Ipsc-derived t cells for solid tumor therapy

Abstract

This invention provides methods and compositions for cancer immunotherapy. In various embodiments, the composition comprises functionally enhanced derived effector cells obtained by directed differentiation of genome-engineered iPSCs. In various embodiments, the derived cells provided herein have stable and functional genome editing that delivers improved or enhanced therapeutic effects. Therapeutic compositions and their uses are also provided, comprising these functionally enhanced derived effector cells alone or in combination therapies.

Description

Related 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 incorporated herein by reference in its entirety. By referencing and incorporating into the sequence list

[0002] The sequence list with the title 184143-652601_SL.xml, created on November 7, 2024 and measuring 21,676 bytes, is hereby incorporated in its entirety by reference. Technical Field

[0003] This disclosure broadly relates to the field of off-the-shelf immune cell products. More specifically, this disclosure relates to strategies for developing multifunctional effector cells capable of delivering therapeutically relevant properties in vivo. Cell products developed according to this disclosure address key limitations of patient-derived cell therapies. Background Technology

[0004] Current focus in the field of adoptive cell therapy is on using patient-derived and donor-derived cells, which makes the sustainable manufacturing of cancer immunotherapies and delivery of the therapy to all potentially benefiting patients particularly challenging. Improvements in the efficacy and survival of adopted lymphocytes are also needed to promote better patient outcomes. Lymphocytes, such as T cells and natural killer (NK) cells, are potent anti-tumor effectors and play a vital role in both innate and adaptive immunity. However, using these immune cells in adoptive cell therapy remains challenging, and the need for improvement has not yet been met. Therefore, there are still significant opportunities to leverage the full potential of T cells and NK cells or other lymphocytes in adoptive immunotherapy. Summary of the Invention

[0005] Functionally enhanced effector cells are needed to address issues ranging from response rate, cell exhaustion, transfusion cell loss (survival and / or retention), tumor escape via target loss or lineage switching, tumor targeting precision, off-target toxicity, detumescent effects to efficacy against solid tumors (e.g., efficacy in the tumor microenvironment and related immunosuppression, recruitment, transport, and invasion).

[0006] The purpose of embodiments of the present invention is for methods and compositions for adoptive cell therapy, wherein the adoptive cell therapy comprises administration of an adoptive cell therapy product produced from derived non-pluripotent cells differentiated from a single-cell-derived iPSC (induced pluripotent stem cell) clonal line, the iPSC line containing one or more gene modifications in its genome. In some embodiments, the one or more gene modifications include one or more of DNA insertions, deletions, and substitutions, and these modifications are retained and remain functional in subsequently derived cells after differentiation, expansion, passage, and / or transplantation.

[0007] In one aspect, this disclosure provides a method of treating a subject's cancer. In some embodiments, the method includes: (a) administering one or more doses of chemotherapy to the subject; and (b) administering one or more doses of an adoptive cell therapy product to the subject in a first effective amount; wherein the adoptive cell therapy product comprises engineered T lineage cells having (i) expression of HER2-CAR (chimeric antigen receptor), IL7 / IL7 receptor fusion protein (IL7RF), TGFβ signal transduction redirection receptor (TGFβ-SRR), CXC motif chemokine receptor 2 (CXCR2), and exogenous CD16; (ii) CD38 knockout; and (iii) T cell receptor (TCR) knockout; and wherein the cancer comprises HER2-positive cancer cells. In some embodiments, the method includes repeating the foregoing steps.

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

[0009] In some embodiments, the HER2-positive cancer cells contain HER2 expression, amplification, or mutation. In some embodiments, the method further includes administering an EGFR inhibitor to the subject; and wherein the cancer cells contain EGFR mutations. In some embodiments, the EGFR inhibitor is administered before and / or after administration of the adoptive cell therapy product. In some embodiments, the EGFR inhibitor includes matuzumab, panitumumab, or necitumumab. In some embodiments, the EGFR inhibitor includes cetuximab. In some embodiments, the chemotherapy includes: (a) one or both of cyclophosphamide (CY) and fludarabine (FLU); (b) bendamustine; or (c) one or both of docetaxel and cisplatin. In some embodiments, chemotherapy is administered one or more days before administration of the adoptive cell therapy product; optionally, chemotherapy is administered at least two, three, four, or five days before administration of the adoptive cell therapy product. In some implementations, chemotherapy (a) comprises (i) a daily dose of approximately 250 mg / m². 2 Approximately 600 mg / m 2 (ii) Cyclophosphamide; and (ii) a daily dose of approximately 20 mg / m 2 Approximately 40 mg / m 2 (a) Fludarabine; and (b) Administered for 3 consecutive days starting approximately 4–6 days prior to administration of the adoptive cell therapy product. In some embodiments, chemotherapy includes bendamustine and administered for 2 consecutive days starting approximately 4–6 days prior to administration of the adoptive cell therapy product at approximately 30 mg / m². 2 Approximately 100 mg / m 2 The daily dose is administered.

[0010] In some embodiments, the engineered T lineage cells are derived from engineered induced pluripotent stem cells (iPSCs) containing polynucleotides encoding HER2-CAR, IL7RF, TGFβ-SRR, CXCR2, exogenous CD16, TCR knockout, and CD38 knockout. In some embodiments, the first and / or second effective amount of the adoptive cell therapy product is about 5 × 10⁻⁶. 7 Approximately 3 × 10 9 The adoptive cell lineage comprises engineered T-lineage cells, and optionally the number of engineered T-lineage cells is increased based on the dose-limiting toxicity rate of the amount of adoptive cell therapy administered. In some embodiments, the first effective amount and / or the second effective amount of the adoptive cell therapy product comprises about 5 × 10⁻⁶ cells. 7 One, approximately 1×10 81, approximately 3 × 10 8 1, approximately 9 x 10 8 One or approximately 2 × 10 9 The adoptive cell therapy product contains engineered T lineage cells. In some embodiments, the number of engineered T lineage cells in the adoptive cell therapy product is increased up to 3-fold at a dose-limiting toxicity (DLT) rate of 25%-35% or lower. In some embodiments, the adoptive cell therapy product is: (i) allogeneic; (ii) (a) administered via intravenous infusion, and / or (b) administered in an outpatient setting; and / or (iii) cryopreserved and then thawed prior to administration. In some embodiments, the adoptive cell therapy product is FT825.

[0011] In some implementations, the subject: (i) has not received prior cancer treatment; or (ii) has received one or more prior HER2-targeted therapies; and / or one or more prior EGFR-targeted therapies. In some implementations, the cancer includes breast cancer, esophageal cancer, gastroesophageal junction (GEJ) adenocarcinoma, non-small cell lung cancer (NSCLC), colorectal cancer (CRC), or head and neck squamous cell carcinoma (HNSCC).

[0012] In some embodiments, the method further includes detecting and comparing one or more of the following after administration of a first effective dose of adoptive cell therapy: (a) the presence of engineered immune cells in the subject's tumor; (b) disease protein markers in the subject's serum; (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 one of (a)-(e) is used to assess tumor burden, tumor immunobiology, and / or tumor treatment response to determine the efficacy of the multi-dose targeted adoptive cell therapy. In some embodiments, the subject has a complete response (CR), partial response (PR), or stable disease (SD) after receiving the adoptive cell therapy product.

[0013] In one aspect, a method of treating cancer includes: (a) administering one or more first doses of an EGFR inhibitor to a subject; (b) administering one or more doses of an adoptive cell therapy product to a subject; and (c) administering one or more second doses of an EGFR inhibitor to a subject; wherein the adoptive cell therapy product comprises engineered T lineage cells expressing (i) HER2-CAR (chimeric antigen receptor), IL7 / IL7 receptor fusion protein (IL7RF), TGFβ signaling redirection receptor (TGFβ-SRR), CXC motif chemokine receptor 2 (CXCR2), and exogenous CD16; (ii) CD38 knockout; and (iii) T cell receptor (TCR) knockout; and wherein the cancer comprises cancer cells expressing EGFR. In some embodiments, the method includes repeating the foregoing steps. In some embodiments, the method includes administering one or more doses of chemotherapy to a subject prior to administering the EGFR inhibitor. In some embodiments, the EGFR inhibitor includes cetuximab, mateuzumab, panitumumab, or necstizumab. In some implementations, EGFR inhibitors include cetuximab. In some implementations, the cancer cells expressing EGFR also express HER2. In some implementations, chemotherapy includes: (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, this disclosure provides a kit for cancer treatment. In some embodiments, the kit comprises FT825, one or more chemotherapy agents, and optionally an EGFR inhibitor; wherein (a) the FT825 comprises engineered T lineage cells expressing: (i) HER2-CAR (chimeric antigen receptor), IL7 / IL7 receptor fusion protein (IL7RF), TGFβ signaling redirection receptor (TGFβ-SRR), CXC motif chemokine receptor 2 (CXCR2), and exogenous CD16; (ii) CD38 knockout; and (iii) T cell receptor (TCR) knockout; (b) the chemotherapy includes: (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 includes cetuximab, mateuzumab, panitumumab, or nectocinumab. In some implementations, the cancer is HER2 positive and / or EGFR positive. Attached Figure Description

[0015] Figure 1An exemplary treatment regimen is illustrated to evaluate FT825 as a combination of allogeneic cell therapy and chemotherapy. The abbreviations are as follows: CY (cyclophosphamide), DLT (dose-limiting toxicity), FLU (fludarabine), LTFU (long-term follow-up), PTFU (post-treatment follow-up), y (years). Follow-up a According to the protocol, subjects were followed up for up to 2 years after the last cycle of FT825 and for up to 15 years after the last cycle of FT825.

[0016] Figure 2 An exemplary dose escalation and dose expansion protocol for FT825 in cancer treatment is shown. The dose level 1 (DL1) shown is set to 1 × 10⁻⁶. 8 Furthermore, additional dose levels >DL1 (≤3 × previously cleared dose level) can be explored. DL0 = 5 × 10 7 Cells / dose (evaluate if DL1 exceeds MTD). Note a Subjects with NSCLC exhibiting HER2 ≥ 2+ positive tumors and HER2 mutations may be given priority consideration. Subjects with HER2 IHC 1+ tumors may also be treated. Note b HER2-positive breast cancer and gastric / GEJ cancer, defined in this protocol as IHC 3+, or IHC 2+ and ISH positive (Bartley et al., 2016; Wolff et al., 2018). Note c Low HER2 breast cancer and gastric / GEJ cancer are defined in this protocol as IHC 1+ or IHC 2+ / ISH-negative. Note d The decision to open the amplification cohort may depend on the review and evaluation of clinical experience and pharmacodynamic activity of FT825 observed during dose escalation. The 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 evaluable dose); MTD (maximum tolerated dose); NSCLC (non-small cell lung cancer).

[0017] Figure 3 It shows according to Figure 1 An exemplary treatment regimen for another treatment cycle (cycle 2) following the treatment shown (cycle 1). Note aFor subjects who tolerated the first cycle of treatment with CY / FLU or bendamustine, those who initially had an objective response or stable disease (SD), and those who subsequently developed progressive disease (PD), a second cycle of docetaxel / cisplatin may be considered for retreatment. Second cycle retreatment can follow the same schedule as the first cycle. When administered with CY / FLU or bendamustine, the FT825 dose for second cycle retreatment can be up to the maximum clearance of FT825. If a subject is ineligible for docetaxel / cisplatin, they may be switched to the same chemotherapy administered in the first cycle. Note b Subjects can restart PTFU and be followed up for up to 2 years after the second retreatment cycle. Note c Follow-up can continue for 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 (progression of disease); PR (partial response); PTFU (post-treatment follow-up); RECIST v1.1 (Responding to Solid Tumor Response Criteria, version 1.1); SD (stable disease). Detailed Implementation

[0018] Genomic modifications of iPSCs (induced pluripotent stem cells) can include one or more of polynucleotide insertions, deletions, and substitutions. Exogenous gene expression in genome-engineered iPSCs often encounters problems, such as gene silencing or reduced gene expression after long-term clonal expansion of initially genome-engineered iPSCs, after cell differentiation, and in dedifferentiated cell types derived from genome-engineered iPSCs. On the other hand, directly engineering primary immune cells such as T cells or NK cells can be challenging and may pose obstacles to the preparation and delivery of engineered immune cells for adoptive cell therapy. In various embodiments, the present invention provides an effective, reliable, and targeted method for stably integrating one or more exogenous genes (such as suicide genes or other functional modalities) into iPSC-derived cells, the one or more exogenous genes providing improved therapeutic properties related to transplantation, transport, homing, migration, cytotoxicity, viability, maintenance, expansion, lifespan, self-renewal, persistence, and / or survival, such iPSC-derived cells including, but not limited to, HSCs (hematopoietic stem cells and progenitor cells), T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, and NK cells.

[0019] definition

[0020] Unless otherwise defined herein, scientific and technical terms used in connection with this application will have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms.

[0021] It should be understood that the present invention is not limited to the specific methods, schemes, and reagents described herein, and therefore is subject to variation. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the claims.

[0022] As used herein, the articles “a,” “a,” and “the” refer to one or more (i.e., at least one) grammatical objects of the article. For example, “an element” means one or more elements.

[0023] The use of alternatives (e.g., "or") should be understood to mean any one, two, or any combination of the alternatives.

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

[0025] As used herein, the term "about" or "approximately" means a variation of up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In one embodiment, the term "about" or "approximately" means a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% in quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length with respect to a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0026] As used herein, the terms "substantially" or "substantially" mean that the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length is approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the terms "substantially identical" or "substantially identical" mean a range of quantities, levels, values, numbers, frequencies, percentages, dimensions, sizes, amounts, weight, or lengths that are approximately the same as the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0027] As used herein, the terms “substantially free” and “substantially free” are used interchangeably and, when used to describe a composition (e.g., a cell population or culture medium), mean a composition that is free of the specified substance or its source, for example, 95%, 96%, 97%, 98%, or 99% free of the specified substance or its source, or undetectable, as measured by conventional methods. The terms “free” or “substantially free” in a composition also mean (1) that the composition does not contain any concentration of such a substance, or (2) that the composition contains a functionally inert, low concentration of such a substance. A similar meaning can be applied to the term “lacking”, which means that the composition lacks a specific substance or its source.

[0028] Throughout this specification, unless the context otherwise requires, the word "comprising" should be understood to imply that it includes the stated steps or elements or a group of steps or elements, but does not exclude any other steps or elements or a group of steps or elements. In certain embodiments, the terms "comprising," "having," "containing," and "including" are used synonymously.

[0029] The phrase “composed of” is intended to include and limit anything that follows the phrase “composed of.” Therefore, the phrase “composed of” indicates that the listed elements are necessary or required, and that no other elements can exist.

[0030] The phrase “consistently of…” is intended to include any element listed following the phrase, and is limited to other elements that do not interfere with or affect the activity or function of the listed elements specified in this disclosure. Thus, the phrase “consistently of…” indicates that the listed elements are necessary or required, but other elements are optional and may be present or absent depending on whether they affect the activity or function of the listed elements.

[0031] Throughout this specification, references to "an embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a certain embodiment," "an additional embodiment," or "another embodiment," or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Therefore, the foregoing phrases appearing throughout this specification do not necessarily all refer to the same embodiment. Furthermore, in one or more embodiments, a particular feature, structure, or characteristic may be combined in any suitable manner.

[0032] The term "ex vivo" generally refers to activities occurring outside of an organism, such as experiments or measurements performed in or on living tissue in an artificial environment outside of an organism, preferably where variations in natural conditions are minimal. In certain embodiments, an "ex vivo" procedure involves obtaining living cells or tissues from an organism and culturing them, typically under sterile conditions, in laboratory equipment for several hours or up to about 24 hours, but including up to 48 hours or 72 hours or longer, depending on the circumstances. In some embodiments, such tissues or cells may be collected and frozen, and later thawed for ex vivo processing. Tissue culture experiments or procedures using living cells or tissues for longer than several days are generally considered "in vitro," but in some embodiments, this term may be used interchangeably with "ex vivo."

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

[0034] As used herein, the terms “reprogramming,” “dedifferentiation,” “enhanced cell efficacy,” or “enhanced developmental efficacy” refer to a method of improving cell efficacy or dedifferentiating cells into a lower differentiation state. For example, cells with enhanced cell efficacy have greater developmental plasticity (i.e., the ability to differentiate into more cell types) compared to the same cells in their unreprogrammed state. In other words, reprogrammed cells are cells with a lower differentiation state compared to the same cells in their unreprogrammed state.

[0035] As used herein, the term “differentiation” is the process by which undifferentiated (“non-specialized”) or weakly specialized cells acquire the characteristics of specialized cells (such as blood cells or muscle cells). Differentiated cells, or differentiation-inducing cells, are cells that are already in a more specialized (“specialized”) position within a cell lineage. The term “specialization,” when applied to the differentiation process, refers to a cell that has progressed along the differentiation pathway to a point where, under normal circumstances, it would continue to differentiate into a specific cell type or a subpopulation of that cell type, and where, under normal circumstances, it cannot differentiate into a different cell type or reverts to a weaker differentiated cell type. As used herein, the term “pluripotency” refers to the ability of a cell to form all lineages of the body or cell body (i.e., the embryo itself). For example, embryonic stem cells are a type of pluripotent stem cell capable of forming cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuous developmental efficiency ranging from incomplete or partially pluripotent cells (such as ectoderm stem cells or EpiSCs) that cannot produce a complete organism to more primitive, multipotent cells (such as embryonic stem cells) that can produce a complete organism.

[0036] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to stem cells generated in vitro from differentiated adult, neonatal, or fetal cells that have been induced or altered, i.e., reprogrammed to differentiate into tissues capable of differentiating from all three germ layers or cortical layers: mesoderm, endoderm, and ectoderm. In some embodiments, the reprogramming method uses reprogramming factors and / or small molecule chemical-driven approaches. The resulting iPSCs 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 within the internal cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and, during development, produce all derived cells from the three primary germ layers: ectoderm, endoderm, and mesoderm. They do not contribute to the outer membranes of the embryo or the placenta (i.e., they are not totipotent).

[0038] As used herein, the term "pluripotent stem cell" refers to a cell that has the developmental potential to differentiate into cells of one or more germ layers (i.e., ectoderm, mesoderm, and endoderm), but not all three. Therefore, pluripotent cells can also be referred to as "partially differentiated cells." Pluripotent cells are known in the field, and examples of pluripotent cells include adult stem cells, such as hematopoietic stem cells and neural stem cells. "Pluripotent" means that the cell can form many types of cells within a specified lineage, rather than cells from other lineages. For example, pluripotent hematopoietic cells can form many different types of blood cells (red blood cells, white blood cells, platelets, etc.), but they cannot form neurons. Therefore, the term "pluripotency" refers to a cell state whose developmental potential is less than that of totipotency and pluripotency.

[0039] Pluripotency can be determined in part by assessing the pluripotent properties of cells. Pluripotent properties include, but are not limited to: (i) pluripotent stem cell morphology; (ii) potential for unlimited self-renewal; (iii) expression of pluripotent stem cell markers, including but not limited to SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30, and / or CD50; (iv) the ability to differentiate into all three somatic cell lineages (ectoderm, mesoderm, and endoderm); (v) teratoma formation composed of the three somatic cell lineages; and (vi) embryomorph formation composed of cells from the three somatic cell lineages.

