IPSC-derived effector cells for treatment of autoimmune diseases

Abstract

Methods and compositions for immunotherapy are provided. In various embodiments, the compositions comprise 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. Also provided are therapeutic compositions comprising derived effector cells, alone or enhanced in these functions in combination therapy, and uses thereof.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 581,612, filed September 8, 2023; U.S. Provisional Application Serial No. 63 / 557,427, filed February 23, 2024; and U.S. Provisional Application Serial No. 63 / 644,427, filed May 8, 2024, the disclosures of which are incorporated herein by reference in their entirety.

[0003] By referencing and incorporating into the sequence list

[0004] The sequence list with the title 184143-654601_SL.xml, created on September 5, 2024 and measuring 20,563 bytes, is hereby incorporated in its entirety by reference. Technical Field

[0005] 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

[0006] 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

[0007] The need is for functionally improved effector cells that address issues ranging from response rate, cell desaturation, loss of infused cells (survival and / or persistence), off-target toxicity, off-target effects to efficacy against various diseases.

[0008] 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.

[0009] In one aspect, the present invention provides a method for treating lupus or its symptoms in a subject. In some embodiments, the method includes (a) administering one or more doses of chemotherapy to the subject; and (b) after step (a), 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 effector cells comprising (i) expression of CD19-CAR (chimeric antigen receptor) at the T cell receptor α constant (TRAC) locus, and (ii) T cell receptor (TCR) knockout.

[0010] In some embodiments, the method further includes (c) after step (b) administering one or more doses of additional chemotherapy to the subject, wherein the additional chemotherapy is the same as or different from the chemotherapy in step (a); and (d) after step (c) administering one or more doses of adoptive cell therapy product to the subject in a second effective amount that is the same as or different from the first effective amount.

[0011] In some implementations, lupus or its symptoms include: (i) cutaneous lupus; (ii) lupus nephritis; (iii) neuropsychiatric lupus; or (iv) pericarditis, atherosclerosis, angina pectoris, Raynaud's syndrome, nephropathy, metabolic syndrome, thyroid disorders, fibromyalgia, respiratory tract infection, skin infection, or urinary tract infection.

[0012] In some embodiments, the chemotherapy includes one or more of (a) cyclophosphamide (CY) or fludarabine (FLU); or (b) bendamustine. In some embodiments, the chemotherapy is administered one or more days prior to the administration of the adoptive cell therapy product; optionally, the chemotherapy is administered in one or more of the following cases: (i) three days prior to the adoptive cell therapy product, (ii) four days prior to the adoptive cell therapy product, or (iii) five days prior to the adoptive cell therapy product. In some embodiments, the chemotherapy comprises starting approximately 4-6 days prior to day 1 of administration of the adoptive cell therapy product, and continuing for 3 days at approximately 250 mg / m². 2 Approximately 600 mg / m 2 Daily dose of cyclophosphamide and at approximately 20 mg / m 2 Approximately 40 mg / m 2 The daily dose of fludarabine is administered. In some implementations, chemotherapy includes bendamustine and is started approximately 4-6 days prior to day 1 of the adoptive cell therapy product, administered for two consecutive days at approximately 30 mg / m². 2 Approximately 150 mg / m 2 The daily dose is administered.

[0013] In some embodiments, the engineered T-lineage effector cells are derived from engineered induced pluripotent stem cells (iPSCs) containing TCR knockout and a polynucleotide encoding CD19-CAR. In some embodiments, the first effective amount of the adoptive cell therapy product is approximately 1.8 × 10⁻⁶. 8 One cell to approximately 9 × 10 9 The number of cells is [number], and optionally increased. In some embodiments, the first effective amount of the adoptive cell therapy product is approximately 3.6 × 10⁻⁶ cells. 8 One cell, approximately 1 × 10 9 1 cell, approximately 3 × 10 9 One cell or approximately 9 × 10 9 Individual cells. In some embodiments, the adoptive cell therapy product is cryopreserved and then thawed prior to application. In some embodiments, the adoptive cell therapy product is FT819.

[0014] In some embodiments, the subject has been diagnosed with lupus. In some embodiments, the subject is positive for at least one of the following: (a) antinuclear antibody; (b) anti-dsDNA antibody; or (c) anti-Smith antibody. In some embodiments, treating lupus includes achieving lupus remission or low-activity in the subject. In some embodiments, remission or reduced disease activity is defined by complete remission (DORIS definition of systemic lupus erythematosus remission), clinical remission on DORIS during treatment, and / or LLDAS (low-disease-activity status of lupus). In some embodiments, treating lupus includes controlling lupus flare-ups. In some embodiments, treatment includes reducing autoantibody production in the subject to a longer disease-free interval without the use of immunosuppressive drugs; and / or remodeling healthy B-cell compartments in the subject compared to B cells under active lupus. In some implementations, the subject has previously received one or more prior treatments, including glucocorticoids, CY, mycophenolate mofetil or derivatives thereof, belimumab, methotrexate, azathioprine, anifrolumab, rituximab, obinutuzumab, cyclosporine, tacrolimus, or voclosporin. In some implementations, the subject has not responded to one or more prior treatments.

[0015] In one aspect, the present invention provides a method for treating a subject with lupus or symptoms thereof. In some embodiments, the method includes (a) administering one or more doses of chemotherapy to the subject; and (b) after step (a), 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 effector cells, the engineered T-lineage effector cells comprising (i) expression of CD19-CAR (chimeric antigen receptor) at the T-cell receptor α constant (TRAC) locus, and (ii) T-cell receptor (TCR) knockout; and wherein the subject has previously received one or more prior treatments and has not responded to the one or more prior treatments, the one or more prior treatments including glucocorticoids, CY, mycophenolate mofetil or derivatives thereof, belimumab, methotrexate, azathioprine, anifrucizumab, rituximab, obbituzumab, cyclosporine, tacrolimus, or vorozoprothiolane.

[0016] In another aspect, the present invention provides a method for treating lupus or its symptoms in a subject, the method comprising administering to the subject one or more doses of an adoptive cell therapy product in a first effective amount; wherein the adoptive cell therapy product comprises engineered NK lineage effector cells comprising CD38 knockout and expression of: (i) CD19-CAR (chimeric antigen receptor); (ii) an allogeneic immune defense receptor (ADR) targeting 4-1BB; (iii) a high-affinity, non-cleavable CD16 (hnCD16); and (iv) an interleukin-15 (IL15) / IL15 receptor fusion protein (IL15RF). In one embodiment, the method does not include administering chemotherapy for lymphotropic purposes to the patient. Attached Figure Description

[0017] Figure 1A and Figure 1B Exemplary treatment regimens are illustrated to evaluate FT819 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), and PTFU (post-treatment follow-up). According to the illustrated regimens, subjects are followed up for up to 2 years after a cycle of FT819 treatment and for up to 15 years after FT819 treatment.

[0018] Figure 2 An exemplary dose escalation and dose expansion protocol for FT819 in the treatment of lupus is shown. The dose level 1 (DL1) shown is set to 3.6 × 10⁻⁶. 8 Furthermore, additional dose levels >DL1 (≤3 × previously cleared dose level) can be explored. DL0 = 1.8 × 10 8 Cells / dose (estimated if DL1 exceeds MTD).

[0019] Figure 3 The study shows CD19+ B cell depletion observed in a 72-hour kill assay of PPMCS (E:T 1:1) from blood samples derived from patients with SLE.

[0020] Figure 4 The results show that FT819 demonstrates better targeting of CD19+ B cells from blood samples from SLE patients compared to autologous CAR T cells. The curves in each figure from left to right correspond to the legend entries from top to bottom.

[0021] Figure 5 The FT819 PK (mean ± SEM) of 90 million and 360 million cells in the Phase 1 clinical trial is shown.

[0022] Figure 6 This study demonstrated B cell depletion in peripheral blood during FT819 treatment cycles in patients, accompanied by persistent B cell suppression.

[0023] Figure 7 This study illustrates B-cell depletion observed after administering a single dose of FT819 to two patients resistant to Cy / Flu conditioning chemotherapy.

[0024] Figure 8 This study demonstrates CD19+ B cell depletion following a single dose of FT819 in combination with bendamustine as an alternative to Cy / Flu conditioning chemotherapy.

[0025] Figure 9 The study showed plasma cell depletion and B cell remodeling from naïve B cells in three patients 6–10 weeks after a single dose of FT819.

[0026] Figure 10 The study showed that FT819 was detected and persisted in the bone marrow on day 9, and that CD19+ cells in the bone marrow and peripheral blood were completely eliminated at the end of a treatment cycle consisting of standard conditioning chemotherapy followed by a single dose of FT819 at 90M cells.

[0027] Figure 11 The study showed depletion of CD19+ B cells in peripheral blood, a reduction of >50% in CD19+ tumor cells in secondary lymphoid tissue after a single dose of FT819, and a complete metabolic response with approximately 80% reduction in target lesion 1 after retreatment with a single dose of FT819 by FDG-PET.

[0028] Figure 12 This study demonstrates the sustained depletion of CD19+ B cells in peripheral blood and the complete elimination of CD19+ clumps in the liver following a single dose of FT819.

[0029] Figure 13 This study demonstrates the depletion of CD19+ B cells in pre-treatment peripheral blood samples from SLE patients following exposure to FT819 in a 24-hour co-culture assay for in vitro translation assessment.

[0030] Figure 14 This study demonstrates rapid, deep, and persistent CD19+ B cell depletion in the peripheral blood of SLE patients throughout the treatment cycle, with T cell compartment recovery following treatment.

[0031] Figure 15The results show that FT522 demonstrated comparable or better CAR-mediated CD19+ B cell killing compared to TCR-free allogeneic primary CAR-T (pCAR-T) cells in 24-hour cytotoxicity assays with various E:T ratios and in two different SLEPBMC donors. The lower curve in each figure corresponds to the results for FT522.

[0032] Figure 16A An in vitro allogeneic re-challenge assay was shown, in which SLE donor PBMCs were co-cultured with pCAR-T cells, FT596 (ADR negative) or FT522 (ADR armed) for 8 days, and then re-drugged with effector cells and re-challenged with SLE donor PBMCs, and co-cultured for a total of 14 days.

[0033] Figure 16B Flow cytometry of CD3+ SLE donor PBMC T cells was demonstrated, showing T cell activation targeting CD25 and 4-1BB markers on days 4 and 6, and allogeneic responses to pCAR-T and FT596 cells but not FT522 cells.

[0034] Figure 16C The total SLE donor PBMC-derived T cells expanded in response to allogeneic challenge on day 6 and the CD19+ cell count prior to re-challenge are shown. Figure 16D This shows that pCAR-T and FT596 cells were exhausted after the allogeneic re-challenge assay, while FT522 cells persisted, and CD19+ cell counts were recorded at the end of the assay. Cell counts were normalized relative to individual PBMCs.

