WO2024102704A2

WO2024102704A2 - Ipsc-derived nk cell targeting mica/b for solid tumor treatment

Info

Publication number: WO2024102704A2
Application number: PCT/US2023/078904
Authority: WO
Inventors: Bahram Valamehr; Yu-Waye CHU
Original assignee: Fate Therapeutics, Inc.
Priority date: 2022-11-07
Filing date: 2023-11-07
Publication date: 2024-05-16

Abstract

Provided are methods and compositions for use in cancer immunotherapies. In various embodiments, the compositions include functionally enhanced derivative effector cells obtained from directed differentiation of genomically engineered iPSCs. In various embodiments, the derivative cells provided herein have stable and functional genome editing that delivers improved or enhanced therapeutic effects. Also provided are therapeutic compositions and the use thereof comprising the functionally enhanced derivative effector cells alone, or with antibodies or checkpoint inhibitors in combination therapies.

Description

iPSC-DERIVED NK CELL TARGETING MICA/B FOR SOLID TUMOR TREATMENT

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63/382,719, filed on November 7, 2022, the disclosure of which is hereby incorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The Sequence Listing titled 184143-650601_SL.xml, which was created on November 6, 2023 and is 15,634 bytes in size, is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present disclosure is broadly concerned with the field of off-the-shelf immunocellular products. More particularly, the present disclosure is concerned with 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-sourced cell therapies.

BACKGROUND OF THE INVENTION

[0004] The field of adoptive cell therapy is currently focused on using patient- and donor- sourced cells, which makes it particularly difficult to achieve consistent manufacturing of cancer immunotherapies and to deliver therapies to all patients who may benefit. There is also the need to improve the efficacy and persistence of adoptively transferred lymphocytes to promote favorable patient outcomes. Lymphocytes such as T cells and natural killer (NK) cells are potent anti-tumor effectors that play an important role in innate and adaptive immunity. However, the use of these immune cells for adoptive cell therapies remains challenging and has unmet needs for improvement. Therefore, significant opportunities remain to harness the full potential of T 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 issues ranging from response rate, cell exhaustion, loss of transfused cells (survival and/or persistence), tumor escape through target loss or lineage switch, tumor targeting precision, off-target toxicity, off- tumor effect, to efficacy against solid tumors, i.e., tumor microenvironment and related immune suppression, recruiting, trafficking and infiltration. [0006] It is an object of embodiments of the present invention to provide methods and compositions for adoptive cell therapy, wherein the adoptive cell therapy includes administering an adoptive cell therapy product generated from derivative non-pluripotent cells differentiated from a single cell derived iPSC (induced pluripotent stem cell) clonal line, which iPSC line comprises one or several genetic modifications in its genome. Said one or several genetic modifications include, in some embodiments, one or more of DNA insertion, deletion, and substitution, and which modifications are retained and remain functional in subsequently derived cells after differentiation, expansion, passaging and/or transplantation.

[0007] The iPSC derived non-pluripotent cells of the present application include, but are not limited to, CD34⁺ cells, hemogenic endothelium cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, and B cells. The iPSC-derived non-pluripotent cells of the present application comprise one or several genetic modifications in their genome through differentiation from an iPSC comprising the same genetic modifications. In some embodiments, the engineered clonal iPSC differentiation strategy for obtaining genetically engineered derivative cells benefits from a developmental potential of the iPSC in a directed differentiation that is not significantly adversely impacted by the engineered modality in the iPSC, and also that the engineered modality functions as intended in the derivative cell. Further, this strategy overcomes the present barrier in engineering primary lymphocytes, such as T cells or NK cells obtained from peripheral blood, as such cells are difficult to engineer, with engineering of such cells often lacking reproducibility and uniformity, resulting in cells exhibiting poor cell persistence with high cell death and low cell expansion. Moreover, this strategy avoids production of a heterogenous effector cell population otherwise obtained using primary cell sources which are heterogenous to start with.

[0008] Accordingly, in one aspect, the present invention provides a method of treating a subject having a solid tumor, the method comprising: administering to the subject at least a first cycle of an adoptive cell therapy product, with the first cycle comprising one or more doses of the adoptive cell therapy product administered in a first effective amount at a preselected frequency, and with an option of administering one or more additional cycles with one or more doses in a second effective amount, during a course of treatment over a period of time; wherein the first and the second effective amounts are the same or different; and wherein the product comprises an engineered natural killer (NK) lineage cell comprising MICA/B-CAR (chimeric antigen receptor) expression, exogenous CD 16 expression, IL15RF expression and CD38 knockout. In various embodiments of the method of treating, the solid tumor comprises cancer cells expressing: (i) MICA/B; (ii) PD-L1; (iii) HER2; (iv) EGFR; and/or (v) both EGFR and MET. In various embodiments, the cancer cells are comprised in at least one of the following cancers and relapsed or refractor forms thereof: (i) breast cancer (BC); (ii) advanced or metastatic colorectal cancer (CRC); (iii) advanced or metastatic non-small cell lung cancer (NSCLC); (iv) gastric cancer or gastroesophageal adenocarcinoma; (v) ovarian cancer; (vi) pancreatic cancer; (vii) head and neck cancer; and (viii) urothelial carcinoma (UC).

[0009] In various embodiments of the method of treating, the course of treatment further comprises administering to the subject an effective amount of a selected therapeutic monoclonal antibody (mAb). In some embodiments, the therapeutic mAb comprises an anti-EGFR antibody, an anti-HER2 antibody, an anti-PD-Ll antibody, or a bi-specific antibody targeting EGFR and MET. In some embodiments, the anti-EGFR antibody comprises cetuximab; wherein the anti- HER2 antibody comprises trastuzumab or biosimilars; wherein the anti-PD-Ll antibody comprises avelumab; or wherein the bi-specific antibody targeting EGFR and MET comprises amivantamab.

[00010] In various embodiments of the method of treating, the course of treatment further comprises administering to the subject an effective amount of initial doses of the same therapeutic monoclonal antibody in an effective amount at a starting time prior to the first cycle of administering the adoptive cell therapy product. In some embodiments, the therapeutic mAb is cetuximab and wherein a single initial dose of about 400 mg/m² to about 500 mg/m² is administered to the subject about 4 days prior to the first cycle of administering the adoptive cell therapy product. In some embodiments, the therapeutic mAb is trastuzumab and wherein: (i) a single dose of about 4 mg/kg is administered to the subject about 4 days prior to the first cycle of administering the adoptive cell therapy product, wherein the subject has HER2⁺ metastatic breast cancer (mBC); or (ii) a single dose of about 8 mg/kg is administered to the subject about 4 days prior to the first cycle of administering the adoptive cell therapy product, wherein the subject has a HER2⁺ solid tumor other than mBC. In some embodiments, the therapeutic mAb is avelumab, and wherein a single initial dose of about 800 mg is administered to the subject about 4 days prior to the first cycle of administering the adoptive cell therapy product. In some embodiments, therapeutic mAb is amivantamab, and wherein the starting time is about 4-10 days prior to the first cycle of administering the adoptive cell therapy product, and wherein the initial doses of the monoclonal antibody comprise 1-2 weekly (QW) doses of: (i) about 1050 mg for subjects weighing <80 kg; or (ii) about 1400 mg for subjects >80 kg, and optionally wherein the first weekly dose of the monoclonal antibody is administered over two consecutive days.

[00011] In various embodiments of the method of treating, the course of treatment further comprises administering to the subject an effective amount of an immune checkpoint inhibitor (ICI). In some embodiments, the ICI comprises an anti-PDl/PDLl mAb, and optionally wherein the anti-PDl/PDLl mAb comprises pembrolizumab, or nivolumab, atezolizumab. In some embodiments, the course of treatment further comprises administering to the subject an effective amount of an initial dose of the same ICI in an effective amount about 4 days prior to the first cycle of administering the adoptive cell therapy product, and wherein: (i) pembrolizumab is in an amount of about 200 mg to about 400 mg; (ii) nivolumab is in an amount of about 240 mg to about 480 mg; or (iii) atezolizumab is in an amount of about 840 mg to about 1680 mg.

[00012] In various embodiments of the method of treating, the course of treatment further comprises administering to the subject an effective amount of IL2 prior to the adoptive cell therapy product. In some embodiments, the administration of IL2 is about 1-3 hours prior to the adoptive cell therapy product. In some embodiments, the effective amount of IL2 is: (i) about 10 MIU; or (ii) about 3 MIU per m² for subjects with a baseline body weight of <45 kg.

[00013] In various embodiments of the method of treating, 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, wherein the duration between the administration of a 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. In some embodiments, the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU); and optionally wherein the CY and FLU are administered daily for three consecutive days, or wherein the dose of CY is at about 300-500 mg/m² and the dose of FLU is at about 25-30 mg/m². In some embodiments, the specified period of time is: (i) about 24-84 hours; or (ii) about 3 days.

[00014] In various embodiments of the method of treating, the engineered NK lineage cell is derived from an engineered induced pluripotent stem cell (iPSC) comprising a polynucleotide encoding a MICA/B-CAR, a polynucleotide encoding an exogenous CD 16, a polynucleotide encoding IL15RF and CD38 knockout. In some embodiments, the MICA/B-CAR comprises: (i) a heavy chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 1; (ii) a light chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 2; and/or (iii) a scFV represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NOs: 3 or 4. In various embodiments, the first and second effective amounts of the adoptive cell therapy product in each dose is about 5 * 10⁷ cells to about 9 * 10⁹ cells. In various embodiments, the first and second effective amounts of the adoptive cell therapy product in each dose is about 1 x 10⁸ cells, about 3 x io⁸ cells, about 1 x 10⁹ cells, about 3 x io⁹ cells or about 9 x io⁹ cells. In some embodiments, if the effective amount in each dose results in a dose limiting toxicity (DLT) rate of <30% with an equivalence interval of 25%-35%, then the effective amount increases to a next higher effective amount.

[00015] In some embodiments of the method of treating, (i) cetuximab is administered weekly (QW) or every two weeks (Q2W) in an amount of: (a) about 400 mg/m² when administered QW; or (b) about 500 mg/m² when administered Q2W; (ii) trastuzumab is administered QW or every three weeks (Q3W) in an amount of: (a) about 2 mg/kg QW for subjects having HER2⁺ mBC; or (b) about 6 mg/kg Q3W for subjects having a HER2⁺ solid tumor other than mBC; (iii) avelumab is administered Q2W in an amount of about 800 mg/dose; and/or (iv) amivantamab is administered Q2W in an amount of about 1000-1500 mg. In some embodiments, when avelumab and the adoptive cell product are administered on the same day, the adoptive cell product is administered first.

[00016] In some embodiments of the method of treating, (i) pembrolizumab is administered Q3W or every six weeks (Q6W) in an amount of about 400 mg; (ii) nivolumab is administered Q2W or every four weeks (Q4W) in an amount of: (a) about 240 mg when administered Q2W; or (b) about 480 mg when administered Q4W; and/or (iii) atezolizumab is administered Q2W, Q3W or Q4W in an amount of: (a) about 840 mg when administered Q2W; (b) about 1200 mg when administered Q3W; or (c) about 1680 mg when administered Q4W.

[00017] In some embodiments of the method of treating, the subject suitable for the adoptive cell therapy product has: (i) advanced or metastatic non-small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer (BC), ovarian cancer, or pancreatic cancer; (ii) advanced or metastatic solid tumors having PD-L1 expression, or advanced CRC whose tumors are microsatellite instability high (MSI-H) and/or mismatch repair deficient (dMMR); (iii) advanced or metastatic HER2⁺ solid tumors or NSCLC with HER2 mutation; (iv) advanced or metastatic squamous NSCLC, CRC, or head and neck squamous cell carcinoma; or (v) advanced or metastatic NSCLC with EGFR driver mutation(s), MET exon 14 skipping mutation, or MET amplification. In some embodiments, (i) tumors having PD-L1 expression comprise: (a) NSCLC with a PD-L1 tumor expression >1%, or tumor-infiltrating immune cells (ICs) covering >10% of tumor area; (b) gastroesophageal adenocarcinoma with a PD-L1 expression >1%; (c) head and neck squamous cell carcinoma with a PD-L1 expression >1%, or >1% tumor cell expression; (d) Triple-negative breast cancer with a PD-L1 expression >10% or ICs covering >1% of tumor area; (e) UC with a PD-L1 expression >10%, >1% tumor cell expression, or ICs covering >5% of tumor area; or (f) locally advanced or metastatic CRC that is MSI-H and/or dMMR; (ii) tumors having HER2⁺ expression comprises tumors having: (a) >2+ immunohistochemistry (IHC); or (b) an average HER2 copy number >4 signals per cell, for example by in situ hybridization (ISH) or >4 copies as determined by, for example, next- generation sequencing (NGS); (iii) advanced or metastatic squamous NSCLC, CRC, or head and neck squamous cell carcinoma comprises: (a) CRC that is KRAS/NRAS wild type and the subject has relapsed or progressed following prior cetuximab or panitumumab treatment; or (b) head and neck cancer wherein the subject has relapsed or progressed following prior cetuximab treatment, or wherein the subject is documented to refuse standard cetuximab-based treatment; or (iv) subject having advanced or metastatic NSCLC comprises having progressed or being intolerant to at least one prior line of EGFR tyrosine kinase inhibitor (TKI) treatment, or not being a candidate for TKI treatment (v) MET amplification defined as MET/CEP7 gene expression ratio >1.8, for example, by FISH; or as gene copy number >5 as determined, for example, by ISH or NGS. In some embodiments, subjects having advanced or metastatic HER2⁺ solid tumors or NSCLC with HER2 mutation must have received: (a) at least one prior line of anti-HER2 antibody -based therapy for subjects having gastric cancer; or (b) at least two prior lines of anti-HER2 antibody -based therapy for subjects having breast cancer.

[00018] In various embodiments of the method of treating, the method comprises administering (i) one cycle of the adoptive cell therapy product over about 3 weeks at a dose frequency of 1 dose per week; (ii) two cycles of the adoptive cell therapy product, with each cycle comprising three doses over about 3 weeks at a dose frequency of 1 dose per week, wherein a second cycle of the two cycles is given within 42 days of a last infusion of the adoptive cell therapy product in a first cycle; or (iii) one, or two, or three or four cycles of the adoptive cell therapy product, with each cycle comprising three doses over about 3 weeks at a dose frequency of 1 dose per week. In various embodiments, the administering of the adoptive cell therapy product is (i) via intravenous infusion, and/or (ii) at a site of an outpatient setting; and/or wherein each dose of the adoptive cell therapy product is cryopreserved, and then thawed prior to administering; and wherein the adoptive cell therapy product is FT536.

[00019] In another aspect, the invention provides a method of slowing progression of and/or treating a solid tumor in a subject, wherein the subject has advanced or metastatic nonsmall cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer (BC), ovarian cancer, or pancreatic cancer, the method comprising: administering to the subject at least one cycle of an adoptive cell therapy product, with the cycle comprising one or more doses of the adoptive cell therapy product administered in a first effective amount at a preselected frequency, and with an option of administering one or more additional cycles with one or more doses in a second effective amount, during a course of treatment over a period of time; wherein the subject has a reduction in lesion size or number after treatment with the adoptive cell therapy product; and wherein the adoptive cell therapy comprises engineered natural killer (NK) lineage cells comprising MICA/B-CAR (chimeric antigen receptor) expression, exogenous CD 16 expression, IL15RF expression and CD38 knockout. In some embodiments, the MICA/B-CAR comprises: (i) a heavy chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 1; (ii) a light chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 2; and/or (iii) a scFV represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NOs: 3 or 4.

[00020] In various embodiments of the method of slowing progression of and/or treating a solid tumor, the method further comprises administering to the subject a daily dose of one or more chemotherapeutic agents for three consecutive days, wherein the duration between the last daily dose administration of the one or more chemotherapeutic agents and a first weekly administration of the adoptive cell therapy product is: (i) about 24-84 hours; or (ii) about 3 days. In some embodiments, the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU), and optionally wherein the daily dose of CY is at about 300-500 mg/m² and the daily dose of FLU is at about 25-30 mg/m².

[00021] In various embodiments of the method of slowing progression of and/or treating a solid tumor, the method further comprises administering to the subject an effective amount of IL2 prior to the adoptive cell therapy product, wherein the administration of IL2 is about 1-3 hours prior to the adoptive cell therapy product. In some embodiments, the effective amount of IL2 is: (i) about 10 MIU; or (ii) about 3 MIU per m² for subjects with a baseline body weight of <45 kg. In some embodiments, the engineered NK lineage cell is derived from an engineered induced pluripotent stem cell (iPSC), and wherein the engineered iPSC comprises a polynucleotide encoding a MICA/B-CAR, a polynucleotide encoding an exogenous CD 16, and optionally, a polynucleotide encoding IL15RF and CD38 knockout. In some embodiments, the first and the second effective amounts of the adoptive cell therapy product are about 5 * 10⁷ cells/dose to about 9 * 10⁹ cells/dose, and wherein the first and the second effective amounts are the same or different. In some embodiments, the effective amount of the adoptive cell therapy product in each dose is about 1 x 10⁸ cells, about 3 * 10⁸ cells, about 1 x 10⁹ cells, about 3 x io⁹ cells or about 3 x io⁹ cells. In some embodiments, if the effective amount in each dose results in a dose limiting toxicity (DLT) rate of <30% with an equivalence interval of 25%-35%, then the effective amount increases to a next higher effective amount.

[00022] In various embodiments of the method of slowing progression of and/or treating a solid tumor, the method further comprises further comprising assessing disease response to the adoptive cell therapy after each cycle of treatment with the adoptive cell therapy product. In some embodiments, assessing disease response comprises measuring target lesions or short axis of pathological lymph nodes of the subject for complete remission or partial remission. In some embodiments, (i) complete remission comprises: (a) disappearance of all target lesions; and/or (b) a short axis reduction of any pathological lymph nodes; and/or (ii) partial remission comprises at least a 30% decrease in a sum of all diameters of target lesions, as compared to that of the target lesions prior to the course of treatment.

[00023] In various embodiments of the method of slowing progression of and/or treating a solid tumor, the administering of the adoptive cell therapy product is (i) via intravenous infusion, and/or (ii) at a site of an outpatient setting; and/or wherein each dose of the adoptive cell therapy product is cryopreserved, and then thawed prior to administering. In some embodiments, the additional cycle during a course of treatment comprises (i) a number of doses, (ii) at a dose frequency of the adoptive cell therapy product, (iii) in an effective cell amount per dose, wherein (i), (ii) or (iii) herein is same or different from that of the at least one cycle administered. In some embodiments, the additional cycle during a course of treatment comprising the adoptive cell therapy product comprises at least 3 doses at a dose frequency of 1 dose per week, and wherein the additional cycle is administered within 42 days of a last infusion of the adoptive cell therapy product of the first cycle.

[00024] In another aspect, the invention provides a method of slowing progression of and/or treating a solid tumor in a subject, wherein the subject has advanced or metastatic solid tumors with PD-L1 expression, or advanced CRC whose tumors are microsatellite instability high (MSI-H) and/or mismatch repair deficient (dMMR), the method comprising: (i) administering to the subject at least one cycle of an adoptive cell therapy product, with the cycle comprising one or more doses of the adoptive cell therapy product administered in a first effective amount at a preselected frequency, and with an option of administering one or more additional cycles with one or more doses in a second effective amount, during a course of treatment over a period of time; and (ii) administering to the subject one or more doses of an effective amount of an anti-PD-Ll monoclonal antibody during the at least one cycle of the adoptive cell therapy product; wherein the subject has a reduction in lesion size and/or number after treatment with the adoptive cell therapy product; and wherein the adoptive cell therapy comprises engineered natural killer (NK) lineage cells comprising MICA/B-CAR (chimeric antigen receptor) expression, exogenous CD 16 expression, IL15RF expression and CD38 knockout.

[00025] In various embodiments, the subject has: (a) NSCLC with a PD-L1 tumor expression >1%, or tumor-infiltrating immune cells (ICs) covering >10% of tumor area using a SP142 assay; (b) gastroesophageal adenocarcinoma with a PD-L1 expression >1%; (c) head and neck squamous cell carcinoma with a PD-L1 expression >1%, or >1% tumor cell expression; (d) triple-negative breast cancer with a PD-L1 expression >10% or ICs covering >1% of tumor area; (e) UC with a PD-L1 expression >10%, >1% tumor cell expression, or ICs covering >5% of tumor area; or (f) locally advanced or metastatic CRC that is MSI-H and/or dMMR. In some embodiments, the engineered NK lineage cell is derived from an engineered induced pluripotent stem cell (iPSC) comprising a polynucleotide encoding a MICA/B-CAR, a polynucleotide encoding an exogenous CD 16, a polynucleotide encoding IL15RF and CD38 knockout. In some embodiments, the MICA/B-CAR comprises: (i) a heavy chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 1; (ii) a light chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 2; and/or (iii) a scFV represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NOs: 3 or 4.

[00026] In various embodiments, the method further comprises administering to the subject a single initial dose of the same anti-PD-Ll monoclonal antibody to the subject about 4 days prior to the first cycle of administering the adoptive cell therapy product. In various embodiments, the method further comprises administering to the subject a daily dose of one or more chemotherapeutic agents for three consecutive days, wherein the duration between the last daily dose administration of the one or more chemotherapeutic agents and a first weekly administration of the adoptive cell therapy product is: (i) about 24-84 hours; or (ii) about 3 days. In some embodiments, the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU), and optionally wherein the daily dose of CY is at about 300-500 mg/m² and the daily dose of FLU is at about 25-30 mg/m². In various embodiments, the method further comprises administering to the subject an effective amount of IL2 prior to the adoptive cell therapy product, wherein the administration of IL2 is about 1-3 hours prior to the adoptive cell therapy product. In some embodiments, the effective amount of IL2 is: (i) about 10 MIU; or (ii) about 2 MIU per m² for subjects with a baseline body weight of <45 kg. In some embodiments, the effective amount of the one or more doses of the adoptive cell therapy product is about 5 * 10⁷ cells/dose to about 9 * 10⁹ cells/dose, and wherein the effective amounts of each of the one or more doses are the same or different. In some embodiments, the effective amount of the adoptive cell therapy product in each dose is about 1 x 10⁸ cells, about 3 * 10⁸ cells, about 1 * 10⁹ cells, about 3 x io⁹ cells or about 9 x io⁹ cells. In some embodiments, if the effective amount in each dose results in a dose limiting toxicity (DLT) rate of <30% with an equivalence interval of 25%-35%, then the effective amount increases to a next higher effective amount.

[00027] In various embodiments, the anti-PD-Ll monoclonal antibody is avelumab, and wherein the avelumab is administered Q2W in an amount of about 800 mg/dose. In some embodiments, when avelumab and the adoptive cell product are administered on the same day, the adoptive cell product is administered first. In some embodiments, the method further comprises further comprising assessing disease response to the adoptive cell therapy after each cycle of treatment with the adoptive cell therapy product. In some embodiments, assessing disease response comprises measuring target lesions or short axis of pathological lymph nodes of the subject for complete remission or partial remission. In some embodiments, (i) complete remission comprises: (a) disappearance of all target lesions; and/or (b) a short axis reduction of any pathological lymph nodes; and/or (ii) partial remission comprises at least a 30% decrease in a sum of all diameters of target lesions, as compared to that of the target lesions prior to the course of treatment. In some embodiments, the administering of the adoptive cell therapy product is (i) via intravenous infusion, and/or (ii) at a site of an outpatient setting; and/or wherein each dose of the adoptive cell therapy product is cryopreserved, and then thawed prior to administering.

[00028] In another aspect, the invention provides a method of slowing progression of and/or treating a solid tumor in a subject, wherein the subject has advanced or metastatic solid tumors with PD-L1 expression, or advanced CRC whose tumors are microsatellite instability high (MSI-H) and/or mismatch repair deficient (dMMR), the method comprising: (i) administering to the subject at least one cycle of an adoptive cell therapy product, with the cycle comprising one or more doses of the adoptive cell therapy product administered in a first effective amount at a preselected frequency, and with an option of administering one or more additional cycles with one or more doses in a second effective amount, during a course of treatment over a period of time; and (ii) administering to the subject one or more doses of an effective amount of an immune checkpoint inhibitor (ICI) during the at least one cycle of the adoptive cell therapy product, wherein the ICI comprises pembrolizumab, or nivolumab, atezolizumab; wherein the subject has a reduction in lesion size and/or number after treatment with the adoptive cell therapy product; and wherein the adoptive cell therapy comprises engineered natural killer (NK) lineage cells comprising MICA/B-CAR (chimeric antigen receptor) expression, exogenous CD 16 expression, IL15RF expression and CD38 knockout. In some embodiments, the subject has: (a) NSCLC with a PD-L1 tumor expression >1%, or tumorinfiltrating immune cells (ICs) covering >10% of tumor area; (b) gastroesophageal adenocarcinoma with a PD-L1 expression >1%; (c) head and neck squamous cell carcinoma with a PD-L1 expression >1%, or >1% tumor cell expression; (d) triple-negative breast cancer with a PD-L1 expression >10% or ICs covering >1% of tumor area; (e) UC with a PD-L1 expression >10%, >1% tumor cell expression, or ICs covering >5% of tumor area; or (f) locally advanced or metastatic CRC that is MSI-H and/or dMMR. In some embodiments, the engineered NK lineage cell is derived from an engineered induced pluripotent stem cell (iPSC) comprising a polynucleotide encoding a MICA/B-CAR, a polynucleotide encoding an exogenous CD 16, a polynucleotide encoding IL15RF and CD38 knockout. In some embodiments, the MICA/B-CAR comprises: (i) a heavy chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 1; (ii) a light chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 2; and/or (iii) a scFV represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NOs: 3 or 4.

[00029] In various embodiments, the method further comprises administering to the subject a single initial dose of the same ICI to the subject about 4 days prior to the first cycle of administering the adoptive cell therapy product, and wherein the ICI is: (i) pembrolizumab and the initial dose is about 200 mg to about 400 mg; (ii) nivolumab and the initial dose is about 240 mg to about 480 mg; or (iii) atezolizumab and the initial dose is about 840 mg to about 1680 mg. In various embodiments, the method further comprises administering to the subject a daily dose of one or more chemotherapeutic agents for three consecutive days, wherein the duration between the last daily dose administration of the one or more chemotherapeutic agents and a first weekly administration of the adoptive cell therapy product is: (i) about 24-84 hours; or (ii) about 3 days. In some embodiments, the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU), and optionally wherein the daily dose of CY is at about 300-500 mg/m² and the daily dose of FLU is at about 25-30 mg/m². In various embodiments, the method further comprises administering to the subject an effective amount of IL2 prior to the adoptive cell therapy product, wherein the administration of IL2 is about 1-3 hours prior to the adoptive cell therapy product. In some embodiments, the effective amount of IL2 is: (i) about 10 MIU; or (ii) about 3 MIU per m² for subjects with a baseline body weight of <45 kg. In some embodiments, the effective amount of the one or more doses of the adoptive cell therapy product is about 5 * 10⁷ cells/dose to about 9 * 10⁹ cells/dose, and wherein the effective amounts of each of the one or more doses are the same or different. In some embodiments, the effective amount of the adoptive cell therapy product in each dose is about 1 x 10⁸ cells, about 3 x io⁸ cells, about 1 x 10⁹ cells, about 3 x io⁹ cells or about 9 x io⁹ cells. In some embodiments, if the effective amount in each dose results in a dose limiting toxicity (DLT) rate of <30% with an equivalence interval of 25%-35%, then the effective amount increases to a next higher effective amount.

[00030] In some embodiments, the ICI is: (i) pembrolizumab and is administered Q3W or every six weeks (Q6W) in an amount of about 400 mg; (ii) nivolumab and is administered Q2W or every four weeks (Q4W) in an amount of: (a) about 240 mg when administered Q2W; or (b) about 480 mg when administered Q4W; and/or (iii) atezolizumab and is administered Q2W, Q3W or Q4W in an amount of: (a) about 840 mg when administered Q2W; (b) about 1200 mg when administered Q3W; or (c) about 1680 mg when administered Q4W. In some embodiments, the method further comprises further comprising assessing disease response to the adoptive cell therapy after each cycle of treatment with the adoptive cell therapy product. In some embodiments, assessing disease response comprises measuring target lesions or short axis of pathological lymph nodes of the subject for complete remission or partial remission. In some embodiments, (i) complete remission comprises: (a) disappearance of all target lesions; and/or (b) a short axis reduction of any pathological lymph nodes; and/or (ii) partial remission comprises at least a 30% decrease in a sum of all diameters of target lesions, as compared to that of the target lesions prior to the course of treatment. In some embodiments, the administering of the adoptive cell therapy product is (i) via intravenous infusion, and/or (ii) at a site of an outpatient setting; and/or wherein each dose of the adoptive cell therapy product is cryopreserved, and then thawed prior to administering.

[00031] In another aspect, the invention provides a method of slowing progression of and/or treating a solid tumor in a subject, wherein the subject has advanced or metastatic HER2⁺ solid tumors or NSCLC with HER2 mutation, the method comprising: (i) administering to the subject at least one cycle of an adoptive cell therapy product, with the cycle comprising one or more doses of the adoptive cell therapy product administered in a first effective amount at a preselected frequency, and with an option of administering one or more additional cycles with one or more doses in a second effective amount, during a course of treatment over a period of time; and (ii) administering to the subject one or more doses of an effective amount of an anti- HER2 monoclonal antibody during the at least one cycle of the adoptive cell therapy product; wherein the subject has a reduction in lesion size and/or number after treatment with the adoptive cell therapy product; and wherein the adoptive cell therapy comprises engineered natural killer (NK) lineage cells comprising MICA/B-CAR (chimeric antigen receptor) expression, exogenous CD 16 expression, IL15RF expression and CD38 knockout. In some embodiments, the subject has tumors having: (a) >2+ immunohistochemistry (IHC); or (b) an average HER2 copy number >4 signals per cell, for example, by in situ hybridization (ISH) or >4 copies as determined, for example, by next-generation sequencing (NGS). In some embodiments, the engineered NK lineage cell is derived from an engineered induced pluripotent stem cell (iPSC) comprising a polynucleotide encoding a MICA/B-CAR, a polynucleotide encoding an exogenous CD 16, a polynucleotide encoding IL15RF and CD38 knockout. In some embodiments, the MICA/B-CAR comprises: (i) a heavy chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 1; (ii) a light chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 2; and/or (iii) a scFV represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NOs: 3 or 4. [00032] In various embodiments, the method further comprises administering to the subject a single initial dose of the anti-HER2 monoclonal antibody to the subject about 4 days prior to the first cycle of administering the adoptive cell therapy product, and wherein: (i) the single initial dose is about 4 mg/kg for subjects having HER2⁺ metastatic breast cancer (mBC); or (ii) the single dose is about 8 mg/kg for subjects having a HER2⁺ solid tumor other than mBC. In various embodiments, the method further comprises administering to the subject a daily dose of one or more chemotherapeutic agents for three consecutive days, wherein the duration between the last daily dose administration of the one or more chemotherapeutic agents and a first weekly administration of the adoptive cell therapy product is: (i) about 24-84 hours; or (ii) about 3 days. In some embodiments, the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU), and optionally wherein the daily dose of CY is at about 300-500 mg/m² and the daily dose of FLU is at about 25-30 mg/m². In various embodiments, the method further comprises administering to the subject an effective amount of IL2 prior to the adoptive cell therapy product, wherein the administration of IL2 is about 1-3 hours prior to the adoptive cell therapy product. In some embodiments, the effective amount of IL2 is: (i) about 10 MIU; or (ii) about 3 MIU per m² for subjects with a baseline body weight of <45 kg. In some embodiments, the effective amount of the one or more doses of the adoptive cell therapy product is about 5 * 10⁷ cells/dose to about 9 * 10⁹ cells/dose, and wherein the effective amounts of each of the one or more doses are the same or different. In some embodiments, the effective amount of the adoptive cell therapy product in each dose is about 1 x 10⁸ cells, about 3 * 10⁸ cells, about 1 * 10⁹ cells, about 3 x io⁹ cells or about 9 x io⁹ cells. In some embodiments, if the effective amount in each dose results in a dose limiting toxicity (DLT) rate of <30% with an equivalence interval of 25%-35%, then the effective amount increases to a next higher effective amount.

[00033] In some embodiments, the anti-HER2 monoclonal antibody is trastuzumab and is administered QW or every three weeks (Q3W) in an amount of: (a) about 2 mg/kg QW for subjects having HER2⁺ mBC; or (b) about 6 mg/kg Q3W for subjects having a HER2⁺ solid tumor other than mBC. In some embodiments, the method further comprises further comprising assessing disease response to the adoptive cell therapy after each cycle of treatment with the adoptive cell therapy product. In some embodiments, assessing disease response comprises measuring target lesions or short axis of pathological lymph nodes of the subject for complete remission or partial remission. In some embodiments, (i) complete remission comprises: (a) disappearance of all target lesions; and/or (b) a short axis reduction of any pathological lymph nodes; and/or (ii) partial remission comprises at least a 30% decrease in a sum of all diameters of target lesions, as compared to that of the target lesions prior to the course of treatment. In some embodiments, the administering of the adoptive cell therapy product is (i) via intravenous infusion, and/or (ii) at a site of an outpatient setting; and/or wherein each dose of the adoptive cell therapy product is cryopreserved, and then thawed prior to administering.