[0040] Two types of pluripotency have been previously described: the “excited” or “metastable” pluripotent state is equivalent to ectodermal stem cells (EpiSCs) of late blastocysts, and the “initial” or “basal” pluripotent state is equivalent to the internal cell mass of early / preimplantation blastocysts. While both pluripotent states exhibit the properties described above, the initial or basal state further exhibits: (i) pre-inactivation or reactivation of the X chromosome in female cells; (ii) improved clonality and viability during single-cell culture; (iii) a general reduction in DNA methylation; (iv) reduced deposition of the H3K27me3 repressive chromatin marker on promoters of developmental regulatory genes; and (v) reduced expression of differentiation markers relative to the excited state. The characteristics of the excited pluripotent state are typically found in standard cell reprogramming methods (where exogenous pluripotent genes are introduced into somatic cells, expressed, and then silenced or removed from all pluripotent cells). Under standard pluripotent cell culture conditions, these cells remain in the excited state unless exogenous transgene expression is maintained (where the characteristics of the basal state are observed).

[0041] As used in this article, the term "pluripotent stem cell morphology" refers to the classic morphological characteristics of embryonic stem cells. Normal embryonic stem cells are characterized by a small, round shape, a high nucleus-to-cytoplasm ratio, a prominent nucleolus, and typical intercellular spacing.

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

[0043] "Pluripotency factors" or "reprogramming factors" refer to agents that, alone or in combination with other agents, enhance the developmental efficacy of cells. Pluripotency factors include, but are not limited to, polynucleotides, peptides, and small molecules that can improve cell developmental efficacy. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0044] “Cultivation” or “cell culture” refers to the maintenance, growth, and / or differentiation of cells in an in vitro environment. “Cell culture medium,” “culture medium” (in all cases, the singular form “medium”), “supplement”, and “culture medium supplement” refer to nutrient compositions for cultivating cell cultures.

[0045] "Cultivation" or "maintenance" refers to the maintenance, proliferation (growth), and / or differentiation of cells outside a tissue or body (e.g., in sterile plastic (or coated plastic) cell culture dishes or flasks). "Cultivation" or "maintenance" can utilize culture media as a source of nutrients, hormones, and / or other factors that contribute to cell proliferation and / or maintenance.

[0046] As used in this article, the term "mesoderm" refers to one of the three germ layers that appears during early embryonic development and produces various specialized cell types, including blood cells of the circulatory system, muscles, heart, dermis, bones, and other supporting and connective tissues.

[0047] As used herein, the terms "permanent hematopoietic endothelial cells" (HE) or "multipotent stem cell-derived permanent hematopoietic endothelial cells" (iHE) refer to a subset of endothelial cells that generate hematopoietic stem cells and progenitor cells during the transformation from endothelial cells to hematopoietic cells. Hematopoietic cell development in the embryo proceeds sequentially: from the lateral plate mesoderm to angiogenic cells to permanent hematopoietic endothelial cells and hematopoietic progenitor cells.

[0048] The terms "hematopoietic stem cells and progenitor cells," "hematopoietic stem cells," "hematopoietic progenitor cells," or "hematopoietic precursor cells" refer to cells that are specialized in the hematopoietic lineage but can further differentiate into hematopoiesis, and include pluripotent hematopoietic stem cells (blood embryonic cells), bone marrow progenitor cells, megakaryocyte progenitor cells, erythrocyte progenitor cells, and lymphoid progenitor cells. Hematopoietic stem cells and progenitor cells (HSCs) are pluripotent stem cells that produce all types of blood cells, including bone marrow (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells). As used herein, the term "permanent hematopoietic stem cells" refers to CD34 cells. + Hematopoietic cells are responsible for producing mature bone marrow cell types and lymphocyte types, including T lineage cells, NK lineage cells, and B lineage cells. Hematopoietic cells also include various subsets of primitive hematopoietic cells, which produce primitive erythrocytes, megakaryocytes, and macrophages.

[0049] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to the primary type of white blood cell that matures in the thymus and plays a variety of roles in the immune system, including identifying specific foreign antigens in the body and activating and deactivating other immune cells. T cells can be any type of T cell, such as cultured T cells, primary T cells, or T cells derived from cultured T cell lines, such as Jurkat, SupT1, etc., or T cells derived from mammals. T cells can be CD3+. + T cells. T cells can be any type of T cell and can be at any developmental stage, including but not limited to CD4. + / CD8 + Double-positive T cells, CD4 + Helper T cells (e.g., Th1 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, regulatory T cells, gamma delta T cells (γδ T cells), and so on. Other types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Other types of memory T cells include cells such as central memory T cells (Tcm cells) and effector memory T cells (Tem cells and TEMRA cells). The term "T cell" can also refer to genetically engineered T cells, such as T cells modified to express T cell receptors (TCRs) or chimeric antigen receptors (CARs). T cells or T cell-like effector cells can also differentiate from stem cells or progenitor cells ("derived T cells" or "derived T cell-like effector cells," or collectively "derived T lineage cells"). Derived T cell-like effector cells may possess some aspects of the T cell lineage, but also have one or more functional features not present in primary T cells. In this application, T cells, T cell-like effector cells, derived T cells, derived T cell-like effector cells, or derived T lineage cells are collectively referred to as "T lineage cells". In some embodiments, derived T lineage cells are iPSC-derived T cells obtained by differentiating iPSCs, which 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 responses. They are characterized by a post-stimulation secretory profile that may include secreting cytokines such as IFN-γ, TNF-α, IL2, IL4, and IL10. The CD4 molecule was initially defined as a differentiation antigen on T lymphocytes but has also been found on other cells, including monocytes / macrophages, as a 55-kD glycoprotein. The CD4 antigen is a member of the immunoglobulin superfamily and is shown to be a relevant recognition element in major histocompatibility complex (MHC) class II restricted immune responses. On T lymphocytes, it defines a subset of helper / inducer factors.

[0051] CD8 + "T cells" refers to a subset of T cells that express CD8 on their surface, are MHC class I restricted, and act as cytotoxic T cells. The CD8 molecule is a differentiation antigen found on thymocytes and on cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin superfamily and a relevant recognition element in major histocompatibility complex class I restricted interactions.

[0052] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). As used herein, the terms "adaptive NK cells" and "memory NK cells" are interchangeable and refer to a subset of NK cells with a CD3 phenotype. - and CD56 + It expresses at least one of NKG2C and CD57 and optionally CD16, but lacks expression of one or more of the following: PLZF, SYK, FceRɣ, and EAT-2. In some embodiments, isolated CD56... + NK cell subsets include expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activated and repressive KIR, NKG2A, and / or DNAM-1. CD56 + The expression can be weak or strong. NK cells or NK cell-like effector cells can differentiate from stem cells or progenitor cells (“derived NK cells” or “derived NK cell-like effector cells,” or collectively “derived NK lineage cells”). Derived NK cell-like effector cells may possess NK cell lineage in some respects, but also have one or more functional characteristics not present in primary NK cells. In this application, NK cells, NK cell-like effector cells, derived NK cells, derived NK cell-like effector cells, or derived NK lineage cells are collectively referred to as “NK lineage cells.” In some embodiments, derived NK lineage cells are iPSC-derived NK cells obtained by differentiating iPSCs, which are also referred to herein as “iNK” cells.

[0053] As used herein, the term "NKT cells" or "natural killer T cells" refers to CD1d-restricted T cells that express the T cell receptor (TCR). Unlike conventional T cells, which detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d (a non-classical MHC molecule). Two types of NKT cells are recognized. Constant or type I NKT cells express a very limited TCR lineage: a classic α chain (Vα24-Jα18 in humans) with a limited spectrum of β chains (Vβ11 in humans). A second population of NKT cells, called non-classical or non-constant type II NKT cells, exhibits a more uneven TCR αβ utilization. Type I NKT cells are considered suitable for immunotherapy. Adaptive or constant (type I) NKT cells can be identified by the expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, αGalCer, CD161, and CD56.

[0054] As used herein, the term "isolated" refers to a cell or cell population that has been separated from its initial environment, i.e., the environment in which the isolated cells are isolated substantially free of at least one component found in the environment where "unisolated" reference cells are present. The term includes cells removed from some or all of the components as if they were found in their natural environment, such as from tissue or biopsy samples. The term also includes cells removed from at least one, some, or all of the components as if they were found in a non-natural environment, such as from cell cultures or cell suspensions. Thus, "isolated cells" are partially or completely separated from at least one component (including other substances, cells, or cell populations) as if they were found in nature or as if they were grown, stored, or survived in a non-natural environment. Specific examples of isolated cells include partially pure cell compositions, substantially pure cell compositions, and cells cultured in non-natural media. Isolated cells can be obtained by separating the desired cells or populations 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 in this article, the term "purification" refers to an increased purity. For example, 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 properties of a specific sequence of nucleotides in a polynucleotide (e.g., a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes, which have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the biological properties acquired therefrom. Thus, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, then the gene encodes that protein. Both the coding strand (whose nucleotide sequence is consistent with the mRNA sequence and is typically provided in a sequence listing) and the non-coding strand (which serves as a template for gene or cDNA transcription) can be referred to as "encoding" the protein or other product of that gene or cDNA.

[0057] "Construction" refers to a macromolecule or molecular complex containing a polynucleotide to be delivered to a host cell, either in vitro or in vivo. As used herein, "vector" refers to any nucleic acid construct capable of guiding the delivery or transfer of foreign genetic material to a target cell, where the construct is capable of replication and / or expression. Therefore, the term "vector" encompasses the construct to be delivered. Vectors can be linear or circular molecules. Vectors can be integrated or non-integrated. Major types of vectors include, but are not limited to, plasmids, free vectors, viral vectors, granules, and artificial chromosomes. Viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, Sendai virus vectors, etc.

[0058] "Integration" refers to the stable insertion of one or more nucleotides of the construct into the cellular genome, i.e., covalently linked to a nucleic acid sequence within the cellular chromosomal DNA. "Targeted integration" refers to the insertion of nucleotides of the construct into the cellular chromosomal or mitochondrial DNA at a pre-selected site or "integration site." As used herein, the term "integration" further refers to a process involving the insertion of one or more exogenous sequences or nucleotides of the construct into the integration site, with or without the deletion of an endogenous sequence or nucleotide. In the case of a deletion at the insertion site, "integration" may also include replacing the deleted endogenous sequence or nucleotide with one or more inserted nucleotides.

[0059] As used herein, the term "exogenous" is intended to mean a reference molecule or activity introduced into the host cell, or that is not native to the host cell. This molecule can be introduced, for example, by introducing the coding nucleic acid into the host genetic material, such as by integration into the host chromosome, or as non-chromosomal genetic material, such as a plasmid. Therefore, when used with respect to the expression of the coding nucleic acid, this term refers to the introduction of the coding nucleic acid into the cell in an expressible form. The term "endogenous" refers to a reference molecule or activity present in the host cell. Similarly, when used with respect to the expression of the coding nucleic acid, this term refers to the expression of the coding nucleic acid contained within the cell, rather than being exogenously introduced.

[0060] As used herein, a “gene of interest” or “polynucleotide sequence of interest” is a DNA sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into RNA in vivo and, in some cases, translated into a polypeptide. Genes of interest or polynucleotide sequences can include, but are not limited to, prokaryotic sequences, cDNA derived from eukaryotic mRNA, genomic DNA sequences derived from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, a gene of interest may encode miRNA, shRNA, native polypeptides (i.e., polypeptides found in nature) or fragments thereof; variant polypeptides (i.e., mutants of native polypeptides with less than 100% sequence identity to native polypeptides) or fragments thereof; engineered polypeptides or peptide fragments, therapeutic peptides or polypeptides, imaging markers, optional markers, etc.

[0061] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides) of any length or similar. A polynucleotide sequence consists of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) (uracil replaces thymine when the polynucleotide is RNA). Polynucleotides can include genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched-chain polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides also refer to double-stranded and single-stranded molecules.

[0062] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to molecules in which amino acid residues are covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, the term refers to short chains (often also referred to in the field as, for example, peptides, oligopeptides, and oligomers) and longer chains (often referred to in the field as polypeptides or proteins). “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and others. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0063] "Operably linked / operatively linked" (the terms "operably connected / operatively connected" are used interchangeably) refers to the association of a nucleic acid sequence with a single nucleic acid fragment (or amino acids in a polypeptide having multiple domains) such that the function of one is influenced by the other. For example, a promoter is operatively linked to a coding sequence or functional RNA when it can influence the expression of that sequence or RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). The coding sequence can be operatively linked to a regulatory sequence in a sense or antisense orientation. As another example, a receptor-binding domain can be operatively linked to an intracellular signal transduction domain such that receptor-ligand binding transduction responds to the binding signal.

[0064] As used herein, a "fusion protein" or "chimeric protein" is a genetically engineered protein used to link two or more partial or complete polynucleotide coding sequences that encode a single protein, and the expression of these linked polynucleotides produces a single peptide or multiple polypeptides having functional properties derived from each of the original protein or fragments thereof. In a fusion protein, a linker (or spacer) peptide may be added between two adjacent polypeptides from different sources.

[0065] As used herein, the term "genetic imprint" refers to genetic or epigenetic information that contributes to preferred therapeutic properties of a source cell or iPSC and is retained in source cell-derived iPSCs and / or iPSC-derived hematopoietic lineage cells. As used herein, a "source cell" is a non-pluripotent cell that can be used to generate iPSCs through reprogramming, and source cell-derived iPSCs can further differentiate into specific cell types, including any hematopoietic lineage cell. Depending on the context, source cell-derived iPSCs and their differentiated cells are sometimes collectively referred to as "derived or derivative cells." For example, as used throughout this application, derived effector cells or derived NK lineage cells or derived T lineage cells are cells differentiated from iPSCs compared to their primary counterparts obtained from natural / native sources (e.g., peripheral blood, cord blood, or other donor tissues). As used herein, genetic imprinting that confers preferred therapeutic properties is incorporated into iPSCs by reprogramming selected source cells that are specific to donor, disease, or treatment response or by introducing gene modification patterns into iPSCs through genome editing. In the case of source cells derived from specially selected donors, disease, or treatment contexts, genetic imprints contributing to preferred therapeutic properties may include any background-specific genes or epigenetic modifications that exhibit a retainable phenotype, i.e., preferred therapeutic properties, which are then transferred to derived cells of the selected source cells, regardless of whether the underlying molecular events are identified. Donor, disease, or treatment-response-specific source cells may include genetic imprints that can be retained in iPSCs and derived hematopoietic lineage cells, including but not limited to pre-arranged single-specific TCRs, such as those from virus-specific T cells or constant-type natural killer T (iNKT) cells; traceable and desired genetic polymorphisms, such as isotype for point mutations encoding high-affinity CD16 receptors in the selected donor; and predetermined HLA requirements, i.e., the selected HLA-matched donor cells exhibiting haplotypes as the population grows. As used herein, preferred therapeutic properties include transplantation, transport, homing, viability, self-renewal, survival, regulation and modulation of immune responses, survival, and improved cytotoxicity of derived cells. Preferred therapeutic attributes may also involve antigen-targeting receptor expression; HLA presentation or its absence; resistance to the tumor microenvironment; induction and immunomodulation of neighboring immune cells; improved target specificity as extratumor effects decrease; and / or resistance to treatments such as chemotherapy. When derived cells possessing one or more therapeutic attributes are obtained by differentiating genetically imprinted iPSCs, such derived cells are also referred to as “synthetic cells,” the genetic imprinting conferring preferred therapeutic attributes integrated into the iPSC. For example, as used throughout this application, synthetic effector cells or synthetic NK cells or synthetic T cells are cells differentiated from genomically modified iPSCs compared to their primary counterparts obtained from natural / native sources (such as peripheral blood, umbilical cord blood, or other donor tissues).In some implementations, the synthetic cell has one or more non-native cell functions when compared to its closest corresponding primary cell.

[0066] As used herein, the term "enhanced therapeutic properties" refers to cells whose therapeutic properties are enhanced compared to typical immune cells of the same general cell type. For example, NK cells with "enhanced therapeutic properties" will have enhanced, improved, and / or strengthened therapeutic properties compared to typical, unmodified, and / or naturally occurring NK cells. Therapeutic properties of immune cells can include, but are not limited to, cell transplantation, transport, homing, viability, self-renewal, survival, regulation and modulation of immune responses, survival, and cytotoxicity. Therapeutic properties of immune cells are also manifested through: antigen-targeting receptor expression; HLA presentation or its absence; resistance to the tumor microenvironment; induction and immunomodulation of neighboring immune cells; improved target specificity as extratumor effects decrease; and / or resistance to treatments such as chemotherapy.

[0067] As used herein, the term "adaptor" refers to a molecule, such as a fusion polypeptide, that enables the formation of a link between immune cells (e.g., T cells, NK cells, NKT cells, B cells, macrophages, neutrophils) and tumor cells, and activates the immune cells. Examples of adaptors include, but are not limited to, bispecific T cell adaptors (BiTE), bispecific killer cell adaptors (BiKE), trispecific killer cell adaptors (TriKE), or multispecific killer cell adaptors, or universal adaptors 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 can be engineered and expressed on effector cells (e.g., T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils). In some embodiments, surface triggering receptors facilitate bispecific or multispecific antibody binding between effector cells and specific target cells (e.g., tumor cells), independent of the effector cell's natural receptor and cell type. Using this approach, iPSCs containing a universal surface triggering receptor can be generated, and these iPSCs can then be differentiated into populations of various effector cell types expressing the universal surface triggering receptor. "Universal" means that the surface triggering receptor can be expressed on any effector cell and activate any effector cell (regardless of cell type), and all effector cells expressing the universal receptor can be coupled or connected to an adaptor recognizable by the surface triggering receptor (regardless of the adaptor's tumor-binding specificity). In some embodiments, adaptors with the same tumor-targeting specificity are used for coupling with the universal surface triggering receptor. In some embodiments, adaptors with different tumor-targeting specificities are used for coupling with the universal surface triggering receptor. Therefore, one or more effector cell types can be conjugated, thereby killing a specific type of tumor cell in some cases and killing two or more types of tumor cells in others. Surface trigger receptors typically contain a co-stimulatory domain for effector cell activation and an antiepitope specific to the epitope of the adaptor. Bispecific adaptors are specific to the antiepitope of the surface trigger receptor at one end and to the tumor antigen at the other end.

[0069] As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent potential toxicity of cell therapy or otherwise prevent side effects. In some cases, safety switch protein expression is conditionally controlled to address safety concerns of transplanted engineered cells that have permanently incorporated the gene encoding the safety switch protein into their genome. This conditional regulation can be variable and may include control via small molecule-mediated post-translational activation and tissue-specific and / or temporal transcriptional regulation. Safety switch proteins can mediate the induction of apoptosis, inhibition of protein synthesis or DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation, and / or antibody-mediated depletion. In some cases, safety switch proteins are activated by exogenous molecules, such as prodrugs, which trigger apoptosis and / or cell death in the therapeutic cells upon activation. 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, the prodrug administered in the event of an adverse event is activated by the product of a suicide gene and kills the transduced cells.