[0035] Figure 17A This demonstrates that, in an in vitro killing assay, FT596 elicited a stronger antitumor effect compared to FT522 CAR iNK cells at the tested E:T ratio. Figure 17B The results showed that FT522 exhibited a higher PK on day 4 compared to an equivalent dose of FT596, and a higher total AUC during the treatment cycle compared to single or triple doses of FT596.

[0036] Figure 18A The activity of FT596 in eliminating peripheral blood B cells was demonstrated in a BCL setting in patients (n=9) as a single therapy. Figure 18B The activity of FT522 in eliminating peripheral blood B cells was demonstrated in combination regimens with rituximab. Detailed Implementation

[0037] 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.

[0038] definition

[0039] 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.

[0040] 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.

[0041] 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.

[0042] The use of alternatives (e.g., "or") should be understood to mean any one, two, or any combination of the alternatives.

[0043] The term “and / or” should be understood to mean one or both of the alternatives.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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."

[0052] The term "body" generally refers to activities that take place inside an organism.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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).

[0057] 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.

[0058] Pluripotency can be determined in part by assessing the pluripotent characteristics of cells. Pluripotent characteristics 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.

[0059] 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).

[0060] 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.

[0061] As used herein, the term “subject” refers to any animal, preferably a human patient, livestock or other domesticated animal.

[0062] "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.

[0063] “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.

[0064] "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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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 optional CD16, but lacks expression of one or more of the following: PLZF, SYK, FceR And EAT-2. In some implementations, the CD56 is separated. + 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.

[0072] 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.

[0073] 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.

[0074] 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%.

[0075] 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.

[0076] "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.

[0077] "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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] "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.

[0083] 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.

[0084] 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, diseases, or therapeutic 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 therapeutic 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 by neighboring immune cells; 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 upon integration 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.

[0085] 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 by neighboring immune cells; and / or resistance to treatments such as chemotherapy.

[0086] As used herein, the term "adaptor" refers to a molecule (e.g., 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 target 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.

[0087] 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., B 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 target-binding specificity). In some embodiments, adaptors with the same target specificity are used for coupling with the universal surface triggering receptor. In some embodiments, adaptors with different target specificities are used for coupling with the universal surface triggering receptor. Therefore, one or more effector cell types can be conjugated, thereby killing one specific type of cell in some cases and killing two or more target cell types in others. Surface trigger receptors typically contain a co-stimulatory domain for effector cell activation and an anti-epitope specific to the epitope of the adaptor. Bispecific adaptors are specific to the anti-epitope of the surface trigger receptor at one end and to the target cell antigen at the other end.

[0088] 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.

[0089] 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, antibodies or fragments thereof, growth factors, and / or cytokines.

[0090] 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.

[0091] 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 specific cells, and (iv) other targeting strategies in the absence of specific antigens or surface molecules.

[0092] 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.

[0093] 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.

[0094] As used herein, “lympholysis depletion” and “lymphoconditioning” are used interchangeably and refer to the destruction of lymphocytes and T cells typically prior to immunotherapy. The purpose of lymphoconditioning 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, lymphoconditioning is typically accomplished by administering one or more chemotherapeutic agents to the subject prior to the first dose of adoptive cell therapy. In various embodiments, lymphoconditioning precedes the first dose of adoptive cell therapy by several hours to several days. Exemplary chemotherapeutic agents that can be used for lymphoconditioning 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 conditioning 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 lymphoconditioning procedures, as further described herein.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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."

[0105] 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.

[0106] "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.

[0107] 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.

[0108] As used herein, “FT819” refers to an engineered T-cell therapy produced from an iPSC line engineered by a clonal master and engineered to have multiple modalities to enhance innate immunity: (1) anti-CD19-CAR at the TRAC locus; and (2) T-cell receptor (TCR) knockout.

[0109] I. Cells and compositions that can be used in adoptive cell therapy with enhancing properties

[0110] 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.

[0111] Furthermore, while CAR-T cells have proven effective and potent in treating several hematologic malignancies, engineered autologous T-cell therapies still face numerous limitations. Besides antigen accessibility, antigen homogeneity, antigen specificity, and antigen heterogeneity are significant obstacles to the successful development of CAR-T cell therapy. Moreover, the inherent genetic engineering variability observed in patient- and donor-derived immune cells limits the widespread application of CAR-T cell therapy. This application provides an off-the-shelf adoptive cell therapy environment using effector cells derived from engineered iPSCs to treat autoimmune diseases, including systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis (RA), and scleroderma.

[0112] 1. TCR knockout

[0113] 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.

[0114] 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.

[0115] 2. Chimeric antigen receptor (CAR) expression

[0116] 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), 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: 7; CD8asp) or MDFQVQIFSFLLISASVIMSR (SEQ ID NO: 8; IgKsp), or any signal peptide sequence or functional variant thereof known in the art.

[0117] 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 encoding a CAR that contain one or more additional modifications as described herein, and the derived T cells may include helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβ T cells, γδ T cells, or combinations thereof.

[0118] 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).

[0119] In some embodiments, the scFV is an scFV selected based on its specific binding affinity to a target (e.g., CD19). In some embodiments, the scFV comprises one or more sequences of an antigen-binding fragment of an antibody selected based on its specific binding affinity to a target (e.g., CD19). In some embodiments, the antigen-binding domain of the CAR comprises a single-chain variable fragment (scFV) containing a heavy chain and a light chain having at least 85%, 90%, 95%, 99%, 100%, or any percentage therebetween, sequence identity when compared to the exemplary sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively. In some embodiments, the scFV comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or 2. In some embodiments, the scFV comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 1 or 2. In some embodiments, the scFV comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the scFV comprises the amino acid sequence of SEQ ID NO: 2. In some implementations, the scFV comprises the amino acid sequences of both SEQ ID NO: 1 and 2.

[0120] SEQ ID NO: 1

[0121] AELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYF

[0122] SEQ ID NO: 2

[0123] KFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTS

[0124] In some embodiments, the CAR further comprises a transmembrane domain, such as the transmembrane domain of CD28. In some embodiments, the CAR further comprises a co-stimulatory domain, such as the co-stimulatory domain of CD28. In some embodiments, the CAR further comprises an activation domain, such as the activation domain of CD3ζ1XX. 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: 3, 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: 3, 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: 3, 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: 3. In some embodiments, the CAR provided herein recognizes the CD19 antigen on B cells. In some embodiments, the CAR provided herein recognizes the CD19 antigen on autoreactive B cells.

[0125] SEQ ID NO: 3

[0126] ESKYGPPCPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM FWVLVVVGGVLACYSL LVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS RVKFSRSADAPAYQQGQNQLYNELN LGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFD ALHMQALPPR

[0127] (Interval - CD28) TM - CD28 co-stimulation - CD3ζ1XX activation )

[0128] 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: 6, 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: 6, 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: 6, 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: 6. In some embodiments, the CAR provided herein recognizes the CD19 antigen.

[0129] SEQ ID NO: 6

[0130] MALPPVTALLLPLALLLHAEVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISS VVDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYF CQQYNRYPYTSGGGTKLEIKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR

[0131] 3. Enter CD16

[0132] 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 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with SEQ ID NO: 4. In some embodiments, hnCD16 comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 4. In some embodiments, hnCD16 comprises an amino acid sequence having at least about 95% identity with SEQ ID NO: 4. In some implementations, hnCD16 contains the amino acid sequence of SEQ ID NO: 4.

[0133] SEQ ID NO: 4

[0134] MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLR CHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVPTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK

[0135] 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.

[0136] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an NK cell-mediated lysis mechanism 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. Non-cleavable CD16 provides antibody-dependent cell-mediated cytotoxicity (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 therapy administration also enable the in vitro loading of ADCC antibodies onto T cells via hnCD16.

[0137] 4. Exogenously introduced cytokine signaling complexes

[0138] 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).

[0139] 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 replaced by 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: 5, 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: 5, 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: 5, 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: 5, 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: 5. In some embodiments, such a construct comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% identity with SEQ ID NO: 5, excluding the amino acids of the exemplary transmembrane domain, signal peptide, and linker shown in SEQ ID NO: 5.

[0140] SEQ ID NO: 5

[0141] MDWTWILFLVAAATRVHSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNNKILMGTKEHS GGGSGGGGSGGGGSGGGGSGGGSLQESGYAQNGDLEDAELDDYSFSCYSQLEVNGSQHSLTCAFEDPDVNITNLEFEICGALVEVKCLNFRKLQEIYFIETKKFLLIGKSNICVKVGEKSLTCKKIDLTTIVK PEAPFDLSVVYREGANDFVVTFNTSHLQKKYVKVLMHDVAYRQEKDENKWTHVNLSSTKLTLLQRKLQPAAMYEIKVRSIPDHYFKGFWSEWSPSYYFRTPEINNSSGEMD PILLTISILSFFSVALLVILACVLW KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0142] (signal peptide-IL7-) connector -IL7R; Transmembrane domain ( TM (Signal peptides and linkers can vary in length and sequence)

[0143] 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.

[0144] 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.

[0145] In some embodiments, the cytokine signaling complex comprises an IL15 receptor fusion (IL15RF) containing a full-length or partial-length IL15 and a full-length or partial-length IL15 receptor. The transmembrane (TM) domain for the IL15 receptor may be native or modified or replaced by the transmembrane domain of any other membrane-binding protein. In some embodiments, IL15 and IL15Rα are co-expressed using a self-cleaving peptide to mimic trans-presentation of IL15 without eliminating cis-presentation. In other embodiments, IL15Rα is fused to IL15 at its C-terminus via a linker to mimic trans-presentation without eliminating cis-presentation and to ensure IL15 membrane binding. In other embodiments, IL15Rα having a truncated intracellular domain is fused to IL15 at its C-terminus via a linker to mimic trans-presentation of IL15, maintain IL15 membrane binding, and further eliminate cis-presentation mediated by normal IL15R through its intracellular domain and / or any other potential signal transduction pathways. In other embodiments, IL15Rα is fused with IL15, which lacks an intracellular domain (IL15Rα). (As described in international publications WO 2019 / 191495 and WO 2019 / 126748, the entire contents of which are incorporated herein by reference.)

[0146] In various embodiments, this truncated construct comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 9. In one embodiment of truncated IL15 / IL15Rα, the construct does not contain the last four amino acid residues (KSRQ) of SEQ ID NO: 9 and comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 10.

[0147] SEQ ID NO: 9

[0148] MDWTWILFLVAAATRVHS GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQ

[0149] (379 amino acids; signal transduction and adaptor peptides are underlined)

[0150] SEQ ID NO: 10

[0151] MDWTWILFLVAAATRVHS GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SGGGSGGGGSGGGGSGGGGSGGGSLQ ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYL

[0152] (375 amino acids; signal transduction and linker peptides are underlined)

[0153] 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.

