Ipsc-derived t cells for solid tumor treatment

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 in combination therapies.

Description

iPSC-DERIVED T CELLS FOR SOLID TUMOR TREATMENTRELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 597,327, filed November 8, 2023, the disclosure of which is hereby incorporated by reference in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The Sequence Listing titled 184143-652601_SL.xml, which was created on November 7, 2024 and is 21,676 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 (e.g., efficacy in a 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] In one aspect, the present disclosure provides a method of treating cancer in a subject. In some embodiments, the method comprises: (a) administering to the subject one or more doses of chemotherapy; and (b) administering to the subject one or more doses of an adoptive cell therapy product in a first effective amount; wherein the adoptive cell therapy product comprises engineered T lineage cells comprising (i) expression of a HER2-CAR (chimeric antigen receptor), an IL7 / IL7 receptor fusion protein (IL7RF), a TGFP signaling redirector receptor (TGFP-SRR), a C-X-C motif chemokine receptor 2 (CXCR2), and an exogenous CD 16; (ii) a CD38 knockout; and (iii) a T cell receptor (TCR) knockout; and wherein the cancer comprises cancer cells that are HER2 positive. In some embodiments, the method comprises repeating the foregoing steps.

[0008] In some embodiments, the method further comprises: (a) administering to the subject one or more doses of an additional chemotherapy that is the same or different as the chemotherapy (e.g., after administering the one or more doses of the adoptive cell therapy product in the first effective amount); and (b) administering to the subject one or more doses of the adoptive cell therapy product in a second effective amount that is the same or different as the first effective amount (e.g., after administering the additional chemotherapy).

[0009] In some embodiments, the cancer cells that are HER2 positive comprise HER2 expression, amplification, or mutation. In some embodiments, the method further comprises administering an EGFR inhibitor to the subject; and wherein the cancer cells comprise an EGFR mutation. In some embodiments, administering the EGFR inhibitor is before and / or after administering the adoptive cell therapy product. In some embodiments, the EGFR inhibitor comprises, matuzumab, panitumumab, or necitumumab. In some embodiments, the EGFR inhibitor comprises cetuximab. In some embodiments, the chemotherapy comprises: (a) one or both of cyclophosphamide (CY) and fludarabine (FLU); (b) bendamustine; or (c) one or both of docetaxel and cisplatin. In some embodiments, the chemotherapy is administered one or moredays prior to administering the adoptive cell therapy product; optionally wherein the chemotherapy is administered at least two days, three days, four days, or five days before administering the adoptive cell therapy product. In some embodiments, the chemotherapy (a) comprises (i) cyclophosphamide at a daily dose of about 250 mg / m2to about 600 mg / m2; and (ii) fludarabine at a daily dose of about 20 mg / m2to about 40 mg / m2; and (b) is administered 3 consecutive days starting from about 4-6 days prior to administering the adoptive cell therapy product. In some embodiments, the chemotherapy comprises bendamustine and is administered at a daily dose of about 30 mg / m2to about 100 mg / m2for 2 consecutive days starting from about 4-6 days prior to administering the adoptive cell therapy product.

[0010] In some embodiments, the engineered T lineage cells are derived from an engineered induced pluripotent stem cell (iPSC) comprising a polynucleotide encoding the HER2-CAR, a polynucleotide encoding the IL7RF, a polynucleotide encoding the TGFP-SRR, a polynucleotide encoding the CXCR2, a polynucleotide encoding the exogenous CD 16, a TCR knockout, and a CD38 knockout. In some embodiments, the first effective amount and / or second effective amount of the adoptive cell therapy product is about 5 * 107to about 3 * 109engineered T lineage cells, and optionally wherein the number of engineered T lineage cells is increased based on a dose limiting toxicity rate of the amount of the adoptive cell therapy administered. In some embodiments, the first effective amount and / or second effective amount of the adoptive cell therapy product comprises about 5 * 107, about 1 x 108, about 3 * 108, about 9 - io8, or about 2 * 109engineered T lineage cells. In some embodiments, at a dose limiting toxicity (DLT) rate of 25%-35% or below, the number of engineered T lineage cells in the adoptive cell therapy product is increased by up to 3-fold. In some embodiments, the adoptive cell therapy product is: (i) allogeneic; (ii) administered (a) via intravenous infusion, and / or (b) in an outpatient setting; and / or (iii) cryopreserved, and then thawed prior to administering. In some embodiments, the adoptive cell therapy product is FT825.

[0011] In some embodiments, the subject has had: (i) no prior treatment of the cancer; or (ii) one or more prior HER2-directed therapies; and / or one or more prior EGFR-directed therapies. In some embodiments, the cancer comprises a breast cancer, an esophagogastric cancer, a gastroesophageal junction (GEJ) adenocarcinoma, a non-small cell lung cancer (NSCLC), a colorectal cancer (CRC), or a head and neck squamous cell cancer (HNSCC).

[0012] In some embodiments, the method further comprises detecting and comparing one or more of the following subsequent to administration of the first effective amount 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; or(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 some embodiments, the subject has a complete response (CR), partial response (PR), stable disease (SD) after receiving the adoptive cell therapy product.

[0013] In one aspect, a method of treating cancer comprises: (a) administering to the subject one or more first doses of an EGFR inhibitor; (b) administering to the subject one or more doses of an adoptive cell therapy product; and (c) administering to the subject one or more second doses of the EGFR inhibitor; wherein the adoptive cell therapy product comprises an engineered T lineage cell comprising (i) expression of a HER2-CAR (chimeric antigen receptor), an IL7 / IL7 receptor fusion protein (IL7RF), a TGFP signaling redirector receptor (TGFP-SRR), a C-X-C motif chemokine receptor 2 (CXCR2), and an exogenous CD 16; (ii) a CD38 knockout; and (iii) a T cell receptor (TCR) knockout; and wherein the cancer comprises cancer cells expressing EGFR. In some embodiments, the method comprises repeating the foregoing steps. In some embodiments, the method comprises administering to the subject one or more doses of chemotherapy before administering the EGFR inhibitor. In some embodiments, the EGFR inhibitor comprises cetuximab, matuzumab, panitumumab, or necitumumab. In some embodiments, the EGFR inhibitor comprises cetuximab. In some embodiments, the cancer cells expressing EGFR also express HER2. In some embodiments, the chemotherapy comprises: (a) one or both of cyclophosphamide (CY) and fludarabine (FLU); (b) bendamustine; or (c) one or both of docetaxel and cisplatin.

[0014] In one aspect, the present disclosure provides kits for cancer treatment. In some embodiments, the kit comprises FT825, one or more chemotherapy agents, and optionally an EGFR inhibitor; wherein (a) FT825 comprises engineered T lineage cells comprising: (i) expression of a HER2-CAR (chimeric antigen receptor), an IL7 / IL7 receptor fusion protein (IL7RF), a TGFP signaling redirector receptor (TGFP-SRR), a C-X-C motif chemokine receptor 2 (CXCR2), and an exogenous CD 16; (ii) a CD38 knockout; and (iii) a T cell receptor (TCR) knockout; (b) the chemotherapy comprises: (i) one or both of cyclophosphamide (CY) and fludarabine (FLU); (ii) bendamustine; or (iii) one or both of docetaxel and cisplatin; and (c) the EGFR inhibitor comprises cetuximab, matuzumab, panitumumab, or necitumumab. In some embodiments, the cancer is HER2 positive and / or EGFR positive.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 shows an exemplary treatment schema to evaluate FT825 as an allogeneic cell therapy in combination with a chemotherapy. Abbreviations are as follows: CY (cyclophosphamide), DLT (dose-limiting toxicity), FLU (fludarabine), LTFU (long-term follow-up), PTFU (post-treatment follow-up), y (year). Follow-Upa: According to the illustrated schema, subjects are followed in post-treatment follow-up for up to 2 years after the last cycle of FT825, and followed in long-term follow-up for up to 15 years after the last cycle of FT825.

[0016] FIG. 2 shows an exemplary dose-escalation and dose expansion schema for the use of FT825 in cancer treatment. Illustrated Dose Level 1 (DL1) is set at 1 x 108, and additional dose levels > DL1 (< 3 x the prior cleared dose level) may be explored. DLO = 5 * 107cells / dose (to be assessed if DL1 exceeds MTD). Notea: Subjects withHER.2 >2+ positive tumors and HER2-mutated NSCLC may be prioritized. Subjects with tumors that are HER2 IHC 1+ may also be treated. Noteb: HER2-positive breast cancer and gastric / GEJ cancer defined in this schema as IHC 3+, or IHC 2+ and ISH-positive (Bartley et al., 2016; Wolff et al., 2018). Notec: HER2-low breast cancer and gastric / GEJ cancer defined in this schema as IHC 1+, or IHC 2+ / ISH-negative. Noted: The decision to open expansion cohort may be contingent on review and evaluation of FT825 clinical experience and pharmacodynamic activity observed during dose escalation. Abbreviations are as follows: Benda (Bendamustine); Cy (Cyclophosphamide); DL (Dose level); DLT (Dose-limiting toxicity); EGFR (Epidermal growth factor receptor); Flu (Fludarabine); GEJ (Gastroesophageal junction); HER2 (Human epidermal growth factor receptor 2); IHC (Immunohistochemistry); ISH (in situ hybridization);MAD (Maximum assessed dose); MTD (Maximum tolerated dose); NSCLC (Non-small cell lung cancer).

[0017] FIG. 3 shows an exemplary treatment schema for a further cycle of treatment (Cycle 2) following treatment according to the schema illustrated in FIG. 1 (Cycle 1). Notea: Cycle 2 retreatment with docetaxel / cisplatin may be considered for subjects who tolerate Cycle 1 treatment with CY / FLU or bendamustine, who initially have an objective response or SD, and who subsequently have PD. Cycle 2 retreatment may follow the same schedule as Cycle 1. The FT825 dose for Cycle 2 retreatment may be up to the highest cleared dose of FT825 when administered with CY / FLU or bendamustine. If a subject is ineligible to receive docetaxel / cisplatin, the subject may instead receive the same chemotherapy as was administered in Cycle 1. Noteb: Subjects may restart PTFU and be followed for up to 2 years after the end of the Cycle 2 retreatment cycle. Notec: Follow-up may continue until up to 15 years after the last dose of FT825. Abbreviations are as follows: CY (Cyclophosphamide); CR (Complete response); FLU (Fludarabine); LTFU (Long-term follow-up); PD (Progressive disease); PR (Partial response); PTFU (Post-treatment follow-up); RECIST vl.l (Response Evaluation Criteria in Solid Tumors, Version 1.1); SD (Stable disease).DETAILED DESCRIPTION OF THE INVENTION

[0018] 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 can be challenging, and may present hurdles 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, such as suicide genes or 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, of 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.

[0019] Definitions

[0020] 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.

[0021] 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.

[0022] 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.

[0023] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0024] The term “and / or” should be understood to mean either one, or both of the alternatives.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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 areoptional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0031] 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.

[0032] 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.

[0033] The term “ / / / vivo" refers generally to activities that take place inside an organism.

[0034] 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.

[0035] 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 normalcircumstances, 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).

[0036] 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.

[0037] 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).

[0038] 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.

[0039] 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 differentiateto 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.

[0040] 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.

[0041] 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.

[0042] As used herein, the term “subject” refers to any animal, preferably a human patient, livestock, or other domesticated animal.

[0043] 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.

[0044] 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.

[0045] 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 coatedplastic) 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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%.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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-chromosomalgenetic 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.

[0060] 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.

[0061] 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.

[0062] 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 polypeptidesinclude natural polypeptides, recombinant polypeptides, synthetic polypeptides, or a combination thereof.

[0063] “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.

[0064] “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.

[0065] 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 aretainable 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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) targetingof cancer stem cells, and (v) other targeting strategies in the absence of a specific antigen or surface molecule.

[0073] 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.

[0074] 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.

[0075] 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 (i.e. chemotherapy) 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 a T lineage 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.

[0076] 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.

[0077] 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.

[0078] 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. A therapeutically sufficient amount may be an amount delivered in a single dose, or refer to a cumulative amount administered over a plurality of doses that may be separated in time according to a dosing schedule.

[0079] 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 celldifferentiation, 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] “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.

[0085] 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.”

[0086] 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 orreceptor-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.

[0087] “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.

[0088] 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 domainscan 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.

[0089] CD 16, a FcyR receptor, has been identified to have two isoforms, Fc receptorsFcyRIIIa (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 CD 16. 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 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.