[00034] In another aspect, the invention provides a method of slowing progression of and/or treating a solid tumor in a subject, wherein the subject has advanced or metastatic squamous NSCLC, CRC, or head and neck squamous cell carcinoma, the method comprising: (i) administering to the subject at least one cycle of an adoptive cell therapy product, with the cycle comprising one or more doses of the adoptive cell therapy product administered in a first effective amount at a preselected frequency, and with an option of administering one or more additional cycles with one or more doses in a second effective amount, during a course of treatment over a period of time; and (ii) administering to the subject one or more doses of an effective amount of an anti-EGFR monoclonal antibody during the at least one cycle of the adoptive cell therapy product; wherein the subject has a reduction in lesion size and/or number after treatment with the adoptive cell therapy product; and wherein the adoptive cell therapy comprises engineered natural killer (NK) lineage cells comprising MICA/B-CAR (chimeric antigen receptor) expression, exogenous CD 16 expression, IL15RF expression and CD38 knockout. In some embodiments, the subject has: (a) CRC that is KRAS/NRAS wild type and the subject has relapsed or progressed following prior cetuximab or panitumumab treatment; or (b) head and neck cancer, and wherein the subject has relapsed or progressed following prior cetuximab treatment, or wherein the subject has refused standard cetuximab-based treatment. In some embodiments, the engineered NK lineage cell is derived from an engineered induced pluripotent stem cell (iPSC) comprising a polynucleotide encoding a MICA/B-CAR, a polynucleotide encoding an exogenous CD 16, a polynucleotide encoding IL15RF and CD38 knockout. In some embodiments, the MICA/B-CAR comprises: (i) a heavy chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 1; (ii) a light chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 2; and/or (iii) a scFV represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NOs: 3 or 4.

[00035] In some embodiments, the method further comprises administering to the subject a single initial dose of the anti-EGFR monoclonal antibody to the subject about 4 days prior to the first cycle of administering the adoptive cell therapy product, and wherein the single initial dose is about 400 mg/m² to about 500 mg/m². In some embodiments, the method further comprises administering to the subject a daily dose of one or more chemotherapeutic agents for three consecutive days, wherein the duration between the last daily dose administration of the one or more chemotherapeutic agents and a first weekly administration of the adoptive cell therapy product is: (i) about 24-84 hours; or (ii) about 3 days. In some embodiments, the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU), and optionally wherein the daily dose of CY is at about 300-500 mg/m² and the daily dose of FLU is at about 25-30 mg/m². In various embodiments, the method further comprises administering to the subject an effective amount of IL2 prior to the adoptive cell therapy product, wherein the administration of IL2 is about 1-3 hours prior to the adoptive cell therapy product. In some embodiments, the effective amount of IL2 is: (i) about 10 MIU; or (ii) about 3 MIU per m² for subjects with a baseline body weight of <45 kg. In some embodiments, the effective amount of the one or more doses of the adoptive cell therapy product is about 5 * 10⁷ cells/dose to about 9 x 10⁹ cells/dose, and wherein the effective amounts of each of the one or more doses are the same or different. In some embodiments, the effective amount of the adoptive cell therapy product in each dose is about 1 x 10⁸ cells, about 3 * 10⁸ cells, about 1 x 10⁹ cells, about 3 x io⁹ cells or about 9 x io⁹ cells. In some embodiments, if the effective amount in each dose results in a dose limiting toxicity (DLT) rate of <30% with an equivalence interval of 25%-35%, then the effective amount increases to a next higher effective amount.

[00036] In some embodiments, the anti-EGFR monoclonal antibody is cetuximab and is administered weekly (QW) or every two weeks (Q2W) in an amount of: (a) about 400 mg/m² when administered QW; or (b) about 500 mg/m² when administered Q2W. In some embodiments, the method further comprises further comprising assessing disease response to the adoptive cell therapy after each cycle of treatment with the adoptive cell therapy product. In some embodiments, assessing disease response comprises measuring target lesions or short axis of pathological lymph nodes of the subject for complete remission or partial remission. In some embodiments, (i) complete remission comprises: (a) disappearance of all target lesions; and/or (b) a short axis reduction of any pathological lymph nodes to <10 mm; and/or (ii) partial remission comprises at least a 30% decrease in a sum of all diameters of target lesions, as compared to that of the target lesions prior to the course of treatment. In some embodiments, the administering of the adoptive cell therapy product is (i) via intravenous infusion, and/or (ii) at a site of an outpatient setting; and/or wherein each dose of the adoptive cell therapy product is cryopreserved, and then thawed prior to administering.

[00037] In another aspect, the invention provides a method of slowing progression of and/or treating a solid tumor in a subject, wherein the subject has advanced or metastatic NSCLC with EGFR driver mutation(s), MET exon 14 skipping mutation, or MET amplification, the method comprising: (i) administering to the subject at least one cycle of an adoptive cell therapy product, with the cycle comprising one or more doses of the adoptive cell therapy product administered in a first effective amount at a preselected frequency, and with an option of administering one or more additional cycles with one or more doses in a second effective amount, during a course of treatment over a period of time; and (ii) administering to the subject one or more doses of an effective amount of a bi-specific antibody targeting EGFR and MET during the at least one cycle of the adoptive cell therapy product; wherein the subject has a reduction in lesion size and/or number after treatment with the adoptive cell therapy product; and wherein the adoptive cell therapy comprises engineered natural killer (NK) lineage cells comprising MICA/B-CAR (chimeric antigen receptor) expression, exogenous CD 16 expression, IL15RF expression and CD38 knockout. In some embodiments, the subject has tumors having: (a) EGFR driver mutation(s), and wherein the subject has progressed on or was intolerant to at least one prior line of EGFR tyrosine kinase inhibitor (TKI) treatment or wherein the subject is not a candidate for TKI treatment; (b) MET exon 14 skipping mutation, and wherein the subject has progressed on or is intolerant of at least one prior line of MET TKI treatment, or wherein the subject was not a candidate for TKI treatment; (c) MET amplification defined as a MET/CEP7 gene expression ratio >1.8, for example by FISH; or (d) MET amplification defined as gene copy number >5, for example as determined by ISH or NGS. In some embodiments, the engineered NK lineage cell is derived from an engineered induced pluripotent stem cell (iPSC) comprising a polynucleotide encoding a MICA/B-CAR, a polynucleotide encoding an exogenous CD 16, a polynucleotide encoding IL15RF and CD38 knockout. In some embodiments, the MICA/B-CAR comprises: (i) a heavy chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 1; (ii) a light chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 2; and/or (iii) a scFV represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NOs: 3 or 4.

[00038] In some embodiments, the method further comprises administering to the subject one or more initial doses of the same bi-specific antibody targeting EGFR and MET to the subject about 4-10 days prior to the first cycle of administering the adoptive cell therapy product, and wherein the initial doses of the bi-specific antibody comprise 1-2 weekly (QW) doses of about 1000-1400mg, for example: (i) about 1050 mg for subjects weighing <80 kg; or (ii) about 1400 mg for subjects >80 kg, and optionally wherein the first weekly dose of the monoclonal antibody is administered over two consecutive days. In some embodiments, the method further comprises administering to the subject a daily dose of one or more chemotherapeutic agents for three consecutive days, wherein the duration between the last daily dose administration of the one or more chemotherapeutic agents and a first weekly administration of the adoptive cell therapy product is: (i) about 24-84 hours; or (ii) about 3 days. In some embodiments, the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU), and optionally wherein the daily dose of CY is at about 300-500 mg/m² and the daily dose of FLU is at about 25-30 mg/m². In various embodiments, the method further comprises administering to the subject an effective amount of IL2 prior to the adoptive cell therapy product, wherein the administration of IL2 is about 1-3 hours prior to the adoptive cell therapy product. In some embodiments, the effective amount of IL2 is: (i) about 10 MIU; or (ii) about 3 MIU per m² for subjects with a baseline body weight of <45 kg. In some embodiments, the effective amount of the one or more doses of the adoptive cell therapy product is about 5 * 10⁷ cells/dose to about 9 * 10⁹ cells/dose, and wherein the effective amounts of each of the one or more doses are the same or different. In some embodiments, the effective amount of the adoptive cell therapy product in each dose is about 1 x 10⁸ cells, about 3 * 10⁸ cells, about 1 * 10⁹ cells, about 3 x io⁹ cells or about 9 x io⁹ cells. In some embodiments, if the effective amount in each dose results in a dose limiting toxicity (DLT) rate of <30% with an equivalence interval of 25%-35%, then the effective amount increases to a next higher effective amount. [00039] In some embodiments, the bi-specific antibody is amivantamab and is administered every two weeks (Q2W) in an amount of about 1000-1400 mg, for example: (i) about 1050 mg for subjects weighing <80 kg; or (ii) about 1400 mg for subjects >80 kg. In some embodiments, the method further comprises further comprising assessing disease response to the adoptive cell therapy after each cycle of treatment with the adoptive cell therapy product. In some embodiments, assessing disease response comprises measuring target lesions or short axis of pathological lymph nodes of the subject for complete remission or partial remission. In some embodiments, (i) complete remission comprises: (a) disappearance of all target lesions; and/or (b) a short axis reduction of any pathological lymph nodes; and/or (ii) partial remission comprises at least a 30% decrease in a sum of all diameters of target lesions, as compared to that of the target lesions prior to the course of treatment. In some embodiments, the administering of the adoptive cell therapy product is (i) via intravenous infusion, and/or (ii) at a site of an outpatient setting; and/or wherein each dose of the adoptive cell therapy product is cryopreserved, and then thawed prior to administering.

[00040] In yet another aspect, the invention provides a method of a multi-dose targeted adoptive cell therapy in a subject in need thereof comprising: (i) weekly administration to the subject of an effective amount of a targeted adoptive cell therapy product for a course of treatment of about three weeks, wherein the product comprises an engineered immune cell expressing a MICA/B-CAR, expressing CD16, expressing IL15RF, and comprising CD38 knockout; and (ii) detecting and comparing one or more of the following at different given time points following administration of a first dose of the adoptive cell therapy: (a) the presence of the engineered immune cell in a tumor of the subject; (b) protein markers of disease in serum of the subject; (c) cytokines in a peripheral blood sample from the subject; (d) circulating tumor DNA in a peripheral blood sample from the subject; and (e) lesion size and/or number, wherein any of (a)-(e) is used to assess tumor burden, tumor immunobiology, and/or tumor therapy response, thereby determining efficacy of the multi-dose targeted adoptive cell therapy. In various embodiments, the subject has breast cancer (BC), advanced or metastatic colorectal cancer (CRC), advanced or metastatic non-small cell lung cancer (NSCLC), gastric cancer or gastroesophageal adenocarcinoma, ovarian cancer, pancreatic cancer, head and neck cancer, or urothelial carcinoma (UC). In some embodiments, the effective amount of the one or more doses of the adoptive cell therapy product is about 5 * 10⁷ cells/dose to about 9 * 10⁹ cells/dose, and wherein the effective amounts of each of the one or more doses are the same or different. In some embodiments, the effective amount of the adoptive cell therapy product in each dose is about 1 x 10⁸ cells, about 3 x io⁸ cells, about 1 x 10⁹ cells, about 3 x io⁹ cells or about 9 x io⁹ cells. In some embodiments, if the effective amount in each dose results in a dose limiting toxicity (DLT) rate of <30% with an equivalence interval of 25%-35%, then the effective amount increases to a next higher effective amount.

[00041] In some embodiments, the method further comprises administering to the subject an effective amount of a selected therapeutic monoclonal antibody (mAb). In some embodiments, the therapeutic mAb comprises an anti-EGFR antibody, an anti-HER2 antibody, an anti-PD-Ll antibody, or a bi-specific antibody targeting EGFR and MET. In some embodiments, the anti-EGFR antibody comprises cetuximab; wherein the anti-HER2 antibody comprises trastuzumab or biosimilars; wherein the anti-PD-Ll antibody comprises avelumab; or wherein the bi-specific antibody comprises amivantamab. In various embodiments, the method further comprises administering to the subject an effective amount of an immune checkpoint inhibitor (ICI). In some embodiments, the ICI comprises an anti-PDl/PDLl mAb, and optionally wherein the anti-PDl/PDLl mAb comprises pembrolizumab, or nivolumab, atezolizumab. In some embodiments, the method further comprises administering to the subject a daily dose of one or more chemotherapeutic agents for three consecutive days, wherein the duration between the last daily dose administration of the one or more chemotherapeutic agents and a first weekly administration of the adoptive cell therapy product is: (i) about 24-84 hours; or (ii) about 3 days. In some embodiments, the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU), and optionally wherein the daily dose of CY is at about 300-500 mg/m² and the daily dose of FLU is at about 25-30 mg/m². In various embodiments, the method further comprises administering to the subject an effective amount of IL2 prior to the adoptive cell therapy product, wherein the administration of IL2 is about 1-3 hours prior to the adoptive cell therapy product. In some embodiments, the effective amount of IL2 is: (i) about 10 MIU; or (ii) about 3 MIU per m² for subjects with a baseline body weight of <45 kg. In some embodiments, the engineered immune cell is derived from an engineered induced pluripotent stem cell (iPSC), and wherein the engineered iPSC comprises a polynucleotide encoding a MICA/B-CAR, a polynucleotide encoding an exogenous CD 16, and optionally, a polynucleotide encoding IL15RF and CD38 knockout. In some embodiments, the MICA/B-CAR comprises: (i) a heavy chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 1; (ii) a light chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 2; and/or (iii) a scFV represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NOs: 3 or 4.

[00042] Various objects and advantages of the compositions and methods as provided herein will become apparent from the following description taken in conjunction with the accompanying drawings wherein are set forth, by way of illustration and example, certain embodiments of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[00043] FIG. l is a graphic representation showing an exemplary iNK cell for use in an immunotherapy described herein.

[00044] FIG. 2 is an exemplary treatment schema to evaluate an iNK cell therapy as a monotherapy and in combination with secondary antigen targeting monoclonal antibodies. As noted in FIG. 2: ^a Additional cycles of FT536 (Cycles 3 and 4) will follow the same schedule as initial FT536 treatment in Cycles 1 and 2. ^b Subjects will proceed to LTFU if progression-free up to 2 years after the last dose of FT536, experience disease relapse or progression prior to 2 years, or begin a new, non-protocol-defined anti-cancer therapy.

IL = Interleukin; LTFU = Long-term follow-up; mAb = Monoclonal antibody; PD = Progressive disease; PTFU = Post-treatment follow-up.

[00045] FIG. 3 shows initial patient phenotypes and observations following an initial cycle of low dose iNK cell therapy.

[00046] FIG. 4 shows illustrative results in patients treated with a cell therapy in accordance with an embodiment. As noted in FIG. 4, DLT = dose limiting toxicity, CRS = cytokine release syndrome, ICANS = immune effector cell-associated neurotoxicity syndrome, BOR = best overall response, PD = progressive disease, NA = not available. Data as of Sep2022. *Disease progression due to new lesion. DETAILED DESCRIPTION OF THE INVENTION

[00047] Genomic modification of iPSCs (induced pluripotent stem cells) can include one or more of polynucleotide insertion, deletion and substitution. Exogenous gene expression in genome-engineered iPSCs often encounters problems such as gene silencing or reduced gene expression after prolonged clonal expansion of the original genome-engineered iPSCs, after cell differentiation, and in dedifferentiated cell types from the cells derived from the genome- engineered iPSCs. On the other hand, direct engineering of primary immune cells such as T or NK cells is challenging, and presents a hurdle to the preparation and delivery of engineered immune cells for adoptive cell therapy. In various embodiments, the present invention provides an efficient, reliable, and targeted approach for stably integrating one or more exogenous genes, including suicide genes and other functional modalities, which provide improved therapeutic properties relating to engraftment, trafficking, homing, migration, cytotoxicity, viability, maintenance, expansion, longevity, self-renewal, persistence, and/or survival, into iPSC derivative cells, including but not limited to HSCs (hematopoietic stem and progenitor cells), T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells.

[00048] Definitions

[00049] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[00050] 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.

[00051] 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.

[00052] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[00053] The term “and/or” should be understood to mean either one, or both of the alternatives.

[00054] As used herein, the term “about” or “approximately” refers to a quantity, 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 quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[00055] As used herein, the term “substantially” or “essentially” refers to a quantity, 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 higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the terms “essentially the same” or “substantially the same” refer a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[00056] As used herein, the terms “substantially free of’ and “essentially free of’ are used interchangeably, and when used to describe a composition, such as a cell population or culture media, refer to a composition that is free of a specified substance or its source thereof, such as, 95% free, 96% free, 97% free, 98% free, 99% free of the specified substance or its source thereof, or is undetectable as measured by conventional means. The term “free of’ or “essentially free of’ a certain ingredient or substance in a composition also means that no such ingredient or substance is (1) included in the composition at any concentration, or (2) included in the composition at a functionally inert, low concentration. Similar meaning can be applied to the term “absence of,” where referring to the absence of a particular substance or its source thereof of a composition.

[00057] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. In particular embodiments, the terms “include,” “has,” “contains,” and “comprise” are used synonymously.

[00058] By “consisting of’ is meant including, and limited to, whatever 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.

[00059] By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[00060] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing 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.

[00061] The term “ex vivo" refers generally to activities that take place outside an organism, such as experimentation or measurements done in or on living tissue in an artificial environment outside the organism, preferably with minimum alteration of the natural conditions. In particular embodiments, “ex vivo" procedures involve living cells or tissues taken from an organism and cultured in a laboratory apparatus, usually under sterile conditions, and typically for a few hours or up to about 24 hours, but including up to 48 or 72 hours or longer, depending on the circumstances. In certain embodiments, such tissues or cells can be collected and frozen, and later thawed for ex vivo treatment. Tissue culture experiments or procedures lasting longer than a few days using living cells or tissue are typically considered to be “zzz vitro " though in certain embodiments, this term can be used interchangeably with ex vivo.

[00062] The term “/// vivo" refers generally to activities that take place inside an organism.

[00063] As used herein, the terms “reprogramming” or “dedifferentiation” or “increasing cell potency” or “increasing developmental potency” refer to a method of increasing the potency of a cell or dedifferentiating the cell to a less differentiated state. For example, a cell that has an increased cell potency has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in the non-reprogrammed state. In other words, a reprogrammed cell is one that is in a less differentiated state than the same cell in a nonreprogrammed state.

[00064] As used herein, the term “differentiation” is the process by which an unspecialized (“uncommitted”) or less specialized cell acquires the features of a specialized cell such as, for example, a blood cell or a muscle cell. A differentiated or differentiation- induced cell is one that has taken on a more specialized (“committed”) position within the lineage of a cell. The term “committed”, when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. As used herein, the term “pluripotent” refers to the ability of a cell to form all lineages of the body or soma (i.e., the embryo proper). For example, embryonic stem cells are a type of pluripotent stem cells that are able to form cells from each of the three germs layers, the ectoderm, the mesoderm, and the endoderm. Pluripotency is a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell (e.g., an epiblast stem cell or EpiSC), which is unable to give rise to a complete organism to the more primitive, more pluripotent cell, which is able to give rise to a complete organism (e.g., an embryonic stem cell). [00065] As used herein, the term “induced pluripotent stem cells” or “iPSCs”, refers to stem cells that are produced in vitro from differentiated adult, neonatal or fetal cells that have been induced or changed, i.e., reprogrammed into cells capable of differentiating into tissues of all three germ or dermal layers: mesoderm, endoderm, and ectoderm. In some embodiments, the reprogramming process uses reprogramming factors and/or small molecule chemical driven methods. The iPSCs produced do not refer to cells as they are found in nature.

[00066] As used herein, the term “embryonic stem cell” refers to naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. They do not contribute to the extra-embryonic membranes or the placenta (i.e., are not totipotent).

[00067] As used herein, the term “multipotent stem cell” refers to a cell that has the developmental potential to differentiate into cells of one or more germ layers (i.e., ectoderm, mesoderm and endoderm), but not all three. Thus, a multipotent cell can also be termed a “partially differentiated cell.” Multipotent cells are known in the art, and examples of multipotent cells include adult stem cells, such as for example, hematopoietic stem cells and neural stem cells. “Multipotent” indicates that a cell may form many types of cells in a given lineage, but not cells of other lineages. For example, a multipotent hematopoietic cell can form the many different types of blood cells (red, white, platelets, etc.), but it cannot form neurons. Accordingly, the term “multipotency” refers to the state of a cell with a degree of developmental potential that is less than totipotent and pluripotent.

[00068] Pluripotency can be determined, in part, by assessing pluripotency characteristics of the cells. Pluripotency characteristics include, but are 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) ability to differentiate to all three somatic lineages (ectoderm, mesoderm and endoderm); (v) teratoma formation consisting of the three somatic lineages; and (vi) formation of embryoid bodies consisting of cells from the three somatic lineages.

[00069] Two types of pluripotency have previously been described: the “primed” or “metastable” state of pluripotency akin to the epiblast stem cells (EpiSC) of the late blastocyst, and the “naive” or “ground” state of pluripotency akin to the inner cell mass of the early/preimplantation blastocyst. While both pluripotent states exhibit the characteristics as described above, the naive or ground state further exhibits: (i) pre-inactivation or reactivation of the X-chromosome in female cells; (ii) improved clonality and survival during single-cell culturing; (iii) global reduction in DNA methylation; (iv) reduction of H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters; and (v) reduced expression of differentiation markers relative to primed state pluripotent cells. Standard methodologies of cellular reprogramming in which exogenous pluripotency genes are introduced to a somatic cell, expressed, and then either silenced or removed from the resulting pluripotent cells are generally seen to have characteristics of the primed-state of pluripotency. Under standard pluripotent cell culture conditions such cells remain in the primed state unless the exogenous transgene expression is maintained, wherein characteristics of the ground-state are observed.

[00070] As used herein, the term “pluripotent stem cell morphology” refers to the classical morphological features of an embryonic stem cell. Normal embryonic stem cell morphology is characterized by being round and small in shape, with a high nucleus-to-cytoplasm ratio, the notable presence of nucleoli, and typical inter-cell spacing.

[00071] As used herein, the term “subject” refers to any animal, preferably a human patient, livestock, or other domesticated animal.

[00072] A “pluripotency factor,” or “reprogramming factor,” refers to an agent capable of increasing the developmental potency of a cell, either alone or in combination with other agents. Pluripotency factors include, without limitation, polynucleotides, polypeptides, and small molecules capable of increasing the developmental potency of a cell. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents. [00073] Culture” or “cell culture” refers to the maintenance, growth and/or differentiation of cells in an in vitro environment. “Cell culture media,” “culture media” (singular “medium” in each case), “supplement” and “media supplement” refer to nutritive compositions that cultivate cell cultures.

[00074] Cultivate” or “maintain” refers to the sustaining, propagating (growing) and/or differentiating of cells outside of tissue or the body, for example in a sterile plastic (or coated plastic) cell culture dish or flask. “Cultivation” or “maintaining” may utilize a culture medium as a source of nutrients, hormones and/or other factors helpful to propagate and/or sustain the cells. [00075] As used herein, the term “mesoderm” refers to one of the three germinal layers that appears during early embryogenesis and which gives rise to various specialized cell types including blood cells of the circulatory system, muscles, the heart, the dermis, skeleton, and other supportive and connective tissues.

[00076] 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-to- hematopoietic transition. The development of hematopoietic cells in the embryo proceeds sequentially from lateral plate mesoderm through the hemangioblast to the definitive hemogenic endothelium and hematopoietic progenitors.

[00077] The terms “hematopoietic stem and progenitor cells,” “hematopoietic stem cells,” “hematopoietic progenitor cells,” or “hematopoietic precursor cells” refer to cells which are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation and include, multipotent hematopoietic stem cells (hematoblasts), myeloid progenitors, megakaryocyte progenitors, erythrocyte progenitors, and lymphoid progenitors. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T cells, B cells, NK cells). The term “definitive hematopoietic stem cell” as used herein, refers to CD34⁺ hematopoietic cells capable of giving rise to both mature myeloid and lymphoid cell types including T lineage cells, NK lineage cells and B lineage cells. Hematopoietic cells also include various subsets of primitive hematopoietic cells that give rise to primitive erythrocytes, megakarocytes and macrophages.

[00078] As used herein, the terms “T lymphocyte” and “T cell” are used interchangeably and refer to a principal type of white blood cell that completes maturation in the thymus and that has various roles in the immune system, including the identification of specific foreign antigens in the body and the activation and deactivation of other immune cells. A T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. The T cell can be a CD3⁺ cell. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4⁺/CD8⁺ double positive T cells, CD4⁺ helper T cells (e.g., Thl and Th2 cells), CD8⁺ 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, regulator T cells, gamma delta T cells (y5 T cells), and the like. Additional types of helper T cells include cells such as Th3 (Treg), Thl7, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tern cells and TEMRA cells). The term “T cell” can also refer to a genetically engineered T cell, such as a T cell modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). A T cell or T cell-like effector cell can also be differentiated from a stem cell or progenitor cell (“a derived T cell” or “a derived T cell like effector cell”, or collectively, “a derivative T lineage cell”). A derived T cell like effector cell may have a T cell lineage in some respects, but at the same time has one or more functional features that are not present in a primary T cell. In this application, a T cell, a T cell like effector cell, a derived T cell, a derived T cell like effector cell, or a derivative T lineage cell, are collectively termed as “a T lineage cell”. In some embodiments, the derivative T lineage cell is an iPSC-derived T cell obtained by differentiating an iPSC, which cells are also referred to herein as “iT” cells.

[00079] CD4⁺ T cells” refers to a subset of T cells that express CD4 on their surface and are associated with cell-mediated immune response. They are characterized by the secretion profiles following stimulation, which may include secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4 and IL10. “CD4” molecules are 55-kD glycoproteins originally defined as differentiation antigens on T-lymphocytes, but also found on other cells including monocytes/macrophages. CD4 antigens are members of the immunoglobulin supergene family and are implicated as associative recognition elements in MHC (major histocompatibility complex) class Il-restricted immune responses. On T-lymphocytes they define the helper/inducer subset.

[00080] CD8⁺ T cells” refers to a subset of T cells which express CD8 on their surface, are MHC class I-restricted, and function as cytotoxic T cells. “CD8” molecules are differentiation antigens found on thymocytes and on cytotoxic and suppressor T-lymphocytes. CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility complex class I-restricted interactions.

[00081] As used herein, the term “NK cell” or “Natural Killer cell” refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD 16 and the absence of the T cell receptor (CD3). As used herein, the terms “adaptive NK cell” and “memory NK cell” are interchangeable and refer to a subset of NK cells that are phenotypically CD3" and CD56⁺, expressing at least one of NKG2C and CD57, and optionally, CD16, but lack expression of one or more of the following: PLZF, SYK, FceRy, and EAT-2. In some embodiments, isolated subpopulations of CD56⁺ NK cells comprise expression of CD 16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A and/or DNAM- 1. CD56⁺ can be dim or bright expression. An NK cell, or an NK cell-like effector cell may be differentiated from a stem cell or progenitor cell (“a derived NK cell” or “a derived NK cell like effector cell”, or collectively, “a derivative NK lineage cell”). A derivative NK cell like effector cell may have an NK cell lineage in some respects, but at the same time has one or more functional features that are not present in a primary NK cell. In this application, an NK cell, an NK cell like effector cell, a derived NK cell, a derived NK cell like effector cell, or a derivative NK lineage cell, are collectively termed as “an NK lineage cell”. In some embodiments, the derivative NK lineage cell is an iPSC-derived NK cell obtained by differentiating an iPSC, which cells are also referred to herein as “iNK” cells.

[00082] As used herein, the term “NKT cells” or “natural killer T cells” refers to CD Id- restricted T cells, which express a T cell receptor (TCR). Unlike conventional T cells that detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD Id, a non-classical MHC molecule. Two types of NKT cells are recognized. Invariant or type I NKT cells express a very limited TCR repertoire - a canonical a-chain (Va24-Jal8 in humans) associated with a limited spectrum of P chains (Vpi l in humans). The second population of NKT cells, called non-classical or non-invariant type II NKT cells, display a more heterogeneous TCR aP usage. Type I NKT cells are considered suitable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified with the expression of at least one or more of the following markers, TCR Va24-Jal8, Vbll, CDld, CD3, CD4, CD8, aGalCer, CD161 and CD56.

[00083] As used herein, the term “isolated” or the like refers to a cell, or a population of cells, which has been separated from its original environment, i.e., the environment of the isolated cells is substantially free of at least one component as found in the environment in which the “un-isolated” reference cells exist. The term includes a cell that is removed from some or all components as it is found in its natural environment, for example, isolated from a tissue or biopsy sample. The term also includes a cell that is removed from at least one, some or all components as the cell is found in non-naturally occurring environments, for example, isolated form a cell culture or cell suspension. Therefore, an “isolated cell” is partly or completely separated from at least one component, including other substances, cells or cell populations, as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments. Specific examples of isolated cells include partially pure cell compositions, substantially pure cell compositions and cells cultured in a medium that is non-naturally occurring. Isolated cells may be obtained by separating the desired cells, or populations thereof, from other substances or cells in the environment, or by removing one or more other cell populations or subpopulations from the environment.

- l- [00084] As used herein, the term “purify” or the like refers to increasing purity. For example, the purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[00085] As used herein, the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or a mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either 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 mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as “encoding” the protein or other product of that gene or cDNA.

[00086] A “construct” refers to a macromolecule or complex of molecules comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo. A “vector,” as used herein refers to any nucleic acid construct capable of directing the delivery or transfer of a foreign genetic material to target cells, where it can be replicated and/or expressed. Thus, the term “vector” comprises the construct to be delivered. A vector can be a linear or a circular molecule. A vector can be integrating or non-integrating. The major 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, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, Sendai virus vectors, and the like.

[00087] By “integration” it is meant that one or more nucleotides of a construct is stably inserted into the cellular genome, i.e., covalently linked to the nucleic acid sequence within the cell's chromosomal DNA. By “targeted integration” it is meant that the nucleotide(s) of a construct is inserted into the cell's chromosomal or mitochondrial DNA at a pre-selected site or “integration site”. The term “integration” as used herein further refers to a process involving insertion of one or more exogenous sequences or nucleotides of the construct, with or without deletion of an endogenous sequence or nucleotide at the integration site. In the case, where there is a deletion at the insertion site, “integration” may further comprise replacement of the endogenous sequence or a nucleotide that is deleted with the one or more inserted nucleotides. [00088] As used herein, the term “exogenous” is intended to mean that the referenced molecule or the referenced activity is introduced into, or is non-native to, the host cell. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the cell. The term “endogenous” refers to a referenced molecule or activity that is present in the host cell. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the cell and not exogenously introduced.

[00089] As used herein, a “gene of interest” or “a polynucleotide sequence of interest” is a DNA sequence that is transcribed into RNA and in some instances translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. A gene or polynucleotide of interest can include, but is not limited to, prokaryotic sequences, cDNAfrom eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, a gene of interest may encode an miRNA, an shRNA, a native polypeptide (i.e., a polypeptide found in nature) or fragment thereof; a variant polypeptide (i.e., a mutant of the native polypeptide having less than 100% sequence identity with the native polypeptide) or fragment thereof; an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, and the like.

[00090] 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 four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. A polynucleotide can include a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), 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- and single-stranded molecules.

[00091] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a molecule having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids of a polypeptide. As used herein, the terms refer to both short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are 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. The polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or a combination thereof.

[00092] “Operably-linked” or “operatively linked,” interchangeable with “operably connected” or “operatively connected,” refers to the association of nucleic acid sequences on a single nucleic acid fragment (or amino acids in a polypeptide with multiple domains) so that the function of one is affected by the other. For example, a promoter is operably-linked with a coding sequence or functional RNA when 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). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. As a further example, a receptor-binding domain can be operatively connected to an intracellular signaling domain, such that binding of the receptor to a ligand transduces a signal responsive to said binding.

[00093] “Fusion proteins” or “chimeric proteins”, as used herein, are proteins created through genetic engineering to join two or more partial or whole polynucleotide coding sequences encoding separate proteins, and the expression of these joined polynucleotides results in a single peptide or multiple polypeptides with functional properties derived from each of the original proteins or fragments thereof. Between two neighboring polypeptides of different sources in the fusion protein, a linker (or spacer) peptide can be added.

[00094] As used herein, the term “genetic imprint” refers to genetic or epigenetic information that contributes to preferential therapeutic attributes in a source cell or an iPSC, and is retainable in the source cell derived iPSCs, and/or the iPSC-derived hematopoietic lineage cells. As used herein, “a source cell” is a non-pluripotent cell that may be used for generating iPSCs through reprogramming, and the source cell derived iPSCs may be further differentiated to specific cell types including any hematopoietic lineage cells. The source cell derived iPSCs, and differentiated cells therefrom are sometimes collectively called “derived” or “derivative” cells depending on the context. For example, derivative effector cells, or derivative NK lineage cells or derivative T lineage cells, as used throughout this application are cells differentiated from an iPSC, as compared to their primary counterpart obtained from natural/native sources such as peripheral blood, umbilical cord blood, or other donor tissues. As used herein, the genetic imprint(s) conferring a preferential therapeutic attribute is incorporated into the iPSCs either through reprogramming a selected source cell that is donor-, disease-, or treatment response- specific, or through introducing genetically modified modalities to iPSCs using genomic editing. In the aspect of a source cell obtained from a specifically selected donor, disease or treatment context, the genetic imprint contributing to preferential therapeutic attributes may include any context-specific genetic or epigenetic modifications which manifest a retainable phenotype, i.e., a preferential therapeutic attribute, that is passed on to derivative cells of the selected source cell, irrespective of the underlying molecular events being identified or not. Donor-, disease-, or treatment response- specific source cells may comprise genetic imprints that are retainable in iPSCs and derived hematopoietic lineage cells, which genetic imprints include but are not limited to, prearranged monospecific TCR, for example, from a viral specific T cell or invariant natural killer T (iNKT) cell; trackable and desirable genetic polymorphisms, for example, homozygous for a point mutation that encodes for the high-affinity CD 16 receptor in selected donors; and predetermined HLA requirements, i.e., selected HLA-matched donor cells exhibiting a haplotype with increased population. As used herein, preferential therapeutic attributes include improved engraftment, trafficking, homing, viability, self-renewal, persistence, immune response regulation and modulation, survival, and cytotoxicity of a derived cell. A preferential therapeutic attribute may also relate to antigen targeting receptor expression; HLA presentation or lack thereof; resistance to tumor microenvironment; induction of bystander immune cells and immune modulations; improved on-target specificity with reduced off-tumor effect; and/or resistance to treatment such as chemotherapy. When derivative cells having one or more therapeutic attributes are obtained from differentiating an iPSC that has genetic imprint(s) conferring a preferential therapeutic attribute incorporated thereto, such derivative cells are also called “synthetic cells”. For example, synthetic effector cells, or synthetic NK cells or synthetic T cells, as used throughout this application are cells differentiated from a genomically modified iPSC, as compared to their primary counterpart obtained from natural/native sources such as peripheral blood, umbilical cord blood, or other donor tissues. In some embodiments, a synthetic cell possesses one or more non-native cell functions when compared to its closest counterpart primary cell.