[0154] 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. 2A peptides derived from FMDV, ERAV, PTV-I, and TaV are sometimes referred to as “F2A,” “E2A,” “P2A,” and “T2A,” respectively.

[0155] 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 includes a polynucleotide encoding CD19-CAR (chimeric antigen receptor), and optionally one or more of CD16 or variants thereof that mediate ADCC when combined with a monoclonal antibody, CD38 knockout and IL7 signaling complex.

[0156] 5. CAR-19 NK cells armed with ADR

[0157] Undesirable activation of T cells and NK cells often promotes allogeneic immune responses, leading to the development of graft-versus-host disease (GvHD). Although steps can be taken to reduce the reactivity of allogeneic cells in the recipient individual, these cells will still be targeted by the recipient's immune system (primarily T cells and NK cells), which recognizes them as foreign, resulting in rejection and limiting treatment benefits. On the other hand, modulating the host immune system to reduce allogeneic immune responses, for example, through lymphomodulation using chemotherapy such as Cy / Flu (cyclophosphamide / fludarabine), often results in associated hematologic toxicities, including increased susceptibility to severe infections due to arbitrary lymphatic depletion and, as a result, a severely compromised immune system. In various implementations, to control pathogenic conditions resulting from unwanted activation of the immune system, the genetically engineered immune cells, iPSCs, and their derived effector cells described herein contain allogeneic immune defense receptors (ADRs) and other components. In some embodiments, this application provides immune cells, iPSCs, and iPSC-derived effector cells that are genetically engineered to include, in addition to other edits as conceived and described herein, 4-1BB or CD38-specific allogeneic immune defense receptors (ADRs) for effector cell enhancement and selective depletion of allogeneic reactive host NK cells and T cells with upregulated 4-1BB and / or CD38 expression, wherein the latter include pathogenic T cells and regulatory T cells, without harming resting cells in the recipient.

[0158] In some embodiments of an ADR specific to 4-1BB (CD137, also known as "41BB"), the ADR comprises an extracellular domain targeting 4-1BB upregulated in host T cells or NK cells when host T cells or NK cells are activated, and a signaling domain promoting effector cell activation. For example, the 41BB-ADR extracellular domain may comprise any suitable ligand for 4-1BB, including 4-1BBL, an antibody targeting 4-1BB (or a functional fragment thereof), a fusion of Fc with 4-1BBL, or a functional derivative or fragment thereof. In some embodiments, the 41BB-ADR extracellular domain comprises 4-1BBL or a fragment thereof that effectively binds to 4-1BB. In some embodiments, the 41BB-ADR extracellular domain comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NO: 11. In some embodiments, the 41BB-ADR extracellular domain comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 11. In some embodiments, the 41BB-ADR extracellular domain comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 11. In some embodiments, the 41BB-ADR extracellular domain comprises the amino acid sequence of SEQ ID NO: 11.

[0159] SEQ ID NO: 11

[0160] GLLDLC

[0161] In certain embodiments, the ADR comprises CD3ζ, which is represented by an amino acid sequence or a functional fragment thereof having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NO: 12, or comprises a CD3ζ derivative (e.g., CD3ζ1XX, which is represented by an amino acid sequence or a functional fragment thereof having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NO: 13). In some embodiments, CD3ζ comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 12. In some embodiments, CD3ζ comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 12. In some embodiments, CD3ζ comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the CD3ζ derivative comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 13. In some embodiments, the CD3ζ derivative comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 13. In some embodiments, the CD3ζ derivative comprises the amino acid sequence of SEQ ID NO: 13. CD3ζ mediates downstream ITAM-derived signaling during effector T cell or NK cell activation.

[0162] SEQ ID NO: 12

[0163] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0164] (CD3ζ)

[0165] SEQ ID NO: 13

[0166] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR

[0167] (CD3ζ1XX - contains 2 mutations in ITM1)

[0168] In one embodiment of the 4-1BB specific ADR, the 41BB-ADR is represented by an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NO: 14 or SEQ ID NO: 15-17. In some embodiments, the 41BB-ADR comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 14 or SEQ ID NO: 15-17. In some embodiments, the 41BB-ADR comprises an amino acid sequence having at least about 95% identity with SEQ ID NO: 14 or SEQ ID NO: 15-17. In some embodiments, the 41BB-ADR comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the 41BB-ADR comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the 41BB-ADR comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the 41BB-ADR comprises the amino acid sequence of SEQ ID NO: 17.

[0169] SEQ ID NO: 14

[0170] MEFGLSWLFLVAILKGVQCGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSE ESKYGPPCPPCPGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYT QKSLSLSPGKKDPK FWVLVVVGGVLACYSLLVTVAFIIFWVRS RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R

[0171] Signal peptide-41BBL- Interval -CD28(TM)- CD3z (The signal peptide, spacer region, and TM / transmembrane domain can vary.)

[0172] SEQ ID NO: 15

[0173] MEFGLSWLFLVAILKGVQCGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSE ESKYGPPCPPCPGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYT QKSLSLSPGKKDPK FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0174] Signal peptide-41BBL- Interval -CD28(TM)-CD28(ICD)- CD3z (The signal peptide, spacer region, and TM / transmembrane domain can vary.)

[0175] SEQ ID NO: 16

[0176] MEFGLSWLFLVAILKGVQCGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSE ESKYGPPCPPCPGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYT QKSLSLSPGKKDPK FWVLVVVGGVLACYSLLVTVAFIIFWVRS RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPP R

[0177] Signal peptide-41BBL- Interval -CD28(TM)- CD3z1xx (The signal peptide, spacer region, and TM / transmembrane domain can vary.)

[0178] SEQ ID NO: 17

[0179] MEFGLSWLFLVAILKGVQCGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSE ESKYGPPCPPCPGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYT QKSLSLSPGKKDPK FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFS EIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR

[0180] Signal peptide-41BBL- Interval -CD28(TM)-CD28(ICD)- CD3z1xx (The signal peptide, spacer region, and TM / transmembrane domain can vary.)

[0181] 6. Antibodies used in immunotherapy

[0182] 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, the antigen activates the applied iPSC-derived effector cells to enhance their cytotoxicity. 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 ® ).

[0183] 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.

[0184] II. Therapeutic uses of derived immune cells for autoimmune diseases

[0185] Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by immune abnormalities, including an excess of pathogenic autoantibodies, overactive B cells, activated T cells, increased cytokines, and the presence of enhanced innate immune function. Overactive B cells function as precursors to autoantibody-producing cells and as antigen-presenting cells for T cells, while immune complexes containing autoantibodies complexed with their own autoantigens stimulate plasmacytoid dendritic cells and neutrophils, where these immune complexes form and deposit in various tissues, including the kidneys, skin, and joints.

[0186] SLE is a chronic, heterogeneous, systemic autoimmune disease affecting many organs. About half of all people with lupus experience high blood pressure, pericarditis (heart inflammation), atherosclerosis (arteriosclerosis), angina, Raynaud's syndrome, and other cardiovascular symptoms. Cutaneous lupus is a skin complication specific to lupus. It is classified into three types: chronic cutaneous lupus, subacute cutaneous lupus, and acute cutaneous lupus. Lupus poses a significant risk to the kidneys because the disease attacks the body's kidney tissue. It is estimated that about 6 out of 10 people with lupus will develop lupus nephritis, and about 1 out of 10 will develop end-stage renal disease due to this complication. People with lupus are also more likely to experience infections and infection-related complications than those without lupus. The most common infections include those affecting the respiratory tract, skin, and urinary tract. Opportunistic infections are also common in lupus patients, such as herpes zoster, Staphylococcus aureus, Escherichia coli, Salmonella, and Candida albicans. Metabolic syndrome, which can lead to the development of type 2 diabetes, is common in lupus. People with lupus are at increased risk for many types of cancer, including bladder cancer, cervical cancer, esophageal cancer, colon cancer, anal cancer, liver cancer, pancreatic cancer, hepatobiliary cancer, lung cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, laryngeal cancer, leukemia, multiple myeloma, non-melanoma skin cancer, oropharyngeal cancer, kidney cancer, thyroid cancer, cervical cancer, vaginal / vulvar cancer, and skin cancer. Hyperthyroidism and thyroid nodules (two types of thyroid disorders) are more common in people with lupus than in the general population, and the rate of thyroid cancer is more than double in lupus patients than in the general population. Furthermore, about one-third of people with lupus develop fibromyalgia (a condition that causes fatigue and widespread muscle pain throughout the body). In addition, 80-90% of people with lupus experience neuropsychiatric lupus (a condition with a range of symptoms including depression, headaches, and cognitive fog).

[0187] SLE is a disease that typically affects young women aged 15 to 45, with women being 10 times more likely to be affected than men. Prevalence and severity are higher in Africans, Hispanics, Asians, and people of Native American descent. Although the 10-year mortality risk has decreased to 5% to 10% (Yen et al., Ann Intern Med, 5 Dec 2017; 167(11):777-85), patients still die from active disease, infections, cardiovascular causes, and treatment-related side effects. Despite improved survival rates, few patients maintain good to excellent disease control for more than one year after starting medication, as the disease is characterized by remissions and flare-ups.

[0188] While the goals of treatment are to reduce inflammation and organ damage, and to prevent or reverse disease progression, most of the agents commonly used are nonspecific immunosuppressants. Combinations of topical or low-dose oral steroids, hydroxychloroquine, nonsteroidal anti-inflammatory drugs (NSAIDs), and methotrexate are used for non-organ-threatening diseases, while combinations of high-dose oral or intravenous steroids, cyclophosphamide (CY), azathioprine (AZA), mycophenolate mofetil (MMF), vorozoprothiolane, anifrucizumab, and belimumab are used for organ-threatening diseases. When vital organs are involved, aggressive treatment is required to prevent organ damage or failure. Many of these agents do not reduce disease activity, and flare-ups are common.

[0189] Therefore, more targeted agents are needed for long-term disease control. Given the presumed role of B cells in SLE, B cell inhibitors may offer a longer-term solution for disease control. However, monoclonal antibodies against B cell antigens may show a significant reduction in circulating B cells but do not appear to affect the large numbers found in affected tissues. Furthermore, recent findings suggest that most pathogenic B cells are extrafollicularly derived and activated in tissues (Jenks et al., Immunity, 16 Oct 2018; 49(4):725-739.e6). Additionally, some classic B cell markers are absent in these subsets, although they all share CD19 marker expression.

[0190] This article presents an iPSC-derived CD19-targeted CAR T-cell product that drives a deep and durable response by targeting and eliminating activated B cells in lupus tissue of active, organ-at-risk lesions. The Phase 1 study described in this article was designed to evaluate the safety, pharmacokinetics, and anti-B-cell activity of FT819 in participants with SLE.