[0090] As used herein, “FT825” refers to a multiplexed engineered T cell therapy generated from a clonal master engineered iPSC line, and is engineered with multiple modalities for enhanced innate immunity: (1) a high-affinity non-cleavable CD16 (hnCD16) Fc receptor;(2) IL7 / IL7R fusion protein (IL7RF); (3) anti-HER2-CAR; (4) TGFP-SRR; (5) CXCR2; (6) CD38 knockout; and (7) T cell receptor (TCR) knockout.I. Cells and Compositions Useful for Adoptive Cell Therapies with Enhanced Properties

[0091] Provided herein is a strategy to systematically engineer the regulatory circuitry of a clonal iPSC without impacting the differentiation potency 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 modified activities and / or properties. 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 the need for reconfiguration of differentiation protocols enabling phenotypic and / or functional change 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. Further, all genomic modifications and combinations thereof as may be described in the context of iPSC and iPSC-derived effector cells are applicable to primary sourced cells, including primary immune cells such as T, NK, or immunregulatory cells, whether cultured or expanded, the modification of which results in engineered immune cells useful for adoptive cell therapy.

[0092] Further, while CAR-T cells have been shown to be effective and potent in treating several hematologic malignancies, engineered T cell therapies have had limited success in addressing solid tumors. Unlike liquid tumors where uniformly-expressed antigens are accessible and can be effectively targeted, tumor access, lack of tumor-exclusive antigen targets, and antigen heterogeneity are significant barriers to the successful development of CAR-T cells in solid tumors. In addition, inherent genetic engineering variability seen with patient- and donor- derived immune cells limits the wide application of CAR-T cell therapy. The present application provides genomic engineering aspects in the form of a solid tumor targetingbackbone, as well as other genetic modalities, to improve on-target specificity with reduced off- tumor effect in the off-the-shelf adoptive cell therapy setting using effector cells derived from engineered iPSCs, to evade allorej ection, as well as to overcome suppressive tumor microenvironment, a heightened challenge especially with solid tumors. / . C-X-C Motif Chemokine Receptor Overexpression

[0093] Chemokines are a family of homogeneous serum proteins of about 7 to about 16 kDa originally characterized by their ability to induce leukocyte migration. Most of chemokines have four characteristic cysteines (Cys) and are classified into C-X-C (or alpha, CXC), C-C (or beta), C (or gamma), and CX3C (or delta) chemokine classes, according to motifs displayed by the first two cysteines. Subfamilies of C-X-C (or alpha, CXC) are further classified, according to the presence of an ELR motif (Glu-Leu-Arg) preceding the first cysteine, into two groups: ELR-CXC chemokines and non-ELR-CXC chemokines.

[0094] CXC chemokine receptor 2 (CXCR2), also known as CD128, interleukin 8 receptor beta (IL8RP), or L8 receptor type B, is a chemokine receptor mostly expressed by neutrophils, mast cells, monocytes, and macrophages. It is known that CD56 dim NK cells express CXCR2, however its expression can be downregulated upon NK cell activation. T cells typically do not express CXCR2. iPSCs and iPSC-derived T cells do not express CXCR2 without transducing exogenous polynucleotides encoding CXCR2 as disclosed in this application. The chemokine IL8 (also known as CXCL8) is secreted by mononuclear macrophages, neutrophils, eosinophils, T lymphocytes, epithelial cells, and fibroblasts, and functions as a chemotactic factor by guiding the neutrophils to the site of infection. CXCL8 is also secreted by tumor cells and promotes tumor migration, invasion, angiogenesis and metastasis. CXCL8 is one of the ligands to multiple CXC chemokine receptors including CXCR1 and CXCR2. Additional chemokines known to bind to CXCR2 include, but are not limited to, CXCL1, GROp (CXCL2), CXCL3, CXCL5, CXCL6, and CXCL7.

[0095] CXC chemokine receptor 3 (CXCR3), also known as G Protein-coupled Receptor 9 (GPR9) and CD183, is a G Protein-coupled receptor that binds to the chemokines CXCL9, CXCL10, and CXCL11. CXCR3 is expressed primarily in activated T-helper type 1 (Thl) lymphocytes, but is also present in natural killer cells, macrophages, dendritic cells, and B lymphocyte subsets. The interaction of CXCR3 and its ligands is involved in guiding receptorbearing cells to specific parts of the body, especially sites of inflammation, immune impairment, and immune dysfunction.

[0096] In various embodiments, the present application provides effector cells or iPSCs genetically engineered to comprise, among other editing as contemplated and described herein, asolid tumor targeting backbone comprising, among other genetic modalities, a C-X-C motif chemokine receptor. In various embodiments, the C-X-C motif chemokine receptor comprises CXCR2 or CXCR3, or variants thereof. A non-limiting example of the amino acid sequence of human CXCR2 is one registered as UniProtKB No: P25025. In one embodiment, the CXCR2 comprises an amino acid sequence of at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 1. In some embodiments, the CXCR2 comprises an amino acid sequence of at least 90% identity to SEQ ID NO: 1. In some embodiments, the CXCR2 comprises an amino acid sequence of at least 95% identity to SEQ ID NO: 1. In some embodiments, the CXCR2 comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variant of CXCR2 comprises a CXCR2 isoform represented by SEQ ID NOs: 2, 3, 4, 5, or 6. In some embodiments, the variant of CXCR2 comprises an amino acid sequence of at least 75%, 80%, 85%, 90%, 95% or 99% identity to any one of SEQ ID NOs: 2, 3, 4, 5, and 6. In some embodiments, the variant of CXCR2 comprises an amino acid sequence of at least 90% identity to any one of SEQ ID NOs: 2, 3, 4, 5, and 6. In some embodiments, the variant of CXCR2 comprises an amino acid sequence of at least 95% identity to any one of SEQ ID NOs: 2, 3, 4, 5, and 6. In some embodiments, the variant of CXCR2 comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the variant of CXCR2 comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the variant of CXCR2 comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the variant of CXCR2 comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the variant of CXCR2 comprises the amino acid sequence of SEQ ID NO: 6. 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 i.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: 1MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVI IYALVFLLSLLGNSLVM LVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCKVVSLLKEVNFYSGILLLACI SVDRYLAIVHATRTLTQKRYLVKFICLSIWGLSLLLALPVLLFRRTVYSSNVSPACYEDMGNNTANWRML LRILPQSFGFIVPLLIMLFCYGFTLRTLFKAHMGQKHRAMRVIFAWLIFLLCWLPYNLVLLADTLMRTQ VIQETCERRNHIDRALDATEILGILHSCLNPLIYAFIGQKFRHGLLKILAIHGLISKDSLPKDSRPSFVG SSSGHTSTTL(360 a.a. CXCR2; UniProtKB No: P25025)SEQ ID NO: 2MEDFNMESDSFEDFW(15 a.a. CXCR2 Isoform 1 (residues 1-15 of CXCR2); UniProtKB No: Q6LCZ7)SEQ ID NO: 3MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVI IYALVFL LSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCK VVSLLKEVNFYSGILLLACISVDRYLAIVHATRTLTQKRYLVKFICLSIWGLSLLLALPV LLFRRTVYSSNVSPACYEDM(200 a.a. CXCR2 Isoform 2 (residues 1-200 of CXCR2); UniProtKB No: C9JW47)SEQ ID NO: 4MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVI IYALVFL LSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCK VVSLLKEVNFYSGIL(135 a.a. CXCR2 Isoform 3 (residues 1-135 of CXCR2); UniProtKB No: C9JG19)SEQ ID NO: 5MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVI IYALVFLLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCKVVSLLKEVNFYSGILLLACISVDRYLAIVHATRTLTQKRYLVKFICLSIWGL(172 a.a. CXCR2 Isoform 4 (residues 1-172 of CXCR2); UniProtKB No: C9J1J7)SEQ ID NO: 6MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVI IYALVFL LSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCK VVSLLKEVNFYSGILLLA(138 a.a. CXCR2 Isoform 5 (residues 1-138 of CXCR2); UniProtKB No: C9J2F9)

[0097] In various embodiments, the polynucleotide encoding the C-X-C motif chemokine receptor or variant thereof is inserted in a selected locus of a primary-sourced effector cell or an iPSC for deriving functional effector cells comprising the same genetic editing through directed differentiation. In some embodiments, the selected locus for insertion of the C-X-C motif chemokine receptor comprises a safe harbor locus, a gene locus intended to be disrupted or knocked out, a gene locus that provides an endogenous promoter that provides spacial and / or temporal control of the exogenous gene expression. In some embodiments, the selected locus for C-X-C motif chemokine receptor insertion comprises AAVS1, CCR5, ROSA26, collagen, HTRP, Hl l, PH12, GAPDH, RUNX1, B2M, TAPI, TAP2, Tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In one embodiment, theselected locus for C-X-C motif chemokine receptor insertion is the TCR locus. In one embodiment, the selected locus for C-X-C motif chemokine receptor insertion is the CD38 locus.

[0098] In some embodiments, the C-X-C motif chemokine receptor is co-expressed with one or more exogenous polynucleotides encoding a polypeptide of interest through separate expression constructs, or a single bi- or tri- cistronic expression cassete. In some embodiments, the single bi- or tri- cistronic expression cassete comprising the C-X-C motif chemokine receptor and one or more exogenous polynucleotides encoding a polypeptide of interest comprises a 2A sequence, such that the C-X-C motif chemokine receptor and the additional polynucleotide(s) are in a single open reading frame (ORF). The bi-cistronic design allows coordinated expression of multiple polynucleotides both in timing and quantity, and under the same control mechanism that may be chosen to incorporate, for example, an inducible promoter for the expression of the single ORF. Self-cleaving peptides are found in members of the Picornaviridae 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, ML, et al, J. Gen. Virol, 82, 1027-101 (2001); Ryan, MD, et al., J. Gen. Virol., 72, 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. In some embodiments, the exogenous polynucleotides that could be co-expressed with the C-X-C motif chemokine receptor encode one or more polypeptides comprising a CAR, a CD 16 or a variant thereof, a cytokine, a cytokine receptor, a cytokine signaling complex, a chimeric fusion receptor, a chimeric Fc receptor, an engager, a checkpoint inhibitor, an Fc receptor, or an antibody or functional variant or fragment thereof. In one embodiment, the exogenous polynucleotides that are co-expressed with the C-X-C motif chemokine receptor in a bi-cistronic cassette do not encode a CAR. In one embodiment, at least one exogenous polynucleotide that is co-expressed in a bi-cistronic cassette with the C-X-C motif chemokine receptor encodes an exogenous CD 16. In some embodiments, the primary-sourced or derived effector cells comprising the C-X-C motif chemokine receptor or variant thereof are T lineage cells.