[00095] The term “enhanced therapeutic property” as used herein, refers to a therapeutic property of a cell that is enhanced as compared to a typical immune cell of the same general cell type. For example, an NK cell with an “enhanced therapeutic property” will possess an enhanced, improved, and/or augmented therapeutic property as compared to a typical, unmodified, and/or naturally occurring NK cell. Therapeutic properties of an immune cell may include, but are not limited to, cell engraftment, trafficking, homing, viability, self-renewal, persistence, immune response regulation and modulation, survival, and cytotoxicity. Therapeutic properties of an immune cell are also manifested by antigen targeting receptor expression; HLA presentation or lack thereof; resistance to tumor microenvironment; induction of bystander immune cells and immune modulations; improved on-target specificity with reduced off-tumor effect; and/or resistance to treatment such as chemotherapy. [00096] As used herein, the term “engager” refers to a molecule, e.g., a fusion polypeptide, which is capable of forming 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 activating the immune cell. Examples of engagers include, but are not limited to, bi-specific T cell engagers (BiTEs), bispecific killer cell engagers (BiKEs), tri-specific killer cell engagers (TriKEs), or multi-specific killer cell engagers, or universal engagers compatible with multiple immune cell types.

[00097] As used herein, the term “surface triggering receptor” refers to a receptor capable of triggering or initiating an immune response, e.g., a cytotoxic response. Surface triggering receptors may be engineered, and may be expressed on effector cells, e.g., a T cell, an NK cell, an NKT cell, a B cell, a macrophage, or a neutrophil. In some embodiments, the surface triggering receptor facilitates bi- or multi- specific antibody engagement between the effector cells and a specific target cell (e.g., a tumor cell) independent of the effector cells’ natural receptors and cell types. Using this approach, one may generate iPSCs comprising a universal surface triggering receptor, and then differentiate such iPSCs into populations of various effector cell types that express the universal surface triggering receptor. By “universal”, it is meant that the surface triggering receptor can be expressed in, and activate, any effector cells irrespective of the cell type, and all effector cells expressing the universal receptor can be coupled or linked to the engagers recognizable by the surface triggering receptor, regardless of the engager’s tumor binding specificities. In some embodiments, engagers having the same tumor targeting specificity are used to couple with the universal surface triggering receptor. In some embodiments, engagers having different tumor targeting specificity are used to couple with the universal surface triggering receptor. As such, one or multiple effector cell types can be engaged to kill one specific type of tumor cells in some cases, and to kill two or more types of tumors in some other cases. A surface triggering receptor generally comprises a co-stimulatory domain for effector cell activation and an anti-epitope that is specific to the epitope of an engager. A bispecific engager is specific to the anti-epitope of a surface triggering receptor on one end, and is specific to a tumor antigen on the other end.

[00098] As used herein, the term “safety switch protein” refers to an engineered protein designed to prevent potential toxicity or otherwise adverse effects of a cell therapy. In some instances, the safety switch protein expression is conditionally controlled to address safety concerns for transplanted engineered cells that have permanently incorporated the gene encoding the safety switch protein into its genome. This conditional regulation could be variable and might include control through a small molecule-mediated post-translational activation and tissue-specific and/or temporal transcriptional regulation. The safety switch protein could mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation and/or antibody-mediated depletion. In some instance, the safety switch protein is activated by an exogenous molecule, e.g., a prodrug, that when activated, triggers apoptosis and/or cell death of a 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 that is administered in the event of an adverse event is activated by the suicide-gene product and kills the transduced cell.

[00099] As used herein, the term “pharmaceutically active proteins or peptides” refers to proteins or peptides that are capable of achieving a biological and/or pharmaceutical effect on an organism. A pharmaceutically active protein has healing curative or palliative properties against a disease and may be administered to ameliorate relieve, alleviate, reverse or lessen the severity of a disease. A pharmaceutically active protein also has prophylactic properties and is used to prevent the onset of a disease or to lessen the severity of such disease or pathological condition when it does emerge. Pharmaceutically active proteins include an entire protein or peptide or pharmaceutically active fragments thereof. The term also includes pharmaceutically active analogs of the protein or peptide or analogs of fragments of the protein or peptide. The term pharmaceutically active protein also refers to a plurality of proteins or peptides that act cooperatively or synergistically to provide a therapeutic benefit. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth suppressing proteins, antibodies or fragments thereof, growth factors, and/or cytokines.

[000100] As used herein, the term “signaling molecule” refers to any molecule that modulates, participates in, inhibits, activates, reduces, or increases, cellular signal transduction. “Signal transduction” refers to the transmission of a molecular signal in the form of chemical modification by recruitment of protein complexes along a pathway that ultimately triggers a biochemical event in the cell. Examples of signal transduction pathways are 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.

[000101] As used herein, the term “targeting modality” refers to a molecule, e.g., a polypeptide, that is genetically incorporated into a cell to promote antigen and/or epitope specificity that includes but is not limited to (i) antigen specificity as it relates to a unique chimeric antigen receptor (CAR) or T cell receptor (TCR), (ii) engager specificity as it relates to monoclonal antibodies or bispecific engagers, (iii) targeting of transformed cells, (iv) targeting of cancer stem cells, and (v) other targeting strategies in the absence of a specific antigen or surface molecule.

[000102] As used herein, the term “specific” or “specificity” can be used to refer to the ability of a molecule, e.g., a receptor or an engager, to selectively bind to a target molecule, in contrast to non-specific or non-selective binding.

[000103] The term “adoptive cell therapy” as used herein refers to a cell-based immunotherapy that relates to the transfusion of autologous or allogeneic lymphocytes, whether the immune cells are isolated from a human donor, or effector cells obtained from in vitro differentiation of a pluripotent cell; whether they are genetically modified or not; or whether they are primary donor cells or cells that have been passaged, expanded, or immortalized, ex vivo, after isolation from a donor.

[000104] As used herein, “lymphodepletion” and “lympho-conditioning” are used interchangeably to refer to the destruction of lymphocytes and T cells, typically prior to immunotherapy. The purpose of lympho-conditioning prior to the administration of an 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, lympho-conditioning is typically accomplished by administering one or more chemotherapeutic agents to the subject prior to a first dose of the adoptive cell therapy. In various embodiments, lympho-conditioning precedes the first dose of the adoptive cell therapy by a few hours to a few days. Exemplary chemotherapeutic agents useful for lympho-conditioning include, but are not limited to, cyclophosphamide (CY), fludarabine (FLU), and those described below. However, a sufficient lymphodepletion through anti-CD38 mAb could provide an alternative conditioning process (e.g., for use in an iNK cell therapy in accordance with various embodiments herein), without or with minimal need of a CY/FLU- based lympho-conditioning procedure, as further described herein.

[000105] As used herein, the term “outpatient” refers to a patient who is not hospitalized overnight, but who visits a hospital, clinic, or associated facility for diagnosis and/or treatment. Thus, an “outpatient setting,” as compared to an “inpatient setting” refers to an environment for providing ambulatory care or outpatient care to a patient where hospitalization for one or more days/nights is not required while receiving treatment and/or diagnosis, thereby reducing overall discomfort to the patient receiving treatment and/or diagnosis, while reducing overall cost for such treatment and/or diagnosis with relative ease in management and coordination. Additionally, an outpatient setting is more readily accessible to a larger population of patients and increases patient availability and patient compliance with a treatment protocol during a trial or course of treatment. [000106] As used herein, “induction therapy,” also called “first-line therapy,” “primary therapy,” or “primary treatment,” refers to a first treatment given to a patient for a particular disease. It is often part of a standard set of treatments, such as surgery followed by chemotherapy and radiation. Thus, an “induction attempt” or “attempt of induction therapy” refers to an initial attempt at treating a particular disease using known and/or conventional therapeutic approaches for the particular disease.

[000107] A “therapeutically sufficient amount,” as used herein, includes within its meaning a non-toxic but sufficient and/or effective amount of a particular therapeutic agent and/or pharmaceutical composition to which it is referring to provide a desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient's general health, the patient's age and the stage and severity of the condition being treated. In particular embodiments, a therapeutically sufficient amount is sufficient and/or effective to ameliorate, reduce, and/or improve at least one symptom associated with a disease or condition of the subject being treated.

[000108] Differentiation of pluripotent stem cells requires a change in the culture system, such as changing the stimuli agents in the culture medium or the physical state of the cells. The most conventional strategy utilizes the formation of embryoid bodies (EBs) as a common and critical intermediate to initiate lineage-specific differentiation. “Embryoid bodies” are three- dimensional clusters that have been shown to mimic embryo development as they give rise to numerous lineages within their three-dimensional area. Through the differentiation process, typically a few hours to days, simple EBs (for example, aggregated pluripotent stem cells elicited to differentiate) continue maturation and develop into a cystic EB at which time, typically days to a few weeks, they are further processed to continue differentiation. EB formation is initiated by bringing pluripotent stem cells into close proximity with one another in three-dimensional multilayered clusters of cells. Typically, this is achieved by one of several methods including allowing pluripotent cells to sediment in liquid droplets, sedimenting cells into “U” bottomed well-plates or by mechanical agitation. To promote EB development, the pluripotent stem cell aggregates require further differentiation cues, as aggregates maintained in pluripotent culture maintenance medium do not form proper EBs. As such, the pluripotent stem cell aggregates need to be transferred to differentiation medium that provides eliciting cues towards the lineage of choice. EB-based culture of pluripotent stem cells typically results in generation of differentiated cell populations (i.e., ectoderm, mesoderm and endoderm germ layers) with modest proliferation within the EB cell cluster. Although proven to facilitate cell differentiation, EBs, however, give rise to heterogeneous cells in variable differentiation states because of the inconsistent exposure of the cells in the three-dimensional structure to the differentiation cues within the environment. In addition, EBs are laborious to create and maintain. Moreover, cell differentiation through EB formation is accompanied with modest cell expansion, which also contributes to low differentiation efficiency.

[000109] In comparison, “aggregate formation,” as distinct from “EB formation,” can be used to expand the populations of pluripotent stem cell derived cells. For example, during aggregate-based pluripotent stem cell expansion, culture media are selected to maintain proliferation and pluripotency. Cell proliferation generally increases the size of the aggregates, forming larger aggregates, which can be mechanically or enzymatically dissociated into smaller aggregates to maintain cell proliferation within the culture and increase numbers of cells. As distinct from EB culture, cells cultured within aggregates in maintenance culture media maintain markers of pluripotency. The pluripotent stem cell aggregates require further differentiation cues to induce differentiation.

[000110] As used herein, “monolayer differentiation” is a term referring to a differentiation method distinct from differentiation through three-dimensional multilayered clusters of cells, i.e., “EB formation.” Monolayer differentiation, among other advantages disclosed herein, avoids the need for EB formation to initiate differentiation. Because monolayer culturing does not mimic embryo development such as is the case with EB formation, differentiation towards specific lineages is deemed to be minimal as compared to all three germ layer differentiation in EB formation.

[000111] As used herein, a “dissociated cell” or “single dissociated cell” refers to a cell that has been substantially separated or purified away 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, such as 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. Dissociation thus can involve breaking cell interactions with extracellular matrix (ECM) and substrates (e.g., culture surfaces), or breaking the ECM between cells.

[000112] As used herein, a “master cell bank” or “MCB” refers to a clonal master engineered iPSC line, which is a clonal population of iPSCs that have been engineered to comprise one or more therapeutic attributes, have been characterized, tested, qualified, and expanded, and have been shown to reliably serve as the starting cellular material for the production of cell-based therapeutics through directed differentiation in manufacturing settings. In various embodiments, an MCB is maintained, stored, and/or cryopreserved in multiple vessels to prevent genetic variation 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 processes.

[000113] “Functional” as used in the context of genomic editing or modification of iPSC, and derived non-pluripotent cells differentiated therefrom, or genomic editing or modification of non-pluripotent cells and derived iPSCs reprogrammed therefrom, refers to (1) at the gene level — successful knocked-in, knocked-out, knocked-down gene expression, transgenic or controlled gene expression such as inducible or temporal expression at a desired cell development stage, which is achieved through direct genomic editing or modification, or through “passing-on” via differentiation from or reprogramming of a starting cell that is initially genomically engineered; or (2) at the cell level — successful removal, addition, or alteration of a cell function/characteristic via (i) gene expression modification obtained in said cell through direct genomic editing, (ii) gene expression modification maintained in said cell through “passing-on” via differentiation from or reprogramming of a starting cell that is initially genomically engineered; (iii) down-stream gene regulation in said cell as a result of gene expression modification that only appears in an earlier development stage of said cell, or only appears in the starting cell that gives rise to said cell via differentiation or reprogramming; or (iv) enhanced or newly attained cellular function or attribute displayed within the mature cellular product, initially derived from the genomic editing or modification conducted at the iPSC, progenitor or dedifferentiated cellular origin.

[000114] The term “ligand” refers to a substance that forms a complex with a target molecule to produce a signal by binding to a site on the target. The ligand may be a natural or artificial substance capable of specific binding to the target. The ligand may be in the form of a protein, a peptide, an antibody, an antibody complex, a conjugate, a nucleic acid, a lipid, a polysaccharide, a monosaccharide, a small molecule, a nanoparticle, an ion, a neurotransmitter, or any other molecular entity capable of specific binding to a target. The target to which the ligand binds, may be a protein, a nucleic acid, an antigen, a receptor, a protein complex, or a cell. A ligand that binds to and alters the function of the target and triggers a signaling response is called “agonistic” or “an agonist”. A ligand that binds to a target and blocks or reduces a signaling response is “antagonistic” or “an antagonist.”

[000115] The term “antibody” encompasses antibodies and antibody fragments that contain at least one binding site that specifically binds to a particular target of interest, wherein the target may be an antigen, or a receptor that is capable of interacting with certain antibodies. The term “antibody” includes, but is not limited to, an immunoglobulin molecule or an antigen-binding or receptor-binding portion thereof. For example, an NK cell can be activated by the binding of an antibody or the Fc region of an antibody to its Fc-gamma receptors (FcyR), thereby triggering the ADCC (antibody-dependent cellular cytotoxicity) mediated effector cell activation. A specific piece or portion of an antigen or receptor, or a target in general, to which an antibody binds is known as an epitope or an antigenic determinant. The term antibody also includes, but is not limited to, native antibodies and variants thereof, fragments of native antibodies and variants thereof, peptibodies and variants thereof, and antibody mimetics that mimic the structure and/or function of an antibody or a specified fragment or portion thereof, including single chain antibodies and fragments thereof. An antibody may be a murine antibody, a human antibody, a humanized antibody, a camel IgG, single variable new antigen receptor (VNAR), shark heavy-chain antibody (Ig-NAR), a chimeric antibody, a recombinant antibody, a singledomain antibody (dAb), an anti-idiotype antibody, a bi-specific-, multi-specific- or multimeric- antibody, or antibody fragment thereof. Anti-idiotype antibodies are specific for binding to an idiotope of another antibody, wherein the idiotope is an antigenic determinant of an antibody. A bi-specific antibody may be a BiTE (bi-specific T cell engager) or a BiKE (bi-specific killer cell engager), and a multi-specific antibody may be a TriKE (tri-specific Killer cell engager). Nonlimiting 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), minibody, diabody, triabody, tetrabody, single-domain antigen binding fragments (sdAb), camelid heavy-chain IgG and Nanobody® fragments, recombinant heavychain-only antibody (VHH), and other antibody fragments that maintain the binding specificity of the antibody.

[000116] “Fc receptors,” abbreviated “FcR”, are classified based on the type of antibody that they recognize. For example, those that bind the most common class of antibody, IgG, are called Fc-gamma receptors (FcyR), those that bind IgA are called Fc-alpha receptors (FcaR) and those that bind IgE are called Fc-epsilon receptors (FcsR). The classes of FcRs 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 (FcyR) includes several members, FcyRI (CD64), FcyRIIA (CD32), FcyRIIB (CD32), FcyRIIIA (CD 16a), FcyRIIIB (CD 16b), which differ in their antibody affinities due to their different molecular structures.

[000117] Chimeric Fc Receptor,” abbreviated as “CFcR,” is a term used to describe engineered Fc receptors having their native transmembrane and/or intracellular signaling domains modified, or replaced with non-native transmembrane and/or intracellular signaling domains. In some embodiments of the chimeric Fc receptor, in addition to having one of, or both, transmembrane and signaling domains being non-native, one or more stimulatory domains can be introduced to the intracellular portion of the engineered Fc receptor to enhance cell activation, expansion and function upon triggering of the receptor. Unlike chimeric antigen receptor (CAR) which contains antigen binding domain to target antigen, the chimeric Fc receptor binds to an Fc fragment, or the Fc region of an antibody, or the Fc region comprised in an engager or a binding molecule and activating the cell function with or without bringing the targeted cell close in vicinity. For example, a Fey receptor can be engineered to comprise selected transmembrane, stimulatory, and/or signaling domains in the intracellular region that respond to the binding of IgG at the extracellular domain, thereby generating a CFcR. In one example, a CFcR is produced by engineering CD16, a Fey receptor, by replacing its transmembrane domain and/or intracellular domain. To further improve the binding affinity of the CD 16 based CFcR, the extracellular domain of CD64 or the high-affinity variants of CD 16 (Fl 76 V, for example) can be incorporated. In some embodiments of the CFcR where high affinity CD 16 extracellular domain is involved, the proteolytic cleavage site comprising a serine at position 197 is eliminated or is replaced such at the extracellular domain of the receptor is non-cleavable, i.e., not subject to shedding, thereby obtaining a hnCD16 based CFcR.

[000118] CD16, a FcyR receptor, has been identified to have two isoforms, Fc receptors

FcyRIIIa (CD16a) and FcyRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells, which binds monomeric IgG attached to target cells to activate NK cells and facilitate antibody-dependent cell-mediated cytotoxicity (ADCC). “High affinity CD 16,” “non-cleavable CD 16,” or “high affinity non-cleavable CD 16” (abbreviated as hnCD16), as used herein, refers to a natural or non-natural variant of CD16. The wildtype CD16 has low affinity and is subject to ectodomain shedding, a proteolytic cleavage process that regulates the cells surface density of various cell surface molecules on leukocytes upon NK cell activation. F176V and F158V are exemplary CD 16 polymorphic variants having high affinity. A CD 16 variant having the cleavage site (position 195-198) in the membrane-proximal region (position 189-212) altered or eliminated is not subject to shedding. The cleavage site and the membrane-proximal region are described in detail in WO2015/148926 and US Pat. No. 10,464,989, the complete disclosures of which are incorporated herein by reference. The CD 16 S197P variant is an engineered non- cleavable version of CD16. A CD16 variant comprising both F158V and S197P has high affinity and is non-cleavable. Another exemplary high affinity and non-cleavable CD 16 (hnCD16) variant is an engineered CD 16 comprising an ectodomain originated from one or more of the 3 exons of the CD64 ectodomain.

[000119] As used herein, “FT536” is a multiplexed engineered NK cell therapy generated from a clonal master engineered iPSC line, and is engineered with four modalities for enhanced innate immunity: (1) a high-affinity non-cleavable CD16 (hnCD16) Fc receptor; (2) IL15/IL15 receptor fusion (IL15RF); (3) anti -MIC A/B CAR; and (4) CD38 knockout to mitigate NK cell fratricide by CD38-directed monoclonal antibodies and to promote higher rates of glycolysis with improved metabolic fitness and resistance to oxidative stress within the tumor microenvironment.

[000120] In addition, the abbreviations in this application and definitions thereof are provided herein in Table 1.

Table 1:

I. Cells and Compositions Useful for Adoptive Cell Therapies with Enhanced Properties

[000121] Provided herein is a strategy to systematically engineer the regulatory circuitry of a clonal iPSC without impacting the differentiation potency of the iPSC and cell development biology of the iPSC and its derivative cells, while enhancing the therapeutic properties of the derivative cells differentiated from the iPSC. The iPSC-derived cells are functionally improved and suitable for adoptive cell therapies following a combination of selective modalities being introduced to the cells at the level of iPSC through genomic engineering. It was previously unclear whether altered iPSCs comprising one or more provided genetic edits still have the capacity to enter cell development, and/or to mature and generate functional differentiated cells while retaining modulated activities. Unanticipated failures during directed cell differentiation from iPSCs have been attributed to aspects including, but not limited to, development stage specific gene expression or lack thereof, requirements for HLA complex presentation, protein shedding of introduced surface expressing modalities, and need for reconfiguration of differentiation protocols enabling phenotypic and/or functional changes in the cell. The present application has shown that the one or more selected genomic modifications as provided herein does not negatively impact iPSC differentiation potency, and the functional effector cells derived from the engineered iPSC have enhanced and/or acquired therapeutic properties attributable to the individual or combined genomic modifications retained in the effector cells following the iPSC differentiation.

/. MICA/B-CAR

[000122] MICA and MICB are expressed family members of human major histocompatibility complex class I chain-related gene (MIC). The members of the MIC family are highly polymorphic (more than 100 human alleles) but with structurally conserved motifs. Applicable to the genetically engineered iPSC and derivative effector cells thereof may be one or more CAR designs. CAR, a chimeric antigen receptor, is a fusion protein generally including an ectodomain that comprises an antigen recognition region, a transmembrane domain, and an endodomain. In some embodiments, the ectodomain can further include a signal peptide or leader sequence and/or a spacer (also called a hinge). In some embodiments, the endodomain can further comprise a signaling domain, where the signaling domain originates from a cytoplasmic domain of a signal transducing protein specific to T and/or NK cell activation or functioning. In some embodiments, the antigen recognition domain can specifically bind an antigen. In some embodiments, the antigen recognition domain can specifically bind an antigen associated with a disease or pathogen. In some embodiments, the disease-associated antigen is a tumor antigen, wherein the tumor may be a liquid or a solid tumor. In some embodiments, the CAR is suitable to activate T, NK or NKT cells expressing the CAR. In some embodiments, the CAR is NK cell specific for comprising NK-specific signaling components. In certain embodiments, the NK cells are derived from iPSCs comprising the CAR. In some embodiments, the CAR is T cell specific for comprising T cell specific signaling components. In certain embodiments, the T cells are derived from an iPSC comprising the CAR, and the derivative T cells may comprise T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, aP T cells, y5 T cells, or a combination thereof.

[000123] In certain embodiments, the antigen recognition domain comprises a murine antibody, a human antibody, a humanized antibody, a camel Ig, a shark heavy-chain-only antibody (VNAR), Ig NAR, a chimeric antibody, a recombinant antibody, or antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, antigen binding single chain variable fragment (scFv), (scFv)2, disulfide stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen binding fragments (sdAb, Nanobody), recombinant heavy-chain-only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody. In some embodiments, the antigen recognition region of a CAR originates from the binding domain of a T cell receptor (TCR) that targets a tumor associated antigen (TAA).

[000124] In one example, the present specification provides a CAR comprising an antigen recognition region that targets tumor antigen MICA and MICB. In some embodiments of the MICA/B-targeting CAR, the antigen recognition region is a scFV that specifically binds to the conserved a3 domain of MICA and MICB (referred to as “MICA/B-CAR” throughout this application). In one embodiment, the scFV comprises a variable region of the heavy chain represented by an amino acid sequence that is of at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identity to SEQ ID NO: 1, and a variable region of the light chain represented by an amino acid sequence that is of at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identity to SEQ ID NO: 2. In one embodiment of the MICA/B scFV, the scFV is represented by an amino acid sequence that is of at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identity to SEQ ID NO: 3, in which the linker and/or signal peptide are exemplary and are replaceable. In another embodiment of the MICA/B scFV, the scFV is represented by an amino acid sequence that is of at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identity to SEQ ID NO: 4, in which the linker and/or signal peptide are exemplary and their length and sequence can vary. As used herein and throughout the application, the percent identity between two sequences is a function of the number of identical positions shared by the sequences fi.e., % identity = # of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm recognized in the art.

SEQ ID NO: 1

QIQLVQSGPELKKPGETVKVSCKASGYMFTNYAMNWVKQAPEKGLKWMGWINTHTGDPTYADDFKGRIAF SLETSASTAYLQINNLKNEDTATYFCVRTYGNYAMDYWGQGTSVTVSS

(118AA. MICA/B scFV heavy chain (HC))

SEQ ID NO: 2

DIQMTQTTSSLSASLGDRVTISCSASQDISNYLNWYQQKPDGTVKLLIYDTSILHLGVPSRFSGSGSGTD YSLTISNLEPEDIATYYCQQYSKFPRTFGGGTTLEIK

(107AA. MICA/B scFV light chain (LC))

SEQ ID NO: 3 (HC-Linker-LC)

MDFQVQIFSFLLISASVIMSRQ I QLVQS GP EL KKPGETVKVS OKAS GYMFTNYAMNWVKQAP EKGL KWMG WINTHTGDPTYADDFKGRIAFSLETSASTAYLQINNLKNEDTATYFCVRTYGNYAMDYWGQGTSVTVSSG GGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCSASQDISNYLNWYQQKPDGTVKLLIYDTSILHL GVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKFPRTFGGGTTLEIK

(Signal peptide - other signal peptides are also possible; Linker - other linkers are also possible)

SEQ ID NO: 4 (LC-Z fer-HC)

MDFQVQIFSFLLISASVIMSRD I QMTQTTS SL S ASLGDRVT I S C S ASQD I SNYLNWYQQKPDGTVKLL I Y DTSILHLGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKFPRTFGGGTTLEIKGGGGSGGGGSGG GGSQIQLVQSGPELKKPGETVKVSCKASGYMFTNYAMNWVKQAPEKGLKWMGWINTHTGDPTYADDFKGR IAFSLETSASTAYLQINNLKNEDTATYFCVRTYGNYAMDYWGQGTSVTVSS

[000125] Another aspect of the present specification provides a genetically engineered iPSC and its derivative cell, wherein the cell comprises MICA/B tumor antigen targeting specificity and comprises an exogenous polynucleotide encoding at least a MICA/B-CAR. In some embodiments, the iPSC-derived effector cell comprising MICA/B tumor antigen targeting specificity and optionally an exogenous polynucleotide encoding at least a MICA/B-CAR is a T cell. In some embodiments, the iPSC-derived effector cell comprising MICA/B tumor antigen targeting specificity and optionally an exogenous polynucleotide encoding at least a MICA/B- CAR is an NK cell. In some other embodiments, the iPSC-derived effector cell comprising MICA/B tumor antigen targeting specificity and optionally an exogenous polynucleotide encoding at least a MICA/B-CAR is an NKT cell.

[000126] MICA/B as a tumor associated antigen is predominantly expressed in GI epithelium, endothelial cells and fibroblasts, and its expression is induced by cellular/genotoxic stress, and is highly expressed on epithelial and melanoma cancers. The shedding of MICA/B on tumor cells, on the other hand, results in increased soluble MICA/B which is not recognized by NKG2D expressed on NK and T cell subsets, possibly enables tumor evasion/escape and inhibits immunosurveillance. As shown in the present specification, the MICA/B tumor antigen targeted by the MICA/B-CAR as provided herein inhibits surface MICA/B shedding observed in many human and murine tumor cell lines, resulting in an increase in MICA/B cell surface density, reduced soluble shedding of MICA/B, and enhanced NK and/or T cell mediated tumor killing. Capable of targeting and stabilizing tumor cell surface MICA/B, the MICA/B-CAR as provided herein does not interfere with NKG2D binding to the tumor MICA and MICB, and is capable of enhancing immunosurveillance and preventing or reducing tumor evasion through tumor antigen shedding, while activating the immune cells expressing the MICA/B-CAR, including, but not limited to, primary T cells, NK cells, iPSC-derived T lineage cells, and iPSC- derived NK lineage cells to carry out MICA/B specific targeted tumor cell killing. Further, the immune cells carrying the provided MICA/B-CAR are capable of pan MICA/B (tumor) targeting and killing as shown by a wide range of tumor cell types expressing various MICA/B alleles.

[000127] In some embodiments of the MICA/B-CAR, there is a spacer/hinge between the MICA/B binding domain and the transmembrane domain of the CAR. Exemplary spacers that may be included in a CAR are commonly known in the art, including, but not limited to, IgG4 spacers, CD28 spacers, CD8 spacers, or combinations of more than one spacer. The length of the spacers could also vary, from about 25 amino acids up to about 300 amino acids or more. In this application, a spacer of less than 100 amino acids, or less than 50 amino acids, is considered short; whereas a spacer of more than 100 amino acids, or more than 200 amino acids is considered long.

[000128] In some embodiments, the transmembrane domain of a CAR comprises a full length or at least a portion of the native (i.e., wildtype) or modified transmembrane region of a transmembrane protein, including, but not limited to, CD35, CD3s, CD3y, CD3(^, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD 166, 4- IBB, 0X40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2.DL4, KIR2DS1, NKp'30, NKp44, NKp46, NKG2C, NKG2D, and T cell receptor polypeptide. In one embodiment, the MICA/B- CAR comprises a transmembrane domain derived from CD28. In one embodiment, the MICA/B-CAR comprises a transmembrane domain derived from NKG2D.

[000129] In some embodiments, the signaling domain of the endodomain (or intracellular domain) comprises a full length or at least a portion of a signaling molecule, including, but not limited to, CD3< 2B4, DAP10, DAP12, DNAM1, CD137 (4-1BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor (TCR) polypeptide. In one embodiment, the signaling peptide of a CAR disclosed herein comprises an amino acid sequence that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to at least one IT AM (immunoreceptor tyrosine-based activation motif) of CD3(^. [000130] In certain embodiments, the endodomain of a CAR further comprises at least one co-stimulatory signaling region. The co-stimulatory signaling region can comprise a full length or at least a portion of a signaling molecule, including, but not limited to, CD27, CD28, 4- IBB, 0X40, ICOS, PD1, LAG3, 2B4, BTLA, DAP10, DAP12, CTLA4, or NKG2D, or any combination thereof.

[000131] In one embodiment, the CAR applicable to the cells provided herein comprises a co-stimulatory domain derived from CD28, and a signaling domain comprising the native or modified ITAM1 of CD3(^, represented by an amino acid sequence of at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 5. In a further embodiment, the CAR comprising a co-stimulatory domain derived from CD28, and a native or modified ITAM1 of CD3(^ also comprises a hinge domain and trans-membrane domain derived from CD28, wherein an scFv may be connected to the trans-membrane domain through the hinge, and the CAR comprises an amino acid sequence of at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 6, wherein the length and/or the sequence of the hinge/spacer can vary.

SEQ ID NO: 5

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKD TFDALHMQALPPR

(153 a.a. CD28 co-stim + CD3^ITAM)

SEQ ID NO: 6

IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVEVVVGGVEACYSEEVTVAF 11 FWVRS KR SRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDA

LHMQALPPR

(219 a.a. CD28 hinge + CD28 TM + CD28 co-stim + CD3^ITAM)

[000132] In another embodiment, the CAR applicable to the cells provided herein comprises a transmembrane domain derived from NKG2D, a co-stimulatory domain derived from 2B4, and a signaling domain comprising the native or modified CD3(^, represented by an amino acid sequence of at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 7. The CAR comprising a transmembrane domain derived from NKG2D, a co-stimulatory domain derived from 2B4, and a signaling domain comprising the native or modified CD3(^ may further comprise a CD8 hinge, wherein the amino acid sequence of such a structure is of at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 8.

SEQ ID NO: 7

SNLFVASWIAVMIIFRIGMAVAIFCCFFFPSWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTF PGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLS RKELENFDVYSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

(263 a.a NKG2D TM + 2B4 + CD3Q

SEQ ID NO: 8

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDSKLFVASW1AVM11FR1GMAVA1FC CFFFPSWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAY TLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYK QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR

(308 a.a CD8 hinge + NKG2D TM + 2B4 + CD3Q

[000133] Non-limiting CAR strategies further include heterodimeric, conditionally activated CAR through dimerization of a pair of intracellular domain (see for example, U.S. Pat. No. 9,587,020); split CAR, where homologous recombination of antigen binding, hinge, and endodomains to generate a CAR (see for example, U.S. Pub. No. 2017/0183407); multi-chain CAR that allows non-covalent link between two transmembrane domains connected to an antigen binding domain and a signaling domain, respectively (see for example, U.S. Pub. No.

2014/0134142); CARs having bispecific antigen binding domain (see for example, U.S. Pat. No. 9,447,194), or having a pair of antigen binding domains recognizing same or different antigens or epitopes (see for example, U.S. Pat No. 8,409,577), a tandem CAR (see for example, Hegde et al., J Clin Invest. 2016;126(8):3036-3052); inducible CAR (see for example, U.S. Pub. Nos. 2016/0046700, 2016/0058857, 2017/0166877); switchable CAR (see for example, U.S. Pub. No. 2014/0219975); and any other designs known in the art.

[000134] The genomic loci suitable for MICA/B-CAR insertion include loci meeting the criteria of a genome safe harbor as provided herein and gene loci where the knock-down or knockout of the gene in the selected locus as a result of the integration is desired. In some embodiments, the genomic loci suitable for MICA/B CAR insertion include, not are not limited to, AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, RUNX1, B2M, TAPI, TAP2, Tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR a or 0 constant region, NKG2A, NKG2D, CD38, CD25, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT.