[0191] 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 disease and / or disease-related adverse effects or symptoms, the effect may be preventative in terms of complete or partial prevention of the disease, and / or therapeutic in terms of partial or complete cure. Preventative benefits include reducing the incidence and / or exacerbation of one or more diseases, conditions, or symptoms during treatment (e.g., between treated and untreated groups, or between a subject’s treated and untreated state). 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 remission, or re-inducing a disease response to therapy) or one or more symptoms associated with the disease. Treatment of developing diseases is also of interest, where treatment stabilizes or reduces undesirable clinical symptoms in patients. In some implementations, the subjects requiring treatment suffer from diseases, conditions, and / or injuries for which at least one related symptom can be contained, improved, and / or aggravated by cell therapy.

[0192] The FT819 drug substance comprises allogeneic T cells derived from cloned T-cell receptor (TCR) knockout human induced pluripotent stem cell (iPSC) lines (master cell bank; MCB) that encode a CD19-targeted CAR whose expression is regulated by the T-cell receptor α constant (TRAC) locus promoter. In some embodiments, manufacturing the drug substance from the iPSC master cell bank involves an iPSC expansion phase followed by a differentiation into T cells and T-cell expansion phases, 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. Manufacturing the FT819 drug product from the MCB directly addresses many limitations associated with current patient- and donor-specific cell therapies. Notably, many doses of the FT819 drug product can be uniformly produced in a single manufacturing activity. These doses of the drug product are uniform and can be: (i) tested to ensure compliance with predefined quality specifications, (ii) cryopreserved in infusion media, and (iii) stored to maintain sustainable inventory. Therefore, the use of FT819 in multi-dose regimens is readily available in a clinical setting, which could prove crucial for driving long-term, durable responses in patients with aggressive disease. The readily available availability of FT819 (which enables multi-dose administration of a homogeneous cell product) also allows for clinical studies of alternative dosing schedules that could potentially convey the benefits of FT819 compared to patient- and donor-specific CAR T-cell immunotherapies.

[0193] FT819 contains a CD19-targeting CAR that has been specifically developed to provide enhanced CAR-T cell efficacy against CD19+ B cells. An anti-CD19 single-stranded variable fragment (scFv) is linked to the CD28 transmembrane domain and signaling domains from both CD28 and CD3ζ. A tyrosine-to-phenylalanine point mutation is introduced within the immune receptor tyrosine-based activation motif (ITAM) of the CD3ζ chain to generate the modified ITAM conformation CD3ζ-1XX. The 1XX ITAM conformation works in combination with the CD28 signaling domain to initiate calibrated cell signaling pathways designed to enhance CAR T-cell effector function and prevent CAR T-cell desaturation. CAR expression in FT819 is controlled by the promoter at the TRAC locus. Disruption of the TRAC locus via biallelic CAR insertion prevents endogenous TCR expression and is designed to minimize the risk of graft-versus-host disease (GvH) in allogeneic cell therapy settings.

[0194] The protocols and dosage designs for cell products take into account both drug efficacy and risk reduction. In some implementations, protocols and dosage designs are tailored to patient subgroups. In other implementations, protocols and dosage designs are tailored to subsets of indications, diseases, or conditions.

[0195] 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).

[0196] 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.

[0197] 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., not related to the progression of an 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 medication. Overdose itself is not considered an AE / SAE (serious adverse event).

[0198] 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.

[0199] Monitoring, assessing, and managing evidence of FT819 immunogenicity and its clinical impact during treatment is crucial, as potentially FT819-induced immune responses may only manifest clinically, for example, as infusion-related reactions of varying severity. FT819

[0200] FT819 is formulated in DMSO for cryopreservation. DMSO side effects and symptoms are monitored, assessed, and managed during treatment. DMSO-related side effects are typically 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.

[0201] Extensive testing of FT819 during manufacturing minimizes the potential risk of disease transmission. However, while FT819 is a human-derived cell therapy, cell products may come into contact with animal-derived reagents during handling. Therefore, close monitoring and management of infectious and / or disease pathogens transmitted during treatment by known or unknown pathogens are essential.

[0202] FT819 has been engineered to express a CD19 CAR. CRS and ICANN refer to toxicities observed in other CD19 CAR T-cell therapies. Low-grade CRS has been observed in patients with B-cell malignancies treated with FT819, but ICANN has not been observed. 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 therapies that can be fatal or life-threatening (Breyanzi USPI; Kymriah USPI; Yescarta USPI). Other clinical manifestations of CRS, besides fever, hypoxia, and hypotension, 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. Since the signs and symptoms of CRS are not specific to CRS, clinical symptoms that are not considered to be unrelated to FT819 (such as bacteremia and other severe infections) are not reported as CRS.

[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 FT819 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 FT819 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 FT819 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] In some embodiments, chemotherapy is administered prior to FT819 administration to create an immune environment suitable for the sustained and amplified proliferation of FT819. This is achieved by promoting the homeostatic proliferation of FT819 and eliminating other components of the immune system that regulate immune cells and compete for homeostatic cytokines. However, chemotherapy drugs, such as cyclophosphamide (CY) and fludarabine (FLU), have been reported to 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 those 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 cases 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] In some implementations, bendamustine can be used as an alternative chemotherapy regimen to CY / FLU. Bendamustine can be administered in participants with severe hemorrhagic cystitis and a history of CY, and may be administered over multiple treatment cycles. Warnings and precautions associated with bendamustine include myelosuppression, 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. Compared to CY / FLU chemotherapy, bendamustine chemotherapy provides similar clinical efficacy and is associated with lower rates of CRS, neurotoxicity, hematologic toxicity, infection, neutropenic fever, and post-infusion hospitalization.

[0210] Compared to autologous CAR T-cell therapy in SLE, FT819 can be administered off-the-shelf / on-demand without requiring leukatomization. Because the immunosuppression required by autologous CAR T-cell therapy can be omitted or immediately discontinued before pretreatment and FT819 infusion, the risk of SLE exacerbations due to immunosuppression is reduced. In some implementations, the reduction in the risk of SLE exacerbations when treating SLE with FT819 is significant.

[0211] In one aspect, the present invention provides a method for treating lupus or its symptoms in a subject. In some embodiments, the method includes (a) administering one or more doses of chemotherapy to the subject; and (b) after step (a), 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 effector cells comprising (i) expression of CD19-CAR (chimeric antigen receptor) at the T cell receptor α constant (TRAC) locus, and (ii) T cell receptor (TCR) knockout.

[0212] In some embodiments, the method further includes (c) after step (b) administering one or more doses of additional chemotherapy to the subject, wherein the additional chemotherapy is the same as or different from the chemotherapy in step (a); and (d) after step (c) administering one or more doses of adoptive cell therapy product to the subject in a second effective amount that is the same as or different from the first effective amount.

[0213] In some implementations, lupus or its symptoms include: (i) cutaneous lupus; (ii) lupus nephritis; (iii) neuropsychiatric lupus; or (iv) pericarditis, atherosclerosis, angina pectoris, Raynaud's syndrome, nephropathy, metabolic syndrome, thyroid disorders, fibromyalgia, respiratory tract infection, skin infection, or urinary tract infection.

[0214] In some embodiments, chemotherapy includes one or more of (a) cyclophosphamide (CY) or fludarabine (FLU); or (b) bendamustine. In some embodiments, CY is administered as an opsonizing agent. In some embodiments, FLU is administered as an opsonizing agent. In some embodiments, both CY and FLU are administered for opsonizing. In some embodiments, CY is not used for opsonizing. In some embodiments, FLU is not used for opsonizing. In some other embodiments, neither CY nor FLU is used for opsonizing. In still other embodiments, bendamustine is used for opsonizing. In some embodiments, chemotherapy is administered one or more days prior to administration of the adoptive cell therapy product; optionally, chemotherapy is administered in one or more of the following cases: (i) three days prior to the adoptive cell therapy product, (ii) four days prior to the adoptive cell therapy product, or (iii) five days prior to the adoptive cell therapy product. In some embodiments, chemotherapy comprises starting approximately 4-6 days prior to day 1 of administration of the adoptive cell therapy product, for 3 consecutive days at approximately 250 mg / m². 2 Approximately 600 mg / m 2 Daily dose of cyclophosphamide and at approximately 20 mg / m 2 Approximately 40 mg / m 2 The daily dose of fludarabine is administered. In some implementations, chemotherapy includes bendamustine and is started approximately 4-6 days prior to day 1 of the adoptive cell therapy product, administered for two consecutive days at approximately 30 mg / m². 2 Approximately 150 mg / m 2 The daily dose is administered.

[0215] In some embodiments, the engineered T-lineage effector cells are derived from engineered induced pluripotent stem cells (iPSCs) containing TCR knockout and a polynucleotide encoding CD19-CAR. In some embodiments, the first effective amount of the adoptive cell therapy product is approximately 1.8 × 10⁻⁶. 8 One cell to approximately 9 × 10 9 The number of cells is [number], and optionally increased. In some embodiments, the first effective amount of the adoptive cell therapy product is approximately 3.6 × 10⁻⁶ cells. 8 One cell, approximately 1 × 10 9 1 cell, approximately 3 × 10 9 One cell or approximately 9 × 10 9 Individual cells. In some embodiments, the adoptive cell therapy product is cryopreserved and then thawed prior to application. In some embodiments, the adoptive cell therapy product is FT819.

[0216] In some embodiments, the subject has been diagnosed with lupus. In some embodiments, the subject is positive for at least one of the following: (a) antinuclear antibody; (b) anti-dsDNA antibody; or (c) anti-Smith antibody. In some embodiments, treating lupus includes achieving lupus remission or low-activity in the subject. In some embodiments, remission or reduced disease activity is defined by DORIS (the definition of systemic lupus erythematosus remission), DORIS clinical remission during treatment, and / or LLDAS (low-disease-activity status of lupus). In some embodiments, treating lupus includes controlling lupus flare-ups. In some embodiments, treatment includes reducing autoantibody production in the subject to a longer disease-free interval without the use of immunosuppressive drugs; and / or remodeling healthy B-cell compartments in the subject compared to B cells under active lupus. In some implementations, the subject has previously received one or more prior treatments, including glucocorticoids, CY, mycophenolate mofetil or derivatives thereof, belimumab, methotrexate, azathioprine, anifrulimumab, rituximab, obbituzumab, cyclosporine, tacrolimus, or vorosaporin. In some implementations, the subject has not responded to one or more prior treatments.

[0217] In one aspect, the present invention provides a method for treating a subject with lupus or symptoms thereof. In some embodiments, the method includes (a) administering one or more doses of chemotherapy to the subject; and (b) after step (a), 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 effector cells, the engineered T-lineage effector cells comprising (i) expression of CD19-CAR (chimeric antigen receptor) at the T-cell receptor α constant (TRAC) locus, and (ii) T-cell receptor (TCR) knockout; and wherein the subject has previously received one or more prior treatments and has not responded to the one or more prior treatments, the one or more prior treatments including glucocorticoids, CY, mycophenolate mofetil or derivatives thereof, belimumab, methotrexate, azathioprine, anifrucizumab, rituximab, obbituzumab, cyclosporine, tacrolimus, or vorozoprothiolane.