[0070] As used herein, the term "medicatically active protein or peptide" refers to a protein or peptide capable of exerting biological and / or pharmaceutical effects on an organism. Medicinally active proteins possess curative or palliative properties against a disease and can be administered to improve, alleviate, slow, reverse, or reduce the severity of a disease. Medicinally active proteins also possess preventative properties and are used to prevent the onset of disease or to reduce the severity of such diseases or pathologies when they manifest. Medicinally active proteins include whole proteins or peptides or their pharmaceutically active fragments. The term also includes pharmaceutically active analogs of proteins or peptides or analogs of fragments of proteins or peptides. The term "medicinally active protein" also refers to a variety of proteins or peptides that act in a cooperative or synergistic manner to provide therapeutic benefits. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth inhibitory proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0071] As used herein, the term "signaling molecule" refers to any molecule that regulates, participates in, inhibits, activates, reduces, or increases cellular signal transduction. "Signal transduction" refers to the transmission of molecular signals in a chemically modified form, achieved by recruiting protein complexes along pathways that ultimately trigger biochemical events in the cell. Examples of signal transduction pathways are well-known in the field 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, the ERK / MAPK signaling pathway, the Wnt signaling pathway, the cAMP-dependent pathway, and the IP3 / DAG signaling pathway.

[0072] As used herein, the term “targeting modality” refers to the genetic incorporation of molecules (e.g., peptides) into cells to promote antigen and / or epitope specificity, including but not limited to (i) antigen specificity (when it involves a unique chimeric antigen receptor (CAR) or T-cell receptor (TCR); (ii) adaptor specificity (when it involves a monoclonal antibody or a bispecific adaptor); (iii) targeting transformed cells; (iv) targeting cancer stem cells; and (v) other targeting strategies in the absence of specific antigens or surface molecules.

[0073] As used herein, the term "specificity" can be used to refer to the ability of a molecule (e.g., a receptor or adaptor) to selectively bind to a target molecule, in contrast to nonspecific or nonselective binding.

[0074] As used herein, the term “adoptive cell therapy” refers to cell-based immunotherapy involving the infusion of autologous or allogeneic lymphocytes, regardless of whether the immune cells are isolated from a human donor or from effector cells obtained through in vitro differentiation of pluripotent cells; regardless of whether these immune cells are genetically modified; or regardless of whether these immune cells are primary donor cells or cells that have been passaged, expanded, or immortalized in vitro after being isolated from a donor.

[0075] As used herein, “lymphocyte depletion” and “lymphomodulation” are used interchangeably and refer to the destruction of lymphocytes and T cells typically prior to immunotherapy. The purpose of lymphomodulation prior to the administration of adoptive cell therapy is to promote the homeostatic proliferation of effector cells and to eliminate other competing elements of regulatory immune cells and the immune system that compete for homeostatic cytokines. Therefore, lymphomodulation is typically performed by administering one or more chemotherapeutic agents (i.e., chemotherapy) to the subject prior to the first dose of adoptive cell therapy. In various embodiments, lymphomodulation precedes the first dose of adoptive cell therapy by several hours to several days. Exemplary chemotherapeutic agents that can be used for lymphomodulation include, but are not limited to, cyclophosphamide (CY), fludarabine (FLU), and those described below. However, sufficient lymphocyte depletion achieved by anti-CD38 mAb can provide alternative modulation procedures (e.g., in T-lineage cell therapy according to various embodiments of this document) without requiring or requiring only minimal amounts of CY / FLU-based lymphomodulation procedures, as further described herein.

[0076] As used herein, the term "outpatient" refers to a patient who does not require overnight hospitalization but travels to a hospital, clinic, or related facility for diagnosis and / or treatment. Therefore, compared to a "hospitalized environment," an "outpatient environment" refers to an environment designed to provide mobile or outpatient care to patients where hospitalization for one or more days / nights is not required for treatment and / or diagnosis, thus reducing overall patient discomfort and, more conveniently, minimizing the overall cost of such treatment and / or diagnosis in terms of management and coordination. Furthermore, a larger patient population is more willing to enter an outpatient environment, increasing patient availability during trials or treatments and improving patient adherence to treatment protocols.

[0077] As used in this article, "induction therapy," also known as "first-line therapy," "primary therapy," or "primary treatment," refers to the first treatment given to a patient for a specific disease. It is often part of a standard treatment group, such as chemotherapy and radiation therapy following surgery. Therefore, "induction attempt" or "attempt of induction therapy" refers to an initial attempt to treat a specific disease using known and / or conventional treatments for that specific disease.

[0078] As used herein, "therapeuticly adequate amount" includes, within its meaning, a non-toxic but sufficient and / or effective amount of a specific therapeutic agent and / or pharmaceutical composition to provide the desired therapeutic effect. The precise amount required will vary from subject to subject, depending on factors such as the patient's overall health status, age, and stage and severity of the condition being treated. In certain embodiments, a therapeutically adequate amount is sufficient and / or effective to improve, reduce, and / or alleviate at least one symptom associated with the disease or condition of the subject being treated. A therapeutically adequate amount can be an amount delivered in a single dose or a cumulative amount administered over multiple doses that may be time-separated according to a dosing schedule.

[0079] Differentiation of pluripotent stem cells requires alterations to the culture system, such as changes to the stimulants in the culture medium or the physical state of the cells. Most conventional strategies utilize embryoid body (EB) formation as a common and crucial intermediate step in initiating lineage-specific differentiation. An EB is a three-dimensional cluster that has been shown to mimic embryonic development because it generates multiple lineages within its three-dimensional region. Through the differentiation process, typically lasting hours to days, simple EBs (e.g., aggregates of pluripotent stem cells that have been induced to differentiate) continue to mature and develop into cystic EBs, at which point they are usually further treated for several days to weeks to continue differentiating. EB formation is initiated by bringing pluripotent stem cells into close proximity to each other within a three-dimensional, multi-layered cell cluster. This is typically achieved through one of several methods, including allowing pluripotent cells to settle in droplets, allowing cells to settle in a U-shaped bottom plate, or by mechanical agitation. Further differentiation cues are needed to promote EB development because aggregates maintained in maintenance media for pluripotent culture do not form suitable EBs. Therefore, pluripotent stem cell aggregates need to be transferred to a differentiation medium that provides inducing cues to the selected lineage. EB-based culture of pluripotent stem cells typically induces the production of differentiated cell populations (i.e., ectoderm, mesoderm, and endoderm germ layers) through moderate proliferation within EB cell clusters. While EB has been shown to promote cell differentiation, it produces heterogeneous cells with variable differentiation states due to inconsistent exposure of cells in the three-dimensional structure to differentiation cues within the environment. Furthermore, EB formation and maintenance are cumbersome. Moreover, cell differentiation via EB formation is accompanied by moderate cell proliferation, which also leads to reduced differentiation efficiency.

[0080] In contrast, "aggregate formation," distinct from "EB formation," can be used to expand pluripotent stem cell-derived cell populations. For example, during aggregate-based pluripotent stem cell expansion, a culture medium that maintains proliferation and pluripotency is selected. Cell proliferation typically increases aggregate size, forming larger aggregates that can dissociate into smaller aggregates mechanically or enzymatically, thus maintaining cell proliferation and increasing cell number within the culture. Unlike EB culture, cells cultured within aggregates in a maintenance medium maintain pluripotency markers. 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 that differs from differentiation through three-dimensional, multi-layered cell clusters, i.e., “EB formation.” In addition to other advantages disclosed herein, monolayer differentiation avoids the need for EB formation to initiate differentiation. Because monolayer culture does not mimic embryonic development, such as in the case of EB formation, differentiation toward a specific lineage is considered minimal compared to all three germ layer differentiations in EB formation.

[0082] As used herein, "dissociated cell" or "single dissociated cell" refers to a cell that has been substantially separated or purified from other cells or surfaces (e.g., a culture plate surface). For example, cells can be dissociated from animals or tissues by mechanical or enzymatic methods. Alternatively, cells aggregated in vitro can be dissociated from each other enzymatically or mechanically, such as by dissociating into clusters, single cells, or a suspension of a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells can be dissociated from a culture plate or other surface. Thus, dissociation may involve disrupting cell interactions with the extracellular matrix (ECM) and the substrate (e.g., a culture surface), or disrupting 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 include one or more therapeutic properties, have been characterized, tested, qualitatively and expanded, and have proven to function reliably as starting cell material for the production of cell-based therapeutics through directed differentiation in a manufacturing environment. In various embodiments, the MCB is maintained, stored, and / or cryopreserved in multiple containers to prevent genetic variation and / or potential contamination by reducing and / or eliminating the total number of passages, thawing, or handling of the iPS cell line during manufacturing.

[0084] As used in the context of genome editing or modification of iPSCs and their derivative non-pluripotent cells or genome editing or modification of non-pluripotent cells and their reprogrammed derivative iPSCs, "function" means (1) at the gene level—successful knock-in, knockout, reduction of gene expression, transgenesis, or controlled gene expression, such as inducible or transient expression at a desired stage of cell development, achieved through direct genome editing or modification or through "transmission," via differentiation or reprogramming of the initiating cell from which the genome was initially engineered; or (2) at the cellular level—successful removal, addition, or alteration of cellular function / characteristics, achieved by: (i) in the cell Gene expression modifications obtained through direct genome editing, (ii) gene expression modifications maintained in the cells by “transmission” via differentiation or reprogramming from the initial cell from which the genome was originally engineered; (iii) downstream gene regulation in the cells as a result of gene expression modifications that occur only in the earlier developmental stages of the cells or only in the initial cells from which the cells were generated by differentiation or reprogramming; or (iv) enhanced or newly acquired cellular functions or properties presented in mature cell products that originally originated from genome editing or modifications performed at iPSC, progenitor cells, or dedifferentiated cell sources.

[0085] The term "ligand" refers to a substance that forms a complex with a target molecule to generate a signal by binding to a site on the target. Ligands can be natural or artificial substances capable of specifically binding to a target. Ligands can be proteins, peptides, antibodies, antibody complexes, conjugates, nucleic acids, lipids, polysaccharides, monosaccharides, small molecules, nanoparticles, ions, neurotransmitters, or any other molecular entity capable of specifically binding to a target. The target bound to the ligand can be a protein, nucleic acid, antigen, receptor, protein complex, or cell. A ligand that binds to a target and alters the target's function to trigger a signal transduction response is called an "agonist" or "activator." A ligand that binds to a target and blocks or reduces a signal transduction response is called an "antagonist" or "antagonist."

[0086] The term "antibody" includes antibodies and antibody fragments containing at least one binding site that specifically binds to a particular target of interest, where the target may be an antigen or a receptor capable of interacting with certain antibodies. The term "antibody" includes, but is not limited to, immunoglobulin molecules or their antigen-binding or receptor-binding portions. For example, NK cells can be activated by the binding of an antibody or the Fc region of an antibody to its Fc-γ receptor (FcγR), thereby triggering ADCC (antibody-dependent cytotoxicity)-mediated effector cell activation. A specific fragment or portion of an antigen, receptor, or target that binds to an antibody is often referred to as an epitope or antigenic determinant. The term "antibody" also includes, but is not limited to, native antibodies and their variants, fragments of native antibodies and their variants, peptide bodies and their variants, and antibody mimics that imitate the structure and / or function of antibodies or specific fragments or portions thereof (including single-chain antibodies and their fragments). Antibodies can be mouse antibodies, human antibodies, humanized antibodies, camel IgG, single variable neoantigen receptor (VNAR), shark heavy chain antibody (Ig-NAR), chimeric antibodies, recombinant antibodies, single-domain antibodies (dAb), anti-idiotype antibodies, bispecific antibodies, multispecific antibodies, or multimeric antibodies, or antibody fragments thereof. Anti-idiotype antibodies are specific for binding to the idiotype of another antibody, where the idiotype is an antigenic determinant of the antibody. Bispecific antibodies can be BiTE (bispecific T-cell adaptor) or BiKE (bispecific cytotoxic cell adaptor), and multispecific antibodies can be TriKE (trispecific cytotoxic cell adaptor). Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, Fac, pFc, Fd, single-chain variable region fragments (scFv), tandem scFv (scFv)2, single-chain Fab (scFab), disulfide-stabilized Fv (dsFv), microantibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, single-domain antigen-binding fragments (sdAb), camel heavy chain IgG, and nanobody. ® Fragments, recombinant antibodies consisting only of heavy chains (VHH), and other antibody fragments that maintain the binding specificity of the antibody.

[0087] "Fc receptors" (abbreviated as "FcRs") are classified based on the types of antibodies they recognize. For example, receptors that bind to the most common class of antibodies (IgG) are called Fc-γ receptors (FcγRs), those that bind to IgA are called Fc-α receptors (FcαRs), and those that bind to IgE are called Fc-ε receptors (FcεRs). FcRs are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T cells, and B cells) and the signal transduction characteristics of each receptor. Fc-γ receptors (FcγRs) include several members: FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b), which have different affinities for their antibodies due to their different molecular structures.

[0088] "Chimeric Fc receptor," abbreviated as "CFcR," is a term used to describe engineered Fc receptors in which their native transmembrane and / or intracellular signaling domains are modified or replaced by non-native transmembrane and / or intracellular signaling domains. In some embodiments of chimeric Fc receptors, in addition to making one or both of the transmembrane and signaling domains non-native, one or more stimulatory domains may be introduced into the intracellular portion of the engineered Fc receptor to enhance cellular activation, expansion, and function upon receptor triggering. Unlike chimeric antigen receptors (CARs) containing an antigen-binding domain to the target antigen, chimeric Fc receptors bind to an Fc fragment, or the Fc region of an antibody, or are contained in an adaptor or binding molecule and activate cellular function upon proximity or absence of a target cell. For example, an Fcγ receptor can be engineered to include selected transmembrane, stimulatory, and / or signaling domains in an intracellular region that responds to IgG binding in the extracellular domain, thereby generating a CFcR. In one example, CFcRs are generated from engineered CD16, Fcγ receptors by replacing their transmembrane and / or intracellular domains. To further enhance the binding affinity of CD16-based CFcRs, the extracellular domain of a high-affinity variant of CD64 or CD16 (e.g., F176V) can be integrated. In some embodiments of CFcRs involving the high-affinity CD16 extracellular domain, the serine-containing proteolytic cleavage site at position 197 is eliminated or replaced, making the receptor's extracellular domain uncleavable, i.e., not subject to shedding, thereby obtaining hnCD16-based CFcRs.

[0089] The FcγR receptor CD16 has been identified as having two isoforms: Fc receptor FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG on target cells to activate NK cells and promote antibody-dependent cell-mediated cytotoxicity (ADCC). As used herein, “high-affinity CD16,” “non-cleavable CD16,” or “non-cleavable high-affinity CD16 (abbreviated as hnCD16)” refers to natural or non-natural CD16 variants. Wild-type CD16 has low affinity and undergoes extracellular domain shedding, a protein cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes after NK cell activation. F176V and F158V are exemplary high-affinity CD16 polymorphic variants. CD16 variants with altered or eliminated cleavage sites (positions 195-198) in regions near the membrane (positions 189-212) do not undergo shedding. The cleavage sites and regions near the membrane are described in detail in WO2015 / 148926 and U.S. Patent No. 10,464,989, the full disclosures of which are incorporated herein by reference. The CD16 S197P variant is an engineered, non-cleavable form of CD16. CD16 variants containing F158V and S197P exhibit high affinity and are non-cleavable. Another exemplary high-affinity and non-cleavable CD16 (hnCD16) variant is an engineered CD16 containing extracellular domains derived from one or more of the three exons of the CD64 extracellular domain.

[0090] As used herein, “FT825” refers to a multi-engineered T-cell therapy derived from a clone-engineered iPSC line and engineered to have multiple modalities to enhance innate immunity: (1) a high-affinity, non-cleavable CD16 (hnCD16) Fc receptor; (2) an IL7 / IL7R fusion protein (IL7RF); (3) an anti-HER2-CAR; (4) a TGFβ-SRR; (5) a CXCR2; (6) a CD38 knockout; and (7) a T-cell receptor (TCR) knockout.

[0091] I. Cells and compositions suitable for adoptive cell therapy with enhancing properties

[0092] This article presents a strategy that systematically engineeres the regulatory circuits of cloned iPSCs without affecting differentiation efficiency or the cell developmental biology of iPSCs and their derivatives, while enhancing the therapeutic properties of derivative cells differentiated from iPSCs. Following the introduction of selective pattern combinations into cells through genetic engineering at the iPSC level, iPSC-derived cells exhibit improved function and are suitable for adoptive cell therapy. It remains unclear whether iPSCs containing one or more of the provided gene-editing alterations still possess the ability to intervene in cell development and / or to mature and generate functionally differentiated cells while retaining the modified activity and / or properties. Unexpected failures during iPSC-directed cell differentiation are attributed to, but are not limited to, aspects including: developmental stage-specific gene expression or lack of gene expression, the need for HLA complex presentation, protein shedding of the introduced surface expression patterns, and the need for reconfiguration of differentiation protocols to achieve phenotypic and / or functional changes in cells. This application has demonstrated that one or more selected genomic modifications provided herein do not negatively affect iPSC differentiation efficacy, and that functional effector cells derived from engineered iPSCs possess enhanced and / or acquired therapeutic properties attributable to individual or combined genomic modifications retained in the effector cells after iPSC differentiation. Furthermore, all genomic modifications and combinations thereof, as described in the context of iPSCs and iPSC-derived effector cells, are applicable to primary-derived cells, including primary immune cells such as T cells, NK cells, or immunomodulatory cells, whether cultured or expanded, whose modifications produce engineered immune cells for adoptive cell therapy.

[0093] Furthermore, while CAR-T cells have proven effective and potent in treating several hematologic malignancies, the success of engineered T-cell therapies in treating solid tumors has been limited. Unlike liquid tumors, where uniformly expressed antigens are accessible and can be effectively targeted, tumor accessibility, tumor-specific antigenic targets, and antigenic heterogeneity are major obstacles to the successful development of CAR-T cells in solid tumors. Moreover, the inherent genetic engineering variability observed with patient- and donor-derived immune cells limits the widespread application of CAR-T cell therapy. This application provides aspects of genetic engineering in the form of a solid tumor targeting scaffold, as well as other genetic modalities, to improve on-target specificity and reduce extratumor effects in off-the-shelf adoptive cell therapy settings using effector cells derived from engineered iPSCs, avoiding rejection, and overcoming the suppressive tumor microenvironment, which is an enhanced challenge, especially for solid tumors.

[0094] 1. Overexpression of CXC motif chemokine receptor

[0095] Chemokines are a family of homogeneous serum proteins ranging from approximately 7 kDa to approximately 16 kDa, initially characterized by their ability to induce leukocyte migration. Most chemokines possess four characteristic cysteine ​​residues (Cys) and are classified into CXC (or α, CXC), CC (or β), C (or γ), and CX3C (or δ) chemokine classes based on the motifs displayed by the first two cysteine ​​residues. The CXC (or α, CXC) subfamily is further classified into two groups based on the presence of the ELR motif (Glu-Leu-Arg) preceding the first cysteine: ELR-CXC chemokines and non-ELR-CXC chemokines.

[0096] CXC chemokine receptor 2 (CXCR2), also known as CD128, interleukin-8 receptor β (IL8Rβ), or type B L8 receptor, is a chemokine receptor primarily expressed by neutrophils, mast cells, monocytes, and macrophages. CXCR2 is known to be expressed on CD56dim NK cells; 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 unless transduced with exogenous polynucleotides encoding CXCR2 as described in this application. Chemokine IL8 (also known as CXCL8) is secreted by monocytes / macrophages, neutrophils, eosinophils, T lymphocytes, epithelial cells, and fibroblasts, and acts as a chemokine by guiding neutrophils to sites of infection. CXCL8 is also secreted by tumor cells and promotes tumor migration, invasion, angiogenesis, and metastasis. CXCL8 is one of the ligands for several CXC chemokine receptors, including CXCR1 and CXCR2. Other known chemokines that bind to CXCR2 include, but are not limited to, CXCL1, GROβ (CXCL2), CXCL3, CXCL5, CXCL6, and CXCL7.