[000135] In one embodiment, the iPSC and its derivative cells comprising MICA/B tumor antigen targeting specificity via a MICA/B-CAR, the cells have the CAR inserted in a TCR constant region (TRAC or TRBC), leading to endogenous TCR knockout (TCR^neg), and optionally placing CAR expression under the control of the endogenous TCR promoter. In one particular embodiment of the iPSC derivative cell comprising MICA/B tumor antigen targeting specificity described herein, the cells comprise endogenous TCR knockout (TCR^neg), wherein the derivative cell is a T lineage cell. In another embodiment, the iPSC and its derivative cells comprising MICA/B tumor antigen targeting specificity described herein, the cells are TCR^neg and the CAR is inserted in the NKG2A locus or NKG2D locus, leading to NKG2A or NKG2D knockout, and optionally placing CAR expression under the control of the endogenous NKG2A or NKG2D promoter. In one particular embodiment of the iPSC derivative cell comprising MICA/B tumor antigen targeting specificity described herein, wherein the cell is NKG2A or NKG2D null and, the derivative cell is an NK lineage cell. In yet another embodiment, the iPSC and its derivative cells comprising MICA/B tumor antigen targeting specificity described herein have the CAR inserted in a CD38 coding region, leading to CD38 knockout, and optionally placing CAR expression under the control of the endogenous CD38 promoter. In one embodiment, the iPSC and its derivative cells comprising a MICA/B tumor antigen targeting specificity described herein have the CAR inserted in a CD58 coding region, leading to CD58 knockout. In one embodiment, the iPSC and its derivative cells comprising MICA/B tumor antigen targeting specificity described herein have the CAR inserted in a CD54 coding region, leading to CD54 knockout. In one embodiment, the iPSC and its derivative cells comprising MICA/B tumor antigen targeting specificity described herein have the CAR inserted in a CIS (Cytokine-Inducible SH2-containing protein) coding region, leading to CIS knockout. In one embodiment, the iPSC and its derivative cells comprising MICA/B tumor antigen targeting specificity described herein have the CAR inserted in a CBL-B (E3 ubiquitin-protein ligase CBL-B) coding region, leading to CBL-B knockout. In one embodiment, the iPSC and its derivative cells comprising MICA/B tumor antigen targeting specificity described herein have the CAR inserted in a SOCS2 (E3 ubiquitin-protein ligase CBL-B) coding region, leading to SOCS2 knockout. In one embodiment, the iPSC and its derivative cells comprising MICA/B tumor antigen targeting specificity described herein have the CAR inserted in a CD56 (NCAM1) coding region, leading to NCAM1 knockout. In another embodiment, the iPSC and its derivative cells comprising MICA/B tumor antigen targeting specificity described herein have the CAR inserted in a coding region of any one of PD1, CTLA4, LAG3 and TIM3, leading to the gene knockout at the insertion site. In a further embodiment, the iPSC and its derivative cells comprising MICA/B tumor antigen targeting specificity herein have the CAR inserted in a coding region of TIGIT, leading to TIGIT knockout.

[000136] Provided herein therefore are effector cells obtained from differentiating genomically engineered iPSCs, wherein both the iPSCs and the derivative cells comprise a polynucleotide encoding a MICA/B-CAR, wherein the effector cells are cells from primary sources or derived from iPSC differentiation, or wherein the genetically engineered iPSCs are capable of differentiating into derived effector cells comprising MICA/B tumor antigen targeting specificity described herein. In some embodiments, the primary-sourced or derived effector cells comprising MICA/B tumor antigen targeting specificity described herein are T lineage cells. In some embodiments, the primary-sourced or derived effector cells comprising MICA/B tumor antigen targeting specificity described herein are NK lineage cells.

[000137] Additionally provided in this application is a master cell bank comprising single cell sorted and expanded clonal engineered iPSCs having at least one phenotype as provided herein, including but not limited to, MICA/B tumor antigen targeting specificity via a MICA/B- CAR, wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapy products, including but not limited to derivative NK and T cells, which are well-defined and uniform in composition, and can be mass produced at significant scale in a cost-effective manner.

2. CD38 knockout

[000138] The cell surface molecule CD38 is highly upregulated in multiple hematologic malignancies derived from both lymphoid and myeloid lineages, including multiple myeloma and a CD20 negative B-cell malignancy, which makes it an attractive target for antibody therapeutics to deplete cancer cells. Antibody mediated cancer cell depletion is usually attributable to a combination of direct cell apoptosis induction and activation of immune effector mechanisms such as ADCC (antibody-dependent cell-mediated cytotoxicity). In addition to ADCC, the immune effector mechanisms in concert with the therapeutic antibody may also include phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC).

[000139] Other than being highly expressed on malignant cells, CD38 is also expressed on plasma cells, as well as on NK cells, and activated T and B cells. During hematopoiesis, CD38 is expressed on CD34⁺ stem cells and lineage-committed progenitors of lymphoid, erythroid, and myeloid, and during the final stages of maturation which continues through the plasma cell stage. As a type II transmembrane glycoprotein, CD38 carries out cell functions as both a receptor and a multifunctional enzyme involved in the production of nucleotide-metabolites. As an enzyme, CD38 catalyzes the synthesis and hydrolysis of the reaction from NAD⁺ to ADP- ribose, thereby producing secondary messengers CADPR and NAADP which stimulate release of calcium from the endoplasmic reticulum and lysosomes, critical for the calcium dependent process of cell adhesion. As a receptor, CD38 recognizes CD31 and regulates cytokine release and cytotoxicity in activated NK cells. CD38 is also reported to associate with cell surface proteins in lipid rafts, to regulate cytoplasmic Ca²⁺ flux, and to mediate signal transduction in lymphoid and myeloid cells.

[000140] In malignancy treatment, systemic use of CD38 antigen binding receptor transduced T cells have been shown to lyse the CD38⁺ fractions of CD34⁺ hematopoietic progenitor cells, monocytes, NK cells, T cells and B cells, leading to incomplete treatment responses and reduced or eliminated efficacy because of the impaired recipient immune effector cell function. In addition, in multiple myeloma patients treated with daratumumab, a CD38 specific antibody, NK cell reduction in both bone marrow and peripheral blood was observed, although other immune cell types, such as T cells and B cells, were unaffected despite their CD38 expression (Casneuf et al., Blood Advances. 2017; l(23):2105-2114).

[000141] Without being limited by theories, the present application includes a strategy to leverage the full potential of CD38 targeted cancer treatment by 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 or B cells, by suppressing and/or eliminating these activated lymphocytes using an anti-CD38 antibody such as daratumumab in the recipient of allogeneic effector cells, host allorej ection against these effector cells would be reduced and/or prevented, thereby increasing effector cell survival and persistency. As such, a CD38 antagonist, such as an anti-CD38 antibody, a secreted CD38 specific engager or a CD38-CAR (chimeric antigen receptor) against activation of recipient T, Treg, NK, and/or B cells can be used as a replacement for lymphodepletion using chemotherapy such as Cy/Flu (cyclophosphamide/fludarabine) prior to adoptive cell transferring.

[000142] In addition, when targeting CD38⁺ T and pbNK cells using CD38" effector cells in the presence of anti-CD38 antibodies or CD38 inhibitors, the depletion of CD38⁺ alloreactive cells increases the NAD⁺ (nicotinamide adenine dinucleotide, a substrate of CD38) availability and decreases NAD⁺ consumption related cell death, which, among other advantages, boosts effector cell responses in an immunosuppressive tumor microenvironment and supports cell rejuvenation in aging, degenerative or inflammatory diseases.

[000143] Embodiments provided herein, e.g. for CD38 knockout, are compatible with other components and processes contemplated herein. Some embodiments include generating an iPSC line having MICA/B tumor antigen targeting specificity described herein and a CD38 knockout, a master cell bank comprising single cell sorted and expanded clonal iPSCs, and obtaining CD38 negative (CD38^neg or CD38'^/_) derivative effector cells comprising the MICA/B tumor antigen targeting specificity described herein through directed differentiation of the engineered iPSC line. In some embodiments, the derivative effector cells are protected against fratricide and allorej ection when CD38 targeted therapeutic moi eties are employed with the effector cells, among other advantages including improved metabolic fitness, increased resistance to oxidative stress and inducing a protein expression program in the effector cell that enhances cell activation and effector function. In addition, anti-CD38 monoclonal antibody therapy significantly depletes a patient’s activated immune system without adversely affecting the patient’s hematopoietic stem cell compartment. A CD38^neg derivative cell has the ability to resist CD38 antibody mediated depletion, and may be effectively administered in combination with an anti-CD38 antibody or CD38-CAR without the use of toxic conditioning agents and thus reduce and/or replace chemotherapy-based lymphodepletion.

[000144] In one embodiment as provided herein, the CD38 knockout in an iPSC line is a bi- allelic knockout. In another embodiment, knocking out CD38 at the same time as inserting one or more transgenes including a MICA/B-CAR, as provided herein, at a selected position in CD38 can be achieved, for example, by a CD38-targeted knock-in/knockout (CD38-KI/KO) construct. In some embodiments of the construct, the construct comprises a pair of CD38 targeting homology arms for position-selective insertion within the CD38 locus. In some embodiments, the preselected targeting site is within an exon of CD38. The CD38-KI/KO constructs provided herein allow the transgene(s) to express either under the CD38 endogenous promoter or under an exogenous promoter comprised in the construct. When two or more transgenes are to be inserted at a selected location in the CD38 locus, a linker sequence, for example, a 2A linker or IRES, is placed between any two transgenes. The 2A linker encodes a self-cleaving peptide derived from FMDV, ERAV, PTV-I, and TaV (referred to as “F2A”, “E2A”, “P2A”, and “T2A”, respectively), allowing for separate proteins to be produced from a single translation. In some embodiments, insulators are included in the construct to reduce the risk of transgene and/or exogenous promoter silencing. The exogenous promoter comprised in a CD38-KI/KO construct may be CAG, or other constitutive, inducible, temporal-, tissue-, or cell type- specific promoters including, but not limited to CMV, EFla, PGK, and UBC. In one embodiment, the MICA/B-CAR is inserted in the CD38 locus to simultaneously knockout CD38 in iPSC.

[000145] As such, this application provides an iPSC and derivative cells therefrom comprising MICA/B tumor antigen targeting specificity via a MICA/B-CAR, where the cells further comprise a CD38 knockout. In various embodiments, said iPSC is capable of directed differentiation to produce functional derivative hematopoietic cells including, but not limited to, mesodermal cells with definitive hemogenic endothelium (HE) potential, definitive HE, CD34⁺ hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitors (MPP), 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, the CD38 negative effector cells are NK lineage cells derived from iPSCs. In some embodiments, the CD38 negative effector cells are T lineage cells derived from iPSCs. In some embodiments, the CD38 negative iPSC and its derivative cells comprise one or more additional genomic edits as described herein, including but not limited to, MICA/B tumor antigen targeting specificity via a MICA/B-CAR, and may further comprise one or more additional engineered modalities described herein.

[000146] Additionally provided in this application is a master cell bank comprising single cell sorted and expanded clonal engineered iPSCs having at least one phenotype as provided herein, including but not limited to, MICA/B tumor antigen targeting specificity via a MICA/B- CAR, wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapy products, including but not limited to derivative NK and T cells, which are well-defined and uniform in composition, and can be mass produced at significant scale in a cost-effective nner.

3. CD16 knock-in

[000147] CD16 has been identified as two isoforms, Fc receptors FcyRIIIa (CD16a; NM_000569.6) and FcyRIIIb (CD16b; NM_000570.4). CD16a is a transmembrane protein expressed by NK cells, which binds monomeric IgG attached to target cells to activate NK cells and facilitate antibody-dependent cell-mediated cytotoxicity (ADCC). CD 16b is exclusively expressed by human neutrophils. “High affinity CD 16,” “non-cleavable CD 16,” “high affinity non-cleavable CD 16,” or “hnCD16,” as used herein, refers to various CD 16 variants. The wildtype CD 16 has low affinity and is subject to ectodomain shedding, a proteolytic cleavage process that regulates the cells surface density of various cell surface molecules on leukocytes upon NK cell activation. F176V (also called F158V in some publications) is an exemplary CD 16 polymorphic variant having high affinity; whereas an S197P variant is an example of a genetically engineered non-cleavable version of CD 16. An engineered CD 16 variant comprising both Fl 76V and S197P has high affinity and is non-cleavable, which was described in greater detail in International Pub. No. WO2015/148926 and US Pat. No. 10,464,989, the complete disclosures of which are incorporated herein by reference. In addition, a chimeric CD 16 receptor with the ectodomain of CD 16 essentially replaced with at least a portion of CD64 ectodomain can also achieve the desired high affinity and non-cleavable features of a CD 16 receptor capable of carrying out ADCC. In some embodiments, the replacement ectodomain of a chimeric CD 16 comprises one or more of ECI, EC2, and EC3 exons of CD64 (UniPRotKB_P12314 or its isoform or polymorphic variant).

[000148] As such, various embodiments of an exogenous CD 16 introduced to a cell include functional CD 16 variants and chimeric receptors thereof. In some embodiments, the functional CD 16 variant is a high-affinity non-cleavable CD 16 receptor (hnCD16). An hnCD16, in some embodiments, comprises both Fl 76V and S197P; and in some embodiments, comprises Fl 76V and with the cleavage region eliminated. In some other embodiments, a hnCD16 comprises a sequence having identity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage in-between, when compared to any of the exemplary sequences, SEQ ID NOs: 9, 10 and 11, each comprises at least a portion of CD64 ectodomain.

SEQ ID NO: 9

MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQ TSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWK DKLVYNVLYYRNGKAFKFFHWNSNLTILKTNI SHNGTYHCSGMGKHRYTSAGI SVTVKELFPAP VLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSG LYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWFHYQ VSFCL VMVLLFA VDTGL YFS VKTNIR SSTRDWKDHKFKWRKDPQDK

(340 a.a. CD64 domain-based construction; CI)16TM CD16ICD

SEQ ID NO: 10

MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQ TSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWK DKLVYNVLYYRNGKAFKFFHWNSNLTILKTNI SHNGTYHCSGMGKHRYTSAGI SVTVKELFPAP VLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSG LYWCEAATEDGNVLKRSPELELQVLGLFF P PG YQ VSFCL VMVLLFA VDTGL YFS VKTNIRSSTR DWKDHKFKWRKDPQDK

(336 a.a. CD64 exon-based construction; CD16TM CD16ICD')

SEQ ID NO: 11

MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQ TSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWK DKLVYNVLYYRNGKAFKFFHWNSNLTILKTNI SHNGTYHCSGMGKHRYTSAGI SVTVKELFPAP VLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSG LYWCEAATEDGNVLKRSPELELQVLGFF P PG YQ VSFCL VMVLLFA VDTGL YFS VKTNIRSSTRD WKDHKFKWRKDPQDK

(335 a.a. CD64 exon-based construction; CD16TM CD16ICD

[000149] Accordingly, provided herein are effector cells or iPSCs genetically engineered to comprise, among other editing as contemplated and described herein, an exogenous CD 16 or variant thereof, wherein the effector cells are cells from primary sources or derived from iPSC differentiation, or wherein the genetically engineered iPSCs are capable of differentiating into derived effector cells comprising the exogenous CD 16 introduced to the iPSCs. In some embodiments, the exogenous CD 16 is a high-affinity non-cleavable CD 16 receptor (hnCD16). In some embodiments, the exogenous CD 16 comprises at least a portion of the CD64 ectodomain. In some embodiments, the exogenous CD 16 is in a form of a CD 16-based chimeric Fc receptor (CFcR) that comprises a transmembrane domain, a stimulatory domain and/or a signaling domain that is not derived from CD 16.

[000150] In some embodiments, the primary-sourced or derived effector cells comprising the exogenous CD 16 or variant thereof are NK lineage cells. In some embodiments, the primary- sourced or derived effector cells comprising the exogenous CD 16 or variant thereof are T lineage cells. In some embodiments, the exogenous CD 16 or functional variants thereof comprised in iPSC or effector cells has high affinity in binding to a ligand that triggers downstream signaling upon such binding. Non-limiting examples of ligands binding to the exogenous CD 16 or functional variants thereof include not only ADCC antibodies or fragments thereof, but also to bi-, tri-, or multi- specific engagers or binders that recognize the CD 16 or CD64 extracellular binding domains of the exogenous CD 16. Examples of bi-, tri-, or multispecific engagers or binders are further described below in this application. As such, at least one of the aspects of the present application provides a derivative effector cell or a cell population thereof, preloaded with one or more pre-selected ADCC antibodies through an exogenous CD 16 expressed on the derivative effector cell, in an amount sufficient for therapeutic use in a treatment of a condition, a disease, or an infection as further detailed this application, wherein the exogenous CD 16 comprises an extracellular binding domain of CD64, or of a CD 16 having Fl 76V and S197P.

[000151] Unlike the endogenous CD 16 expressed by primary NK cells which gets cleaved from the cellular surface following NK cell activation, the various non-cleavable versions of CD 16 in derivative NK avoid CD 16 shedding and maintain constant expression. In derivative NK cells, non-cleavable CD16 increases expression of TNFa and CD107a, indicative of improved cell functionality. Non-cleavable CD 16 also enhances the antibody-dependent cell- mediated cytotoxicity (ADCC), and the engagement of bi-, tri-, or multi- specific engagers. ADCC is a mechanism of NK cell mediated lysis through the binding of CD16 to antibody- coated target cells. The additional high affinity characteristics of the introduced hnCD16 in derived NK cells also enables in vitro loading of an ADCC antibody to the NK cell through hnCD16 before administering the cell to a subject in need of a cell therapy.

[000152] In some embodiments, the derived NK cells comprising MICA/B tumor antigen targeting specificity as described herein, MICA/B tumor antigen targeting specificity is via an exogenous CD 16 or a variant thereof that mediates ADCC when in combination with an anti- MICA/B antibody, or additionally via a MICA/B-CAR. In some embodiments, the derived NK cells comprising MICA/B tumor antigen targeting specificity described herein further comprise CD38 knockout. In some embodiments, the derived NK cells comprising MICA/B tumor antigen targeting specificity described herein and CD38 knockout, are in combination with a CD38 antibody. In some embodiments, the CD38 antibody is daratumumab. In some embodiments, the derived NK cells comprising MICA/B tumor antigen targeting specificity described herein and CD38 knockout, are in combination with one or more of an anti-EGFR antibody (e.g., cetuximab, amivantamab), an anti-HER2 antibody (e.g., trastuzumab or biosimilars, pertuzumab), an anti-PDLl antibody (e.g., avelumab), or a bi-specific antibody targeting EGFR and MET (e.g., amivantamab). In some embodiments, the derived NK cells comprising MICA/B tumor antigen targeting specificity described herein and CD38 knockout, are preloaded with one or more anti-MICA/B antibodies.

[000153] Unlike primary NK cells, mature T cells from a primary source (i.e., natural/native sources such as peripheral blood, umbilical cord blood, or other donor tissues) do not express CD16. It was unexpected that an iPSC comprising an expressed exogenous non-cleavable CD16 did not impair the T cell developmental biology and was able to differentiate into functional derivative T lineage cells that not only express the exogenous CD 16, but also are capable of carrying out function through an acquired ADCC mechanism. This acquired ADCC in the derivative T lineage cell can additionally be used as an approach for dual targeting and/or to rescue antigen escape often occurred with CAR-T cell therapy, where the tumor relapses with reduced or lost CAR-T targeted antigen expression or expression of a mutated antigen to avoid recognition by the CAR (chimerical antigen receptor). When said derivative T lineage cell comprises acquired ADCC through exogenous CD 16, including functional variants, expression, and when an antibody targets a different tumor antigen from the one targeted by the CAR, the antibody can be used to rescue CAR-T antigen escape and reduce or prevent relapse or recurrence of the targeted tumor often seen in CAR-T treatment. Such a strategy to reduce and/or prevent antigen escape while achieving dual targeting is equally applicable to NK cells expressing one or more CARs.

4. Exogenously introduced cytokine signaling complex

[000154] By avoiding systemic high-dose administration of clinically relevant cytokines, the risk of dose-limiting toxicities due to such a practice is reduced while cytokine mediated cell autonomy being established. To achieve lymphocyte autonomy without the need to additionally administer soluble cytokines, a cytokine signaling complex comprising a partial or full peptide of one or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and/or their respective receptors may be introduced to the cell to enable cytokine signaling with or without the expression of the cytokine itself, thereby maintaining or improving cell growth, proliferation, expansion, and/or effector function with reduced risk of cytokine toxicities. In some embodiments, the introduced cytokine and/or its respective native or modified receptor for cytokine signaling (signaling complex) are expressed on the cell surface. In some embodiments, the cytokine signaling is constitutively activated. In some embodiments, the activation of the cytokine signaling is inducible. In some embodiments, the activation of the cytokine signaling is transient and/or temporal. In some embodiments, the transient/temporal expression of a cell surface cytokine/cytokine receptor is through an expression construct carried by a retrovirus, Sendai virus, an adenovirus, an episome, mini-circle, or RNAs, including mRNA.

[000155] For example, in embodiments where the signaling complex is for IL15, the transmembrane (TM) domain can be native to the IL15 receptor or may be modified or replaced with transmembrane domain of any other membrane bound proteins. In various embodiments, the cytokine signaling complex comprises an IL 15 receptor fusion (IL15RF) comprising a full or partial length of IL 15 and a full or partial length of IL 15 receptor (IL15R). In some embodiments, IL 15 and IL15Ra are co-expressed by using a self-cleaving peptide, mimicking trans-presentation of IL 15, without eliminating cis-presentation of IL15. In other embodiments, IL15Ra is fused to IL15 at the C-terminus through a linker, mimicking trans-presentation without eliminating cis-presentation of IL15 as well as ensuring that IL15 is membrane-bound. In other embodiments, IL15Ra with truncated intracellular domain is fused to IL15 at the C- terminus through a linker, mimicking trans-presentation of IL 15, maintaining IL 15 membranebound, and additionally eliminating cis-presentation and/or any other potential signal transduction pathways mediated by a normal IL15R through its intracellular domain. In other embodiments, IL15Ra is fused to IL 15 without an intracellular domain (IL 15 A), as described in International Pub. Nos. WO 2019/191495 and WO 2019/126748, the entire disclosure of each of which is incorporated herein by reference.

[000156] In various embodiments, such a truncated construct comprises an amino acid sequence of at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 12. In one embodiment of the truncated IL15/IL15Ra, the construct does not comprise the last 4 amino acid residues (KSRQ) of SEQ ID NO: 12, and comprises an amino acid sequence of at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 13.

SEQ ID NO: 12

MDWTWILFLVAAATRVHSGIHVFILGCFSAGLPKTEANWVNVI SDLKKIEDLIQSMHIDATLYT ESDVHPSCKVTAMKCFLLELQVI SLESGDASIHDTVENLI ILANNSLSSNGNVTESGCKECEEL EEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIW VKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPST VTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEI SSHESSHG TPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAI STSTVLLCGLSAVSLLACYLKSRQ (379 a.a.; signal and linker peptides are underlined)

SEQ ID NO: 13

MDWTWILFLVAAATRVHSGIHVFILGCFSAGLPKTEANWVNVI SDLKKIEDLIQSMHIDATLYT ESDVHPSCKVTAMKCFLLELQVI SLESGDASIHDTVENLI ILANNSLSSNGNVTESGCKECEEL EEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIW VKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPST VTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEI SSHESSHG TPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAI STSTVLLCGLSAVSLLACYL (375 a.a.; signal and linker peptides are underlined)

[000157] One having ordinary skill in the art would appreciate that the signal peptide and the linker sequences above are illustrative and in no way limit their variations suitable for use as a signal peptide or linker. There are many suitable signal peptide or linker sequences known and available to those in the art. The ordinary skilled in the art understands that the signal peptide and/or linker sequences may be substituted for another sequence without altering the activity of the functional peptide led by the signal peptide or linked by the linker.

[000158] In iPSCs and derivative cells therefrom comprising both CAR and exogenous cytokine and/or cytokine receptor signaling (signaling complex or “IL”), the CAR and IL may be expressed in separate constructs, or may be co-expressed in a bi-cistronic construct comprising both CAR and IL, or both hnCD16 and IL. In some further embodiments, the signaling complex can be linked to either the 5’ or the 3’ end of a CAR or hnCD16 expression construct through a self-cleaving 2A coding sequence. As such, an IL signaling complex (e.g., IL15 or IL7 signaling complex) and CAR may be in a single open reading frame (ORF). In one embodiment, the signaling complex is comprised in a CAR-2A-IL or an IL-2A-CAR construct. In one embodiment, the signaling complex is comprised in a hnCD16-2A-IL or an IL-2A- hnCD16 construct. When CAR-2A-IL or IL-2A-CAR, or hnCD16-2A-IL or IL-2A-hnCD16, is expressed, the self-cleaving 2 A peptide allows the expressed CAR and IL or hnCD16 and IL, to dissociate, and the dissociated IL can then be presented at the cell surface, with the transmembrane domain anchored in the cell membrane. The CAR-2A-IL or IL-2A-CAR bi- cistronic design, or the hnCD16-2A-IL or IL-2A-hnCD16 bi-cistronic design, allows for coordinated IL signaling complex expression with CAR or hnCD16 both in timing and quantity, and under the same control mechanism that may be chosen to incorporate, for example, an inducible promoter or promoter with temporal or spatial specificity for the expression of the single ORF. Self-cleaving peptides are found in members of the Picomaviridae virus family, including aphthoviruses such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV) and porcine tescho virus- 1 (PTV-I) (Donnelly, et al, J. Gen. Virol, 82, 1027-101 (2001); Ryan, et al., J. Gen. Virol., 'll, 2727-2732 (2001)), and cardioviruses such as Theilovirus (e.g., Theiler’s murine encephalomyelitis) and encephalomyocarditis viruses. The 2A peptides derived from FMDV, ERAV, PTV-I, and TaV are sometimes also referred to as “F2A”, “E2A”, “P2A”, and “T2A”, respectively.

[000159] As such, in various embodiments, the cytokine signaling complex, may be introduced to iPSC using one or more of the construct designs described above, and to its derivative cells upon iPSC differentiation. In addition, provided herein is an induced pluripotent cell (iPSC), a clonal iPSC, a clonal iPS cell line, or iPSC-derived cells comprising a polynucleotide encoding a MICA/B tumor antigen targeting specificity described herein and at least one phenotype as provided herein, including but not limited to, CD38 knockout (CD38 negative), exogenous CD 16 or a variant thereof, a cytokine signaling complex, and/or one or more engineered modalities as disclosed herein, wherein the cytokine signaling complex comprises a partial or full peptide of a cell surface expressed exogenous cytokine and/or a receptor thereof, as described in this section, and wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapy products, which are well-defined and uniform in composition, and can be mass produced at a significant scale in a cost-effective manner. 5. Genetically engineered iPSC line and derivative cells provided herein

[000160] In light of the above, the present application provides an iPSC, an iPS cell line cell, or a population thereof, or a derivative functional cell obtained from differentiating the iPSC, wherein each cell comprises a polynucleotide encoding a MICA/B tumor antigen targeting specificity, and wherein the cell comprises one or more polynucleotides encoding a MICA/B- CAR (chimeric antigen receptor), a CD 16 or a variant thereof that mediates ADCC when in combination with an anti-MICA/B antibody, wherein the cell may further comprise one or more additional genetic modifications as described in the application, and wherein the cell is an eukaryotic cell, an animal cell, a human cell, an induced pluripotent cell (iPSC), an iPSC derived effector cell, an immune cell, or a feeder cell.

[000161] In some embodiments, the functional derivative cells are hematopoietic cells include, but are not limited to, mesodermal cells with definitive hemogenic endothelium (HE) potential, definitive HE, CD34⁺ hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitors (MPP), 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 some embodiments, the functional derivative hematopoietic cells comprise effector cells having one or more functional features that are not present in a counterpart primary T, NK, NKT, and/or B cell.

[000162] Also provided herein is an iPSC comprising a MICA/B tumor antigen targeting specificity described herein, CD38 knockout, and a polynucleotide encoding an exogenous CD 16 or a variant thereof, wherein the iPSC is capable of directed differentiation to produce functional derivative hematopoietic cells. In some embodiments, when an anti-CD38 antibody is used to induce CD 16 mediated enhanced ADCC, the iPSC and/or its derivative effector cells can target the CD38 expressing (tumor) cells without causing effector cell elimination, i.e., reduction or depletion of CD38 expressing effector cells, thereby increasing persistence and/or survival of the iPSC and its effector cell. In some embodiments, the effector cell has increased persistence and/or survival in vivo in the presence of anti-CD38 therapeutic agents, which may be an anti-CD38 antibody. In addition, since CD38 is upregulated on activated lymphocytes such as T or B cells, an anti-CD38 antibody can be used for lymphodepletion thereby eliminating those activated lymphocytes, overcoming allorej ection, and increasing survival and persistency of the CD38 negative effector cells without fratricide in the recipient of the allogeneic effector cell therapy. In some embodiments, the effector cells comprise NK lineage cells. iPSC-derived NK lineage cells comprising CD38 negative and exogenous CD16 or a variant thereof have enhanced cytotoxicity and have reduced NK cell fratricide in the presence of anti-CD38 antibodies.

[000163] Additionally provided is an iPSC comprising a MICA/B tumor antigen targeting specificity described herein, CD38 knockout, exogenous CD 16 or a variant thereof, and a polynucleotide encoding at least one exogenous cytokine signaling complex (IL) to enable cytokine signaling contributing to cell survival, persistence and/or expansion, wherein the iPSC line is capable of hematopoietic differentiation to produce functional derivative effector cells having improved survival, persistency, expansion, and effector function. The exogenously introduced cytokine signaling(s) comprise the signaling of any one, or two, or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In some embodiments, the introduced partial or full peptide of cytokine and/or its respective receptor for cytokine signaling are expressed on the cell surface. In some embodiments, the cytokine signaling is constitutively activated. In some embodiments, the activation of the cytokine signaling is inducible. In some embodiments, the activation of the cytokine signaling is transient and/or temporal. In some embodiments, the transient/temporal expression of a cell surface cytokine/cytokine receptor is through a retrovirus, Sendai virus, an adenovirus, an episome, mini-circle, or RNAs including mRNA. In some embodiments, the exogenous cell surface cytokine and/or receptor comprised in the iPSC or derivative cells thereof enables IL15 signaling.

[000164] In view of the above, also provided herein is an iPSC comprising a MICA/B tumor antigen targeting specificity described herein, a CD38 knockout, exogenous CD 16 or a variant thereof, and an exogenous cytokine signaling complex, and wherein the iPSC is capable of directed differentiation to produce functional derivative hematopoietic cells, wherein the derivative hematopoietic cells include, but are not limited to, mesodermal cells with definitive hemogenic endothelium (HE) potential, definitive HE, CD34⁺ hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitors (MPP), T cell progenitors, NK cell progenitors, myeloid cells, neutrophil progenitors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, macrophages, or a derivative effector cell having one or more functional features that are not present in a counterpart primary T, NK, NKT, and/or B cell.

6. Antibodies for immunotherapy

[000165] In some embodiments, in addition to the genomically engineered effector cells as provided herein, additional therapeutic agents comprising an antibody, or an antibody fragment that targets an antigen associated with a condition, a disease, or an indication may be used with these effector cells in a combinational therapy. Exemplary antigens associated with cancers that may be treated with the combination therapy are shown in Table 2.

Table 2:

[000166] In some embodiments, the antibody is used in combination with a population of the effector cells described herein by concurrent or consecutive administration to a subject. In other embodiments, such antibody or a fragment thereof may be expressed by the effector cells by genetically engineering an iPSC using an exogenous polynucleotide sequence encoding said antibody or fragment thereof, and directing differentiation of the engineered iPSC. In some embodiments, the effector cell expresses an exogenous CD 16 variant, wherein the cytotoxicity of the effector cell 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, the tumor or viral specific antigen activates the administered iPSC-derived effector cells to enhance their killing ability. In some embodiments, the antibodies suitable for combinational treatment as an additional therapeutic agent to the administered iPSC-derived effector cells include, but are not limited to, an anti-EGFR antibody (cetuximab, matuzumab, panitumumab and necitumumab), an anti-HER.2 antibody (trastuzumab or biosimilars, pertuzumab, 4B5, ertumaxomab), an anti- PDL1 antibody (avelumab, durvalumab, pembrolizumab, nivolumab, or atezolizumab), a bi- specific antibody targeting EGFR and MET (amivantamab), and their humanized or Fc modified variants or fragments or their functional equivalents and biosimilars. Exemplary trastuzumab biosimilars include, but are not limited to, trastuzumab-anns (Kanjinti™), trastuzumab-dkst (Ogivri®), trastuzumab -qyyp (Trazimera™), trastuzumab-pkrb (Herzuma®), trastuzumab -dttb (Ontruzant®).

[000167] In some embodiments, antibody for combinational therapy is cetuximab is administered weekly (QW) or every two weeks (Q2W) in an amount of in an amount of (a) about 400 mg/m² when administered QW; or (b) about 500 mg/m² when administered Q2W. In some embodiments, antibody for combinational therapy is trastuzumab is administered QW or every three weeks (Q3W) in an amount of (a) about 2 mg/kg QW for subjects having HER2⁺ mBC; or (b) about 6 mg/kg Q3W for subjects having a HER2⁺ solid tumor other than mBC. In some embodiments, antibody for combinational therapy is avelumab is administered Q2W in an amount of about 800 mg/dose. In some embodiments, antibody for combinational therapy is amivantamab is administered Q2W in an amount of (i) about 1050 mg for subjects weighing <80 kg; or (ii) about 1400 mg for subjects >80 kg. In some embodiments, when avelumab and the effector cells are administered on the same day, the effector cells are administered first. [000168] In some embodiments, an initial dose of the monoclonal antibody is administered in an effective amount at a starting time prior to a first cycle of administering the iPSC-derived effector cells. In some embodiments, the starting time is about 4-10 days prior to the first cycle of administering the iPSC-derived effector cells. In some embodiments, the antibody for combination treatment is cetuximab and wherein the initial dose is a single initial dose of about 400 mg/m² to about 500 mg/m² administered to the subject about 4 days prior to the first cycle of administering the effector cells. In some embodiments, the antibody for combination treatment is trastuzumab and wherein the initial dose is: (i) a single dose of about 4 mg/kg administered to the subject about 4 days prior to the first cycle of administering the effector cells, wherein the subject has HER2⁺ metastatic breast cancer (mBC); or (ii) a single dose of about 8 mg/kg administered to the subject about 4 days prior to the first cycle of administering the effector cells, wherein the subject has a HER2⁺ solid tumor other than mBC. In some embodiments, the antibody for combination treatment is avelumab, and wherein the initial dose is a single initial dose of about 800 mg administered to the subject about 4 days prior to the first cycle of administering the effector cells. In some embodiments, the antibody for combination treatment is amivantamab, and wherein the starting time of the initial dose is about 4-10 days prior to the first cycle of administering the effector cells, and wherein the initial doses of the monoclonal antibody comprise 1-2 weekly (QW) doses of (i) about 1050 mg for subjects weighing <80 kg; or (ii) about 1400 mg for subjects >80 kg, and optionally wherein the first weekly dose of the monoclonal antibody is administered over two consecutive days.