[0218] Lupus typically involves alternating periods of marked increase in disease activity (referred to as a “seizure”) and suppression or reduction in disease activity. A seizure is defined as a measurable increase in disease activity in one or more organ systems, involving new or worsening clinical signs and symptoms and / or laboratory measurements. Seizures are generally considered an indication for initiating or changing treatment (e.g., escalating doses of treatment already being taken by the subject, or the introduction of a new therapeutic intervention). In some implementations, a seizure may be characterized or defined by hospitalization due to SLE. In some implementations, for example, a seizure may be, or include, a measurable increase in disease activity in one or more organ systems, involving new or worsening clinical signs and symptoms and / or laboratory measurements, compared to previously performed measurements. Various clinical measurements have been used to determine whether a subject with lupus has a clinical seizure. One such measurement is the SLENA-SLEDA Disease Activity Index (SLENA-SLEDA ... Other examples of disease measures used to classify lupus activity and seizure identification include the Physician Overall Assessment (PGA), SLEDAI-2K, and BILAG-2004. In some implementations, a seizure is characterized by, or defined as, a change of greater than or equal to 1.0 in the Physician Overall Assessment of Disease Activity (measured on a 0-3 scale) from a previous visit or from visits within the most recent 200 days (e.g., the most recent 100 days, the most recent 93 days, the most recent 75 days, the most recent 50 days, the most recent 25 days, the most recent 10 days, the most recent 5 days, or the most recent 1 day). In some implementations, a lupus seizure is defined as >1 new BILAG-2004 A or >2 new (worsening) BILAG-2004 B domain scores within a predetermined observation period (e.g., one month).

[0219] In one aspect, the methods for treating lupus disclosed herein include controlling lupus flare-ups. Controlling lupus flare-ups may include shortening the duration of flare-ups in subjects, reducing the severity of flare-ups in subjects, and / or reducing the incidence of flare-ups in subjects. Improvements achieved in controlling lupus flare-ups can be achieved through comparison with a reference group of subjects who received treatment prior to treatment or untreated subjects with lupus.

[0220] Exemplary implementation plan

[0221] This disclosure provides the following exemplary implementation schemes.

[0222] Implementation Scheme 1. A method for treating lupus or its symptoms in a subject, the method comprising:

[0223] (a) administering one or more doses of chemotherapy to the subject; and

[0224] (b) After step (a), administer one or more doses of the adoptive cell therapy product to the subject in a first effective amount;

[0225] The adoptive cell therapy product comprises engineered T lineage effector cells, which include (i) expression of CD19-CAR (chimeric antigen receptor) at the T cell receptor α constant (TRAC) locus and (ii) T cell receptor (TCR) knockout.

[0226] Implementation Scheme 2. The method according to Implementation Scheme 1, further comprising:

[0227] (c) Following step (b), administer one or more doses of additional chemotherapy to the subject, wherein the additional chemotherapy is the same as or different from the chemotherapy of step (a); and

[0228] (d) After step (c), one or more doses of the adoptive cell therapy product are administered to the subject at a second effective amount that is the same as or different from the first effective amount.

[0229] Implementation Scheme 3. The method according to Implementation Scheme 1, wherein the lupus or its symptoms include: (i) cutaneous lupus; (ii) lupus nephritis; (iii) neuropsychiatric lupus; or (iv) pericarditis, atherosclerosis, angina pectoris, Raynaud's syndrome, nephropathy, metabolic syndrome, thyroid disorders, fibromyalgia, respiratory tract infection, skin infection, or urinary tract infection.

[0230] Implementation Scheme 4. The method according to any one of Implementation Schemes 1 to 3, wherein the chemotherapy comprises one or more of (a) cyclophosphamide (CY) or fludarabine (FLU); or (b) bendamustine.

[0231] Implementation Scheme 5. The method according to any one of Implementation Schemes 1 to 4, wherein the chemotherapy is administered one or more days prior to the administration of the adoptive cell therapy product; optionally wherein the chemotherapy is administered in one or more of the following cases: (i) three days prior to the administration of the adoptive cell therapy product, (ii) four days prior to the administration of the adoptive cell therapy product, or (iii) five days prior to the administration of the adoptive cell therapy product.

[0232] Implementation Scheme 6. The method according to any one of Implementation Schemes 1 to 5, wherein the chemotherapy comprises starting approximately 4 to 6 days prior to the first day of administration of the adoptive cell therapy product, and continuing for 3 days at approximately 250 mg / m². 2 Approximately 600 mg / m2 Daily dose of cyclophosphamide and at approximately 20 mg / m 2 Approximately 40 mg / m 2 The daily dose of fludarabine.

[0233] Implementation Scheme 7. The method according to any one of Implementation Schemes 1 to 6, wherein the chemotherapy comprises bendamustine and administered for two consecutive days at approximately 30 mg / m², starting approximately 4-6 days prior to day 1 of administration of the adoptive cell therapy product. 2 Approximately 150 mg / m 2 The daily dose is administered.

[0234] Implementation Scheme 8. The method according to any one of Implementation Schemes 1 to 7, wherein the engineered T lineage effector cells are derived from engineered induced pluripotent stem cells (iPSCs) containing TCR knockout and a polynucleotide encoding the CD19-CAR.

[0235] Implementation Scheme 9. The method according to any one of Implementation Schemes 1 to 8, wherein the first effective amount of the adoptive cell therapy product is about 1.8 × 10⁻⁶. 8 One cell to approximately 9 × 10 9 Cells, and optionally increasing in number.

[0236] Implementation Scheme 10. The method according to Implementation Scheme 9, wherein the first effective amount of the adoptive cell therapy product is approximately 3.6 × 10⁻⁶. 8 One cell, approximately 1 × 10 9 1 cell, approximately 3 × 10 9 One cell or approximately 9 × 10 9 Each cell.

[0237] Implementation Scheme 11. The method according to any one of Implementation Schemes 1 to 10, wherein the adoptive cell therapy product is cryopreserved and then thawed before application.

[0238] Implementation Scheme 12. The method according to any one of Implementation Schemes 1 to 11, wherein the adoptive cell therapy product is FT819.

[0239] Implementation Scheme 13. The method according to any one of Implementation Schemes 1 to 12, wherein the subject has been diagnosed with lupus.

[0240] Implementation Scheme 14. The method according to any one of Implementation Schemes 1 to 12, wherein the subject is positive for at least one of: (a) antinuclear antibody; (b) anti-dsDNA antibody; or (c) anti-Smith antibody.

[0241] Implementation Scheme 15. A method for achieving lupus remission or low activity in a subject according to any one of Implementation Schemes 1 to 14.

[0242] Implementation Scheme 16. The method according to Implementation Scheme 15, wherein the lupus remission or low activity is defined by DORIS (the definition of systemic lupus erythematosus remission), DORIS clinical remission during treatment, and / or LLDAS (low disease activity status of lupus).

[0243] Implementation Scheme 17. A method for controlling lupus flare-ups according to any one of Implementation Schemes 1 to 14.

[0244] Implementation Scheme 18. The method according to any one of Implementation Schemes 1 to 14, wherein the treatment includes reducing the production of autoantibodies in the subject during a longer disease-free interval without the use of immunosuppressive drugs; and / or reconstructing healthy B cell compartments in the subject compared to B cells under active lupus.

[0245] Implementation Scheme 19. The method according to any one of Implementation Schemes 1 to 18, wherein the subject has previously received one or more prior treatments, the one or more prior treatments including glucocorticoids, CY, mycophenolic acid or derivatives thereof, belimumab, methotrexate, azathioprine, anifurumab, rituximab, obbituzumab, cyclosporine, tacrolimus or vorosaporin.

[0246] Implementation Scheme 20. The method according to Implementation Scheme 19, wherein the subject has not responded to the one or more prior treatments.

[0247] Implementation Scheme 21. A method for treating lupus or its symptoms in a subject, the method comprising:

[0248] (a) administering one or more doses of chemotherapy to the subject; and

[0249] (b) After step (a), administer one or more doses of the adoptive cell therapy product to the subject in a first effective amount;

[0250] The adoptive cell therapy product comprises engineered T-lineage effector cells, wherein the engineered T-lineage effector cells express (i) CD19-CAR (chimeric antigen receptor) at the T-cell receptor α constant (TRAC) locus, and (ii) T-cell receptor (TCR) knockout; and

[0251] The subject has previously received one or more prior treatments and has not responded to the one or more prior treatments, including glucocorticoids, CY, mycophenolic acid or derivatives thereof, belimumab, methotrexate, azathioprine, anifrucizumab, rituximab, obbituzumab, cyclosporine, tacrolimus or vorosaponin.

[0252] Implementation Scheme 22. A method for treating lupus or its symptoms in a subject, the method comprising:

[0253] Administer one or more doses of the adoptive cell therapy product to the subject at a first effective amount;

[0254] The adoptive cell therapy product described herein comprises engineered NK lineage effector cells comprising CD38 knockout and expression of: (i) CD19-CAR (chimeric antigen receptor); (ii) an allogeneic immune defense receptor (ADR) targeting 4-1BB; (iii) a high-affinity, non-cleavable CD16 (hnCD16); and (iv) an interleukin-15 (IL15) / IL15 receptor fusion protein (IL15RF).

[0255] Implementation Scheme 23. The method according to Implementation Scheme 22, wherein the method does not include the application of chemotherapy for lymphatic regulation.

[0256] Example

[0257] The following examples are provided for illustrative purposes and not for limitation.

[0258] Example 1 - Activity and biodistribution of FT819

[0259] Autoreactive B cells are a key driver of various autoimmune diseases, such as systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis (RA), and scleroderma. Management of autoimmune diseases typically involves long-term use of immunosuppressants and B-cell-directing agents. For example, the anti-CD20 antibody rituximab has been shown to deplete peripheral blood B cells. However, the clinical efficacy of rituximab is limited and varies widely among different autoimmune patient populations due to incomplete clearance of autoreactive B cells from secondary lymphoid tissues. FT819 co-cultured with PBMCs from five healthy donors demonstrated potent and specific CD19+ B cell killing in an in vitro cytotoxicity assay. Similarly, in the same assay, PBMCs derived from SLE and RA patients showed durable CD19+ B cell elimination.

