polypeptide (i.e., a polypeptide found in nature) or a fragment thereof, a variant polypeptide (i.e., a variant of a natural polypeptide having less than 100% sequence identity with the natural polypeptide) or a fragment thereof, an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selection marker, etc.

[0068] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The sequence of a polynucleotide is composed of the four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T); if the polynucleotide is RNA, thymine is uracil (U). Polynucleotides can include genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. "Polynucleotide" also refers to both double-stranded and single-stranded molecules.

[0069] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to molecules having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, these terms refer to both short chains, also generally referred to in the art as peptides, oligopeptides and oligomers, and longer chains, generally referred to in the art as polypeptides or proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0070] As used herein, the term "subunit" refers to each separate polypeptide chain of a protein complex, each separate polypeptide chain being capable of forming a stable folded structure by itself. Many protein molecules are composed of two or more subunits, where the amino acid sequence can be either identical, similar, or completely different for each subunit. For example, the CD3 complex is composed of CD3α, CD3ε, CD3δ, CD3γ, and CD3ζ subunits, which form CD3ε / CD3γ, CD3ε / CD3δ, and CD3ζ / CD3ζ dimers. Within a single subunit, contiguous portions of the polypeptide chains are often folded into compact, localized, semi-independent units called "domains". Many protein domains may further comprise independent "structural subunits", also called subdomains, that contribute to a common function of the domain. Thus, as used herein, the term "subdomain" refers to a protein domain inside a larger domain, for example, a binding domain in the ectodomain of a cell surface receptor, or a stimulatory or signaling domain in the endodomain of a cell surface receptor.

[0071] "Operably-linked" or "operatively linked" are interchangeable with "operably connected" or "operatively connected" and refer to the association of nucleic acid sequences (or amino acids in a polypeptide having multiple domains) on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence or functional RNA if it is capable of affecting the expression of that coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in a sense or antisense orientation. As a further example, a receptor binding domain can be operably connected to an intracellular signaling domain such that binding of the receptor to a ligand transduces a signal in response to said binding.

[0072] A "fusion protein" or "chimeric protein," as used herein, is a protein created through genetic engineering to join two or more partial or complete polynucleotides encoding sequences coding for separate proteins, such that expression of these joined polynucleotides results in a single peptide or multiple polypeptides having functional properties derived from each of the original proteins or fragments thereof. A linker (or spacer) peptide can be added between the two adjacent polypeptides of different sources in a fusion protein.

[0073] As used herein, the term "genetic imprint" refers to genetic or epigenetic information that contributes to the preferential therapeutic properties of source cells or iPSCs and can be retained in source cell-derived iPSCs and / or iPSC-derived hematopoietic lineage cells. As used herein, a "source cell" is a non-pluripotent cell that can be used to generate iPSCs through reprogramming, which can be further differentiated into a specific cell type, including any hematopoietic lineage cell. Source cell-derived iPSCs, and cells differentiated therefrom, can be collectively referred to as "derived" or "derived" cells, depending on the context. For example, derived effector cells, or derived NK cells or derived T cells, as used throughout this application, are cells differentiated from iPSCs, when compared to their primary counterparts obtained from natural / native sources, such as peripheral blood, umbilical cord blood, or other donor tissues. As used herein, genetic imprints conferring preferential therapeutic attributes are incorporated into iPSCs by reprogramming selected source cells that are donor, disease, or therapeutic response specific, or by introducing genetic modification modalities into iPSCs using genome editing. In aspects of source cells obtained from specifically selected donors, diseases, or therapeutic contexts, genetic imprints that contribute preferential therapeutic attributes may include context-specific genetic or epigenetic modifications that represent a retainable phenotype, i.e., preferential therapeutic attributes, that are passed on to descendants of the selected source cells, regardless of whether the underlying molecular events have been identified. Source cells that are donor, disease, or therapeutic response specific may contain genetic imprints that can be retained in iPSCs and derived hematopoietic lineage cells, including, but not limited to, pre-positioned monospecific TCRs, e.g., from virus-specific T cells or invariant natural killer T (iNKT) cells, traceable and desirable genetic polymorphisms, e.g., homozygosity for a point mutation encoding the high affinity CD16 receptor in the selected donor, and selected HLA-matched donor cells that exhibit predetermined HLA requirements, i.e., haplotypes in an expanded population.As used herein, preferential therapeutic properties include improved engraftment, trafficking, homing, viability, self-renewal, persistence, control and regulation of immune responses, survival rate, and cytotoxicity of derived cells. Preferential therapeutic properties may also be expressed by antigen-targeting receptor expression, HLA presentation or lack thereof, resistance to tumor microenvironment, induction of bystander immune cells and immune modulation, improved on-target specificity with reduced extratumoral effects, resistance to treatments such as chemotherapy. When derived cells with one or more therapeutic properties are obtained from differentiating iPSCs that incorporate genetic imprints that confer preferential therapeutic properties, such derived cells are also referred to as "synthetic cells". For example, synthetic effector cells, or synthetic NK cells or synthetic T cells as used throughout this application are cells differentiated from genomically modified iPSCs compared to their primary counterparts obtained from natural / native sources such as peripheral blood, umbilical cord blood, or other donor tissues. In some embodiments, the synthetic cells have one or more non-native cell functions when compared to their closest corresponding primary cells.

[0074] As used herein, the term "enhanced therapeutic properties" refers to therapeutic properties of a cell that are enhanced compared to typical immune cells of the same general cell type. For example, NK cells with "enhanced therapeutic properties" have enhanced, improved, and / or increased therapeutic properties compared to typical unmodified and / or naturally occurring NK cells. Therapeutic properties of immune cells may include, but are not limited to, cell engraftment, trafficking, homing, viability, self-renewal, persistence, control and regulation of immune responses, viability, and cytotoxicity. Therapeutic properties of immune cells are also represented by antigen targeting receptor expression, HLA presentation or lack thereof, resistance to tumor microenvironment, induction of bystander immune cells and immune modifications, improved on-target specificity with reduced extratumoral effects, resistance to treatments such as chemotherapy.

[0075] As used herein, the term "engager" refers to a molecule, e.g., a fusion polypeptide, that can form a link between an immune cell, e.g., a T cell, a NK cell, a NKT cell, a B cell, a macrophage, a neutrophil, and a tumor cell and activate the immune cell. Examples of engagers include, but are not limited to, bi-specific T cell engagers (BiTEs), bi-specific killer cell engagers (BiKEs), tri-specific killer cell engagers (TriKEs), or multispecific killer cell engagers, or universal engagers that can be compatible with multiple immune cell types.

[0076] As used herein, the term "surface triggering receptor" refers to a receptor that can induce or initiate an immune response, e.g., a cytotoxic response. Surface triggering receptors can be engineered and expressed on effector cells, e.g., T cells, NK cells, NKT cells, B cells, macrophages, neutrophils. In some embodiments, the surface triggering receptor promotes bispecific or multispecific antibody binding between an effector cell and a specific target cell, e.g., a tumor cell, regardless of the effector cell's native receptor and cell type. Using this approach, it is possible to generate iPSCs that contain a universal surface triggering receptor and differentiate such iPSCs into a population of different effector cell types that express the universal surface triggering receptor. "Universal" means that the surface triggering receptor can be expressed and activated on any effector cell regardless of cell type, and all effector cells that express the universal receptor can bind or link to an engager that has the same epitope that can be recognized by the surface triggering receptor, regardless of the tumor binding specificity of the engager. In some embodiments, engagers with the same tumor targeting specificity are used to bind to the universal surface triggering receptor. In some embodiments, engagers with different tumor targeting specificities are used to bind to the universal surface triggering receptor. Thus, one or more effector cell types may be used to kill one specific type of tumor cell or two or more types of tumors. Surface triggering receptors generally contain a costimulatory domain for effector cell activation and an anti-epitope specific to the epitope of the engager. Bispecific engagers are specific to the anti-epitope of the surface triggering receptor on one end and specific to the tumor antigen on the other end.

[0077] As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent possible toxicity or other adverse effects of a cell therapy. In some examples, expression of the safety switch protein is conditionally controlled to address safety concerns of transplanted engineered cells that have a gene encoding the safety switch protein permanently integrated into their genome. This conditional regulation can vary and can include post-translational activation via small molecules and control by tissue-specific and / or transient transcriptional control. The safety switch can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene control, and / or antibody-mediated depletion. In some examples, the safety switch protein is activated by an exogenous molecule, e.g., a prodrug, and upon activation, induces apoptosis and / or cell death of the therapeutic cell. Examples of safety switch proteins include, but are not limited to, suicide genes such as caspase 9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase, B cell CD20, modified EGFR, and any combination thereof. In this strategy, a prodrug administered upon the occurrence of an adverse event is activated by the suicide gene product and kills the transduced cells.

[0078] As used herein, the term "pharmaceutical active protein or peptide" refers to a protein or peptide capable of achieving a biological and / or pharmaceutical effect on an organism. Pharmaceutically active proteins have healing, curative or palliative properties for a disease and can be administered to ameliorate, relieve, alleviate, reverse or lessen the severity of the disease. Pharmaceutically active proteins also have prophylactic properties and are used to prevent the onset of a disease or to reduce the severity of such a disease or pathological condition once it appears. A "pharmaceutical active protein" includes an entire protein or peptide or a pharmaceutical active fragment thereof. The term also includes a pharmaceutical active analog of a protein or peptide or an analog of a fragment of a protein or peptide. The term "pharmaceutical active protein" also refers to multiple proteins or peptides that act cooperatively or synergistically to provide a therapeutic benefit. Examples of pharma- ceutical active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth suppressor proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0079] As used herein, the term "signaling molecule" refers to any molecule that regulates, participates in, inhibits, activates, reduces, or increases cell signaling. "Signaling" refers to the transmission of a molecular signal in the form of a chemical modification by the recruitment of protein complexes along a pathway that ultimately leads to a biochemical event in the cell. Signaling pathways are well known in the art and include, but are not limited to, G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, toll gate signaling, ligand-gated ion channel signaling, ERK / MAPK signaling pathway, Wnt signaling pathway, cAMP-dependent pathway, and IP3 / DAG signaling pathway.

[0080] As used herein, the term "targeting modality" refers to molecules, e.g., polypeptides, that are genetically incorporated into cells to promote antigen and / or epitope specificity, including, but not limited to, (i) antigen specificity when associated with a unique chimeric antigen receptor (CAR) or T cell receptor (TCR), (ii) engager specificity when associated with a monoclonal antibody or bispecific engager, (iii) transformed cell targeting, (iv) cancer stem cell targeting, and (v) other targeting strategies in the absence of a specific antigen or surface molecule.

[0081] As used herein, the terms "specific" or "specificity" can be used to refer to the ability of a molecule, e.g., a receptor or engager, to selectively bind to a target molecule, as opposed to non-specific or non-selective binding.

[0082] As used herein, the term "adoptive cell therapy" refers to cell-based immunotherapy involving the infusion of autologous or allogeneic lymphocytes, whether the immune cells are isolated from a human donor or are effector cells obtained from in vitro differentiation of pluripotent cells, whether they are genetically modified, or whether they are primary donor cells that have been passaged, expanded or immortalized ex vivo after isolation from the donor.

[0083] As used herein, "lymphodepletion" and "lymphatic conditioning" are used interchangeably to refer to the destruction of lymphocytes and T cells, typically prior to immunotherapy. The purpose of lymphatic conditioning prior to administration of adoptive cell therapy is to promote homeostatic proliferation of effector cells, as well as to eliminate regulatory immune cells and other competing elements of the immune system that compete for homeostatic cytokines. Thus, lymphatic conditioning is typically accomplished by administering one or more chemotherapeutic agents to the subject prior to the first dose of adoptive cell therapy. In various embodiments, lymphatic conditioning is performed hours to days prior to the first dose of adoptive cell therapy. Exemplary chemotherapeutic agents useful for lymphatic conditioning include, but are not limited to, cyclophosphamide (CY), fludarabine (FLU), and those described below. However, sufficient lymphodepletion with anti-CD38 mAb may provide an alternative conditioning process for the present iNK cell therapy without or at a minimum the need for a CY / FLU-based lymphoid conditioning procedure, as further described herein.

[0084] As used herein, "homing" or "trafficking" refers to the active navigation (migration) of a cell to a target site (e.g., a cell, a tissue (e.g., a tumor), or an organ). A "homing molecule" refers to a molecule that directs a cell to a target site. In some embodiments, a homing molecule functions to recognize and / or initiate the interaction of a cell to a target site.

[0085] As used herein, the term "outpatient" refers to a patient who is not admitted overnight but visits a hospital, clinic, or related facility for diagnosis or treatment. Thus, an "outpatient environment" refers to an environment for providing ambulatory or outpatient treatment to a patient that does not require hospitalization for one or more days / nights while undergoing treatment or diagnosis, as compared to an "inpatient environment," thereby reducing the overall discomfort to the patient undergoing treatment and / or diagnosis, while reducing the overall cost of such treatment and / or diagnosis with relative ease of management and coordination. Additionally, an outpatient environment is more easily accessible to a larger patient population, increasing patient availability during a clinical trial or treatment course and patient compliance with treatment protocols.

[0086] As used herein, "induction therapy," also referred to as "first-line therapy," "primary therapy," or "first-line treatment," refers to the first treatment given to a patient for a particular disease. It is often part of a standard course of treatment, such as surgery followed by chemotherapy and radiation. Thus, an "induction attempt" or "induction therapy attempt" refers to the first attempt to treat a particular disease using a known and / or conventional treatment approach for the particular disease.

[0087] As used herein, a "therapeutically sufficient amount" includes within its meaning a non-toxic but sufficient and / or effective amount of the particular therapeutic and / or pharmaceutical composition to which it refers to provide the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient's overall health, the patient's age, and the stage and severity of the condition. In certain embodiments, a "therapeutically sufficient amount" is sufficient and / or effective to ameliorate, reduce, and / or improve at least one symptom associated with the disease or condition of the subject being treated.

[0088] Differentiation of pluripotent stem cells requires changes in the culture system, including changes in stimuli in the medium and the physical state of the cells. The most common strategy utilizes the formation of embryoid bodies (EBs) as a common and important intermediate for initiating lineage-specific differentiation. "Embryoid bodies" are three-dimensional clusters that have been shown to mimic embryonic development, giving rise to multiple lineages within a three-dimensional area. Throughout the differentiation process, which typically takes hours to days, simple EBs (e.g., aggregated pluripotent stem cells induced to differentiate) continue to mature and grow into cystic EBs, at which point they are further processed to continue differentiation, typically for days to weeks. EB formation is initiated by bringing pluripotent stem cells into close proximity to each other in a three-dimensional multi-layered cluster of cells. Typically, this is accomplished by one of several methods, including settling the pluripotent cells in droplets, settling the cells in "U" bottom well plates, or by mechanical agitation. Because aggregates maintained in pluripotency culture maintenance medium do not form proper EBs, pluripotent stem cell aggregates require further differentiation cues to promote EB growth. Therefore, pluripotent stem cell aggregates need to be transferred to differentiation medium that provides cue induction to the selected lineage. EB-based culture of pluripotent stem cells typically generates differentiated cell populations (i.e., ectoderm, mesoderm, and endoderm germ layers) with moderate proliferation within the EB cell clusters. Although EBs have been proven to promote cell differentiation, they give rise to heterogeneous cells with various differentiation states due to inconsistent exposure of cells in three-dimensional structures to differentiation cues from the environment. In addition, EBs are laborious to create and maintain. Furthermore, cell differentiation by EBs is accompanied by moderate cell proliferation, which also leads to reduced differentiation efficiency.

[0089] In contrast, "aggregate formation", unlike "EB formation", can be used to expand a population of pluripotent stem cell-derived cells. For example, during aggregate-based pluripotent stem cell expansion, the culture medium is selected to maintain proliferation and pluripotency. Cell proliferation generally increases the size of the aggregates forming larger aggregates that can be mechanically or enzymatically dissociated into smaller aggregates to maintain cell proliferation and increase cell number in culture. Unlike EB culture, cells cultured within aggregates in maintenance culture maintain markers of pluripotency. Pluripotent stem cell aggregates require further differentiation cues to induce differentiation.