[0099] Additionally provided in this application is a master cell bank comprising single cell sorted and expanded clonal engineered iPSCs having at least one modification or phenotype as provided herein, including but not limited to, a C-X-C motif chemokine receptor or a variant thereof, wherein the cell bank provides clonal engineered iPSCs for additional engineering and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapyproducts, including but not limited to derivative T cells, which are well-defined and uniform in composition, and can be mass produced at significant scale in a cost-effective manner.2. Exogenously introduced TGFp Redirector Receptor[000100] Transforming growth factor beta (TGFP) is a multipotent immunosuppressive cytokine with complex roles in tumorigenesis including epithelial to mesenchymal transition, angiogenesis, tumor cell motility and metastasis, cancer associated fibroblast (CAF) proliferation, and immunosuppression. TGFP exists in its latent form in the tumor microenvironment, and is known to suppress T cell effector function, in part, through Smad- mediated downregulation of the target genes granzyme, perforin, and interferon. Furthermore, the detection of a TGFP gene expression signature correlates with T cell exclusion from tumors and resistance to immunotherapy. One aspect of the present application provides a multielement solid tumor targeting backbone design that incorporates a synthetic transforming growth factor beta receptor (TGFpR) signaling redirector receptor, among other editing as contemplated and described herein, to equip allogeneic effector cells, including those derived from genetically engineered iPSCs for better efficacy in tumors in general, and in solid tumors in particular. In general, a “signaling (or signal) redirector receptor” or “SRR” redirects the signaling of an extracellular domain from a first receptor (e.g., a TGFP receptor) through an intracellular domain from a different receptor (e.g., a cytokine receptor) by joining the extracellular domain of the first receptor and intracellular domains of the different receptor. In the context of TGFpR, the signaling redirector receptor may be referred to as a “TGFpR redirector” or “TGFpR redirector receptor” or “TGFP signal redirector receptor” or “TGFP-SRR” throughout this application.[000101] In some embodiments, iPSCs and derivative cells therefrom comprise a polynucleotide encoding a TGFP redirector receptor (TGFP-SRR), which comprises a partial or full peptide of an extracellular domain (ECD) of TGFpR. In some embodiments, the TGFp redirector receptor comprises: (i) an extracellular domain, or a fragment thereof, of transforming growth factor beta receptor (TGFpR); and (ii) an intracellular domain (ICD), or a fragment thereof, of a cytokine receptor comprising IL2R, IL12R, IL18R, IL21R, or any combination thereof. In some embodiments, the intracellular domain is an intracellular domain of IL18R. [000102] In some embodiments, the TGFP redirector receptor comprising the ECD and ICD as described above further comprises a transmembrane domain (TM). In various embodiments, the transmembrane (TM) domain of the TGFP redirector receptor can: (i) originate from the same molecule providing the intracellular domain, (ii) originate from the same molecule providing the extracellular domain, or (iii) may be modified or replaced with a transmembranedomain of any other membrane bound proteins. In some embodiments, the cytokine receptor providing an intracellular domain or a fragment thereof of the TGFP redirector receptor comprises at least one of IL2R, IL4R, IL6R, IL7R, IL9R, IL10R, IL11R, IL12R, IL15R, IL18R and IL21R.[000103] In some embodiments, the extracellular domain (ECD) of TGFpR comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 7. In some embodiments, the extracellular domain (ECD) of TGFpR comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 7. In some embodiments, the extracellular domain (ECD) of TGFpR comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 7. In some embodiments, the extracellular domain (ECD) of TGFpR comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the intracellular domain (ICD) of IL18RP comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 8. In some embodiments, the intracellular domain (ICD) of IL18RP comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 8. In some embodiments, the intracellular domain (ICD) of IL18RP comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 8. In some embodiments, the intracellular domain (ICD) of IL18RP comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the ECD and ICD are joined by a TM. In some embodiments, the TM comprises SEQ ID NO: 9.SEQ ID NO: 7TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKND ENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDLLL VIFQ( ECD of TGFpR)SEQ ID NO: 8 YRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVV PGGAVVDEIHSLIEKSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKI ILIEFTPVTDFTFLPQSLK LLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES ( ICD of IL8Rp )SEQ ID NO: 9VTGISLLPPLGVAISVI IIFYCYRVN( exemplary TM domain)[000104] Accordingly, in some embodiments, the present invention provides immune cells, iPSCs, and iPSC derived cells comprising a solid tumor targeting backbone comprising a polynucleotide encoding a TGFP redirector receptor (“TGFP-SRR”), among other genetic modalities, wherein the cells, such as derivative T cells, are useful for overcoming or reducing tumor microenvironment suppression associated with a tumor, and particularly, a solid tumor. In some embodiments, the iPSC and derivative cells thereof comprise a solid tumor targeting backbone comprising two or more of: a polynucleotide encoding a C-X-C motif chemokine receptor or a variant thereof, a polynucleotide encoding a TGFP redirector receptor, and and / or one or more additional genomic edits as described herein, without adversely impacting the differentiation potential of the iPSC and function of the derived effector cells, such as derivative T cells.3. CD38 knockout[000105] 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).[000106] 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.[000107] 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).[000108] 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.[000109] 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.[000110] Moreover, the strategy as provided herein, i.e., CD38 knockout, is compatible with other components and processes contemplated herein, thereby generating an iPSC line having CD 19 targeting specificity described herein and a CD38 knockout, a master cell bank comprising single cell sorted and expanded clonal iPSCs, and obtaining CD38 negative (CD38negor CD38z) derivative effector cells comprising the CD 19 targeting specificity described herein through directed differentiation of the engineered iPSC line, wherein the derivative effector cells are protected against fratricide and allorej ection when CD38 targeted therapeutic moieties are employed with the effector cells among other advantages including improved metabolic fitness, increased resistance to oxidative stress and inducing a proteinexpression 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 CD38negderivative 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.[000111] In one embodiment as provided herein, the CD38 knockout in an iPSC line is a bi- allelic knockout. 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.4. TCR Knockout[000112] In some embodiments, a polynucleotide encoding one or more modalities described herein (e.g., one or more of a CAR, CXCR2, IL7RF, or TGFP-SRR) inserted in a TCR constant region (e.g., TRAC or TRBC), leading to TCR knockout, and optionally placing expression of the one or more modalities under the control of the endogenous TCR promoter. The disruption of the constant region of TCRa or TCRP (TRAC or TRBC) produces a TCRnegcell. TCRnegcells do not require HLA matching, have reduced alloreactivity, and are able to prevent GvHD (Graft versus Host Disease) when used in allogeneic adoptive cell therapies. In some embodiments, polynucleotides encoding different modalities described herein are inserted at different cites. Additional insertion sites include, but are not limited to, AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, PH12, GAPDH, RUNX1, B2M, TAPI, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, NKG2A, NKG2D, CD25, CD38, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT.[000113] In one embodiment as provided herein, the TCR knockout in an iPSC line is a bi- allelic knockout. When two or more transgenes are to be inserted at a selected location in a TCR 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 may be included in the construct to reduce the risk of transgene and / or exogenous promoter silencing.5. CD16 knock-in[000114] 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 some embodiments, the hnCD16 comprises an amino acid sequence of at least about 90% identity to SEQ ID NO: 10. In some embodiments, the hnCD16 comprises an amino acid sequence of at least about 95% identity to SEQ ID NO: 10. In some embodiments, the hnCD16 comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, an 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. 11, 12 and 13, and each comprises at least a portion of CD64 ectodomain. In some embodiments, the hnCD16 comprises an amino acid sequence of at least 90% identity to any of SEQ ID NOs. 11-13, and optionally one or more of Fl 76V, S197P, and at least a portion of CD64 ectodomain. In some embodiments, the hnCD16 comprises an amino acid sequence of at least 95% identity to any of SEQ ID NOs. 11-13, and optionally one or more of F176V, S197P, and at least a portion of CD64 ectodomain. In some embodiments, the hnCD16 comprises the amino acid sequence ofSEQ ID NO 11. In some embodiments, the hnCD16 comprises the amino acid sequence of SEQ ID NO 12. In some embodiments, the hnCD16 comprises the amino acid sequence of SEQ ID NO 13.SEQ ID NO: 10MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQ ASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVT YLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVPTISSFFPPGYQVS FCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDKSEQ ID NO: 11MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSY RITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRN GKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTL SCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQV LGLQLPYPYV1FRYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK ( 340 a . a . CD64 domain-based construction ; CD16TM; CD16ICD)SEQ ID NO: 12MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSY RITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRN GKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTL SCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVL GL F F P P GYQ VSFCL VMVLLFAVDTGL YFSVKTNIRSS TRD WKDHKFKWRKDPQDK ( 336 a . a . CD64 exon-based construction ; CD16TM; CD16ICD)SEQ ID NO: 13MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSY RITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRN GKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTL SCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQV 1YGFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK ( 335 a . a . CD64 exon-based construction ; CD16TM; CD16ICD)[000115] 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 extracellular binding domains of the exogenous CD 16. Examples of bi-, tri-, or multi- specific engagers or binders are further described below in this application. As such, at least one of the aspects of the present application provides a derivativeeffector 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 a CD 16 having Fl 76V and S197P.[000116] Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism of NK cell mediated lysis through the binding of CD 16 to antibody-coated target cells. However, the endogenous CD 16 expressed by primary NK cells gets cleaved from the cellular surface following NK cell activation. Various non-cleavable versions of CD 16 avoid CD 16 shedding and maintain constant expression. In some embodiments, non-cleavable CD 16 provides the ADCC, and the engagement of bi-, tri-, or multi- specific engagers to T cells. In derivative T cells, non-cleavable CD16 increases expression of TNFa and CD107a, indicative of improved cell functionality. The additional high affinity characteristics of the introduced hnCD16 in derived T cells also enables in vitro loading of an ADCC antibody to the T cell through hnCD16 before administering the cell to a subject in need of a cell therapy.[000117] In some embodiments, the derived T cells comprising HER2 tumor antigen targeting specificity as described herein, have additional tumor antigen targeting specificity via an exogenous CD 16 or a variant thereof that mediates ADCC when in combination with an antibody. In some embodiments, the derived T cells comprising HER2 tumor antigen targeting specificity described herein further comprise CD38 knockout. In some embodiments, the derived T cells comprising HER2 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 T cells comprising HER2 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 bispecific antibody targeting EGFR and MET (e.g., amivantamab).6. Exogenously introduced cytokine signaling complex[000118] 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 withoutthe 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.[000119] In other various embodiments, the cytokine signaling complex comprises an IL7 receptor fusion (IL7RF) comprising a full or partial length of IL7 and a full or partial length of IL7 receptor. The transmembrane (TM) domain can be native to the IL7 receptor or may be modified or replaced with a transmembrane domain of any other membrane bound proteins. In one embodiment, a native (or wildtype) or modified IL7R may be fused to IL7 at the C-terminus through a linker, enabling constitutive signaling and maintaining membrane-bound IL7. In some embodiments, such a construct comprises an amino acid sequence of at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 14, with transmembrane domain, signal peptide and linker being flexible and varying in length and / or sequences. In some embodiments, the IL7 construct comprises an amino acid sequence of at least 85% identity to SEQ ID NO: 14, with transmembrane domain, signal peptide and linker being flexible and varying in length and / or sequences. In some embodiments, the IL7 construct comprises an amino acid sequence of at least 90% identity to SEQ ID NO: 14, with transmembrane domain, signal peptide and linker being flexible and varying in length and / or sequences. In some embodiments, the IL7 construct comprises an amino acid sequence of at least 95% identity to SEQ ID NO: 14, with transmembrane domain, signal peptide and linker being flexible and varying in length and / or sequences. In some embodiments, the IL7 construct comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, such a construct comprises an amino acid sequence of at least 85%, 90%, 95% or 99% identity to SEQ ID NO: 14, excluding the amino acids of the exemplary transmembrane domain, signal peptide and linker indicated in SEQ ID NO: 14.SEQ ID NO: 14MDWTWILFLVAAATEVHSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKE GMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQ KKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGGGSGGGGSGGGGSGGGGSGGGSLOESGYAQNGDLEDAE LDDYSFSCYSQLEVNGSQHSLTCAFEDPDVNITNLEFEICGALVEVKCLNFRKLQEIYFIETKKFLLIGK SNICVKVGEKSLTCKKIDLTTIVKPEAPFDLSVVYREGANDFVVTFNTSHLQKKYVKVLMHDVAYRQEKD ENKWTHVNLSSTKLTLLQRKLQPAAMYEIKVRS IPDHYFKGFWSEWSPSYYFRTPEINNSSGEMDPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDI QARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDWITPESFGRDSSLTCLAGNVSACDAPILSS SRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSS FYQNQ(Signal peptide-TLl -linker-IL7R; transmembrane domain (TM), signal peptide and linker can vary in length and sequences)[000120] In another embodiment, a native or modified common receptor yC is fused to IL7 at the C-terminus through a linker for constitutive and membrane-bound cytokine signaling complex. In addition, engineered IL7R that forms a homodimer in the absence of IL7 is useful for producing constitutive signaling of the cytokine as well.