[0260] Biodistribution studies in immunodeficient mice demonstrated the ability of FT819 to distribute well throughout the mouse population, including in tertiary tissues associated with autoimmune diseases, such as bone marrow and spleen. The FT819-mediated B-cell effect was assessed by flow cytometry and immunohistochemistry in blood and tumor samples obtained from lymphoma patients treated with FT819. Peripheral blood B cells were barely detectable in most patients following FT819 infusion, with persistent suppression lasting up to 29 days. Importantly, B-cell depletion was also observed in lymph node biopsies, illustrating FT819's ability to reach secondary tissues. Recovery of peripheral B cells occurred 1–3 months after treatment cycles, indicating that prolonged B-cell hypoplasia was avoided through B-cell remodeling. Overall, FT819 demonstrates unique potential as a promising therapeutic paradigm for autoimmune diseases. FT819’s excellent safety profile in oncology and its readily available renewable source make it ideal for combination therapy with standard care regimens (including, but not limited to, glucocorticoids, CY, mycophenolate mofetil or their derivatives, belimumab, methotrexate, azathioprine, anifurumab, rituximab, obbituzumab, cyclosporine, tacrolimus, and vorozoprothiolane) in autoimmune diseases.

[0261] Example 2 - FT819 Study Design

[0262] Patients with lupus, an organ-critical disease, frequently experience cycles of remission and flare-ups; however, despite the availability of most medications, disease activity persists. This persistence leads to end-organ damage, resulting in conditions such as the need for a kidney transplant. Autoimmune therapy requires treatments capable of resetting the immune system. This study was designed to evaluate the safety, tolerability, and clinical activity of FT819 in a lupus population with organ-critical disease activity. It explored whether FT819 could provide a longer disease-free interval without the use of all immunosuppressive drugs, and / or reduce autoantibody production in patients, and / or remodel healthy B-cell compartments compared to B cells in active lupus.

[0263] The FT819 drug substance used in this embodiment comprises allogeneic T cells derived from a T-cell receptor (TCR) knockout iPSC lineage and expressing a CD19-targeting CAR. 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 phases. Cells harvested after T-cell expansion are then washed and designated as the drug substance. The composition of FT819 is homogeneous. The study described herein was designed to evaluate the safety, pharmacokinetics (PK), and anti-B-cell activity of FT819 in patients with SLE. Subjects were enrolled in two phases: a dose-escalation phase and a dose-expansion phase. After assessing safety and tolerability to define the maximum tolerated dose (MTD) for the dose-escalation phase, the dose-expansion phase further evaluated the safety and activity of FT819.

[0264] Human clinical studies consist of a screening period, followed by 2 or 3 days of chemotherapy, depending on the chemotherapy regimen (including standard of care regimens for autoimmune diseases), and then FT819 infusion on day 1. The DLT (dose-limiting toxicity) assessment period is from day 1 of FT819 administration to day 29, followed by post-treatment follow-up (Figure 1). As shown in Figure 1, bendamustine can be used as an alternative to CY / FLU. In the timeline of Figure 1, there is no day 0; instead, the number of days in the cycle progresses from day -5 to day -1, followed by day 1, at which time FT819 is infused. The FT819 treatment cycle continues until day 29. After completing the FT819 treatment cycle (defined as treatment cycle completion / early treatment discontinuation visit), participants enter the study's post-treatment follow-up (PTFU) period and may additionally enter long-term follow-up (LTFU), with regular monitoring for new and / or resolved treatment-related adverse events (TEAEs) and exploratory assessments. Participants who develop progressive disease (PD) during PTFU may be eligible for an additional retreatment cycle.

[0265] Standard lupus assessments were performed throughout treatment, including the mixed SLEDAI and Physician Global Assessment (PGA). Additionally, the Functional Assessment of Chronic Disease Therapy (FACIT) scale was used to assess fatigue. Standard laboratory measurements, as well as immunomarkers and autoantibodies, were obtained, and exploratory markers were collected for assay development.

[0266] Example 3 - FT819 Dosage Escalation and Dosage Amplification

[0267] The optimal dose of CAR T-cell therapy in lupus and other autoimmune diseases is unknown and has so far been based primarily on dose and schedules studied in oncology indications. The goal of the dose escalation phase in this study was to determine a dose level of FT819 not exceeding the MTD, suitable for further evaluation of dose expansion to determine the recommended phase 2 dose (RP2D). Continuous dose levels were evaluated according to the dose escalation rules provided herein to determine the continuation of dose escalation.

[0268] Dosing was based on CD19 CAR expression, with ≥90.0% of administered live FT819 cells expressing CD19 CAR. Dosage escalation began at DL1. Figure 2 As shown, the initial FT819 dose level (dose level 1 [DL1]) in the dose escalation was 3.6 × 10⁻⁶. 8 Start with VC / dose (living cells per dose). As the DL1 dose-escalation group clears DLT assessment, dose escalation proceeds to the next dose level (DL2). When DL1 does not exceed the MTD in the lupus setting, successive dose levels will not exceed 3 times (±15% variance) of the previously cleared highest dose level. If DL1 shows evidence of clinical activity (SLEDAI of 0 within 3 months), a lower FT819 dose is explored. In some implementations, if DL1 is intolerable or exceeds the MTD (maximum tolerated dose), DL0, 1.8 × 10⁻⁶, is evaluated. 8 One VC / dose; and when DL0 does not exceed MTD, dose amplification at DL0 is performed under the same amplification protocol. In the absence of dose-limiting toxicities (DLT), dose levels can be further explored based on observed safety, tolerability, activity, PK, and pharmacodynamic data.

[0269] The FT819 dose to be 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. One or more dose levels with cleared DLT assessments were 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.

[0270] Example 4 - FT819 Treatment Protocol

[0271] The goal of chemotherapy prior to FT819 administration is to create an immune environment suitable for the sustained and proliferative activity of FT819. Although the mechanism is not fully understood, the combination of CY and FLU may induce cytokines that promote homeostatic proliferation and eliminate regulatory immune cells and other immune system elements that compete for homeostatic cytokines, thereby promoting the homeostatic proliferation of FT819.

[0272] In some implementations, bendamustine is an alternative to CY / FLU treatment prior to administration of the cell product in subjects experiencing severe hemorrhagic cystitis with a history of CY. Bendamustine is an alkylating agent with well-characterized and favorable safety profiles, used as a component of immunochemotherapy regimens. The planned doses for CY / FLU and bendamustine are the same as those used for approved CAR T-cell therapies (Breyanzi). ® USPI; Kymriah USPI; Yescarta USPI).

[0273] In patients with lupus, the starting dose of FT819 is approximately 3.6 × 10⁻⁶ after a chemotherapy course consisting of CY / FLU or bendamustine. 8 One VC / dose (Figure 1). Dose escalation is performed by increasing relative dose levels, starting at DL2, and consisting of intervals no greater than 3 times the previously cleared dose level. The relative dose intervals are designed to allow flexibility in dose escalation in smaller increments in any non-DLT safety observation, making smaller escalation increments more beneficial or associated with lower safety risks. Multiple dose levels that have been cleared in the DLT assessment during dose escalation can be evaluated in dose expansion to further define the risk-benefit profile of each dose level to determine the dose at which FT819 confers the greatest clinical benefit in lupus. In some embodiments, the dose is the amount described herein. In some examples, lower doses of FT819 may be explored if a patient has demonstrated safety and disease remission after receiving a first dose of FT819. Lower doses of FT819 for lupus treatment are possible compared to those used for B-cell malignancies because the B-cell burden in SLE may be much lower than that in B-cell malignancies.

[0274] Example 5 - Patient populations for FT819 treatment of lupus in each study group

[0275] Patients eligible for FT819 treatment include those diagnosed with SLE according to the European League Against Rheumatism (EULAR) / American College of Rheumatology (ACR) classification criteria and who are positive for at least one autoantibody, including but not limited to (1) positive antinuclear antibody (ANA) ≥1:160 on immunofluorescence assay, exhibiting a homogeneous or ring pattern characteristic of lupus; (2) positive anti-dsDNA, confirmed by a Crithidia test; and (3) positive anti-SmithKline. In some implementations, patients have active SLE at screening.

[0276] Active SLE is defined as a SLEDAI of 8 or higher (where the clinical SLEDAI is 4 or higher, excluding alopecia, mucosal ulceration, and fever); and one or more major organ systems with a British Isles Lupus Assessment Group (BILAG) A score, excluding musculoskeletal, mucocutaneous, and / or systemic organ systems. In some embodiments, SLE patients eligible for FT819 treatment are previously untreated. In some embodiments, SLE patients eligible for FT819 treatment have previously failed to respond to glucocorticoids (e.g., despite treatment, disease symptoms have not significantly improved or have worsened). In some embodiments, SLE patients eligible for FT819 treatment have previously failed at least one treatment, including but not limited to: CY, mycophenolate mofetil or derivatives thereof, belimumab, methotrexate, azathioprine, anifrucizumab, rituximab, obbituzumab, cyclosporine, tacrolimus, and vorozoprothiolane. In some implementations, SLE patients eligible for FT819 treatment have failed at least two of the aforementioned treatments.

[0277] Example 6 - Research Intervention and Companion Therapy

[0278] Research interventions are all pre-designated research and non-research medical products intended for administration to research participants during the research period. These medical products include FT819 (designated as a research medical product (IMP)) and authorized adjunctive medical products (AxMPs) as non-IMPs, such as cyclophosphamide (CY), fludarabine (FLU), and bendamustine. Research interventions are defined according to Table 1 below:

[0279]

[0280] The FT819 drug product comprises allogeneic T cells derived from a cloned TCR knockout iPSC line and expressing a CD19-targeting CAR regulated by the TRAC locus promoter. FT819 cells are suspended in an infusion medium containing albumin (human) and DMSO. In some embodiments, FT819 cells are contained in cryopreserved bags and thawed at the administration site. In some embodiments, FT819 is administered using an IV (intravenous) administration kit with an in-line filter.

[0281] Prior to FT819 administration, participants received a pre-administration of 650 mg of acetaminophen orally (PO) 4 to 6 hours before and after administration, and a pre-administration of 25 to 50 mg of diphenhydramine via PO or IV. Corticosteroids were not used as pre-administration of FT819.

[0282] Prior to CY administration, participants immediately received 500 mL of IV saline according to institutional standards. Following assessment of hydration status, participants may receive additional IV saline after CY administration. Dosage adjustments may also be made based on body weight / creatinine ratio. Administered at approximately 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:

[0283]

[0284] On days -5, -4, and -3 of the treatment cycle, at approximately 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).

[0285] In some implementations, the CY / FLU dosage and schedule can be modified. For participants with moderately impaired renal function (creatinine clearance of 30 mL / min / 1.73 mcg), this may be necessary. 2 -70mL / min / 1.73m 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.

[0286] In some implementations, the dosage and schedule for bendamustine may be modified. 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.

[0287] In some implementations of FT819 treatment for SLE, concomitant therapies are given in conjunction with the intervention. Concomitant therapies include any medications used by the patient other than the intervention specified in the protocol (e.g., prescription drugs, over-the-counter drugs, vaccines, herbal or homeopathic medications, nutritional supplements). In some implementations, concomitant medications are used for SLE. In some implementations, concomitant medications are used for non-SLE cases. In some implementations, concomitant medications include those used for lupus flare-ups. In some implementations, concomitant medications include those used for SAE.