[0097] 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 primarily expressed in activated T helper 1 (Th1) lymphocytes, but is also present in natural killer cells, macrophages, dendritic cells, and B lymphocyte subsets. The interaction between CXCR3 and its ligands involves guiding receptor-carrying cells to specific parts of the body, particularly sites of inflammation, immune injury, and immune dysfunction.

[0098] In various embodiments, this application provides effector cells or iPSCs genetically engineered to include a solid tumor targeting scaffold in addition to the editing considered and described herein, which includes a CXC motif chemokine receptor in addition to other gene patterns. In various embodiments, the CXC motif chemokine receptor comprises CXCR2 or CXCR3 or variants thereof. A non-limiting example of the amino acid sequence of human CXCR2 is the amino acid sequence registered under UniProtKB: P25025. In one embodiment, CXCR2 comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 1. In some embodiments, CXCR2 comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 1. In some embodiments, CXCR2 comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 1. In some embodiments, CXCR2 comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, variants of CXCR2 comprise CXCR2 isomers represented by SEQ ID NO: 2, 3, 4, 5, or 6. In some embodiments, variants of CXCR2 comprise amino acid sequences having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 2, 3, 4, 5, or 6. In some embodiments, variants of CXCR2 comprise amino acid sequences having at least 90% identity with SEQ ID NO: 2, 3, 4, 5, and 6. In some embodiments, variants of CXCR2 comprise amino acid sequences having at least 95% identity with SEQ ID NO: 2, 3, 4, 5, and 6. In some embodiments, variants of CXCR2 comprise the amino acid sequence of SEQ ID NO: 2. In some embodiments, variants of CXCR2 comprise the amino acid sequence of SEQ ID NO: 3. In some embodiments, variants of CXCR2 comprise the amino acid sequence of SEQ ID NO: 4. In some embodiments, variants of CXCR2 comprise the amino acid sequence of SEQ ID NO: 5. In some embodiments, variants of CXCR2 comprise the amino acid sequence of SEQ ID NO: 6. As used herein and throughout this application, considering the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences, the percentage of identity between the two sequences is a function of the number of coherent positions shared by the sequences (i.e., identity % = number of coherent positions / total number of positions × 100). The comparison of sequences and the determination of the percentage of identity between the two sequences can be achieved using mathematical algorithms recognized in the art.

[0099]

[0100] (360 amino acids CXCR2; UniProtKB No.: P25025)

[0101]

[0102] (15 amino acid CXCR2 isomer 1 (residues 1-15 of CXCR2); UniProtKB number: Q6LCZ7)

[0103]

[0104] (200 amino acids CXCR2 isomer 2 (CXCR2 residues 1-200); UniProtKB No.: C9JW47)

[0105]

[0106] (CXCR2 isomer 3 (residues 1-135 of CXCR2); UniProtKB No.: C9JG19)

[0107]

[0108] (172 amino acids, CXCR2 isomer 4 (residues 1-172 of CXCR2); UniProtKB number: C9J1J7)

[0109]

[0110] (138 amino acids, CXCR2 isomer 5 (CXCR2 residues 1-138); UniProtKB number: C9J2F9)

[0111] In various embodiments, a polynucleotide encoding a CXC motif chemokine receptor or a variant thereof is inserted into a selected locus in primary effector cells or iPSCs to derive functional effector cells containing the same gene edit through directed differentiation. In some embodiments, the selected locus for the CXC motif chemokine receptor insertion includes a safe harbor locus, a locus designed to be disrupted or knocked out, or a locus providing an endogenous promoter that provides spatial and / or temporal control over the expression of the exogenous gene. In some embodiments, the selected loci for CXC motif chemokine receptor insertion include AAVS1, CCR5, ROSA26, collagen, HTRP, H11, PH12, GAPDH, RUNX1, B2M, TAP1, TAP2, TAP-associated protein, 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, the selected locus for CXC motif chemokine receptor insertion is the TCR locus. In one embodiment, the selected locus for CXC motif chemokine receptor insertion is the CD38 locus.

[0112] In some embodiments, the CXC motif chemokine receptor is co-expressed with one or more exogenous polynucleotides encoding a polypeptide of interest via a separate expression construct or a single bicistronic or tricistronic expression cassette. In some embodiments, a single bicistronic or tricistronic expression cassette containing the CXC motif chemokine receptor and one or more exogenous polynucleotides encoding a polypeptide of interest includes a 2A sequence such that the CXC motif chemokine receptor and the additional polynucleotide are within a single open reading frame (ORF). The bicistronic design allows for the coordinated expression of multiple polynucleotides in time and number, and under the same control mechanisms that can be optionally incorporated, for example, into an inducible promoter to express a single ORF. Self-cleaving peptides have been found in members of the Picornaviridae family, including the genus *Aphthoviruses*, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), *TaV*, and porcine tussock virus 1 (PTV-I) (Donnelly, ML et al., *J. Gen. Virol*, Vol. 82, 1027-101 (2001); Ryan, MD et al., *J. Gen. Virol*, Vol. 72, 2727-2732 (2001)), and the genus *Cardioviruses*, such as Theylvirus (e.g., Theyl rodent encephalomyelitis) and encephalocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-I, and TaV are sometimes referred to as "F2A," "E2A," "P2A," and "T2A," respectively. In some embodiments, the exogenous polynucleotide co-expressed with the CXC motif chemokine receptor encodes one or more polypeptides, including CARs, CD16 or variants thereof, cytokines, cytokine receptors, cytokine signaling complexes, chimeric fusion receptors, chimeric Fc receptors, adaptors, checkpoint inhibitors, Fc receptors, or antibodies or functional variants or fragments thereof. In one embodiment, the exogenous polynucleotide co-expressed with the CXC motif chemokine receptor in a bicistronic cassette does not encode a CAR. In one embodiment, at least one exogenous polynucleotide co-expressed with the CXC motif chemokine receptor in a bicistronic cassette encodes exogenous CD16. In some embodiments, the primary or derived effector cells containing the CXC motif chemokine receptor or variants thereof are T lineage cells.

[0113] This application also provides a master cell library comprising single-cell sorted and expanded clonal engineered iPSCs having at least one modification or phenotype as provided herein, including but not limited to CXC motif chemokine receptor or variants thereof, wherein the cell library provides engineered iPSCs for further engineered clones and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapy products, including but not limited to derived T cells, which are compositionally well-defined and homogeneous and can be mass-produced in a cost-effective manner.

[0114] 2. Exogenously introduced TGFβ-redirected receptor

[0115] Transforming growth factor β (TGFβ) 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. TGFβ exists latently in the tumor microenvironment and is known to suppress T cell effector function, partly through Smad-mediated downregulation of target genes granzymes, perforin, and interferon. Furthermore, detection of TGFβ gene expression tags is associated with T cell exclusion from tumors and resistance to immunotherapy. One aspect of this application provides a multi-element solid tumor-targeting scaffold design that incorporates a synthetic transforming growth factor β receptor (TGFβR) signaling retargeting receptor, in addition to other edits as considered and described herein, to equip allogeneic effector cells (including those derived from genetically engineered iPSCs) for enhanced efficacy in general tumors, particularly solid tumors. Typically, a "signal transduction (or signal) redirection receptor" or "SRR" redirects signal transduction from the extracellular domain of a first receptor (e.g., a TGFβ receptor) to the intracellular domain of a different receptor (e.g., a cytokine receptor) by connecting the extracellular domain of a first receptor to the intracellular domain of a different receptor. In the context of TGFβR, this signal transduction redirection receptor may be referred to throughout this application as a "TGFβR redirector," "TGFβR redirection receptor," "TGFβ signal redirection receptor," or "TGFβ-SRR."

[0116] In some embodiments, iPSCs and their derived cells contain a polynucleotide encoding a TGFβ-redirecting receptor (TGFβ-SRR), which includes a portion or all of the peptide of the extracellular domain (ECD) of TGFβR. In some embodiments, the TGFβ-redirecting receptor comprises: (i) an extracellular domain or a fragment thereof of a transforming growth factor β receptor (TGFβR); and (ii) an intracellular domain (ICD) or a fragment thereof of a cytokine receptor including IL2R, IL12R, IL18R, IL21R, or any combination thereof. In some embodiments, the intracellular domain is the intracellular domain of IL18R.

[0117] In some embodiments, the TGFβ redirecting receptor comprising the ECD and ICD as described above further comprises a transmembrane domain (TM). In various embodiments, the transmembrane (TM) domain of the TGFβ redirecting receptor may: (i) be derived from the same molecule providing the intracellular domain, (ii) be derived from the same molecule providing the extracellular domain, or (iii) be modified or substituted by the transmembrane domain of any other membrane-binding protein. In some embodiments, the cytokine receptor providing the intracellular domain of the TGFβ redirecting receptor or a fragment thereof comprises at least one of IL2R, IL4R, IL6R, IL7R, IL9R, IL10R, IL11R, IL12R, IL15R, IL18R, and IL21R.

[0118] In some embodiments, the extracellular domain (ECD) of TGFβR comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with SEQ ID NO: 7. In some embodiments, the extracellular domain (ECD) of TGFβR comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 7. In some embodiments, the extracellular domain (ECD) of TGFβR comprises an amino acid sequence having at least about 95% identity with SEQ ID NO: 7. In some embodiments, the extracellular domain (ECD) of TGFβR comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the intracellular domain (ICD) of IL18Rβ comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with SEQ ID NO: 8. In some embodiments, the intracellular domain (ICD) of IL18Rβ comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 8. In some embodiments, the intracellular domain (ICD) of IL18Rβ comprises an amino acid sequence having at least about 95% identity with SEQ ID NO: 8. In some embodiments, the intracellular domain (ICD) of IL18Rβ comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the ECD and ICD are linked by a TM. In some embodiments, the TM comprises SEQ ID NO: 9.

[0119]

[0120] (ECD of TGFβR)

[0121]

[0122] (IL8Rβ ICD)

[0123]

[0124] (Example TM domain)

[0125] Therefore, in some embodiments, the present invention provides immune cells, iPSCs, and iPSC-derived cells comprising a solid tumor targeting cytoskeleton that, among other genetic patterns, contains a polynucleotide encoding a TGFβ-redirecting receptor (“TGFβ-SRR”), wherein the cells (such as derived T cells) can be used to overcome or reduce tumor microenvironment suppression associated with tumors, particularly solid tumors. In some embodiments, iPSCs and their derived cells comprise a solid tumor targeting cytoskeleton that contains two or more of the following: a polynucleotide encoding a CXC motif chemokine receptor or a variant thereof, a polynucleotide encoding a TGFβ-redirecting receptor, and / or one or more additional genome edits as described herein, without adversely affecting the differentiation potential of the iPSCs and the function of the derived effector cells (such as derived T cells).

[0126] 3. CD38 knockout

[0127] The cell surface molecule CD38 is highly upregulated in a variety of hematologic malignancies derived from both lymphoid and bone marrow lineages, including multiple myeloma and CD20-negative B-cell malignancies, making antibody-based therapeutics aimed at exhausting cancer cells an attractive target. Antibody-mediated cancer cell exhaustion is generally attributable to a combination of direct apoptosis induction and activation by immune effector mechanisms such as ADCC (antibody-dependent cell-mediated cytotoxicity). In addition to ADCC, immune effector mechanisms, along with therapeutic antibodies, may also include phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

[0128] In addition to its high expression on malignant cells, CD38 is also expressed on plasma cells, NK cells, and activated T and B cells. During hematopoiesis, CD38 is expressed on CD34. + It is expressed on lineage-specific progenitor cells of stem cells and lymphocytes, erythrocytes, and bone marrow, and continues throughout the final stages of maturation, up to the plasma cell stage. As a type II transmembrane glycoprotein, CD38 functions both as a receptor and as a multifunctional enzyme involved in the production of nucleotide metabolites. As an enzyme, CD38 catalyzes the production of NAD+ from NAD+. + The synthesis and hydrolysis of ADP-ribose via a reaction produce the secondary messengers CADPR and NAADP, which stimulate calcium release from the endoplasmic reticulum and lysosomes, crucial for calcium-dependent processes of cell adhesion. As a receptor, CD38 recognizes CD31 and regulates cytokine release and cytotoxicity in activated NK cells. CD38 association with cell surface proteins in lipid rafts has also been reported, thereby regulating cytoplasmic calcium release. 2+ It increases blood flow and mediates signal transduction in lymphocytes and bone marrow cells.

[0129] In the treatment of malignant tumors, systemic use of CD38 antigen-binding receptor transduced T cells has demonstrated CD34 lysis. + CD38 of hematopoietic progenitor cells, monocytes, NK cells, T cells, and B cells + In some cases, impaired function of the recipient's immune effector cells leads to incomplete treatment response and reduced or eliminated efficacy. Furthermore, in multiple myeloma patients treated with daratumumab or CD38-specific antibodies, although other immune cell types (e.g., T cells and B cells) were unaffected regardless of their CD38 expression, a decrease in NK cells was observed in both bone marrow and peripheral blood (Casneuf et al., *Blood Advances*, 2017; Vol. 1, No. 23, pp. 2105-2114).

[0130] Not limited by theory, this application includes a strategy to fully utilize the full potential of CD38-targeted cancer therapy by overcoming effector cell depletion or reduction induced by CD38-specific antibodies and / or CD38 antigen-binding domains through self-mutilation. Furthermore, because CD38 is upregulated on activated lymphocytes (such as T cells or B cells) by using anti-CD38 antibodies (such as daratumumab) in recipients of allogeneic effector cells to inhibit and / or eliminate these cells, host allogeneic rejection against these effector cells is reduced and / or prevented, thereby increasing effector cell survival and retention. Therefore, CD38 antagonists targeting recipient T cells, Treg cells, NK cells, and / or B cells, such as anti-CD38 antibodies, secreted CD38-specific adaptors, or CD38-CARs (chimeric antigen receptors), can be used as alternatives to lymphocyte depletion induced by chemotherapy such as Cy / Flu (cyclophosphamide / fludarabine) prior to adoptive cell transfer.

[0131] Furthermore, this application also discloses the use of CD38 in the presence of anti-CD38 antibodies or CD38 inhibitors. - Effector cells target CD38 + When T cells and pbNK cells are present, CD38 + Depletion of allogeneic reactive cells increases NAD. + (Nicotinamide adenine dinucleotide, a substrate of CD38) availability and reduced interaction with NAD + Consumption-related cell death, among other benefits, enhances effector cell responses in the immunosuppressive tumor microenvironment and supports cell regeneration in aging, degenerative, or inflammatory diseases.

[0132] Furthermore, the strategy presented herein, namely CD38 knockout, is compatible with other components and methods considered herein, thereby generating iPSC lines with CD19 targeting specificity and CD38 knockout as described herein, a master cell library of cloned iPSCs including single-cell sorting and expansion, and obtaining CD38-negative (CD38 knockout) cells with CD19 targeting specificity as described herein through directed differentiation of engineered iPSC lines. neg Or CD38 - / - CD38-derived effector cells, in which the therapeutic portion targeting CD38 is used in conjunction with effector cells, are protected from self-harm and allogeneic rejection, and other benefits include improved metabolic adaptation, increased resistance to oxidative stress, and induction of protein expression programs that enhance cell activation and effector function in effector cells. Furthermore, anti-CD38 monoclonal antibody therapy significantly depletes the patient's activated immune system without adversely affecting the patient's hematopoietic stem cell compartment. 阴性 The derived cells are able to resist CD38 antibody-mediated depletion and can be effectively combined with anti-CD38 antibodies or CD38-CARs without the use of toxic opsonizers, thus reducing and / or replacing chemotherapy-based lymphocyte depletion.

[0133] In one embodiment provided herein, CD38 knockout in the iPSC line is biallelic knockout. In some embodiments of the construct, the construct includes a pair of CD38 targeting homologous arms for position-selective insertion within the CD38 locus. In some embodiments, the pre-selected target site is within an exon of CD38. The CD38-KI / KO construct provided herein allows transgene expression under an endogenous CD38 promoter or under an exogenous promoter contained within the construct. When two or more transgenes are inserted at selected locations within the CD38 locus, a linker sequence, such as a 2A adapter or IRES, is placed between any two transgenes. The 2A adapter encodes a self-cleaving peptide derived from FMDV, ERAV, PTV-I, and TaV (referred to as “F2A,” “E2A,” “P2A,” and “T2A,” respectively), enabling the production of a single protein from a single translation. In some embodiments, the construct includes an insulator to reduce the risk of transgene and / or exogenous promoter silencing. The exogenous promoter contained in the CD38-KI / KO construct may be CAG or other constitutive, inducible, time-specific, tissue-specific and / or cell-type-specific promoters, including but not limited to CMV, EF1α, PGK and UBC.

[0134] 4. TCR knockout

[0135] In some implementations, a polynucleotide encoding one or more of the modes described herein (e.g., CAR, CXCR2, IL7RF, or TGFβ-SRR) is inserted into the TCR constant region (e.g., TRAC or TRBC), resulting in TCR knockout, and optionally placing the expression of one or more modes under the control of an endogenous TCR promoter. Disruption of the constant region (TRAC or TRBC) of TCRα or TCRβ produces TCR neg Cells. TCR neg The cells do not require HLA matching, have reduced allogeneic reactivity, and can prevent GvHD (graft-versus-host disease) when used for allogeneic adoptive cell therapy. In some embodiments, polynucleotides encoding different patterns described herein are inserted at different sites. Additional insertion sites include, but are not limited to, AAVS1, CCR5, ROSA26, collagen, HTRP, H11, PH12, GAPDH, RUNX1, B2M, TAP1, TAP2, TAP-associated protein, 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.

[0136] In one embodiment as provided herein, TCR knockout in the iPSC line is biallelic knockout. When two or more transgenes are inserted at selected sites in the TCR locus, an adapter sequence, such as a 2A adapter or IRES, is placed between any two transgenes. The 2A adapter encodes a self-cleaving peptide derived from FMDV, ERAV, PTV-I, and TaV (referred to as “F2A,” “E2A,” “P2A,” and “T2A,” respectively), allowing for the production of a single protein from a single translation. In some embodiments, the construct may include an insulator to reduce the risk of transgene and / or exogenous promoter silencing.

[0137] 5. Enter CD16

[0138] CD16 has been identified as two isoforms: Fc receptor FcγRIIIa (CD16a; NM_000569.6) and FcγRIIIb (CD16b; NM_000570.4). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG on target cells to activate NK cells and promote antibody-dependent cell-mediated cytotoxicity (ADCC). CD16b is expressed only by human neutrophils. As used herein, “high-affinity CD16,” “non-cleavable CD16,” “high-affinity non-cleavable CD16,” or “hnCD16” refers to various CD16 variants. Wild-type CD16 has low affinity and undergoes extracellular domain shedding, a protein cleavage process that, upon NK cell activation, regulates the cell surface density of various cell surface molecules on leukocytes. F176V (also referred to as F158V in some publications) is an exemplary high-affinity CD16 polymorphic variant; while the S197P variant is an example of a genetically engineered, non-cleavable form of CD16. The engineered CD16 variant comprising both F176V and S197P is high-affinity and non-cleavable, and is described in more detail in International Publication No. WO2015 / 148926 and U.S. Patent No. 10,464,989, the full disclosures of which are incorporated herein by reference. In some embodiments, hnCD16 comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 10. In some embodiments, hnCD16 comprises an amino acid sequence having at least about 95% identity with SEQ ID NO: 10. In some embodiments, hnCD16 comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, hnCD16 comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage thereof, when compared to any of the exemplary sequences SEQ ID NO: 11, 12, and 13, and each of these comprises at least a portion of the CD64 extracellular domain. In some embodiments, hnCD16 comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 11-13, and optionally one or more of F176V and S197P, and at least a portion of the CD64 extracellular domain. In some embodiments, hnCD16 comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 11-13, and optionally one or more of F176V and S197P, and at least a portion of the CD64 extracellular domain. In some embodiments, hnCD16 comprises the amino acid sequence of SEQ ID NO: 11.In some embodiments, hnCD16 comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, hnCD16 comprises the amino acid sequence of SEQ ID NO: 13.