[000169] In some embodiments of the combinational therapy, the iPSC-derived effector cells comprise NK cells comprising a MICA/B tumor antigen targeting specificity described herein, CD38 knockout, exogenous CD 16 or a variant thereof, and exogenous cytokine/receptor, where the cytokine is IL15. In some other embodiments of the combination therapy comprising the derivative cells provided herein and at least one antibody, said antibody is not produced by, or in, the derivative cells and is additionally administered before, with, or after the administering of the derivative cells.

7. Checkpoint inhibitors

[000170] Checkpoints are cell molecules, often cell surface molecules, capable of suppressing or downregulating immune responses when not inhibited. It is now clear that tumors co-opt certain immune-checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumor antigens. Immune checkpoint inhibitors (ICIs) are antagonists capable of reducing checkpoint gene expression or gene products, or deceasing activity of checkpoint molecules, thereby block inhibitory checkpoints, restoring immune system function. The development of checkpoint inhibitors targeting PD1/PDL1 or CTLA4 has transformed the oncology landscape, with these agents providing long term remissions in multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and relapse remains a significant concern. One aspect of the present application provides a therapeutic approach to overcome ICI resistance by including genomically-engineered functional derivative cells as provided herein in a combination therapy with ICI. In some embodiments, the checkpoint inhibitor is used in combination with a population of the effector cells described herein by concurrent or consecutive administration thereof to a subject. In some other embodiments, the checkpoint inhibitor is expressed by the effector cells by genetically engineering an iPSC using an exogenous polynucleotide sequence encoding said checkpoint inhibitor, or a fragment or variant thereof, and directing differentiation of the engineered iPSC.

[000171] In one embodiment of the combination therapy, the derivative cells are NK cells. In addition to exhibiting direct antitumor capacity, the derivative NK cells provided herein have been shown to resist PDL1-PD1 mediated inhibition, and to have the ability to enhance T cell migration, to recruit T cells to the tumor microenvironment, and to augment T cell activation at the tumor site. Therefore, the tumor infiltration of T cells facilitated by the functionally potent genomically-engineered derivative NK cells indicate that said NK cells are capable of synergizing with T cell targeted immunotherapies, including the checkpoint inhibitors, to relieve local immunosuppression and to reduce tumor burden.

[000172] Some embodiments of the combination therapy with the provided derivative NK cells comprise at least one checkpoint inhibitor to target at least one checkpoint molecule. In one embodiment, the derived NK cell for checkpoint inhibitor combination therapy comprises MICA/B tumor antigen targeting specificity as described herein, CD38 knockout, exogenous CD 16 or a variant thereof, and an exogenous cytokine signaling complex. In some embodiments, the above derivative NK cell additionally comprises deletion, disruption, or reduced expression in at least one of TAPI, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene in the chromosome 6p21 region; or introduction of at least one of HLA-E, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A₂AR, CAR, TCR, FC receptor, an engager, and surface triggering receptor for coupling with bi-, multi- specific or universal engagers.

[000173] The above described derivative NK cell may be obtained from differentiating an iPSC clonal line comprising MICA/B tumor antigen targeting specificity, CD38 knockout, exogenous CD 16 or a variant thereof, and an exogenous cell surface cytokine expression, and optionally HLA modification. In some embodiments, above said iPSC clonal line further comprises deletion, disruption, or reduced expression in at least one of TAPI, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene in the chromosome 6p21 region; or introduction of at least one of HLA-E, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, CAR, TCR, Fc receptor, an engager, and surface triggering receptor for coupling with bi-, multi- specific or universal engagers.

[000174] Suitable checkpoint inhibitors for combination therapy with the derivative NK cells as provided herein include, but are not limited to, antagonists of PD1 (Pdcdl, CD279), PDL-1 (CD274), TIM3 (Havcr2), TIGIT (WUCAM and Vstm3), LAG3 (CD223), CTLA4 (CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A₂AR, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD 160, 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 (for example, 2DL1, 2DL2, 2DL3, 3DL1, and 3DL2).

[000175] In some embodiments, the antagonist inhibiting any of the above checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibodies may be murine antibodies, human antibodies, humanized antibodies, a camel Ig, a single variable new antigen receptor (VNAR), a shark heavy-chain antibody (Ig NAR), chimeric antibodies, recombinant antibodies, or antibody fragments thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, single chain antigen binding fragments (scFv), (scFv)2, disulfide stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen binding fragments (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, more easily used, 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 pembrolizumab, nivolumab, or atezolizumab (anti-PDl/anti-PDLl mAb), avelumab (anti-PDLl mAb), durvalumab (anti-PDLl mAb), and any derivatives, functional equivalents, or biosimilars thereof.

[000176] In some embodiments, ICI for combinational therapy is pembrolizumab administered every three weeks (Q3W) or every six weeks (Q6W) in an amount of about 400 mg. In some embodiments, ICI for combinational therapy is nivolumab administered every two weeks (Q2W) or every four weeks (Q4W) in an amount of: (a) about 240 mg when administered Q2W; or (b) about 480 mg when administered Q4W. In some embodiments, ICI for combinational therapy is atezolizumab administered Q2W, Q3W or Q4W in an amount of: (a) about 840 mg when administered Q2W; (b) about 1200 mg when administered Q3W; or (c) about 1680 mg when administered Q4W.

[000177] In some embodiments, an initial dose of the ICI is administered in an effective amount at a starting time prior to a first cycle of administering the iPSC-derived effector cells. In some embodiments, the starting time is about 4-10 days prior to the first cycle of administering the iPSC-derived effector cells. In some embodiments, the ICI for combination treatment is pembrolizumab and is administered in an initial amount of about 200 mg to about 400 mg. In some embodiments, the ICI for combination treatment is nivolumab and is administered in an initial amount of about 240 mg to about 480 mg. In some embodiments, the ICI for combination treatment atezolizumab and is administered in an initial amount of about 840 mg to about 1680 mg.

[000178] In some embodiments, the exogenous polynucleotide sequence encoding the antibody, or a fragment or a variant thereof that inhibits a checkpoint is co-expressed with a chimeric antigen receptor (CAR), either in separate constructs or in a bi-cistronic construct comprising both CAR and the sequence encoding the antibody, or the fragment thereof. In some further embodiments, the sequence encoding the antibody or the fragment thereof can be linked to either the 5’ or the 3’ end of a CAR expression construct through a self-cleaving 2 A coding sequence, illustrated as, for example, CAR-2A-CI or CI-2A-CAR. As such, the coding sequences of the checkpoint inhibitor and the CAR are in a single open reading frame (ORF). When the checkpoint inhibitor is delivered, expressed and secreted as a payload by the derivative effector cells capable of infiltrating the tumor microenvironment (TME), it counteracts the inhibitory checkpoint molecule upon engaging the TME, allowing activation of the effector cells by activating modalities such as CAR or activating receptors.

IL Therapeutic Use of Derivative Immune Cells For Solid Tumors

[000179] Key advancements in cancer immunotherapy include the development of immune checkpoint inhibitor (ICI) monoclonal antibodies (mAbs) that block key inhibitory pathways on T cells and the development of adoptive transfer of immune cells as exemplified by CAR T-cell therapy. Both approaches have led to the development of novel therapeutic regimens that have increased survival in patients with a variety of solid and hematologic tumors. However, the majority of patients will either not respond to treatment or will eventually experience disease relapse. Particularly in solid tumors, the mechanisms of immunotherapy tumor resistance are diverse and include the ability of tumors to form physical and immunologic barriers to immune effector cells such as T cells and NK cells. For example, treatment with anti-programmed cell death- 1/programmed death-ligand 1 (anti-PD-l/PD-Ll) antibodies has shown promising and durable responses either as monotherapy or in combination with chemotherapy in a growing number of solid tumor indications including, but not limited to, melanoma, renal cell carcinoma, lung cancer, head and neck squamous cell carcinoma, and urothelial carcinoma (UC). However, most patients do not have responses or ultimately experience disease progression on anti-PD- l/PD-Ll therapy. Treatment with mAbs targeting human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), and/or mesenchymal -epithelial transition (MET) receptor is standard of care for certain tumors that express these targets, but once patients progress beyond approved mAbs for their disease there are limited treatment options available. Thus, therapies that enhance and potentially add anti-tumor activity to these existing mAb therapeutics are an urgent unmet need.

[000180] FT536 is an off-the-shelf NK-cell product candidate that is manufactured from a clonal master iPSC line that has the potential to address the shortcomings of current adoptive cell therapy. The functional attributes of FT536 include expression of the hnCD16 Fc receptor, IL-15RF, and an anti-MICA/B CAR (FIG. 1). As provided herein, the MICA/B-CAR comprises: (i) a heavy chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 1; (ii) a light chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 2; and/or (iii) a scFV represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NOs: 3 or 4. To date, there are limited clinical data for FT536, including its short-term or long-term efficacy and safety effects. [000181] Pertinent to mechanism of action of FT536, cancer cells evade recognition by NK cells via proteolytic shedding of the natural killer group 2D (NKG2D) ligands MICA and MICB (MICA/B). The released soluble MICA/B protein can interfere with NKG2D signaling and further inhibit cancer cell recognition and elimination by NK cells. MICA/B expression has been detected in approximately 90%-100% of tumortissue samples in multiple tumor indications including, but not limited to, BC, CRC, NSCLC, gastric, ovarian, and pancreatic cancers with little to no expression found in adjacent nonmalignant tissue. Although the observed differential expression between tumor and nontumor tissue allows tumor cells to be marked for NK-cell- mediated destruction upon expression of MICA/B, which is upregulated by many human cancers in response to cellular stress pathways associated with malignant transformation such as DNA damage and accumulation of misfolded proteins, tumor cells can escape NK-mediated recognition and destruction via proteolytic shedding of the MICA/B extracellular domain. Proteolytic shedding of the MICA/B extracellular domain increases the serum soluble MICA/B (i.e., cleaved MICA/B), and there is an inverse correlation between serum soluble MICA/B and survival. In addition, MICA/B expression is associated with significantly longer median overall survival (OS) and recurrence free survival but is also inversely correlated with stage of disease, which contributes to the clinical uncertainty of MICA/B targeted immunotherapy including FT536.

1. Methods of treatment using FT536

[000182] The treatment using the derived hematopoietic lineage cells of embodiments disclosed herein, or the compositions provided herein, could be carried out upon symptom presentation, or for relapse prevention. The terms “treating,” “treatment,” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment” as used herein covers any intervention of a disease in a subject and includes: preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; and inhibiting the disease, i.e., arresting its development; or relieving the disease, i.e., causing regression of the disease, or reinduction of disease response to the therapy. Treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is also of particular interest. In some embodiments, the subject in need of a treatment has a disease, a condition, and/or an injury that can be contained, ameliorated, and/or improved in at least one associated symptom by a cell therapy. [000183] In some embodiments, FT536 is provided herein as a monotherapy or combinational therapy in subjects with advanced solid tumors including, but not limited to, NSCLC, CRC, BC, and ovarian or pancreatic cancer. As described herein, FT536 has been specifically designed to leverage the aforementioned role of MICA/B in modulating NK-cell mediated anti-tumor responses. It is hypothesized that an NK cell expressing a MICA/B-CAR and optimized for direct tumor cell cytotoxicity as well as inhibiting MICA/B shedding is capable of facilitating effective anti-tumor immune responses with well designed and tested regimens and procedures to deliver efficacious clinical outcomes. The selection of patient population and combinations of FT536 with mAbs are based on mechanistic features of FT536 described herein.

[000184] Given that NK cells utilize ADCC as a mechanism of anti-tumor activity, and with the expression of hnCD16 to enhance ADCC, FT536 can be used in combination with mAbs that have the capacity to facilitate ADCC and that have shown to be relevant to selected solid tumor indications. These antibodies include avelumab (targeting PD-L1, which, in addition to acting as an immune checkpoint inhibitor (ICI), retains ADCC), trastuzumab and trastuzumab biosimilars (targeting HER2), cetuximab (targeting EGFR) and amivantamab (IgGl bispecific antibody targeting EGFR and MET, and exhibiting ADCC capability through Fc).

[000185] Furthermore, FT536 cells have been shown to express checkpoint molecules including PD-1, and therefore could retain their cytotoxic function through combinations with ICIs. Moreover, resistance to ICIs may occur through loss of MHC-I expression on tumors, which is required for CD8 T-cell activity but would not hinder the anti-tumor activity of FT536. Clinical activity of FT536 when combined with ADCC-competentand non-competent anti- PD1/PD-L1 mAbs (FT536 plus pembrolizumab, nivolumab, or atezolizumab), including in subjects who have failed prior ICI treatment, provides more therapeutic use of FT536 and methods of treatment of a specific indication. Upon showing of the capacity of FT536 in interacting with effector cells of the adaptive immune system, i.e., T cells, and any potential to reverse mechanisms of resistance of tumors to T-cell-based therapy, specifically ICIs targeting the PD-1/PD-L1 axis, FT536 administration in combination with such an ICI can be used as a method for reinduction of an objective response or sustained stable disease in patients who failed to respond in a prior line of treatment using ICI only.

[000186] The present application provides methods of administering FT536 for treatment of a broad array of oncology indications including: (i) as a monotherapy against, e.g., advanced non-small cell lung cancer (NSCLC), advanced or metastatic colorectal cancer (CRC), breast cancer (BC), ovarian cancer, or pancreatic cancer, for greater clinical benefit than current allogeneic NK cell-based therapies; (ii) in combination with approved and investigational tumor- targeting mAbs, including (a) avelumab (anti-PDLl mAb) and is approved for treatment of patients with advanced or metastatic urethelial carcinoma (UC); (b) trastuzumab or biosimilars (anti-HER2 mAb) and is approved for treatment of patients with breast cancer; (c) cetuximab (anti-EGFR mAb) and is approved for treatment of patients with CRC or head and neck squamous cell carcinoma; (d) amivantamab (bi-specific antibody targeting EGFR and MET) and is approved for treatment of patients with advanced NSCLC with documented EGFR driver mutation(s), MET exon 14 skipping mutation, or MET amplification; or (iii) in combination with approved and investigational tumor-targeting, non-ADCC-capable mAbs, including (a) pembrolizumab (anti-PDl/PD-Ll antibody) which is approved for treatment of patients with endometrial carcinoma that is microsatellite instability high (MSI-H) and/or mismatch repair deficient (dMMR); (b) nivolumab (anti-PDl/PD-Ll antibody) which is approved for treatment of patients with NSCLC; and (c) atezolizumab (anti-PDl/PD-Ll antibody) which is approved for treatment of patients with NSCLC. Depending on the mAb used in the combination therapy, said iNK cells could also provide clinical benefit in treating solid cancers.

[000187] The present application provides a method of treatment to patients with advanced HER2⁺ disease, which includes HER2⁺ disease with HER2 overexpression as assessed by IHC or by elevated gene copy number by providing FT536 combined with trastuzumab or approved biosimilars. Patient populations suitable for this FT536 treatment include, for example, those who have disease/cancer cells having a level of surface expression that is lower than is required for approved trastuzumab indications, because the lower HER2 expression threshold is sufficient to elicit anti-tumor activity through enhanced ADCC when FT536 is combined with trastuzumab. The patient population for this treatment could also include those with HER2- mutated non-small cell lung cancer (NSCLC) regardless of HER2 expression level using the combinational treatment comprising FT536 and the HER2-directed antibody drug conjugate trastuzumab-deruxtecan, taking advantage of the ability of FT536 to elicit anti-tumor activity through enhanced ADCC.

[000188] The present application provides a method of treatment to patients with tumors where EGFR overexpression is prevalent by providing FT536 combined with cetuximab. Patient populations suitable for this FT536 treatment include, for example, those who have locally advanced or metastatic colorectal cancer (CRC) irrespective of KRAS/NRAS mutational status. For this patient population, the combination of cetuximab with FT536 would rely on the ability of cetuximab to direct FT536 through hnCD16 to sites of EGFR⁺ disease and elicit antitumor activity via ADCC and not through inhibition of EGFR signaling that is influenced by RAS mutations. The patient population for this treatment could also include those have CRC or head and neck squamous cell carcinoma following progression on prior anti-EGFR antibody therapy for re-induction of an objective response or sustained stable disease in these patients. The patient population for this treatment could further include those having squamous NSCLC where overexpression of EGFR is prevalent by providing FT536 and the anti-EGFR mAb necitumumab in combination with gemcitabine and cisplatin. Although the activity of cetuximab as monotherapy in NSCLC has been modest at approximately 5%, the combination with FT536 could enhance the activity of anti-EGFR mAbs which have ADCC activity leading to higher rates of objective responses.

[000189] The present application provides a method of treatment by providing FT536 combined with amivantamab, a bi-specific antibody that targets both EGFR and MET and has ADCC activity. Amivantamab was recently granted accelerated approval from the FDA for the treatment of NSCLC with exon 20 insertion mutation, which typically has been resistant to EGFR-targeted tyrosine kinase inhibitors (TKIs). Additionally, subjects with MET exon 14 skipping mutation also have demonstrated preliminary responses on amivantamab treatment with 4 of 9 response-evaluable subjects having confirmed partial responses. It is hypothesized that FT536 would further enhance the activity of amivantamab through ADCC in subjects with EGFR or MET driver mutations.

2. Treatment response evaluation

[000190] When evaluating responsiveness to the treatment comprising the derived hematopoietic lineage cells of embodiments disclosed herein, the response can be measured by criteria comprising at least one of: clinical benefit rate, survival until mortality, pathological complete response, semi-quantitative measures of pathologic response, clinical complete remission, clinical partial remission, clinical stable disease, recurrence-free survival, metastasis free survival, disease free survival, circulating tumor cell decrease, circulating marker response, and RECIST (Response Evaluation Criteria In Solid Tumors) criteria.

[000191] A. RECIST Response Criteria:

[000192] Under the Response Evaluation Criteria in Solid Tumors, Version 1.1 (RECIST vl. l), measurable disease is defined as the presence of at least one measurable lesion of >10 mm in diameter by computed tomography (CT) scan. When >1 measurable lesion is present at baseline, all lesions up to a maximum of 5 lesions total (and a maximum of 2 lesions per organ system) are identified as target lesions and will be recorded and measured at baseline. Target lesions are selected based on their size (lesions with the longest diameter) and should be representative of all involved organs.

[000193] Pathological lymph nodes that are defined as measurable and may be identified as target lesions must meet the criterion of a short axis of >15 mm by CT scan. Only the short axis of these nodes will contribute to the baseline sum. All other pathological nodes (those with short axis >10 mm but <15 mm) should be considered nontarget lesions. Nodes that have a short axis <10 mm are considered nonpathological and should not be recorded or followed.

[000194] A sum of diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and reported as the baseline sum diameters. If lymph nodes are to be included in the sum, then as noted above, only the short axis is to be added into the sum. The baseline sum diameters will be used as reference to further characterize any objective tumor regression in the measurable dimension of the disease.

[000195] All other lesions (or sites of disease) including pathological lymph nodes should be identified as nontarget lesions and should also be recorded at baseline. Measurements are not required, and these lesions should be followed as “present,” “absent,” or in rare cases “unequivocal progression.”

[000196] Evaluation of Target Lesions: Under RECIST vl .1, the definition of the criteria used to determine objective tumor response for target lesions are as follows, with abbreviations used throughout this application.

[000197] Complete response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether target or nontarget) must have a reduction in short axis to <10 mm. [000198] Partial response (PR): At least a 30% decrease in the sum of the diameter of target lesions taking as reference the baseline sum diameters.

[000199] Progressive disease (PD): At least a 20% increase, in the sum of diameters of target lesions taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (NOTE: The appearance of one or more new lesions is also considered progression.)

[000200] Stable disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD taking as references the smallest sum diameters while on study.

[000201] Evaluation of Non-Target Lesions: Under RECIST vl.l, the definitions of the criteria used to determine the tumor response for the group of nontarget lesions are as follows, with the understanding that while some nontarget lesions may be measurable, they need not be measured and instead should be assessed only qualitatively at the timepoints of radiographic assessments.

[000202] CR: Disappearance of all nontarget lesions. All lymph nodes must be nonpathological in size (<10 mm short axis).

[000203] Noncomplete response/non-progressive disease: Persistence of 1 or more nontarget lesion(s). [000204] PD: Unequivocal progression of existing non-target lesions. (NOTE: The appearance of 1 or more new lesions is also considered progression.)

[000205] Evaluation of New Lesions: New measurable lesions may be identified if the new lesions meet criteria as defined for baseline target lesion selection and meet the same minimum RECIST vl .1 size requirements of 10 mm in long diameter for non-nodal lesions and a minimum of 15 mm in short axis for nodal lesions. New measurable lesions shall be prioritized according to size with the largest lesions selected as new measured lesions.

[000206] All new lesions not selected as new measurable lesions are considered new nonmeasurable lesions and are followed qualitatively. Only unequivocal progression of new nonmeasurable lesions leads to an overall assessment of PD for the timepoint.

[000207] The finding of a new lesion should be unequivocal, i.e., not attributable to differences in scanning technique, change in imaging modality or findings thought to represent something other than tumor (e.g., some “new” bone lesions may be simply healing or flare of pre-existing lesions). This is particularly important when the patient’s baseline lesions show PR or CR. For example, necrosis of a liver lesion may be reported on a CT scan report as a “new” cystic lesion, which it is not.

[000208] If a new lesion is equivocal, for example because of its small size, continued therapy and follow-up evaluation will clarify if it represents truly new disease. If repeat scans confirm a definite new lesion, then progression is declared using the date of the initial scan.

[000209] Evaluation of Overall Response: The best overall response (BOR) for each patient is the best response recorded from the start of treatment until disease progress! on/recurrence, the initiation of subsequent anti cancer therapy, death, or end of study/ study termination, whichever occurs first (taking reference for PD the smallest measurements recorded since the treatment started). The patient’s best response assignment will depend on the achievement of both measurement and confirmation criteria. The assignment of response for an individual patient, based on both target and non-target lesions, at each assessment timepoint is shown in Table 3.

Table 3: Response at Each Assessment Timepoint for Patients

a If the investigator’s response assessment is difficult to determine due to presence of confounding factors (i.e., tumor flare), then overall response is SD until proven otherwise.

CR = Complete response; NE = Not evaluable; PD = Progressive disease; PR = Partial response; SD = Stable disease

[000210] B. iRECIST Response Criteria'.

[000211] In addition to RECIST Response Criteria, the Modified Response Evaluation Criteria in Solid Tumors, Version 1.1, is used for evaluation of Immune-Based Therapeutics (iRECIST).

[000212] Measurable Disease: Measurable lesions are defined as those that can be accurately measured in >1 dimension (longest diameter to be recorded) as >10 mm with computed tomography (CT) scan (with minimum slice thickness of 5 mm), or >10 mm caliper measurement by clinical exam, or >20 mm by chest X-ray.

[000213] Pathological lymph nodes may also be considered as target on-target lesions. To be considered pathologically enlarged and measurable (target lesion), a lymph node must be >15 mm in short axis when assessed by CT scan (minimum slice thickness of 5 mm). Lymph nodes with a short axis >10 mm but <15 mm should be considered non-target lesions. Lymph nodes that have a short axis <10 mm are considered nonpathologic and should not be recorded as target lesions at baseline.

[000214] Nonmeasurable Disease: Nonmeasurable disease comprises all other lesions (or sites of disease), including small lesions (longest diameter <10 mm or pathological lymph nodes with >10 to <15 mm short axis) as well as leptomeningeal disease, ascites, pleural or pericardial effusions, inflammatory breast disease, lymphangitis cutis or pulmonis, abdominal masses, or organomegaly identified by physical exam that is not measurable by reproducible imaging techniques.

[000215] Bone lesions: Bone scan, positron emission tomography (PET) scan or plain films are not considered adequate imaging techniques to measure bone lesions. However, these techniques can be used to confirm the presence or disappearance of bone lesions. Lytic bone lesions or mixed lytic-blastic lesions, with identifiable soft tissue components, that can be evaluated by cross-sectional imaging techniques such as CT or magnetic resonance imaging (MRI) can be considered as measurable lesions if the soft-tissue component meets the definition of measurability described herein. Blastic bone lesions are considered nonmeasurable. [000216] Cystic lesions that meet the criteria for radiographically defined simple cysts should not be considered as malignant lesions (neither measurable nor nonmeasurable) since they are, by definition, simple cysts. Cystic lesions thought to represent cystic metastases can be considered as measurable lesions, if they meet the definition of measurability described herein. However, if non-cystic lesions are present in the same subject, these are preferred for selection as target lesions.

[000217] Tumor lesions situated in a previously irradiated area, or in an area subjected to other locoregional therapy, are not considered measurable unless there has been demonstrated progression in the lesion. Such lesions should not be selected as target lesions when other measurable lesions are available.

[000218] Target Lesions: Up to a maximum of 5 measurable lesions total (and a maximum of 2 lesions per organ) representative of all involved organs should be identified as target lesions and recorded and measured at baseline. Target lesions should be selected on the basis of their size (lesions with the longest diameter) and their suitability for accurate and reproducible repeated measurements (either by imaging techniques or clinically).

[000219] Non-Target Lesions: All other lesions (or sites of disease) should be identified as non-target lesions and should also be recorded at baseline. Measurements are not required, and these lesions should be followed as “present,” “absent,” or “unequivocal progression.” Non- target lesions include measurable lesions that exceed the maximum number per organ or total of all involved organs as well as non-measurable lesions. It is possible to record multiple non-target lesions involving the same organ as a single item on the case report form (e.g., “multiple enlarged pelvic lymph nodes” or “multiple liver metastases”). Measurements of these lesions are not required, but the presence or absence of each should be noted throughout follow-up.

[000220] Methods of Assessment: CT is the best currently available and reproducible method to measure lesions selected for response assessment. MRI is also acceptable in certain situations (e.g., for body scans). The minimum slice thickness should be 5 mm. If slice thickness is >5 mm, the minimum size for a measurable lesion should be twice the slice thickness.

[000221] Other methods other than CT or MRI to assess response include, but are not limited to: in the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion; Chest X-ray to follow measurable lesions when they are clearly defined and surrounded by aerated lung; functional fluorodeoxyglucose (FDG)-PET data to complement CT data when assessing progressive disease (PD); endoscopy or laparoscopy to confirm complete pathological response or to determine disease relapse; tumor markers to assist the assessment of response or progression; cytology or histology to differentiate between partial response (PR) and CR in residual lesions, or to confirm the neoplastic origin of any effusion for differentiation between CR, PR, stable disease (SD), and PD.

[000222] Determination of Tumor Response and Progression: Determination of tumor responses and disease progression follows the following guidelines.

[000223] All baseline evaluations are performed as closely as possible to the beginning of treatment within the protocol-defined screening period.

[000224] All sites of disease are followed as either target or non-target lesions, as categorized at baseline. All measurable lesions up to a maximum of 2 lesions per organ or 5 lesions in total, representative of all involved organs, are identified as target lesions, while all other lesions (either additional measurable lesions or non-measurable lesions) are classified as non-target lesions.

[000225] All measurements are taken and recorded in metric notation using a ruler or calipers. A sum of the diameters for all target lesions is calculated and reported as the baseline sum of the diameters. For solid tumor lesions, only the long axis is added to the sum and for lymph nodes, only the short axis is added to the sum.

[000226] All lesions (nodal and non-nodal) recorded at baseline should have their actual measurements recorded at each subsequent evaluation, even when very small (e.g., 2 mm). If the lesion has likely disappeared, the measurement is recorded as 0 mm. If a target lesion (nodal or non-nodal) becomes so faint on radiographic imaging that an exact measurement cannot be assigned, then a default value of 5 mm (minimum slice thickness) is assigned.

[000227] The short axis measurement of any lymph node that is considered a target lesion should continue to be recorded even if the node regresses to <10 mm. However, because this may prevent the sum of lesions from being zero even if CR criteria are met, target lymph nodes that regress to <10 mm can be considered to have become normal for purposes of CR calculation.

[000228] At each post-baseline tumor assessment, the sum of the diameters of the index lesions is added together to calculate the sum of target lesions. Comparison of subsequent assessments to the smallest sum of the diameters (nadir tumor burden), including the baseline sum if that is the smallest sum of the diameters during the study, is used to characterize objective tumor progression in the measurable dimensions of the disease.

[000229] New lesions are assessed and categorized as measurable or nonmeasurable. Any new, measurable lesions (as defined herein) (up to a maximum of 5 measurable new lesions total (2 new lesions per organ)) representative of all involved organs, are measured and recorded separately on the case report form but not included in the sum of lesions for target lesions identified at baseline. Other measurable and nonmeasurable lesions are recorded as new lesions

-n- non-target. If a new lesion is identified (thus meeting the criteria for immune-unconfirmed progressive disease (iUPD)) and the subject is clinically stable, treatment is continued. New lesions do not have to resolve for subsequent immune stable disease (iSD) or immune partial response (iPR) providing that the next assessment did not confirm progression. New lesions do not need to meet the criteria for new target lesion to result in iUPD (or immune-confirmed progressive disease (iCPD)). New lesions that are either target or non-target can drive iUPD or iCPD. Progressive disease is confirmed (iCPD) in the new lesion category if the next imaging assessment confirms additional new lesions or a further increase in new lesion size from iUPD (sum of measures increase in new target lesion >5 mm, any increase for new non- target lesions). [000230] Tumor Response and Progression Criteria: Responses are categorized as immune complete response (iCR), immune partial response (iPR), immune stable disease (iSD), immune- unconfirmed progressive disease (iUPD), or immune-confirmed progressive disease (iCPD). In addition, a response category of non-evaluable (NE) is provided for situations in which there is inadequate information to otherwise categorize response status.

[000231] Target Lesions: Response and progression for target lesions is as follows.

[000232] iCR: Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm.

[000233] iPR: At least a 30% decrease in the sum of diameters of target lesions, taking as reference the screening (baseline) sum diameters.

[000234] iUPD: At least a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on study (this includes the screening (baseline) sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. iUPD can be assigned multiple times as long as iCPD is not confirmed at the next assessment.

[000235] iCPD: if the target lesion response was iUPD at the last timepoint and shows a further increase in tumor burden as evidenced (as applicable) by a >5 mm increase in sum of measures of target lesions. However, the criteria for iCPD (after iUPD) are notconsidered to have been met if iCR, iPR, or iSD criteria (compared with baseline and as defined by RECIST vl .1) are met at the next assessment after iUPD. The status is reset and iCR, iPR, or iSD should then be assigned; and if no change is detected, then the timepoint response is iUPD.

[000236] iSD: Neither sufficient shrinkage to qualify for iPR nor sufficient increase to qualify for iUPD.

[000237] NE: In a subject who does not have iUPD or iCPD, the inability to perform a response assessment due to missing data regarding target lesions. [000238] Target Lesions: While some non-target lesions may be measurable, they need not be measured and instead may be assessed only qualitatively at the timepoints specified in the protocol. Response and progression for non-target lesions are as follows.

[000239] iCR: Disappearance of all non-target lesions, normalization of an elevated tumor marker level, and all lymph nodes nonpathologic in size (<10 mm in the short axis).

[000240] Non-iCPD/non-iUPD: Persistence of >1 non-target lesion and/or maintenance of tumor marker level above the upper limit of normal (ULN).

[000241] iUPD: Unequivocal progression of existing non-target lesion representing substantial worsening in non-target disease such that, even in the presence of stable or decreasing target disease, the overall tumor burden appears to have increased. iUPD can be assigned several times as long as iCPD is not confirmed at the next assessment.

[000242] iCPD: Progressive disease in the non-target lesion category is confirmed if subsequent imaging shows a further increase from iUPD. The criteria for iCPD are not judged to have been met if RECIST vl. l criteria for complete response or non-iCR/non-iUPD are met after a previous iUPD. The status is reset and iCR or non-iCR/non-iUPD is assigned. If no change is detected, the timepoint response is iUPD.

[000243] NE: In a subject who does not have iUPD or iCPD, the inability to perform a response assessment due to missing data regarding non-target lesions

[000244] Determination of Response or Progression at Each Timepoint: The occurrence of tumor response or progression will be determined at each timepoint. Table 4 provides a summary of the overall response or progression status at each timepoint.

Table 4: Assignment of Timepoint Response Using iRECIST

a Using RECIST vl.l principles. If no PD occurs, RECIST vl.l and iRECIST categories for CR, PR, and SD would be the same. b In any lesion category. c Previously identified in assessment immediately prior to this timepoint.

[000245] Best Overall Response (BOR): For iRECIST, the immune best overall response (iBOR) recorded from the start of treatment until the end of treatment will be determined. An iUPD will not override a subsequent best overall response of iSD, iPR, or iCR, meaning that iPR or iSD can be assigned even if new lesions have not regressed, or if unequivocal progression (non-target lesions) remains unchanged, providing that the criteria for iCPD are not met.

3. Uncertainties Associated with FT536-Based Mono- or Combination Therapies

[000246] The preclinical data demonstrated the functional profile of FT536 in a non-human setting. There are additional unknown features, uncertainties or risks for human therapeutic use of FT536 that could not be addressed in non-clinical settings. [000247] An adverse event (AE) is any untoward medical occurrence in a patient or clinical study subject temporally associated with the use of study treatment, whether or not considered related to the study treatment. An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease (new or exacerbated) temporally associated with the use of a medicinal (investigational) product, whether or not related to the medicinal (investigational) product.