[000121] 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.[000122] 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. In some embodiments, IL 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 2 A 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 some embodiments, bi-cistronic designs allow 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. 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. 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 porcinetescho virus- 1 (PTV-I) (Donnelly, et al, J. Gen. Virol, 82, 1027-101 (2001); Ryan, et al., J. Gen. Virol., 72, 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.[000123] 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 HER2 tumor antigen targeting specificity, and wherein the cell comprises polynucleotides encoding a HER2-CAR (chimeric antigen receptor), a CD 16 or a variant thereof that mediates ADCC when in combination with an monoclonal antibody, a CD38 knockout, and an IL7 signaling complex.[000124] 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 T lineage cells. iPSC-derived T lineage cells comprising CD38 negative and exogenous CD 16 or a variant thereof have enhanced cytotoxicity and have reduced susceptibility to host alloreactive immune cell cytotoxicity in the presence of anti-CD38 antibodies.[000125] The iPSC, an iPS cell line cell, or a population thereof, or a derivative functional cell obtained from differentiating the iPSC provided herein, comprising a CD38 knockout and polynucleotides encoding a HER2-CAR (chimeric antigen receptor), a CD 16 or a variant thereof, a CXCR2, a TGFP-SRR, and an IL7 signaling complex, wherein the exogenous cytokine signaling complex (IL) enables 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. 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, theactivation of the cytokine signaling is inducible. In some embodiments, the activation of the cytokine signaling is transient and / or temporal. In some embodiments, the exogenous cell surface cytokine and / or receptor comprised in the iPSC or derivative cells thereof enables IL7 signaling.7. Chimeric Antigen Receptor (CAR) expression[000126] Applicable to the genetically engineered immune cells, iPSCs and derivative effector cells thereof may be any CAR design known in the art. CAR is a fusion protein generally including an ectodomain that comprises a target binding region (for example, an antigen recognition domain), a transmembrane domain, and an endodomain. In some embodiments, the ectodomain can further include a signal peptide or leader sequence and / or a spacer. In some embodiments, the endodomain can further comprise a signaling peptide that activates the effector cell expressing the CAR. In some embodiments, the signaling peptide of the endodomain (or intracellular domain) comprises a full length or at least a portion of a polypeptide of 2B4, CD2, CD3< CDS^IXX, CD8, CD28, CD28H, CD137 (4-1BB), CS1, DAP10, DAP12, DNAM1, FcERIy, IL2Ry, IL7R, IL21R, IL2RP (IL15RP), IL21, IL7, IL12, IL15, IL21, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, or NKG2D. In one embodiment, the signaling peptide of a CAR 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 ITAM (immunoreceptor tyrosine-based activation motif) of CD3(^. Exemplary N-terminal signal peptides include MALPVTALLLPLALLLHA (SEQ ID NO: 15; CD8asp) or MDFQVQIFSFLLISASVIMSR (SEQ ID NO: 16; IgKsp), or any signal peptide sequence or functional variants thereof known in the art.[000127] In some embodiments, the antigen recognition domain can specifically bind an antigen. In some embodiments, the CAR is suitable to activate T, NK or NKT cells expressing said CAR. In some embodiments, the CAR is T cell specific for comprising T-specific signaling components. In certain embodiments, said T cells are derived from CAR-encoding iPSCs comprising a solid tumor targeting backbone as described herein, 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.[000128] In various embodiments, the antigen recognition region comprises a murine antibody, a human antibody, a humanized antibody, a camel Ig, a single variable new antigen receptor (VNAR), a shark heavy-chain antibody (Ig-NAR), a chimeric antibody, a recombinant antibody, a single-domain antibody (dAb), an anti-idiotype antibody, a bi-specific-, multi- specific- or multimeric- antibody, or antibody fragment thereof. Anti-idiotype antibodies arespecific for binding to an idiotope of another antibody, wherein the idiotope is an antigenic determinant of an antibody. Abi-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). Non-limiting examples of antibody fragments include Fab, Fab’, F(ab’)2, F(ab’)3, Fv, Fabc, pFc, Fd, single chain fragment variable (scFv), tandem scFv (scFv)2, single chain Fab (scFab), disulfide stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen binding fragments (sdAb), camelid heavy-chain IgG and Nanobody® fragments, recombinant heavy-chain-only antibody (VHH), and other antibody fragments that maintain the binding specificity of the antibody. In some embodiments an antigen binding domain of a CAR comprises CDR1, CDR2, and CDR3 of a heavy chain (H-CDRs) of an antibody or fragments thereof. In some embodiments, the antigen binding domain of a CAR comprising the H-CDRs of an antibody further comprises the CDRs of a light chain (L-CDRs) of the antibody.[000129] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor associated HER2, wherein an effector cell comprising said CAR and a solid tumor targeting backbone as disclosed is useful for treating one or more cancers comprising at least bladder cancer, breast cancer, breast lung cancer, colorectal cancer, esophageal cancer, gastric / stomach cancer, head and neck cancer, lung cancer, ovarian cancer, or salivary gland cancer.[000130] In some embodiments the antigen binding domain of the CAR comprises a single chain variable fragment (scFV) having a sequence identity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage in-between, when compared to the exemplary sequences represented by SEQ ID NO: 17 or SEQ ID NO: 18, wherein each of SEQ ID NOs: 17 and 18 comprise a linker that can vary in length and / or sequence. In some embodiments, the scFV comprises an amino acid sequence of at least 90% identity to SEQ ID NO: 17 or 18. In some embodiments, the scFV comprises an amino acid sequence of at least 95% identity to SEQ ID NO: 17 or 18. In some embodiments, the scFV comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the scFV comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the CAR further comprises a transmembrane domain, such as a transmembrane domain of CD28. In some embodiments, the CAR further comprises a costimulatory domain, such as a co-stimulatory domain of CD28. In some embodiments, the CAR further comprises an activation domain, such as an activation domain of CD3p XX.SEQ ID NO: 17EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYGMSWVRQTPDRRLELVATINNNGGGTYYPDSVKGRFTI SRDNAKNTLYLQMSSLKSEDTAMYYCTSPGLLWDAWGAGTTVTVSSGSTSGGGSGGGSGGGGSSDWMTQ TPLTLSVSIGQPASISCKSSQSLLDSDGRTYLNWLLQRPGQSPKRLIYLVSKLDSGAPDRFTGSGSGTDF TLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIKSEQ ID NO: 18DWMTQTPLTLSVSIGQPASISCKSSQSLLDSDGRTYLNWLLQRPGQSPKRLIYLVSKLDSGAPDRFTGS GSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLVESGGG LVQPGGSLKLSCAASGFTFSNYGMSWVRQTPDRRLELVATINNNGGGTYYPDSVKGRFTISRDNAKNTLY LQMSSLKSE DT AM Y Y CT S P GL L WDAWGAGT V VS S[000131] In one embodiment, the CAR provided herein 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: 19, wherein the linker in the ectodomain and the spacer between the ectodomain and transmembrane domain may vary in length and sequence. In some embodiments, the CAR comprises an amino acid sequence of at least about 90% identity to SEQ ID NO: 19, wherein the linker in the ectodomain and the spacer between the ectodomain and transmembrane domain may vary in length and sequence. In some embodiments, the CAR comprises an amino acid sequence of at least about 95% identity to SEQ ID NO: 19, wherein the linker in the ectodomain and the spacer between the ectodomain and transmembrane domain may vary in length and sequence. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the CAR provided herein recognizes a HER2 antigen specific to cells of solid tumors. In some embodiments, the CAR provided herein recognizes a HER2 antigen of a tumor comprising breast cancer, ovary cancer, endometrium cancer, lung cancer, esophageal cancer, salivary gland cancer, bladder cancer, gastric cancer, colorectal cancer, or head and neck cancer. In yet some other embodiments, the CAR provided herein recognizes a HER2 antigen of a tumor and does not respond, or has a low level of response, to HER2 expressed on non-cancer or normal cells.SEQ ID NO: 19EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYGMSWVRQTPDRRLELVATINNNGGGTYYPDSVKGRFTI S RDNAKNTL YLQMS S LK S EDT AMYYCT S P GLLWDAWGAGTT VT VS SGSTSGGGSGGGSGGGGSSDWMTQ TPLTLSVSIGQPASISCKSSQSLLDSDGRTYLNWLLQRPGQSPKRLIYLVSKLDSGAPDRFTGSGSGTDF TLK I S RVEAEDLGVYYCWQGTHFPQTFGGGTKLE I KESKYGPPCPPCPGGGSSGGGSGGQPREPQ VYTLP PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSWHEAL HYTQKSLSLSLGKMFWLVWGGVLACYSLLVTVAFI IFWVRSKRSRLLHSDYMNMTP RRPGPTRKRYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR(anti-HER2 scFV[linker]- spacer- CD28 TM- CD28 Costim-CD3^1XX activation)[000132] As described herein, in some embodiments, the cells comprising a solid tumor targeting backbone comprising polynucleotides encoding a CXCR2, a TGFP-SRR, a HER2- CAR and / or one or more additional modified modalities provided herein. In iPSCs and derivative cells therefrom comprising both an exogenous cytokine signaling complex (“IL”) and a HER2-CAR, the IL and HER2-CAR may be expressed in separate constructs, or may be coexpressed in a bi-cistronic construct comprising both IL and CAR. 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, a solid tumor targeting backbone as described herein and a HER2-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 T cells, which are well-defined and uniform in composition, and can be mass produced at significant scale in a cost-effective manner.8. Antibodies for immunotherapy[000133] 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. 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, anti-CD20 antibody (e.g. rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab), an anti-EGFR antibody (cetuximab, matuzumab, panitumumab and necitumumab), an anti-HER2 antibody(trastuzumab or biosimilars, pertuzumab, 4B5, ertumaxomab), an anti-PDLl 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®).[000134] 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 2-6 days prior to the first cycle of administering the iPSC-derived effector cells. In some embodiments, the antibody for combination treatment is rituximab and wherein the initial dose is a single initial dose of about 300 mg / m2to about 450 mg / m2administered to the subject about 4 days prior to the first cycle of administering the effector cells.9. Checkpoint inhibitors[000135] 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.[000136] Some embodiments of the combination therapy with the provided derivative T cells comprise at least one checkpoint inhibitor to target at least one checkpoint molecule. Suitable checkpoint inhibitors for combination therapy with the derivative T 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 (CD 152), 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).[000137] 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 -PD1 / anti-PDLl mAb), avelumab (anti-PDLl mAb), durvalumab (anti-PDLl mAb), and any derivatives, functional equivalents, or biosimilars thereof.[000138] 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.[000139] 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 ofadministering 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.IL Therapeutic Use of Derivative Immune Cells For Solid Tumors[000140] Prior to the present disclosure, therapeutic benefit of CAR T-cell therapies in the treatment of solid tumors has been modest. Among the challenges facing clinical application of CAR T cell therapy in solid tumors is the heterogeneity of putative solid tumor target antigen expression on normal tissues, which increases the risk of on-target, off-tumor toxicity, especially in the absence of differential expression between tumors and normal tissues. Efforts to maximize the therapeutic index of CAR T cells in solid tumors have therefore focused on targeting single antigens and / or combinations of antigens whose expression on tumor cells, which frequently arise from mutated oncogenic drivers, are distinguished from their expression in normal tissues (Sterner and Sterner, 2021). Additional challenges facing CAR T-cell therapies that are unique to solid tumors, such as facilitating the trafficking of the T-cell product into tumors and overcoming the suppressive tumor immune microenvironment, may call for further modifications of the T cell product.[000141] Mutations driving altered expression and / or signaling of the ErbB family of receptor tyrosine kinases, which include epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2) / neu (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4), are recognized oncogenic drivers in a broad range of solid tumors (Zhang et al., 2007). Agents targeting EGFR and HER2 are key components of some standard anticancer treatment regimens (e.g., Enhertu® USPI; Erbitux® USPI; Gilotrif® USPI; Herceptin® USPI; Iressa® USPI; Kadcyla® USPI; Margenza® USPI; Nerlynx® USPI; Perjeta® USPI; Rybrevant® USPI; Tagrisso® USPI; Tarceva® USPI; Tukysa® USPI; Tykerb® USPI; Vectibix® USPI). However, despite the availability of approved therapies for advanced metastatic HER2- and EGFR-positive malignancies, novel treatments to improve survival have been scarce.[000142] The efficacy of current HER2- and EGFR-targeting agents are often limited by tumor resistance mechanisms and levels of expression that are insufficient to result in meaningful clinical benefit. For example, the clinical activity of HER2 -targeting agents is largely predicated on demonstrating sufficient levels of HER2 expression based on established diagnostic testing criteria (Bartley et al., 2016; Wolff et al., 2018). Similarly, tumor-specificfactors impact the clinical benefit with EGFR-targeting agents. The efficacy of EGFR-targeting monoclonal antibodies (mAbs) in colorectal cancer (CRC), for example, has been limited to K- RAS wild-type tumors (Erbitux USPI; Vectibix USPI) and is not correlated to EGFR expression (Hecht et al., 2010). In non-small cell lung cancer (NSCLC), EGFR-targeting agents have provided clinical benefit only to the approximately one-third of patients whose tumors contain EGFR driver mutations (Zhang et al., 2016). Moreover, therapeutic options have been limited upon progression due to the development of tumor resistance.[000143] The clinical activity of HER2- and EGFR- directed therapies in tumors that have recently been shown to express HER2 and / or EGFR are relatively unknown. For example, HER2 expression has been demonstrated in subsets of multiple solid malignancies, including cancers of the salivary gland and duct, and ovarian, pancreatic, endometrial, cervical, bladder, and biliary / gall-bladder cancers (Dumbrava et al., 2019; Omar et al., 2015). In addition, advanced EGFR mutated lung cancer that has progressed following prior treatment with tyrosine kinase inhibitors (TKIs) may acquire a resistance mechanism through HER2 gene amplification (Yu et al., 2013), making HER2 a targetable tumor antigen.[000144] Unlike lineage-specific surface proteins, e.g., CD19 and BCMAthat are highly selective targets in hematologic malignancies, surface proteins including EGFR and HER2 are expressed on normal cells as well as cancer cells, resulting in the potential for on-target, off- tumor toxicity (Sterner and Sterner, 2021). For example, reported toxi cities associated with HER2-directed therapies, such as cardiomyopathy and cardiac failure (Herceptin USPI; Kadcyla USPI; Perjeta USPI), diarrhea (Nerlynx USPI; Tukysa USPI), and interstitial lung disease (ILD) and pneumonitis (Enhertu USPI) are the direct consequence of low-level HER2 expression in normal tissue.[000145] An additional important consideration for the clinical application of CAR T-cell therapies for solid tumors involves the need to generate “immunologic space” for the CAR T- cell product to persist and expand. Approved CAR T-cell therapies for B-cell malignancies are typically administered in combination with lymphodepleting regimens including cyclophosphamide (CY) and fludarabine (FLU) or bendamustine, which in addition to facilitating CAR T-cell persistence and expansion also provide anti-tumor activity. While there has been early demonstration of clinical activity, the optimal chemotherapy to combine with CAR T cells remains unclear (Bechman and Maher, 2021; Owen et al., 2023), and a greater understanding of the ability of chemotherapy and other agents to enhance the pharmacokinetic (PK) and clinical activity of CAR T cells in solid tumors is needed. Regimens for providing improved therapeutic benefit in treating solid tumors are provided herein.[000146] Treatment using 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; inhibiting the disease (e.g., arresting its development); or relieving the disease (e.g., 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.[000147] FT825, as described herein, refers to a human induced pluripotent stem cell(iPSC) derived CAR T-cell therapy that incorporates multiple engineered modalities designed to optimize anti-tumor activity against multiple oncogenic targets, including HER2-expressing cancers. In addition to a CAR that exhibits preferential targeting of HER2 on tumor cells relative to normal cells, FT825 incorporates an IL7 / IL7 receptor fusion protein (IL7RF) designed to enhance cell persistence, a C-X-C motif chemokine receptor 2 (CXCR2) to optimize trafficking of cells to tumors, a transforming growth factor beta signal redirection receptor (TGFP-SRR) to overcome the immunosuppressive tumor microenvironment, and a high affinity, non-cleavable CD 16 (hnCD16) to enable multi-antigen targeting via antibody-dependent cellular cytotoxicity (ADCC) through combinations with mAbs, including, but not limited to, cetuximab (an anti-EGFR monoclonal antibody). hnCD16 enhances the activity of FT825 when combined with therapeutic antibodies against tumor cells with minimal HER2 expression, supporting clinical application against tumors agnostic of HER2 status. A Phase 1 study of FT825 for the treatment of participants with advanced HER2 expressing solid tumors is designed to utilize the functional attributes of FT825 in a treatment regimen intended to overcome barriers to effective immune-cell therapies in solid tumors.[000148] The Phase 1 study described herein assesses the clinical activity of FT825 in participants with HER2 expressing tumors where there is known clinical activity with HER2- targeting agents (e.g., breast cancer, esophagogastric cancer and gastroesophageal junction [GEJ] adenocarcinoma, HER2 mutated NSCLC, and CRCs) and in HER2-expressing tumors forwhich there are no currently approved HER2 targeting agents, such as salivary gland cancers and endometrial cancers. In addition, the study provides initial evaluation of FT825 combined with cetuximab in participants with tumors that express EGFR and those that co-express EGFR and HER2. The study also assesses docetaxel and low-dose cisplatin chemotherapy with FT825 as Cycle 2 retreatment in participants with disease progression following Cycle 1 FT825 treatment with CY / FLU chemotherapy.[000149] In some embodiments, FT825 drug substance comprises allogeneic T cells, derived from a T-cell receptor (TCR) and CD38 knockout iPSC line, that express a HER2 targeted CAR, IL7RF, TGFP-SRR, hnCD16, and CXCR2. In some embodiments, manufacture of drug substance from an iPSC master cell bank involves stages of iPSC expansion, followed by stages of differentiation to T cells and T cell expansion, which may include sequential coculture with two separate irradiated feeder cell lines. Cells that are harvested after T-cell expansion may then be washed and designated as the drug substance. In some embodiments, FT825 cells are intended to primarily mediate anti-tumor activity via the following pathways: cytotoxicity of HER2-expressing target cells following recognition by a HER2-directed CAR; and ADCC, through which hnCD16-expressing drug product cells actively lyse a target cell whose membrane-surface antigens have been bound by specific mAbs administered in combination with the drug product cells.[000150] In some embodiments, FT825 cells support anti-tumor activity via multiple pathways. For example, FT825 expresses an IL7RF containing an IL7 covalently linked to an IL7Ra by a flexible linker, which is designed to enhance T cell survival and persistence; FT825 expresses a TGFP-SRR in which extracellular and transmembrane domains of TGFPR2 have been fused with an activating intracellular domain from IL18R, enabling sustained anti -turn or activity in the presence of TGFP; and FT825 expresses a CXCR2 to enhance specific migration to CXCR2-ligand expressing tumors. CXCR2 ligands, including CXCL1, CXCL2, CXCL5, and CXCL8 / IL-8, are enriched within the tumor microenvironment of multiple tumor types, including breast, gastric, lung, and colorectal, and associate with poor patient outcomes. Furthermore, disruption of the CD38 gene enhances metabolic fitness and further prevents anti- CD38 mAb binding, therefore preventing depletion of CD38-knockout effector cells via fratricide induced by anti-CD38 mAbs, which can facilitate the use of anti-CD38 mAbs in scenarios of tumor targeting and lympho-conditioning. Moreover, CD38 is a validated locus for genetic engineering in iPSCs, supporting high and homogenous gene expression of inserted transgenes. In various embodiments, the regimen and dosage design of the cell product takes into consideration drug efficacy and risk reduction. In some embodiments, the regimen anddosage design is towards a subpopulation of the patients. In some embodiments, the regimen and dosage design is towards a subset of an indication, a disease, or a condition.[000151] Specific conditions potentially related to engineered cellular immunotherapy products in general include, but are not limited to, new malignancies, new or worsening neurologic disorders, new or worsening autoimmune or rheumatologic disorders, or new hematologic disorders. Subjects may undergo safety monitoring during treatment, including assessment of the nature, frequency, and severity of adverse events (AEs).[000152] 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 product, whether or not related to the medicinal product.[000153] 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).[000154] Acute allergic / infusion reactions may occur with any treatment, including with the use of CY, FLU, bendamustine, docetaxel, cisplatin, and mAbs. Subjects may 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. Acute allergic / infusion reactions may also be a manifestation of immunogenicity of an allogeneic cell product.[000155] Evidence of FT825 immunogenicity and its clinical impact is monitored, assessed, and managed over the course of treatment, given that potential FT825 induced immune response may manifest only clinically, e.g., as infusion-related reactions with varying degrees of severity.[000156] FT825 is formulated in DMSO to enable cryopreservation. DMSO side effects and symptoms are monitored, assessed, and managed over the course of treatment. DMSO related side effects are generally associated with histamine release and include coughing, flushing, rash, chest tightness and wheezing, nausea and vomiting, and cardiovascular instability. In some embodiments, the method includes slowing the rate of infusion, and / or medicating with antihistamines.[000157] FT825 is extensively tested during manufacturing to minimize the potential risk of disease transmission. However, as a cell therapy of human origin, the cell products may be in contact with reagents of animal origin during processing. Therefore, transmission of infectious disease and / or disease agents by known or unknown agents during the treatment is closely monitored and managed.[000158] Cytokine Release Syndrome (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. CRS is a clearly defined syndrome following treatment with autologous and allogeneic CAR T cell therapies, which can be fatal or life-threatening (Breyanzi USPI; Kymriah USPI; Yescarta USPI). In addition to fever, hypoxia, and hypotension, other clinical manifestations of CRS may include cardiac, gastrointestinal, hepatic, coagulation, renal, respiratory, skin, and constitutional (fever, rigors, headaches, malaise, fatigue, arthralgia, nausea, and vomiting) signs and symptoms. To consistently characterize its severity, CRS are defined and graded according to the ASCO / ASTCT CRS consensus grading. The signs and symptoms of CRS are not unique to CRS. Clinical symptoms, such as bacteremia and other severe infections, that are not considered related to FT825 are not reported as CRS.[000159] Tumor Lysis Syndrome (TLS) is a possible fatal risk associated with anti-tumor therapy in both hematologic and solid tumors, especially with large tumor burden. TLS has been reported to occur within 7 days following chemotherapy across various solid tumor settings, with many 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. TLS symptoms include 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. In some embodiments, methods include prophylaxis for and management of TLS, such as by methods known in the art.[000160] Neurologic toxicities arising as a result of immune therapies has been termed Immune Effector Cell-Associated Neurotoxicity Syndromes, or ICANS, defined as a disordercharacterized by a pathologic process involving the CNS 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 bispecific antibodies such as blinatumomab (Blincyto® USPI). The exact mechanism of toxicity in these settings is not known, but has generally improved with treatment discontinuations and corticosteroids. Central nervous system toxi cities following CD 19 CAR T-cell therapy are characterized by encephalopathy, confusion, delirium, aphasia, obtundation, and seizures (Kymriah® USPI; Yescarta® USPI). Cases of cerebral edema have also been reported in CAR-T therapies.[000161] Because FT825 is an allogeneic immune effector cell product, there is a potential risk of Acute Graft-versus-Host Disease (GvHD). In some embodiments, acute GvHD assessments are performed with assignment of the overall severity based on the CIBMTR acute GvHD grading scale, and the method may include management of GvHD, such as by methods known in the art.[000162] In addition, FT825 may considered a xenotransplantation product when it comes in contact with cells of animal origin (e.g., mouse cells) during the manufacturing process. In some embodiments, the cells of animal origin serve as an ancillary material in the manufacture of drug product but are not intended to be in the drug product. Risks of receiving a xenotransplantation product may include, but are not limited to, developing infections from agents that may be associated with cells of animal origin, transmitting these infectious agents to others, and the growth of tumors. Animal cells that come into contact with the FT825 cells during the manufacturing process may originate from a master cell bank that has been extensively tested to reduce these risks. In some embodiments, subjects are monitored for clinical manifestations of xenotransplantation products throughout the course of treatment, including long-term follow-up, in case of rare associated condition or illness develops.[000163] Some adoptive cell therapies delivered with supportive medications, such as Cyclophosphamide (CY) and Fludarabine (FLU) for conditioning, have been reported to cause myelosuppression (neutropenia and / or thrombocytopenia), immunosuppression, infections, 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 may be undertaken. In some embodiments, the method includes management of cytopenias and infections, including transfusion support, antimicrobial prophylaxis, and use of growth factors, such as by methods known in the art.[000164] Warnings and precautions related to cyclophosphamide (CY) include: myelosuppression, immunosuppression, bone marrow failure, and infections; urinary tract andrenal toxicity including hemorrhagic cystitis, pyelitis, ureteritis, and hematuria; cardiotoxicity including myocarditis, myopericarditis, pericardial effusion, arrythmias, and congestive heart failure; pulmonary toxicity including pneumonitis, pulmonary fibrosis, and pulmonary veno-occlusive disease leading to respiratory failure; secondary malignancies; veno-occlusive liver disease; and embryo-fetal toxicity. Adverse reactions reported most often include neutropenia, febrile neutropenia, fever, alopecia, nausea, vomiting, and diarrhea. In some embodiments, dose modifications due to toxicity are taken into consideration during a course of treatment.[000165] Warnings and precautions related to fludarabine (FLU) include severe bone marrow suppression, notably anemia, thrombocytopenia, and neutropenia; transfusion- associated GvHD; severe CNS toxicity; infections; renal insufficiency; TLS; and embryo-fetal toxicity. Severe CNS toxicity was observed in patients treated at FLU doses of 96 mg / m2for 5 to 7 days. This toxicity was observed in <0.2% of patients treated at FLU doses of 25 mg / m2. Adverse reactions occurring in >30% of participants treated with FLU include myelosuppression (neutropenia, thrombocytopenia, and anemia), fever, infection, nausea and vomiting, fatigue, anorexia, cough, and weakness.[000166] Warnings and precautions related to bendamustine include myelosuppression, infections, infusion reactions and anaphylaxis TLS; skin reactions including rash, toxic skin reactions (such as Stevens- Johnson syndrome and toxic epidermal necrolysis), and bullous exanthema; other malignancies; and fetal harm.[000167] Risks associated with docetaxel include secondary myelodysplasia or acute myeloid leukemia; cutaneous reactions, including erythema of the extremities with edema followed by desquamation; neurologic reactions, including paresthesia, dysesthesia, and pain; asthenia; embryo-fetal toxicity; and common adverse reactions including infections, neutropenia, anemia, febrile neutropenia, hypersensitivity, thrombocytopenia, neuropathy, dysgeusia, dyspnea, constipation, anorexia, nail disorders, fluid retention, asthenia, pain, nausea, diarrhea, vomiting, mucositis, alopecia, skin reactions, and myalgia.[000168] Risks associated with cisplatin include hypersensitivity reactions, which may result in anaphylaxis and death; cumulative ototoxicity; ocular toxicity including optic neuritis, papilledema and cortical blindness; secondary leukemia; embryo-fetal toxicity; and common adverse reactions include nephrotoxicity, peripheral neuropathy, myelosuppression, nausea and vomiting.[000169] In addition to the known and potential risks of FT825, CY, FLU, bendamustine, docetaxel, and cisplatin, additional risks of the combination treatment due to drug interaction including, but not limited to, increased frequency and / or severity of risks known to the mAbmay occur. Therefore evidence of toxicities of approved mAbs whose frequency and severity may be affected by combination treatment with FT825 may be collected and dealt with when risks present over a course of treatment.[000170] In one aspect, the present disclosure provides a method of treating cancer in a subject. In some embodiments, the method comprises: (a) administering to the subject one or more doses of chemotherapy; and (b) administering to the subject one or more doses of an adoptive cell therapy product in a first effective amount; wherein the adoptive cell therapy product comprises engineered T lineage cells comprising (i) expression of a HER2-CAR (chimeric antigen receptor), an IL7 / IL7 receptor fusion protein (IL7RF), a TGFP signaling redirector receptor (TGFP-SRR), a C-X-C motif chemokine receptor 2 (CXCR2), and an exogenous CD 16; (ii) a CD38 knockout; and (iii) a T cell receptor (TCR) knockout; and wherein the cancer comprises cancer cells that are HER2 positive. In some embodiments, the method comprises repeating the foregoing steps.[000171] In some embodiments, the method further comprises: (a) administering to the subject one or more doses of an additional chemotherapy that is the same or different as the chemotherapy (e.g., after administering the one or more doses of the adoptive cell therapy product in the first effective amount); and (b) administering to the subject one or more doses of the adoptive cell therapy product in a second effective amount that is the same or different as the first effective amount (e.g., after administering the additional chemotherapy).[000172] In some embodiments, the cancer cells that are HER2 positive comprise HER2 expression, amplification, or mutation. In some embodiments, the method further comprises administering an EGFR inhibitor to the subject; and wherein the cancer cells comprise an EGFR mutation. In some embodiments, administering the EGFR inhibitor is before and / or after administering the adoptive cell therapy product. In some embodiments, the EGFR inhibitor comprises, matuzumab, panitumumab, or necitumumab. In some embodiments, the EGFR inhibitor comprises cetuximab. In some embodiments, the chemotherapy comprises: (a) one or both of cyclophosphamide (CY) and fludarabine (FLU); (b) bendamustine; or (c) one or both of docetaxel and cisplatin. In some embodiments, the chemotherapy is administered one or more days prior to administering the adoptive cell therapy product; optionally wherein the chemotherapy is administered at least two days, three days, four days, or five days before administering the adoptive cell therapy product. In some embodiments, the chemotherapy (a) comprises (i) cyclophosphamide at a daily dose of about 250 mg / m2to about 600 mg / m2; and (ii) fludarabine at a daily dose of about 20 mg / m2to about 40 mg / m2; and (b) is administered 3 consecutive days starting from about 4-6 days prior to administering the adoptive cell therapy product. In some embodiments, the chemotherapy comprises bendamustine and is administeredat a daily dose of about 30 mg / m2to about 100 mg / m2for 2 consecutive days starting from about 4-6 days prior to administering the adoptive cell therapy product.[000173] In some embodiments, the engineered T lineage cells are derived from an engineered induced pluripotent stem cell (iPSC) comprising a polynucleotide encoding the HER2-CAR, a polynucleotide encoding the IL7RF, a polynucleotide encoding the TGFP-SRR, a polynucleotide encoding the CXCR2, a polynucleotide encoding the exogenous CD 16, a TCR knockout, and a CD38 knockout. In some embodiments, the first effective amount and / or second effective amount of the adoptive cell therapy product is about 5 * 107to about 3 * 109engineered T lineage cells, and optionally wherein the number of engineered T lineage cells is increased based on a dose limiting toxicity rate of the amount of the adoptive cell therapy administered. In some embodiments, the first effective amount and / or second effective amount of the adoptive cell therapy product comprises about 5 * 107, about 1 x 108, about 3 * 108, about 9 - io8, or about 2 * 109engineered T lineage cells. In some embodiments, at a dose limiting toxicity (DLT) rate of 25%-35% or below, the number of engineered T lineage cells in the adoptive cell therapy product is increased by up to 3-fold. In some embodiments, the adoptive cell therapy product is: (i) allogeneic; (ii) administered (a) via intravenous infusion, and / or (b) in an outpatient setting; and / or (iii) cryopreserved, and then thawed prior to administering. In some embodiments, the adoptive cell therapy product is FT825.[000174] In some embodiments, the subject has had: (i) no prior treatment of the cancer; or (ii) one or more prior HER2-directed therapies; and / or one or more prior EGFR-directed therapies. In some embodiments, the cancer comprises a breast cancer, an esophagogastric cancer, a gastroesophageal junction (GEJ) adenocarcinoma, a non-small cell lung cancer (NSCLC), a colorectal cancer (CRC), or a head and neck squamous cell cancer (HNSCC).[000175] In some embodiments, the method further comprises detecting and comparing one or more of the following subsequent to administration of the first effective amount 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; or (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 some embodiments, the subject has a complete response (CR), partial response (PR), stable disease (SD) after receiving the adoptive cell therapy product. [000176] In one aspect, a method of treating cancer comprises: (a) administering to the subject one or more first doses of an EGFR inhibitor; (b) administering to the subject one or more doses of an adoptive cell therapy product; and (c) administering to the subject one or moresecond doses of the EGFR inhibitor; wherein the adoptive cell therapy product comprises an engineered T lineage cell comprising (i) expression of a HER2-CAR (chimeric antigen receptor), an IL7 / IL7 receptor fusion protein (IL7RF), a TGFP signaling redirector receptor (TGFP-SRR), a C-X-C motif chemokine receptor 2 (CXCR2), and an exogenous CD 16; (ii) a CD38 knockout; and (iii) a T cell receptor (TCR) knockout; and wherein the cancer comprises cancer cells expressing EGFR. In some embodiments, the method comprises repeating the foregoing steps. In some embodiments, the method comprises administering to the subject one or more doses of chemotherapy before administering the EGFR inhibitor. In some embodiments, the EGFR inhibitor comprises cetuximab, matuzumab, panitumumab, or necitumumab. In some embodiments, the EGFR inhibitor comprises cetuximab. In some embodiments, the cancer cells expressing EGFR also express HER2. In some embodiments, the chemotherapy comprises: (a) one or both of cyclophosphamide (CY) and fludarabine (FLU); (b) bendamustine; or (c) one or both of docetaxel and cisplatin.[000177] In one aspect, the present disclosure provides kits for cancer treatment. In some embodiments, the kit comprises FT825, one or more chemotherapy agents, and optionally an EGFR inhibitor; wherein (a) FT825 comprises engineered T lineage cells comprising: (i) expression of a HER2-CAR (chimeric antigen receptor), an IL7 / IL7 receptor fusion protein (IL7RF), a TGFP signaling redirector receptor (TGFP-SRR), a C-X-C motif chemokine receptor 2 (CXCR2), and an exogenous CD 16; (ii) a CD38 knockout; and (iii) a T cell receptor (TCR) knockout; (b) the chemotherapy comprises: (i) one or both of cyclophosphamide (CY) and fludarabine (FLU); (ii) bendamustine; or (iii) one or both of docetaxel and cisplatin; and (c) the EGFR inhibitor comprises cetuximab, matuzumab, panitumumab, or necitumumab. In some embodiments, the cancer is HER2 positive and / or EGFR positive.EXAMPLES[000178] The following examples are offered by way of illustration and not by way of limitation.EXAMPLE 1 - FT825 Study Design[000179] FT825 drug substance utilized in the present example comprises allogeneic T cells, derived from a T-cell receptor (TCR) and CD38 knockout iPSC line, and express a HER2 targeted CAR, IL7RF, TGFP-SRR, hnCD16, and CXCR2. Manufacture of drug substance from the iPSC master cell bank involved stages of iPSC expansion, followed by stages of differentiation to T cells and T cell expansion. Cells that were harvested after T-cell expansion were then washed and designated as the drug substance. FT825 is uniform in composition.[000180] The study as described herein was designed to evaluate the safety, PK, and antitumor activity of FT825 with or without cetuximab following chemotherapy in participants with advanced HER2-positive or other advanced solid tumors. The study was also designed to determine the recommended Phase 2 dose (RP2D) and schedule for FT825 with or without cetuximab following chemotherapy in participants with advanced HER2 and / or EGFR expressing solid tumors. In addition, the study was designed to assess the association of baseline clinical and tumor characteristics with safety and efficacy of FT825; to correlate the PK / pharmacodynamic parameters of FT825 with safety and efficacy of FT825; and to assess pharmacodynamic biomarkers of FT825, with or without cetuximab following CY / FLU or bendamustine. Moreover, the study provides opportunities for evaluating the safety, tolerability, and anti -turn or activity of FT825; and characterizing PK and pharmacodynamic biomarkers of FT825, with or without cetuximab following docetaxel / ci splatin (Cycle 2 retreatment).[000181] Cycle 1 study has two regimens. Regimen A: Single-dose FT825 following chemotherapy with CY / FLU or bendamustine in participants with advanced HER2-expressing solid tumors. Regimen B: Single-dose FT825 following chemotherapy in combination with cetuximab following chemotherapy with CY / FLU or bendamustine in participants with advanced EGFR-expressing and EGFR and HER2 co-expressing solid tumors.[000182] During Cycle 1, participants receive CY / FLU chemotherapy prior to infusion of FT825 on Day 1. Bendamustine is considered for use instead of CY / FLU in instances where CY or FLU are unavailable or where a participant may be unable to receive CY or FLU.[000183] Participants are enrolled in 2 stages: a dose-escalation stage and a dose-expansion stage. After the safety and tolerability have been assessed in the dose-escalation stage, the dose-expansion stage further evaluates the safety and activity of FT825 in indicationspecific cohorts.[000184] In one embodiment, the treatment using FT825 comprises a 29-day treatment cycle. In some embodiments, the 29-day treatment cycle follows a chemotherapy comprising CY / FLU or bendamustine. As shown in FIG. 1, chemotherapy is administered starting Day -5 (i.e., 5 days prior to Day 1 FT825 administration), followed by administering FT825 on Day 1. The dose limiting toxicity (DLT) assessment period during dose escalation is from Day 1 to Day 29. Following the treatment cycle completion (i.e., passing Day 29), participants enter the posttreatment follow-up (PTFU) phase and are regularly monitored for new and / or resolving treatment-emergent adverse events (TEAEs) and exploratory assessments. Participants with progressive disease (PD) during the PTFU who are eligible to receive Cycle 2 retreatment receive Cycle 2 study intervention. Cycle 2 retreatment may be administered at any time during the post-treatment follow-up period after Day 29.[000185] For each regimen, progressively higher dose levels of FT825 are evaluated using a modified toxicity probability interval (mTPI)-2 dose-escalation design. The progression of FT825 dose escalation is as follows: (1) dose escalation is initiated with Regimen A, dose level (DL)1; (2) initiation of Regimen B dose escalation is contingent on the clearance of at least one dose level in Regimen A and may proceed starting at the highest cleared dose level in Regimen A; (3) dose escalation of Regimen A and Regimen B may be conducted independently until the MTD / maximum assessed dose (MAD) is identified for each regimen; (4) For Regimen A and Regimen B, if DL1 is not tolerable or exceeds the MTD, Regimen A or Regimen B DLO may be explored.[000186] The starting FT825 dose levels in dose escalation are as follows: (1) DLO:5 x io7cells per dose (to be assessed if DL1 is not tolerable or exceeds the MTD); (2) DL1 : 1 x 108cells per dose. If the initial dose level does not exceed the MTD, successive dose levels are tested; none to exceed 3 times (±15% variance) the highest cleared dose level. In the event that an evaluated dose level exceeds the MTD, intermediate dose levels may be explored. In the absence of dose-limiting toxi cities (DLTs), dose levels may be explored further based on observed safety, tolerability, activity, and PK and pharmacodynamic data.[000187] The FT825 dose for each regimen administered in dose expansion is determined based on the clinical and available PK and pharmacodynamic data from the respective doseescalation stage and does not exceed the MTD or the MAD. Enrollment into dose expansion cohort(s) begins once a given dose level is cleared in dose escalation. One or more dose levels that have cleared DLT assessment in dose escalation in Regimen A and / or Regimen B may be evaluated in dose expansion to further define clinical activity. Combined safety and efficacy analyses from both dose escalation and dose expansion are used to determine the RP2D for each regimen. Regimen A dose expansion includes: HER2-positive breast cancer defined as IHC 3±, or IHC 2+ and ISH-positive (Wolff et al. 2018); HER2-positive gastric / GEJ cancer defined as IHC 3±, or IHC 2+ and ISH-positive (Bartley et al. 2016); HER2-low breast cancer (IHC1+ or IHC2± / ISH-negative); HER2-low gastric / GEJ cancer (IHC1+ or IHC2± / ISH-negative); HER2- mutated NSCLC; and Other HER2 expressing cancers (including salivary gland and endometrial cancers) with HER2 IHC >1+ or HER2 amplification. Regimen B dose expansion includes: HNSCC with HER2 IHC >1+ or HER2 amplification; CRC with EGFR IHC >1+ or KRAS wild type or BRAFV600E mutated, and HER2 IHC >1+ or HER2 amplification; NSCLC with EGFR IHC >1+ or EGFR mutation, and HER2 IHC >1+ or HER2 amplification.[000188] The purpose of chemotherapy prior to the administration of FT825 is to create an immune environment amenable to FT825 persistence, expansion, and function. While the mechanism is not fully understood, the combination of CY and FLU induces cytokines thatpromote homeostatic proliferation and eliminate of regulatory immune cells and other elements of the immune system that compete for homeostatic cytokines.[000189] Bendamustine is an alkylating agent with a well-characterized and favorable safety profile as a component of immunochemotherapy regimens, administered in multiple treatment cycles, for the treatment of B-cell malignancies. Bendamustine is listed as an alternative to CY / FLU treatment before administration of tisagenlecleucel in subjects who experienced severe hemorrhagic cystitis with prior CY or whose tumor demonstrates resistance to a previous CY-containing regimen.[000190] Treatment of patients with Regimen A is designed to assess the clinical activity of FT825, specifically with respect to the HER2-CAR, in patients with HER2-expressing tumors. One aspect of the study is to investigate the effect of FT825, containing a HER2- targeting CAR, on HER2-expressing tumor cells as compared to HER2-expressing normal cells, and as compared to preclinical observation of the anti-tumor activity against tumor cell lines expressing a broad range of HER2 expression, including HER2 low.[000191] FT825 incorporates the hnCD16 to enable multi-antigen targeting via ADCC through combinations with mAbs to broaden FT825 anti-tumor targeting. Another aspect of the study is for the clinical assessment of FT825 in combination with cetuximab in EGFR- expressing tumors with varying levels of HER2 co-expression (such as HNSCC, NSCLC and CRC) in view of the preclinical enhanced anti -tumor activity of FT825 when combined with cetuximab against tumor cell lines that have low-level HER2 expression, but high-level EGFR expression. Participants are enrolled to receive Regimen B in different phases of the study. To assess the safety of FT825 combined with cetuximab, participants with known EGFR-positive tumors and agnostic to known a priori HER2 status (which is characterized retrospectively) are enrolled in the dose-escalation phase of the study. During the dose expansion phase of the study, participants with tumors with documented expression of both EGFR and HER2 are enrolled.[000192] The starting dose for FT825 in Regimen A is 1 x 108cells per dose following a course of chemotherapy, such as CY / FLU or bendamustine in Cycle 1 (FIG. 2). FT825 dose escalation proceeds by relative dose level increases rather than prespecified dosing levels, with dose escalation intervals not exceeding 3 times (±15% variance) the highest cleared dose level. The relative dose interval is designed to allow flexibility to dose escalate by a smaller increment when needed.[000193] FIG. 2 illustrates the exemplary dose-escalation and dose expansion schema. As noted above, dose level 1 (DL1) is 1 x 108cells / dose. Dose escalation may continue at <3 x the previous cleared dose (±15% variance). DL0 is 5 x io7cells / dose (to be assessed if DL1exceeds MTD). Initiation of dose escalation occurs with Regimen A, DL1. Designation of DL1 through DL4 is for illustrative purposes only.EXAMPLE 2 - FT825 Cycle 2 Retreatment[000194] Based on the safety, tolerability, and clinical response to treatment in Cycle 1, participants may be considered for an additional treatment cycle (Cycle 2 retreatment) with docetaxel and cisplatin instead of CY / FLU as pre-treatment chemotherapy.[000195] FT825 administration and monitoring activities are performed following the same schedule as Cycle 1. FIG. 3 shows an exemplary treatment schema for the Cycle 2 retreatment.[000196] The reasons for the failure of existing CAR T-cell therapies to consistently achieve deep and durable responses are unknown. Unlike in hematologic malignancies, CY / FLU and bendamustine are not broadly used for the treatment of solid tumors due in part to the lack of robust anti-tumor activity. Combinations of taxanes and platins such as docetaxel and cisplatin are active anti-tumor agents and have been shown to induce positive immunologic anti-cancer effects to facilitate T-cell infiltration into the solid tumor microenvironment.[000197] This study tests the combination of docetaxel and cisplatin (docetaxel / cisplatin) as a combination with FT825 as Cycle 2 retreatment in patients whose disease has progressed following initial (Cycle 1) treatment with CY / FLU or bendamustine and FT825 combinations, for both Regimen A and Regimen B. In addition to evaluating the safety and tolerability of FT825 combined with docetaxel / cisplatin, the ability of the different chemotherapy combination with FT825 to re-induce an objective response is also tested. Finally, Cycle 2 retreatment with docetaxel / cisplatin combined with FT825 enables intra-patient comparisons of Cycle 1 and Cycle 2 FT825 PK and pharmacodynamic assessments. It is yet another objective to evaluate the Cycle 2 treatment for improving patient outcomes in the treatment of solid tumors.[000198] Participants meeting the following criteria are selected for Cycle 2 retreatment: (1) initial confirmed stable disease (SD), partial response (PR), or CR, with a subsequent tumor assessment demonstrating PD per RECIST, vl.l; (2) resolution of non-hematologic AEs from Grade 2-3 to Grade <1 or baseline grade, whichever is higher, with the exception of alopecia and dermatologic changes such as Grade 2 dry skin and / or rash; and (3) Eastern Cooperative Oncology Group (ECOG) Performance Status 0 or 1.EXAMPLE 3 - Study Population for Each Study Cohort[000199] The patients selected for treatments described herein have histopathological or cytologically confirmed locally advanced or metastatic cancer based on HER2, EGFR, KRAS, and BRAF testing as determined by a Clinical Laboratory Improvement Amendment (CLIA)- approved assay or otherwise appropriately validated assay.[000200] For treatment comprising single-dose FT825 following chemotherapy (Regimen A) with dose escalation, suitable patients include those having: (a) HER2-positive breast cancer and gastric / GEJ cancer, which is defined in the present example as IHC3+, or IHC2+ and ISH- positive per established guidelines (American Society of Clinical Oncology [ASCO] / College of American Pathologists [CAP] guidelines for breast cancer; CAP / American Society for Clinical Pathology [ASCP] / ASCO guidelines for gastric / GEJ cancer); or (b) NSCLC with HER2 mutation, which is identified from tissue (fresh or archival) or plasma (ctDNA); or (c) HER2- expressing cancers other than breast cancer, gastric / GEJ cancer or NSCLC, as defined as >2+ by IHC or HER2 / CEP17 ratio >2.[000201] For treatment comprising single-dose FT825 following chemotherapy with dose expansion, suitable patients include those having: (a) HER2-positive breast cancer and gastric / GEJ cancer, which is defined in the present example as IHC 3+, or IHC 2+ and ISH- positive; (b) HER2-low breast cancer and gastric / GEJ cancer, which is defined in the present example as IHC 1+, or IHC 2+ / ISH-negative; (c) NSCLC with HER2 mutation, which is identified from tissue (fresh or archival) or plasma (ctDNA) regardless of HER2 expression level; or (d) HER2-expressing cancers other than breast cancer, gastric / GEJ cancer or NSCLC, defined in the present example as >1+ by IHC or HER2 / CEP17 ratio >2.[000202] For treatment comprising single-dose FT825 with cetuximab following chemotherapy (Regimen B) with dose escalation, suitable patients include those having: (a) HNSCC, with HER2 expression, amplification, or mutation; (b) NSCLC, with HER2 expression, amplification, or mutation; and EGFR driver mutation; or (c) CRC, with HER2 expression, amplification, or mutation; and any of the following — KRAS wild-type, EGFR >1+ by IHC, or BRAF V600E mutation.[000203] For treatment comprising single-dose FT825 with cetuximab following chemotherapy with dose expansion, suitable patients include those having: (a) HNSCC, with HER2-expression defined in the present example as >1+ by IHC or HER2 / CEP17 ratio >2; (b) CRC, with HER2-expression defined in the present example as >1+ by IHC or HER2 / CEP17 ratio >2, and any of the following — KRAS wild-type, EGFR >1+ by IHC, or BRAF V600E mutation; or (c) NSCLC, with HER2-expression defined in the present example as >1+ by IHCor HER2 / CEP17 ratio >2, or with HER2 mutation identified from tissue or plasma (ctDNA); and EGFR driver mutation.[000204] Patients selected for treatments comprising Regimen A also include those having: (a) breast cancer that has relapsed or progressed, with or without one or more HER2-directed therapies; (b) Gastric or GEJ cancer that has relapsed or progressed, with or without one or more HER2-directed therapies; or (c) HER2-expressing cancers other than breast cancer, gastric cancer, or GEJ cancer that have relapsed or progressed, with or without one or more lines of standard-of-care therapy.[000205] Patients selected for treatments comprising Regimen B also include those having (a) HNSCC that has relapsed or progressed, with or without following one or more lines of prior systemic therapy, including EGFR-directed therapy; (b) NSCLC expressing EGFR driver mutation(s) that has progressed, with or without at least one prior line of EGFR-directed therapy; or (c) CRC that has relapsed or progressed, with or without at least one prior EGFR- based therapy and / or at least one prior HER2 -based therapy.EXAMPLE 4 - Study Interventions and Concomitant Therapy[000206] Study interventions are all pre-specified investigational and non-investigational medicinal products intended to be administered to the study participants during the study, which include FT825 (designated as Investigational Medicinal Product (IMP)), and authorized auxiliary medicinal product (AxMP) that are non-IMP, such as cetuximab, cyclophosphamide (CY), fludarabine (FLU), and bendamustine. Study interventions are according to Table 1 below:Table 1: Study Interventionsa Participants who are otherwise eligible to receive Cycle 2 retreatment but who are not eligible to receive docetaxel / cisplatin may receive CY / FLU or bendamustine with medical monitor approval; the same chemotherapy administered in Cycle 1 is administered with Cycle 2 retreatment. b Pre-medication and anti-emetic therapy and hydration as per institutional standard of care. Modification to CY / FLU or bendamustine dose or schedule is possible based on medical monitor approval. c Pre-medication, anti-emetic therapy, and hydration as per institutional standard of care. Dose reduction in the event of renal insufficiency as guidelines for palliative anti-cancer therapy (Krens et al., 2019). Cisplatin is not administered if creatinine clearance <50 mL / min. Modification to docetaxel / cisplatin dose or schedule is possible based on medical monitor approval.[000207] FT825 drug product comprises allogeneic T cells, derived from a TCR and CD38 knockout iPSC line, that express a HER2 -targeted CAR, IL7RF, TGFP-SRR, hnCD16, and CXCR2, as described above in Example 1. Immediately prior to CY administration, participants receive 500 mL of IV normal saline per institutional standards. Participants may receive additional IV normal saline following CY administration following assessment of hydration status. Dose adjustments for weight / creatinine may also be made. CY is administered as an IV infusion at a dose of 500 mg / m2for 3 consecutive days on Day -5, Day -4, and Day -3 of the treatment cycle. 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)[000208] FLU is administered as an IV infusion at a dose of 30 mg / m2for 3 consecutive days on Day -5, Day -4, and Day -3 of the treatment cycle. Dose adjustments for weight and / or renal function (e.g., as assessed by creatinine clearance) may be made. Bendamustine is administered as an IV infusion at a dose of 90 mg / m2for 2 consecutive days (Days -5 and -4). Docetaxel is administered as an IV infusion at a dose of 60 mg / m2or 75 mg / m2on Day -5. Cisplatin is administered as an IV infusion at a dose of 25 mg / m2(for renal clearance 60 mL / min) or 19 mg / m2(for renal clearance 50 to 59 mL / min) on Days -5, -4, and -3.[000209] Cetuximab may be administered every week (QW) or every 2 weeks (Q2W). For example, for QW dosing, cetuximab is administered at an initial loading dose of 400 mg / m2as a 120-minute IV infusion; subsequent doses are administered at 250 mg / m2as a 60-minute infusion every week. For Q2W dosing, cetuximab is administered at 500 mg / m2as a 120- minute IV infusion every 2 weeks. Treatment continues for up to 2 years or until disease progression or unacceptable toxicity. Premedication may include a histamine- 1 receptor antagonist IV 30-60 minutes prior to the first dose or subsequent doses if necessary.Preventative measures for EGFR antibody-associated rash may be also provided.EXAMPLE 5 - Dose and Schedule Modifications of the Study[000210] Dose and schedule modifications may be made for CY / FLU. For participants 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 administered to participants with severely impaired renal function (creatinine clearance <30 mL / min / 1.73 m2).[000211] For participants who complete the first treatment cycle (Cycle 1) but are still recovering from AEs that preclude directly proceeding to Cycle 2, additional weekly monitoring visits are performed until the criteria to start Cycle 2 are met. Determination of the need for dose reduction is based on CBC on Day 29 and / or the presence of treatment-related AEs during Cycle 1.[000212] If Grade >3 AEs of 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 by Day 29, and / or for participants who experience significant treatment-related AEs (i.e., serious infections requiring hospitalization) where dose adjustment is considered appropriate, the following dose modifications are made: the daily dose of CY is reduced to 300 mg / m2on Days -5, -4, and -3; and the daily dose of FLU is reduced to 25 mg / m2on Days -5, -4, and -3.[000213] Dose and schedule modifications may be made for bendamustine. Bendamustine is not administered to participants who have renal or hepatic impairment as defined by:creatinine clearance <40 mL / min; AST or ALT that is 2.5 to 10 x ULN, and total bilirubin that is 1.5 to 3 x ULN; or total bilirubin >3 x ULN. Bendamustine administration is delayed in the event of Grade 4 hematologic toxicity or clinically significant Grade >2 non-hematologic toxicity. Once non-hematologic toxicity has recovered to Grade <1 or baseline grade, whichever is higher, and / or blood counts have improved, e.g., ANC >1000 / pL, platelets >75000 / pL or baseline grade, bendamustine may be administered. In addition, the following dose modifications may be made for toxicities attributable to bendamustine: for Grade 4 toxicity, bendamustine dose may be reduced to 60 mg / m2on Days -5 and -4 in Cycle 2; for non- hematologic toxicity: For Grade >3 toxicity, reduce bendamustine dose to 60 mg / m2on Days -5 and -4 in Cycle 2.EXAMPLE 6 - Tumor Response Assessment and Efficacy Analyses[000214] Tumor response is assessed using Response Evaluation Criteria in Solid Tumors, Version 1.1 (RECIST vl.l; Eisenhauer et al., 2009). Participants are classified into the following tumor response categories: complete response (CR), partial response (PR), stable disease (SD), progressive disease (PD), or not evaluable (NE).[000215] Approximately 48 participants per regimen are enrolled in the dose-escalation stage for Regimens A and B for determination of the maximum tolerated dose (MTD) using a modified toxicity probability interval (mTPI)-2 design, where 30% represents the target toxicity. A sample size of 15 participants for each dose-expansion cohort enables identification of early signs of promising clinical activity based primarily on objective responses, for subsequent Phase 2 expansion. Table 2 displays the exact 95% CI for true response rates ranging from 40% to 73%.Table 2:[000216] 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.[000217] 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.[000218] 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

CLAIMSWhat is claimed is:

1. A method of treating cancer in a subject, the method comprising:(a) administering to the subject one or more doses of chemotherapy; and(b) administering to the subject one or more doses of an adoptive cell therapy product in a first effective amount; wherein the adoptive cell therapy product comprises engineered T lineage cells comprising (i) expression of a HER2-CAR (chimeric antigen receptor), an IL7 / IL7 receptor fusion protein (IL7RF), a TGFP signaling redirector receptor (TGFP-SRR), a C-X-C motif chemokine receptor 2 (CXCR2), and an exogenous CD 16; (ii) a CD38 knockout; and (iii) a T cell receptor (TCR) knockout; and wherein the cancer comprises cancer cells that are HER2 positive.

2. The method of claim 1, further comprising:(a) administering to the subject one or more doses of an additional chemotherapy that is the same or different as the chemotherapy; and(b) administering to the subject one or more doses of the adoptive cell therapy product in a second effective amount that is the same or different as the first effective amount.

3. The method of claim 1, wherein the cancer cells that are HER2 positive comprise HER2 expression, amplification, or mutation.

4. The method of claim 1, further comprising administering an EGFR inhibitor to the subject; and wherein the cancer cells comprise an EGFR mutation.

5. The method of claim 4, wherein administering the EGFR inhibitor is before and / or after administering the adoptive cell therapy product.

6. The method of claim 4, wherein the EGFR inhibitor comprises, matuzumab, panitumumab, or necitumumab.

7. The method of claim 4, wherein the EGFR inhibitor comprises cetuximab.

8. The method of claim 1, wherein the chemotherapy comprises: (a) one or both of cyclophosphamide (CY) and fludarabine (FLU); (b) bendamustine; or (c) one or both of docetaxel and cisplatin.

9. The method of claim 1, wherein the chemotherapy is administered one or more days prior to administering the adoptive cell therapy product; optionally wherein the chemotherapy is administered at least two days, three days, four days, or five days before administering the adoptive cell therapy product.

10. The method of claim 1, wherein the chemotherapy:(a) comprises (i) cyclophosphamide at a daily dose of about 250 mg / m2to about 600 mg / m2; and (ii) fludarabine at a daily dose of about 20 mg / m2to about 40 mg / m2; and(b) is administered 3 consecutive days starting from about 4-6 days prior to administering the adoptive cell therapy product.

11. The method of claim 1, wherein the chemotherapy comprises bendamustine and is administered at a daily dose of about 30 mg / m2to about 100 mg / m2for 2 consecutive days starting from about 4-6 days prior to administering the adoptive cell therapy product.

12. The method of claim 1, wherein the engineered T lineage cells are derived from an engineered induced pluripotent stem cell (iPSC) comprising a polynucleotide encoding the HER2-CAR, a polynucleotide encoding the IL7RF, a polynucleotide encoding the TGFP-SRR, a polynucleotide encoding the CXCR2, a polynucleotide encoding the exogenous CD 16, a TCR knockout, and a CD38 knockout.

13. The method of claim 1, wherein the first effective amount and / or second effective amount of the adoptive cell therapy product is about 5 * 107to about 3 * 109engineered T lineage cells, and optionally wherein the number of engineered T lineage cells is increased based on a dose limiting toxicity rate of the amount of the adoptive cell therapy administered.

14. The method of claim 2, wherein the first effective amount and / or second effective amount of the adoptive cell therapy product comprises about 5 * 107, about 1 x 108, about 3 x 108, about 9 x io8, or about 2 x io9engineered T lineage cells.

15. The method of claim 1, wherein at a dose limiting toxicity (DLT) rate of 25%-35% or below, the number of engineered T lineage cells in the adoptive cell therapy product is increased by up to 3 -fold.

16. The method of claim 1, wherein the adoptive cell therapy product is:(i) allogeneic;(ii) administered (a) via intravenous infusion, and / or (b) in an outpatient setting; and / or(iii) cryopreserved, and then thawed prior to administering.

17. The method of claim 1, wherein the adoptive cell therapy product is FT825.

18. The method of claim 1, wherein the subject has had:(i) no prior treatment of the cancer; or(ii) one or more prior HER2-directed therapies; and / or one or more prior EGFR-directed therapies.

19. The method of claim 1, wherein the cancer comprises a breast cancer, an esophagogastric cancer, a gastroesophageal junction (GEJ) adenocarcinoma, a non-small cell lung cancer (NSCLC), a colorectal cancer (CRC), or a head and neck squamous cell cancer (HNSCC).

20. The method of claim 1, further comprising detecting and comparing one or more of the following subsequent to administration of the first effective amount 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; or(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.

21. The method of claim 1, the subject has a complete response (CR), partial response (PR), stable disease (SD) after receiving the adoptive cell therapy product.

22. A method of treating cancer in a subject, the method comprising:(a) administering to the subject one or more first doses of an EGFR inhibitor;(b) administering to the subject one or more doses of an adoptive cell therapy product; and(c) administering to the subject one or more second doses of the EGFR inhibitor; wherein the adoptive cell therapy product comprises an engineered T lineage cell comprising (i) expression of a HER2-CAR (chimeric antigen receptor), an IL7 / IL7 receptor fusion protein (IL7RF), a TGFP signaling redirector receptor (TGFP-SRR), a C-X-C motif chemokine receptor 2 (CXCR2), and an exogenous CD 16; (ii) a CD38 knockout; and (iii) a T cell receptor (TCR) knockout; and wherein the cancer comprises cancer cells expressing EGFR.

23. The method of claim 22, wherein the method comprises administering to the subject one or more doses of chemotherapy before administering the EGFR inhibitor.

24. The method of claim 22, wherein the EGFR inhibitor comprises cetuximab, matuzumab, panitumumab, or necitumumab.

25. The method of claim 22, wherein the EGFR inhibitor comprises cetuximab.

26. The method of claim 22, wherein the cancer cells expressing EGFR also express HER2.

27. The method of claim 23, wherein the chemotherapy comprises: (a) one or both of cyclophosphamide (CY) and fludarabine (FLU); (b) bendamustine; or (c) one or both of docetaxel and cisplatin.

28. A method of treating cancer in a subject, wherein the method comprises repeating the steps according to the method of claim 1 or 22.

29. A kit for cancer treatment comprising FT825, one or more chemotherapy agents, and optionally an EGFR inhibitor; wherein:(a) FT825 comprises engineered T lineage cells comprising: (i) expression of a HER.2- CAR (chimeric antigen receptor), an IL7 / IL7 receptor fusion protein (IL7RF), a TGFP signaling redirector receptor (TGFP-SRR), a C-X-C motif chemokine receptor 2 (CXCR2), and an exogenous CD 16; (ii) a CD38 knockout; and (iii) a T cell receptor (TCR) knockout;(b) the chemotherapy comprises: (i) one or both of cyclophosphamide (CY) and fludarabine (FLU); (ii) bendamustine; or (iii) one or both of docetaxel and cisplatin; and(c) the EGFR inhibitor comprises cetuximab, matuzumab, panitumumab, or necitumumab.

30. The kit of claim 29, wherein the cancer is HER2 positive and / or EGFR positive.

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