[0139]

[0140]

[0141] (340 amino acids) Construction based on CD64 domain CD16TM; CD16ICD )

[0142]

[0143] (336 amino acids) Construction based on CD64 exon CD16TM; CD16ICD )

[0144]

[0145] (335 amino acids) Construction based on CD64 exon CD16TM; CD16ICD )

[0146] In some embodiments, the primary or derived effector cells comprising exogenous CD16 or variants thereof are T lineage cells. In some embodiments, the exogenous CD16 or functional variants thereof contained in iPSCs or effector cells have high affinity for a ligand upon binding, which triggers downstream signaling upon such binding. Non-limiting examples of ligands binding to exogenous CD16 or functional variants thereof include not only ADCC antibodies or fragments thereof, but also bispecific, trispecific, or multispecific adaptors or binders that recognize the extracellular binding domain of the exogenous CD16. Examples of bispecific, trispecific, or multispecific adaptors or binders are further described below in this application. Therefore, at least one aspect of this application provides a derived effector cell or a cell population thereof, which is preloaded with one or more preselected ADCC antibodies by expressing exogenous CD16 on the derived effector cell in an amount sufficient for therapeutic use in treating conditions, diseases or infections as further detailed in this application, wherein the exogenous CD16 comprises an extracellular binding domain of CD16 having F176V and S197P.

[0147] Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism of NK cell-mediated lysis that involves the binding of CD16 to antibody-coated target cells. However, endogenous CD16 expressed by primary NK cells cleaves from the cell surface upon NK cell activation. Various non-cleavable forms of CD16 prevent CD16 shedding and maintain constant expression. In some embodiments, non-cleavable CD16 provides ADCC, as well as bispecific, trispecific, or multispecific adaptors for binding to T cells. In derived T cells, non-cleavable CD16 increases the expression of TNFα and CD107a, indicating improved cellular function. The additional high-affinity properties of hnCD16 introduced into derived T cells prior to cell administration to subjects requiring cell therapy also enable the in vitro loading of ADCC antibodies onto T cells via hnCD16.

[0148] In some embodiments, the HER2 tumor antigen-targeting-specific T cells described herein have additional tumor antigen-targeting specificity via exogenous CD16 or a variant thereof, which mediates ADCC when combined with an antibody. In some embodiments, the HER2 tumor antigen-targeting-specific T cells described herein further comprise CD38 knockout. In some embodiments, the HER2 tumor antigen-targeting-specific and CD38 knockout-specific T cells described herein are combined with a CD38 antibody. In some embodiments, the CD38 antibody is daratumumab. In some embodiments, the HER2 tumor antigen-targeting-specific and CD38 knockout-specific T cells described herein are combined with one or more of the following: anti-EGFR antibody (e.g., cetuximab, amivantamab), anti-HER2 antibody (e.g., trastuzumab or a biosimilar, pertuzumab), anti-PDL1 antibody (e.g., avelumab), or a bispecific antibody targeting EGFR and MET (e.g., amivantamab).

[0149] 6. Exogenously introduced cytokine signaling complexes

[0150] By avoiding systemic high-dose administration of clinically relevant cytokines, the risk of dose-limiting toxicity associated with such practices is reduced, while simultaneously establishing cytokine-mediated cell autonomy. To achieve lymphocyte autonomy without the need for additional administration of soluble cytokines, a cytokine signaling complex comprising partial or full-length peptides of one or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21 and / or their respective receptors can be introduced into cells to achieve cytokine signaling with or without the expression of the cytokines themselves, thereby maintaining or improving cell growth, proliferation, expansion, and / or effector function, and reducing the risk of cytokine toxicity. In some embodiments, the introduced cytokines and / or their corresponding native or modified receptors for cytokine signaling (the signaling complex) are expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, the activation of cytokine signaling is inducible. In some embodiments, the activation of cytokine signaling is transient and / or transient. In some implementations, transient / temporary expression of cell surface cytokines / cytokine receptors is achieved via retroviruses, Sendai viruses, adenoviruses, episomes, microcircles, or expression constructs carried by RNA (including mRNA).

[0151] In various other embodiments, the cytokine signaling complex comprises an IL7 receptor fusion (IL7RF) comprising a full-length or partial-length IL7 and a full-length or partial-length IL7 receptor. The transmembrane (TM) domain for the IL7 receptor may be native or modified or substituted with the transmembrane domain of any other membrane-binding protein. In one embodiment, a native (or wild-type) or modified IL7R is fused to IL7 at its C-terminus via a linker to achieve constitutive signaling and maintain membrane-bound IL7. In some embodiments, such a construct comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 14, wherein the transmembrane domain, signal peptide, and linker are flexible and vary in length and / or sequence. In some embodiments, the IL7 construct comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 14, wherein the transmembrane domain, signal peptide, and linker are flexible and vary in length and / or sequence. In some embodiments, the IL7 construct comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 14, wherein the transmembrane domain, signal peptide, and linker are flexible and vary in length and / or sequence. In some embodiments, the IL7 construct comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 14, wherein the transmembrane domain, signal peptide, and linker are flexible and vary in length and / or sequence. 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 having at least 85%, 90%, 95%, or 99% identity with SEQ ID NO: 14, excluding the amino acids of the exemplary transmembrane domain, signal peptide, and linker shown in SEQ ID NO: 14.

[0152]

[0153]

[0154] (signal peptide-IL7-) connector -IL7R; Transmembrane domain ( TM (Signal peptides and linkers can vary in length and sequence)

[0155] In another embodiment, a native or modified co-receptor γC is fused to IL7 at its C-terminus via a linker for constitutive and membrane-bound cytokine signaling complexes. Furthermore, engineered IL7Rs that form homodimers in the absence of IL7 are also suitable for generating constitutive cytokine signaling.

[0156] Those skilled in the art will understand that the above-described signal peptide and adapter sequences are illustrative and in no way limit the applicability of their variants as signal peptides or adapters. Many suitable signal peptide or adapter sequences are known and available to those skilled in the art. Those skilled in the art will understand that signal peptide and / or adapter sequences can replace another sequence without altering the activity of the functional peptide guided by the signal peptide or linked by the adapter.

[0157] In iPSCs and their derived cells containing both CAR and exogenous cytokines and / or cytokine receptor signaling (signaling complex or "IL"), CAR and IL can be expressed in separate constructs. In some embodiments, IL can be co-expressed in a bicistronic construct containing both CAR and IL or both hnCD16 and IL. In some other embodiments, the signaling complex can be linked to the 5' or 3' end of the CAR or hnCD16 expression construct via a self-cleaving 2A coding sequence. Thus, the IL signaling complex (e.g., the IL15 or IL7 signaling complex) and CAR can be in a single open reading frame (ORF). In some embodiments, the bicistronic design allows for the expression of the IL signaling complex coordinated with CAR or hnCD16 in terms of time and quantity, and under the same control mechanisms that can be selectively incorporated, for example, into an inducible promoter or a promoter with time or space specificity to express a single ORF. In one embodiment, the signaling complex is contained in a CAR-2A-IL or IL-2A-CAR construct. In one embodiment, the signal transduction complex is contained in a hnCD16-2A-IL or IL-2A-hnCD16 construct. When CAR-2A-IL or IL-2A-CAR, or hnCD16-2A-IL or IL-2A-hnCD16, the self-cleaving 2A peptide allows the expressed CAR and IL or hnCD16 and IL to dissociate, and the dissociated IL is then presented at the cell surface, wherein the transmembrane domain is anchored in the cell membrane. Self-cleaving peptides have been found in members of the Picornaviridae family, including the genus *Aphthovirus*, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), *Thosea asigna virus* (TaV), and porcine tescho virus-1 (PTV-I) (Donnelly et al., J. Gen. Virol, 82, 1027-101 (2001); Ryan et al., J. Gen. Virol., 72, 2727-2732 (2001)), and the genus *Cardiovirus*, such as Theilovirus (e.g., Theilovirus encephalomyelitis) and encephalocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-I, and TaV are sometimes referred to as “F2A,” “E2A,” “P2A,” and “T2A,” respectively.

[0158] In view of the above, this application provides iPSCs, iPS cell lines or populations thereof, or derived functional cells obtained by differentiating iPSCs, wherein each cell contains a polynucleotide encoding HER2 tumor antigen targeting specificity, and wherein the cell contains a polynucleotide encoding HER2-CAR (chimeric antigen receptor) that mediates CD16 or a variant thereof for ADCC when combined with a monoclonal antibody, CD38 knockout and IL7 signaling complex.

[0159] In some embodiments, when an anti-CD38 antibody is used to induce CD16-mediated enhanced ADCC, iPSCs and / or their derived effector cells can target CD38-expressing (tumor) cells without causing effector cell elimination, i.e., a reduction or depletion of CD38-expressing effector cells, thereby increasing the survival and / or survival of iPSCs and their effector cells. In some embodiments, effector cells have increased survival and / or survival in vivo in the presence of an anti-CD38 therapeutic agent, which may be an anti-CD38 antibody. Furthermore, since CD38 is upregulated on activated lymphocytes such as T cells or B cells, anti-CD38 antibodies can be used for lymphocyte depletion, thereby eliminating those activated lymphocytes, overcoming allogeneic rejection, and increasing the survival and survival of CD38-negative effector cells without self-mutilation in recipients of allogeneic effector cell therapy. In some embodiments, these effector cells include T lineage cells. iPSC-derived T lineages containing CD38-negative and exogenous CD16 or variants thereof exhibit enhanced cytotoxicity and reduced susceptibility to host alloreactive immune cells in the presence of anti-CD38 antibodies.

[0160] iPSCs, iPS cell lines or populations thereof, or derivative functional cells derived from iPSCs provided herein, comprising CD38 knockout and polynucleotides encoding HER2-CAR (chimeric antigen receptor), CD16 or variants thereof, CXCR2, TGFβ-SRR, and the IL7 signaling complex, wherein the exogenous cytokine signaling complex (IL) enables cytokine signaling to contribute to cell survival, retention, and / or expansion, wherein the iPSC line is capable of hematopoietic differentiation to produce functional derivative effector cells with improved survival, retention, expansion, and effector functions. In some embodiments, some or all of the introduced peptides of cytokines and / or their corresponding receptors for cytokine signaling are expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, activation of cytokine signaling is inducible. In some embodiments, activation of cytokine signaling is transient and / or transient. In some embodiments, exogenous cell surface cytokines and / or receptors contained in iPSCs or their derivatives allow IL7 signaling.

[0161] 7. Chimeric antigen receptor (CAR) expression

[0162] Genetically engineered immune cells, iPSCs, and their derived effector cells can be any CAR design known in the art. A CAR is a fusion protein that typically comprises an extracellular domain, a transmembrane domain, and an intracellular domain, the extracellular domain containing a target-binding region (e.g., an antigen recognition domain). In some embodiments, the extracellular domain may also include a signal peptide or leader sequence and / or a spacer. In some embodiments, the intracellular domain may also contain a signal transduction peptide that activates effector cells expressing the CAR. In some embodiments, the signal transduction peptide of the intracellular domain (or intracellular structural domain) comprises the full length or at least a portion of a polypeptide of the following: 2B4, CD2, CD3ζ, CD3ζ1XX, CD8, CD28, CD28H, CD137 (4-1BB), CS1, DAP10, DAP12, DNAM1, FcERIγ, IL2Rγ, IL7R, IL21R, IL2Rβ (IL15Rβ), IL21, IL7, IL12, IL15, IL21, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, or NKG2D. In one embodiment, the CAR signal transduction peptide comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with at least one ITAM (immunoreceptor tyrosine-based activation motif) of CD3ζ. Exemplary N-terminal signal peptides include MALPVTLPLALLLHA (SEQ ID NO: 15; CD8asp) or MDFQVQIFSFLLISASVIMSR (SEQ ID NO: 16; IgKsp), or any signal peptide sequence or functional variant thereof known in the art.

[0163] In some embodiments, the antigen recognition domain can specifically bind to an antigen. In some embodiments, the CAR is adapted to activate T cells, NK cells, or NKT cells expressing the CAR. In some embodiments, the CAR is T cell specific due to containing a T-specific signaling component. In some embodiments, the T cells are derived from iPSCs containing an encoding CAR as described herein as a solid tumor targeting cytokine, and the derived T cells may comprise T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβ T cells, γδ T cells, or combinations thereof.

[0164] In various implementations, the antigen recognition region comprises mouse antibodies, human antibodies, humanized antibodies, camel Ig, single variable neoantigen receptor (VNAR), shark heavy chain antibody (Ig-NAR), chimeric antibodies, recombinant antibodies, single-domain antibodies (dAb), anti-idiotype antibodies, bispecific antibodies, multispecific antibodies, or multimeric antibodies, or antibody fragments thereof. Anti-idiotype antibodies are specific for binding to the idiotype of another antibody, where the idiotype is an antigenic determinant of the antibody. Bispecific antibodies may be BiTE (bispecific T-cell adaptor) or BiKE (bispecific cytotoxic cell adaptor), and multispecific antibodies may be TriKE (trispecific cytotoxic cell adaptor). Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, Fac, pFc, Fd, single-chain variable region fragments (scFv), tandem scFv (scFv)2, single-chain Fab (scFab), disulfide-stabilized Fv (dsFv), microantibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, single-domain antigen-binding fragments (sdAb), camel heavy chain IgG, and nanobody. ® The CAR comprises a fragment, a recombinant antibody consisting only of the heavy chain (VHH), and other antibody fragments that maintain the binding specificity of the antibody. In some embodiments, the antigen-binding domain of the CAR includes CDR1, CDR2, and CDR3 (H-CDR) of the heavy chain of the antibody or a fragment thereof. In some embodiments, the antigen-binding domain of the CAR containing the H-CDR of the antibody also includes the CDR of the light chain of the antibody (L-CDR).

[0165] In some implementations, the antigen recognition domain of the CAR specifically binds to tumor-associated HER2, and the CAR and effector cells containing the disclosed solid tumor-targeting cytoskeleton can be used to treat one or more cancers, including at least bladder cancer, breast cancer, breast lung cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, lung cancer, ovarian cancer, or salivary gland cancer.

[0166] In some embodiments, the antigen-binding domain of the CAR includes a single-stranded variable fragment (scFV) having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage thereof, of sequence identity when compared to the exemplary sequences represented by SEQ ID NO: 17 or SEQ ID NO: 18, wherein each of SEQ ID NO: 17 and 18 includes a linker that may vary in length and / or sequence. In some embodiments, the scFV includes an amino acid sequence having at least 90% identity with SEQ ID NO: 17 or 18. In some embodiments, the scFV includes an amino acid sequence having at least 95% identity with SEQ ID NO: 17 or 18. In some embodiments, the scFV includes the amino acid sequence of SEQ ID NO: 17. In some embodiments, the scFV includes the amino acid sequence of SEQ ID NO: 18. In some embodiments, the CAR also includes a transmembrane domain, such as the transmembrane domain of CD28. In some implementations, the CAR also includes a co-stimulatory domain, such as the co-stimulatory domain of CD28. In some implementations, the CAR also includes an activation domain, such as the activation domain of CD3ζ1XX.

[0167]

[0168]

[0169] In one embodiment, the CAR provided herein comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with SEQ ID NO: 19, wherein the linker in the extracellular domain and the spacer region between the extracellular domain and the transmembrane domain may vary in length and sequence. In some embodiments, the CAR comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 19, wherein the linker in the extracellular domain and the spacer region between the extracellular domain and the transmembrane domain may vary in length and sequence. In some embodiments, the CAR comprises an amino acid sequence having at least about 95% identity with SEQ ID NO: 19, wherein the linker in the extracellular domain and the spacer region between the extracellular domain and the 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 the HER2 antigen, which is specific to cells of solid tumors. In some embodiments, the CAR provided herein recognizes the HER2 antigen of a tumor, including breast cancer, ovarian cancer, endometrial cancer, lung cancer, esophageal cancer, salivary gland cancer, bladder cancer, gastric cancer, colorectal cancer, or head and neck cancer. In still other embodiments, the CAR provided herein recognizes the HER2 antigen of a tumor and is unresponsive or has a low level of response to HER2 expressed on non-cancer cells or normal cells.

[0170]

[0171] (Anti-HER2 scFV) connector -Interval - CD28 TM -CD28 co-stimulation-CD3ζ1XX activation )

[0172] As described herein, in some embodiments, cells containing a solid tumor targeting cytokine complex (“IL”) contain polynucleotides encoding CXCR2, TGFβ-SRR, HER2-CAR, and / or one or more other modified forms provided herein. In iPSCs and their derived cells containing both exogenous cytokine signaling complexes (“IL”) and HER2-CAR, IL and HER2-CAR may be expressed in separate constructs or co-expressed in bicistronic constructs containing both IL and CAR. Additionally, a master cell library is provided herein containing single-cell sorted and expanded clonal engineered iPSCs having at least one phenotype as described herein, including but not limited to the solid tumor targeting cytokine complex and HER2-CAR as described herein. This cell library provides a platform for further iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapy products, including but not limited to derived T cells, which are compositionally well-defined and homogeneous and can be mass-produced in a cost-effective manner.

[0173] 8. Antibodies used in immunotherapy

[0174] In some embodiments, in addition to the genomically engineered effector cells as provided herein, additional therapeutic agents comprising antibodies or antibody fragments targeting antigens associated with a condition, disease, or indication may be used with these effector cells in combination therapy. In some embodiments, antibodies are used in combination with the effector cell population described herein by simultaneous or sequential administration to a subject. In other embodiments, such antibodies or fragments thereof may be expressed by effector cells by genetically engineering iPSCs using exogenous polynucleotide sequences encoding said antibody or fragments thereof and guiding the differentiation of the engineered iPSCs. In some embodiments, effector cells express exogenous CD16 variants, wherein the cytotoxicity of effector cells is enhanced by the antibody via ADCC. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, a tumor or virus-specific antigen activates the administered iPSC-derived effector cells to enhance their killing capacity. In some implementations, antibodies suitable as adjunctive therapeutic agents in combination with the administered iPSC-derived effector cells include, but are not limited to, anti-CD20 antibodies (e.g., rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obbituzumab), anti-EGFR antibodies (cetuximab, matuzumab, panitumumab, and necitumumab), and anti-HER2 antibodies (such as...). Trastuzumab or biosimilars, pertuzumab, 4B5, ertumaxomab, anti-PDL1 antibodies (avelumab, durvalumab, pembrolizumab, nivolumab, or atezolizumab), bispecific antibodies 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 (Herzumab) ® ), Trastuzumab-dttb (Ontruzant ® ).

[0175] In some embodiments, an initial dose of the monoclonal antibody is administered at an effective amount at the start time prior to the first cycle of iPSC-derived effector cell administration. In some embodiments, the start time is approximately 2 to 6 days prior to the first cycle of iPSC-derived effector cell administration. In some embodiments, the antibody used for combination therapy is rituximab, and the initial dose is approximately 300 mg / m² administered to the subject approximately 4 days prior to the first cycle of effector cell administration. 2 Approximately 450 mg / m 2 A single initial dose.