[0090] As used herein, "monolayer differentiation" refers to a differentiation method that is different from the differentiation of three-dimensional multi-layered clusters of cells, i.e. "EB formation".Monolayer differentiation, among other advantages disclosed herein, avoids the need for EB formation to initiate differentiation.Since monolayer culture does not mimic embryonic development as in the case of EB formation, differentiation into specific lineages is considered to be minimal compared to the differentiation of all three germ layers in EB formation.

[0091] As used herein, "dissociated cells" or "single dissociated cells" refers to cells that are substantially separated or purified from other cells or from a surface (e.g., a culture plate surface). For example, cells can be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro can be enzymatically or mechanically dissociated from each other, for example, by dissociation into a suspension of clusters, single cells, or a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells can be dissociated from a culture plate or other surface. Thus, dissociation includes disrupting the extracellular matrix (ECM) and cellular interactions with the substrate (such as the culture surface) or disrupting the ECM between cells.

[0092] As used herein, "master cell bank" or "MCB" refers to a clonal master engineered iPSC line that is a clonal population of iPSCs that have been engineered to contain one or more therapeutic properties, characterized, tested, qualified, expanded, and shown to reliably serve as starting cell material for the production of cell-based therapeutics by directed differentiation in a manufacturing environment. In various embodiments, the MCB is maintained, stored, and / or cryopreserved in multiple containers to prevent genetic mutations and / or potential contamination by reducing and / or eliminating the total number of times the iPS cell line is passaged, thawed, or handled during the manufacturing process.

[0093] As used herein, "feeder cells" or "feeders" is a term that refers to one type of cell that is co-cultured with a second type of cell to provide an environment in which the second type of cell can grow, proliferate, or differentiate, where the feeder cells provide stimuli, growth factors, nutrients, and support the second cell type. Feeder cells may be derived from a different species than the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. In another example, peripheral blood-derived cells or transformed leukemia cells support the proliferation and maturation of natural killer cells. Feeder cells can typically be inactivated by irradiation or treatment with antimitotic agents such as mitomycin to prevent them from outgrowing the cells they support when co-cultured with other cells. Feeder cells can include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the foregoing, one particular feeder cell type can be a human feeder, such as human dermal fibroblasts. Another feeder cell type can be mouse embryonic fibroblast (MEF). In general, various feeder cells can be used in part to maintain pluripotency, direct differentiation to specific lineages, enhance proliferation capacity, and promote maturation into specialized cell types such as effector cells.

[0094] As used herein, a "feeder-free" (FF) environment refers to an environment, such as a culture condition, cell culture, or culture medium, that is essentially free of feeder or stromal cells and / or has not been preconditioned by culturing feeder cells. A "preconditioned" medium refers to a medium that is harvested after feeder cells have been cultured in the medium for a period of time, such as at least one day. Preconditioned medium contains many mediator substances, including growth factors and cytokines, secreted by feeder cells cultured in the medium. In some embodiments, the feeder-free environment does not contain either feeder cells or stromal cells, and has not been preconditioned by culturing feeder cells.

[0095] "Functional" as used in the context of genome editing or modification of iPSCs and derived non-pluripotent cells differentiated therefrom, or genome editing or modification of non-pluripotent cells and derived iPSCs reprogrammed therefrom, means (1) at the genetic level, successful transgenic or controlled gene expression, such as inducible or transient expression, at a desired stage of cellular development, achieved by knock-in, knock-out, knock-down gene expression, direct genome editing or modification, or by "passaging" through differentiation or reprogramming from an initially genomically engineered starting cell, or (2) at the cellular level, (i) the ability to achieve a desired level of function in a cell through direct genome editing. " refers to the successful removal, addition or modification of a cellular function / characteristic by gene expression modification, (ii) gene expression modification that is maintained in the cell through "passage" via differentiation or reprogramming from an original genomically engineered starting cell, (iii) downstream gene regulation in the cell as a result of gene expression modifications that are only apparent in an earlier developmental stage of the cell or only apparent in the starting cell that gives rise to the cell via differentiation or reprogramming, or (iv) enhanced or newly achieved cellular function or attribute exhibited in a mature cell product originally derived from genomic editing or modification performed on an iPSC, precursor or de-differentiated cellular source.

[0096] "HLA-deficient," including HLA class I-deficient or HLA class II-deficient, or both, refers to cells that lack or no longer maintain or have reduced levels of surface expression of complete MHC complexes comprising HLA class I protein heterodimers and / or HLA class II heterodimers, the reduced or decreased levels being lower than those naturally detectable by other cells or synthetic methods.

[0097] As used herein, "modified HLA-deficient iPSCs" refers to HLA-deficient iPSCs that are further modified by introducing genes expressing proteins associated with improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to inhibition, proliferation, costimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cytotoxicity, such as, but not limited to, non-classical HLA class I proteins (e.g., HLA-E and HLA-G), chimeric antigen receptors (CARs), T cell receptors (TCRs), CD16 Fc receptors, BCL11b, NOTCH, RUNX1, IL15, 41BB, DAP10, DAP12, CD24, CD3zeta, 4-1BBL, CD47, CD113, and PDL1. "Modified HLA-deficient" cells also include cells other than iPSCs.

[0098] The term "ligand" refers to a substance that forms a complex with a target molecule and generates a signal by binding to a site on the target. A ligand may be a natural or artificial substance that can specifically bind to a target. A ligand may be in the form of a protein, peptide, antibody, antibody complex, conjugate, nucleic acid, lipid, polysaccharide, monosaccharide, small molecule, nanoparticle, ion, neurotransmitter, or any other molecular entity that can specifically bind to a target. The target to which the ligand binds may be a protein, nucleic acid, antigen, receptor, protein complex, or cell. A ligand that binds to a target and changes its function, inducing a signaling response, is called "agonistic" or "agonist". A ligand that binds to a target and blocks or reduces a signaling response is "antagonistic" or "antagonist".

[0099] The term "antibody" is used herein in the broadest sense and generally refers to an immune response generating molecule that contains at least one binding site that specifically binds to a target, which may be an antigen or a receptor that can interact with a particular antibody. For example, NK cells can be activated by the binding of an antibody or the Fc region of an antibody to its Fc-gamma receptor (FcγR), thereby triggering ADCC (antibody-dependent cellular cytotoxicity)-mediated effector cell activation. The particular fragment or portion of an antigen or receptor that an antibody binds to, or generally the target, is known as an epitope or antigenic determinant. The term "antibody" includes, but is not limited to, antibody mimetics that mimic the structure and / or function of an antibody or a particular fragment or portion thereof, including natural antibodies and variants thereof, fragments of natural antibodies and variants thereof, peptibodies and variants thereof, and single chain antibodies and fragments thereof. The antibody may be a murine antibody, a human antibody, a humanized antibody, a camelid IgG, a single variable new antigen receptor (VNAR), a shark heavy chain antibody (Ig-NAR), a chimeric antibody, a recombinant antibody, a single domain antibody (dAb), an anti-idiotypic antibody, a bispecific, multispecific, or multimeric antibody, or a fragment thereof. An anti-idiotypic antibody is specific for binding to the idiotope of another antibody, an idiotope being an antigenic determinant of an antibody. A bispecific antibody may be a BiTE (bispecific T cell engager) or BiKE (bispecific killer cell engager), and a multispecific antibody may be a TriKE (trispecific killer cell engager).Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, Fabc, pFc, Fd, single chain fragment variable (scFv), tandem scFv (scFv)2, single chain Fab (scFab), disulfide stabilized Fv (dsFv), minibodies, diabodies, triabodies, tetrabodies, single-domain antigen binding fragments (sdAb), camelid heavy chain IgG and Nanobody® fragments, recombinant heavy-chain-only antibodies (VHH), and other antibody fragments which maintain the binding specificity of the whole antibody.

[0100] "Fc receptors", abbreviated as FcR, are classified based on the type of antibody they recognize. For example, those that bind the most common class of antibody IgG are called Fc-gamma receptors (FcγR), those that bind IgA are called Fc-alpha receptors (FcαR), and those that bind IgE are called Fc-epsilon receptors (FcεR). Classes of FcR are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T and B cells) and the signaling properties of each receptor. Fc-gamma receptors (FcγR) include several members with different molecular structures and therefore different antibody affinities, such as FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b).

[0101] "Chimeric receptor" is a general term used to describe engineered artificial or hybrid receptor protein molecules that are made to contain two or more portions of amino acid sequence from at least two different proteins. Chimeric receptor proteins are engineered to confer the cell the ability to initiate signal transduction and perform downstream functions upon binding of an agonistic ligand to the receptor. Exemplary "chimeric receptors" include, but are not limited to, chimeric antigen receptors (CARs), chimeric fusion receptors (CFRs), chimeric Fc receptors (CFcRs), and fusions of two or more receptors.

[0102] "Chimeric Fc receptor", abbreviated as "CFcR", is a term used to describe engineered Fc receptors in which the native transmembrane domain and / or intracellular signaling domain have been modified or replaced with a non-native transmembrane domain and / or intracellular signaling domain. In some embodiments of chimeric Fc receptors, in addition to one or both of the transmembrane and signaling domains being non-native, one or more stimulatory domains can be introduced into the intracellular portion of the engineered Fc receptor to enhance receptor-induced cell activation, proliferation, and function. Unlike chimeric antigen receptors (CARs), which contain an antigen-binding domain to a target antigen, chimeric Fc receptors bind to an Fc fragment, or Fc region of an antibody, or Fc region contained in an engager or binding molecule that activates a cell function, with or without bringing the target cell into close proximity. For example, Fcγ receptors can be engineered to contain selected transmembrane domains, stimulatory domains, and / or signaling domains in the intracellular region that respond to binding of IgG at the extracellular domain, thereby generating CFcR. In one example, CFcR is produced by engineering the Fcγ receptor CD16 by replacing its transmembrane and / or intracellular domains. To further improve the binding affinity of CD16-based CFcR, the extracellular domain of CD64 or a high affinity variant of CD16 (e.g., F176V) can be incorporated. In some embodiments of CFcRs that include a high affinity CD16 extracellular domain, the proteolytic cleavage site containing serine at position 197 is removed or the extracellular domain of the receptor is replaced to be non-cleavable, i.e., not shed, thereby obtaining hnCD16-based CFcR.

[0103] Two isoforms of the FcγR receptor CD16 have been identified: Fc receptors FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells to activate NK cells and promote antibody-dependent cell-mediated cytotoxicity (ADCC). As used herein, "high affinity CD16", "non-cleavable CD16", or "high affinity non-cleavable CD16 (abbreviated as hnCD16)" refers to natural or non-natural variants of CD16. Wild-type CD16 has low affinity and upon NK cell activation is subject to ectodomain shedding, a proteolytic cleavage process that controls the cell surface density of various cell surface molecules on leukocytes. F176V and F158V are exemplary CD16 polymorphic variants with high affinity. CD16 variants in which the cleavage site (positions 195-198) in the membrane proximal region (positions 189-212) has been altered or eliminated are not shed. The cleavage site and membrane proximal region are described in detail in WO 2015 / 148926, the full disclosure of which is incorporated herein by reference. The S197P variant of CD16 is a non-cleavable version of CD16. CD16 variants containing both F158V and S197P are high affinity and non-cleavable. Another exemplary high affinity and non-cleavable CD16 (hnCD16) variant is an engineered CD16 that contains an ectodomain derived from one or more of the three exons of the CD64 ectodomain.

[0104] As used herein, "FT576" refers to an iPSC-derived, off-the-shelf, BCMA-directed chimeric antigen receptor (CAR) natural killer (NK) cell therapy that has four engineered modalities: (1) a high affinity non-cleavable CD16 Fc receptor for enhanced antibody-dependent cellular cytotoxicity (ADCC), (2) an IL-15 / IL-15 receptor fusion, (3) a CD38 knockout, and (4) a BCMA-directed CAR targeting clonal plasma cells.

[0105] I. Cells and Compositions Useful for Adoptive Cell Therapy with Enhanced Properties Provided herein is a strategy to systematically manipulate the regulatory circuitry of clonal iPSCs without affecting the differentiation potential and cell developmental biology of iPSCs and their derived cells while enhancing the therapeutic properties of derived cells differentiated from iPSCs. iPSC-derived cells are functionally improved and suitable for adoptive cell therapy after a combination of selective modalities is introduced into the cells at the iPSC level through genomic manipulation. Previously, it was unclear whether modified iPSCs containing one or more gene edits provided would still have the ability to enter cell development and / or mature to generate functional differentiated cells while retaining the regulated activity and / or properties. Unexpected failures during directed cell differentiation from iPSCs have been attributed to aspects including, but not limited to, developmental stage specific gene expression or lack thereof, requirement for HLA complex presentation, protein shedding of introduced surface expression modalities, and the need for reconstitution of differentiation protocols to allow for changes in phenotype and / or function within the cells. The present application has demonstrated that one or more selected genomic modifications provided herein do not adversely affect iPSC differentiation potential, and that functional effector cells derived from engineered iPSCs have enhanced and / or acquired therapeutic properties resulting from individual or combined genomic modifications that are retained in the effector cells following iPSC differentiation.

[0106] 1.CD38 knockout The cell surface molecule CD38 is highly upregulated in multiple hematological malignancies of both lymphoid and myeloid origin, including multiple myeloma and CD20-negative B-cell malignancies, making it an attractive target for antibody therapy to deplete cancer cells. Antibody-mediated cancer cell depletion usually results from a combination of direct induction of cell apoptosis and activation of immune effector mechanisms such as ADCC (antibody-dependent cellular cytotoxicity). In addition to ADCC, immune effector mechanisms in conjunction with therapeutic antibodies may also include phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

[0107] In addition to being highly expressed on malignant cells, CD38 is also expressed on plasma cells, as well as on NK cells, activated T cells, and B cells. During hematopoiesis, CD38 is expressed in association with CD34 + It is expressed in stem cells and progenitor cells committed to the lymphoid, erythroid, and myeloid lineages, as well as in the final stages of maturation up to the plasma cell stage. As a type II transmembrane glycoprotein, CD38 serves cellular functions both as a receptor and a multifunctional enzyme involved in the production of nucleotide metabolites. As an enzyme, CD38 mediates the synthesis and upregulation of NAD + CD38 catalyzes the hydrolysis of acetylcholine to ADP-ribose, thereby generating the second messengers CADPR and NAADP, which stimulate calcium release from the endoplasmic reticulum and lysosomes, which is calcium-dependent and important for the process of cell adhesion. CD38 recognizes CD31 as a receptor and controls cytokine release and cytotoxicity of activated NK cells. CD38 associates with cell surface proteins in lipid rafts and mediates the release of cytoplasmic Ca. 2+ It has also been reported to regulate flux and mediate signaling in lymphoid and myeloid cells.

[0108] In the treatment of malignancies, the systemic use of T cells transduced with the CD38 antigen-binding receptor has been shown to inhibit the expression of CD34 + CD38 on hematopoietic progenitor cells, monocytes, NK cells, T cells, and B cells +Fraction lysis and impaired immune effector cell function in the recipient leads to incomplete therapeutic response and reduced or eliminated efficacy. In addition, in multiple myeloma patients treated with the CD38-specific antibody daratumumab, a reduction in NK cells was observed in both bone marrow and peripheral blood, whereas other immune cell types such as T cells and B cells were unaffected despite CD38 expression (Casneuf et al., Blood Advances. 2017;1(23):2105-2114).

[0109] Without being limited by theory, the present application provides a strategy to maximize the potential of CD38-targeted cancer therapy by knocking out CD38 in effector cells, thereby overcoming CD38-specific antibody and / or CD38 antigen binding domain-induced effector cell depletion or reduction through fratricide. In addition, since CD38 is upregulated on activated lymphocytes, such as T cells or B cells, suppressing the activation of these recipient lymphocytes using a CD38-specific antibody, such as daratumumab, in recipients of allogeneic effector cells reduces and / or prevents host allorejection of these effector cells, thereby increasing the survival and persistence of effector cells. Thus, CD38-specific antibodies, secreted CD38-specific engagers, or CD38-CARs (chimeric antigen receptors) for activation of recipient T, Treg, NK, and / or B cells can be used as an alternative to lymphodepletion using chemotherapy such as Cy / Flu (cyclophosphamide / fludarabine) prior to adoptive cell transfer. In addition, the use of hnCD16a in the presence of anti-CD38 antibodies or CD38 inhibitors can be used to inhibit lymphocyte depletion. + / CD38 - Effector cells were used to identify CD38 + For targeting T and pbNK cells, CD38 + Depletion of alloreactive cells increases NAD + (nicotinamide adenine dinucleotide, a substrate for CD38) availability, increasing NAD +It reduces consumption-associated cell death, which, among other benefits, boosts effector cell responses in the immunosuppressive tumor microenvironment and supports cellular rejuvenation in aging, degenerative, or inflammatory diseases.