[0288] In some implementations, lupus patients receiving FT819 may also receive prednisone and / or hydroxychloroquine. In some implementations, prednisone is administered at a low dose of up to 10 mg daily or the equivalent of a PO dose. In some implementations, systemic corticosteroids are avoided via intramuscular or intravenous routes.

[0289] Example 7 - Disease Response Assessment and Efficacy Analysis

[0290] The disease response was assessed using a combination of SLEDAI (Systemic Lupus Erythematosus Disease Activity Index), FACIT (Functional Assessment of Chronic Disease Therapy), and PGA.

[0291] The hybrid SLEDAI assay measures the amount of lupus disease activity, identifies disease improvement and exacerbation, and assesses dozens of lupus manifestations. Each parameter / manifestation is scored if present, with scores from 1 to 8 weighted. Parameters with a weight of 8 include epilepsy, mental disorders, organic brain syndromes, visual disturbances, neurological disorders, lupus headache, cerebrovascular accident, and vasculitis. Parameters with a weight of 4 include arthritis, myositis, urethral discharge, hematuria, proteinuria, and pyuria. Parameters with a weight of 2 include rash, alopecia, mucosal ulcers, pleurisy, pericarditis, low complement, and increased DNA binding. Parameters with a weight of 1 include fever, thrombocytopenia, and leukopenia. Scores of 1 to 5 reflect mild disease, 6 to 12 indicate moderate disease, and 13 to 20 indicate severe disease. The inclusion criteria for the study required a mixed SLEDAI score of 8 or higher, with a clinical SLEDAI score of 4 or higher excluding alopecia, mucosal ulcers, and fever; and one or more major organ systems with a BILAG A score, excluding musculoskeletal, mucocutaneous, and / or systemic organ systems (involving kidney, peripheral / myocarditis, pleurisy / lung involvement, and / or non-CNS vasculitis must be moderate to severe and considered reversible by the investigator).

[0292] FACIT is an assessment scale that measures an individual's level of fatigue during daily activities over the past week. The scale measures twelve items, and each item is answered on a scale of 0 (none at all) to 4 (very many). A lower total score reflects more fatigue.

[0293] The Visual Analogue Scale (PAG) is a physician-assessed visual analog scale ranging from 0 (“no disease activity”) to 3 (“most severe disease activity”) used to measure disease activity in patients with SLE during the preceding month. The scale incorporates values ​​1 and 2 as internal markers to categorize disease activity as mild (≥0.5 to 1), moderate (>1 to ≤2), and severe (>2 to 3). The PGA aims to assess overall disease activity considering the severity of activity manifestations and clinical laboratory findings, but excluding organ damage, serological findings, and subjective findings unrelated to disease activity.

[0294] Efficacy analysis was conducted based on populations with evaluable efficacy. SLE remission was defined in three ways: DORIS (the definition of systemic lupus erythematosus remission) complete remission, DORIS clinical remission during treatment, and LLDAS (low disease activity status of lupus).

[0295] The clinical remission of DORIS during treatment is described as follows:

[0296] • Clinical SLE Disease Activity Index (SLEDAI) = 0, excluding measurements of autoantibodies and C3 / C4 levels.

[0297] • Permissible serological activity

[0298] • SELENA-SLEDAI Physician Overall Assessment (PGA) ≤ 0.5 (0-3 scale)

[0299] • Permissible low doses of glucocorticoids (e.g., prednisone ≤ 5 mg / day)

[0300] • Permitted maintenance antimalarial drugs, immunosuppressants, and / or stabilizing (maintenance) biological agents

[0301] DORIS complete relief is described as follows:

[0302] • Clinical SLEDAI=0, excluding measurements of autoantibodies and C3 / C4 levels.

[0303] • No serum activity

[0304] • SELENA-SLEDAI PGA ≤0.5 (0-3 scale)

[0305] • Glucocorticoid-free

[0306] • Permitted maintenance antimalarial drugs, but not immunosuppressants and / or biological agents.

[0307] The operational definition of low disease activity status (LLDAS) for lupus is met when all of the following criteria are met:

[0308] • Mixed systemic lupus erythematosus disease activity index (SLEDAI) ≤4, with no activity in major organ systems;

[0309] • No new features of lupus disease activity were observed compared to previous assessments;

[0310] • Permissible serological activity (provided that total mixed SLEDAI ≤ 4).

[0311] • SELENA-SLEDIA Physician Overall Assessment (PGA) ≤1 (0-3 scale);

[0312] • The current dosage of prednisolone (or equivalent) is ≤7.5 mg; and

[0313] • Standard maintenance doses of immunosuppressive drugs and approved biologics, excluding investigational drugs.

[0314] The percentage of participants who met these criteria was calculated at the 3rd, 6th, 9th, and 12th months, as well as overall. The denominator for the percentage of participants achieving DORIS or LLDAS was based on the number of participants who completed the necessary assessments at that visit.

[0315] As a secondary endpoint, the pharmacokinetic (PK) of FT819 was assessed by detecting FT819 in peripheral blood (PB) after FT819 administration. To characterize the PK of FT819, PB samples were collected before and after FT819 infusion on the day of FT819 administration. Descriptive summaries of the maximum observed concentration (Cmax) and area under the curve (AUC) are provided, including sample size (n), mean, standard deviation, standard error, median, Q1, Q3, and minimum and maximum values.

[0316] The pharmacokinetic assessment of FT819 for one cycle is measured by changes in serum immunoglobulin levels (i.e., IgG, IgM, IgA), serum complement components (i.e., C3, C4), and blood lymphocyte subsets (e.g., T, B, NK) relative to baseline at each scheduled assessment. Exploratory analyses are descriptive and include assessment of potential predictive and prognostic biomarkers in PB, serum, or urine. The association between FT819 and other pharmacokinetic markers (PK) and safety and clinical activity will be descriptively assessed.

[0317] The association between baseline clinical and disease characteristics and the safety and clinical activity of FT819 was also explored. Blood samples were collected for allogeneic immunization against human leukocyte antigen (HLA) of the FT819 product via histological antibody testing. Additional blood, serum, and urine samples were collected for exploratory biomarker analysis, including but not limited to:

[0318] • Measurement of PB cytokine levels

[0319] • Measurement of autoantibody and complement levels

[0320] • Measurement of protein biomarkers of disease activity in exploratory immune surveillance

[0321] • PBMC functional characterization and immunophenotyping, such as T cell subset analysis

[0322] • Cellular immunogenicity

[0323] • Assessment of urinary protein biomarkers for disease activity

[0324] In some implementations, assays and other exploratory analyses include, but are not limited to, analyses of lymphocytes, T cell activation, TCR repertoire, inflammation-related cytokines, complement activation markers, and B cell subsets and activation.

[0325] Example 8 - FT819 demonstrates rapid and deep extraction of CD19+ B cells from peripheral blood of mismatched SLE patients. Exhaustion

[0326] Rapid and deep depletion of CD19+ B cells was observed in a 72-hour kill assay of PBMCs (E:T 1:1) from mismatched blood samples from patients with SLE. Figure 3 In patients with autoimmune diseases, the estimated population of diseased CD19+ B cells in peripheral tissues and tissues is as high as 300 million cells. Figure 4 As shown, FT819 exhibited enhanced targeting of CD19+ B cells from mismatched blood samples from SLE patients compared to autologous or allogeneic CAR T cells. In an E:T ratio analysis of this specific assay, results indicated that FT819 adequately and completely depleted the CD19+ B cell load in SLE patients at doses of 100-300 million CAR T cells. In this analysis, autologous CAR T cells were primary T cells transduced with a lentivirus containing the CAR transgene and cultured for 4 days for recovery and expansion; and allogeneic CAR T cells were primary T cells engineered with CRISPR targeting the TRAC locus containing the CAR transgene and cultured for 11 days for recovery and expansion.

[0327] Example 9-FT819 demonstrates dose-dependent PK, rapid and deep B cell exhaustion, and initial B cell remodeling. Key treatment mechanisms

[0328] In vivo CAR T cell expansion and persistence were measured by ddPCR based on the CAR transgene copy number per microgram of genomic DNA in blood samples. The starting cell dose for the FT819 clinical trial in patients with SLE was 360 million cells (NCT06308978). Figure 5The FT819 PK (mean ± SEM) of 90 million and 360 million cells were shown in a patient treatment setting for B-cell lymphoma (BCM). The FT819 profile shows the expansion kinetics reaching Cmax at approximately day 8, dose-dependent PK increase, and its controlled and defined persistence in peripheral blood, which enables more rapid remodeling of B-cell compartments and reduces complications associated with B-cell dysplasia.

[0329] Figure 6 The data presented include 23 patients with baseline B-cell counts within the normal range (up to 1,000 cells / μL; mean ± SEM). Baseline represents peripheral blood B-cell counts prior to standard conditioning chemotherapy and a single-dose FT819 treatment. During a single-dose FT819 treatment cycle in these patients, rapid and profound B-cell depletion was observed in peripheral blood using flow cytometry, accompanied by persistent B-cell suppression. Figure 6 ).like Figure 7 As shown, following a single dose of FT819, two patients in the case study who were resistant to Cy / Flu opsonization chemotherapy also experienced rapid and deep B-cell depletion, demonstrating that FT819's ability to deplete B-cells is independent of Cy / Flu opsonization. Furthermore, Figure 8 This includes four patients who received bendamustine as an alternative to Cy / Flu conditioning chemotherapy and a single dose of FT819. These patients had baseline B-cell counts within the normal range (up to 1,000 cells / μL), and their peripheral blood B-cell depletion was rapid and deep, and / or persistent. Figure 7 and Figure 8 Together, it was shown that FT819 can rapidly, deeply, and persistently deplete CD19+ B cells without the use of one or both of cyclophosphamide and fludarabine as opsonizing agents, and that bendamustine can be used as an alternative opsonizing agent for FT819.

[0330] Figure 9 This study presents a case study of plasma cell depletion and B cell remodeling in three patients with different B cell repositories at baseline. As shown, the remodeled cell population consisted of naïve B cells during 6–10 weeks following a single dose of FT819.

[0331] Example 10-FT819 demonstrates the achievement of immune repositioning in autoimmune diseases in secondary and tertiary tissues. Key treatment mechanisms

[0332] In a case study of patients treated with opsonization chemotherapy followed by a single dose of FT819 (90M cells), FT819 was detected on day 9 and persisted in the bone marrow. CD19+ cells were completely eliminated from the bone marrow and peripheral blood at the end of the treatment cycle. Figure 10 ).