[0176] 9. Checkpoint inhibitors

[0177] Checkpoints are cellular molecules, typically cell surface molecules, that can suppress or downregulate immune responses when not inhibited. It is now clear that tumors select certain immune checkpoint pathways as the primary mechanism of immune resistance, particularly targeting T cells specific to tumor antigens. Immune checkpoint inhibitors (ICIs) are antagonists that reduce checkpoint gene expression or gene products, or decrease the activity of checkpoint molecules, thereby blocking inhibitory checkpoints and restoring immune system function. The development of checkpoint inhibitors targeting PD1 / PDL1 or CTLA4 has transformed the oncology landscape, with these agents providing long-term remission for multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapies, and relapse remains a significant problem. One aspect of this application provides a treatment method to overcome ICI resistance by including genomically engineered functionally derived cells, as described herein in combination therapies with ICIs. In some embodiments, the checkpoint inhibitor is used in combination with the effector cell population described herein, administered simultaneously or sequentially to the subject. In some other embodiments, the checkpoint inhibitor is expressed by effector cells by genetically engineering iPSCs using an exogenous polynucleotide sequence encoding the checkpoint inhibitor or a fragment or variant thereof and guiding the differentiation of the engineered iPSCs.

[0178] Some implementations of combination therapies with the provided derived T cells include at least one checkpoint inhibitor to target at least one checkpoint molecule. Checkpoint inhibitors suitable for combination therapies with derived T cells as described herein include, but are not limited to, PD1 (Pdcdl, CD279), PDL-1 (CD274), TIM3 (Havcr2), TIGIT (WUCAM and Vstm3), LAG3 (CD223), CTLA4 (CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), and A. 2A Antagonists of R, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor α (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIRs (e.g., 2DL1, 2DL2, 2DL3, 3DL1, and 3DL2).

[0179] In some embodiments, the antagonist that inhibits any of the above checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibody may be a murine antibody, a human antibody, a humanized antibody, a camel Ig, a single variable neoantigen receptor (VNAR), a shark heavy chain antibody (Ig NAR), a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab′, F(ab′)2, F(ab′)3, Fv, single-chain antigen-binding fragments (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), microantibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, single-domain antigen-binding fragments (sdAb, nanobodies), recombinant antibodies with only heavy chains (VHH), and other antibody fragments that maintain the binding specificity of the entire antibody, which may be more cost-effective to produce, easier to use, or more sensitive than the entire antibody. In some embodiments, the one, two, three, or more checkpoint inhibitors include at least one of pembrolizumab, nivolumab or atezolizumab (anti-PD1 / anti-PDL1 mAb), avelumab (anti-PDL1 mAb), durvalumab (anti-PDL1 mAb), and any derivatives, functional equivalents, or biosimilars thereof.

[0180] In some embodiments, the ICI used in combination therapy is pembrolizumab administered at a dose of about 400 mg every three weeks (Q3W) or every six weeks (Q6W). In some embodiments, the ICI used in combination therapy is nivolumab administered at the following doses every two weeks (Q2W) or every four weeks (Q4W): (a) about 240 mg when administered at Q2W; or (b) about 480 mg when administered at Q4W. In some embodiments, the ICI used in combination therapy is atezolizumab administered at the following doses every Q2W, Q3W, or Q4W: (a) about 840 mg when administered at Q2W; (b) about 1200 mg when administered at Q3W; or (c) about 1680 mg when administered at Q4W.

[0181] In some embodiments, an initial dose of the ICI is administered at an effective amount prior to the first cycle of iPSC-derived effector cell administration. In some embodiments, the initiation time is approximately 4 to 10 days prior to the first cycle of iPSC-derived effector cell administration. In some embodiments, the ICI used for combination therapy is pembrolizumab and is administered at an initial dose of approximately 200 mg to approximately 400 mg. In some embodiments, the ICI used for combination therapy is nivolumab and is administered at an initial dose of approximately 240 mg to approximately 480 mg. In some embodiments, the ICI used for combination therapy is atezolizumab and is administered at an initial dose of approximately 840 mg to approximately 1680 mg.

[0182] II. Therapeutic use of derived immune cells for solid tumors

[0183] Prior to this disclosure, the therapeutic benefits of CAR T-cell therapy in solid tumors have been modest. A challenge to the clinical application of CAR T-cell therapy in solid tumors is the heterogeneity of putative solid tumor target antigen expression in normal tissues, which increases the risk of target localization and detumescent toxicity, especially when there is no differential expression between tumor and normal tissues. Therefore, efforts to maximize the therapeutic index of CAR T-cells in solid tumors have focused on targeting single antigens and / or combinations of antigens, often caused by mutated oncogenic drivers, which are expressed differently on tumor cells than in normal tissues (Sterner and Sterner, 2021). Other challenges specific to solid tumor-specific CAR T-cell therapy, such as facilitating the delivery of T-cell products to the tumor and overcoming the suppressive tumor immune microenvironment, may require further modification of the T-cell products.

[0184] Mutations driving altered expression and / or signaling of the ErbB receptor tyrosine kinase family (including epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2) / neu (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4)) are considered oncogenic drivers in a wide 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, new treatments to improve survival remain scarce.

[0185] The efficacy of current HER2 and EGFR-targeting agents is often limited by tumor resistance mechanisms and expression levels that are insufficient to generate meaningful clinical benefit. For example, the clinical activity of HER2-targeting agents is primarily based on established diagnostic testing criteria demonstrating sufficient levels of HER2 expression for prediction (Bartley et al., 2016; Wolff et al., 2018). Similarly, tumor-specific factors influence the clinical benefit of EGFR-targeting agents. For instance, the efficacy of EGFR-targeting monoclonal antibodies (mAbs) in colorectal cancer (CRC) has been limited to K-RAS wild-type tumors (Erbitux USPI; Vectibix USPI) and is not correlated with EGFR expression (Hecht et al., 2010). In non-small cell lung cancer (NSCLC), EGFR-targeting agents provide clinical benefit only in about one-third of patients whose tumors contain EGFR driver mutations (Zhang et al., 2016). Furthermore, treatment options remain limited after progression due to the development of tumor resistance.

[0186] Recent studies have shown that the clinical activity of HER2 and EGFR-targeted therapies in tumors expressing HER2 and / or EGFR is relatively unknown. For example, HER2 expression has been demonstrated in a subset of various solid malignancies, including salivary gland and ductal carcinoma, as well as ovarian, pancreatic, endometrial, cervical, bladder, and bile duct / gallbladder cancers (Dumbrava et al., 2019; Omar et al., 2015). Furthermore, advanced EGFR-mutant lung cancer that has progressed after prior treatment with tyrosine kinase inhibitors (TKIs) can acquire resistance through HER2 gene amplification (Yu et al., 2013), making HER2 a targetable tumor antigen.

[0187] Unlike lineage-specific surface proteins (e.g., CD19 and BCMA, which are highly selective targets in hematologic malignancies), surface proteins including EGFR and HER2 are expressed in both normal and cancer cells, leading to the potential for targeted, detumescent toxicity (Sterner et al., 2021). For example, reported toxicities associated with HER2-targeted therapy, such as cardiomyopathy and heart failure (Herceptin USPI; Kadcyla USPI; Perjeta USPI), diarrhea (Nerlynx USPI; Tukysa USPI), and interstitial lung disease (ILD) and pneumonia (Enhertu USPI), are direct consequences of low levels of HER2 expression in normal tissues.

[0188] Another important consideration for the clinical application of CAR T-cell therapy in solid tumors involves the need to create an “immune space” for the survival and expansion of CAR T-cell products. Approved CAR T-cell therapies for B-cell malignancies are often administered in combination with lymphocyte elimination regimens, including cyclophosphamide (CY) and fludarabine (FLU) or bendamustine, which, in addition to facilitating CAR T-cell survival and expansion, also provide antitumor activity. While early clinical activity has been demonstrated, the optimal chemotherapy regimen for combination with CAR T-cells remains unclear (Bechman and Maher, 2021; Owen et al., 2023), and a deeper understanding of the ability of chemotherapy and other agents to enhance the pharmacokinetics (PK) and clinical activity of CAR T-cells in solid tumors is needed. This article provides regimens for providing improved therapeutic benefit in the treatment of solid tumors.

[0189] Treatment using hematopoietic lineage cells derived from the embodiments disclosed herein or the compositions provided herein may be administered after symptom onset or to prevent relapse. The terms “treatment”, “treating,” etc., are generally used herein to mean achieving the desired pharmacological and / or physiological effect. For diseases and / or adverse effects attributable to said diseases, said effects may be preventative in terms of complete or partial prevention of the disease, and / or therapeutic in terms of partial or complete cure. As used herein, “treatment” encompasses any intervention on a subject’s disease and includes: preventing the onset of the disease in subjects who may be susceptible but have not yet been diagnosed with it; inhibiting the disease (e.g., blocking its development); or alleviating the disease (e.g., causing disease regression, or re-inducing a disease response to therapy). Treatment of developing diseases is also of interest, where treatment stabilizes or reduces undesirable clinical symptoms in patients. In some embodiments, the subject requiring treatment suffers from a disease, condition, and / or lesion for which at least one related symptom can be contained, improved, and / or resolved by cell therapy.

[0190] As described in this article, FT825 refers to a human induced pluripotent stem cell (iPSC)-derived CAR T-cell therapy that incorporates multiple engineered modalities designed to optimize antitumor activity against a variety of oncogenic targets, including cancers expressing HER2. In addition to exhibiting preferential targeting of HER2 on tumor cells relative to normal cells, FT825 also incorporates an IL7 / IL7 receptor fusion protein (IL7RF) designed to enhance cell survival, CXC motif chemokine receptor 2 (CXCR2) designed to optimize cell transport to tumors, transforming growth factor β signal redirection receptor (TGFβ-SRR) designed to overcome the immunosuppressive tumor microenvironment, and a high-affinity, non-cleavable CD16 (hnCD16) for multi-antigen targeting via antibody-dependent cytotoxicity (ADCC) through combination with mAbs (including but not limited to cetuximab, an anti-EGFR monoclonal antibody). hnCD16 enhances the activity of FT825 when combined with therapeutic antibodies targeting tumor cells with minimal HER2 expression, supporting clinical application against tumors unrelated to HER2 status. A phase 1 study of FT825 in participants with advanced solid tumors expressing HER2 was designed to utilize the functional properties of FT825 in a treatment regimen aimed at overcoming barriers to effective immunocellular therapy in solid tumors.

[0191] The Phase 1 study described in this article evaluated the clinical activity of FT825 in participants with HER2-expressing tumors where HER2-targeting agents have known clinical activity (e.g., breast cancer, esophageal and gastric cancer, gastroesophageal junction [GEJ] adenocarcinoma, HER2-mutant NSCLC, and CRC), as well as in HER2-expressing tumors for which no currently approved HER2-targeting agents exist (such as salivary gland cancer and endometrial cancer). Furthermore, this study provides a preliminary evaluation of the combination of FT825 and cetuximab in participants with EGFR-expressing tumors and tumors co-expressing EGFR and HER2. The study also evaluated docetaxel and low-dose cisplatin chemotherapy with FT825 as a second-cycle retreatment in participants whose disease progressed after the first cycle of CY / FLU chemotherapy.

[0192] In some embodiments, the FT825 active pharmaceutical ingredient comprises allogeneic T cells derived from T cell receptor (TCR) and CD38 knockout iPSC lines, which express HER2-targeting CARs, IL7RF, TGFβ-SRR, hnCD16, and CXCR2. In some embodiments, manufacturing the drug substance from an iPSC master cell bank involves an iPSC expansion phase followed by differentiation into T cells and T cell expansion, which may include sequential co-culturing with two separate irradiated feeder cell lines. Cells harvested after T cell expansion can then be washed and designated as the drug substance. In some embodiments, FT825 cells are designed to mediate antitumor activity primarily via: cytotoxicity of HER2-expressing target cells after recognition by a HER2-targeted CAR; and ADCC, through which drug product cells expressing hnCD16 actively lyse target cells whose membrane surface antigens have been bound to a specific mAb administered in combination with the drug product cells.

[0193] In some implementations, FT825 cells support antitumor activity through multiple pathways. For example, FT825 expresses IL7RF containing IL7 covalently linked to IL7Rα via a flexible linker, IL7RF being engineered to enhance T cell survival and retention; FT825 expresses TGFβ-SRR, where the extracellular and transmembrane domains of TGFβR2 are fused with an activated intracellular domain from IL18R, thereby achieving sustained antitumor activity in the presence of TGFβ; and FT825 expresses CXCR2 to enhance specific migration to tumors expressing CXCR2 ligands. CXCR2 ligands, including CXCL1, CXCL2, CXCL5, and CXCL8 / IL-8, are enriched in the tumor microenvironment of various tumor types, including breast, stomach, lung, and colorectal, and are associated with adverse patient outcomes. Furthermore, disruption of the CD38 gene enhances metabolic adaptation and further prevents the binding of anti-CD38 mAb, thus preventing self-destructive depletion of CD38 knockout effector cells induced by anti-CD38 mAb. This can facilitate the use of anti-CD38 mAb in tumor-targeting and lymphomodulation scenarios. Additionally, CD38 is a validated locus for genetic engineering in iPSCs, supporting high and uniform gene expression of the inserted transgene. In various embodiments, the regimen and dosage design of the cell product takes into account drug efficacy and risk reduction. In some embodiments, the regimen and dosage design is targeted at a patient subgroup. In some embodiments, the regimen and dosage design is targeted at a subset of indications, diseases, or conditions.

[0194] Specific conditions that may involve engineered cell immunotherapy products typically include, but are not limited to, new malignancies, new or worsening neurological disorders, new or worsening autoimmune diseases, rheumatic diseases, or new hematologic disorders. Subjects may undergo safety monitoring during treatment, including assessment of the nature, frequency, and severity of adverse events (AEs).

[0195] An adverse event (AE) is any adverse medical event in a patient or clinical study subject that is temporarily associated with the use of the study treatment, whether or not it is considered to be related to the study treatment. Therefore, an AE can be any unfavorable and unexpected symptom (including abnormal laboratory findings), condition, or illness (new or worsening) that is temporarily associated with the use of a medical product, whether or not it is related to the medical product.

[0196] Events that meet the definition of an 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, radiographic scans, vital sign measurements), including those that deteriorate from baseline and are considered clinically significant in the investigator's medical and scientific judgment (i.e., unrelated to the progression of the underlying disease); (ii) exacerbation of a pre-existing chronic or intermittent condition, including an increase in the frequency and / or intensity of the condition; (iii) a new condition detected or diagnosed after administration of the study treatment, even if it may have existed before the start of the study; (iv) signs, symptoms, or clinical sequelae of suspected drug-drug interactions; and (v) signs, symptoms, or clinical sequelae of suspected overdose of the study treatment or concomitant drug. Overdose itself is not considered an AE / SAE (serious adverse event).

[0197] Acute anaphylactic / infusion reactions can occur with any treatment, including those using CY, FLU, bendamustine, docetaxel, cisplatin, and mAb. Subjects should be closely monitored for the occurrence of acute anaphylactic / allergic infusion reactions, such as chills and shivering, rash, urticaria, hypotension, dyspnea, and angioedema, during and after infusion. Acute anaphylactic / infusion reactions can also be a manifestation of the immunogenicity of allogeneic cell products.

[0198] Evidence on monitoring, assessing and managing the immunogenicity of FT825 and its clinical impact during treatment is crucial, as potential FT825-induced immune responses may only manifest clinically, for example, as infusion-related reactions of varying severity.

[0199] FT825 is formulated in DMSO for cryopreservation. DMSO side effects and symptoms are monitored, assessed, and managed during treatment. DMSO-related side effects are commonly associated with histamine release and include cough, flushing, rash, chest tightness and wheezing, nausea and vomiting, and cardiovascular instability. In some implementations, methods include slowing the infusion rate and / or administering an antihistamine.

[0200] FT825 is extensively tested during manufacturing to minimize the potential risk of disease transmission. However, as a human-derived cell therapy, the cell product may come into contact with animal-derived reagents during handling. Therefore, close monitoring and management of infectious diseases and / or disease pathogens transmitted by known or unknown pathogens during treatment are essential.

[0201] Cytokine release syndrome (CRS) is defined as a supraphysiological response following any immunotherapy that results in the activation or conjugation of endogenous or infused immune effector cells. CRS is a well-defined syndrome following treatment with autologous and allogeneic CAR T-cell therapy that 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, stiffness, headache, asthenia, fatigue, arthralgia, nausea, and vomiting) signs and symptoms. To consistently characterize its severity, CRS is defined and graded according to the ASCO / ASTCT CRS consensus grading. Signs and symptoms of CRS are not specific to CRS. Clinical symptoms not considered associated with FT825, such as bacteremia and other serious infections, are not reported as CRS.

[0202] Tumor lysis syndrome (TLS) is a potentially fatal risk associated with antitumor therapy for hematologic malignancies and solid tumors, especially in cases of large tumor burden. TLS has been reported to occur within 7 days after chemotherapy in various solid tumor settings, with numerous cases reported in patients with gynecologic cancers. One case of fatal metabolic syndrome compatible with TLS was reported in an ovarian cancer patient following NK cell therapy 5 days after CY administration. Symptoms of TLS include nausea, vomiting, diarrhea, muscle cramps or twitching, weakness, numbness or tingling, fatigue, decreased urination, irregular heartbeat, restlessness, irritability, delirium, hallucinations, and seizures. TLS is composed of abnormal laboratory changes, including hyperuricemia, hyperkalemia, hyperphosphatemia, and hypocalcemia. In some implementations, methods include the prevention and management of TLS, such as those known in the art.

[0203] Neurotoxicity caused by immunotherapy is known as immune effector cell-associated neurotoxicity syndrome, or ICANS, defined as a condition characterized by a pathological process in the CNS following any immunotherapy involving the activation or conjugation of endogenous or infused immune effector cells. ICANS has been reported with CAR T-cell therapies and bispecific antibodies such as blinatumomab. ® The exact mechanisms of toxicity in these settings are unknown, but generally improve with treatment discontinuation and corticosteroids. Central nervous system toxicity following CD19 CAR T-cell therapy is characterized by encephalopathy, confusion, delirium, aphasia, lethargy, and seizures (Kymriah). ® USPI; Yescarta ® Cases of cerebral edema have also been reported with CAR-T therapy.

[0204] 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 assessment is performed based on the CIBMTR Acute GvHD Grading Scale to assign overall severity, and the method may include the management of GvHD, such as by methods known in the art.

[0205] Furthermore, when FT825 comes into contact with animal-derived cells (such as mouse cells) during the manufacturing process, it can be considered a xenograft product. In some embodiments, animal-derived cells serve as auxiliary materials in the manufacture of the pharmaceutical product but are not intended to be present in the pharmaceutical product. Risks of receiving a xenograft product may include, but are not limited to, developing infections from pathogens that may be associated with animal-derived cells, spreading these infectious pathogens to other people, and tumor growth. Animal cells that come into contact with FT825 cells during the manufacturing process may be derived from a master cell bank that has been extensively tested to mitigate these risks. In some embodiments, the clinical performance of the xenograft product in subjects is monitored throughout treatment, including long-term follow-up, in rare cases of relevant conditions or disease development.