[000248] Events meeting the definition of AE include, but are not limited to, the following: (i) Any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis) or other safety assessments (e.g., ECG, radiological scans, vital signs measurements), including those that worsen from baseline, considered clinically significant in the medical and scientific judgment of the investigator (i.e., not related to progression of underlying disease), (ii) Exacerbation of a chronic or intermittent pre-existing condition including either an increase in frequency and/or intensity of the condition, (iii) New conditions detected or diagnosed after study treatment administration even though it may have been present before the start of the study, (iv) Signs, symptoms, or the clinical sequelae of a suspected drug-drug interaction, (v) Signs, symptoms, or the clinical sequelae of a suspected overdose of either study treatment s) or a concomitant medication. Overdose per se is not considered an AE/SAE (serious adverse event).

[000249] As described herein, FT536 is engineered to eliminate CD38 expression and to constitutively express hnCD16 and IL-15RF. FT536 expresses an anti-MICA/B-CAR utilizing the antigen binding domain derived from a mAb, which was observed in an immunohistochemistry (IHC)-based tissue cross reactivity assay to bind to the cell membranes of normal tissues including mononuclear cells, hematopoietic precursor cells and epithelial cells in the adrenal cortex, parathyroid, and seminiferous tubules of the testis. To enable cryopreservation FT536 is formulated in dimethyl sulfoxide (DMSO), which has side effects and symptoms generally associated with histamine release. In addition, there may be contributory risks that are driven by the specific mAb in combination with FT536. Therefore, any impact of FT536 associated treatment to hematologic, adrenal, and parathyroid function in human is unknown and needs to be monitored, determined, and managed.

[000250] Acute Allergic/infusion Reaction: Acute allergic/infusion reactions may occur with any treatment, including with the use of CY, FLU, IL-2, and mAbs. Subjects should be closely monitored for the occurrence of acute allergic/anaphylactoid infusion reactions such as rigors and chills, rash, urticaria, hypotension, dyspnea, and angioedema during and following completion of the infusion. Clinical assessments, including vital signs, are described herein. Acute allergic/infusion reactions may also be a manifestation of FT536 immunogenicity given that FT536 is an allogeneic cell product.

[000251] Immunogenicity Risks: FT536 induced immune response may manifest only through laboratory assessments, or may manifest clinically, e.g., as infusion-related reactions with varying degrees of severity, including serious life-threatening anaphylactic reactions. In addition, FT536 immunogenicity may have an impact on FT536 PK, which in turn may have an impact on FT536 anti-tumor activity. Evidence of FT536 immunogenicity and its clinical impact, including various levels of adverse events (AEs) arising from FT536 immunogenicity are monitored.

[000252] DMSO-Related Risks: FT536 is formulated in DMSO to enable cryopreservation. DMSO side effects and symptoms are generally associated with histamine release and include coughing, flushing, rash, chest tightness and wheezing, nausea and vomiting, and cardiovascular instability. Treat by slowing the rate of infusion, medicating with antihistamines, and treating symptoms per institutional practice (AABB 2016).

[000253] Infection: FT536 is cell therapy of human origin. During processing, the cells are in contact with reagents of animal origin, and FT536 has a final formulation that contains albumin (human). As with any product of human and/or animal origin, transmission of infectious disease and/or disease agents by known or unknown agents may occur. FT536 has been extensively tested to minimize the potential risk of disease transmission. However, these measures do not completely eliminate the risk.

[000254] Cytokine Release Syndrome (CRS): CRS is defined as a supraphysiologic response following any immune therapy that results in the activation or engagement of endogenous or infused immune effector cells. Clinical manifestations of CRS include, but are not limited to, cardiac, gastrointestinal, hepatic, coagulation, renal, respiratory, skin, and constitutional (fever, rigors, headaches, malaise, fatigue, arthralgia, nausea, and vomiting) signs and symptoms. Treatment-emergent adverse events (TEAEs) that may be attributed at least in part to CRS include fever, febrile neutropenia, hypotension, acute vascular leak syndrome, renal failure, hypoxia, and pleural effusion. Because the signs and symptoms of CRS are not unique to CRS, other causes of fever, hypotension, and/or hypoxia must be excluded. Notably, bacteremia and other severe infections have been reported concurrent with and even mistaken for CRS.

[000255] While CRS is a clearly defined syndrome with CAR T-cell therapy, it has generally not been observed as a toxicity associated with NK-cell therapies unless administered with systemic cytokines that may independently drive the proliferation and activation of CD8⁺ T cells, e.g., exogenous IL-15 [000256] To consistently characterize its severity, CRS is defined and graded according to ASTCT CRS consensus grading and not by NCI CTCAE. Clinical symptoms as noted above that are not considered related to FT536 should not be reported as CRS. In addition to clinical manifestations, if CRS is suspected, C-reactive protein (CRP) and ferritin levels should be assessed locally, and blood samples are collected for central cytokine analysis.

[000257] Tumor Lysis Syndrome (TLS): TLS is a possible fatal risk associated with antitumor therapy in both hematologic and solid tumors, especially with large tumor burden. TLS symptoms include, but are not limited to, nausea, vomiting, diarrhea, muscle cramps or twitches, weakness, numbness or tingling, fatigue, decreased urination, irregular heart rate, restlessness, irritability, delirium, hallucinations, and seizures. TLS is comprised of abnormal laboratory changes that include hyperuricemia, hyperkalemia, hyperphosphatemia, and hypocalcemia. TLS has been reported to occur within 7 days following chemotherapy across various solid tumor settings, with 10 published reports of TLS cases in patients with gynecological cancer. One case of fatal metabolic syndrome compatible with TLS was reported following NK-cell therapy in a patient with ovarian cancer 5 days after receiving CY. Prophylaxis for and management of TLS should be done in accordance with standard institutional practice.

[000258] Following FT536 administration, subjects are followed closely for signs and symptoms of TLS, with regular clinical (including telemetry where applicable) and laboratory monitoring as described herein. Laboratory abnormalities suggestive of TLS should prompt immediate action by the treating clinicians, and TLS may be treated aggressively per institutional practice.

[000259] Immune Cell-Associated Neurotoxicity Syndrome (ICANS): Neurologic toxicities arising as a result of immune therapies has been termed ICANS, defined as a disorder characterized by a pathologic process involving the central nervous system following any immune therapy that results in the activation or engagement of endogenous or infused immune effector cells. ICANS has been reported with CAR T-cell therapy and bi-specific antibodies such as blinatumomab (Blincyto USPI). The exact mechanism of toxicity in these settings is not known and may not be responsive to cytokine-directed therapy such as tocilizumab but has generally improved with treatment discontinuations and corticosteroids (Blincyto USPI).

[000260] While ICANS is a clearly defined syndrome associated with CAR T-cell-based therapies, it is generally not a toxicity associated with NK-cell therapies. Neurotoxicity resembling ICANS was reported in one trial of adoptively transferred NK cells given with subcutaneous IL- 15, but the mechanism of the toxicity was not well defined. Central nervous system toxicities following CD19-CAR T-cell therapy is characterized by encephalopathy, confusion, delirium, aphasia, obtundation, and seizures (Kymriah USPI; Yescarta USPI). Cases of cerebral edema have also been reported.

[000261] To consistently characterize its severity, ICANS is graded using ASTCT consensus guidelines.

[000262] Neurotoxicity not considered related to FT536 is not considered ICANS. Any neurotoxicity not meeting the criteria for ICANS is graded according to NCI CTCAE.

[000263] Acute Graft-versus Host Disease: Because FT536 is an allogeneic immune effector cell product, there is a potential risk of GvHD even though allogeneic NK-cell therapies have not been associated with GvHD. Acute GvHD assessments are performed with assignment of the overall severity based on the CIBMTR acute GvHD grading scale. Management of GvHD is done in accordance with local institutional practice.

[000264] Risks Associated with Conditioning - Cyclophosphamide (Cl): Warnings and precautions ascribed to CY include: (i) Myelosuppression, immunosuppression, bone marrow failure, and infections; (ii) Urinary tract and renal toxicity including hemorrhagic cystitis, pyelitis, ureteritis, and hematuria (urinary tract obstructions must be corrected prior to receipt of CY); (iii) Cardiotoxicity including myocarditis, myopericarditis, pericardial effusion, arrythmias, and congestive heart failure, which may be fatal (subjects are closely monitored for cardiotoxicity, especially those with risk factors for cardiotoxicity or pre-existing cardiac disease); (iv) Pulmonary toxicity including pneumonitis, pulmonary fibrosis, and pulmonary veno-occlusive disease leading to respiratory failure; (v) Secondary malignancies; (vi) Venoocclusive liver disease, which can be fatal; and (vii) Embryo-fetal toxicity. Adverse reactions reported most often include neutropenia, febrile neutropenia, fever, alopecia, nausea, vomiting, and diarrhea. For the complete safety profile of CY, as well as information regarding supportive care and management of associated toxicities, refer to the current local prescribing information. Considerations of dose modifications due to toxicity should be discussed with the Medical Monitor.

[000265] Risks Associated with Conditioning - Fludarabine (FLU): Warnings and precautions ascribed to FLU include: (i) Severe bone marrow suppression, notably anemia, thrombocytopenia, and neutropenia; (ii) Transfusion-associated GvHD (use only irradiated blood products for transfusions); (iii) Severe central nervous system (CNS) toxicity (severe CNS toxicity was observed in patients treated at FLU doses of 96 mg/m² for 5-7 days. This toxicity was observed in <0.2% of patients treated at FLU doses of 25 mg/m²); (iv) Infections; (v) Renal insufficiency (the subject’s renal function should be monitored closely); (vi) TLS; and (vii) Embryo-fetal toxicity. [000266] Adverse reactions occurring in >30% of subjects treated with FLU include myelosuppression (neutropenia, thrombocytopenia, and anemia), fever, infection, nausea and vomiting, fatigue, anorexia, cough, and weakness. For the complete safety profile of FLU, as well as information regarding supportive care and management of associated toxicities, refer to the current local prescribing information. Considerations of dose modifications due to toxicity should be discussed with the Medical Monitor.

[000267] Myelosuppression, Immunosuppression, Bone Marrow Failure, and

Infections: Some adoptive cell therapies delivered with supportive medications, such as CY and FLU for conditioning, have been reported to cause myelosuppression (neutropenia and/or thrombocytopenia), leukopenia, anemia, and in some cases, bone marrow failure. Hematologic cytopenias could be further compounded by other factors such as underlying disease, concurrent illnesses, and concomitant medications. Close monitoring of complete blood count for the development of cytopenias and infections is strongly recommended. Management of cytopenias and infections, including transfusion support, antimicrobial prophylaxis, and use of growth factors, are done in accordance with standard institutional practice.

[000268] Risks Associated with Interleukin-2: Risks associated with IL-2 (aldesleukin) include constitutional symptoms (flu-like symptoms) including, but not limited to, fever, rash, fatigue, arthralgias, myalgias, and capillary leak syndrome and with injection site nodules and erythema. IL-2 is associated with delayed adverse reactions to iodinated contrast media (Proleukin USPI). Symptoms usually occurred within hours (most commonly 1 to 4 hours) following the administration of contrast media. These reactions include fever, chills, nausea, vomiting, pruritus, rash, diarrhea, hypotension, edema, and oliguria. Management is per institutional practice. For the complete safety profile of aldesleukin, as well as information regarding supportive care and management of associated toxicities, refer to the current local prescribing information. Considerations of dose modifications due to toxicity should be discussed with the Medical Monitor.

[000269] Risks Associated with Monoclonal Antibodies: The information presented herein for combined mAb in Study FT536-101 is based on the respective current local prescribing information for each approved mAb. Refer to the respective product’s current local prescribing information for updated comprehensive safety information. Safety monitoring is performed using institutional standard clinical safety and laboratory assessments and study assessments as outlined in the protocol.

[000270] Avelumab: Avelumab may cause immune-mediated adverse events (AEs) in any organ system, including but not limited to, pneumonitis, liver enzyme elevations and hepatitis, colitis, endocrinopathies (adrenal insufficiency, thyroid disorders, type 1 diabetes), nephritis, exfoliative dermatologic disorders, myocarditis, and neurological toxicities. Infusion reactions may also occur, and there is also a potential for embryo-fetal toxicity. For the complete safety profile of avelumab, refer to the current local prescribing information.

[000271] Pembrolizumab: Pembrolizumab may cause immune-mediated pneumonitis, immune-mediated hepatitis, immune-mediated colitis, immune-mediated nephritis, immune- mediated dermatological reactions, immune-mediated endocrinopathies (hypophysitis, thyroid disorders, adrenal insufficiency, type 1 diabetes), infusion reactions, and embryo-fetal toxicity. For the complete safety profile of pembrolizumab, refer to the current local prescribing information.

[000272] Nivolumab: Nivolumab may cause immune-mediated pneumonitis, immune- mediated hepatitis, immune-mediated colitis, immune-mediated nephritis, immune-mediated dermatological reactions, immune-mediated endocrinopathies (hypophysitis, thyroid disorders, adrenal insufficiency, type 1 diabetes), infections, infusion reactions, and embryo-fetal toxicity. For the complete safety profile of nivolumab, refer to the current local prescribing information. [000273] Atezolizumab: Atezolizumab may cause immune-mediated pneumonitis, immune- mediated hepatitis, immune-mediated colitis, immune-mediated endocrinopathies (hypophysitis, thyroid disorders, adrenal insufficiency, type 1 diabetes), infections, infusion reactions, and embryo-fetal toxicity. For the complete safety profile of atezolizumab, refer to the current local prescribing information.

[000274] Trastuzumab: Trastuzumab may cause cardiomyopathy, infusion reactions, embryo-fetal toxicity, pulmonary toxicity, and exacerbation of chemotherapy-induced neutropenia. For the complete safety profile of trastuzumab, refer to the current local prescribing information.

[000275] Cetuximab: Cetuximab may cause infusion reactions, cardiopulmonary arrest, pulmonary and dermatologic toxicity, and hypomagnesemia/electrolyte abnormalities. For the complete safety profile of cetuximab, refer to the current local prescribing information.

[000276] Amivantamab: Amivantamab may cause infusion-related reactions, interstitial lung disease/pneumonitis, dermatological adverse reactions (e.g., rash including acneiform dermatitis and toxic epidermal necrolysis), ocular toxicity and embryo-fetal toxicity. For the complete safety profile of amivantamab, refer to the current local prescribing information. [000277] Drug Interactions: There are no known interactions with FT536 with other therapies. Refer to the current local prescribing information for study-specific therapies for drug interactions with these agents. In addition to the known and potential risks of FT536, CY, FLU, IL-2, and mAbs, additional risks of the combination treatment including, but not limited to, increased frequency and/or severity of risks known to the mAb may occur. Additional information on the toxicities of approved mAbs whose frequency and severity may be affected by combination treatment with FT536 are described in the respective current local prescribing information. The nature, frequency, and severity of these toxicities in the context of FT536 administration is not currently known.

EXAMPLES

[000278] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1 - Overall Study Design:

[000279] FT536 is an off-the-shelf NK-cell product candidate that is manufactured from a clonal master iPSC line that has the potential to address the shortcomings of current adoptive cell therapy. The functional attributes of FT536 include expression of the hnCD16 Fc receptor, IL-15RF, and an anti-MICA/B CAR. As provided herein, the MICA/B-CAR comprises: (i) a heavy chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 1; (ii) a light chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 2; and/or (iii) a scFV represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NOs: 3 or 4. The study as described herein evaluates FT536 as a monotherapy and in combination with a variety of mAbs, including those that facilitate antibody-dependent cellular cytotoxicity (ADCC; e.g., avelumab targeting PD-L1, trastuzumab targeting HER2, cetuximab targeting EGFR, amivantamab targeting EGFR and MET), as well as non-ADCC competent ICI (e.g., pembrolizumab, nivolumab, atezolizumab and other anti-PD-l/PD-Ll mAbs).

[000280] There are six studies with FT536, with 6 initial cohorts A-F. Cohorts A-F are given FT536 without IL-2 support. Based on data from Cohorts A-F, selected Cohorts AA-FF are open and given FT536 with IL-2 support.

[000281] Study 1: FT536 Monotherapy - Cohorts A/AA is for the following indications: Advanced non-small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer (BC), ovarian cancer, or pancreatic cancer.

[000282] Study 2: FT536 plus Avelumab (ADCC-competent anti-PD-Ll Antibody) - Cohorts B/BB is for the following indications: Select advanced solid tumors for which anti PD- 1/PD-L1 antibodies are approved and whose tumors have documented PD-L1 expression, or advanced CRC whose tumors are microsatellite instability high (MSI-H) and/or mismatch repair deficient (dMMR). [000283] Study 3: FT536 plus Pembrolizumab, Nivolumab, or Atezolizumab (non- ADCC-Competent Anti-PD-l/PD-Ll Antibodies) - Cohorts C/CC is for the following indications: Select advanced solid tumors for which anti-PD-l/PD-Ll antibodies are approved and whose tumors have documented PD-L1 expression, or advanced CRC whose tumors are MSI-H and/or dMMR.

[000284] Study 4: FT536 plus Trastuzumab - Cohorts D/DD is for the following indications: Advanced solid tumors with documented HER2+ expression or NSCLC with documented HER2 mutation. An FDA-approved trastuzumab biosimilar may be used for Cohort D.

[000285] Study 5: FT536 plus Cetuximab - Cohorts E/EE is for the following indications: Advanced squamous NSCLC, CRC, or head and neck squamous cell carcinoma.

[000286] Study 6: FT536 plus Amivantamab - Cohorts F/FF is for the following indications: Advanced NSCLC with documented EGFR driver mutation(s), MET exon 14 skipping mutation, or MET amplification.

[000287] The study begins with Cohort A at dose level 1 (DL 1). Initiation of dose escalation in Cohorts B-F begins at a dose level as low as DL 1 or at a dose level that does not exceed the highest Cohort A dose level. Additional cohorts (AA-FF), in which FT536 is evaluated at a dose level no greater than the maximum tolerated dose/maximum assessed dose (MTD/MAD) as determined in Cohorts A-F combined with the respective mAb and IL-2, are opened.

[000288] Each of the studies includes an up to 28-day screening period and a treatment period consisting of conditioning followed by FT536 as monotherapy or in combination with mAbs. Atotal of 3 doses of FT536 is administered in each cycle. Subjects return to the clinic for a treatment completion visit on Day 29 of each cycle.

[000289] Subjects who have an initial partial response (PR) or complete response (CR) by Response Evaluation Criteria in Solid Tumors, Version 1.1 (RECIST vl.l) but subsequently experience progressive disease (PD), additional treatment cycles (Cycles 3 and 4) consisting of conditioning followed by FT536, following the same schedule as Cycles 1 and 2, are considered. If the subject does not meet criteria for PR/CR followed by PD after the first 2 cycles but demonstrates signs of clinical benefit per the investigator’s judgment, additional treatment with Cycles 3 and 4 are discussed with for approval by the Medical Monitor.

EXAMPLE 2 - Study Population for Each Study Cohort

[000290] Subjects for the FT536 studies described herein have progressed/relapsed, are refractory or intolerant to standard therapies (e.g., surgery, radiation therapy, chemotherapy, hormonal therapy, targeted therapy, and immunotherapy) approved for the specific tumor types specified below, which includes at least one line of therapy, or two lines of therapy for breast cancer (BC) in Cohorts D/DD. Subjects who actively decline standard therapy(s) for the treatment of advanced disease (unresectable or metastatic) are eligible with documented refusal. [000291] The subjects in Cohort A/AA for FT536 Monotherapy have locally advanced or metastatic NSCLC, CRC, BC, ovarian cancer, or pancreatic cancer.

[000292] The subjects in Cohorts B/BB and C/CC for FT536 plus anti-PD-l/PD-Ll Antibodies have locally advanced or metastatic solid tumor indications with documented PD-L1 expression as determined by the appropriate FDA-approved test. The defined cutoffs for PD-L1 expression are as follows:

[000293] (i) NSCLC with a PD-L1 tumor proportion score/tumor cell expression >1% using the 22C3 assay (22C3), >1% tumor cell expression using the 28-8 pharmDx assay (28-8), or tumor-infiltrating immune cells (ICs) covering >10% of tumor area using the SP142 assay (SP142);

[000294] (ii) Gastroesophageal adenocarcinoma with a PD-L1 combined positive score (CPS) >1% (22C3);

[000295] (iii) Head and neck squamous cell carcinoma with a PD-L1 CPS >1% (22C3) or >1% tumor cell expression (28-8);

[000296] (iv) Triple-negative breast cancer with a PD-L1 CPS >10% (22C3) or ICs covering >1% of tumor area (SP142);

[000297] (v) UC with a PD-L1 CPS >10% (22C3), >1% tumor cell expression (28-8), or

ICs covering >5% of tumor area (SP142); or

[000298] (vi) Locally advanced or metastatic CRC that is MSI-H and/or dMMR. If PD-L1 expression test results are only available with an assay not specifically mentioned above, please consult with the Medical Monitor about whether the result meets criteria.

[000299] The subjects in Cohort D/DD for FT536 plus Trastuzumab have locally advanced or metastatic disease and meet one of the two following criteria:

[000300] (i) Locally advanced or metastatic NSCLC with documented expression of HER2 mutation regardless of HER2 expression level; or

[000301] (ii) Any locally advanced or metastatic solid tumor (including NSCLC) with documented HER2⁺ expression level as determined by: (a) >2+ immunohistochemistry (IHC); or (b) average HER2 copy number >4 signals per cell by in situ hybridization (ISH) or >4 copies as determined by next-generation sequencing (NGS).

[000302] In addition, all subjects in Cohort D/DD have received at least one prior line of standard-of-care therapy. For subjects with breast or gastric cancer eligible for standard-of-care anti-HER2 antibody -based therapy, they have also received an anti-HER2 antibody in two or one prior lines of therapy, respectively.

[000303] The subjects in Cohort E/EE for FT536 plus Cetuximab have locally advanced or metastatic squamous NSCLC, head and neck cancer, or CRC. Subjects with CRC that is KRAS/NRAS wild type have relapsed or progressed following prior cetuximab or panitumumab treatment. Subj ects with head and neck cancer have relapsed or progressed following prior cetuximab treatment, or are documented to refuse standard cetuximab-based treatment.

[000304] The subjects in Cohort F/FF for FT536 plus Amivantamab have locally advanced or metastatic NSCLC having at least one of the following:

[000305] (i) EGFR driver mutation(s) and have progressed on or were intolerant to at least one prior line of EGFR tyrosine kinase inhibitor (TKI; e.g., osimertinib, afatinib, gefitinib, erlotinib, dacomitinib) or were not candidates for or declined TKI;

[000306] (ii) MET exon 14 skipping mutation that has progressed on or intolerant of at least one prior line of MET TKI (e.g., capmatinib or tepotinib) or were not candidates for or declined TKI;

[000307] (iii) MET amplification defined as MET/CEP7 ratio >1.8 by FISH; and

[000308] (iv) MET amplification defined as gene copy number >5 as determined by ISH or

NGS.

[000309] For Cohorts B/BB and C/CC, subjects who experienced an anti-PD-l/PD-Ll- related adverse reaction that resulted in discontinuation of the anti-PD-l/PD-Ll antibody, have received an allograft organ transplant or with presence or history of autoimmune disease (e.g., lupus erythematosus, rheumatoid arthritis, Addison’s disease, autoimmune disease associated with lymphoma, Crohn’s disease, ulcerative colitis), except for subjects with isolated vitiligo, atopic dermatitis, controlled hypoadrenalism or hypopituitarism, and controlled thyroid disease are not included.

[000310] Similarly, subjects who experienced a trastuzumab (or biosimilarsj-related adverse reaction that resulted in discontinuation of trastuzumab are not included in Cohorts D/DD.

[000311] Subj ects who experienced a cetuximab-related adverse reaction that resulted in discontinuation of cetuximab are not included in Cohorts EZEE.

[000312] Subjects who experienced an amivantamab-related adverse reaction that resulted in discontinuation of amivantamab are not included in Cohorts F/FF. EXAMPLE 3 - Study Treatments

[000313] The investigational medicinal product for this study is FT536, which is iNK cells suspended in an infusion medium containing albumin (human) and DMSO that is provided in a cryopreserved bag and thawed at the site of administration. The iNK cell therapy is administered as an IV infusion via gravity with an in-line filter. Additional treatments used in this study include cyclophosphamide (CY), fludarabine (FLU), IL-2, avelumab, pembrolizumab, nivolumab, atezolizumab, trastuzumab (or biosimilars), cetuximab, and amivantamab. An overview of the Study Treatments is shown in Table 5.

Table 5: Study Treatments Overview

NOTES: Cohorts A/AA is FT536 monotherapy. Refer to the protocol for the planned dose levels for FT536. a Conditioning will be given prior to treatment with FT536 in Cycle 1. Dose adjustments may be undertaken for conditioning starting with Cycle 2 (refer to the protocol). b Subjects with baseline weight <45 kg will be administered IL-2 at 3 MIU/m².

0

Upon Sponsor approval, an FDA-approved biosimilar may be used.

ADCC = Antibody-dependent cellular cytotoxicity; CAR = Chimeric antigen receptor; CY = Cyclophosphamide; FLU = Fludarabine; IL-2 = Interleukin-2; IV = Intravenous; mAb = Monoclonal antibody; PD-1 = Programmed cell death- 1; PD-L1 = Programmed cell deathligand 1; SC = Subcutaneous [000314] The overall study design schema is shown in FIG. 2 and the study treatment schema is shown in FIG. 3. There is no Day 0, i.e., days within each cycle progress from Day -5 to Day -1, followed by Day 1. Cohort A evaluates FT536 as monotherapy. For Cohorts B, C, D, and E, mAh treatment starts on Day -4, and for Cohort F, mAh treatment starts on Day -11. All mAh treatments may continue for up to 2 years or until progression or unacceptable toxicity. Additional expansion arms (Cohorts AA-FF; FT536 with IL-2 support) are opened after completion of the respective dose escalation of Cohorts A-F (see Example 4). Optional treatment beyond Cycle 2 (Cycles 3 and 4) follows the same schedule as initial FT536 treatment in Cycles 1 and 2. Criteria for treatment beyond Cycle 1 and additional treatment cycle(s) beyond Cycle 2 are provided herein.

[000315] Prior to each treatment cycle with FT536, all subjects receive cyclophosphamide (CY) and fludarabine (FLU) as conditioning. IL-2 support with each dose of FT536 will be explored in dedicated Cohorts AA-FF after MTD/MAD for Cohorts A-F without IL-2 support has been determined.

[000316] Prior to administration of FT536, subjects are pre-medicated with acetaminophen 650 mg orally and diphenhydramine 25 to 50 mg orally or IV before and 4 hours after FT536 administration. Corticosteroids as pre-medication for CY and FLU should be avoided, because of their deleterious effect on iNK cell-based therapy.

[000317] Dosing of FT536 is based on MICA/B-CAR expression of the iNK cells to be administered. Where >80% of administered iNK cells express MICA/B-CAR (which is often the drug product release criteria in manufacturing), the starting dose for iNK cell monotherapy and in combination with the monoclonal antibodies is set to be about 5 * 10⁷ cells per dose to about 3 * 10⁹ cells per dose. A dose level ranging from approximately 3 x 10⁷ to approximately 1 x 10¹⁰ cells is considered well tolerated as the starting dose of allogeneic NK cell therapies. Thus, the planned dosing levels (DL) of the NK cell therapy are: DL0: 5 x io⁷ cells, DLL 1 x 10⁸ cells, DL2: 3 x io⁸ cells, DL3: 1 x io⁹ cells, and DL4: 3 x io⁹ cells, each of which is suitable for repeat administration with an expected tolerance and no dose-dependent toxicities.

[000318] Lympho-conditioning'. The purpose of lympho-conditioning prior to the administration of iNK cell therapy is to promote homeostatic proliferation of iNK cells as well as to eliminate regulatory immune cells and other competing elements of the immune system that compete for homeostatic cytokines. Cyclophosphamide (CY) is administered as an IV infusion at a dose of 500 mg/m² for three consecutive days (Days -5, -4, and -3). CY dosing is calculated based on actual body weight (ABW). If ABW is >150% of the ideal body weight (IBW), then the dose is computed using adjusted body weight as follows: Adjusted body weight = IBW + 0.5(ABW-IBW). Fludarabine (FLU) is administered as an IV infusion at a dose of 30 mg/m² for three consecutive days (Days -5, -4, and -3). The duration between the last dose of FLU and the infusion of iNK cell therapy is between 40 and 84 hours. In some cases, the iNK cell therapy may be administered after the 84-hour timepoint. Dose adjustments for weight/creatinine are per institutional guidelines. Unless considered necessary, corticosteroids should not be administered within 24 hours before or after iNK cell therapy administration. [000319] Interleukin-2 (IL2): Although the iNK cell therapy product FT536 expresses IL15RF, which is designed to provide an endogenous survival and proliferation signal, IL2 has been shown to promote NK-cell cytotoxicity and activates a distinct receptor signaling pathway relative to IL15. The IL2 dose and schedule in this study is designed to optimize the exposure of the iNK cells to IL2 in the 6-hour window after iNK cell therapy infusion. Aldesleukin (IL2) is administered subcutaneously (SC) at a dose of 10 MIU on Days 1, 8, and 15 of each treatment cycle, 2 hours (± 15 minutes) prior to infusion of iNK cell therapy. Subjects with baseline body weight <45 kilograms are administered IL2 at a dose of 3 MIU per m². Subjects are monitored for weight change at each treatment study visit, and for pulmonary edema during and after the IL2 administration. Pre-medication with acetaminophen 650 mg given orally and diphenhydramine 25 mg given orally or IV before and 4 hours after each dose of IL2 may be provided. Acetaminophen and diphenhydramine may be administered prior to administration of the iNK cell therapy and after IL2 administration if the interval between acetaminophen and diphenhydramine doses does not exceed 6 hours, i.e., IL2 is administered 2 hours after iNK cell therapy. Otherwise, an additional acetaminophen and diphenhydramine dose should be administered prior to IL2 administration.

[000320] Cetuximab: Cetuximab is an ADCC-compatible anti-EGFR monoclonal antibody. Cetuximab is administered at the initial dose of about 400 mg/m² as a 120-minute IV infusion; subsequent doses are administered at about 250 mg/m² as a 60-minute infusion QW. Alternatively, cetuximab is administered at about 500 mg/ m² as a 120-minute IV infusion Q2W. Cetuximab is continued for up to 2 years or until disease progression or unacceptable toxicity. Subjects can be pre-medicated with a histamine-1 receptor antagonist IV 30-60 minutes prior to the first dose or subsequent doses of cetuximab as deemed necessary. Preventative measures for EGFR antibody-associated rash is provided per institutional standards (e.g., moisturizer, sunscreen, topical steroid, and a tetracycline).

[000321] Traztuzumab: Traztuzumab is an ADCC-compatible anti-HER2 mAb. For subjects with HER2⁺ metastatic breast cancer (mBC), trastuzumab is administered at an initial dose of about 4 mg/kg as a 90-minute IV infusion, followed by subsequent doses of about 2 mg/kg as 30-minute IV infusions every week (QW) for up to 2 years or until disease progression or unacceptable toxicity. For subjects with all other tumor types expressing HER2, including metastatic gastric cancer (mGC) or subjects with NSCLC with HER2 mutation, trastuzumab is administered at an initial dose of 8 mg/kg as a 90-minute IV infusion, followed by subsequent doses of 6 mg/kg as an IV infusion over 30-90 minutes Q3W until disease progression or unacceptable toxicity.

[000322] Avelumab: Avelumab is an ADCC-compatible anti-PDLl monoclonal antibody. Avelumab is administered by IV infusion at a dose of about 800 mg every 2 weeks (Q2W) for up to 2 years or until disease progression or unacceptable toxicity. Subjects can be pre-medicated with an antihistamine and with acetaminophen prior to the first four infusions of avelumab as needed. When administration of the iNK cell therapy and avelumab administration occurs on the same day, the iNK cell therapy should be administered first.

[000323] Amivantamab: Amivantamab is an ADCC-compatible bi-specific antibody targeting EGFR and MET. Amivantamab is administered as an IV infusion at a dose of about 1050 mg for subjects weighing <80 kg or 1400 mg for subjects weighing >80 kg, weekly for 4 weeks, with the first weekly dose separated over 2 consecutive days, then administered Q2W thereafter for up to 2 years or until disease progression or unacceptable toxicity. The first dose can be split over 2 days. The first doses of amivantamab is administered on Days -11, -10, -4, 4, 11 and 18 of Cycle 1 and every 2 weeks thereafter starting from Cycle 1 Day 18. Subjects may be pre-medicated with aqbout 25 to 50 mg diphenhydramine or equivalent and 650 to 1000 mg acetaminophen 15-30 minutes (IV) or 30-60 minutes (oral) prior to each administration of amivantamab. Prior to the split infusions given on Days -11 and -10 only, glucocorticoid premedication such as methylprednisolone (40 mg), dexamethasone (10 mg) may be given IV 45-60 minutes prior to amivantamab administration. Steroids should be avoided altogether as pre-medications at subsequent infusions starting Cycle 1 Day -4 or later in order to avoid possible suppression of FT536 activity.

[000324] Pembrolizumab: Pembrolizumab is an immune checkpoint inhibitor (ICI) and anti-PDl/PDLl mAb. Pembrolizumab is administered as an IV infusion at a dose of about 200 mg over 30 minutes Q3W or 400 mg over 30 minutes Q6W until disease progression or unacceptable toxicity, or up to 2 years in subjects without disease progression.

[000325] Nivolumab: Nivolumab is an immune checkpoint inhibitor (ICI) and anti- PDl/PDLl mAb. Nivolumab is administered as an IV infusion at a dose of about 240 mg over 30 minutes Q2W or 480 mg Q4W for up to 2 years or until disease progression or unacceptable toxicity.

[000326] Atezolizumab: Atezolizumab is an immune checkpoint inhibitor (ICI) and anti- PDl/PDLl mAb. Atezolizumab is administered as an IV infusion at a dose of 840 mg Q2W, 1200 mg Q3W, or 1680 mg every 4 weeks (Q4W) for up to 2 years or until disease progression or unacceptable toxicity. The initial infusion is administered over 60 minutes, with all subsequent infusions delivered over 30-60 minutes.