[0333] Figure 11 This study presents a case study of a patient treated with opsonization chemotherapy followed by a single dose of FT819 containing 180M cells. The patient had three prior lines of therapy, including autologous CD19-targeted CAR T-cell therapy. Baseline lymph node biopsy showed DLBCL cells with high CD19 expression. After one cycle, a single dose of FT819 resulted in rapid, deep, and sustained depletion of CD19+ B cells in peripheral blood and a reduction of >50% in CD19+ tumor cells in secondary lymphoid tissue. Retreatment with a single dose of FT819 resulted in a complete metabolic response in FDG-PET, with a reduction of approximately 80% in target lesion 1 and complete elimination of target lesion 2 by the end of cycle 2.

[0334] Figure 12 This study presents a case study of a patient treated with opsonization chemotherapy followed by a single dose of FT819 containing 90M cells. The patient had six prior lines of therapy and was refractory to autologous CD19-targeted CAR T-cell therapy. A single dose of FT819 resulted in sustained depletion of CD19+ B cells in the peripheral blood and complete elimination of CD19+ clumps in the liver, indicating the potential of FT819 for transport, infiltration, and function in secondary and tertiary tissues.

[0335] Example 11 - Analysis of peripheral blood samples from SLE patients before and after treatment

[0336] In a phase 1 autoimmune study of single-dose FT819 administration, pre-treatment peripheral blood samples obtained from patients prior to clinical administration of opsonization chemotherapy and FT819 were used for 24-hour co-culture assays. Figure 13 The results showed that, in this in vitro translation assessment, patients’ CD19+ B cells were deeply depleted upon exposure to 360 million cells of FT819, indicating potent in vitro CD19+ B cell depletion after a single dose of FT819 treatment.

[0337] Patients from the FT819 Phase 1 Autoimmune Study received bendamustine as a modulatory chemotherapy treatment, followed by a single dose of FT819. Figure 14 As shown, post-treatment analysis of peripheral blood from the first SLE patient revealed rapid, deep, and sustained depletion of peripheral blood B cells by day 8, as well as initial recovery of T cell compartments after bendamustine conditioning.

[0338] Example 12 - FT522 Disease Response Assessment and Efficacy Analysis

[0339] "FT522" refers to an engineered NK cell therapy derived from a clone-engineered iPSC lineage, engineered to enhance innate immunity in multiple modalities: (1) anti-CD19-CAR; (2) an allogeneic immune defense receptor (ADR) targeting 4-1BB; (3) a high-affinity, non-cleavable CD16 (hnCD16); and (4) an interleukin-15 (IL15) / IL15 receptor fusion protein (IL15RF), and CD38 knockout. The potential of FT522 to induce CD19+ B cell depletion in a range of autoimmune diseases, including without modulating patients with intensive chemotherapy, has been evaluated. In a diffuse Nalm6 leukemia model composed of CD19+ target cells resistant to T-cell killing, ADR-armed CD19-targeting CAR NK cells exhibited robust CD19+ target cell killing in vivo in the presence of allogeneic reactive T cells. This suggests that FT522 has the potential to deplete CD19+ B cells and drive a clinical response without requiring intensive chemotherapy to modulate the patient's response. Further studies were conducted to evaluate the potential of combining FT522 with monoclonal antibody therapy in depleting lineages that generate autoantibodies against both CD19+ B cells and CD38+ plasma cells.

[0340] like Figure 15 As shown, FT522 demonstrated better CAR-mediated CD19+ B cell killing compared to TCR-free allogeneic primary CAR-T (pCAR-T) cells in 24-hour cytotoxicity assays with various E:T ratios and in two different SLEPBMC donors.

[0341] In the in vitro allogeneic re-challenge assay, SLE donor PBMCs were co-cultured with pCAR-T cells (primary CAR-T cells), FT596 (ADR-negative), or FT522 (ADR-armed) for 8 days, and then re-administered with effector cells and re-challenged with SLE donor PBMCs, allowing for an additional 6 days of co-culture. Figure 16A On days 4 and 6, flow cytometry analysis of CD3+ SLE donor PBMC T cells with CD25 and 4-1BB demonstrated T cell activation and allogeneic response to pCAR-T and FT596 cells, but not to FT522 cells. Figure 16B The image shows the total SLE donor PBMC-derived T cells expanded on day 6 in response to allogeneic challenge, and the CD19+ cell count normalized relative to individual PBMCs before re-challenge. Figure 16C After the allogeneic re-challenge assay was completed, pCAR-T and FT596 cells were exhausted, with FT522 cells demonstrating sustained and effective control over CD19+ cell counts at the end of the assay. Figure 16D (Normalized relative to individual PBMCs). In summary, FT522 exhibits rapid and deep CD19+ B cell depletion and eliminates allogeneic reactive 4-1BB+ T cells while maintaining functional persistence against SLE donor PBMCs.

[0342] like Figure 17A As shown, in the in vitro killing assay of the WIL2S cell line, FT522 elicited a stronger antitumor effect compared to FT596 at the tested E:T ratio. Figure 17B As shown, in a phase 1 study in the B-cell lymphoma (BCL) setting, FT522 demonstrated higher PK on day 4 than an equivalent dose of FT596 administered on day 1 at 3e8 cells, and also demonstrated higher total AUC over the treatment cycle (3e8 cells per single dose of FT596 or 1.8e9 cells per three doses of FT596). Figure 18A and Figure 18B In this context, baseline represents the peripheral blood B-cell count prior to standard conditioning chemotherapy. The 30-day treatment cycle was divided into multiple phases. Clinical activity of FT596 monotherapy (n=9) was... Figure 18A The clinical activity (n=2) of the FT522 combined with rituximab was shown in [the figure]. Figure 18B The study demonstrates that FT522 rapidly, deeply, and continuously depletes CD19+ B cells.

[0343] 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.

[0344] 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.

[0345] 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 lupus or its symptoms in a subject, the method comprising: (a) Administering one or more doses of chemotherapy to the subject; as well as (b) After step (a), 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 effector cells, which include (i) expression of CD19-CAR (chimeric antigen receptor) at the T cell receptor α constant (TRAC) locus and (ii) T cell receptor (TCR) knockout.

2. The method according to claim 1, further comprising: (c) Following step (b), administer one or more doses of additional chemotherapy to the subject, wherein the additional chemotherapy is the same as or different from the chemotherapy of step (a); and (d) After step (c), one or more doses of the adoptive cell therapy product are administered to the subject at a second effective amount that is the same as or different from the first effective amount.

3. The method according to claim 1, wherein the lupus or its symptoms include: (i) cutaneous lupus; (ii) lupus nephritis; (iii) neuropsychiatric lupus; or (iv) pericarditis, atherosclerosis, angina pectoris, Raynaud's syndrome, nephropathy, metabolic syndrome, thyroid disorders, fibromyalgia, respiratory infection, skin infection, or urinary tract infection.

4. The method of claim 1, wherein the chemotherapy comprises one or more of (a) cyclophosphamide (CY) or fludarabine (FLU); or (b) bendamustine.

5. 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 in one or more of the following cases: (i) three days prior to the administration of the adoptive cell therapy product, (ii) four days prior to the administration of the adoptive cell therapy product, or (iii) five days prior to the administration of the adoptive cell therapy product.

6. The method of claim 1, wherein the chemotherapy comprises starting approximately 4-6 days prior to day 1 of administration of the adoptive cell therapy product, and continuing for 3 days at approximately 250 mg / m². 2 Approximately 600 mg / m 2 Daily dose of cyclophosphamide and at approximately 20 mg / m 2 Approximately 40 mg / m 2 The daily dose of fludarabine.

7. 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 day 1 of administration of the adoptive cell therapy product. 2 Approximately 150 mg / m 2 The daily dose is administered.

8. The method of claim 1, wherein the engineered T lineage effector cells are derived from engineered induced pluripotent stem cells (iPSCs) containing TCR knockout and a polynucleotide encoding the CD19-CAR.

9. The method of claim 1, wherein the first effective amount of the adoptive cell therapy product is about 1.8 × 10⁻⁶. 8 One cell to approximately 9 × 10 9 Cells, and optionally increasing in number.

10. The method of claim 9, wherein the first effective amount of the adoptive cell therapy product is about 3.6 × 10⁻⁶. 8 One cell, approximately 1 × 10 9 1 cell, approximately 3 × 10 9 One cell or approximately 9 × 10 9 Each cell.

11. The method of claim 1, wherein the adoptive cell therapy product is cryopreserved and then thawed prior to application.

12. The method of claim 1, wherein the adoptive cell therapy product is FT819.

13. The method of claim 1, wherein the subject has been diagnosed with lupus.

14. The method of claim 1, wherein the subject is positive for at least one of: (a) antinuclear antibody; (b) anti-dsDNA antibody; or (c) anti-Smith antibody.

15. A method for achieving lupus remission or low activity in a subject according to any one of claims 1 to 14.

16. The method of claim 15, wherein the lupus remission or low activity is defined by DORIS (the definition of systemic lupus erythematosus remission), DORIS clinical remission during treatment, and / or LLDAS (low disease activity status of lupus).

17. A method for controlling lupus flare-ups according to any one of claims 1 to 14.

18. The method of any one of claims 1 to 14, wherein the treatment comprises reducing the production of autoantibodies in the subject during a prolonged disease-free interval without the use of immunosuppressive drugs; and / or reconstructing healthy B cell compartments in the subject compared to B cells under active lupus.

19. The method according to any one of claims 1 to 14, wherein the subject has previously received one or more prior treatments, the one or more prior treatments including glucocorticoids, CY, mycophenolic acid or derivatives thereof, belimumab, methotrexate, azathioprine, anifurumab, rituximab, obbituzumab, cyclosporine, tacrolimus or vorosaponin.

20. The method of claim 19, wherein the subject has not responded to the one or more prior treatments.

21. A method for treating lupus or its symptoms in a subject, the method comprising: (a) Administering one or more doses of chemotherapy to the subject; as well as (b) After step (a), 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 effector cells, wherein the engineered T-lineage effector cells express (i) CD19-CAR (chimeric antigen receptor) at the T-cell receptor α constant (TRAC) locus and (ii) T-cell receptor (TCR) knockout; and The subject has previously received one or more prior treatments and has not responded to the one or more prior treatments, including glucocorticoids, CY, mycophenolic acid or derivatives thereof, belimumab, methotrexate, azathioprine, anifrucizumab, rituximab, obbituzumab, cyclosporine, tacrolimus or vorosaponin.

22. A method for treating lupus or its symptoms in a subject, the method comprising: Administer one or more doses of the adoptive cell therapy product to the subject at a first effective amount; The adoptive cell therapy product described herein comprises engineered NK lineage effector cells comprising CD38 knockout and expression of: (i) CD19-CAR (chimeric antigen receptor); (ii) an allogeneic immune defense receptor (ADR) targeting 4-1BB; (iii) a high-affinity, non-cleavable CD16 (hnCD16); and (iv) an interleukin-15 (IL15) / IL15 receptor fusion protein (IL15RF).

23. The method of claim 22, wherein the method does not include the application of chemotherapy for lymphatic regulation.

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