[0206] There have been reports of adoptive cell therapies delivered with supportive medications such as cyclophosphamide (CY) and fludarabine (FLU) for conditioning, which can cause bone marrow suppression (neutropenia and / or thrombocytopenia), immunosuppression, infection, leukopenia, anemia, and in some cases, bone marrow failure. Hematologic cytopenia may be further exacerbated by other factors such as underlying diseases, comorbidities, and concomitant medications. Close monitoring of complete blood counts is necessary to monitor the development of cytopenia and infection. In some embodiments, methods include managing cytopenia and infection, including transfusion support, antimicrobial prophylaxis, and the use of growth factors, such as by methods known in the art.

[0207] Warnings and precautions associated with cyclophosphamide (CY) include: bone marrow suppression, immunosuppression, bone marrow failure, and infection; urethral and nephrotoxicity, including hemorrhagic cystitis, pyelonephritis, urethritis, and hematuria; cardiotoxicity, including myocarditis, myocardial pericarditis, pericardial effusion, arrhythmias, and congestive heart failure; pulmonary toxicity, including pneumonia, pulmonary fibrosis, and pulmonary venous occlusive disease leading to respiratory failure; secondary malignancies; venous occlusive liver disease; and embryotoxicity. The most frequently reported adverse reactions include neutropenia, febrile neutropenia, fever, alopecia, nausea, vomiting, and diarrhea. In some implementations, dose changes due to toxicity are considered during the treatment process.

[0208] Warnings and precautions associated with fludarabine (FLU) include severe myelosuppression, particularly anemia, thrombocytopenia, and neutropenia; transfusion-related GvHD; severe CNS toxicity; infections; renal insufficiency; TLS; and embryotoxicity. At a dose of 96 mg / m² 2 Severe CNS toxicity was observed in patients treated with FLU at doses of 25 mg / m² for 5 to 7 days. In patients with ≤0.2% of these patients treated at 25 mg / m², severe CNS toxicity was observed. 2 This toxicity was observed in patients treated with FLU at doses that were not specified. Adverse reactions that occurred in >30% of participants treated with FLU included bone marrow suppression (neutropenia, thrombocytopenia, and anemia), fever, infection, nausea and vomiting, fatigue, anorexia, cough, and weakness.

[0209] Warnings and precautions associated with bendamustine include bone marrow suppression, infection, infusion reactions and anaphylactic TLS; skin reactions, including rash, toxic skin reactions (such as Stevens-Johnson syndrome and toxic epidermal necrolysis) and bullous eruptions; other malignancies; and fetal harm.

[0210] Risks associated with docetaxel include secondary myelodysplastic syndromes or acute myeloid leukemia; skin reactions, including erythema and edema of the extremities followed by desquamation; neurological reactions, including paresthesia, hypoesthesia, and pain; asthenia; embryotoxicity; and common adverse reactions, including infections, neutropenia, anemia, febrile neutropenia, hypersensitivity, thrombocytopenia, neuropathy, taste disturbances, dyspnea, constipation, anorexia, nail disorders, fluid retention, asthenia, pain, nausea, diarrhea, vomiting, mucositis, alopecia, skin reactions, and myalgia.

[0211] Risks associated with cisplatin include hypersensitivity reactions that can lead to allergic reactions and death; cumulative ototoxicity; ocular toxicity, including optic neuritis, papilledema, and cortical blindness; secondary leukemia; embryotoxicity; and common adverse reactions include nephrotoxicity, peripheral neuropathy, myelosuppression, nausea, and vomiting.

[0212] In addition to the known and potential risks of FT825, CY, FLU, bendamustine, docetaxel, and cisplatin, other risks associated with combination therapy due to drug interactions include, but are not limited to, an increased frequency and / or severity of risks known to occur with mAbs. Therefore, when risks exist during treatment, evidence of toxicity of approved mAbs that may be affected by FT825 combination therapy in terms of frequency and severity can be collected and addressed.

[0213] In one aspect, this disclosure provides a method of treating a subject's cancer. In some embodiments, the method includes: (a) administering one or more doses of chemotherapy to the subject; and (b) administering one or more doses of an adoptive cell therapy product to the subject in a first effective amount; wherein the adoptive cell therapy product comprises engineered T lineage cells having (i) expression of HER2-CAR (chimeric antigen receptor), IL7 / IL7 receptor fusion protein (IL7RF), TGFβ signal transduction redirection receptor (TGFβ-SRR), CXC motif chemokine receptor 2 (CXCR2), and exogenous CD16; (ii) CD38 knockout; and (iii) T cell receptor (TCR) knockout; and wherein the cancer comprises HER2-positive cancer cells. In some embodiments, the method includes repeating the foregoing steps.

[0214] In some embodiments, the method further includes: (a) administering to the subject one or more doses of an additional chemotherapy that is the same as or different from the chemotherapy (e.g., after administering one or more doses of the adoptive cell therapy product at a first effective amount); and (b) administering to the subject one or more doses of the adoptive cell therapy product at a second effective amount that is the same as or different from the first effective amount (e.g., after administering the additional chemotherapy).

[0215] In some embodiments, the HER2-positive cancer cells contain HER2 expression, amplification, or mutation. In some embodiments, the method further includes administering an EGFR inhibitor to the subject; and wherein the cancer cells contain an EGFR mutation. In some embodiments, the EGFR inhibitor is administered before and / or after administration of the adoptive cell therapy product. In some embodiments, the EGFR inhibitor includes mateuzumab, panitumumab, or necrotizing murine acetaminophen. In some embodiments, the EGFR inhibitor includes cetuximab. In some embodiments, the chemotherapy includes: (a) one or both of cyclophosphamide (CY) and fludarabine (FLU); (b) bendamustine; or (c) one or both of docetaxel and cisplatin. In some embodiments, chemotherapy is administered one or more days before administration of the adoptive cell therapy product; optionally, chemotherapy is administered at least two, three, four, or five days before administration of the adoptive cell therapy product. In some embodiments, chemotherapy (a) comprises (i) a daily dose of about 250 mg / m². 2 Approximately 600 mg / m 2 (ii) Cyclophosphamide; and (ii) a daily dose of approximately 20 mg / m 2 Approximately 40 mg / m 2 (a) Fludarabine; and (b) Administered for 3 consecutive days starting approximately 4–6 days prior to administration of the adoptive cell therapy product. In some embodiments, chemotherapy includes bendamustine and administered for 2 consecutive days starting approximately 4–6 days prior to administration of the adoptive cell therapy product at approximately 30 mg / m². 2 Approximately 100 mg / m 2 The daily dose is administered.

[0216] In some embodiments, the engineered T lineage cells are derived from engineered induced pluripotent stem cells (iPSCs) containing polynucleotides encoding HER2-CAR, IL7RF, TGFβ-SRR, CXCR2, exogenous CD16, TCR knockout, and CD38 knockout. In some embodiments, the first and / or second effective amount of the adoptive cell therapy product is about 5 × 10⁻⁶. 7 Approximately 3 × 10 9 The adoptive cell lineage comprises engineered T-lineage cells, and optionally the number of engineered T-lineage cells is increased based on the dose-limiting toxicity rate of the amount of adoptive cell therapy administered. In some embodiments, the first effective amount and / or the second effective amount of the adoptive cell therapy product comprises about 5 × 10⁻⁶ cells. 7 One, approximately 1×10 8 1, approximately 3 × 10 8 1, approximately 9 x 108 One or approximately 2 × 10 9 The adoptive cell therapy product contains engineered T lineage cells. In some embodiments, the number of engineered T lineage cells in the adoptive cell therapy product is increased up to 3-fold at a dose-limiting toxicity (DLT) rate of 25%-35% or lower. In some embodiments, the adoptive cell therapy product is: (i) allogeneic; (ii) (a) administered via intravenous infusion, and / or (b) administered in an outpatient setting; and / or (iii) cryopreserved and then thawed prior to administration. In some embodiments, the adoptive cell therapy product is FT825.

[0217] In some implementations, the subject: (i) has not received prior cancer treatment; or (ii) has received one or more prior HER2-targeted therapies; and / or one or more prior EGFR-targeted therapies. In some implementations, the cancer includes breast cancer, esophageal cancer, gastroesophageal junction (GEJ) adenocarcinoma, non-small cell lung cancer (NSCLC), colorectal cancer (CRC), or head and neck squamous cell carcinoma (HNSCC).

[0218] In some embodiments, the method further includes detecting and comparing one or more of the following after administration of a first effective dose of adoptive cell therapy: (a) the presence of engineered immune cells in the subject's tumor; (b) disease protein markers in the subject's serum; (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 one of (a)-(e) is used to assess tumor burden, tumor immunobiology, and / or tumor treatment response to determine the efficacy of the multi-dose targeted adoptive cell therapy. In some embodiments, the subject has a complete response (CR), partial response (PR), or stable disease (SD) after receiving the adoptive cell therapy product.

[0219] In one aspect, a method of treating cancer includes: (a) administering one or more first doses of an EGFR inhibitor to a subject; (b) administering one or more doses of an adoptive cell therapy product to a subject; and (c) administering one or more second doses of an EGFR inhibitor to a subject; wherein the adoptive cell therapy product comprises engineered T lineage cells expressing (i) HER2-CAR (chimeric antigen receptor), IL7 / IL7 receptor fusion protein (IL7RF), TGFβ signaling redirection receptor (TGFβ-SRR), CXC motif chemokine receptor 2 (CXCR2), and exogenous CD16; (ii) CD38 knockout; and (iii) T cell receptor (TCR) knockout; and wherein the cancer comprises cancer cells expressing EGFR. In some embodiments, the method includes repeating the foregoing steps. In some embodiments, the method includes administering one or more doses of chemotherapy to a subject prior to administering the EGFR inhibitor. In some embodiments, the EGFR inhibitor includes cetuximab, mateuzumab, panitumumab, or necstizumab. In some implementations, EGFR inhibitors include cetuximab. In some implementations, the cancer cells expressing EGFR also express HER2. In some implementations, chemotherapy includes: (a) one or both of cyclophosphamide (CY) and fludarabine (FLU); (b) bendamustine; or (c) one or both of docetaxel and cisplatin.

[0220] In one aspect, this disclosure provides a kit for cancer treatment. In some embodiments, the kit comprises FT825, one or more chemotherapy agents, and optionally an EGFR inhibitor; wherein (a) the FT825 comprises engineered T lineage cells expressing: (i) HER2-CAR (chimeric antigen receptor), IL7 / IL7 receptor fusion protein (IL7RF), TGFβ signaling redirection receptor (TGFβ-SRR), CXC motif chemokine receptor 2 (CXCR2), and exogenous CD16; (ii) CD38 knockout; and (iii) T cell receptor (TCR) knockout; (b) the chemotherapy includes: (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 includes cetuximab, mateuzumab, panitumumab, or nectocinumab. In some implementations, the cancer is HER2 positive and / or EGFR positive.

[0221] Example

[0222] The following examples are provided for illustrative purposes and not for limitation.

[0223] Example 1 - FT825 Study Design

[0224] The FT825 active pharmaceutical ingredient used in this embodiment comprises allogeneic T cells derived from the T cell receptor (TCR) and CD38 knockout iPSC lineage, expressing HER2-targeting CAR, IL7RF, TGFβ-SRR, hnCD16, and CXCR2. Manufacturing the drug substance from the iPSC master cell bank involves an iPSC expansion phase, followed by differentiation into T cells and T cell expansion. Cells harvested after T cell expansion are then washed and designated as the drug substance. The composition of FT825 is homogeneous.

[0225] As described in this article, the study was designed to evaluate the safety, pharmacokinetic (PK) and antitumor activity of FT825 in participants with or without cetuximab following chemotherapy. The study was also designed to determine the recommended phase 2 dose (RP2D) and schedule for FT825 in participants with advanced solid tumors expressing HER2 and / or EGFR following chemotherapy, with or without cetuximab. Furthermore, the study was designed to assess the association between baseline clinical and tumor characteristics and the safety and efficacy of FT825; to correlate FT825 PK / pharmacodynamic parameters with the safety and efficacy of FT825; and to evaluate pharmacodynamic biomarkers of FT825 following CY / FLU or bendamustine, with or without cetuximab. In addition, this study provides an opportunity to evaluate the safety, tolerability, and antitumor activity of FT825; and to characterize the PK and pharmacodynamic biomarkers of FT825 with or without cetuximab after docetaxel / cisplatin (cycle 2 retreatment).

[0226] The first cycle of the study had two options. Option A: In participants with advanced solid tumors expressing HER2, a single dose of FT825 was administered after chemotherapy with CY / FLU or bendamustine. Option B: In participants with advanced solid tumors expressing EGFR or co-expressing EGFR and HER2, a single dose of FT825 was administered after chemotherapy with CY / FLU or bendamustine, followed by chemotherapy in combination with cetuximab.

[0227] During cycle 1, participants received CY / FLU chemotherapy prior to the FT825 infusion on day 1. Bendamustine may be considered as an alternative to CY / FLU if CY or FLU is unavailable or if the participant may be unable to tolerate CY or FLU.

[0228] Participants were enrolled in two phases: a dose escalation phase and a dose expansion phase. After safety and tolerability were assessed in the dose escalation phase, the dose expansion phase further evaluated the safety and activity of FT825 in a specific indication cohort.

[0229] In one implementation, treatment with FT825 comprises a 29-day treatment cycle. In some implementations, the 29-day treatment cycle follows chemotherapy containing CY / FLU or bendamustine. Figure 1 As shown, chemotherapy was initiated on day -5 (i.e., 5 days prior to the administration of FT825 on day 1), followed by the administration of FT825 on day 1. The dose-limiting toxicity (DLT) assessment period during dose escalation was from day 1 to day 29. Upon completion of the treatment cycle (i.e., after day 29), participants entered the treatment-to-follow-up (PTFU) period, where they were regularly monitored for new adverse events and / or resolution of treatment-emergent adverse events (TEAEs) and exploratory assessments. Eligible participants who experienced disease progression (PD) during PTFU and received a second cycle of retreatment received the second cycle of study intervention. The second cycle of retreatment could be administered at any time during the post-treatment retreatment period after day 29.

[0230] For each regimen, a modified toxicity probability interval (mTPI)-2 dose escalation design was used to evaluate progressively higher FT825 dose levels. The FT825 dose escalation was performed as follows: (1) Dose escalation began with regimen A, dose level (DL) 1; (2) Dose escalation for regimen B was initiated based on clearance at at least one dose level in regimen A and could begin from the highest clearance dose level in regimen A; (3) Dose escalation for regimens A and B could be performed independently until the MTD / maximum evaluable dose (MAD) for each regimen was determined; (4) For regimens A and B, if DL1 was intolerable or exceeded the MTD, DL0 for regimen A or regimen B could be explored.

[0231] The initial FT825 dose levels during dose escalation were as follows: (1) DL0: 5 × 10 7 (1) Cells / dose (evaluate if DL1 is intolerable or exceeds MTD); (2) DL1: 1×10 8 Cells / dose. If the initial dose level does not exceed the MTD, test successive dose levels; not exceeding 3 times (±15% variance) of the highest clearance dose level. If the evaluated dose level exceeds the MTD, intermediate dose levels can be explored. In the absence of dose-limiting toxicities (DLTs), further dose levels can be explored based on observed safety, tolerability, activity, PK, and pharmacodynamic data.

[0232] The FT825 dose for each regimen administered in dose expansion was determined based on clinical and available PK and pharmacodynamic data from the corresponding dose escalation phases, and did not exceed the MTD or MAD. Enrollment in the dose expansion cohort began once a given dose level had been cleared in dose escalation. One or more dose levels that had been cleared in dose escalation in regimen A and / or regimen B, as assessed by DLT, could be evaluated in dose expansion to further define clinical activity. The RP2D for each regimen was determined using combined safety and efficacy analyses from both dose escalation and dose expansion. The dose amplification scheme A 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); low HER2 breast cancer (IHC 1+ or IHC 2+ / ISH negative); low HER2 gastric / GEJ cancer (IHC 1+ or IHC 2+ / ISH negative); HER2-mutant NSCLC; and other HER2-expressing cancers (including salivary gland cancer and endometrial cancer) with HER2 IHC ≥1+ or HER2 amplification. The dose amplification regimen B includes: HNSCC with HER2 IHC ≥1+ or HER2 amplification; CRC with EGFR IHC ≥1+ or KRAS wild-type or BRAFV600E mutation, and HER2 IHC ≥1+ or HER2 amplification; and NSCLC with EGFR IHC ≥1+ or EGFR mutation and HER2 IHC ≥1+ or HER2 amplification.

[0233] The goal of chemotherapy prior to FT825 administration is to create an immune environment conducive to the survival, amplification, and function of FT825. Although the mechanism is not fully understood, the combination of CY and FLU induces cytokines that promote homeostatic proliferation and eliminates regulatory immune cells and other immune system elements that compete for homeostatic cytokines.

[0234] Bendamustine is an alkylating agent with a well-characterized and favorable safety profile, administered as a component of an immunochemotherapy regimen over multiple treatment cycles for the treatment of B-cell malignancies. In subjects experiencing severe hemorrhagic cystitis, prior CY therapy, or whose tumors have shown resistance to previous CY-containing regimens, bendamustine is considered an alternative to CY / FLU therapy prior to tisagenlecleucel administration.

[0235] Treatment of patients with regimen A was designed to evaluate the clinical activity of FT825 in patients with HER2-expressing tumors, particularly regarding the clinical activity of HER2-CARs. One aspect of the study was to investigate the effect of FT825 containing a HER2-targeting CAR on HER2-expressing tumor cells compared to normal cells expressing HER2, and compared to preclinical observations of antitumor activity against tumor cell lines expressing a broad range of HER2, including low-HER2.

[0236] FT825 was incorporated into hnCD16 to broaden its antitumor targeting by achieving multi-antigen targeting via ADCC through combination with mAbs. Another aspect of this study was the clinical evaluation of the combination of FT825 and cetuximab in EGFR-expressing tumors with varying HER2 co-expression levels (such as HNSCC, NSCLC, and CRC), given the preclinically enhanced antitumor activity of FT825 in combination with cetuximab against tumor cell lines with low HER2 expression but high EGFR expression. Participants were enrolled and received regimen B at different times during the study. To assess the safety of the FT825 and cetuximab combination, participants with known EGFR-positive tumors, independent of known prior HER2 status (retrospective characterization), were enrolled during the dose escalation phase of the study. During the dose expansion phase of the study, participants with tumors showing documented expression of both EGFR and HER2 were enrolled.

[0237] In regimen A, the starting dose of FT825 is 1×10⁻⁶ after a chemotherapy cycle, such as CY / FLU or bendamustine in cycle 1. 8 Cells / dose ( Figure 2 FT825 dose escalation is performed by increasing relative dose levels rather than by pre-specified dose levels, with escalation intervals not exceeding three times (±15% variance) of the highest clearance dose level. The relative dose intervals are designed to allow for flexibility in escalating doses in smaller increments as needed.

[0238] Figure 2 Exemplary dose escalation and dose amplification protocols are illustrated. As described above, dose level 1 (DL1) is 1 × 10⁻⁶. 8 Cells / dose. Dose escalation can continue with ≤3 × the previously cleared dose (±15% variance). DL0 is 5 × 10⁻⁶. 7 Cells / dose (evaluation is performed if DL1 exceeds MTD). Dose escalation begins with Protocol A, DL1. The designation of DL1 through DL4 is for illustrative purposes only.