[000327] Permitted Therapy: Subjects are permitted to use the following therapies during the study: (i) supportive care; and (ii) palliative radiotherapy.

[000328] Supportive care: Throughout the study, the investigator may prescribe any concomitant medications not otherwise described as cautionary therapy or prohibited therapy or treatment deemed necessary to provide adequate supportive care. Supportive care may include anti-microbial agents, analgesics, transfusions, growth factors, etc. Only irradiated blood products are used to minimize the risk of transfusion-associated GvHD.

[000329] Palliative radiotherapy: Subjects may receive palliative radiotherapy at any time and with schedules at the discretion of the investigator provided that the schedule of palliative radiotherapy does not interfere with protocol-specified assessments. Palliative radiation to target lesions is not recommended and must be discussed with the Medical Monitor unless the subject already has PD by RECIST and is not planning further study treatments. Subjects who have irradiated target lesions should not be considered evaluable for any responses other than progressive disease following receiving radiotherapy. Radiation therapy may create a more immunogenic microenvironment, and may potentiate and/or enhance anti-tumor activity of FT536.

[000330] Anti-Microbial Prophylaxis: According to the National Comprehensive Cancer Network (NCCN) guidelines for Prevention and Treatment of Cancer-Related Infections (Version 1.2021; NCCN 2021), patients with cancer receiving FLU are considered to be of intermediate risk for overall infection as it relates to bacterial, fungal and viral infection. Accordingly, it is strongly recommended that patients receive antiviral prophylaxis (acyclovir, valacyclovir or famciclovir) for prevention of herpes simplex virus (HSV) and varicella-zoster virus (VZV) according to institutional guidelines. Consideration should also be given for antibacterial as well as antifungal prophylaxis according to institutional guidelines. In the absence of appropriate institutional guidelines, the following guidance is provided: (i) Antiviral prophylaxis for HSV/VZV: Initiate acyclovir, valacyclovir, or famciclovir immediately prior to the start of conditioning until resolution of neutropenia; (ii) Antibacterial prophylaxis: Initiate fluoroquinolone (levofloxacin is preferred) immediately prior to the start of conditioning until resolution of neutropenia; and (iii) Antifungal prophylaxis: Initiate immediately prior to the start of conditioning until resolution of neutropenia. Fluconazole or micafungin are recommended but voriconazole, posaconazole, or amphotericin B can also be considered.

[000331] Cautionary Therapy: Systemic corticosteroids are avoided during the treatment cycle, unless absolutely required, because they may inhibit NK-cell function. Because of their deleterious effect on NK-cell-based therapy, corticosteroids as pre-medication for CY and FLU are also avoided unless considered necessary by the investigator and should not be administered within 24 hours before or after FT536 administration.

[000332] Intravenous glucocorticoid as pre-medication for CY, FLU, IL-2, and mAbs may be administered per the USPI or institutional guidelines. Methylprednisolone should be used as the preferred glucocorticoid pre-medication given its shorter half-life. Long-acting corticosteroids, such as dexamethasone, should not be used. Glucocorticoids must not be used as pre-medication for FT536.

[000333] Exploratory Analyses: The exploratory analyses of potential predictive and prognostic biomarkers associated with the mechanism of action of FT536 and underlying disease immunobiology are described herein. Such biomarkers may correlate with clinical outcomes. These associations may differ by indication and study subject population. Changes in immune-related biomarkers in the peripheral blood and within tumors may provide evidence for the biologic activity of FT536. An exploratory objective of this study is to assess potential pharmacodynamic biomarkers including, but not limited to, cytokines, PK of FT536, PK of selected mAbs, and NK and T-cell numbers and function and any potential associations with dose-dependent safety and anti-tumor activity.

[000334] In addition to peripheral blood sampling, tumor biopsies are obtained from subjects following initial treatment with FT536. Demonstrating the ability of FT536 to infiltrate sites of tumor, as well as evaluating changes to the tumor microenvironment is of importance in understanding potential mechanisms of FT536 resistance. In this regard, effort is made in this study to obtain tumor biopsies following disease progression and relapse. Information from on- treatment and post-progression biopsies may direct the development of future cell therapies that address these mechanisms of resistance.

[000335] Retreatment following progression: Subjects whose disease has an objective response to FT536, and later relapse or progress, may be eligible to receive retreatment based on a review of clinical data demonstrating evidence of clinical benefit. A retreatment option (i) provides evidence of whether an objective response can be achieved with additional courses of treatment with FT536 monotherapy or in combination with mAbs, which would provide evidence supportive of longer treatment duration, e.g., to achieve deeper responses that may drive longer durations of clinical benefit and/or incorporation of retreatment as part of the standard dosing schedule; and (ii) provides initial insights into potential mechanisms of resistance to treatment with FT536 monotherapy or in combination with a mAb and to characterize treatment-emergent changes in the tumor microenvironment. As part of considerations for retreatment, subjects electing to undergo FT536 retreatment will be required to undergo a pre-retreatment tumor biopsy from a safely accessible site to assess changes/status of the tumor and immune microenvironment.

[000336] End of Study: The end of the study is defined as the date of the last subject’s last assessment (scheduled or unscheduled). Follow-up visits, as defined in the schedule of activities (SoAs) for post-treatment follow-up and for long-term follow-up), may continue until up to 15 years following the completion of the last dose of FT536, all subjects have withdrawn consent, died, or enrolled into a separate long-term follow-up study before the prescribed 15-year followup is achieved in the current study, whichever occurs first. The expected duration of the study is approximately 15 years from the time of the last enrolled subject treated with FT536. The Sponsor may terminate the study at any time.

EXAMPLE 4 - Dose-Escalation and Dose-Expansion Stages of the Study

[000337] As described herein, MICA/B is commonly expressed across various solid tumors and plays an essential role in NK-cell mediated anti-tumor immunity. Importantly, most studies also showed minimal to no MICA/B expression in matched adjacent nonmalignant tissue. One exception is a study that involved a novel anti-MICA/B detection antibody that showed expression in several nonmalignant tissue types. However, 80%-95% of MICA/B expression was not localized at the cell surface, and when using an established and thoroughly validated control anti-MICA/B as a comparator, no MICA/B expression was observed in normal tissue. Specific to FT536, a tissue cross-reactivity study conducted under Good Laboratory Practice (GLP) using an anti-MICA/B detection antibody that recognizes the same epitope as that of the MICA/B- CAR in FT536 confirmed that the expression of MICA/B on the membrane surface of nonmononuclear cells in nonmalignant tissue is primarily limited to bone marrow hematopoietic precursor cells and endocrine organs (adrenal, parathyroid and testis, which is immune- privileged) as well as tissue localized immune mononuclear cells. Based on the observed expression profile, additional safety laboratory assessments will be performed to monitor endocrine function (e.g., calcium, thyroid stimulating hormone, adrenocorticotropic hormone (ACTH), cortisol) while the standard hematology laboratory assessments will be utilized to monitor bone marrow function following CY/FLU conditioning. In summary, based on the limited MICA/B expression observed in normal tissue, combined with the safety reported for other NK-cell therapies, and with the protocol safety monitoring plan FT536 can be safely evaluated in subjects with advanced relapsed/refractory (r/r) disease and who have limited therapeutic options.

[000338] Because a key mechanism of action of FT536 involves the enhancement of ADCC through hnCD16, the current study will also evaluate the clinical risk/benefit profile of FT536 in combination with ADCC-competent mAbs. As described herein, the combination of NK cell therapies, including both nonengineered NK cell and iPSC-derived NK-cell therapies engineered to express hnCD16, have shown to be safe and tolerable with ADCC competent mAbs (e.g., avelumab, cetuximab, or trastuzumab), as well as non-ADCC competent mAbs (e.g., pembrolizumab or nivolumab). Importantly, the observed/reported safety data for the combinations does not suggest any type of additive toxicity. Despite the encouraging historical combination safety data, in the current study, the starting dose level of FT536 in combination with a mAb will not be initiated until the first dose level (i.e., DL 1) of monotherapy FT536 has been evaluated to be safe. Given this precaution as well as the favorable historical safety profile for NK cell therapy combinations, there is sufficient rationale that the combination of FT536 with ADCC competent and non-competent mAbs can be safely evaluated in patients with limited therapeutic options.

[000339] Given the unique mechanism(s) of action between trastuzumab, cetuximab, amivantamab and anti-PD-l/PD-Ll antibodies, the combinations could have different toxi cities when combined with FT536 as well as when compared to FT536 monotherapy. Therefore, the individual maximum tolerated dose/maximum assessed dose (MTD/MAD) of FT536 monotherapy and of each FT536 combination will be determined independently.

[000340] The study begins with Cohort A at dose level 1 (DL 1). Upon clearance of Cohort ADL 1, initiation of dose escalation in Cohorts B-F begins at a dose level as low as DL 1 or at a dose level that does not exceed the highest Cohort A dose level.

[000341] The study consists of two stages: a dose-escalation stage and a dose-expansion stage. The dose-escalation stage defines the individual MTD, or MAD in the absence of DLT(s) defining the MTD, for FT536 monotherapy (Cohort A) or in combination with avelumab (Cohort B), pembrolizumab (Cohort C), trastuzumab (Cohort D), cetuximab (Cohort E), and amivantamab (Cohort F). Additional Cohorts AA-FF evaluates the addition of IL-2 support for each of Cohorts A-F at a dose level not exceeding the MAD/MTD. After the safety and tolerability have been assessed to define the individual MTD/MAD for each cohort in dose escalation, dose expansion subsequently further evaluates the safety and activity to determine the recommended Phase II dose (RP2D) of each respective cohort in specific disease indications.

[000342] FT536 Dose-Escalation Levels and Cohorts: The dose levels of FT536 for each cohort are as the follows: Dose Level 0 (DL 0): 5 x 10⁷ cells; this dose level is assessed only if DL 1 is deemed to be unacceptably toxic; DL 1: 1 x 10⁸ cells; DL 2: 3 x 10⁸ cells; DL 3: 1 x 10⁹ cells; and DL 4: 3 x 10⁹ cells. Dosing is based on MICA/B-CAR expression, where >80% of administered FT536 viable cells express MICA/B-CAR. [000343] Dose escalation begins with Cohort A at DL 1. After Cohort A DL 1 is cleared, dose escalation of Cohorts B-F starting at DL 1 begins. FT536 is administered on Days 1, 8, and 15 of each cycle.

[000344] When the 3 x 10⁹ cells per dose level does not exceed the MTD, additional higher dose levels are explored. Subsequent dose levels do not exceed 3 times the highest cleared dose level. In the event that an evaluated dose level exceeds the MTD, or based upon emerging clinical data (e.g., safety, PK, biomarker, etc.), dose levels intermediate to the planned dose levels is evaluated.

[000345] Dose escalation/de-escalation is conducted following a modified toxicity probability interval (mTPI) algorithm with a target dose limiting toxicity (DLT) rate of 30% and an equivalence interval of (25%, 35%). For each cohort, a minimum of 3 DLT evaluable subjects are enrolled to a dose level. A dose level for a given Cohort is considered as unacceptably toxic if it has an estimated probability of >95% exceeding the target DLT rate of 30% with at least 3 DLT evaluable subjects treated at that dose level. A DLT is defined as any adverse event (AE) that is at least possibly related to FT536 that occurs after the first FT536 infusion through the end of the DLT assessment period on Cycle 1 Day 29 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). Grading of acute GvHD is based on the Center for International Blood and Marrow Transplant Research (CIBMTR) acute GvHD scoring scale. Grading of laboratory AEs is assessed relative to baseline laboratory values defined as the last assessment prior to the start of protocol-defined study medication.

[000346] Dose-Expansion Stage: After the safety and tolerability have been assessed to define the individual MTD/MAD for each cohort in dose escalation, the dose-expansion stage of the study is conducted to further evaluate the safety and activity and to determine the RP2D of each respective cohort in specific disease indications. The dose expansion stage also further assesses safety and tolerability of FT536 monotherapy and in combination with mAbs and, where applicable, IL-2 to identify clinical activity signals.

[000347] Dose and schedule modifications for FT536 in Cycle 1 as described can be applied to additional treatment cycle(s) beyond Cycle 1 (including retreatment). After retreatment or if retreatment is not warranted, subjects proceed to post-treatment follow-up followed by long-term follow-up. [000348] Post-treatment follow-up visits, continue until up to 2 years following the last dose of FT536 or until one of the following occurs: disease progression or relapse, initiation of a new anti-cancer therapy, withdrawal of consent, or lost to follow-up, whichever occurs first.

[000349] Following post-treatment follow-up, subjects who enter the long-term follow-up phase are followed for up to 15 years after the last dose of FT536. Information related to the following are collected: (i) Subsequent anti-cancer therapies; (ii) Long-term follow-up safety assessments; (iii) Cell therapy safety monitoring; (iv) Relevant serious adverse events (SAEs) and associated concomitant therapy; (v) Disease response assessments based on RECIST vl.l and iRECIST may be collected or performed only in subjects who have not already experienced disease progression prior to entering long-term follow-up until disease progression occurs or end of study. The interval of response assessments during long-term follow-up are as clinically indicated per standard of care.

EXAMPLE 5 - Efficacy Assessments

[000350] Tumor Response Assessments: Tumor response are assessed according to RECIST vl.l.

[000351] As an exploratory endpoint, tumor response is also assessed according to modified RECIST (iRECIST) for immune-based therapeutics. It was recognized that some subjects receiving immunotherapy have an initial increase in tumor size that met the criteria for disease progression based on RECIST vl.l yet were noted to have late but deep and durable responses. This initial increase in tumor size was the result of the influx of immune cells termed “pseudoprogression.” To address this issue, iRECIST was introduced. The key difference between iRECIST and RECIST vl. l is the introduction of immune-unconfirmed PD (iUPD), which enables continued treatment if the subject is clinically stable and requires repeat imaging within 4-8 weeks to demonstrate immune-confirmed PD (iCPD). A response of iUPD but not iCPD can also be followed by immune complete response (iCR), immune partial response (iPR), or immune stable disease (iSD).

[000352] Tumor Tissue Biopsy: To characterize the baseline tumor microenvironment, assess the infiltration of FT536 into tumors and explore potential resistance mechanisms to FT536, subjects who have more than one measurable lesion at baseline are expected to undergo tumor biopsies from a safely accessible tumor site. Tumor biopsies are collected before cycle 1 treatment (for example, between the completion of last prior therapy and the initiation of conditioning (Day -5)), during treatment and before cycle 3 retreatment, for exploratory analyses or disease status evaluation following relapse or progression. [000353] Pharmacokinetics of FT536: Peripheral blood samples are collected to characterize the pharmacokinetics (PK) of FT536. Samples are collected before and after FT536 infusion on days when the cells are administered. FT536 quantification is also performed on tumor samples.

[000354] CRS Cytokines: Peripheral blood samples are collected for measurement of CRS cytokines prior to FT536 administration and in cases of clinically suspected CRS. Optionally, c-reactive protein (CRP) and ferritin are also tested.

[000355] Immunogenicity of FT536: Peripheral blood samples are collected for detection of alloimmunization to the FT536 product human leukocyte antigen (HLA) by panel-reactive antibody.

[000356] Additional Exploratory Analyses: In addition, peripheral blood and/or serum samples are collected for exploratory biomarker and immune monitoring analysis that include, but is not limited to: (i) HLA and killer-cell immunoglobulin-like receptor (KIR) typing; (ii) Exploratory serum biomarkers (measurement of peripheral blood cytokine levels); (iii) Exploratory immune monitoring (peripheral blood mononuclear cell (PBMC) functional characterization and immunophenotyping, e.g., T-cell subset analysis and determination of regulatory T-cell (T-reg) frequency); and (iv) mAb PK.

[000357] Exploratory biomarker analysis is also performed on archived tumor tissue samples obtained while the subject is on study, which includes, but is not limited to: (i) Tumor somatic mutation profiling, e.g., tumor mutation burden analysis; (ii) Tumor microenvironment characterization, e.g., tumor infiltrating T-cell characterization, immune inhibitory molecule expression by tumor cells; and (iii) Gene expression profiling that determines baseline and posttreatment effects of FT536 on tumor microenvironment.

[000358] Assays and other exploratory analyses include, but are not limited to, analysis of lymphocytes, T-cell activation, T-cell receptor repertoire, cytokines associated with inflammation, circulating tumor DNA (ctDNA) or minimum residual disease (MRD), cell of origin, and genes or gene signatures associated with tumor immunobiology. Exploratory analyses are conducted on any extra material obtained for clinical purposes (pleural fluid, biopsy, cerebrospinal fluid (CSF), blood, etc.), and related assessments may include analysis of mutations, single nucleotide polymorphisms, and other genomic variants; genomic profiling; as well as additional assay development, validation, and characterization.

[000359] Dose and Schedule Modification of FT536 and mAbs: Dose and schedule modification of FT536 for AE(s) considered related or at least possibly related to FT536, are as follows. [000360] If a Grade 3 or Grade 4 DLT is observed, then the subsequent FT536 infusions is not administered.

[000361] If a Grade 3 non-hematologic AE that is not a DLT is observed and recovers to baseline or Grade <1 by the subsequent scheduled FT536 infusion, then FT536 is dose-reduced to at least one lower dose level. Dose reduction is not needed for Grade 3 laboratory abnormalities that are not clinically significant.

[000362] If a Grade 3 non-hematologic AE that is not a DLT is observed and ongoing at the time of the subsequent scheduled FT536 infusion, FT536 infusion is delayed until resolution of the AE to baseline or Grade <1, at which time FT536 is dose-reduced to at least one lower dose level. In the situation where the dose-delaying AE occurs after the first FT536 infusion, if recovery to baseline or Grade <1 is not observed by Day 8, the scheduled Day 8 FT536 infusion is skipped. However, if recovery to baseline or Grade <1 is not observed by Day 15, the scheduled Day 15 FT536 infusion is skipped. In the situation where the dose-delaying AE occurs after the second FT536 infusion, if recovery to baseline or Grade <1 is not observed by Day 15, the scheduled FT536 infusion is skipped. Dose reduction is not needed for Grade 3 laboratory abnormalities that are not clinically significant.

[000363] If a Grade <3 hematologic AE that is not a DLT is observed, then FT536 dosing continues according to schedule and without dose modification.

[000364] Refer to the respective United States Prescribing Information (USPI) infusionreactions for all mAbs, anti-PD-l/Ll antibodies and associated immune-mediated adverse reactions, trastuzumab and associated cardiomyopathy, or cetuximab and associated dermatologic and pulmonary toxicities that may need dosage modifications to avoid adverse reactions.

[000365] Dose and Schedule Modification of Conditioning: For patients with moderate impairment of renal function (creatinine clearance of 30-70 mL/min/1.73 m²), the FLU dose is reduced by 20%. FLU is not recommended for patients with severely impaired renal function (creatinine clearance <30 mL/min/1.73 m²).

[000366] Dose and schedule modifications of conditioning therapy for AEs considered at least possibly related to CY/FLU within a treatment cycle are described below.

[000367] For subjects who complete a treatment cycle but are still recovering from AEs that preclude directly proceeding to the subsequent treatment cycle, additional monitoring visits (Days 36, 43, 50, and 57) are performed leading up to the start of the subsequent treatment cycle to allow for continued recovery from AEs. Initiation of the subsequent treatment cycle may occur up to 42 days after the last prior dose of FT536 in the prior treatment cycle. [000368] If Grade >3 neutropenia and thrombocytopenia that are considered at least possibly related to CY/FLU do not recover to Grade 2 (ANC of >1000 and platelets of >50,000) or baseline grade, whichever grade is higher, on Day 29 and/or where dose adjustment is feasible, the following dose modifications to CY and FLU are provided: (i) Reduce the daily dose of CY to 300 mg/m² on Days -5, -4, and -3; and reduce the daily dose of FLU to 25 mg/mg² on Days -5, -4, and -3 in Cycle 2; (ii) where additional CY/FLU is not in the subject’s best interest based on prior therapies or observed toxi cities, CY/FLU may be omitted; (iii) If the criteria for a CY/FLU dose reduction are met for a second time during Cycle 2 or later, reduce the daily dose of CY to 300 mg/m² on Days -5 and -4 (2 days only); and reduce the daily dose of FLU to 25 mg/mg² on Days -5 and -4 (2 days only); and (iv) If the criteria for a CY/FLU dose reduction are again met for a third time during Cycle 3, CY/FLU is omitted.

EXAMPLE 6 - Efficacy Analyses

[000369] The anti-tumor activity of FT536 is evaluated per RECIST vl .1 based on assessed objective response rate (ORR), duration of response (DOR), progression-free survival (PFS), and overall survival (OS).

[000370] Tumor response categories per RECIST vl. l include complete response (CR), partial response (PR), stable disease (SD), progressive disease (PD), or not evaluable (NE).

[000371] The best overall response (BOR) based on RECIST vl.l is summarized for the efficacy- evaluable population. The secondary endpoint of objective response rate (ORR) (i.e., the proportion of subjects who achieve a PR or CR) is summarized descriptively for the doseescalation cohorts and for the dose-expansion cohorts, separately by treatment cohort and by disease indication where applicable. In addition, the exact 95% confidence interval (CI) is presented. The time-to-event endpoints (DOR, PFS, and OS) based on RECIST vl.l is summarized using Kaplan-Meier methods. Kaplan-Meier plots are presented by treatment cohort. The number of events and the number of censored subjects are summarized, along with the quartiles, including the median time-to-event and their respective 95% Cis. Censoring methodology is described in the statistical analysis plan (SAP).

[000372] The summaries of BOR, ORR, DOR, and progression-free survival (PFS) includes data for all subjects based on the tumor burden baseline value obtained at screening. Additional tumor assessments after retreatment are based on a pre-retreatment tumor burden baseline value, which serve as the last tumor assessment within 28 days prior to the first dose of FT536 in retreatment. Results based on the original screening versus pre-retreatment tumor burden baseline values are reported separately in the summaries of BOR, ORR, and DOR. The retreatment efficacy endpoints serve as exploratory endpoints. Refer to the SAP for additional information.

[000373] Under RECIST vl. l, for subjects experiencing PD, the date of disease progression to be used for calculation of progression related time-to-event endpoints is the first date at which progression criteria are met (i.e., the date of PD). If a subject experiences clinical progression, prior to or in the absence of documented radiological progression, the date of clinical progression is used for calculation of progression related time-to-event endpoints. [000374] Under iRECIST, for subjects experiencing immune-unconfirmed progressive disease (iUPD), the date of disease progression to be used for calculation of progression related time-to-event endpoints is the first date at which progression criteria are met (i.e., the date of iUPD), provided that immune-confirmed progressive disease (iCPD) is confirmed at the next assessment. If iUPD occurs but is disregarded because of later immune stable disease (iSD), immune partial response (iPR), or immune complete response (iCR), then that iUPD date is not used as the progression event date. If progression is not confirmed and there is no subsequent iSD, iPR, or iCR, then the iUPD date is still used in the following scenarios: if the subject stops protocol treatment because they were not judged to be clinically stable, or no further response assessments are done (because of subject refusal, protocol noncompliance, or subject death); the next timepoint responses are all iUPD, and iCPD never occurs; or the subject dies from their cancer. If a subject experiences clinical progression in the absence of a documented radiological progression, the date of clinical progression is used for calculation of progression related time-to- event endpoints.

[000375] FT536 Pharmacokinetics: The PK of FT536 is assessed by the detection of

FT536 in peripheral blood following FT536 administration as a monotherapy and in combination with mAbs in subjects with advanced solid tumors. The summary is done per PK population, which is defined as subjects in the safety population who provide at least 1 post-FT536 dosing evaluable sample.

[000376] Exploratory Analyses: The anti-tumor activity of FT536 in combination with mAbs is evaluated using iRECIST and RECIST vl. l, as applicable. The assessment is carried out based on the immune objective response rate (iORR), immune duration of response (iDOR), immune progression-free survival (iPFS), immune time to progression (iTTP), immune disease control rate (iDCR), and OS in all regimens using iRECIST. Time to progression (TTP) and disease control rate (DCR) is also evaluated using RECIST.

[000377] Tumor response is assessed using iRECIST. Each response criteria is summarized separately. Subjects are classified into the best of the following tumor response categories for iRECIST: iCR, iPR, iSD, iUPD, iCPD, or NE, as defined in Section II above. [000378] The BOR based on iRECIST (i.e., immune best overall response (iBOR)) is summarized for the efficacy-evaluable population. The exploratory endpoints of iORR (i.e., the proportion of subjects who achieve an iPR or iCR) and iDCR (i.e., the proportion of subjects with an iBOR of iCR or iPR, or iSD >8 weeks) is summarized descriptively for the doseescalation cohorts and for the dose-expansion cohorts by treatment cohort and dose cohort and by disease indication where applicable. In addition, the exact 95% CI is presented. DCR (i.e., the proportion of subjects with a BOR of CR or PR, or SD >8 weeks) is also summarized similarly. [000379] The time-to-event endpoints (iDOR, iPFS, iTTP, TTP) is summarized using Kaplan-Meier methods. Kaplan-Meier plots are presented by treatment cohort. The number of events and the number of censored subjects are summarized, along with the quartiles, including the median time-to-event and their respective 95% Cis. Censoring methodology is described in the SAP.

[000380] Exploratory analyses include both descriptive summaries and assessments of potential predictive and prognostic biomarkers in peripheral blood or serum and tumor biopsies, as well as changes in the tumor microenvironment as appropriate. The association of PK and pharmacodynamics of FT536 as monotherapy and in combination with mAbs with safety and anti-tumor activity are assessed. Additionally, the association of pharmacodynamics and the PK of selected mAbs that may include, but are not limited to, avelumab, trastuzumab (including biosimilars), cetuximab, and amivantamab when administered in combination with FT536 are assessed.

[000381] The association between baseline clinical and tumor characteristics, safety, and anti-tumor activity ofFT536 is also assessed.

EXAMPLE 7 - First Monotherapy FT536 Escalation Cohort A

[000382] Three patients (2 having pancreatic cancer and 1 having colon cancer) were treated with one cycle of FT536 as a monotherapy at a starting dose of 100 million cells/dose according to the treatment schema of FIG. 3. As of September 27, 2022, no DLTs, CRS or ICANS were reported (FIG. 4) and one pancreatic cancer patient experienced a 22.7% reduction in target lesion size in accordance with the study parameters. Monotherapy escalation is continuing at 300 million cells/dose. Moreover, dose escalation in combination with mAbs initiated at 100 million cells/dose based on the safety profile and efficacy of FT536 in cohort A.

[000383] One skilled in the art would readily appreciate that the methods, compositions, and products described herein are representative of exemplary embodiments, and not intended as limitations on the scope of the invention. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the present disclosure disclosed herein without departing from the scope and spirit of the invention.

[000384] All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the present 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 as incorporated by reference.

[000385] The present disclosure illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising,” “consisting essentially of,” and “consisting of’ may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present disclosure claimed. Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Claims

CLAIMS What is claimed is:

1. A method of treating a subject having a solid tumor, comprising: administering to the subject at least a first cycle of an adoptive cell therapy product, with the first cycle comprising one or more doses of the adoptive cell therapy product administered in a first effective amount at a preselected frequency, and with an option of administering one or more additional cycles with one or more doses in a second effective amount, during a course of treatment over a period of time; wherein the first and the second effective amounts are the same or different; and wherein the product comprises an engineered natural killer (NK) lineage cell comprising MICA/B-CAR (chimeric antigen receptor) expression, exogenous CD 16 expression, IL15RF expression and CD38 knockout.

2. The method of claim 1, wherein the solid tumor comprises cancer cells expressing:

(i) MICA/B;

(ii) PD-L1;

(iii) HER2;

(iv) EGFR; and/or

(v) both EGFR and MET.

3. The method of claim 2, wherein the cancer cells are comprised in at least one of the following cancers and relapsed or refractory forms thereof:

(i) breast cancer (BC);

(ii) advanced or metastatic colorectal cancer (CRC);

(iii) advanced or metastatic non-small cell lung cancer (NSCLC);

(iv) gastric cancer or gastroesophageal adenocarcinoma;

(v) ovarian cancer;

(vi) pancreatic cancer;

(vii) head and neck cancer; and

(viii) urothelial carcinoma (UC).

4. The method of claim 1, wherein the course of treatment further comprises administering to the subject an effective amount of a selected therapeutic monoclonal antibody (mAb).

5. The method of claim 4, wherein the therapeutic mAb comprises an anti-EGFR antibody, an anti-HER2 antibody, an anti-PD-Ll antibody, or a bi-specific antibody targeting EGFR and MET.

6. The method of claim 5, wherein the anti-EGFR antibody comprises cetuximab; wherein the anti-HER2 antibody comprises trastuzumab or biosimilars; wherein the anti-PD-Ll antibody comprises avelumab; or wherein the bi-specific antibody targeting EGFR and MET comprises amivantamab.

7. The method of claim 6, wherein the course of treatment further comprises administering to the subject an effective amount of initial doses of the same therapeutic monoclonal antibody in an effective amount at a starting time prior to the first cycle of administering the adoptive cell therapy product.

8. The method of claim 7, wherein the therapeutic mAb is cetuximab, and wherein a single initial dose of about 400 mg/m² to about 500 mg/m² is administered to the subject about 4 days prior to the first cycle of administering the adoptive cell therapy product.

9. The method of claim 7, wherein the therapeutic mAb is trastuzumab, and wherein:

(i) a single dose of about 4 mg/kg is administered to the subject about 4 days prior to the first cycle of administering the adoptive cell therapy product, wherein the subject has HER2⁺ metastatic breast cancer (mBC); or

(ii) a single dose of about 8 mg/kg is administered to the subject about 4 days prior to the first cycle of administering the adoptive cell therapy product, wherein the subject has a HER2⁺ solid tumor other than mBC.

10. The method of claim 7, wherein the therapeutic mAb is avelumab, and wherein a single initial dose of about 800 mg is administered to the subject about 4 days prior to the first cycle of administering the adoptive cell therapy product.

11. The method of claim 7, wherein the therapeutic mAb is amivantamab, and wherein the starting time is about 4-10 days prior to the first cycle of administering the adoptive cell therapy product, and wherein the initial doses of the monoclonal antibody comprise 1-2 weekly (QW) doses of: (i) about 1050 mg for subjects weighing <80 kg; or

(ii) about 1400 mg for subjects >80 kg, and optionally wherein the first weekly dose of the monoclonal antibody is administered over two consecutive days.

12. The method of claim 1, wherein the course of treatment further comprises administering to the subject an effective amount of an immune checkpoint inhibitor (ICI).

13. The method of claim 12, wherein the ICI comprises an anti-PDl/PDLl mAb, and optionally wherein the anti-PDl/PDLl mAb comprises pembrolizumab, nivolumab, or atezolizumab.

14. The method of claim 13, wherein the course of treatment further comprises administering to the subject an effective amount of an initial dose of the same ICI in an effective amount about 4 days prior to the first cycle of administering the adoptive cell therapy product, and wherein:

(i) pembrolizumab is in an amount of about 200 mg to about 400 mg;

(ii) nivolumab is in an amount of about 240 mg to about 480 mg; or

(iii) atezolizumab is in an amount of about 840 mg to about 1680 mg.

15. The method of claim 1, wherein the course of treatment further comprises administering to the subject an effective amount of IL2 prior to the adoptive cell therapy product.

16. The method of claim 15, wherein the administration of IL2 is about 1-3 hours prior to the adoptive cell therapy product.

17. The method of claim 16, wherein the effective amount of IL2 is:

(i) about 10 MIU; or

(ii) about 3 MIU per m² for subjects with a baseline body weight of <45 kg.

18. The method of any one of claims 1-17, further comprising 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, wherein the duration between the administration of a 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.

19. The method of claim 18, wherein the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU); and optionally wherein the CY and FLU are administered daily for three consecutive days, or wherein the dose of CY is at about 300-500 mg/m² and the dose of FLU is at about 25-30 mg/m².

20. The method of claim 19, wherein the specified period of time is: (i) about 24-84 hours; or (ii) about 3 days.

21. The method of claim 1, wherein the engineered NK lineage cell is derived from an engineered induced pluripotent stem cell (iPSC) comprising a polynucleotide encoding a MICA/B-CAR, a polynucleotide encoding an exogenous CD 16, a polynucleotide encoding IL15RF and CD38 knockout.

22. The method of claim 1, wherein the MICA/B-CAR comprises:

(i) a heavy chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 1;

(ii) a light chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 2; and/or

(iii) a scFV represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NOs: 3 or 4.

23. The method of claim 1, wherein the first and second effective amounts of the adoptive cell therapy product in each dose is about 5 * 10⁷ cells to about 9 * 10⁹ cells.

24. The method of claim 23, wherein the first and second effective amounts of the adoptive cell therapy product in each dose is about 1 x 10⁸ cells, about 3 * 10⁸ cells, about 1 x 10⁹ cells, about 3 x io⁹ cells or about 9 x io⁹ cells.

25. The method of claim 24, wherein if the effective amount in each dose results in a dose limiting toxicity (DLT) rate of <30% with an equivalence interval of 25%-35%, then the effective amount increases to a next higher effective amount.

26. The method of claim 6, wherein:

(i) cetuximab is administered weekly (QW) or every two weeks (Q2W) in an amount of:

-no- (a) about 400 mg/m² when administered QW; or

(b) about 500 mg/m² when administered Q2W;

(ii) trastuzumab is administered QW or every three weeks (Q3W) in an amount of:

(a) about 2 mg/kg QW for subjects having HER2⁺ mBC; or

(b) about 6 mg/kg Q3W for subjects having a HER2⁺ solid tumor other than mBC;

(iii) avelumab is administered Q2W in an amount of about 800 mg/dose; and/or

(iv) amivantamab is administered Q2W in an amount of about 1000-1500 mg.

27. The method of claim 26, wherein when avelumab and the adoptive cell product are administered on the same day, the adoptive cell product is administered first.