[0110] Thus, strategies according to some embodiments include the generation of CD38 knockout iPSC lines and directed differentiation of engineered iPSC lines to CD38 negative (CD38 - / - ) to obtain derived effector cells. In one embodiment provided herein, the CD38 knockout in the iPSC line is a biallelic knockout. As disclosed herein, the provided CD38 - / - iPSC lines undergo directed differentiation to produce mesodermal cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34 + Functional derived hematopoietic cells can be produced, including, but not limited to, hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPPs), T cell progenitors, NK cell progenitors, myeloid cells, neutrophil progenitors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. In some embodiments, anti-CD38 antibodies are used to induce ADCC, or CD38 is used when anti-CD38 CARs are used for targeted cell killing. - / - The iPSCs and / or their derived effector cells are not eliminated by anti-CD38 antibodies or anti-CD38 CARs, thereby increasing the persistence and / or viability of the iPSCs and their derived effector cells in the presence of and / or after exposure to such therapeutic agents. In some embodiments, the effector cells have increased persistence and / or viability in vivo in the presence of and / or after exposure to such therapeutic agents. In some embodiments, the CD38 - / - The effector cells are NK cells derived from iPSCs. In some embodiments, CD38 - / - The iPSCs and derived cells comprise one or more additional genome edits as described herein, including, but not limited to, CD16 expression, and cytokine / cytokine receptor expression, and optionally, additional modalities as provided.

[0111] 2.CD16 knock-in CD16 has been identified as two isoforms, the Fc receptors FcγRIIIa (CD16a; NM_000569.6) and FcγRIIIb (CD16b; NM_000570.4). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells to activate NK cells and promote antibody-dependent cellular cytotoxicity (ADCC). CD16b is exclusively expressed by human neutrophils. As used herein, "high affinity CD16", "non-cleavable CD16", "high affinity non-cleavable CD16", or "hnCD16" refer to various CD16 variants. Wild-type CD16 has low affinity and upon NK cell activation is subject to ectodomain shedding, a proteolytic cleavage process that controls the cell surface density of various cell surface molecules on leukocytes. F176V (also referred to as F158V in some publications) is an exemplary CD16 polymorphic variant with high affinity, while the S197P variant is an example of an engineered non-cleavable version of CD16. Engineered CD16 variants, including both F176V and S197P, have high affinity and are non-cleavable, as described in more detail in WO 2015 / 148926, the full disclosure of which is incorporated herein by reference. In addition, a chimeric CD16 receptor, in which the ectodomain of CD16 is essentially replaced with at least a portion of the ectodomain of CD64, can also achieve the desired high affinity and non-cleavable properties of a CD16 receptor capable of performing ADCC. In some embodiments, the replaced ectodomain of the chimeric CD16 comprises one or more of the EC1, EC2, and EC3 exons of CD64 (UniPRotKB_P12314 or an isoform or polymorphic variant thereof).

[0112] Unlike the endogenous CD16 receptor expressed by primary NK cells, which is cleaved from the cell surface following NK cell activation, the various non-cleavable versions of CD16 on derived NK cells avoid CD16 shedding and maintain constant expression. In derived NK cells, non-cleavable CD16 increases the expression of TNFα and CD107a, indicators of improved cell function. Non-cleavable CD16 also enhances antibody-dependent cellular cytotoxicity (ADCC) and the binding of bi-, tri-, or multi-specific engagers. ADCC is a mechanism of NK cell-mediated lysis via binding of CD16 to antibody-coated target cells. The additional high affinity properties of hnCD16 introduced into derived NK cells also allow in vitro loading of ADCC antibodies via hnCD16 prior to administration of the cells to a subject in need of cell therapy. As provided herein, in some embodiments, hnCD16 may comprise F176V and S197P in some embodiments, or may comprise a complete or partial ectodomain from CD64, or may further comprise at least one of a non-native transmembrane domain, a stimulatory domain, and a signaling domain. As disclosed, the present application also provides derived NK cells or cell populations thereof preloaded with one or more preselected ADCC antibodies in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection, as described in more detail below.

[0113] Thus, in some embodiments, the derived NK cells are used in a combination therapy with an antibody. In some embodiments, the antibody in the combination therapy or the antibody with which the derived effector cells are preloaded specifically targets CD38. In some embodiments, the antibody in the combination therapy or the antibody with which the derived effector cells are preloaded specifically targets an antigen different from CD38. In some embodiments, the anti-CD38 antibody is daratumumab.

[0114] Unlike primary NK cells, mature T cells from primary sources (i.e., native / natural sources such as peripheral blood, umbilical cord blood, or other donor tissues) do not express CD16. It was unexpected that iPSCs with expressed exogenous uncleavable CD16 could be differentiated into functional derived T lineage cells that not only express exogenous CD16 but also can perform functions through acquired ADCC mechanisms without compromising the developmental biology of T cells. This acquired ADCC in derived T lineage cells can additionally be used as an approach to rescue antigen escape, which often occurs in dual targeting and / or CAR-T cell therapy, where tumors relapse with reduced or lost expression of CAR-T target antigens, or mutated antigens that avoid recognition by CAR (chimeric antigen receptor). If the derived T lineage cells contain ADCC acquired via exogenous CD16 (including functional variants and CD16-based CFcR) expression, and the antibody targets a tumor antigen different from that targeted by the CAR, the antibody can be used to rescue CAR-T antigen escape and reduce or prevent the recurrence or relapse of the targeted tumor commonly seen with CAR-T therapy. Such strategies to reduce and / or prevent antigen escape while achieving dual targeting are similarly applicable to NK cells expressing one or more CARs.

[0115] Thus, various embodiments of exogenous CD16 introduced into cells include functional CD16 variants and their chimeric receptors. In some embodiments, the functional CD16 variant is a high affinity non-cleavable CD16 receptor (hnCD16). In some embodiments, hnCD16 includes both F176V and S197P, and in some embodiments, F176V, excluding the cleavage region. In some other embodiments, hnCD16 includes a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween identity when compared to any of the exemplary sequences SEQ ID NOs: 1, 2, and 3, each of which includes at least a portion of the CD64 ectodomain. As used herein and throughout this application, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of the percent identity between two sequences can be performed using art-recognized mathematical algorithms.

[0116] [Table 1]

[0117] [Table 2]

[0118] [Table 3]

[0119] Thus, provided herein, among other editing contemplated and described herein, are iPSCs genetically engineered to contain exogenous CD16, i.e., high affinity non-cleavable CD16 receptor (hnCD16), which can be differentiated into effector cells that contain the hnCD16 introduced into the iPSC. In some embodiments, the derived effector cells that contain hnCD16 are NK cells. The exogenous hnCD16 expressed in the iPSCs or their derivatives exhibits high affinity binding to ADCC antibodies or fragments thereof as well as bispecific, trispecific, or multispecific engagers or binders that recognize the extracellular binding domain of the hnCD16 or CD64. The present application therefore provides derived effector cells or cell populations thereof pre-loaded with one or more preselected ADCC antibodies via high affinity binding to the extracellular domain of hnCD16 expressed on the derived effector cells, the hnCD16 comprising the extracellular binding domain of CD64, or of CD16 having F176V and S197P, in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection as described in more detail below.

[0120] In some other embodiments, the native CD16 transmembrane and / or intracellular domains of hnCD16 are further modified or replaced such that a chimeric Fc receptor (CFcR) is produced that includes a non-native transmembrane domain, a non-native stimulatory domain, and / or a non-native signaling domain. As used herein, the term "non-native" means that the transmembrane domain, stimulatory domain, or signaling domain is derived from a different receptor than the receptor that provides the extracellular domain. Exemplary herein, a CFcR based on CD16 or a variant thereof does not have a transmembrane domain, stimulatory domain, or signaling domain derived from CD16. In some embodiments, the exogenous CD16-based CFcR comprises a non-native transmembrane domain derived from CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or a T cell receptor polypeptide. In some embodiments, the exogenous CD16-based CFcR comprises a non-natural stimulatory / inhibitory domain derived from a CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide. In some embodiments, the exogenous CD16-based CFcR comprises a non-natural signaling domain derived from a CD3zeta, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In some embodiments, the CD16-based Fc receptor comprises a transmembrane domain and a signaling domain, both derived from one of an IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, and NKG2D polypeptide.One specific exemplary embodiment of a CD16-based chimeric Fc receptor comprises the transmembrane domain of NKG2D, the stimulatory domain of 2B4, and the signaling domain of CD3ζ, the extracellular domain of CFcR being derived from the full length or a partial sequence of the extracellular domain of CD64 or CD16, the extracellular domain of CD16 comprising F176V and S197P. Another exemplary embodiment of a CD16-based chimeric Fc receptor comprises the transmembrane domain and signaling domain of CD3ζ, the extracellular domain of hnCD16 being derived from the full length or a partial sequence of the extracellular domain of CD64 or CD16, the extracellular domain of CD16 comprising F176V and S197P.

[0121] Various embodiments of CD16-based chimeric Fc receptors as described above can bind with high affinity to the Fc region of an antibody or fragment thereof, or to the Fc region of a bispecific, trispecific, or multispecific engager or binder. Upon binding, the stimulatory and / or signaling domains of the chimeric receptor allow for effector cell activation and cytokine secretion, as well as killing of tumor cells targeted by the antibody, or by the bispecific, trispecific, or multispecific engager or binder having a tumor antigen binding moiety and an Fc region. Without being limited by theory, through the non-native transmembrane, stimulatory, and / or signaling domains of the CD16-based chimeric Fc receptor, or through the binding of the engager to the ectodomain, the CFcR can contribute to the killing ability of the effector cells, increasing the proliferation and / or proliferation potential of the effector cells. Antibodies and engagers can bring antigen-expressing tumor cells and CFcR-expressing effector cells into close proximity, which also contributes to enhanced tumor cell killing. Exemplary tumor antigens for bispecific, trispecific, or multispecific engagers or binders include, but are not limited to, B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. Some non-limiting exemplary bispecific, trispecific, multispecific engagers or binders suitable for binding effector cells expressing CD16-based CFcR in attacking tumor cells include CD16 (or CD64)-CD30, CD16 (or CD64)-BCMA, CD16 (or CD64)-IL15-EPCAM, and CD16 (or CD64)-IL15-CD33.

[0122] Thus, in some embodiments, the present invention relates to a method for treating exogenous CD16 and CD38 knockout (CD38 - / - In some embodiments, the derived cells include derived NK cells (iNK cells) that include exogenous CD16 and CD38 knockout. In some embodiments, the iNK cells that include exogenous CD16 and CD38 knockout are preloaded with an anti-CD38 antibody. In some embodiments, the preloaded anti-CD38 antibody is daratumumab. In some embodiments, the iNK cells that include exogenous CD16 and CD38 knockout further include one or more additional genome edits described herein, including but not limited to cytokine / cytokine receptor expression, and optionally additional modalities provided herein.

[0123] 3. Exogenously introduced cytokine signaling complexes By avoiding systemic high-dose administration of clinically relevant cytokines, the risk of dose-limiting toxicity from such actions is reduced and cytokine-mediated cell autonomy is established. To achieve lymphocyte autonomy without the need for additional administration of soluble cytokines, a cytokine signaling complex comprising one or more partial or full-length peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their corresponding receptors can be introduced into cells to allow cytokine signaling with or without expression of the cytokine itself, thereby reducing the risk of cytokine toxicity and maintaining or improving cell growth, proliferation, expansion, and / or effector function. In some embodiments, the introduced cytokine and / or its respective native or modified receptor (signaling complex) for cytokine signaling is expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, activation of cytokine signaling is inducible. In some embodiments, activation of cytokine signaling is transient and / or temporary.

[0124] Various construct designs are provided herein for introducing protein complexes for cytokine signaling into cells, including but not limited to IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In embodiments where the signaling complex is for IL15, the transmembrane (TM) domain may be negative for the IL15 receptor or may be modified or replaced with the transmembrane domain of any other membrane-bound protein. In some embodiments, IL15 and IL15Rα are co-expressed using a self-cleaving peptide that mimics the trans-presentation of IL15 without eliminating the cis-presentation of IL15. In other embodiments, IL15Rα is fused to IL15 at the C-terminus via a linker, mimicking the trans-presentation without eliminating the cis-presentation of IL15 and ensuring that IL15 is membrane-bound. In another embodiment, IL15Rα with a truncated intracellular domain is fused to IL15 at its C-terminus via a linker to mimic trans-presentation of IL15, maintain membrane association of IL15, and additionally eliminate cis-presentation and / or other potential signaling pathways mediated by normal IL15R via its intracellular domain.

[0125] In some embodiments, the truncated construct comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 4. In one embodiment of truncated IL15 / IL15Rα, the construct does not include the last four amino acid residues (KSRQ) of SEQ ID NO: 4 and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 5. In some embodiments, the sequence identity is at least 80%. In some embodiments, the sequence identity is at least 90%. In some embodiments, the sequence identity is at least 95%. In some embodiments, the sequence identity is 100%.

[0126] [Table 4]

[0127] [Table 5]

[0128] In yet other embodiments, the cytoplasmic domain of IL15Rα can be omitted without adversely affecting the autonomous properties of IL15-equipped effector cells. In other embodiments, the entire IL15Rα is removed, with the exception of the Sushi domain, which is fused to IL15 at one end and to a transmembrane domain at the other (mb-Sushi), with optional linker between the Sushi domain and the transmembrane domain. The fused IL15 / mb-Sushi is expressed on the cell surface via the transmembrane domain of the membrane-bound protein. Unwanted signaling through IL15Rα, including cis-presentation, is eliminated when only the desired trans-presentation of IL15 is retained. In some embodiments, the component comprising IL15 fused to a Sushi domain comprises an amino acid sequence of at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:6.

[0129] [Table 6]

[0130] In other embodiments, native or modified IL15Rβ is fused to IL15 at the C-terminus via a linker to allow constitutive signaling and maintain IL15 membrane binding and trans presentation. In other embodiments, native or modified common receptor γC is fused to IL15 at the C-terminus via a linker for constitutive signaling and membrane-bound trans presentation of cytokines. Common receptor γC is also known as common gamma chain or CD132, and IL2 receptor subunit gamma or IL2RG. γC is a cytokine receptor subunit common to the receptor complexes of many interleukin receptors, including but not limited to IL2, IL4, IL7, IL9, IL15, and IL21 receptors. In other embodiments, engineered IL15Rβ, which forms homodimers in the absence of IL15, is useful for generating constitutive signaling of cytokines.

[0131] Those skilled in the art will understand that the signal peptides and linker sequences described above are exemplary and in no way limit the variations thereof suitable for use as signal peptides or linkers. There are many suitable signal peptide or linker sequences known and available to those skilled in the art. Those skilled in the art will understand that the signal peptide and / or linker sequence can be substituted with another sequence without altering the activity of the functional peptide led by the signal peptide or linked by the linker.

[0132] Thus, in various embodiments, the cytokine IL15 and / or its receptor may be introduced into iPSCs using one or more of the construct designs described above, or into their derived cells upon iPSC differentiation. In addition to induced pluripotent stem cells (iPSCs), clonal iPSCs, clonal iPS cell lines, or iPSC-derived cells are provided that include at least one engineered modality disclosed herein. Also provided are master cell banks that include sorted single cells and expanded clonally engineered iPSCs with at least exogenously introduced cytokine and / or cytokine receptor signaling as described in this section, which provide a platform for further iPSC manipulation and a renewable source for manufacturing off-the-shelf engineered homogenous cell therapy products that are compositionally defined and uniform and can be mass-produced at scale in a cost-effective manner.

[0133] In some embodiments, CD38 knockout (CD38 - / - ) and exogenous CD16, further comprising a cytokine signaling complex. In some embodiments, the derived cells include derived NK cells (iNK cells) comprising a CD38 knockout, exogenous CD16, and a cytokine signaling complex. In some embodiments, the iNK cells comprising a CD38 knockout, exogenous CD16, and a cytokine signaling complex further comprise one or more additional genome edits described herein.

[0134] 4. HLA-I and HLA-II deficiency To avoid the problem of allogeneic rejection, multiple HLA class I and class II proteins must be matched for histocompatibility of the allogeneic recipient. Provided herein are iPSC cell lines in which expression of both HLA class I ("HLA-I") and HLA class II ("HLA-II") proteins is optionally eliminated or substantially reduced. HLA class I deficiency can be achieved by functional deletion of any region of the HLA class I locus (chromosome 6p21), or deletion or reduced expression levels of HLA class I-associated genes, including but not limited to the beta 2 microglobulin (B2M) gene, the TAP1 gene, the TAP2 gene, and tapasin. For example, the B2M gene encodes a common subunit essential for cell surface expression of all HLA class I heterodimers. B2M-negative cells are HLA-I deficient.

[0135] HLA class II deficiency can be achieved by functional deletion or reduction of HLA-II associated genes, including but not limited to RFXANK, CIITA, RFX5, and RFXAP. CIITA is a transcriptional coactivator and functions through activation of the transcription factor RFX5, which is required for the expression of class II proteins. CIITA-negative cells are HLA-II deficient. However, lack of HLA class I expression increases susceptibility to lysis by NK cells. Thus, the present application provides iPSCs and derived cells therefrom that contain HLA-I and / or HLA-II deficiency, e.g., by lack of B2M and / or CIITA expression, and the resulting derived effector cells enable allogeneic cell therapy by eliminating the need for MHC (major histocompatibility complex) matching to avoid recognition and killing by the host's (allogeneic) T cells.

[0136] Furthermore, lack of HLA class I expression leads to lysis by host NK cells. Thus, in addition to the above-mentioned approach of CD38 conditioning to remove activated CD38-expressing host NK cells, to overcome this "loss of self" response, HLA-G can be optionally knocked in to avoid NK cell recognition and killing of HLA-I-deficient effector cells derived from engineered iPSCs. In one embodiment, the provided HLA-I-deficient iPSCs and derived cells thereof further comprise an HLA-G knock-in.

[0137] In some embodiments, iPSCs, as well as CD38 knockout (CD38 - / - ), exogenous CD16, and cytokine signaling complexes, the derived cells further comprise HLA-I and / or HLA-II deficiencies without adversely affecting the differentiation potential of the iPSCs and the function of derived effector cells, including derived T cells and derived NK cells.

[0138] 5. Genetically engineered iPSC lines and derived cells provided herein In light of the above, the present application relates to CD38 - / - iPSCs, cell line cells, or populations thereof, and CD38 (also referred to herein as "CD38 negative" or CD38 knockout) - / - Provided are derived functional derivative cells comprising a CD38 knockout obtained from differentiation of iPSCs. In some embodiments, the functional derivative cells are hematopoietic cells, mesodermal cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34 + These include, but are not limited to, hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPPs), T cell progenitors, NK cell progenitors, myeloid cells, neutrophil progenitors, T lineage cells, NKT lineage cells, NK lineage cells, B lineage cells, neutrophils, dendritic cells, and macrophages. In other embodiments, functional derivative hematopoietic cells include effector cells that have one or more functional attributes not present in the corresponding primary T, NK, NKT, and / or B cells.

[0139] Further provided herein are iPSCs comprising a CD38 knockout and a polynucleotide encoding exogenous CD16, which iPSCs can undergo directed differentiation to produce functional derived hematopoietic cells. In some embodiments, when an anti-CD38 antibody is used to induce CD16-mediated enhanced ADCC, the iPSCs and / or their derived effector cells can target CD38-expressing (tumor) cells without causing effector cell elimination, i.e., reduction or depletion of CD38-expressing effector cells, thereby increasing persistence and / or viability of the iPSCs and their effector cells. In some embodiments, the effector cells have increased persistence and / or viability in vivo in the presence of an anti-CD38 therapeutic agent, which may be an anti-CD38 antibody. In addition, because CD38 is upregulated on activated lymphocytes, such as T cells or B cells, CD38-specific antibodies can be used for lymphodepletion, thereby eliminating those activated lymphocytes, overcoming allogeneic rejection, and inhibiting CD38 without fratricide in recipients of allogeneic effector cell therapy. - / - Increases survival and persistence of effector cells. In some embodiments, the effector cells comprise NK lineage cells. CD38 - / - and exogenous CD16, iPSC-derived NK lineage cells have enhanced cytotoxicity and reduced NK cell fratricide in the presence of anti-CD38 antibody.

[0140] Additionally provided are iPSCs that contain a CD38 knockout, exogenous CD16, and a polynucleotide encoding at least one exogenous cytokine signaling complex (IL) to allow cytokine signaling that contributes to cell viability, persistence, and / or proliferation, and the iPSC line can undergo hematopoietic differentiation to produce functional derived effector cells with improved viability, persistence, proliferation, and effector function. The exogenously introduced cytokine signaling includes any one, or two, or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21 signaling. In some embodiments, the introduced cytokine signaling complex is expressed on the cell surface. In some embodiments, the cytokine signaling is constitutively activated. In some embodiments, the activation of cytokine signaling is inducible. In some embodiments, the activation of cytokine signaling is transient and / or temporary. In some embodiments, the transient / transient expression of cell surface cytokine / cytokine receptor is via retrovirus, Sendai virus, adenovirus, episome, minicircle, or RNA, including mRNA. - / - The exogenous cytokine signaling complex contained within the iPSC or derived cells enables IL15 signaling. - / - The iPSCs and derived cells thereof, containing exogenous CD16 and IL, can autonomously maintain or improve cell growth, proliferation, expansion, and / or effector function without contact with additionally supplied soluble cytokines in vitro or in vivo, and can be used with ADCC capable antibodies for targeted killing. Anti-CD38 antibodies can be used in combination therapy with the CD38 - / - When used in conjunction with cells, the cells have synergistically increased persistence, viability, and effector function.

[0141] Also provided are iPSCs comprising a polynucleotide encoding a CD38 knockout, a B2M knockout, and / or a CIITA knockout, and optionally an HLA-G, the iPSCs being capable of directed differentiation to produce functional derived hematopoietic cells. - / - B2M - / - CIITA - / - The iPSCs and their derived effector cells are both HLA-I and HLA-II deficient and can be used with anti-CD38 antibodies to induce ADCC without causing effector cell elimination, thereby synergistically increasing the persistence and / or survival of the iPSCs and their effector cells. In some embodiments, the effector cells have increased persistence and / or survival in vivo.

[0142] In view of the above, provided herein are iPSCs comprising a CD38 knockout, exogenous CD16, and an exogenous cytokine signaling complex, and optionally a B2M / CIITA knockout, where when B2M is knocked out, a polynucleotide encoding HLA-G is optionally introduced, and the iPSCs are capable of directed differentiation to produce functional derived hematopoietic cells, including mesodermal cells with committed hemogenic endothelial (HE) potential, committed HE, CD34 + These include, but are not limited to, hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPPs), T cell progenitors, NK cell progenitors, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, macrophages, or derived effector cells that have one or more functional attributes not present in the corresponding primary T, NK, NKT, and / or B cells.

[0143] 6. Antibodies for immunotherapy In some embodiments, in addition to the genomically engineered effector cells provided herein, additional therapeutic agents including antibodies or antibody fragments targeting antigens associated with a condition, disease, or indication can be used in combination therapy with these effector cells. In some embodiments, antibodies are used in combination with the population of effector cells described herein by simultaneous or sequential administration to a subject. In other embodiments, such antibodies or fragments thereof can be expressed by effector cells by genetically engineering iPSCs with an exogenous polynucleotide sequence encoding the antibody or fragment thereof and directing differentiation of the engineered iPSCs. In some embodiments, the effector cells additionally express an exogenous CD16 variant, and the cytotoxicity of the effector cells is enhanced by the antibody via ADCC. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, tumor- or virus-specific antigens activate the administered iPSC-derived effector cells, enhancing their killing capacity. In some embodiments, antibodies suitable for combination therapy as additional therapeutic agents to the administered iPSC-derived effector cells include, but are not limited to, anti-CD20 (rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab), anti-HER2 (trastuzumab, pertuzumab), anti-CD52 (alemtuzumab), anti-EGFR (cetuximab), anti-GD2 (dinutuximab), anti-PDL1 (avelumab), anti-CD38 (daratumumab, isatuximab, MOR202), anti-CD123 (7G3, CSL362), anti-SLAMF7 (elotuzumab), and humanized or Fc-modified variants or fragments thereof, or functional equivalents and biosimilars thereof.

[0144] In some embodiments of the combination therapy comprising derivative cells and at least one antibody provided herein, the antibody is not produced by the derivative cells or in the derived cells and is additionally administered prior to, concurrently with, or following administration of the derivative cells, hi some embodiments, the antibody administered prior to, concurrently with, or following administration of the derivative cells is an anti-CD38 monoclonal antibody or an anti-SLAMF7 monoclonal antibody.

[0145] 7. Checkpoint Inhibitors Checkpoints are cellular molecules, often cell surface molecules, that can suppress or downregulate immune responses if not inhibited. It has become clear that tumors co-opt certain immune checkpoint pathways as a primary mechanism of immune resistance, especially to T cells specific for tumor antigens. Checkpoint inhibitors (CIs) are antagonists that can reduce checkpoint gene expression or gene products or decrease the activity of checkpoint molecules, thereby blocking inhibitory checkpoints and restoring immune system function. The development of checkpoint inhibitors targeting PD1 / PDL1 or CTLA4 has transformed the oncology landscape, and these agents have led to long-term remission in multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and recurrence remains a significant concern.

[0146] Thus, in some embodiments, the present application provides a therapeutic approach to overcome CI resistance by including functional derivative cells engineered to be provided in combination therapy with CI. In one embodiment of the combination therapy, the derivative cells are NK cells. In addition to exhibiting direct antitumor capabilities, the derived NK cells provided herein have been shown to have the ability to resist PDL1-PD1 mediated inhibition, enhance T cell migration, recruit T cells to the tumor microenvironment, and enhance T cell activation at tumor sites. Thus, the tumor infiltration of T cells promoted by functionally potent genetically engineered derived NK cells indicates that the NK cells can synergize with T cell targeted immunotherapy, including checkpoint inhibitors, to alleviate local immune suppression and reduce tumor burden.

[0147] In one embodiment, the derived NK cells for checkpoint inhibitor combination therapy comprise a CD38 knockout, exogenous CD16, and exogenous cytokine signaling complex, and optionally a B2M / CIITA knockout, optionally including a polynucleotide encoding HLA-G if B2M is knocked out. In some embodiments, the derived NK cells comprise deleted or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene in the chromosome 6p21 region, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A Additionally includes induced or increased expression of at least one of a surface triggering receptor for binding to an R, a CAR, a TCR, an Fc receptor, an engager, and a bispecific, multispecific, or universal engager.

[0148] The derived NK cells can be obtained by differentiating iPSC clonal line cells comprising CD38 knockout, exogenous CD16, and exogenous cytokine signaling complex, and optionally B2M / CIITA knockout, where if B2M is knocked out, a polynucleotide encoding HLA-G is optionally introduced. In some embodiments, the iPSC clonal line cells comprise deletion or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene in the chromosome 6p21 region, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A Further comprising induced or increased expression of at least one of a surface triggering receptor for binding to an R, a CAR, a TCR, an Fc receptor, an engager, and a bispecific, multispecific, or universal engager.

[0149] Suitable checkpoint inhibitors for combination therapy with derived NK cells provided herein include PD-1 (Pdcdl, CD279), PDL-1 (CD274), TIM-3 (Havcr2), TIGIT (WUCAM and Vstm3), LAG-3 (Lag3, CD223), CTLA-4 (Ctla4, CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A 2A These include, but are not limited to, antagonists of R, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR (e.g., 2DL1, 2DL2, 2DL3, 3DL1, and 3DL2).

[0150] In some embodiments, the antagonist that inhibits any of the above-mentioned checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibody can be a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a shark heavy chain only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab)'2, F(ab)'3, Fv, single chain antigen binding fragment (scFv), (scFv)2, disulfide stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single domain antigen binding fragment (sdAb, nanobody), recombinant heavy chain only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody, which may be more cost-effective to produce, easier to use, or more sensitive than the whole antibody. In some embodiments, the one or two or three or more checkpoint inhibitors comprise at least one of atezolizumab (anti-PDL1 mAb), avelumab (anti-PDL1 mAb), durvalumab (anti-PDL1 mAb), tremelimumab (anti-CTLA4 mAb), ipilimumab (anti-CTLA4 mAb), IPH4102 (anti-KIR), IPH43 (anti-MICA), IPH33 (anti-TLR3), lilimumab (anti-KIR), monalizumab (anti-NKG2A), nivolumab (anti-PD1 mAb), pembrolizumab (anti-PD1 mAb), and derivatives, functional equivalents, or biosimilars thereof.

[0151] In some embodiments, the antagonists that inhibit any of the above-mentioned checkpoint molecules are microRNA-based, as many miRNAs are found as regulators that control the expression of immune checkpoints (Dragomir et al., Cancer Biol Med. 2018, 15(2):103-115). In some embodiments, checkpoint antagonistic miRNAs include, but are not limited to, miR-28, miR-15 / 16, miR-138, miR-342, miR-20b, miR-21, miR-130b, miR-34a, miR-197, miR-200c, miR-200, miR-17-5p, miR-570, miR-424, miR-155, miR-574-3p, miR-513, and miR-29c.

[0152] Some embodiments of the combination therapy with the provided derivative NK cells include at least one checkpoint inhibitor that targets at least one checkpoint molecule. In some embodiments of the combination therapy including at least one checkpoint inhibitor, the checkpoint inhibitor is an antibody, or a humanized or Fc-modified variant or fragment, or a functional equivalent or biosimilar thereof, and the checkpoint inhibitor is produced by the derivative cells by expressing an exogenous polynucleotide sequence encoding the antibody, or a fragment or variant thereof that inhibits a checkpoint. In some embodiments, the exogenous polynucleotide sequence encoding the antibody, or a fragment or variant thereof that inhibits a checkpoint, is co-expressed with a chimeric antigen receptor (CAR), either in a separate construct or in a bicistronic construct that includes both the CAR and the antibody or fragment thereof encoding sequence. In some further embodiments, the antibody or fragment thereof encoding sequence may be linked to either the 5' or 3' end of the CAR expression construct via a self-cleaving 2A coding sequence, exemplified as, for example, CAR-2A-CI or CI-2A-CAR. Thus, the coding sequences for the checkpoint inhibitor and the CAR are in a single open reading frame (ORF). When the checkpoint inhibitor is delivered and expressed and secreted as a payload by derived effector cells that can infiltrate the tumor microenvironment (TME), it counteracts inhibitory checkpoint molecules upon binding to the TME and activates modalities such as the CAR or activates the effector cells by activating receptors. In some embodiments, the checkpoint inhibitor co-expressed with the CAR is a checkpoint molecule, PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2AInhibits at least one of R, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR.

[0153] In some embodiments, the checkpoint inhibitor co-expressed with the CAR in the derivative cells described herein is selected from the group including atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and humanized or Fc-modified variants, fragments, and functional equivalents or biosimilars thereof. In some embodiments, the checkpoint inhibitor co-expressed with the CAR is atezolizumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof. In some other embodiments, the checkpoint inhibitor co-expressed with the CAR is nivolumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof. In some other embodiments, the checkpoint inhibitor co-expressed with the CAR is pembrolizumab, or a humanized, or Fc-engineered variant, fragment, or functional equivalent or biosimilar thereof.

[0154] In some other embodiments of the combination therapy comprising the derived cells and at least one antibody that inhibits a checkpoint molecule provided herein, the antibody is not produced by or in the derived cells and is additionally administered prior to, simultaneously with, or after administration of the derived cells described herein. In some embodiments, the administration of one, two, three, or more checkpoint inhibitors in the combination therapy with the derived NK cells provided is simultaneous or sequential. In one embodiment of the combination therapy comprising the derived NK cells described herein, the checkpoint inhibitor included in the treatment is one or more of atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and humanized or Fc-modified variants, fragments, and functional equivalents or biosimilars thereof. In some embodiments of the combination therapy comprising the derived NK cells described herein, the checkpoint inhibitor included in the treatment is atezolizumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof. In some embodiments of the combination therapy comprising the derived NK cells described herein, the checkpoint inhibitor included in the treatment is nivolumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof. In some embodiments of the combination therapy comprising the derived NK cells described herein, the checkpoint inhibitor included in the treatment is pembrolizumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof.

[0155] II. Therapeutic Use of Derived Immune Cells with Functional Modalities Differentiated from Genetically Engineered iPSCs The invention provides, in some embodiments, compositions comprising isolated populations or subpopulations of functionally enhanced derived immune cells differentiated from genomically engineered iPSCs using the disclosed methods and compositions, in some embodiments, the iPSCs comprise one or more targeted gene edits that are capable of being retained in the iPSC-derived immune cells, and the genetically engineered iPSCs and their derived cells are suitable for cell-based adoptive therapy.

[0156] As shown in Figure 1, the derivative immune cells included in the allogeneic natural killer (NK) cell immunotherapy compositions described herein, according to some embodiments, are produced from a genomically engineered clonal master human induced pluripotent stem cell (iPSC) line and have exogenous CD16, which is a high affinity non-cleavable CD16 receptor (hnCD16) (also referred to herein as "FT516"). According to some other embodiments, the iPSC-derived NK cells included in the allogeneic cell immunotherapy compositions contain the following engineered elements: a) CD38 knockout, b) exogenous CD16, which is a high affinity non-cleavable CD16 receptor (hnCD16), and c) a cytokine signaling complex comprising interleukin (IL)-15 / IL-15 receptor alpha fusion protein (IL15RF) (also referred to herein as "FT538"). For FT538, a clonal master cell bank (MCB) used for the generation of iNK cell therapy was generated by selecting and expanding a single well-characterized iPSC clone in which a single IL15RF / hnCD16 expression cassette was inserted into the CD38 locus through non-viral-mediated targeted transgene integration.

[0157] The use of clonal MCBs as starting material for current Good Manufacturing Practice (cGMP) manufacturing of iNK cell therapy is intended to directly address many of the limitations associated with parental and donor-specific cell therapies. Notably, many doses of iNK cell therapy product can be manufactured uniformly in a single manufacturing campaign. These doses of drug product are uniform, (i) tested to ensure compliance with predefined quality specifications, (ii) cryopreserved in infusion media, and (iii) stockpiled to maintain sustainable inventory. Thus, iNK cell therapy in the clinical setting has off-the-shelf availability for use in multiple dose regimens, which may prove critical for driving long-term sustained responses in patients with progressive disease.

[0158] The engineered properties of iNK cell therapy are designed to provide increased activity against target tumor cells both as monotherapy and when combined with monoclonal antibodies (mAbs) capable of mediating antibody-dependent cellular cytotoxicity (ADCC). Functional attributes of iNK cell therapy according to some embodiments include the following: a) iNK cell therapy products have superior effector function compared to a patient's endogenous NK cells, which are typically reduced in number and function due to previous treatment regimens (e.g., chemotherapy) and tumor suppression mechanisms. Thus, iNK cell therapy mediates "innate cytotoxicity" that is potent and specific against transformed cells. b) CD38 gene KO in iNK cell products prevents anti-CD38 antibody-mediated NK cell fratricide, thereby enhancing ADCC when iNK cell therapy is administered simultaneously with anti-CD38 mAb therapy. In addition, NK cells with CD38 KO are more resistant to oxidative stress and exhibit enhanced effector function and persistence. c) iNK cells express the hnCD16 Fc receptor. The high affinity CD16 variant resulting from the naturally occurring 158V polymorphism has enhanced ADCC when combined with IgG mAbs, including but not limited to rituximab, cetuximab, and trastuzumab. In addition, hnCD16 contains a genetic modification (S179P) that prevents cleavage of CD16 by the metalloproteinase ADAM17, a mechanism in the regulation and attenuation of NK cell activity by the tumor microenvironment. d) iNK cells express IL15RF, which is designed to provide endogenous activation and proliferation signals, reducing dependency on exogenous cytokine administration such as IL-2 and IL15, both of which are associated with significant toxicities that may limit clinical use if incorporated into clinical trials of peripheral blood NK cells.

[0159] These attributes justify the investigation of iNK cells in a broad set of oncology indications, including but not limited to: (i) as monotherapy for, for example, acute myelogenous leukemia (AML), where iNK cells may provide greater clinical benefit than current allogeneic NK cell-based therapies, and (ii) in combination with approved investigational tumor-targeted ADCC-capable mAbs, including daratumumab and elotuzumab, which target CD38 and SLAMF7, respectively, and are approved for the treatment of patients with multiple myeloma (MM). Depending on the mAb used in the combination therapy, the iNK cells may also provide clinical benefit in the treatment of solid tumors.

[0160] Previously, a biodistribution and persistence study of iNK cells in immunodeficient NOD SCID-IL2rγ null (NSG) mice was performed using 3 × 10 iNK cells administered at 7-day intervals on study days 1, 8, and 15. 6 cells / mouse or 1.2 x 10 7Cells were assessed after three IV injections of cells / mouse. The data demonstrated that iNK cell products were detectable in most tissues and that their persistence generally decreased over time to levels approaching or below the lower limit of detection by day 67 post-infusion.

[0161] Thus, various diseases can be ameliorated by introducing the immune cells of the present invention into a subject suitable for adoptive cell therapy. In some embodiments, the iPSC-derived immune cells (e.g., iNK cells) provided are for allogeneic adoptive cell therapy. Additionally, the present invention provides, in some embodiments, therapeutic use of the therapeutic composition described above by introducing the composition into a subject suitable for adoptive cell therapy, the subject having an autoimmune disorder, hematological malignancy, solid tumor, or infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus. Examples of hematological malignancies include acute and chronic leukemias (acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), lymphomas, non-Hodgkin's lymphomas, and the like. Examples of solid cancers include, but are not limited to, cancers of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testis, bladder, kidney, head, neck, stomach, cervix, rectum, larynx, and esophagus. Examples of various autoimmune diseases include alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), some forms of juvenile idiopathic arthritis, and others. These include, but are not limited to, some forms of glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multiple sclerosis, pemphigoid / bullous pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, and granulomatosis with polyangiitis (Wegener's disease).Examples of viral infections include, but are not limited to, HIV- (human immunodeficiency virus), HSV- (herpes simplex virus), KSHV- (Kaposi's sarcoma-associated herpes virus), RSV- (respiratory syncytial virus), EBV- (Epstein-Barr virus), CMV- (cytomegalovirus), VZV (varicella zoster virus), adenovirus-, lentivirus-, BK polyomavirus-associated diseases. In some embodiments, the hematological malignancy is refractory or relapsed. In certain embodiments, the patient receiving adoptive cell therapy has acute myeloid leukemia (AML), refractory or relapsed AML, secondary AML from myelodysplastic syndrome, multiple myeloma (MM), relapsed or refractory MM, non-Hodgkin's lymphoma (NHL), or relapsed or refractory NHL.

[0162] Treatment using the derived hematopoietic lineage cells of the embodiments disclosed herein can be on a symptomatic basis or to prevent recurrence. The terms "treating", "treatment" and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic, in terms of completely or partially preventing the disease, and / or therapeutic, in terms of partially or completely curing the disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses any intervention of a disease in a subject, including: preventing the disease from occurring, inhibiting the disease, i.e. arresting its development, or alleviating the disease, i.e. reversing the disease, in a subject who may be susceptible to the disease but has not yet been diagnosed as having it. Therapeutic agents or compositions can be administered before, during, or after the onset of the disease or injury. Treatment of ongoing diseases, where treatment stabilizes or reduces undesirable clinical symptoms in the patient, is also of particular interest. In certain embodiments, the subject in need of treatment has a disease, condition, and / or injury that can be suppressed, ameliorated, and / or ameliorated by cell therapy at least one associated symptom. Certain embodiments contemplate that subjects in need of cell therapy include, but are not limited to, bone marrow or stem cell transplant candidates, subjects who have undergone chemotherapy or radiation therapy, subjects having or at risk of having a hyperproliferative disorder or cancer, e.g., a hyperproliferative disorder or cancer of the hematopoietic system, subjects having or at risk of developing a tumor, e.g., a solid tumor, subjects having or at risk of having a viral infection or a disease associated with a viral infection.

[0163] When assessing response to a treatment comprising the derived hematopoietic lineage cells (e.g., iNK cells) of the embodiments disclosed herein, response can be measured by criteria including at least one of the following: clinical benefit rate, survival to death, pathologic complete response, semi-quantitative measurement of pathologic response, clinical complete response, clinical partial response, clinical stable disease, recurrence-free survival, metastasis-free survival, disease-free survival, circulating tumor cell reduction, circulating marker response, and RECIST (Response Evaluation Criteria In Solid Tumors) criteria.

[0164] In some embodiments, patients are monitored for any adverse events (AEs) that may occur during the course of treatment. Exemplary adverse events include, but are not limited to, new malignancies, new or worsening neurological disorders, new or worsening autoimmune or rheumatic disorders, or new hematological disorders. In some embodiments, patients are monitored for cytokine release syndrome (CRS), neurotoxicity (ICAN), and / or GvHD before, during, and after the course of treatment.

[0165] Therapeutic compositions comprising derived hematopoietic lineage cells as disclosed can be administered to a subject before, during, and / or after other treatments. Thus, methods of combination therapy can include administration or preparation of iPSC-derived immune cells (e.g., iNK cells) before, during, and / or after the use of one or more additional therapeutic agents. As provided herein, the one or more additional therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNA), mononuclear blood cells, feeder cells, feeder cell components or supplements thereof, vectors comprising one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (IMiDs). Immunomodulatory drugs (IMiDs), such as thalidomide, lenalidomide, and pomalidomide, stimulate both NK cells and T cells. As provided herein, IMiDs can be used with iPSC-derived therapeutic immune cells for cancer treatment. Additionally or alternatively, administration can be combined with other bioactive agents or modalities, such as, but not limited to, anti-tumor agents, or non-pharmacological therapies, such as surgery.

[0166] In some embodiments of the combined cell therapy, the therapeutic combination comprises iPSC-derived hematopoietic lineage cells (e.g., iNK cells) provided herein and an additional therapeutic agent that is an antibody or fragment thereof. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody may be a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, the tumor or virus-specific antigen activates the administered iPSC-derived hematopoietic lineage cells to enhance their killing capacity. In some embodiments, the antibody is a tumor-targeting ADCC-capable monoclonal antibody (mAb). In some embodiments, as an additional therapeutic agent to the iPSC-derived hematopoietic lineage cells administered, antibodies suitable for combination therapy include, but are not limited to, anti-CD20 (e.g., rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab), anti-HER2 (e.g., trastuzumab, pertuzumab), anti-CD52 (e.g., alemtuzumab), anti-EGFR (e.g., certuximab), anti-GD2 (e.g., dinutuximab), anti-PDL1 (e.g., avelumab), anti-CD38 (e.g., daratumumab, isatuximab, MOR202), anti-CD123 (e.g., 7G3, CSL362), anti-SLAMF7 (elotuzumab), and humanized or Fc-modified variants or fragments thereof, or functional equivalents or biosimilars thereof. In certain embodiments, the antibody suitable for combination therapy as an additional therapeutic agent to the iPSC-derived hematopoietic lineage cells administered is daratumumab or elotuzumab.

[0167] In some embodiments, the additional therapeutic agent comprises one or more checkpoint inhibitors. Checkpoints refer to cellular molecules, often cell surface molecules, that, if uninhibited, can suppress or downregulate an immune response. Checkpoint inhibitors are antagonists that can reduce the gene expression or gene products of a checkpoint or decrease the activity of a checkpoint molecule. Checkpoint inhibitors suitable for combination therapy with derived effector cells provided herein include PD-1 (Pdcdl, CD279), PDL-1 (CD274), TIM-3 (Havcr2), TIGIT (WUCAM and Vstm3), LAG-3 (Lag3, CD223), CTLA-4 (Ctla4, CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A2aR, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5 E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR (e.g., 2DL1, 2DL2, 2DL3, 3DL1, and 3DL2) antagonists.

[0168] Some embodiments of combination therapies including the derived effector cells provided further include at least one inhibitor targeting a checkpoint molecule. Some other embodiments of combination therapies with the derived effector cells provided include two, three, or more inhibitors such that two, three, or more checkpoint molecules are targeted. In some embodiments, the effector cells for the combination therapy described herein are derived NK cells as provided. In some embodiments, the derived NK cells for the combination therapy are functionally enhanced as provided herein. In some embodiments, two, three, or more checkpoint inhibitors can be administered in the combination therapy simultaneously, before, or after administration of the derived effector cells. In some embodiments, two or more checkpoint inhibitors are administered simultaneously or one at a time (sequentially).

[0169] In some embodiments, the antagonist that inhibits any of the above-mentioned checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibody can be a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a shark heavy chain only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab)'2, F(ab)'3, Fv, single chain antigen binding fragment (scFv), (scFv)2, disulfide stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single domain antigen binding fragment (sdAb, nanobody), recombinant heavy chain only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody, which may be more cost-effective to produce, easier to use, or more sensitive than the whole antibody. In some embodiments, the one or two or three or more checkpoint inhibitors comprise at least one of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof.

[0170] In some embodiments, other than the derived effector cells as provided herein, the combination for therapeutic use includes one or more additional therapeutic agents, including chemotherapeutic agents or radioactive moieties. Chemotherapeutic agents refer to cytotoxic anti-tumor agents, i.e., chemical agents that are found to preferentially kill tumor cells, or disrupt the cell cycle of rapidly proliferating cells, or eradicate stem cancer cells, and are used therapeutically to prevent or reduce the proliferation of neoplastic cells. Chemotherapeutic agents may also be referred to as anti-tumor or cytotoxic drugs or agents, and are well known in the art.

[0171] In some embodiments, chemotherapeutic agents include anthracyclines, alkylating agents, alkyl sulfonates, aziridines, ethylenimines, methylmelamine, nitrogen mustards, nitrosoureas, antibiotics, antimetabolites, folic acid analogs, purine analogs, pyrimidine analogs, enzymes, podophyllotoxins, platinum-containing drugs, interferons, and interleukins. Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents (cyclophosphamide (CY), mechlorethamine, mephalin, chlorambucil, heparinmethylmelamine, thiotepa, busulfan, carmustine, lomustine, semustine), antimetabolites (methotrexate, fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, thioguanine, pentostatin), vinca alkaloids (vincristine, vinblastine, vindesine), epipodophyllotoxins (etoposide, etoposide orthoquinone, and teniposide), antibiotics (daunorubicin, doxorubicin, mitoxantrone, bisantrene, actinomycin D, plicamycin, puromycin, and gramicidin D), paclitaxel, colchicine, cytochalasin B, emetine, maytansine, and amsacrine.Additional agents include amine glutethimide, cisplatin, carboplatin, mitomycin, altretamine, cyclophosphamide, lomustine (CCNU), carmustine (BCNU), irinotecan (CPT-11), alemtuzamab, altretamine, anastrozole, L-asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan, calcitabine, capecitabine, celecoxib, cetuximab, and cetaxel. , cladribine, cloflavine, cytarabine, dacarbazine, denileukin diftitox, diethylstilbestrol, docetaxel, dromostanolone, epirubicin, erlotinib, estramustine, etoposide, ethinyl estradiol, exemestane, floxuridine, 5-fluorouracil, fludarabine (FLU), flutamide, fulvestrant, gefitinib, gemcitabine, goserelin, hydroxyurea, ibritumoma , idarubicin, ifosfamide, imatinib, interferon alpha (2a, 2b), irinotecan, letrozole, leucovorin, leuprolide, levamisole, mechlorethamine, megestrol, melphalin, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nofetumomab, oxaliplatin, paclitaxel, pamidronate, pemetrexed, pegadome , pegaspargase, pentostatin, pipobroman, plicamycin, porifeprosan, porfimer, procarbazine, quinacrine, rituximab, sargramostim, streptozocin, tamoxifen, temozolomide, teniposide, testolactone, thioguanine, thiotepa, topetecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinorelbine, and zoledronate. Other suitable agents are those approved for human use, including those approved as chemotherapeutic or radiotherapeutic agents and known in the art.Such agents can be found in any of many standard physician and oncologist references (e.g., Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, NY, 1995) or at the National Cancer Institute website (fda.gov / cder / cancer / druglistfrarne.htm), both of which are updated from time to time. In certain embodiments, as additional therapeutic agents to the iPSC-derived hematopoietic lineage cells administered, suitable chemotherapeutic agents for combination therapy are cyclophosphamide and fludarabine.

[0172] Combination therapies comprising derived effector cells and monoclonal antibodies, optionally in combination with one or more chemotherapeutic agents, are used to treat cutaneous T-cell lymphoma, non-Hodgkin's lymphoma (NHL), mycosis fungoides, Pagetoid reticulosis, Sezary syndrome, granulomatous lax skin, lymphomatoid papulosis, chronic pityriasis lichenoides, acute pityriasis lichenoides, CD30+ cutaneous T-cell lymphoma, secondary cutaneous CD30+ large cell lymphoma, non-mycosis fungoides CD30 cutaneous large T-cell lymphoma, pleomorphic T-cell lymphoma, Lennert lymphoma, subcutaneous T-cell lymphoma, angiocentric lymphoma, blastic NK-cell lymphoma, B-cell lymphoma, Hodgkin's lymphoma (HL), head and neck tumors, squamous cell carcinoma, rhabdomyosarcoma, Lewis lung cancer, The combination therapy may be applicable to the treatment of liquid and solid cancers, including, but not limited to, non-small cell lung cancer, squamous cell carcinoma (LLC), non-small cell lung cancer, squamous cell carcinoma of the esophagus, adenocarcinoma of the esophagus, renal cell carcinoma (RCC), colorectal cancer (CRC), acute myeloid leukemia (AML), multiple myeloma (MM), breast cancer, gastric cancer, prostatic small cell neuroendocrine carcinoma (SCNC), liver cancer, glioblastoma, liver cancer, oral squamous cell carcinoma, pancreatic cancer, papillary thyroid cancer, intrahepatic cholangiocarcinoma, hepatocellular carcinoma, bone cancer, metastasis, and nasopharyngeal carcinoma. In certain embodiments, the patient receiving the combination therapy has multiple myeloma (MM), or relapsed or refractory MM. In some embodiments, the patient receiving the combination therapy has AML, or relapsed or refractory AML. In some other embodiments, the patient receiving the combination therapy has NHL, or relapsed or refractory NHL.

[0173] The administration of iPSC-derived immune cells can be separated in time by hours, days, or even weeks from the administration of any of the additional therapeutic agents. In some embodiments, the administration of iPSC-derived immune cells can precede the administration of one or more chemotherapeutic agents, alone or in combination with one or more antibodies. In certain embodiments, the subject can be administered a course of treatment comprising one or more chemotherapeutic agents daily for about 1-5 consecutive days, followed by administration of a first or more doses or cycles of iPSC-derived immune cells (e.g., iNK cells) of the present invention. In some embodiments, the iNK cells are administered once a week for at least three weeks, followed by daily administration of one or more chemotherapeutic agents for three consecutive days. In one embodiment, the duration between the last dose of one or more chemotherapeutic agents and the first dose of iNK cells is about 40-84 hours.

[0174] In other embodiments of the combination cell therapy, the subject may be administered a course of treatment comprising an anti-CD38 monoclonal antibody or an anti-SLAMF7 monoclonal antibody, followed by administration of one or more chemotherapeutic agents, followed by administration of the iPSC-derived immune cells (e.g., iNK cells) of the invention. In some embodiments, the anti-CD38 monoclonal antibody is daratumumab and the anti-SLAMF7 monoclonal antibody is elotuzumab. In some embodiments, the monoclonal antibody is administered weekly for 8 doses, followed by 2 doses every week ± 1 day. In some other embodiments, the monoclonal antibody is administered every 2 weeks. In certain embodiments, the first dose of the monoclonal antibody administered weekly occurs about 4-6 days prior to administration of the one or more chemotherapeutic agents. In some embodiments, the subject may be administered a course of treatment comprising one or more chemotherapeutic agents daily for about 1-5 consecutive days, followed by weekly administration of the iPSC-derived immune cells (e.g., iNK cells) of the invention. In some embodiments, iNK cells are administered once a week for three weeks, followed by daily administration of one or more chemotherapeutic agents for three consecutive days. In one embodiment, the duration between the last dose of the one or more chemotherapeutic agents and the first dose of iNK cells is about 40-84 hours or about three days.

[0175] In addition to the isolated population of iPSC-derived hematopoietic lineage cells contained in the therapeutic composition, a composition suitable for administration to a patient can further include one or more pharma- ceutically acceptable carriers (additives) and / or diluents (e.g., a pharma- ceutically acceptable medium, e.g., cell culture medium), or other pharma- ceutically acceptable components. Pharmaceutically acceptable carriers and / or diluents will be determined, in part, by the particular composition being administered, as well as by the particular method used to administer the therapeutic composition. Thus, there are a wide variety of suitable formulations of the therapeutic compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., the disclosure of which is incorporated herein by reference in its entirety). th (see ed. 1985).

[0176] These pharma- ceutically acceptable carriers and / or diluents can be present in an amount sufficient to maintain the pH of the therapeutic composition at about 3 to about 10. Thus, the buffer can be as much as about 5% on a weight-by-weight basis of the total composition. Electrolytes, such as, but not limited to, sodium chloride and potassium chloride, can also be included in the therapeutic composition. In one aspect, the pH of the therapeutic composition ranges from about 4 to about 10. Alternatively, the pH of the therapeutic composition ranges from about 5 to about 9, about 6 to about 9, or about 6.5 to about 8. In another embodiment, the therapeutic composition includes a buffer having a pH in one of the above pH ranges. In another embodiment, the therapeutic composition has a pH of about 7. Alternatively, the therapeutic composition has a pH in the range of about 6.8 to about 7.4. In yet another embodiment, the therapeutic composition has a pH of about 7.4.

[0177] The present invention also provides, in part, the use of pharma- ceutically acceptable cell culture media in certain compositions and / or cultures of the invention. Such compositions are suitable for administration to a human subject. Generally speaking, any medium that supports the maintenance, growth, and / or health of iPSC-derived immune cells according to embodiments of the invention is suitable for use as a pharmaceutical cell culture medium. In certain embodiments, the pharma- ceutically acceptable cell culture medium is a serum-free and / or feeder-free medium. In various embodiments, the serum-free medium is free of animal components and may optionally be protein-free. Optionally, the medium may include recombinant proteins that are biopharmaceutical acceptable. Animal component-free medium refers to a medium in which components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free media, and nutrients are obtained from synthetic, plant, or microbial sources. In contrast, protein-free medium is defined as being substantially free of protein. Those skilled in the art will appreciate that the examples of media described above are illustrative and in no way limiting of the formulation of media suitable for use in the present invention, and there are many suitable media known and available to those skilled in the art.

[0178] The isolated pluripotent stem cell-derived hematopoietic lineage cells may have at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% NK lineage cells. In some embodiments, the isolated pluripotent stem cell-derived hematopoietic lineage cells have about 95% to about 100% NK cells. In some embodiments, the invention provides therapeutic compositions having purified NK cells, such as compositions having an isolated population of about 95% NK cells, for treating a subject in need of cell therapy.

[0179] In one embodiment, the combination cell therapy comprises an anti-CD38 therapeutic protein or peptide and a population of iNK cells derived from genomically engineered iPSCs, the derived NK cells expressing CD38 - / - In another embodiment, the combination cell therapy comprises an anti-CD38 therapeutic protein or peptide and a population of iNK cells derived from genomically modified iPSCs, the derived NK cells expressing CD38- / - In some embodiments, the combination cell therapy comprises daratumumab, isatuximab, or cyclophosphamide, and a population of NK cells derived from genomically engineered iPSCs, wherein the derived NK cells express CD38 - / - , hnCD16, and IL15RF.

[0180] As will be appreciated by those skilled in the art, both autologous and allogeneic hematopoietic lineage cells derived from iPSCs based on the methods and compositions herein can be used in cell therapy as described above. In the case of autologous transplantation, the isolated population of derived hematopoietic lineage cells is fully or partially HLA-matched with the patient. In another embodiment, the derived hematopoietic lineage cells are not HLA-matched with the subject, and the derived hematopoietic lineage cells are NK cells.

[0181] In some embodiments, the number of derived NK lineage cells in a therapeutic composition is at least 0.1 x 10 per dose. 5 Cells, at least 1 x 10 5 Cells, at least 5 x 10 5 Cells, at least 1 x 10 6 Cells, at least 5 x 10 6 Cells, at least 1 x 10 7 Cells, at least 5 x 10 7 Cells, at least 1 x 10 8 Cells, at least 3 x 10 8 Cells, at least 5 x 10 8 Cells, at least 1 x 10 9 Cells, at least 1.5 x 10 9 cells, or at least 5 × 10 9 In some embodiments, the number of derived NK lineage cells in the therapeutic composition is about 0.1 x 10 per dose. 5 cells ~ approx. 1×10 6 Cells, per dose, approximately 0.5 x 10 6 cells ~ approx. 1×10 7 Cells, per dose, approximately 0.5 x 10 7 cells ~ approx. 1×10 8 Cells, per dose, approximately 0.5 x 10 8 cells ~ approx. 1×109 Cells, per dose, approximately 1 x 10 9 Cells ~ approx. 5 x 10 9 Cells, per dose, approximately 0.5 x 10 9 cells ~ approx. 8 x 10 9 Cells, per dose, approximately 3 x 10 9 cells ~ approx. 3 x 10 10 cells, or any range in between. Generally, for a 60 kg patient, 1 x 10 8 Cells / dose is approximately 1.67 x 10 6 Converted to cells / kg.

[0182] In some embodiments, the number of derived hematopoietic lineage cells per dose administered to a patient is calculated using dose splitting, where the total number of cells administered is divided by the number of doses expected to be administered. In some embodiments, the number of cells per dose is the same for each dose (e.g., a 1:1:1 ratio for three doses). In other embodiments, the ratio of the number of cells per dose is different for two or more doses (e.g., a 2:1:1, 1:1:2, or 1:2:1 ratio for three doses).

[0183] In one embodiment, the number of derived hematopoietic lineage cells in the therapeutic composition is the number of immune cells in a fraction of blood or a single umbilical cord, or at least 0.1 x 10 5 At least 0.5 × 10 cells / kg body weight 5 Cells / kg body weight, at least 1 x 10 5 Cells / kg body weight, at least 5 × 10 5 Cells / kg body weight, at least 10 x 10 5 At least 0.75 x 10 cells / kg body weight 6 At least 1.25 x 10 cells / kg body weight 6 Cells / kg body weight, at least 1.5 x 10 6 Cells / kg body weight, at least 1.75 x 10 6 At least 2 x 10 cells / kg body weight 6 Cells / kg body weight, at least 2.5 x 10 6 Cells / kg body weight, at least 3 × 10 6 Cells / kg body weight, at least 4 x 106 Cells / kg body weight, at least 5 × 10 6 Cells / kg body weight, at least 10 x 10 6 Cells / kg body weight, at least 15 x 10 6 Cells / kg body weight, at least 20 x 10 6 Cells / kg body weight, at least 25 x 10 6 Cells / kg body weight, at least 30 x 10 6 cells / kg body weight, 1×10 8 cells / kg body weight, 5×10 8 cells / kg body weight or 1 x 10 9 cells / kg body weight.

[0184] In one embodiment, a dose of derived hematopoietic lineage cells is delivered to the subject. In an exemplary embodiment, the effective amount of cells provided to the subject is at least 2×10 6 Cells / kg, at least 3 x 10 6 Cells / kg, at least 4 x 10 6 Cells / kg, at least 5 × 10 6 Cells / kg, at least 6 × 10 6 Cells / kg, at least 7 × 10 6 Cells / kg, at least 8 x 10 6 Cells / kg, at least 9 × 10 6 cells / kg, or at least 10 x 10 6 cells / kg, or more cells / kg, including all intervening cell doses.

[0185] In another exemplary embodiment, the effective amount of cells provided to a subject is about 2×10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, or approximately 10 x 10 6 cells / kg, or more cells / kg, including all intervening cell doses.

[0186] In another exemplary embodiment, the effective amount of cells provided to a subject is about 2×10 6 cells / kg ~ approx. 10×10 6 cells / kg, approximately 3×10 6 cells / kg ~ approx. 10×10 6 cells / kg, approximately 4×10 6 cells / kg ~ approx. 10×10 6 cells / kg, approximately 5×10 6 cells / kg ~ approx. 10×10 6 cells / kg, 2×10 6 cells / kg~about 6×10 6 cells / kg, 2×10 6 cells / kg~about 7×10 6 cells / kg, 2×10 6 cells / kg ~ approx. 8×10 6 cells / kg, 3×10 6 cells / kg~about 6×10 6 cells / kg, 3×10 6 cells / kg~about 7×10 6 cells / kg, 3×10 6 cells / kg ~ approx. 8×10 6 cells / kg, 4×10 6 cells / kg~about 6×10 6 cells / kg, 4×10 6 cells / kg~about 7×10 6 cells / kg, 4×10 6 cells / kg ~ approx. 8×10 6 cells / kg, 5×10 6 cells / kg~about 6×10 6 cells / kg, 5×10 6 cells / kg~about 7×10 6 cells / kg, 5×10 6 cells / kg ~ approx. 8×10 6 cells / kg or 6 x 10 6 cells / kg ~ approx. 8×10 6 Cells / kg and includes all intervening cell doses.

[0187] In some embodiments, the therapeutic use of the derived hematopoietic lineage cells is a single dose treatment. In some embodiments, the therapeutic use of the derived hematopoietic lineage cells is a multiple dose treatment. In some embodiments, the multiple dose treatment is a dose every day, every 3rd day, every 5th day, every 7th day, every 10th day, every 15th day, every 20th day, every 25th day, every 30th day, every 35th day, every 40th day, every 45th day, or every 50th day, or any number of days in between. In some embodiments, the determination of the length and duration of the multiple dose treatment regimen is contingent on clinical observation of signs and / or symptoms (e.g., tumor size) of the disease or disorder being treated.

[0188] In some embodiments, the course of treatment may be repeated based on review of clinical data demonstrating evidence of clinical benefit. In some embodiments, patients whose disease has an objective response to iNK cell therapy and subsequently relapses or progresses may receive a second course of treatment.

[0189] Compositions comprising populations of derived hematopoietic lineage cells of the invention may be sterile, suitable for administration to a human patient, and may be administered immediately (i.e., without further treatment). In some embodiments, compositions comprising populations of derived hematopoietic lineage cells of the invention are frozen in bags or vials under specified conditions prior to administration, ready to be thawed at the site of treatment. A cell-based composition that is ready to be administered means that the composition does not require any further processing or manipulation prior to implantation or administration to a subject. In other embodiments, the invention provides isolated populations of derived hematopoietic lineage cells that are expanded and / or regulated prior to administration of one or more therapeutic agents. Therapeutic compositions comprising populations of iPSC-derived hematopoietic lineage cells disclosed herein may be administered individually or in combination with other suitable compounds by intravenous, intraperitoneal, enteral, or tracheal administration methods to affect a desired therapeutic goal. In some embodiments, an adoptive cell therapy product is included in a composition that is administered via intravenous infusion and / or at the site of outpatient settings. In some embodiments, the adoptive cell therapy product is included in a cryopreservation container and thawed at the site of administration.

[0190] Some variation in dosage, frequency, and protocol will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose, frequency, and protocol for the individual subject within the range deemed safe and effective. EXAMPLES

[0191] The following examples are offered by way of illustration and not by way of limitation.

[0192] Example 1 - Materials and Methods Adoptive cell therapy: The treatment regimen is based on administration of an adoptive cell therapy product including allogeneic iPSC-derived NK cell immunotherapy (i.e., iNK cell therapy) in which the iPSC-derived NK (iNK) cells lack CD38 and express CD16 and IL15RF. Also disclosed herein is a treatment regimen based on allogeneic iPSC-derived NK cells expressing CD16 without CD38 knockout or IL15RF expression. The iNK cells are suspended in an infusion medium containing albumin (human) and DMSO, provided in a cryopreservation bag, and thawed at the site of administration. The iNK cell therapy is administered as a gravity-fed IV infusion using an in-line filter.

[0193] Prior to administration of iNK cell therapy, subjects are premedicated with acetaminophen 650 mg orally and diphenhydramine 25-50 mg orally or IV prior to and 4 hours after administration. Corticosteroids are not used as premedications for iNK cell therapy. Dosing is based on CD16 expression. If 90% ± 10% of administered iNK cells express hnCD16, the starting dose for iNK cell monotherapy and combination with monoclonal antibodies is approximately 1 x 10 per dose. 8 Set the number of cells to be approximately 1 x 10 8 ~Approx. 1×10 10 The range of dose levels of cells is believed to be well tolerated as starting doses for allogeneic NK cell therapy. Thus, the planned dosing levels (DL) for NK cell therapy are: DL0: 5×10 7 Cells, DL1:1×108 Cells, DL2:3×10 8 Cells, DL3:1×10 9 Cells, and DL4: 1.5 × 10 9 cells, each of which is expected to be well tolerated, has no dose-dependent toxicity, and is suitable for repeated administration.

[0194] Lymphatic conditioning: The purpose of lymphatic conditioning prior to administration of iNK cell therapy is to promote homeostatic proliferation of iNK cells and to eliminate regulatory immune cells and other competing components of the immune system that compete for homeostatic cytokines. CY is administered at a dose of 500 mg / m per in-house standard treatment. 2 CY dosing is calculated based on actual body weight (ABW). If ABW is >150% of ideal body weight (IBW), dose is calculated using adjusted body weight as follows: adjusted body weight = IBW + 0.5 (ABW-IBW). FLU is 30 mg / m per institutional standard. 2 CY and FLU are administered as an IV infusion at a dose of 0.5 mg / kg / day. The duration between the last dose of FLU and the infusion of iNK cell therapy is usually 40-84 hours. In some cases, iNK cell therapy may be administered after the 84 hour time point. Dose adjustments for body weight / creatinine follow institutional guidelines. Due to their detrimental effects on iNK cell-based therapy, corticosteroids as pre-medications for CY and FLU should be avoided unless deemed necessary by the investigator and should not be administered within 24 hours before or after iNK cell therapy administration.

[0195] One iNK cell therapy product described herein includes a CD38 knockout. The dosing scheme of anti-CD38 mAb exemplified in combination therapy with this iNK cell product (e.g., QW doses on days prior to iNK cell infusion in Table 2) provides lymphodepletion in which activated lymphocytes are eliminated for their upregulated CD38. However, sufficient lymphodepletion with anti-CD38 mAb may provide an alternative conditioning process for the present iNK cell therapy without or at a minimum, a CY / FLU-based lymphoid conditioning procedure, as further described herein. Thus, in various embodiments in which the course of treatment includes administering an anti-CD38 monoclonal antibody to a subject, the method (i) does not require CY / FLU-based lymphoid conditioning, or (ii) at a minimum, requires CY / FLU-based lymphoid conditioning. As used herein, the term "minimally necessary" in this context refers to (i) a substantially lower dose that is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the dose used without anti-CD38 lymphodepletion, and / or (ii) one or two fewer administrations of the same or lower dose compared to three consecutive administrations of CY / FLU-based lymphatic conditioning.

[0196] Daratumumab: Daratumumab (Darzalex) is an anti-cancer drug that binds to CD38, a surface protein overexpressed in multiple myeloma cells. iNK cells engineered with enhanced CD16 potency and CD38 depletion are resistant to CD38-targeted antibody-induced fratricide and more potently mediate anti-myeloma activity in combination with daratumumab. Daratumumab is administered by IV infusion at a dose of approximately 16 mg / kg actual body weight on a prescribed schedule, for example, starting on day -11, then once weekly (QW) for a total of 8 doses, then every 2 weeks (Q2W) for a total of 8 doses, then every 4 weeks (Q4W) until disease progression or unacceptable toxicity.

[0197] Because infusion-related reactions have been observed with daratumumab, pre- and post-infusion medications may be given according to the general guidelines provided in Table 1 to minimize the potential effects of corticosteroids on NK cell function. Antiviral prophylaxis is initiated within 1 week of initiating daratumumab to prevent reactivation of herpes zoster and continued for 3 months after treatment. Additional modifications to the prophylaxis of infusion-related reactions may be made based on case evaluation. In all cases, long-acting corticosteroids, including dexamethasone, are not administered.

[0198] [Table 7]

[0199] Elotuzumab (Combination Therapy 2): Elotuzumab is administered by IV infusion at a dose of approximately 10 mg / kg starting on day -11, then QW for a total of 8 doses, then Q2W until disease progression or unacceptable toxicity.

[0200] Because infusion-related reactions have been observed with elotuzumab, pre- and post-infusion medications may be given according to the general guidelines provided in Table 2 to minimize the potential impact of corticosteroids on iNK cell function. Additional modifications to the prophylaxis of infusion-related reactions can be made based on case evaluation. Antiviral prophylaxis is recommended to be initiated within 1 week of starting elotuzumab to prevent reactivation of herpes zoster and continued for 3 months after treatment. In all cases, long-acting corticosteroids such as dexamethasone are not administered.

[0201] [Table 8]

[0202] Permitted Treatments: Throughout the study, the investigator may prescribe any concomitant medications or treatments deemed necessary to provide adequate supportive care. Supportive care may include antibiotics, checkpoint inhibitors, analgesics, transfusions, growth factors, etc. Only irradiated blood products should be used to minimize the risk of transfusion-associated GvHD. Biologically, radiation therapy may create a more immunogenic microenvironment that enhances iNK cell antitumor activity. Subjects may receive palliative radiation therapy at any time and on a schedule at the investigator's discretion, provided that the palliative radiation therapy schedule does not interfere with the protocol-specified activity schedule.

[0203] Caution in therapy: To avoid inhibition of NK cell function, systemic corticosteroids are avoided during treatment cycles unless absolutely required.

[0204] Prohibited Therapies: Any anti-neoplastic agent for therapeutic purposes other than the protocol-indicated study treatment is prohibited, with the following exceptions: (i) any prior therapy leading to administration of the study treatment described in the inclusion / exclusion criteria, (ii) anti-cancer therapy administered due to disease progression after the iNK cell therapy treatment period, or (c) palliative radiation therapy.

[0205] Exploratory Analysis: Perform exploratory analysis of potential predictive and prognostic biomarkers related to the mechanism of action of iNK cell therapy and underlying disease immunobiology. Biomarkers that correlate with clinical outcomes can be used to correlate with various indications and patient subpopulations. Immune-related biomarkers, such as but not limited to cytokines, iNK cell PK, and T cell numbers and function, pharmacodynamic biomarkers, and any potential correlations with dose-dependent safety and anti-tumor activity in peripheral blood and within tumors, will provide evidence of biological activity and efficacy of iNK cell therapy.

[0206] Sample Collection and Analysis: Tumor samples, including but not limited to bone marrow biopsies and aspirates, along with peripheral blood sampling, are obtained from subjects prior to treatment, after initial treatment with iNK cell therapy, and at later time points, including but not limited to, at the time of disease progression or recurrence. Peripheral blood samples are collected before and after cell infusion on the day cells are administered to characterize the pharmacokinetics (PK) of the iNK cell therapy. iNK cell quantification can also be performed on tumor samples. These samples are useful to assess the ability of iNK cell therapy to infiltrate tumor sites, minimal residual disease (MRD) status, changes to the tumor microenvironment, and potential mechanisms of iNK cell resistance.

[0207] Collected peripheral blood samples are used to measure cytokine release syndrome (CRS) cytokines prior to iNK cell therapy administration and if CRS is clinically suspected. In addition to CRS, C-reactive protein (CRP) and ferritin are also tested. Collected peripheral blood samples are also used to detect alloimmunization against iNK cell product human leukocyte antigen (HLA) by panel reactive antibodies and by evaluation of T cell function, thereby evaluating the immunogenicity of iNK cell therapy. In addition, collected peripheral blood and / or serum samples are used for HLA and killer-cell immunoglobulin-like receptor (KIR) typing, as well as exploratory biomarker analysis, including but not limited to measurement of protein biomarkers of disease progression and / or response to therapy, mAb PK, if applicable, and circulating tumor DNA / profiling.

[0208] Exploratory biomarker analyses are further performed on bone marrow biopsy / aspiration samples obtained while the subject is on study, including, but not limited to, MRD status by flow cytometry and / or DNA sequencing and clonal differentiation, tumor somatic mutational profiling, e.g., tumor mutational burden, microsatellite instability, and / or ploidy analysis, tumor microenvironment characterization, e.g., tumor infiltrating T cell characterization, tumor infiltrating innate immune cell characterization, and / or immune inhibitory molecule expression by tumor cells, and pre-treatment gene polymorphism and expression panels to explore correlations between alterations and outcome.

[0209] In addition, collected peripheral blood and / or serum samples are used to monitor one or more of the following: peripheral blood mononuclear cell (PBMC) functional characterization and immunophenotyping, e.g., T cell subset analysis and determination of T-reg frequencies, non-diagnostic exploratory MRD assessment, and peripheral blood immune cell functional assays.

[0210] Various assays for exploratory analysis include, but are not limited to, analysis of lymphocytes, T cell activation, T cell receptor repertoire, cytokines associated with inflammation, circulating tumor DNA or MRD, cell of origin, and genes or gene signatures associated with tumor immunobiology. Exploratory analysis may include extraction of DNA, cell-free DNA, or RNA, analysis of mutations, single nucleotide polymorphisms, and other genomic variants, and genomic profiling using next generation sequencing of a comprehensive panel of genes.

[0211] Post-treatment follow-up visits will continue for up to 15 years after initiation of study treatment administration or until withdrawal of consent, whichever occurs first. Follow-up activities after iNK cell therapy treatment and before disease recurrence or progression will include clinical and laboratory evaluations, as well as disease response assessments.

[0212] Example 2 - Selection of Study Patients Subjects are enrolled in two phases, a dose escalation phase and a dose expansion phase. After evaluating safety and tolerability and defining the maximum tolerated dose (MTD) of the dose escalation phase (or through the highest evaluated dose in the absence of dose-limiting toxicity (DLT) that defines the MTD), the dose expansion phase is further evaluated for the safety and activity of the adoptive cell therapy. Patient recruitment is performed by considering the following important inclusion / exclusion criteria for the purpose of clinical trials that generate data that clearly demonstrate the clinical benefit of the therapy without potential confounding by residual therapeutic effects of previous therapies. As understood in the art, the inclusion / exclusion criteria for clinical trial subject enrollment are not intended to set limits on the label that is subsequently approved for the therapy.

[0213] Inclusion Criteria - Combination Therapy 1 - Multiple Myeloma (MM) Patients must have a diagnosis of MM that has relapsed or progressed after at least two lines of therapy, including proteasome inhibitors (e.g., bortezomib, carfilzomib, or ixazomib), immunomodulatory agents (e.g., thalidomide, lenalidomide, or pomalidomide), anti-CD38 mAb therapy (e.g., daratumumab or isatuximab), and CAR-T therapy. Planned sequential therapy (e.g., induction therapy followed by stem cell transplant (SCT)) is considered one line of therapy. Patients must have measurable disease defined by at least one of the following: (i) serum M-protein ≥ 1.0 g / dL, ii) urinary M-protein ≥ 200 mg / 24 hr, and (iii) serum free light chain (FLC) level ≥ 10 mg / dL if serum M-protein < 1.0 g / dL and urinary M-protein < 200 mg / 24 hr.

[0214] Exclusion criteria specific to combination therapy for multiple myeloma (MM) Patients on any of the combination therapy regimens who meet any of the following criteria will be excluded: (a) plasma cell count ≥ 2000 / mm 3(b) plasma cell leukemia defined as >100%; (c) leptomeningeal involvement of MM; (d) any nonpalliative biologic therapy, chemotherapy, or radiation therapy within 2 weeks or 5 half-lives prior to Day 1, whichever is shorter, or any investigational therapy within 28 days prior to the first dose of monoclonal antibody (mAb); or (e) allergy or hypersensitivity to antibodies or antibody-related proteins.

[0215] Example 3 - Phase I Study of FT576 as Monotherapy and in Combination with Daratumumab in Subjects with Relapsed / Refractory Multiple Myeloma FT576 is a multiply engineered NK cell therapy generated from a clonal master engineered induced pluripotent stem cell (iPSC) line that can be used as a renewable source for mass production of NK cells of uniform composition for off-the-shelf availability and broad patient access. FT576 is engineered in four modalities to combine multifaceted innate immunity with multi-antigen targeting capabilities: (1) high affinity 158V, a non-cleavable CD16 (hnCD16) Fc receptor for enhanced antibody-dependent cellular cytotoxicity (ADCC), (2) IL-15 / IL-15 receptor fusion to promote NK cell persistence, (3) CD38 knockout to reduce NK cell fratricide with CD38-directed monoclonal antibody (mAb) and promote higher glycolytic rates with improved metabolic fitness and resistance to oxidative stress found in the tumor microenvironment, and (4) BCMA-directed CAR to target clonal plasma cells. These modalities are designed to enhance the potency and durability of FT576 and enable multi-antigen targeting. As used herein, a BCMA-directed CAR comprises an Ig kappa chain variable leader peptide, a BCMA scFV, a human IgG4 Fc, a transmembrane domain derived from NKG2D, a costimulatory domain derived from 2B4, and a signaling domain comprising native or modified ITAM1 (immunoreceptor tyrosine-based motif of activation) of CD3ζ, the amino acid sequence of such structure being at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO:7.In certain embodiments, the BCMA scFv is characterized in that the antigen binding domain comprises a variable heavy chain (VH), the VH comprising a heavy chain complementarity determining region 1 (H-CDR1) having at least 80% sequence identity (e.g., 90% or 100%) to SEQ ID NO:8 (GFTFSRYW), a heavy chain complementarity determining region 2 (H-CDR2) having at least 80% sequence identity (e.g., 90% or 100%) to SEQ ID NO:9 (INPSSSTI), and a heavy chain complementarity determining region 3 (H-CDR4) having at least 80% sequence identity (e.g., 90% or 100%) to SEQ ID NO:10 (ASLYYDYGDAYDY). In some embodiments, the BCMA-directed CAR comprises an amino acid sequence of SEQ ID NO:7, a light chain complementarity determining region 1 (L-CDR1) having at least 80% (e.g., 90% or 100%) sequence identity with SEQ ID NO:11 (QSVESN), a light chain complementarity determining region 2 (L-CDR2) having at least 80% (e.g., 90% or 100%) sequence identity with SEQ ID NO:12 (SAS), and a light chain complementarity determining region 3 (L-CDR3) having at least 80% (e.g., 90% or 100%) sequence identity with SEQ ID NO:13 (QQYNNYPLT). In some embodiments, the BCMA-directed CAR comprises the amino acid sequence of SEQ ID NO:7.

[0216] [Table 9]

[0217] Multiple myeloma (MM) is a cancer formed in a type of white blood cell called plasma cells. Cancerous plasma cells accumulate in the bone marrow and crowd out healthy blood cells, causing complications. This study evaluates the clinical activity of iNK cells in combination with monoclonal antibodies (mAbs) in subjects with advanced hematological malignancies, particularly r / r MM, which remains an area of ​​significant unmet medical need and is potentially amenable to NK cell-based therapy. ADCC, resulting from engagement of the Fc portion of the mAb with CD16 on NK cells, is the primary mechanism of action contributing to the clinical efficacy of currently approved mAbs targeting CD38 (e.g., Daratumumab, Darzalex USPI) and SLAMF7 (e.g., Elotuzumab, Empliciti USPI) in MM. Given that NK cells utilize ADCC as a mechanism of antitumor activity and express hnCD16 to enhance ADCC, the iNK cell therapy is used in combination with mAbs as a way to enhance ADCC.

[0218] Study Design and Methods: This is a multicenter Phase I clinical trial of FT576 in patients with R / R MM. The primary objectives are to determine the recommended Phase II dose of FT576 given as a single or divided dose as monotherapy and in combination with daratumumab in R / R MM, and to evaluate safety and tolerability. Key secondary objectives include antitumor activity and pharmacokinetics as monotherapy and in combination with daratumumab in R / R MM. Exploratory objectives include characterization of FT576 pharmacodynamics, assessment of minimal residual disease, and characterization of the tumor microenvironment pre- and post-treatment.

[0219] The dose escalation phase of the trial has four arms (Table 3): FT576 monotherapy with a single dose on day 1 (regimen A), FT576 monotherapy with multiple doses on days 1 and 15 (regimen A1), FT576 + daratumumab with a single dose on day 1 (regimen B), and FT576 + daratumumab with multiple doses on days 1 and 15 (regimen B1). Up to five dose levels of FT576 ranging from 100 million to 1.5 billion cells per dose will be tested using a modified toxicity probability interval (mTPI) dose escalation design. Daratumumab will be administered according to the approved dose and schedule. Conditioning chemotherapy consisting of fludarabine and cyclophosphamide for 3 consecutive days will be administered prior to the first dose of FT576. Key inclusion criteria include R / R disease and measurable disease following standard approved therapy. Previous BCMA and anti-CD38 targeted therapies, including CAR T cells, are permitted.

[0220] [Table 10]

[0221] The dose escalation phase will determine the appropriate dose level of adoptive cell therapy that does not exceed the maximum tolerated dose (MTD) for further evaluation, either as monotherapy or in combination with monoclonal antibodies (mAbs), and will determine the recommended dose to use in combination with each of the mAbs tested in the dose expansion.

[0222] Dose escalation will proceed independently between iNK cell monotherapy and combination therapy to identify the MTD / MAD of each of the treatment regimens. Initiation of combination 1 (iNK cells + daratumumab) will be contingent on clearance of the first iNK cell monotherapy dose escalation cohort at a dose level corresponding to the dose level of the cleared iNK cell monotherapy dose escalation cohort. Dose escalation will proceed independently between iNK cell monotherapy and the combination through DL2 dose escalation, at which point dose escalation will be paused to consider safety, preliminary efficacy, and iNK cell PK data.

[0223] Dose-limiting toxicity (DLT) was defined as any adverse event (AE) at least potentially related to iNK cell therapy occurring after the first iNK cell infusion through the end of the DLT evaluation period on day 29, with the extension of the DLT evaluation period beyond day 29 to allow for AE recovery, as defined below, that meets one of the following criteria based on the National Cancer Institute Common Terminology Criteria for Adverse Events Version 5.0 (NCI CTCAE, v5.0) or the American Society for Transplantation and Cellular Therapy (ASTCT) Consensus Grading Guidelines for Cytokine Release Syndrome and Neurological Toxicity Associated with Immune Effector Cells (Lee et al. 2019): Acute GvHD grading was based on the Center for International Blood and Marrow Transplant Research (CIBMTR) Acute GvHD scoring scale. Grading of experimental AEs will be assessed relative to baseline laboratory values, defined as the last assessment prior to the start of protocol-defined study drug. Use Table 4 to assess the severity of AEs not listed in the NCI CTCAE, v5.0.

[0224] [Table 11]

[0225] For all patients, DLTs include, but are not limited to, any non-hematological AE of grade 4 or greater (e.g., grade 4 infusion-related reactions), grade 3 pulmonary or cardiac AEs of any duration, any non-hematological AE of grade 3 (except grade 3 pulmonary or cardiac toxicity of greater than 72 hours duration), grade 3 immune cell associated neurotoxicity syndrome (ICANS) of any duration, and any grade 2 or greater acute GvHD that requires systemic steroid administration and does not resolve to grade 1 or less within 7 days. However, fever associated with cytokine release syndrome (CRS) occurring in the setting of CRS less than grade 3 is not considered a DLT. Additional exceptions that are not considered DLTs include grade 3 renal or hepatic AEs lasting less than 7 days, grade 3 laboratory abnormalities that are asymptomatic and not clinically significant as determined by the treating investigator unless otherwise specified, and grade 3 fatigue lasting 3 days or less.

[0226] For patients receiving iNK cell monotherapy, hematologic AEs included prolonged myelosuppression up to 42 days after administration of the first dose of iNK cell therapy, defined as grade 4 neutropenia refractory to granulocyte-colony stimulating factor (G-CSF), in the absence of morphologic evidence of acute leukemia, assessed by evidence of hematopoietic recovery defined by circulating blasts in peripheral blood or leukemic blasts in bone marrow biopsy, and / or greater than 5% cellularity in the bone marrow.

[0227] For patients receiving combination therapy, hematological AEs include any Grade 3 or higher hematological AEs. The following are exceptions and therefore not considered DLTs: (i) Grade 3-4 neutropenia refractory to G-CSF in the absence of documented infection that improves to Grade 2 or lower or 80% or lower of baseline, whichever is lower, within 21 days of neutrophil count nadir, (ii) Grade 3-4 anemia in the absence of ongoing red blood cell transfusions and association with clinical signs and symptoms that improves to Grade 2 or lower or 80% or higher of baseline, whichever is lower, within 21 days of hemoglobin concentration nadir, and (iii) Grade 3-4 thrombocytopenia in the absence of Grade 2 or higher bleeding that improves to Grade 2 or lower or 80% or higher of baseline, whichever is lower, within 21 days of platelet count nadir.

[0228] Example 4 - Dose Expansion Phase The purpose of dose expansion is to further evaluate the safety and tolerability of adoptive cell therapy as monotherapy in subjects with r / r MM (relapsed / refractory multiple myeloma) and to identify clinical activity signals to guide and support future development. Enrollment in dose expansion will occur independently between regimens (1 or 2 doses of iNK cell monotherapy, and 1 or 2 doses of combination therapy).

[0229] The dose of adoptive cell therapy for dose expansion with each of the three regimens will be determined based on clinical and available pharmacokinetic (PK) and pharmacodynamic (PD) data from dose escalation and will not exceed the MTD or MAD for that regimen. Enrollment into the dose expansion cohorts can begin after a given dose level clears dose escalation. For each regimen, two or more dose expansion cohorts of subjects can be opened to (i) more fully characterize safety / tolerability at a given dose level, including evaluation of alternative doses and schedules of adoptive cell therapy to optimize safety and tolerability, provided that the dose does not exceed the MTD or MAD, and (ii) more fully characterize clinical activity in a particular indication or patient population defined by a particular regimen.

[0230] For subjects with r / r MM, disease response will be assessed through analysis of bone marrow biopsy / aspirate and peripheral blood for enumeration of leukemic blasts and classified into the best of the following response categories using the criteria shown in Table 5: stringent complete response (sCR), complete response (CR), very good partial response (VGPR), partial response (PR), minimal response (MR), stable disease (SD), or progressive disease (PD).

[0231] [Table 12-1]

[0232] [Table 12-2]

[0233] ASCT, autologous stem cell transplant; CR, complete response; CRAB trait; elevated calcium; renal failure; anemia; lytic bone lesions; CT, computed tomography (scan); 18 F-FDG-PET, 18 F-fluorodeoxyglucose-PET (scan), FCM, flow cytometry, FLC, free light chain, IMWG, International Myeloma Working Group, M-protein, myeloma protein, MFC, multiparameter flow cytometry, MR, minimal response, MRD, minimal residual disease, MRI, magnetic resonance imaging (scan), NGF, next generation flow, NGS, next generation sequencing, PD, progressive disease, PET / CT, positron emission tomography / computed tomography (scan), PR, partial response, sCR, stringent complete response, SD, stable disease, SPD, sum of the products of the greatest perpendicular diameters of measured lesions, SUV max, maximum standardized uptake value; VGPR, very good partial response.

[0234] As part of best overall response, the secondary endpoint of ORR will be used to summarize the proportion of subjects achieving a PR or better response by regimen and dose level. In addition, exact 95% CIs will be determined. For subjects with sCR or CR responses, the proportion of subjects with MRD-negative responses, subjects with MRD-negative responses lasting at least 1 year, and subjects with MRD-negative responses by imaging based on IMWG MRD criteria will be summarized descriptively with 95% CIs by regimen and dose level as exploratory endpoints. Time-to-event endpoints include duration of response (DOR), PFS, RFS from CR, OS as secondary endpoints, and time to MRD-negative response, and RFS from MRD-negative response as exploratory endpoints.

[0235] Because evidence of clinical benefit has been demonstrated, additional treatment cycles consisting of lymphoid conditioning followed by adoptive cellular therapy following the same schedule as the first treatment cycle may be considered based on review of the clinical data demonstrating evidence of clinical benefit.

[0236] Thus, in some embodiments, a course of treatment comprises (i) administration of one cycle of an adoptive cell therapy product, (ii) administration of one, two, or three cycles of an adoptive cell therapy product, each cycle comprising one or two doses at a dose frequency of one dose per 15 days, or (iii) administration of more than three cycles of an adoptive cell therapy product over an extended period of time at a dose frequency based on clinical evaluation of disease response as set forth above. In some embodiments, in the context of a treatment regimen comprising multiple doses of a cell therapy product, a treatment cycle is said to be completed after each dose of cells has been infused at a predetermined time point for a predetermined duration, and in some embodiments, two or more such treatment cycles may be required, as medically determined.

[0237] As of data cutoff in July 2022, nine patients with R / R MM were treated and evaluable for safety and efficacy at the first two dose levels of regimen A (multiple-dose monotherapy, n=6) and at the first dose level of regimen B (single dose + anti-CD38, n=3). No dose-limiting toxicities or events of any grade of cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS) or graft-versus-host disease (GvHD) were observed. There is evidence of anti-myeloma activity according to IMWG 2016 based on initial data (Tables 6-8).

[0238] [Table 13]

[0239] [Table 14]

[0240] [Table 15]

[0241] sCR: stringent complete response, CR: complete response, VGPR: very good partial response, PR: partial response, MR: minimal response, SD: stable disease, or PD: progressive disease.

[0242] Example 5 - Retreatment after progression Subjects whose disease has an objective response to iNK cell therapy and subsequently relapses or progresses because evidence of clinical benefit has been demonstrated are eligible to receive a second course of treatment. Objective responses achieved with a second course of treatment with iNK cell therapy plus mAb support a longer duration of treatment to achieve a deeper response that may drive, for example, a longer duration of clinical benefit and / or the incorporation of iNK cell therapy with mAb combination retreatment as part of a standard dosing schedule. To understand potential mechanisms of resistance to treatment with iNK cell therapy plus mAb, treatment-emerged changes in the tumor microenvironment are characterized. Evidence of clinical benefit includes, but is not limited to, (i) the absence of signs and symptoms, including worsening laboratory values, indicative of overt disease progression, (ii) no decline in Eastern Cooperative Oncology Group (ECOG) activity indices, and / or (iii) no clear evidence of progressive disease. Exceptions may be made if pseudoprogression due to influx of immune cells to the tumor site is suspected, provided there is an absence of signs and symptoms indicating overt clinical disease progression (including laboratory deterioration) and an absence of tumor progression at critical anatomical sites where organ dysfunction may increase the acute risk of severe and / or irreversible disability or death.

[0243] Those skilled in the art will readily appreciate that the methods, compositions, and products described herein are representative of exemplary embodiments and are not intended as limitations on the scope of the invention. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the disclosure disclosed herein without departing from the scope and spirit of the invention.

[0244] All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0245] The present disclosure illustratively described herein can be practiced without any element or elements, limitations, or limitations not specifically disclosed herein. Thus, for example, in each example herein, any of the terms "comprise", "consist essentially of" and "consist of" can be replaced with any of the other two terms. The terms and expressions used are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents of the features shown and described or portions thereof, but rather, it is recognized that various modifications are possible within the scope of the disclosure as claimed. Thus, although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may occur to those skilled in the art, and such modifications and variations should be understood to be within the scope of the present invention as defined by the appended claims.

Claims

1. 1. A composition comprising engineered natural killer (NK) lineage cells for use in treating a hematological cancer in a subject, comprising: (i) the engineered NK lineage cells comprise exogenous CD16 expression, IL15RF expression, CD38 knockout, and a BCMA-directed CAR (chimeric antigen receptor); (ii) said use comprises a course of treatment comprising at least a first cycle of an adoptive cell therapy product comprising said engineered NK lineage cells, said first cycle comprising one or more doses of said adoptive cell therapy product at a preselected frequency in a first effective amount, with the option of one or more additional cycles having one or more doses in a second effective amount over a period of time; (iii) the first and second effective amounts are the same or different; and (iv) the hematological cancer comprises multiple myeloma (MM) or relapsed or refractory MM (r / r MM); and optionally, the subject is being treated with one or more therapies for MM.

2. 2. The composition for use of claim 1, wherein the engineered NK lineage cells are derived from engineered induced pluripotent stem cells (iPSCs) comprising a polynucleotide encoding exogenous CD16, IL15RF, a CD38 knockout, and a polynucleotide encoding a BCMA-directed CAR.

3. the course of treatment further comprises an initial dose of a monoclonal antibody provided in an effective amount at a starting time prior to the first cycle of the adoptive cell therapy product, wherein the monoclonal antibody is an anti-CD38 monoclonal antibody or an anti-SLAMF7 monoclonal antibody. A composition for use according to claim 1.

4. A composition for use as described in claim 3, wherein the initiation time of the monoclonal antibody is approximately 8 to 12 days before the first cycle of the adoptive cell therapy product, and the initial dose of the monoclonal antibody comprises 6 to 10 weekly (QW) doses, optionally followed by 6 to 10 biweekly (Q2W ± 1 day) doses.

5. 5. The composition for use of claim 4, wherein the anti-CD38 monoclonal antibody comprises daratumumab; and the anti-SLAMF7 monoclonal antibody comprises elotuzumab.

6. 2. The composition for use of claim 1, wherein the course of treatment further comprises at least one daily dose of one or more chemotherapeutic agents prior to the first cycle of the adoptive cell therapy product, and wherein the duration between administration of the last daily dose of the one or more chemotherapeutic agents and the first cycle of the adoptive cell therapy product comprises a specified period of time.

7. The one or more chemotherapeutic agents include cyclophosphamide (CY) and fludarabine (FLU), and optionally, the CY and FLU are administered daily for three consecutive days, or the dose of CY is about 500 mg / m ２ and the dose of FLU is about 30 mg / m ２ 7. The composition for use according to claim 6, wherein

8. The composition for use described in claim 6, wherein the duration is (i) about 40 to 84 hours; or (ii) about 3 days.

9. A composition for use as described in claim 3, wherein the therapeutic course includes an anti-CD38 monoclonal antibody, and optionally, the therapeutic course does not include lymphatic conditioning.

10. The composition for use of claim 1, wherein the effective amount of the adoptive cell therapy product is from about 5 x 10 7 cells to about 3 x 10 9 cells. (i) the daratumumab is in an effective amount of about 15 mg / kg to about 17 mg / kg; and (ii) the elotuzumab is in an effective amount of about 9 mg / kg to about 11 mg / kg; A composition for use according to claim 5.

12. The treatment process comprises: (i) one cycle of said adoptive cell therapy product over about 29 days at one or two doses per cycle; (ii) two or more cycles of said adoptive cell therapy product, each cycle comprising one or two doses; (iii) one or more cycles of the adoptive cell therapy product are administered over an extended period based on clinical assessment of disease response, with a single dose per cycle lasting about 29 days; or (iv) one or more cycles of said adoptive cell therapy product are administered over an extended period based on clinical assessment of disease response, at two doses per cycle over a period of about 29 days; 2. The composition for use according to claim 1, comprising:

13. The composition (i) formulated for intravenous infusion, or (ii) frozen. A composition for use according to any one of claims 1 to 12, which is stored and then thawed before use.

14. A composition for use according to any one of claims 1 to 12, wherein the composition is formulated without the need for IL2 cytokine support during the course of treatment.

15. The BCMA-directed CAR is (i) a variable heavy chain (VH) and a variable light chain (VL), (a) the VH comprises a heavy chain complementarity determining region 1 (H-CDR1) having at least 80% sequence identity to SEQ ID NO: 8 (GFTFSRYW), a heavy chain complementarity determining region 2 (H-CDR2) having at least 80% sequence identity to SEQ ID NO: 9 (INPSSSTI), and a heavy chain complementarity determining region 3 (H-CDR3) having at least 80% sequence identity to SEQ ID NO: 10 (ASLYYDYGDAYDY); (b) a variable light chain (VL), a variable heavy chain (VH), and a variable light chain (VL), wherein the VL comprises a light chain complementarity determining region 1 (L-CDR1) having at least 80% sequence identity to SEQ ID NO: 11 (QSVESN), a light chain complementarity determining region 2 (L-CDR2) having at least 80% sequence identity to SEQ ID NO: 12 (SAS), and a light chain complementarity determining region 3 (L-CDR3) having at least 80% sequence identity to SEQ ID NO: 13 (QQYNNYPLT); or (ii) an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to SEQ ID NO:7; A composition for use according to any one of claims 1 to 12, comprising:

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