[0239] Example 2 - FT825 Second Cycle Retreatment

[0240] Based on the safety, tolerability, and clinical response of the treatment in cycle 1, participants may be considered for an additional cycle of retreatment (cycle 2 retreatment), in which docetaxel and cisplatin replace CY / FLU as pretreatment chemotherapy.

[0241] FT825 was administered and its activity monitored according to the same schedule as in the first cycle. Figure 3 An exemplary treatment regimen for a second cycle of retreatment is shown.

[0242] The reasons why existing CAR T-cell therapies fail to consistently achieve deep and durable responses are unknown. Unlike hematologic malignancies, CY / FLU and bendamustine are not widely used to treat solid tumors due to a lack of robust antitumor activity. Combinations of taxanes and platinum-based agents such as docetaxel and cisplatin are active antitumor agents and have been shown to induce positive immunomodulatory effects to facilitate T-cell infiltration into the solid tumor microenvironment.

[0243] This study tested the combination of docetaxel and cisplatin (docetaxel / cisplatin) with FT825 as a second-cycle retreatment in patients whose disease had progressed after initial (cycle 1) treatment with either CY / FLU or bendamustine and FT825. In addition to evaluating the safety and tolerability of the FT825 combination with docetaxel / cisplatin, the ability of different chemotherapy regimens combined with FT825 to re-induce objective responses was also tested. Finally, the second-cycle retreatment with docetaxel / cisplatin and FT825 enabled intra-patient comparisons and pharmacodynamic assessments of FT825 pharmacokinetics between cycles 1 and 2. Another objective was to evaluate second-cycle therapy for improving patient outcomes in the treatment of solid tumors.

[0244] Participants were selected for a second cycle of retreatment if they met the following criteria: (1) initially confirmed disease stability (SD), partial response (PR), or complete remission (CR), followed by disease progression (PD) as demonstrated by subsequent oncology assessment according to RECIST, v1.1; (2) non-hematologic adverse events (AEs) regressed from grade 2-3 to ≤ grade 1 or baseline grade (whichever is higher), excluding alopecia and dermatological changes such as grade 2 dry skin and / or rash; and (3) Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.

[0245] Example 3 - Research Groups for Each Research Cohort

[0246] Based on HER2, EGFR, KRAS, and BRAF testing, patients selected for the treatment described herein have histopathologically or cytologically confirmed locally advanced or metastatic cancer, as determined by assays approved by the Clinical Laboratory Improvement Amendments (CLIA) or otherwise appropriately validated.

[0247] For treatments including a single dose of FT825 (Regimen A) followed by chemotherapy with dose escalation, suitable patients include those who: (a) HER2-positive breast cancer and gastric / GEJ cancer, defined in this embodiment as IHC 3+, or IHC 2+ and ISH positive, according to established guidelines (American Society of Clinical Oncology [ASCO] / College of American Pathologists [CAP] Breast Cancer Guidelines; CAP / American Society of Clinical Pathologists [ASCP] / ASCO Gastric / GEJ Cancer Guidelines); or (b) NSCLC with a HER2 mutation, identified from tissue (fresh or archived tissue) or plasma (ctDNA); or (c) HER2-expressing cancers other than breast cancer, gastric / GEJ cancer, or NSCLC, defined by IHC ≥2+ or a HER2 / CEP17 ratio ≥2.

[0248] For treatments including administration of a single dose of FT825 followed by dose escalation after chemotherapy, suitable patients include those who: (a) HER2-positive breast cancer and gastric / GEJ cancer, defined in this embodiment as IHC 3+, or IHC 2+ and ISH-positive; (b) low-HER2 breast cancer and gastric / GEJ cancer, defined in this embodiment as IHC 1+, or IHC 2+ / ISH-negative; (c) NSCLC with HER2 mutations, identified from tissue (fresh or archived tissue) or plasma (ctDNA), regardless of HER2 expression levels; or (d) HER2-expressing cancers other than breast cancer, gastric / GEJ cancer, or NSCLC, defined in this embodiment by IHC ≥1+ or a HER2 / CEP17 ratio ≥2.

[0249] For treatment including a single dose of FT825 and cetuximab followed by chemotherapy (Regimen B) with dose escalation, suitable patients include those with: (a) HNSCC with HER2 expression, amplification, or mutation; (b) NSCLC with HER2 expression, amplification, or mutation; and EGFR driver factor mutation; or (c) CRC with HER2 expression, amplification, or mutation; and any of the following—KRAS wild-type, EGFR ≥1+ as determined by IHC, or BRAF V600E mutation.

[0250] For treatment including a single dose of FT825 and cetuximab followed by dose amplification after chemotherapy, suitable patients include those who: (a) HNSCC, wherein HER2 expression is defined in this embodiment as ≥1+ by IHC or the HER2 / CEP17 ratio is ≥2; (b) CRC, wherein HER2 expression is defined in this embodiment as ≥1+ by IHC or the HER2 / CEP17 ratio is ≥2, and has any of the following: KRAS wild-type, EGFR ≥1+ as determined by IHC, or BRAFV600E mutation; or (c) NSCLC, wherein HER2 expression is defined in this embodiment as ≥1+ by IHC or the HER2 / CEP17 ratio is ≥2, or has a HER2 mutation identified from tissue or plasma (ctDNA); and an EGFR driver factor mutation.

[0251] Patients selected for treatment including regimen A also include those who: (a) have recurrent or advanced breast cancer with or without one or more HER2-targeted therapies; (b) have recurrent or advanced gastric or GEJ cancer with or without one or more HER2-targeted therapies; or (c) other than breast cancer, gastric or GEJ cancer, have recurrent or advanced HER2-expressing cancer with or without one or more lines of standard care.

[0252] Patients who choose to receive treatment including Option B also include those who: (a) have relapsed or progressed HNSCC with or without one or more prior lines of systemic therapy (including EGFR-targeted therapy); (b) have progressed NSCLC expressing EGFR driver mutations with or without at least one prior line of EGFR-targeted therapy; or (c) have relapsed or progressed CRC with or without at least one prior EGFR-based therapy and / or at least one prior HER2-based therapy.

[0253] Example 4 - Research Intervention and Companion Therapy

[0254] Study interventions are all pre-designated research and non-research medical products intended to be administered to study participants during the study period. These medical products include FT825 (designated as a research medical product (IMP)) and authorized adjunctive medical products (AxMPs) as non-IMPs, such as cetuximab, cyclophosphamide (CY), fludarabine (FLU), and bendamustine. Study interventions are defined according to Table 1 below:

[0255] Table 1: Research Intervention

[0256]

[0257] a In addition, participants who are eligible for a second cycle of retreatment but are ineligible for docetaxel / cisplatin may receive CY / FLU or bendamustine with the approval of the medical monitor; the same chemotherapy administered in the first cycle shall be administered together with the second cycle of retreatment.

[0258] b Pre-treatment medication and antiemetic therapy, as well as hydration, should be administered according to institutional care standards. Dosage or schedules for CY / FLU or bendamustine may be modified based on the approval of the medical monitor.

[0259] c Pre-treatment medication, antiemetic therapy, and hydration should be administered according to institutional care standards. In cases of renal insufficiency, dose reduction should be used as a guideline for palliative anticancer therapy (Krens et al., 2019). Cisplatin should not be administered if creatinine clearance is <50 mL / min. The dosing or schedule for docetaxel / cisplatin may be modified based on the approval of the medical monitor.

[0260] The FT825 drug product comprises allogeneic T cells derived from TCR and CD38 knockout iPSC lineages. These allogeneic T cells express HER2-targeting CAR, IL7RF, TGFβ-SRR, hnCD16, and CXCR2, as described in Example 1 above. Participants received an IV of 500 mL of saline immediately prior to CY administration, according to institutional standards. Additional IV of saline may be administered after CY administration following assessment of hydration status. Dosage adjustments may also be made based on body weight / creatinine levels. The drug is administered at 500 mg / mL on days -5, -4, and -3 of the treatment cycle. 2 The CY dose is administered via IV infusion for 3 consecutive days. The CY dose is calculated based on actual body weight (ABW). If ABW > 150% of ideal body weight (IBW), the dose is calculated using the adjusted body weight as follows:

[0261] Adjusted weight = IBW + 0.5(ABW - IBW)

[0262] On days -5, -4, and -3 of the treatment cycle, at 30 mg / m² 2 FLU was administered via intravenous infusion for 3 consecutive days at a dose of 90 mg / m². Dosage adjustments may be made based on body weight and / or renal function (e.g., as assessed by creatinine clearance). 2 Bendamustine was administered via IV infusion for two consecutive days (days -5 and -4). On day -5, it was administered at a dose of 60 mg / m². 2 Or 75mg / m 2 The dosage of docetaxel was administered via intravenous infusion. On days 5, 4, and 3, it was administered at a dose of 25 mg / m². 2(For renal clearance of 60 mL / min) or 19 mg / m 2 Cisplatin was administered via IV infusion at a dose (for renal clearance of 50 mL / min to 59 mL / min).

[0263] Cetuximab can be administered weekly (QW) or every two weeks (Q2W). For example, for QW dosing, cetuximab is administered at a dose of 400 mg / m². 2 The initial loading dose was administered via intravenous infusion over 120 minutes; subsequent doses were administered at 250 mg / m². 2 Administered weekly via 60-minute infusion. For Q2W dosing, cetuximab is administered at 500 mg / m². 2 Administered every 2 weeks via intravenous infusion over 120 minutes. Treatment may continue for up to 2 years or until disease progression or unacceptable toxicity. Premedication may include intravenous administration of a histamine-1 receptor antagonist 30-60 minutes before the first or subsequent doses (if necessary). Prophylaxis for EGFR antibody-associated rash may also be provided.

[0264] Example 5 - Modification of Dosage and Schedule for the Study

[0265] Dosage and schedule adjustments for CY / FLU can be made. For participants with moderately impaired renal function (creatinine clearance of 30 mL / min / 1.73 m²–70 mL / min / 1.73 m²), [further adjustments may be made]. 2 The FLU dose was reduced by 20%. It is not indicated for patients with severely impaired renal function (creatinine clearance <30 mL / min / 1.73 mcg). 2 Participants were given FLU.

[0266] For participants who have completed the first treatment cycle (Cycle 1) but are still in the process of recovering from adverse events (AEs) and proceeding directly to Cycle 2 from exclusion, additional weekly monitoring visits will be performed until the criteria for starting Cycle 2 are met. The determination of the need for dose reduction is based on the CBC on day 29 and / or the presence of treatment-related AEs during Cycle 1.

[0267] If a grade 3 adverse event (AE) of neutropenia and thrombocytopenia considered at least potentially associated with CY / FLU does not recover to grade 2 (ANC ≥1000 and platelets ≥50,000) or baseline by day 29, and / or for participants experiencing significant treatment-related AEs (i.e., severe infections requiring hospitalization), where dose adjustment is deemed appropriate, the following dose modification shall be made: the daily dose of CY shall be reduced to 300 mg / m² on days -5, -4, and -3. 2 Furthermore, on days -5, -4, and -3, the daily dose of FLU was reduced to 25 mg / m². 2 .

[0268] Dosage and schedule modifications for bendamustine may be made. Bendamustine should not be administered to participants with renal or hepatic impairment as defined below: creatinine clearance <40 mL / min; AST or ALT of 2.5 to 10 × ULN and total bilirubin of 1.5 to 3 × ULN; or total bilirubin >3 × ULN. In cases of Grade 4 hematologic toxicity or clinically significant Grade ≥2 nonhematologic toxicity, bendamustine administration should be delayed. Bendamustine may be administered once nonhematologic toxicity has recovered to Grade ≤1 or baseline grade (whichever is higher), and / or blood counts have improved, e.g., ANC ≥1000 / µL, platelets ≥75000 / µL, or have reached baseline grade. Furthermore, for toxicities attributable to bendamustine, the following dosage modifications may be made: For Grade 4 toxicity, the bendamustine dose may be reduced to 60 mg / min on days -5 and -4 of cycle 2. 2 For non-hematologic toxicities: For ≥ grade 3 toxicities, reduce the bendamustine dose to 60 mg / m² on days -5 and -4 of cycle 2. 2 .

[0269] Example 6 - Tumor Response Assessment and Efficacy Analysis

[0270] Tumor response was assessed using the Solid Tumor Response Evaluation Criteria, version 1.1 (RECIST v1.1; Eisenhauer et al., 2009). Participants were categorized into the following tumor response categories: complete response (CR), partial response (PR), stable disease (SD), progressive disease (PD), or non-evaluable (NE).

[0271] Approximately 48 participants were enrolled in the dose escalation phases of both Protocol A and Protocol B to determine the maximum tolerated dose (MTD) using a modified toxicity probability interval (mTPI)-2 design, where 30% represented target toxicity. The sample size of 15 participants in each dose expansion cohort enabled the identification of early signs of promising clinical activity primarily based on objective response for subsequent Phase 2 expansion. Table 2 shows the precise 95% CI for true response rates ranging from 40% to 73%.

[0272] Table 2.

[0273]

[0274] Those skilled in the art will readily understand that the methods, compositions, and products described herein represent exemplary embodiments and are not intended to limit the scope of the invention. It will be apparent to those skilled in the art that various substitutions and modifications can be made to this disclosure without departing from the scope and spirit of the invention.

[0275] All patents and publications mentioned in this specification indicate the skill level of a person skilled in the art to which this disclosure pertains. All patents and publications are incorporated by reference to the extent that each individual publication is specifically and individually designated as incorporated by reference.

[0276] The disclosure described herein illustratively may be practiced in any manner where no particular element or limitation is specifically disclosed herein. Thus, for example, in each instance herein, any one of the terms “comprising,” “mainly consisting of,” and “consisting of” may be replaced by any of the other two terms. The terms and expressions used are descriptive rather than limiting, and their use is not intended to exclude any equivalents or portions thereof of the shown and described features, but rather to recognize that various modifications may be possible within the scope of the claimed disclosure. Therefore, it should be understood that while this disclosure has been particularly disclosed by way of preferred embodiments and optional features, modifications and variations may be made to the ideas disclosed herein by those skilled in the art, and such modifications and variations are considered to fall within the scope of the invention as defined by the appended claims.

Claims

1. A method for treating cancer in a subject, the method comprising: (a) Administering one or more doses of chemotherapy to the subject; as well as (b) Administer one or more doses of the adoptive cell therapy product to the subject in a first effective amount; The adoptive cell therapy product comprises engineered T lineage cells that express (i) HER2-CAR (chimeric antigen receptor), IL7 / IL7 receptor fusion protein (IL7RF), TGFβ signaling redirection receptor (TGFβ-SRR), CXC motif chemokine receptor 2 (CXCR2), and exogenous CD16; (ii) CD38 knockout; and (iii) T cell receptor (TCR) knockout; and The cancer described therein contains HER2-positive cancer cells.

2. The method according to claim 1, further comprising: (a) Administering one or more doses of additional chemotherapy to the subject, the additional chemotherapy being the same as or different from the chemotherapy; as well as (b) Administer one or more doses of the adoptive cell therapy product to the subject at a second effective amount that is the same as or different from the first effective amount.

3. The method of claim 1, wherein the HER2-positive cancer cells contain 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 contain an EGFR mutation.

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

6. The method of claim 4, wherein the EGFR inhibitor comprises mateuzumab, panitumumab, or nexituzumab.

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 the administration of the adoptive cell therapy product; optionally, the chemotherapy is administered at least two, three, four, or five days prior to the administration of the adoptive cell therapy product.

10. The method of claim 1, wherein the chemotherapy: (a) Includes (i) a daily dose of approximately 250 mg / m 2 Approximately 600 mg / m 2 (ii) Cyclophosphamide; and (ii) a daily dose of approximately 20 mg / m 2 Approximately 40 mg / m 2 Fludarabine; and (b) Begin application for 3 consecutive days approximately 4-6 days prior to application of the adoptive cell therapy product.

11. The method of claim 1, wherein the chemotherapy comprises bendamustine and administered for two consecutive days at approximately 30 mg / m², starting approximately 4-6 days prior to administration of the adoptive cell therapy product. 2 Approximately 100 mg / m 2 The daily dose is administered.

12. The method of claim 1, wherein the engineered T lineage cells are derived from engineered induced pluripotent stem cells (iPSCs), the iPSCs comprising polynucleotides encoding the HER2-CAR, polynucleotides encoding the IL7RF, polynucleotides encoding the TGFβ-SRR, polynucleotides encoding the CXCR2, polynucleotides encoding the exogenous CD16, TCR knockout, and CD38 knockout.

13. The method of claim 1, wherein the first effective amount and / or the second effective amount of the adoptive cell therapy product is about 5 × 10⁻⁶. 7 Approximately 3 × 10 9 The number of engineered T lineage cells is optionally increased based on the dose-limiting toxicity rate of the amount of adoptive cell therapy administered.

14. The method of claim 2, wherein the first effective amount and / or the second effective amount of the adoptive cell therapy product comprises about 5 × 10⁻⁶. 7 One, approximately 1×10 8 1, approximately 3 × 10 8 1, approximately 9 x 10 8 One or approximately 2 × 10 9 A modified T-lineage cell.

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

16. The method of claim 1, wherein the adoptive cell therapy product: (i) are all different species; (ii)(a) administered via intravenous infusion, and / or (b) administered in an outpatient setting; and / or (iii) Store frozen and then thaw before application.

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

18. The method of claim 1, wherein the subject: (i) No prior treatment for the cancer has been received; or (ii) Has undergone one or more prior HER2-targeted therapies; and / or one or more prior EGFR-targeted therapies.

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

20. The method of claim 1, further comprising detecting and comparing one or more of the following after administration of the first effective amount of adoptive cell therapy: (a) The presence of the engineered immune cells in the tumor of the subject; (b) Disease protein markers in the serum of the subjects; (c) Cytokines from peripheral blood samples of the subject; (d) Circulating tumor DNA from a peripheral blood sample of the subject; or (e) Size and / or number of lesions; Any one of (a)-(e) is used to assess tumor burden, tumor immunobiology, and / or tumor treatment response to determine the efficacy of multi-dose targeted adoptive cell therapy.

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

22. A method for treating cancer in a subject, the method comprising: (a) Administering one or more first doses of an EGFR inhibitor to the subject; (b) administering one or more doses of the adoptive cell therapy product to the subject; and (c) Administer one or more second doses of the EGFR inhibitor to the subject; The adoptive cell therapy product comprises engineered T lineage cells that express (i) HER2-CAR (chimeric antigen receptor), IL7 / IL7 receptor fusion protein (IL7RF), TGFβ signaling redirection receptor (TGFβ-SRR), CXC motif chemokine receptor 2 (CXCR2), and exogenous CD16; (ii) CD38 knockout; and (iii) T cell receptor (TCR) knockout; and The cancers mentioned include cancer cells that express EGFR.

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

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

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 for treating a subject's cancer, wherein the method comprises repeating the steps of the method according to claim 1 or 22.

29. A kit for cancer treatment, the kit comprising FT825, one or more chemotherapy agents, and optionally an EGFR inhibitor; wherein: (a) FT825 comprises engineered T lineage cells that include: (i) expression of HER2-CAR (chimeric antigen receptor), IL7 / IL7 receptor fusion protein (IL7RF), TGFβ signaling redirection receptor (TGFβ-SRR), CXC motif chemokine receptor 2 (CXCR2), and exogenous CD16; (ii) CD38 knockout; and (iii) T cell receptor (TCR) knockout; (b) The chemotherapy regimen 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 inhibitors include cetuximab, mateuzumab, panitumumab or nexituzumab.

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

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