28. The method of claim 13, wherein:

(i) pembrolizumab is administered Q3W or every six weeks (Q6W) in an amount of about 400 mg;

(ii) nivolumab is administered Q2W or every four weeks (Q4W) in an amount of:

(a) about 240 mg when administered Q2W; or

(b) about 480 mg when administered Q4W; and/or

(iii) atezolizumab is administered Q2W, Q3W or Q4W in an amount of:

(a) about 840 mg when administered Q2W;

(b) about 1200 mg when administered Q3W; or

(c) about 1680 mg when administered Q4W.

29. The method of claim 1, wherein the subject has:

(i) advanced or metastatic non-small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer (BC), ovarian cancer, or pancreatic cancer;

(ii) advanced or metastatic solid tumors having PD-L1 expression, or advanced CRC whose tumors are microsatellite instability high (MSI-H) and/or mismatch repair deficient (dMMR);

(iii) advanced or metastatic HER2⁺ solid tumors or NSCLC with HER2 mutation;

(iv) advanced or metastatic squamous NSCLC, CRC, or head and neck squamous cell carcinoma; or

(v) advanced or metastatic NSCLC with EGFR driver mutation(s), MET exon 14 skipping mutation, or MET amplification.

30. The method of claim 29, wherein:

(i) tumors having PD-L1 expression comprise:

(a) NSCLC with PD- LI tumor cell expression >1%, or tumor-infiltrating immune cells (ICs) covering >10% of tumor area;

(b) gastroesophageal adenocarcinoma with a PD-L1 expression >1%;

(c) head and neck squamous cell carcinoma with a PD-L1 expression >1%, or >1% tumor cell expression;

(d) Triple-negative breast cancer with a PD-L1 expression >10% or ICs covering >1% of tumor area;

(e) UC with a PD-L1 expression >10%, >1% tumor cell expression, or ICs covering >5% of tumor area; or

(f) locally advanced or metastatic CRC that is MSLH and/or dMMR;

(ii) tumors having HER2⁺ expression comprises tumors having:

(a) >2+ immunohistochemistry (IHC); or

(b) an average HER2 copy number >4;

(iii) advanced or metastatic squamous NSCLC, CRC, or head and neck squamous cell carcinoma comprises:

(a) CRC that is KRAS/NRAS wild type and the subject has relapsed or progressed following prior cetuximab or panitumumab treatment; or

(b) head and neck cancer wherein the subject has relapsed or progressed following prior cetuximab treatment, or wherein the subject is documented to refuse standard cetuximab-based treatment; or

(iv) subject having advanced or metastatic NSCLC comprises having progressed or being intolerant to at least one prior line of EGFR tyrosine kinase inhibitor (TKI) treatment, or not being a candidate for TKI treatment;

(v) MET amplification defined as a MET/CEP7 gene expression ratio >1.8; or as gene copy number >5.

31. The method of claim 30, wherein subjects having advanced or metastatic HER2⁺ solid tumors with HER2 mutation have previously received at least one line of anti-HER2 antibodybased therapy, optionally wherein, the advanced or metastatic HER2⁺ solid tumor is a gastric cancer or a breast cancer.

32. The method of claim 1, wherein the method comprises administering (i) one cycle of the adoptive cell therapy product over about 3 weeks at a dose frequency of 1 dose per week; (ii) two cycles of the adoptive cell therapy product, with each cycle comprising three doses over about 3 weeks at a dose frequency of 1 dose per week, wherein a second cycle of the two cycles is given within 42 days of a last infusion of the adoptive cell therapy product in a first cycle; or (iii) one, or two, or three or four cycles of the adoptive cell therapy product, with each cycle comprising three doses over about 3 weeks at a dose frequency of 1 dose per week.

33. The method of any one of claims 1-32, wherein the administering of the adoptive cell therapy product is (i) via intravenous infusion, and/or (ii) at a site of an outpatient setting; and/or wherein each dose of the adoptive cell therapy product is cryopreserved, and then thawed prior to administering; and wherein the adoptive cell therapy product is FT536.

34. A method of slowing progression of and/or treating a solid tumor in a subject, wherein the subject has advanced or metastatic non-small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer (BC), ovarian cancer, or pancreatic cancer, the method comprising: administering to the subject at least one cycle of an adoptive cell therapy product, with the cycle comprising one or more doses of the adoptive cell therapy product administered in a first effective amount at a preselected frequency, and with an option of administering one or more additional cycles with one or more doses in a second effective amount, during a course of treatment over a period of time; wherein the subject has a reduction in lesion size or number after treatment with the adoptive cell therapy product; and wherein the adoptive cell therapy comprises engineered natural killer (NK) lineage cells comprising MICA/B-CAR (chimeric antigen receptor) expression, exogenous CD 16 expression, IL15RF expression and CD38 knockout.

35. The method of claim 34, further comprising administering to the subject a daily dose of one or more chemotherapeutic agents for three consecutive days, wherein the duration between the last daily dose administration of the one or more chemotherapeutic agents and a first weekly administration of the adoptive cell therapy product is: (i) about 24-84 hours; or (ii) about 3 days.

36. The method of claim 35, wherein the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU), and optionally wherein the daily dose of CY is at about 300-500 mg/m² and the daily dose of FLU is at about 25-30 mg/m².

37. The method of claim 34, further comprising administering to the subject an effective amount of IL2 prior to the adoptive cell therapy product, wherein the administration of IL2 is about 1-3 hours prior to the adoptive cell therapy product.

38. The method of claim 37, wherein the effective amount of IL2 is:

(i) about 10 MIU; or

(ii) about 3 MIU per m² for subjects with a baseline body weight of <45 kg.

39. The method of claim 34, wherein the engineered NK lineage cell is derived from an engineered induced pluripotent stem cell (iPSC), and wherein the engineered iPSC comprises a polynucleotide encoding a MICA/B-CAR, a polynucleotide encoding an exogenous CD 16, and optionally, a polynucleotide encoding IL15RF and CD38 knockout.

40. The method of claim 39, wherein the MICA/B-CAR comprises:

41. The method of claim 34, wherein the first and the second effective amounts of the adoptive cell therapy product are about 5 * 10⁷ cells/dose to about 9 * 10⁹ cells/dose, and wherein the first and the second effective amounts are the same or different.

42. The method of claim 41, wherein the effective amount of the adoptive cell therapy product in each dose is about 1 x 10⁸ cells, about 3 * 10⁸ cells, about 1 x 10⁹ cells, about 3 x io⁹ cells or about 9 x io⁹ cells.

43. The method of claim 42, wherein if the effective amount in each dose results in a dose limiting toxicity (DLT) rate of <30% with an equivalence interval of 25%-35%, then the effective amount increases to a next higher effective amount.

44. The method of claim 34, further comprising assessing disease response to the adoptive cell therapy after each cycle of treatment with the adoptive cell therapy product.

45. The method of claim 44, wherein assessing disease response comprises measuring target lesions or short axis of pathological lymph nodes of the subject for complete remission or partial remission.

46. The method of claim 45, wherein:

(i) complete remission comprises:

(a) disappearance of all target lesions; and/or

(b) a short axis reduction of any pathological lymph nodes; and/or

(ii) partial remission comprises at least a 30% decrease in a sum of all diameters of target lesions, as compared to that of the target lesions prior to the course of treatment.

47. The method of any one of claims 34-46, wherein the administering of the adoptive cell therapy product is (i) via intravenous infusion, and/or (ii) at a site of an outpatient setting; and/or wherein each dose of the adoptive cell therapy product is cryopreserved, and then thawed prior to administering.

48. The method of claim 34, wherein the additional cycle during a course of treatment comprises (i) a number of doses, (ii) at a dose frequency of the adoptive cell therapy product,

(iii) in an effective cell amount per dose, wherein (i), (ii) or (iii) herein is same or different from that of the at least one cycle administered.

49. The method of claim 48, wherein the additional cycle during a course of treatment comprising the adoptive cell therapy product comprises at least 3 doses at a dose frequency of 1 dose per week, and wherein the additional cycle is administered within 42 days of a last infusion of the adoptive cell therapy product of the first cycle.

50. A method of slowing progression of and/or treating a solid tumor in a subject, wherein the subject has advanced or metastatic solid tumors with PD-L1 expression, or advanced CRC whose tumors are microsatellite instability high (MSI-H) and/or mismatch repair deficient (dMMR), the method comprising: (i) administering to the subject at least one cycle of an adoptive cell therapy product, with the cycle comprising one or more doses of the adoptive cell therapy product administered in a first effective amount at a preselected frequency, and with an option of administering one or more additional cycles with one or more doses in a second effective amount, during a course of treatment over a period of time; and

(ii) administering to the subject one or more doses of an effective amount of an anti- PD-L1 monoclonal antibody during the at least one cycle of the adoptive cell therapy product; wherein the subject has a reduction in lesion size and/or number after treatment with the adoptive cell therapy product; and wherein the adoptive cell therapy comprises engineered natural killer (NK) lineage cells comprising MICA/B-CAR (chimeric antigen receptor) expression, exogenous CD 16 expression, IL15RF expression and CD38 knockout.

51. The method of claim 50, wherein the subject has:

(a) NSCLC with a PD-L1 tumor expression >1%, or tumor-infiltrating immune cells (ICs) covering >10% of tumor area;

(b) gastroesophageal adenocarcinoma with a PD-L1 expression >1% (22C3);

(e) UC with a PD-L1 expresion >10%, >1% tumor cell expression, or ICs covering >5% of tumor area; or

(f) locally advanced or metastatic CRC that is MSI-H and/or dMMR.

52. The method of claim 50, wherein the engineered NK lineage cell is derived from an engineered induced pluripotent stem cell (iPSC) comprising a polynucleotide encoding a MICA/B-CAR, a polynucleotide encoding an exogenous CD 16, a polynucleotide encoding IL15RF and CD38 knockout.

53. The method of claim 52, wherein the MICA/B-CAR comprises:

(ii) a light chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 2; and/or (iii) a scFV represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NOs: 3 or 4.

54. The method of claim 50, further comprising administering to the subject a single initial dose of the same anti-PD-Ll monoclonal antibody to the subject about 4 days prior to the first cycle of administering the adoptive cell therapy product.

55. The method of claim 50, further comprising administering to the subject a daily dose of one or more chemotherapeutic agents for three consecutive days, wherein the duration between the last daily dose administration of the one or more chemotherapeutic agents and a first weekly administration of the adoptive cell therapy product is: (i) about 24-84 hours; or (ii) about 3 days.

56. The method of claim 55, wherein the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU), and optionally wherein the daily dose of CY is at about 300-500 mg/m² and the daily dose of FLU is at about 25-30 mg/m².

57. The method of claim 50, further comprising administering to the subject an effective amount of IL2 prior to the adoptive cell therapy product, wherein the administration of IL2 is about 1-3 hours prior to the adoptive cell therapy product.

58. The method of claim 57, wherein the effective amount of IL2 is:

(i) about 10 MIU; or

(ii) about 3 MIU per m² for subjects with a baseline body weight of <45 kg.

59. The method of claim 50, wherein the effective amount of the one or more doses of the adoptive cell therapy product is about 5 * 10⁷ cells/dose to about 9 * 10⁹ cells/dose, and wherein the effective amounts of each of the one or more doses are the same or different.

60. The method of claim 59, wherein the effective amount of the adoptive cell therapy product in each dose is about 1 x 10⁸ cells, about 3 * 10⁸ cells, about 1 x 10⁹ cells, about 3 x io⁹ cells or about 9 x io⁹ cells.

61. The method of claim 60, wherein if the effective amount in each dose results in a dose limiting toxicity (DLT) rate of <30% with an equivalence interval of 25%-35%, then the effective amount increases to a next higher effective amount.

62. The method of claim 50, wherein the anti-PD-Ll monoclonal antibody is avelumab, and wherein the avelumab is administered Q2W in an amount of about 800 mg/dose.

63. The method of claim 62, wherein when avelumab and the adoptive cell product are administered on the same day, the adoptive cell product is administered first.

64. The method of claim 50, further comprising assessing disease response to the adoptive cell therapy after each cycle of treatment with the adoptive cell therapy product.

65. The method of claim 64, wherein assessing disease response comprises measuring target lesions or short axis of pathological lymph nodes of the subject for complete remission or partial remission.

66. The method of claim 65, wherein:

(i) complete remission comprises:

(a) disappearance of all target lesions; and/or

(b) a short axis reduction of any pathological lymph nodes; and/or

67. The method of any one of claims 50-66, wherein the administering of the adoptive cell therapy product is (i) via intravenous infusion, and/or (ii) at a site of an outpatient setting; and/or wherein each dose of the adoptive cell therapy product is cryopreserved, and then thawed prior to administering.

68. A method of slowing progression of and/or treating a solid tumor in a subject, wherein the subject has advanced or metastatic solid tumors with PD-L1 expression, or advanced CRC whose tumors are microsatellite instability high (MSI-H) and/or mismatch repair deficient (dMMR), the method comprising: (i) administering to the subject at least one cycle of an adoptive cell therapy product, with the cycle comprising one or more doses of the adoptive cell therapy product administered in a first effective amount at a preselected frequency, and with an option of administering one or more additional cycles with one or more doses in a second effective amount, during a course of treatment over a period of time; and

(ii) administering to the subject one or more doses of an effective amount of an immune checkpoint inhibitor (ICI) during the at least one cycle of the adoptive cell therapy product, wherein the ICI comprises pembrolizumab, or nivolumab, atezolizumab; wherein the subject has a reduction in lesion size and/or number after treatment with the adoptive cell therapy product; and wherein the adoptive cell therapy comprises engineered natural killer (NK) lineage cells comprising MICA/B-CAR (chimeric antigen receptor) expression, exogenous CD 16 expression, IL15RF expression and CD38 knockout.

69. The method of claim 68, wherein the subject has:

(b) gastroesophageal adenocarcinoma with a PD-L1 expression >1%;

(d) triple-negative breast cancer with a PD-L1 expression >10% (22C3) or ICs covering >1% of tumor area;

(f) locally advanced or metastatic CRC that is MSI-H and/or dMMR.

70. The method of claim 68, wherein the engineered NK lineage cell is derived from an engineered induced pluripotent stem cell (iPSC) comprising a polynucleotide encoding a MICA/B-CAR, a polynucleotide encoding an exogenous CD 16, a polynucleotide encoding IL15RF and CD38 knockout.

71. The method of claim 70, wherein the MICA/B-CAR comprises:

(i) a heavy chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 1; (ii) a light chain variable region represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NO: 2; and/or

72. The method of claim 68, further comprising administering to the subject a single initial dose of the same ICI to the subject about 4 days prior to the first cycle of administering the adoptive cell therapy product, and wherein the ICI is:

(i) pembrolizumab and the initial dose is about 200 mg to about 400 mg;

(ii) nivolumab and the initial dose is about 240 mg to about 480 mg; or

(iii) atezolizumab and the initial dose is about 840 mg to about 1680 mg.

73. The method of claim 68, further comprising administering to the subject a daily dose of one or more chemotherapeutic agents for three consecutive days, wherein the duration between the last daily dose administration of the one or more chemotherapeutic agents and a first weekly administration of the adoptive cell therapy product is: (i) about 24-84 hours; or (ii) about 3 days.

74. The method of claim 73, wherein the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU), and optionally wherein the daily dose of CY is at about 300-500 mg/m² and the daily dose of FLU is at about 25-30 mg/m².

75. The method of claim 68, further comprising administering to the subject an effective amount of IL2 prior to the adoptive cell therapy product, wherein the administration of IL2 is about 1-3 hours prior to the adoptive cell therapy product.

76. The method of claim 75, wherein the effective amount of IL2 is:

(i) about 10 MIU; or

(ii) about 3 MIU per m² for subjects with a baseline body weight of <45 kg.

77. The method of claim 68, wherein the effective amount of the one or more doses of the adoptive cell therapy product is about 5 * 10⁷ cells/dose to about 9 * 10⁹ cells/dose, and wherein the effective amounts of each of the one or more doses are the same or different.

78. The method of claim 77, wherein the effective amount of the adoptive cell therapy product in each dose is about 1 x 10⁸ cells, about 3 * 10⁸ cells, about 1 x 10⁹ cells, about 3 x io⁹ cells or about 9 x io⁹ cells.

79. The method of claim 78, wherein if the effective amount in each dose results in a dose limiting toxicity (DLT) rate of <30% with an equivalence interval of 25%-35%, then the effective amount increases to a next higher effective amount.

80. The method of claim 68, wherein the ICI is:

(i) pembrolizumab and is administered Q3W or every six weeks (Q6W) in an amount of about 400 mg;

(ii) nivolumab and is administered Q2W or every four weeks (Q4W) in an amount of:

(a) about 240 mg when administered Q2W; or

(b) about 480 mg when administered Q4W; and/or

(iii) atezolizumab and is administered Q2W, Q3W or Q4W in an amount of:

(a) about 840 mg when administered Q2W;

(b) about 1200 mg when administered Q3W; or

(c) about 1680 mg when administered Q4W.

81. The method of claim 68, further comprising assessing disease response to the adoptive cell therapy after each cycle of treatment with the adoptive cell therapy product.

82. The method of claim 81, wherein assessing disease response comprises measuring target lesions or short axis of pathological lymph nodes of the subject for complete remission or partial remission.

83. The method of claim 82, wherein:

(i) complete remission comprises:

(a) disappearance of all target lesions; and/or

(b) a short axis reduction of any pathological lymph nodes; and/or

84. The method of any one of claims 68-83, wherein the administering of the adoptive cell therapy product is (i) via intravenous infusion, and/or (ii) at a site of an outpatient setting; and/or wherein each dose of the adoptive cell therapy product is cryopreserved, and then thawed prior to administering.

85. A method of slowing progression of and/or treating a solid tumor in a subject, wherein the subject has advanced or metastatic HER2⁺ solid tumors with or NSCLC with HER2 mutation, the method comprising:

(i) administering to the subject at least one cycle of an adoptive cell therapy product, with the cycle comprising one or more doses of the adoptive cell therapy product administered in a first effective amount at a preselected frequency, and with an option of administering one or more additional cycles with one or more doses in a second effective amount, during a course of treatment over a period of time; and

(ii) administering to the subject one or more doses of an effective amount of an anti- HER2 monoclonal antibody during the at least one cycle of the adoptive cell therapy product; wherein the subject has a reduction in lesion size and/or number after treatment with the adoptive cell therapy product; and wherein the adoptive cell therapy comprises engineered natural killer (NK) lineage cells comprising MICA/B-CAR (chimeric antigen receptor) expression, exogenous CD 16 expression, IL15RF expression and CD38 knockout.

86. The method of claim 85, wherein the subject has tumors having:

(a) >2+ immunohistochemistry (IHC); or

(b) an average HER2 copy number >4.

87. The method of claim 85, wherein the engineered NK lineage cell is derived from an engineered induced pluripotent stem cell (iPSC) comprising a polynucleotide encoding a MICA/B-CAR, a polynucleotide encoding an exogenous CD 16, a polynucleotide encoding IL15RF and CD38 knockout.

88. The method of claim 87, wherein the MICA/B-CAR comprises:

89. The method of claim 85, further comprising administering to the subject a single initial dose of the anti-HER2 monoclonal antibody to the subject about 4 days prior to the first cycle of administering the adoptive cell therapy product, and wherein:

(i) the single initial dose is about 4 mg/kg for subjects having HER2⁺ metastatic breast cancer (mBC); or

(ii) the single dose is about 8 mg/kg for subjects having a HER2⁺ solid tumor other than mBC.

90. The method of claim 85, further comprising administering to the subject a daily dose of one or more chemotherapeutic agents for three consecutive days, wherein the duration between the last daily dose administration of the one or more chemotherapeutic agents and a first weekly administration of the adoptive cell therapy product is: (i) about 24-84 hours; or (ii) about 3 days.

91. The method of claim 90, wherein the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU), and optionally wherein the daily dose of CY is at about 300-500 mg/m² and the daily dose of FLU is at about 25-30 mg/m².

92. The method of claim 85, further comprising administering to the subject an effective amount of IL2 prior to the adoptive cell therapy, wherein the administration of IL2 is about 1-3 hours prior to the adoptive cell therapy product .

93. The method of claim 92, wherein the effective amount of IL2 is:

(i) about 10 MIU; or

(ii) about 3 MIU per m² for subjects with a baseline body weight of <45 kg.

94. The method of claim 85, wherein the effective amount of the one or more doses of the adoptive cell therapy product is about 5 * 10⁷ cells/dose to about 9 * 10⁹ cells/dose, and wherein the effective amounts of each of the one or more doses are the same or different.

95. The method of claim 94, wherein the effective amount of the adoptive cell therapy product in each dose is about 1 x 10⁸ cells, about 3 * 10⁸ cells, about 1 x 10⁹ cells, about 3 x io⁹ cells or about 9 x 1Q⁹ cells.

96. The method of claim 95, wherein if the effective amount in each dose results in a dose limiting toxicity (DLT) rate of <30% with an equivalence interval of 25%-35%, then the effective amount increases to a next higher effective amount.

97. The method of claim 85, wherein the anti-HER2 monoclonal antibody is trastuzumab and is administered QW or every three weeks (Q3W) in an amount of:

(a) about 2 mg/kg QW for subjects having HER2⁺ mBC; or

(b) about 6 mg/kg Q3W for subjects having a HER2⁺ solid tumor other than mBC.

98. The method of claim 85, further comprising assessing disease response to the adoptive cell therapy after each cycle of treatment with the adoptive cell therapy product.

99. The method of claim 98, wherein assessing disease response comprises measuring target lesions or short axis of pathological lymph nodes of the subject for complete remission or partial remission.

100. The method of claim 99, wherein:

(i) complete remission comprises:

(a) disappearance of all target lesions; and/or

(b) a short axis reduction of any pathological lymph nodes; and/or

101. The method of any one of claims 85-100, wherein the administering of the adoptive cell therapy product is (i) via intravenous infusion, and/or (ii) at a site of an outpatient setting; and/or wherein each dose of the adoptive cell therapy product is cryopreserved, and then thawed prior to administering.

102. A method of slowing progression of and/or treating a solid tumor in a subject, wherein the subject has advanced or metastatic squamous NSCLC, CRC, or head and neck squamous cell carcinoma, the method comprising:

(ii) administering to the subject one or more doses of an effective amount of an anti- EGFR monoclonal antibody during the at least one cycle of the adoptive cell therapy product; wherein the subject has a reduction in lesion size and/or number after treatment with the adoptive cell therapy product; and wherein the adoptive cell therapy comprises engineered natural killer (NK) lineage cells comprising MICA/B-CAR (chimeric antigen receptor) expression, exogenous CD 16 expression, IL15RF expression and CD38 knockout.

103. The method of claim 102, wherein the subject has:

(b) head and neck cancer, and wherein the subject has relapsed or progressed following prior cetuximab treatment, or wherein the subject has refused standard cetuximabbased treatment.

104. The method of claim 102, wherein the engineered NK lineage cell is derived from an engineered induced pluripotent stem cell (iPSC) comprising a polynucleotide encoding a MICA/B-CAR, a polynucleotide encoding an exogenous CD 16, a polynucleotide encoding IL15RF and CD38 knockout.

105. The method of claim 102, wherein the MICA/B-CAR comprises:

106. The method of claim 102, further comprising administering to the subject a single initial dose of the anti-EGFR monoclonal antibody to the subject about 4 days prior to the first cycle of administering the adoptive cell therapy product, and wherein the single initial dose is about 400 mg/m² to about 500 mg/m².

107. The method of claim 102, further comprising administering to the subject a daily dose of one or more chemotherapeutic agents for three consecutive days, wherein the duration between the last daily dose administration of the one or more chemotherapeutic agents and a first weekly administration of the adoptive cell therapy product is: (i) about 24-84 hours; or (ii) about 3 days.

108. The method of claim 107, wherein the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU), and optionally wherein the daily dose of CY is at about 300-500 mg/m² and the daily dose of FLU is at about 25-30 mg/m².

109. The method of claim 102, further comprising administering to the subject an effective amount of IL2 prior to the adoptive cell therapy product, wherein the administration of IL2 is about 1-3 hours prior to the adoptive cell therapy product.

110. The method of claim 109, wherein the effective amount of IL2 is:

(i) about 10 MIU; or

(ii) about 3 MIU per m² for subjects with a baseline body weight of <45 kg.

111. The method of claim 102, wherein the effective amount of the one or more doses of the adoptive cell therapy product is about 5 * 10⁷ cells/dose to about 9 * 10⁹ cells/dose, and wherein the effective amounts of each of the one or more doses are the same or different.

112. The method of claim 111, wherein the effective amount of the adoptive cell therapy product in each dose is about 1 x 10⁸ cells, about 3 * 10⁸ cells, about 1 x 10⁹ cells, about 3 x io⁹ cells or about 9 x io⁹ cells.

113. The method of claim 112, wherein if the effective amount in each dose results in a dose limiting toxicity (DLT) rate of <30% with an equivalence interval of 25%-35%, then the effective amount increases to a next higher effective amount.

114. The method of claim 102, wherein the anti-EGFR monoclonal antibody is cetuximab and is administered weekly (QW) or every two weeks (Q2W) in an amount of:

(a) about 400 mg/m2 when administered QW; or

(b) about 500 mg/m2 when administered Q2W.

115. The method of claim 102, further comprising assessing disease response to the adoptive cell therapy after each cycle of treatment with the adoptive cell therapy product.

116. The method of claim 115, wherein assessing disease response comprises measuring target lesions or short axis of pathological lymph nodes of the subject for complete remission or partial remission.

117. The method of claim 116, wherein:

(i) complete remission comprises:

(a) disappearance of all target lesions; and/or

(b) a short axis reduction of any pathological lymph nodes; and/or

118. The method of any one of claims 102-117, wherein the administering of the adoptive cell therapy product is (i) via intravenous infusion, and/or (ii) at a site of an outpatient setting; and/or wherein each dose of the adoptive cell therapy product is cryopreserved, and then thawed prior to administering.

119. A method of slowing progression of and/or treating a solid tumor in a subject, wherein the subject has advanced or metastatic NSCLC, the method comprising:

(ii) administering to the subject one or more doses of an effective amount of a bispecific antibody targeting EGFR and MET during the at least one cycle of the adoptive cell therapy product; wherein the subject has a reduction in lesion size and/or number after treatment with the adoptive cell therapy product; and wherein the adoptive cell therapy comprises engineered natural killer (NK) lineage cells comprising MICA/B-CAR (chimeric antigen receptor) expression, exogenous CD 16 expression, IL15RF expression and CD38 knockout.

120. The method of claim 119, wherein the subject has tumors having:

(a) EGFR driver mutation(s), and wherein the subject has progressed on or is intolerant to at least one prior line of EGFR tyrosine kinase inhibitor (TKI) treatment or wherein the subject is not a candidate for TKI treatment;

(b) MET exon 14 skipping mutation, and wherein the subject has progressed on or is intolerant of at least one prior line of MET TKI treatment, or wherein the subject was not a candidate for TKI treatment;

(c) MET amplification defined as a MET/CEP7 gene expression ratio >1.8; or

(d) MET amplification defined as gene copy number >5.

121. The method of claim 120, wherein the engineered NK lineage cell is derived from an engineered induced pluripotent stem cell (iPSC) comprising a polynucleotide encoding a MICA/B-CAR, a polynucleotide encoding an exogenous CD 16, a polynucleotide encoding IL15RF and CD38 knockout.

122. The method of claim 121, wherein the MICA/B-CAR comprises:

(iii) a scFV represented by an amino acid sequence that is of at least about 80% identity to SEQ ID NOs: 3 or 4

123. The method of claim 119, further comprising administering to the subject one or more initial doses of the same bi-specific antibody targeting EGFR and MET to the subject about 4-10 days prior to the first cycle of administering the adoptive cell therapy product, and wherein the initial doses of the bi-specific antibody comprise 1-2 weekly (QW) doses of about 1000-1400 mg.

124. The method of claim 119, further comprising administering to the subject a daily dose of one or more chemotherapeutic agents for three consecutive days, wherein the duration between the last daily dose administration of the one or more chemotherapeutic agents and a first weekly administration of the adoptive cell therapy product is: (i) about 24-84 hours; or (ii) about 3 days.

125. The method of claim 124, wherein the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU), and optionally wherein the daily dose of CY is at about 300-500 mg/m² and the daily dose of FLU is at about 25-30 mg/m².

126. The method of claim 119, further comprising administering to the subject an effective amount of IL2 prior to the adoptive cell therapy product, wherein the administration of IL2 is about 1-3 hours prior to the adoptive cell therapy product.

127. The method of claim 126, wherein the effective amount of IL2 is:

(i) about 10 MIU; or

(ii) about 3 MIU per m² for subjects with a baseline body weight of <45 kg.

128. The method of claim 119, wherein the effective amount of the one or more doses of the adoptive cell therapy product is about 5 * 10⁷ cells/dose to about 9 * 10⁹ cells/dose, and wherein the effective amounts of each of the one or more doses are the same or different.

129. The method of claim 128, wherein the effective amount of the adoptive cell therapy product in each dose is about 1 x 10⁸ cells, about 3 * 10⁸ cells, about 1 x 10⁹ cells, about 3 x io⁹ cells or about 9 x io⁹ cells.

130. The method of claim 129, wherein if the effective amount in each dose results in a dose limiting toxicity (DLT) rate of <30% with an equivalence interval of 25%-35%, then the effective amount increases to a next higher effective amount.

131. The method of claim 119, wherein the bi-specific antibody is amivantamab and is administered every two weeks (Q2W) in an amount of about 1000-1500 mg.

132. The method of claim 119, further comprising assessing disease response to the adoptive cell therapy after each cycle of treatment with the adoptive cell therapy product.

133. The method of claim 132, wherein assessing disease response comprises measuring target lesions or short axis of pathological lymph nodes of the subject for complete remission or partial remission.

134. The method of claim 133, wherein:

(i) complete remission comprises:

(a) disappearance of all target lesions; and/or

(b) a short axis reduction of any pathological lymph nodes; and/or

135. The method of any one of claims 119-134, wherein the administering of the adoptive cell therapy product is (i) via intravenous infusion, and/or (ii) at a site of an outpatient setting; and/or wherein each dose of the adoptive cell therapy product is cryopreserved, and then thawed prior to administering.

136. A method of a multi-dose targeted adoptive cell therapy in a subject in need thereof comprising:

(i) weekly administration to the subject of an effective amount of a targeted adoptive cell therapy product for a course of treatment of about three weeks, wherein the product comprises an engineered immune cell expressing a MICA/B-CAR, expressing CD16, expressing IL15RF, and comprising CD38 knockout; and

(ii) detecting and comparing one or more of the following at different given time points following administration of a first dose of the adoptive cell therapy:

(a) the presence of the engineered immune cell in a tumor of the subject;

(b) protein markers of disease in serum of the subject;

(c) cytokines in a peripheral blood sample from the subject;

(d) circulating tumor DNA in a peripheral blood sample from the subject; and

(e) lesion size and/or number, wherein any of (a)-(e) is used to assess tumor burden, tumor immunobiology, and/or tumor therapy response, thereby determining efficacy of the multi-dose targeted adoptive cell therapy.

137. The method of claim 136, wherein the subject has breast cancer (BC), advanced or metastatic colorectal cancer (CRC), advanced or metastatic non-small cell lung cancer (NSCLC), gastric cancer or gastroesophageal adenocarcinoma, ovarian cancer, pancreatic cancer, head and neck cancer, or urothelial carcinoma (UC).

-ISO-

138. The method of claim 136, wherein the effective amount of the adoptive cell therapy product is about 5 x 1Q⁷ cells/dose to about 9 x 1Q⁹ cells/dose.

139. The method of claim 138, wherein the effective amount of the adoptive cell therapy product is about 1 x 10⁸ cells, about 3 * 10⁸ cells, about 1 x 10⁹ cells, about 3 x io⁹ cells or about 9 x io⁹ cells.

140. The method of claim 139, wherein if the effective amount in each dose results in a dose limiting toxicity (DLT) rate of <30% with an equivalence interval of 25%-35%, then the effective amount increases to a next higher effective amount.

141. The method of claim 136, further comprising administering to the subject an effective amount of a selected therapeutic antibody (mAb).

142. The method of claim 141, wherein the therapeutic mAb comprises an anti-EGFR antibody, an anti-HER2 antibody, an anti-PD-Ll antibody, or a bi-specific antibody targeting EGFR and MET.

143. The method of claim 142, wherein the anti-EGFR antibody comprises cetuximab; wherein the anti-HER2 antibody comprises trastuzumab or biosimilars; wherein the anti-PD-Ll antibody comprises avelumab; or wherein the bi-specific antibody comprises amivantamab.

144. The method of claim 136, further comprising administering to the subject an effective amount of an immune checkpoint inhibitor (ICI).

145. The method of claim 144, wherein the ICI comprises an anti-PDl/PDLl mAb, and optionally wherein the anti-PDl/PDLl mAb comprises pembrolizumab, or nivolumab, atezolizumab.

146. The method of claim 136, further comprising administering to the subject a daily dose of one or more chemotherapeutic agents for three consecutive days, wherein the duration between the last daily dose administration of the one or more chemotherapeutic agents and a first weekly administration of the adoptive cell therapy product is: (i) about 24-84 hours; or (ii) about 3 days.

147. The method of claim 146, wherein the one or more chemotherapeutic agents comprise cyclophosphamide (CY) and fludarabine (FLU), and optionally wherein the daily dose of CY is at about 300-500 mg/m² and the daily dose of FLU is at about 25-30 mg/m².

148. The method of claim 136, further comprising administering to the subject an effective amount of IL2 prior to the adoptive cell therapy product, wherein the administration of IL2 is about 1-3 hours prior to the adoptive cell therapy product.

149. The method of claim 148, wherein the effective amount of IL2 is:

(i) about 10 MIU; or

(ii) about 3 MIU per m² for subjects with a baseline body weight of <45 kg.

150. The method of claim 136, wherein the engineered immune cell is derived from an engineered induced pluripotent stem cell (iPSC), and wherein the engineered iPSC comprises a polynucleotide encoding a MICA/B-CAR, a polynucleotide encoding an exogenous CD 16, and optionally, a polynucleotide encoding IL15RF and CD38 knockout.

151. The method of claim 136, wherein the MICA/B-CAR comprises: