B cell lineages derived from pluripotent cells

Abstract

The present disclosure provides an efficient ex vivo process for generating B cell lineages from human induced pluripotent stem cells (iPSCs). In various embodiments, cells generated according to the present disclosure are functional and / or more closely resemble corresponding lineages isolated from peripheral blood or lymphoid organs. The present invention in some aspects provides isolated cells and cell compositions produced by the methods disclosed herein, as well as methods for cell therapy.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 413,454, filed October 5, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted via EFS-Web in XML format and is incorporated herein by reference in its entirety. The XML copy, created on September 26, 2023, is named GRU-017PC_Sequence_Listing.xml and is 30,036 bytes in size. [Background technology]

[0003] B cell lineages play a key role in tissue maintenance and help coordinate immune responses with effectors and regulators. However, their clinical use as cell therapy is hindered by the small number of such cells that can be isolated from typical leukapheresis products. Therefore, the development of large-scale, commercially available B lymphocyte lineages would be an attractive tool for combating cancer and infectious diseases, among other things. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure, in various aspects and embodiments, provides methods for generating hematopoietic lineages for cell therapy, including B cells or their progenitors, and their precursors. In various embodiments, the present invention provides an efficient ex vivo process for developing such hematopoietic lineages from human induced pluripotent stem cells (iPSCs), including gene-edited iPSCs. In various embodiments, cells generated according to the present disclosure are functional and / or more closely resemble corresponding lineages isolated from peripheral blood or bone marrow. The present invention also provides isolated cells and cell compositions produced by the methods disclosed herein, as well as methods for cell therapy.

[0005] In one aspect, the present disclosure provides a method for preparing a cell population containing myeloid cells of the innate immune system. The method includes preparing a pluripotent stem cell (PSC) population, such as an induced pluripotent stem cell (iPSC) population differentiated into embryoid bodies, and enriching for CD34+ cells, thereby preparing a CD34+-enriched population. Endothelial-hematopoietic transformation (EHT) is induced in the CD34+-enriched population, thereby preparing a hematopoietic stem cell (HSC) population, optionally followed by further enrichment of CD34+ cells. The resulting HSC population (or a fraction thereof) can differentiate into myeloid lineages of the innate immune system (e.g., phagocytes or their precursors). In some embodiments, the present disclosure provides a method for generating B cells, B-CAR cells, and immature and mature B cells (or their precursors) from the HSC population ex vivo.

[0006] In various embodiments, iPSCs are prepared by reprogramming somatic cells. In some embodiments, iPSCs are generated from somatic cells, such as (but not limited to) fibroblasts or PBMCs (or cells isolated therefrom). In some embodiments, iPSCs are derived from CD34+ cells isolated from peripheral blood.

[0007] In various embodiments, iPSCs are genetically edited to support HLA matching, such as deletion of one or more HLA class I and / or class II alleles. For example, iPSCs can be genetically edited to delete one or more of HLA-A, HLA-B, and HLA-C, and to delete one or more of HLA-DP, HLA-DQ, and HLA-DR. In certain embodiments, iPSCs retain expression of at least one HLA class I and at least one HLA class II complex. In certain embodiments, iPSCs are homozygous for at least one retained class I and class II locus. In some embodiments, iPSCs are genetically edited to delete one or more of HLA-A, HLA-B, and HLA-C alleles, and to delete one or more of HLA-DP, HLA-DQ, and HLA-DR alleles. In certain embodiments, iPSCs are homozygous for at least one retained class I and class II locus. In some embodiments, iPSCs are genetically edited to delete one or more of HLA-A, HLA-B, and HLA-C alleles. neg , homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg In some embodiments, the iPSCs are further homozygous for HLA-DRB1.

[0008] In various embodiments, iPSCs are prepared and expanded using a culture system. The expanded iPSCs can be harvested from the culture to generate embryoid bodies (EBs). EBs, generated by differentiation of iPSCs, are three-dimensional aggregates of iPSCs that contain three (or alternatively, two or one) embryonic germ cell layers, depending on the differentiation method(s). In some embodiments, the process includes harvesting CD34+ enriched cells from the EBs and inducing endothelial and hematopoietic differentiation.

[0009] In some embodiments, iPSC differentiation proceeds until the cells are at least about 20% CD34+ or at least about 30% CD34+. In some embodiments, CD34 enrichment and EHT can be induced between days 7 and 14 of iPSC differentiation. Differentiation of iPSCs can be by known techniques. In some embodiments, iPSC differentiation involves factors such as, but not limited to, a combination of Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3.

[0010] Induction of EHT can be by any known process. In some embodiments, induction of EHT generates a hematopoietic stem cell (HSC) population including LT-HSC. In some embodiments, EHT generates HSCs through endothelial or hemogenic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological, and / or genetic means (e.g., via stimulation, inhibition, and / or genetic modification). In some embodiments, EHT generates a stem cell population including one or more of long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC), and hematopoietic stem progenitor cells.

[0011] In some embodiments, the method involves increasing the expression or activity of DNA (cytosine-5-)-methyltransferase 3 beta (Dnmt3b) in PSCs, embryoid bodies, CD34+ enriched cells, ECs, HECs, or HSCs, which may be by mechanical, genetic, biochemical, or pharmacological means. For example, in some embodiments, the cells are contacted with an effective amount of an agonist of a mechanosensitive receptor or mechanosensitive channel that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezol. An exemplary Piezol agonist is Yodal. In various embodiments, pharmacological Piezol activation is applied to CD34+ cells harvested from EBs. In some embodiments, this process does not involve increasing the expression of dnmt3b, such as by using a Piezo1 agonist.

[0012] In various embodiments, CD34+ cells (e.g., suspension and / or adherent cells) are harvested from cultures undergoing endothelial-hematopoietic transition between days 10 and 20 of iPSC differentiation, such as between days 12 and 17 of iPSC differentiation. Hematopoietic stem cells (HSCs), which can give rise to innate myeloid, erythroid, and lymphoid lineages, can be identified based on the expression of CD34 and the absence of lineage-specific markers (termed Lin-).

[0013] In various embodiments, the HSC population or a fraction thereof is differentiated into a hematopoietic lineage, which may be selected from, in particular, the B cell lineage and their progenitors and progeny, including multipotent progenitors (MPPs), common lymphoid progenitors (CLPs), common lymphoid 2 progenitors (LCA-2), and B cells. The B cells produced may be early pre-B cells, late pre-B cells, pre-B cells, and immature B cells capable of producing B cells. In various embodiments, the present disclosure provides methods for the ex vivo production of cell populations corresponding to transitional B cells, regulatory B cells, marginal zone B cells, follicular B cells, activated B cells, memory B cells, and plasma B cells (collectively referred to as "B cells").

[0014] In some embodiments, the B cells are further modified to express a chimeric antigen receptor (CAR). Additionally, or optionally, the B-CAR can be engineered to express and / or secrete cytokines (e.g., IL-4, IL-6, IL-15, etc., or interferons) to make the CAR-expressing cells more potent in targeting tumors (for example). In a non-limiting example, the cells can be efficiently transduced by a vector, such as, but not limited to, a retroviral or non-integrating viral vector carrying the CAR, or a non-viral vector. In some embodiments, the CAR can target a tumor-associated antigen or marker.

[0015] In another aspect, the present invention provides cell populations or pharmaceutically acceptable compositions thereof, comprising B cell lineages or precursors thereof, which may be produced by the methods described herein. In some embodiments, the cell populations are capable of engraftment in the thymus, spleen, or secondary lymphoid organs upon administration to a subject in need thereof. In various embodiments, compositions for cell therapy are prepared comprising the cell population and a pharmaceutically acceptable vehicle. In some embodiments, the cell populations are HLA-A neg , homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 negIn some embodiments, the cell population is further homozygous for HLA-DRB1. In various embodiments, the composition comprises a myeloid lineage selected from one or more of monocytes, macrophages, dendritic cells, neutrophils, and myeloid progenitor cells.

[0016] In another aspect, the present invention provides methods for cell therapy comprising administering a cell population described herein or a pharmaceutically acceptable composition thereof to a human subject in need thereof. In various embodiments, the methods described herein are used to treat hematological (malignant and non-malignant), bone marrow, immune, and infectious diseases. In various embodiments, the human subject has a condition comprising one or more of lymphopenia, cancer, immunodeficiency, and autoimmune disease.

[0017] Other aspects and embodiments of the present disclosure will be apparent from the following detailed disclosure and examples. [Brief explanation of the drawings]

[0018] [Figure 1] Figure 1 shows that ETV2 overexpression (OE) does not affect pluripotency. Figure 1 shows a FACS plot showing the transduction efficiency of iPSCs using an adenoviral vector to overexpress ETV2 and GFP sequences. ETV2 overexpression does not affect iPSC stemness, as indicated by the expression of the TRA-1-60 stemness marker. [Figure 2] ETV2 overexpression (OE) increases the yield of hemogenic endothelial cells. Representative flow cytometry analysis and relative quantification of hemogenic endothelial cells (described as CD235a-CD34+CD31+) demonstrates that ETV2-OE enhances the formation of hemogenic endothelial cells. [Figure 3] Figure 1 shows that ETV2 overexpression (OE) enhances CD34+ cell formation during iPSC differentiation. Representative flow cytometry analysis and relative quantification of CD34+ cells demonstrates that ETV2-OE enhances CD34+ cell formation. [Figure 4A]Figure 1 shows that iPSC-derived HSCs derived by EHT of CD34+ cells (using Piezo1 activation) undergo pre-T cell differentiation similar to bone marrow (BM)-HSCs. Figure 2 shows FACS plots of the differentiation efficiency of bone marrow (BM) HSCs and iPSC-HSCs derived by EHT of CD34+ cells (using Piezo1 activation) into CD34+CD7+ pre-T cells. [Figure 4B] Figure 1 shows that iPSC-derived HSCs derived from EHT of CD34+ cells (with Piezo1 activation) undergo pre-T cell differentiation similar to bone marrow (BM)-HSCs. Quantification (%) of CD34+CD7+ cells derived from (1) BM-HSCs and (2) iPSC-HSCs (EHT of CD34+ cells with Piezo1 activation). Averages of three experiments are shown. [Figure 5A] Figure 1 shows that iPSC-derived HSCs generated by EHT of CD34+ cells (in this example, using Piezo1 activation) can undergo T cell differentiation and be activated with CD3 / CD28 beads, similar to BM-HSCs. Figure 2 shows FACS plots of the activation efficiency (CD3+CD69+ expression) of T cells differentiated from BM-HSCs and iPSC-derived HSCs (generated by EHT of CD34+ cells using Piezo1 activation). [Figure 5B] Figure 1 shows that iPSC-derived HSCs generated by EHT of CD34+ cells (in this example, using Piezo1 activation) can undergo T cell differentiation and be activated with CD3 / CD28 beads, similar to BM-HSCs. Quantification (%) of CD3+CD69+ cells derived from (1) BM-HSCs and (2) iPSC-HSCs (derived by EHT of D8 CD34+ cells using Piezo1 activation) is shown. Averages of three experiments are shown. [Figure 6]We show that iPSC-derived HSCs generated by EHT of CD34+ cells (in this example, using Piezo1 activation) can differentiate into functional T cells. IFNγ expression is a consequence of T cell activation after T cell receptor (TCR) stimulation via CD3 / CD28 beads. IFNγ expression in T cells differentiated from iPSC-derived HSCs (in this case, using EHT of CD34+ cells generated by Piezo1 activation) enhances their ability to further differentiate into functional lymphocytes. Averages of three experiments are shown. [Figure 7A] Figure 1 shows phenotypic analysis of HLA-edited (e.g., triple knockout) cells performed by FACS and immunofluorescence. Global expression of HLA class I molecules (HLA-A, HLA-B, and HLA-C) on the cell surface is shown, with HLA-edited cells being positive for global HLA class I expression to a similar extent as wild-type cells. [Figure 7B] FIG. 1 shows phenotypic analysis of HLA-edited (e.g., triple knockout) cells performed by FACS and immunofluorescence. Cellular expression of HLA-A via immunofluorescence is shown, where HLA-A is not expressed in HLA-edited clones. [Figure 8] We show that the HLA-edited clones retain their pluripotency (maintain tri-lineage differentiation) as illustrated by immunofluorescence, where ectodermal differentiation is indicated by NESTIN-488 and PAX6-594 staining, mesodermal differentiation is indicated by GATA-488 staining, and endodermal differentiation is indicated by CXCR4-488 and FOX2A-594 staining. [Figure 9] Immunocompatibility of HLA-edited HSCs: HLA-edited HSCs and control HSCs (WT, B2M KO, and HLA class II null) were cocultured with peripheral blood mononuclear cells (PBMCs) bearing HLA-B and HLA-C matched but mismatched HLA-A, and PBMC-mediated cytotoxicity was measured by Annexin V staining assay. [Figure 10]Figure 1 shows the in vivo engraftment potential of HLA-edited HSCs. Equal proportions of mCherry HLA-edited and unedited HSCs were mixed for competitive transplantation into mice, and bone marrow (BM) and peripheral blood samples were assessed by FACS to compare the relative amounts of each cell type present in the samples. [Figure 11A] Figure 1 shows that HLA-A deletion does not affect class I peptide presentation. Schematic diagram of immunopeptidome analysis. [Figure 11B] Figure 1 shows that HLA-A deletion does not affect class I peptide presentation. Figure 1 shows the results of immunopeptidome analysis, revealing little difference in the number of peptides and representative proteins presented by class I molecules in WT and HLA-edited cells. [Figure 12A] Figure 1 shows that deletion of HLA-DP and DQ does not affect class II peptide presentation. Figure 2 shows the immunopeptidome analysis scheme. [Figure 12B] Figure 1 shows that deletion of HLA-DP and DQ does not affect class II peptide presentation. Figure 2 shows that despite deletion of HLA-DP and DQ, cells retain the ability to present a wide range of peptides through HLA class II. [Figure 13] Schematic diagram of an in vivo test of antigen-mediated immune responses: delayed-type hypersensitivity assay (DTH), sensitization phase, and elimination phase. [Figure 14A] This shows that HLA-edited HSCs reconstitute a functional immune system, as evidenced by DTH responses in immunodeficient mice. A delayed-type hypersensitivity assay, an assay involving crosstalk between different types of immune cells, was performed on transplanted mice. Mice were sensitized by subcutaneous injection of sheep red blood cells (antigen). A functional immune system resulted in swelling of the left paw, as measured with a microcaliper. As can be seen in Figure 14A, non-transplanted mice, due to their immunodeficiency, did not exhibit swelling of the left paw. Conversely, mice transplanted with umbilical cord blood CD34+ cells exhibited tissue swelling, doubling the diameter of their left paw. [Figure 14B]14A-B are graphical evaluations of the results shown in Figure 14A, showing that HLA-edited HSCs reconstitute a functional immune system as demonstrated by DTH responses in immunodeficient mice. [Figure 15] Figure 15 shows the differentiation potential of HSCs into T cell subtypes. After a 35-day differentiation period, pre-T cells were assessed by cell sorting for the presence of CD4+, CD8+, and AB+ T cell populations. Figure 15 compares the differentiation potential of bone marrow-derived CD34+ cells, embryoid body CD34+ cells, and HSCs prepared according to the present disclosure (e.g., using Piezo1 activation) ("gHSCs"). [Figure 16]

[0023] Figure 1 shows the degree of T cell-mediated cytotoxicity measured from co-culture of HSC-derived T cells with CD19+ lymphoma cells in the presence of an anti-CD3 / CD-19 bispecific antibody. T cells prepared from HSCs according to the present disclosure ("gHSCs") exhibit high levels of cytotoxicity against target cells. [Figure 17] The ability of HSCs to evolve into pre-T cells as measured by their CD34-CD7+ markers is shown. [Figure 18A] 10 demonstrates increased expression of T cell-specific transcription factors and thymic engraftment molecules using pre-T cells derived from HSCs according to the present disclosure. TCF7 mRNA expression is shown. [Figure 18B] 10 demonstrates increased expression of T cell-specific transcription factors and thymic engraftment molecules using pre-T cells derived from HSCs according to the present disclosure. CCR7 mRNA expression is shown. [Figure 19A] 1 shows that HSC-derived pre-T cells engraft and differentiate in the thymus. Engraftment and analysis procedures are illustrated. [Figure 19B] Figure 1 shows that HSC-derived pre-T cells engraft and differentiate in the thymus. Figure 2 shows FACS analysis of the CD3 cell population of cells gated on the CD45+ cell population, demonstrating the superior engraftment and differentiation potential of HSC-derived pre-T cells in the thymus. [Figure 20]Figure 1 shows that HSC-derived T cells can be activated in vitro. The top panel shows FACS analysis of activated T cells from different sources, including from HSCs prepared according to the present disclosure. T cells of the present disclosure show comparable or superior activation, as measured by increased CD107 expression. The bottom panel shows Dynabeads activation, where activated T cells express inflammatory cytokines. HSC-derived T cells express higher levels of inflammatory cytokines, as exemplified by TNF-α and interferon gamma expression levels. [Figure 21A] We show that WT and HLA-edited HSCs can differentiate into the monocyte / macrophage lineage, which also preserves the overall expression of both class I and class II molecules, as identified by the CD11b+CD14+ marker. [Figure 21B] Analysis of HLA-I and HLA-II on cells gated on CD11b+CD14+ is shown. [Figure 22A] 1 shows that deletion of HLA-DQB1 and HLA-DPB1 does not affect the expression of other HLA class II molecules. 2 is a schematic diagram showing differentiation of HLA-edited iPSCs into macrophages. [Figure 22B] 1 shows that deletion of HLA-DQB1 and HLA-DPB1 does not affect the expression of other HLA class II molecules. Immunofluorescence experiments confirm the specific deletion of DPB1 and DQB1 molecules. [Figure 22C] Figure 1 shows that deletion of HLA-DQB1 and HLA-DPB1 does not affect the expression of other HLA class II molecules, and that the same cells preserve class II DRB1 expression. [Figure 23] This shows that HLA-edited HSCs can differentiate into megakaryocytes (MKs), which can further differentiate into platelets. The image on the left shows the increased percentage of platelets among HSCs by light microscopy at 1000x magnification. The graph on the right shows a statistically significant increase in the percentage of platelets differentiated from HLA-edited HSCs compared to BM CD34+ and iPSC-34+ cell populations. [Figure 24]Figure 1 shows the ability of HSCs to effectively differentiate into NK cells as evidenced by fluorescence-activated cell sorting (FACS) experiments gated on the basis of CD56 expression. [Figure 25A] 1 shows that HSC-derived NK cells effectively kill tumor cells. A schematic diagram of an experiment is shown in which HSC-derived NK cells are co-cultured with K562 HLA-null cells and the degree of NK cell degranulation is measured using Annexin V staining and a cytotoxicity assay. [Figure 25B] Figure 1 shows that HSC-derived NK cells effectively kill tumor cells. Figure 2 shows the results of NK cell degranulation as measured by fluorescence-activated cell sorting (FACS) using Annexin V staining. [Figure 25C] Figure 1 shows that HSC-derived NK cells effectively kill tumor cells. Figure 2 shows the results of a tumor cell cytotoxicity assay using lactate dehydrogenase (LDH) as a measure of cell death.

[0019] The term "gHSC" is used herein to refer to the iPSC-derived hematopoietic stem cells of the present disclosure.

[0020] The terms "wild-type" (WT), "unedited," and "non-HLA edited" are used interchangeably herein to refer to the non-gene-edited cells of the present disclosure.

[0021] EB34+ cells refer to embryonic body-derived CD34+ cells, which contain hemogenic endothelial cells. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present disclosure, in various aspects and embodiments, provides methods for generating hematopoietic lineages ex vivo for cell therapy, particularly B cell lineages and their progeny, including, in various embodiments, multipotent progenitor cells (MPPs), common lymphoid progenitors (CLPs), common lymphoid 2 progenitors (LCA-2), and B cells. The B cells produced can be immature B cells capable of generating early pre-B cells, late pre-B cells, pre-B cells, and B cells. In various embodiments, the present disclosure provides methods for the ex vivo production of cell populations corresponding to transitional B cells, regulatory B cells, marginal zone B cells, follicular B cells, activated B cells, memory B cells, and plasma B cells (collectively referred to as "B cells"). In various embodiments, the present invention provides an efficient ex vivo process for developing such hematopoietic lineages from human induced pluripotent stem cells (iPSCs). In various embodiments, cells generated according to the present disclosure are functional and / or more closely resemble corresponding lineages isolated from peripheral blood or lymphoid organs. The present invention also provides isolated cells and cell compositions produced by the methods disclosed herein, as well as methods for cell therapy.

[0023] According to aspects and embodiments of the present disclosure, the ability of human induced pluripotent stem cells (hiPSCs) to produce essentially unlimited pluripotent stem cells (PSCs) is exploited to generate an unlimited supply of B cell lineages or their modified forms (e.g., genetically engineered B-CAR cells). The use of B cells as therapeutic lymphocytes is limited by their limited availability, cell number, limited proliferation capacity, and histocompatibility issues. Furthermore, compared to primary cells, hiPSCs undergo genetic modification more readily in vitro, thereby providing opportunities for improved cell targeting specificity, cell number, and avoiding, for example, HLA matching issues. In addition, fully engineered hiPSC clones can serve as a stable and safe source compared to primary cells (Nianias and Themeli, 2019). Furthermore, unlike human embryonic stem cells (hESCs), hiPSCs are of non-embryonic origin, thereby eliminating ethical concerns and providing consistent quality. Therefore, the use of hiPSCs according to the present disclosure offers several advantages over primary cells for generating therapeutic hematopoietic lineages, such as B cell lineages.

[0024] In one aspect, the present disclosure provides a method for preparing a cell population of a B-cell lineage (e.g., ex vivo). The method includes preparing a pluripotent stem cell (PSC) population, such as an induced pluripotent stem cell (iPSC) population, differentiated into embryoid bodies and enriching for CD34+ cells, thereby preparing a CD34+ enriched population. Endothelial-hematopoietic transition (EHT) is induced in the CD34+ enriched population, thereby preparing a hematopoietic stem cell (HSC) population, optionally followed by further enrichment of CD34+ cells. The resulting HSC population (or a fraction thereof) can be differentiated into a B-cell lineage.

[0025] In various embodiments, the B cell population comprises transitional B cells, regulatory B cells, marginal zone B cells, follicular B cells, activated B cells, memory B cells, or plasma B cells, or derivatives thereof.

[0026] In some embodiments, the cell population comprises transitional B cells (TrB cells), which are immature B cells and precursors to mature B cells. TrB cells account for approximately 4% of all CD19+ B lymphocytes in healthy individuals. They reside in peripheral blood, umbilical cord blood, bone marrow, and secondary lymphoid tissues such as lymph nodes, spleen, tonsils, and gut-associated lymphoid tissue (GALT). Human TrB cells often express CD24 hi CD38 hi TrB cells are characterized by their phenotype. TrB cells can be separated into subsets based on the expression of CD27, IgM, IgD, CD10, CD21, and CD32. While the T1-T3 B cell subset expresses low levels of CD27, CD27+ TrB cells express high levels of CD27, CD24, and CD38. T1 B cells express high levels of IgM, CD10, and CD32, while IgD and CD21 expression is low. T2 B cells show intermediate expression of IgM, IgD, CD10, and CD32, and low expression of CD21. T3 B cells express low levels of IgM, IgD, CD10, CD21, and CD32.

[0027] TrB cells suppress autoreactive CD4+ T cell proliferation and limit the proliferation of CD4+ Th1 cells (IFN-γ and TNF-α production) and CD4+ Th17 cells (IL-17 production), thereby suppressing the production of proinflammatory cytokines, preventing CD4+ T cells from differentiating into Th1 and Th17 cells, and promoting the conversion of effector CD4+ T cells into CD4+ FoxP3+ Tregs while limiting excessive proinflammatory cytokine production. TrB cells also inhibit CD8+ T cell responses and maintain invariant natural killer T (iNKT) cells. In addition to producing anti-inflammatory factors, TrB cells can also secrete proinflammatory cytokines such as IL-6 and TNF-α. In some aspects and embodiments, TrB cells are closely related to IL-10-producing regulatory B cells (Bregs) in terms of phenotypic and functional similarities. TrB cells also produce IL-10 and can regulate CD4+ T cell proliferation and differentiation towards T helper (Th) effector cells.

[0028] In some embodiments, the cell population comprises regulatory B (Breg) cells. Bregs are immunosuppressive cells that support immune tolerance. Breg cells are also involved in inhibiting excessive inflammation. Through the production of IL-10, TGF-β, and IL-35, Breg cells can suppress the differentiation of proinflammatory lymphocytes, such as tumor necrosis factor α (TNF-α)-producing monocytes, IL-12-producing dendritic cells, Th17 cells, Th1 cells, and cytotoxic CD8+ T cells. Breg cells can also induce the differentiation of immunosuppressive T cells, Foxp3+ T cells, and T regulatory 1 (Tr1) cells. Breg cells also support the maintenance of iNKT cells. Common markers for human Breg cells include CD19+CD24 hi CD38 hi CD1d hi , CD19+CD24 hi CD27+, CD24 hi CD27+, CD19+CD24 hi CD27 int , CD19+CD24 hi CD38 hi , and CD19+CD25 hi CD71 hi Examples include:

[0029] In various embodiments, the B cells have a phenotype consistent with marginal zone B (MZB) cells. MZB cells provide a first line of defense in response to infections by blood-borne viruses and encapsulated bacteria, rapidly producing IgM and class-switched IgG antibodies. MZB cells can also produce IgM and class-switched IgG and IgA antibodies in response to commensal antigens. MZB cells mediate T cell-dependent antibody production. For example, MZB cells can mount T cell-dependent responses to microbial protein antigens. In some embodiments, iPSC-derived MZB-like cells are CD27+IgM+IgD+ cells, or express high levels of IgM, CD21, CD1, and CD9, and are low to negative for IgD, CD23, CD5, and CD11b, or are CD27+IgM+IgD+ cells. - CD45RB +(defining MZ progenitor cells).

[0030] In some embodiments, the B cells have a phenotype consistent with follicular B cells. Follicular B cells are involved in T cell-dependent antibody responses. In addition, follicular B cells respond to blood-borne pathogens in a T cell-independent manner. After activation, follicular B cells differentiate into short-lived plasma cells in the periphery or participate in T cell-dependent germinal center responses. Follicular B cells express high levels of IgD and CD23, lower levels of CD21 and IgM, and do not express CD1 or CD5. Other cell surface markers that identify follicular B cells include CD10, CD19, CD20, CD22, CD23, and CD38. LOW , CXCR5+, and IgD high These include, but are not limited to:

[0031] B cells express the B cell receptor (BCR), which activates B cells upon binding to either soluble or membrane-bound antigens. Activated BCRs form microclusters and trigger downstream signaling cascades. Microclusters eventually undergo contraction to form an immune synapse, which allows stable interactions between B cells and T cells and provides bidirectional activation signals. Upon encountering antigen, mature activated B cells proliferate and become blastoid B cells. These B cells form germinal centers. Germinal center B cells undergo somatic hypermutation and class switch recombination. Plasma cells and memory B cells with high affinity for the original antigen stimulus are produced. These cells are long-lived, and plasma cells can secrete antibodies for several weeks after initial infection. One of the major transcriptional activators associated with B cell activation is nuclear factor (NF)-κB. Some common markers identifying activated B cells are CD19, CD25, and CD30.

[0032] B cells generated according to the present disclosure can be differentiated (ex vivo or in vivo) into plasma cells. Plasma cells are specialized, terminally differentiated B cells that synthesize and secrete antibodies to maintain humoral immunity. When plasma B cells encounter their own antigen, they ingest the antigen through receptor-mediated endocytosis. The antigen particles are transferred to the cell surface, loaded onto MHC II molecules, and presented to helper T cells. Binding of the helper T cell to the MHC II-antigen complex activates the B cell. Activated B cells undergo a period of rapid proliferation and somatic hypermutation. Selection occurs for cells that produce antibodies with high affinity to that particular antigen. Once terminally differentiated, plasma B cells secrete only antibodies specific to that antigen and can no longer produce antibodies against other antigens.

[0033] B cells generated according to the present disclosure can be differentiated (ex vivo or in vivo) into memory B cells. Memory B cells are B lymphocytes that remember a specific antigen during the initial B cell response. Memory B cells are kept in reserve in the germinal centers of the lymphoid system for when the immune system re-encounters a particular antigen. A hallmark of memory B cells is that they present and secrete antibodies with significantly higher affinity than those produced by primary plasma cells. Upon any repeated exposure, follicular helper T cells differentiate memory cells into plasma B cells with higher sensitivity to that specific antigen. This primes the immune system to mount a more rapid and potent response than previously possible. In humans, memory B cells are commonly identified by their expression of CD27, coupled with low levels of CD23 / FcεRI or the lack of expression of the plasma cell marker syndecan-1 / CD138. DEP-1 / CD148 is also frequently used to identify human memory B cells, as is high-level expression of B7-1 / CD80, B7-2 / CD86, and CD95. Different subsets of memory B cells and plasma cells can be identified based on their expression of Ig isotypes (IgM, IgD, IgG, IgA), which is well understood by those skilled in the art.

[0034] In various embodiments, iPSCs are prepared by reprogramming somatic cells. The term "induced pluripotent stem cells" or "iPSCs" refers to cells derived from somatic cells, such as skin or blood cells, that have been reprogrammed back to an embryonic-like pluripotent state. In some embodiments, iPSCs are generated from somatic cells, such as (but not limited to) fibroblasts or PBMCs (or cells isolated therefrom). In some embodiments, iPSCs are derived from B lymphocytes, umbilical cord blood cells (e.g., CD34+ cells), PBMCs or fractions thereof, CD34+ cells, or other primary human tissues. In some embodiments, iPSCs are derived from CD34+ cells isolated from peripheral blood. In various embodiments, iPSCs are autologous or allogeneic (e.g., HLA-matched at one or more loci) to the recipient (the subject in need of the treatment described herein). In various embodiments, iPSCs can be genetically edited to support HLA matching (such as deletion of one or more HLA class I and / or HLA class II alleles or their master regulators, including, but not limited to, beta-2-microglobulin (B2M), CIITA, etc.), or to delete or express other functions. For example, iPSCs can be genetically edited to delete one or more of HLA-A, HLA-B, and HLA-C, and one or more of HLA-DP, HLA-DQ, and HLA-DR. In certain embodiments, iPSCs retain expression of at least one HLA class I and at least one HLA class II complex. In certain embodiments, iPSCs are homozygous for at least one retained class I and class II locus. In some embodiments, iPSCs are prepared from B cells or other cells that encode a defined BCR or antibody with a predetermined antigen specificity (e.g., for an infectious disease antigen, such as a bacterial or viral surface protein). iPSCs prepared from such B cells, when differentiated into the B cell lineage, produce B cells with defined antigen specificity.

[0035] In various embodiments, iPSCs are gene-edited to be one of: (i) HLA-A-B+C+DP-DR+DQ+, (ii) HLA-A-B+C+DP+DR+DQ-, (iii) HLA-A-B+C+DP-DR+DQ-, (iv) HLA-AB-C+DP-DR+DQ+, (v) HLA-AB-C+DP+DR+DQ-, or (vi) HLA-AB-C+DP-DR+DQ-. For retained HLA (e.g., HLA-B, HLA-C, and HLA-DR), the cells can be homozygous or can retain only a single copy of the gene. For example, the modified cells are identified as at least (a) HLA-C+ and HLA-DR+, and optionally as one or more of (b) HLA-B-, (c) HLA-DP-, and (d) HLA-DQ-. In an exemplary embodiment, the modified cells are HLA-B+, HLA-DP-, and HLA-DQ-.

[0036] In some embodiments, the iPSCs are HLA-A neg and is homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg In some embodiments, the iPSCs are further homozygous for HLA-DRB1.

[0037] As used herein, the term "neg," (-), or "negative" with respect to a particular HLA class I or HLA class II molecule indicates that both copies of the gene have been disrupted in a cell line or population, and therefore the cell line or population does not exhibit significant functional expression of the gene. Such cells can be generated by complete or partial gene deletion or disruption, or alternatively by other techniques such as siRNA. As used herein, the term "deletion" in the context of genetic modification (i.e., gene editing) of a targeted gene refers to the elimination of functional expression of the corresponding gene product (i.e., the corresponding polypeptide). Such gene editing includes complete or partial gene deletion or disruption of a coding sequence or deletion of a critical cis-acting expression control sequence.

[0038] Somatic cells can be reprogrammed by expression of reprogramming factors selected from Sox2, Oct3 / 4, c-Myc, Nanog, Lin28, and klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc, Nanog, Lin28, and klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc, and klf4. Methods for preparing iPSCs are described, for example, in U.S. Pat. Nos. 10,676,165, 9,580,689, and 9,376,664, which are incorporated by reference in their entireties. In various embodiments, reprogramming factors are expressed using well-known viral vector systems, such as lentivirus, Sendai, or measles virus systems. Alternatively, reprogramming factors can be expressed by introducing mRNA(s) encoding the reprogramming factors into somatic cells. Furthermore, iPSCs can be generated by introducing non-integrating episomal plasmids expressing reprogramming factors, i.e., for the generation of transgene-free and virus-free iPSCs. Known episomal plasmids that have limited replicative capacity and are therefore lost over several cell generations can be employed.

[0039] In some embodiments, human pluripotent stem cells (e.g., iPSCs) are gene-edited. Gene editing may include, but is not limited to, modification of HLA genes (e.g., deletion of one or more HLA class I and / or HLA class II genes), deletion of β2 microglobulin (β2M), deletion of CIITA, deletion or addition of a B cell receptor gene, or addition of a chimeric antigen receptor (CAR) gene. Exemplary CAR-B cells may be tissue-specific for inflamed or infected tissues or specific for a target pathogen or cell. For example, iPSCs may be B cell receptor-transduced iPSCs. Such embodiments enable large-scale production of regenerative B lymphocytes with desired antigen specificity. Alternatively, engineered iPSCs with one or more HLA knockouts can be placed in a bioreactor for feeder-free and serum-free differentiation under GMP-grade conditions to generate fully functional B cells (e.g., TrB cells, Bregs, plasma B cells, memory B cells, or B cell progenitors).

[0040] In some embodiments, iPSCs are gene-edited using gRNAs that are 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more nucleotides in length. In some embodiments, the gRNAs comprise modifications at or near the 5' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5' end) and / or at or near the 3' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 3' end). In some embodiments, the modified gRNAs exhibit increased resistance to nucleases. In some embodiments, the gRNAs comprise two separate RNA molecules (i.e., "duplex gRNAs"). Duplex gRNAs comprise two separate RNA molecules, "crispr RNA" (or "crRNA") and "tracr RNA," and are well known to those of skill in the art.

[0041] Generally, various gene editing technologies are known that can be applied in accordance with various embodiments of the present disclosure, including, but not limited to, zinc finger (ZF), transcription activator-like effector (TALE), etc. Fusion proteins comprising one or more of these DNA binding domains and the cleavage domain of a Fokl endonuclease can be used to generate double-stranded breaks in desired regions of DNA within a cell (see, e.g., U.S. Patent Application Publication No. 2012 / 0064620, U.S. Patent Application Publication No. 2011 / 0239315, U.S. Patent No. 8,470,973, U.S. Patent Application Publication No. 2013 / 0217119, U.S. Patent No. 8,420,782, U.S. Patent Application Publication No. 2011 / 0301073, U.S. Patent Application Publication No. 2011 / 0145940, U.S. Patent No. 8,450,471, U.S. Patent No. 8,440,431, U.S. Patent No. 8,440,432, and U.S. Patent Application Publication No. 2013 / 0122581, the entire contents of which are incorporated herein by reference). In some embodiments, gene editing is performed using a CRISPR-associated Cas system (e.g., CRISPR-Cas9) known in the art. See, for example, US8,697,359, US8,906,616, and US8,999,641, each of which is incorporated herein by reference in its entirety. In various embodiments, gene editing employs a type II Cas endonuclease (such as Cas9) or a type V Cas endonuclease (such as Cas12a). Type II and type V Cas endonucleases are guide RNA dependent. Design of gRNAs to guide desired gene editing (while limiting or avoiding off-target editing) is known in the art. See, for example, Mohr SE, et al., CRISPR guide RNA design for research applications, FEBSJ. 2016 Sep; 283(17):3232-3238.In still other embodiments, non-canonical Type II or Type V Cas endonucleases with homology (albeit low primary sequence homology) to S. pyogenes Cas9 or Prevotella and Francisella 1 (Cpf1 or Cas12a) can be employed. Many such non-canonical Cas endonucleases are known in the art. Nidhi S, et al. Novel CRISPR-Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives, Int J Mol Sci. 2021 Apr;22(7):3327. In still other embodiments, gene editing employs base editing or prime editing to incorporate mutations without creating double-strand breaks. See, for example, Antoniou P, et al., Base and Prime Editing Technologies for Blood Disorders, Front. Genome Ed., 28 January 2021; Matsuokas IG, Prime Editing: Genome Editing for Rare Genetic Diseases Without Double-Strand Breaks or Donor DNA, Front. Genet., 9 June 2020. Various other gene editing processes are known, including the use of dead Cas (dCas) systems (e.g., Cas fusion proteins) to target DNA-modifying enzymes to desired targets using dCas as a guide RNA-dependent system. Brezgin S, Dead Cas Systems: Types, Principles, and Applications, Int J Mol Sci. 2019 Dec;20(23):6041.

[0042] Base editors that can introduce precise genome modifications without generating double-stranded DNA breaks can also be used for gene editing in cells (e.g., iPSCs) (e.g., designing gene therapy vectors). Base editors inherently cannot create DSBs and contain a catalytically inactive nuclease, such as Cas9 nickase (nCas9), fused to a nucleic acid base deaminase enzyme, possibly a DNA glycosylase inhibitor. Currently, there are two major categories of base editors: cytidine base editors (CBEs) and adenine base editors (ABEs), which catalyze C>T and A>G transversions. Base editors can be delivered, for example, via HDAd5 / 35++ vectors, to efficiently edit promoters and enhancers to activate or inactivate genes. Exemplary methods are described in U.S. Patent Nos. 9,840,699, 10,167,457, 10,113,163, 11,306,324, 11,268,082, 11,319,532, and 11,155,803. Also contemplated are prime editors comprising a reverse transcriptase conjugated to (e.g., fused with) a Cas endonuclease and a polynucleotide useful as a DNA synthesis template conjugated to (e.g., fused with) a guide RNA, as described in WO2020 / 191153.

[0043] Exemplary vectors that can be used for genome editing applications include plasmids, retroviral vectors, lentiviral vectors, adenoviral vectors (e.g., Ad5 / 35, Ad5, Ad26, Ad34, Ad35, Ad48), parvoviruses (e.g., adeno-associated virus (AAV) vectors), herpes simplex virus vectors, baculovirus vectors, negative-stranded RNA viruses such as coronaviruses, orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-stranded RNA viruses such as picornaviruses and alphaviruses, and herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses. Examples of vectors that can be used include, but are not limited to, double-stranded DNA viruses, including viruses (e.g., canarypox, vaccinia, or modified vaccinia viruses). Vectors containing a nucleic acid molecule of interest can be delivered to cells (e.g., iPS cells, endothelial cells, hemogenic endothelial cells, HSCs (ST-HSCs or LT-HSCs) via any method known in the art, including, but not limited to, transduction, transfection, infection, and electroporation. Any of these vectors may contain a transposable element (such as a piggyBac transposon or a Sleeping Beauty transposon). Transposons insert specific sequences of DNA into the vertebrate genome. A gene of interest can be integrated into the genome of a mammalian cell by transposase-catalyzed cleavage of a similar excision site present in the cell's nuclear genome.

[0044] For increased efficiency, in some embodiments, Cas and gRNA can be combined before delivery into cells. The Cas-gRNA complex is known as a ribonucleoprotein (RNP). Several methods have been developed for direct delivery of RNP into cells. For example, RNP can be delivered to cells in culture by lipofection or electroporation. Electroporation using a nucleofection protocol can be employed, which allows RNP to rapidly enter the cell's nucleus and immediately begin cleaving the genome. See, for example, Zhang S, Shen J, Li D, Cheng Y. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR / Cas9 genome editing. Theranostics. 2021 Jan 1;11(2):614-648, incorporated herein by reference in its entirety. In some embodiments, Cas9 and gRNA are electroporated into donor iPSCs and / or HSCs as RNP.

[0045] Generally, a protospacer adjacent motif (PAM) is required for Cas nuclease cleavage and is typically found 3-4 nucleotides downstream of the cleavage site. The PAM is a short DNA sequence (usually 2-6 base pairs in length) following the DNA region targeted for cleavage by a CRISPR system such as CRISPR-Cas9. In some embodiments, the PAM sequence, sgRNA, or base editing tool targeting a haplotype or polymorphism at an HLA locus does not contain four Gs, four Cs, a GC repeat, or a combination thereof.

[0046] In some embodiments, a CRISPR / Cas9 system specific for a unique HLA haplotype can be developed by using the gRNAs described herein to design a single gRNA that targets each of the donor-specific HLA-A, HLA-DPB1, and HLA-DQB1 genes (for example). To perform gene knockout, the gRNA targets the Cas9 protein to the appropriate site for editing. The Cas9 protein can then perform a double-strand break (DSB), which repairs the DNA through a non-homologous end joining (NHEJ) mechanism, generating an indel that results in a frameshift mutation and terminates the function of the resulting protein. However, off-target gene recombination can occur, altering the function of an otherwise intact gene. For example, the Cas9 endonuclease can generate DSBs at undesired off-target locations, even in the presence of some degree of mismatch. This off-target activity can result in genomic instability events such as point mutations and genomic structural perturbations. In various embodiments, an sgRNA targeting HLA-A can target a region of chromosome 6 defined as 29942532-29942626. In various embodiments, an sgRNA targeting HLA-DQB1 can target a region of chromosome 6 defined as 32665067-32664798. In various embodiments, an sgRNA targeting HLA-DPB1 can target a region of chromosome 6 defined as 33080672-33080935.

[0047] gRNAs can be used to develop clonal iPSCs. Such iPSC lines can be assessed for (i) on-target editing, (ii) off-target editing, and (iii) translocation editing, for example, using sequencing as described herein. Specifically, such assays can be performed by multiplex PCR using primers designed to target and enrich for the region of interest, followed by next-generation sequencing (e.g., Amplicon sequencing, AMP-seq). On-target and translocation panels can amplify the intended edited region and allow for the selection of iPSC clones with the expected edits that do not contain chromosomal translocations resulting from unintended DSB cleavage site fusions. Off-target panels can enrich for any potential off-target regions identified through sequencing, allowing for the selection of iPSC clones with negligible off-target mutations. Together, these assays enable the screening of iPSC clones to select clones with the desired edits while ruling out potential CRISPR / Cas9-related genome integrity issues.

[0048] In some embodiments, to further ensure the genomic stability and integrity of the reprogrammed and edited iPSCs, genetic and genomic assays can be performed to select clones that have not undergone translocation and mutation events and have not integrated the episomal vector. For example, whole genome sequencing (WGS) can be performed on CD34+ cells and iPSC clones after reprogramming, and the genomes can be compared for differences resulting from editing. These analyses provide an assessment of which iPSC clone genomes differ from the CD34+ starting material, allowing for the informed selection of iPSC clones that have not undergone mutations during reprogramming.

[0049] In some embodiments, karyotyping using a system such as the KARYOSTAT assay is used to select iPSC clones that did not develop indels or translocations during reprogramming, as described, for example, in Ramme AP, et al., "Supporting dataset of two integration-free induced pluripotent stem cell lines from related human donors," Data Brief. 2021 May 15;37:107140, incorporated herein by reference in its entirety. The KARYOSTAT assay allows visualization of chromosomal abnormalities with a resolution similar to G-banding karyotyping. The size of structural abnormalities that can be detected is >2 Mb for chromosome gains and >1 Mb for chromosome losses. The KARYOSTAT array is functionalized for balanced whole-genome coverage with low-resolution DNA copy number analysis, and the array covers all 36,000 RefSeq genes, including 14,000 OMIM targets. The assay allows for the detection of aneuploidy, submicroscopic abnormalities, and mosaic events.

[0050] In some embodiments, array comparative genomic hybridization (aCGH) analysis is used to select iPSC clones that have not developed copy number aberrations (CNAs) during reprogramming, as described, for example, in Wiesner et al. "Molecular Techniques," Editor(s): Klaus J. Busam, Pedram Gerami, Richard A. Scolyer, "Pathology of Melanocytic Tumors," Elsevier, 2019, pp. 364-373, ISBN 9780323374576, and Hussein SM, et al. "Copy number variation and selection during reprogramming to pluripotency," Nature. 2011 Mar 3;471(7336):58-62, which are incorporated herein by reference in their entireties. aCGH is a technique that analyzes the entire genome for CNAs by comparing sample DNA with reference DNA.

[0051] In some embodiments, a targeted hematologic malignancy NGS panel analysis is used to select iPSC clones that did not develop hematologic malignancy mutations during reprogramming. For example, a targeted hematologic malignancy NGS panel focuses on genes associated with myeloid leukemia, lymphoma, and / or other hematologic malignancies, generating smaller, more manageable datasets than more broad methods. A targeted hematologic malignancy NGS panel analysis involves the use of highly multiplexed PCR to amplify regions associated with hematologic malignancies, followed by next-generation sequencing.

[0052] In some embodiments, droplet digital PCR (ddPCR) is used to select iPSC clones that have not integrated the episomal vector and have been passaged sufficiently for episomal vector clearance. As discussed herein, iPSC reprogramming of CD34+ cells can be achieved by delivering episomal vectors encoding reprogramming factors. However, episomal vectors can randomly integrate into the cellular genome, although this is rare, which can disrupt developmental processes, homeostasis, and the like. Therefore, ddPCR can be used to detect residual episomal vectors in iPSC cultures, allowing for the selection of iPSC clones that have not integrated the episomal vector.

[0053] In some embodiments, after assessing that selected clones do not contain editing-related genomic abnormalities, the clones can be further tested for spontaneous mutations that may arise during expansion, such as mutations affecting hematologic malignancies, indels, translocations, and numerical abnormalities, as described for pre-edited reprogrammed clones. Analysis of spontaneous mutations can include whole genome sequencing (WGS), KARYOSTAT analysis, array comparative genomic hybridization (aCGH) analysis, targeted hematologic malignancies NGS panel AMP-Seq analysis, and / or droplet digital PCR (ddPCR).

[0054] In various embodiments, iPSCs are prepared and expanded using a culture system. The expanded iPSCs can be used to generate embryoid bodies (EBs). EBs generated by differentiation of iPSCs are three-dimensional aggregates of iPSCs and contain three (or alternatively, two or one) embryonic germ cell layers based on the differentiation method(s). EB preparation is described, for example, in US 2019 / 0177695, which is incorporated herein by reference in its entirety. In some embodiments, EBs prepared by differentiation of iPSCs are expanded in a bioreactor, as described, for example, in Abecasis B. et al., Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches. J. of Biotechnol. 246 (2017) 81-93. Other methods involving 3D suspension culture for the growth or differentiation of EBs are described in WO2020 / 086889, which is incorporated herein by reference in its entirety.

[0055] According to the present disclosure, CD34+ cells are isolated from pluripotent stem cells (e.g., EBs) and induced to undergo endothelial-hematopoietic differentiation to prepare hematopoietic stem cell (HSC) populations. HSCs, including relatively high frequencies of LT-HSCs, can be generated from these cell populations using a variety of stimuli or factors, including mechanical, biochemical, metabolic, and / or local stimuli, as well as factors such as extracellular matrix, niche factors, and cell-extrinsic factors, induction of cell-intrinsic properties, and pharmacological and / or genetic means.

[0056] In some embodiments, the method includes preparing hematopoietic endothelial cells from pluripotent stem cells prior to induction of EHT. In some embodiments, combined overexpression of GATA2 / ETV2, GATA2 / TAL1, or ER71 / GATA2 / SCL can lead to the formation of hematopoietic endothelial cells from PSC sources. In some embodiments, the method includes overexpression of the E26 transformation-specific variant 2 (ETV2) transcription factor in iPSCs. ETV2 can be expressed by introduction of an encoding non-integrating episomal plasmid for constitutive or inducible expression of ETV2 and for the production of transgene-free hematopoietic ECs. In some embodiments, ETV2 is expressed from mRNA introduced into iPSCs. The mRNA can be introduced using any available method, including electroporation or lipofection. Differentiation of ETV2-expressing cells can include the addition of VEGF-A. See Wang K, et al., Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with mRNA. Sci. Adv. Vol. 6 (2020). Cells generated in this manner can be used to produce CD34+ cells and induce EHT according to embodiments of the present disclosure.

[0057] After CD34+ enrichment, HSCs are then generated from endothelial cells using mechanical, biochemical, pharmacological, and / or genetic stimulation or modification.

[0058] In some embodiments, iPSC differentiation proceeds until the cells are at least about 10% CD34+, or at least about 20% CD34+, or at least about 25% CD34+, or at least about 30% CD34+. In some embodiments, CD34 enrichment and EHT can be induced on days 7-14 of iPSC differentiation, such as days 8, 9, 10, 11, 12, 13, or 14. Differentiation of iPSCs can be by known techniques. In some embodiments, iPSC differentiation involves factors such as, but not limited to, combinations of bFGF, Y27632, BMP4, VEGF, SCF, EPO, TPO, IL-6, IL-11, and / or IGF-1. In some embodiments, hPSCs are differentiated using feeder-free, serum-free, and / or GMP-compatible materials. Serum-free cultures typically include a cytokine / growth factor / small molecule cocktail.

[0059] In a non-limiting example, isolated iPSCs can be cultured under conditions that promote lymphopoiesis. In some embodiments, feeder cells, such as a feeder layer of STO mouse fibroblasts, can be used to expand B cells. In some embodiments, hPSCs are co-cultured in serum-containing medium with mouse bone marrow-derived feeder cells, such as OP9, or blood-derived peripheral blood mononuclear cells (PBMCs), or cord blood-derived mesenchymal stem cells, or lymphocyte-derived cancer cell line cells. The culture can contain growth factors and cytokines to support differentiation into embryoid bodies or monolayer systems. Feeder cell co-culture systems can be used to generate multipotent HSPCs that can be further differentiated into several hematopoietic lineages, including B lymphocytes, monocytes or macrophages, dendritic cells, neutrophils, NK cells, T lymphocytes, megakaryocytes, and erythrocytes. See Netsrithong R. et al., Multilineage differentiation potential of hematoendothelial progenitors derived from human induced pluripotent stem cells, Stem Cell Research & Therapy Vol. 11 Art. 481 (2020). Alternatively, a stepwise process using defined conditions in conjunction with specific signals can be used. For example, expression of HOXA9, ERG, RORA, SOX4, and MYB in human PSCs favors direct differentiation into multipotent CD34+ / CD45+ progenitors. Furthermore, expression of factors such as HOXB4, CDX4, SCL / TAL1, or RUNX1a supports the hematopoietic program in human PSCs. See Doulatov S. et al., Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via re-specification of lineage-restricted precursors, Cell Stem Cell. 2013 Oct 3; 13(4).

[0060] Differentiation of iPSCs (e.g., into EBs) can employ a WNT agonist, such as CHIR99021. A WNT agonist is a molecule that mimics or increases WNT signaling. Non-limiting examples of WNT agonists include the small molecule CHIR-99021 (CAS 252917-06-9), 2-amino-4,6-disubstituted pyrimidines such as BML 284 (CAS 853220-52-7), SKL 2001 (CAS 909089-13-0), WAY 262611 (CAS 1123231-07-1), WAY 316606 (CAS 915759-45-4), SB 216763 (CAS 280744-09-4), IQ 1 (CAS 331001-62-8), QS 11 (CAS 944328-88-5), deoxycholic acid (CAS 83-44-3), BIO (CAS 667463-62-9), Kenpaullone (CAS 142273-20-9), or (hetero)arylpyrimidines. In some embodiments, the WNT agonist is an agonist antibody or functional fragment thereof, or an antibody-like polypeptide.

[0061] Differentiation of iPSCs (e.g., into EBs) can employ ROCK inhibitors. Exemplary ROCK inhibitors used in establishing and differentiating iPSCs include, but are not limited to, thiazovivin, Y27632, fasudil, AR122-86, RevitaCell™ supplement, H-1152, Y-30141, Wf-536, HA-1077, hydroxyl-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea, 3-(4-pyridyl)-1H-indole, and (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, H-100, as well as the ROCK inhibitors disclosed in U.S. Pat. No. 8,044,201, which is incorporated herein by reference in its entirety.

[0062] Induction of EHT can be by any known process. In some embodiments, induction of EHT generates a hematopoietic stem cell (HSC) population, including LT-HSC. In some embodiments, EHT generates HSCs through endothelial or hemogenic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological, and / or genetic means (e.g., via stimulation, inhibition, and / or genetic modification). In some embodiments, EHT generates a stem cell population, including one or more of long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC), and hematopoietic stem progenitor cells. In some embodiments, EHT cultures include one or more (e.g., combinations) of Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3.

[0063] In some embodiments, the method comprises increasing the expression or activity of dnmt3b in PSCs, embryoid bodies, CD34+ enriched cells, ECs, HECs, or HSCs, which may be by mechanical, genetic, biochemical, or pharmacological means. In some embodiments, the method comprises increasing the activity or expression of DNA (cytosine-5-)-methyltransferase 3β (Dnmt3b) and / or GTPase IMAP family member 6 (Gimap6) in the cells. See WO2019 / 236943 and WO2021 / 119061, which are incorporated by reference in their entireties. In some embodiments, inducing EHT comprises increasing the expression or activity of dnmt3b.

[0064] In some embodiments, the cells are contacted with an effective amount of an agonist of a mechanosensitive receptor or mechanosensitive channel that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezol. An exemplary Piezol agonist is Yodal. In some embodiments, the mechanosensitive receptor is Trpv4. An exemplary Trpv4 agonist is GSK1016790A. Yodal (2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine) is a small molecule agonist developed for the mechanosensitive ion channel Piezol. Syeda R, Chemical activation of the mechanotransduction channel Piezol. eLife (2015).

[0065] Derivatives of Yodal can be employed in various embodiments. For example, derivatives containing a 2,6-dichlorophenyl core are employed in some embodiments. Exemplary agonists are disclosed in Evans EL, et al., "Yoda1 analogue (Dooku1) which antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation," British Journal of Pharmacology 175(1744-1759):2018. Still other Piezo1 agonists include Jedi1, Jedi2, single-stranded (ss) RNA (e.g., ssRNA40), and derivatives and analogs thereof. See Wang Y., et al., "A lever-like transduction pathway for long-distance chemical- and mechano-gating of the mechanosensitive Piezo1 channel," Nature Communications (2018) 9:1300; and Sugisawa, et al., "RNA Sensing by Gut Piezo1 Is Essential for Systemic Serotonin Synthesis," Cell, Volume 182, Issue 3, 2020, Pages 609-624, which are incorporated herein by reference in their entireties. These Piezo1 agonists are commercially available. In various embodiments, the effective amount of the Piezo1 agonist or derivative ranges from about 1 μM to about 500 μM, or from about 5 μM to about 200 μM, or from about 5 μM to about 100 μM, or in some embodiments, from about 25 μM to about 150 μM, or from about 25 μM to about 100 μM, or from about 25 μM to about 50 μM.

[0066] In various embodiments, pharmacological Piezo1 activation is applied to CD34+ cells (i.e., CD34+ enriched cells). In certain embodiments, pharmacological Piezo1 activation may further be applied to iPSCs, embryoid bodies, ECs, hemogenic endothelial cells (HECs), HSCs, hematopoietic progenitor cells, and hematopoietic lineage(s). In certain embodiments, Piezo1 activation is applied to at least EBs generated from iPSCs, CD34+ cells isolated from EBs, and / or combinations thereof, which, according to various embodiments, enables superior generation of B-cell lineage cells compared to other methods for inducing EHT. In a non-limiting example, EHT CD34+ cells are treated with Yoda1 under culture conditions for 2-7 days, 3-7 days, 4-7 days, 5-7 days, or 6-7 days to generate superior HSCs with a greater ability to generate B-cell lineages or their progenitors.

[0067] Alternatively, or in addition, Dnmt3b activity or expression can be increased directly in cells, for example, in CD34+ enriched cells. For example, Dnmt3b mRNA expression can be increased by, but not limited to, delivering a transcript encoding Dnmt3b to cells, or by introducing a transgene encoding Dnmt3b, or by transgene-free methods such as introducing a non-integrating episome into cells. In some embodiments, gene editing is employed to introduce genetic modifications into Dnmt3b-expressing elements in cells, such as, but not limited to, increasing promoter strength, ribosome binding, RNA stability, and / or affecting RNA splicing.

[0068] In some embodiments, the method includes increasing the activity or expression of Gimap6 in a cell, alone or in combination with Dnmt3b and / or other genes that are up- or down-regulated upon cyclic strain or Piezol activation. To increase the activity or expression of Gimap6, an mRNA transcript encoding Gimap6 can be introduced into the cell; a transgene-free approach can be employed, including, but not limited to, introducing an episome into the cell; or, alternatively, a transgene encoding Gimap6 can be introduced. In some embodiments, gene editing is employed to introduce genetic modifications (such as one or more modifications to increase promoter strength, ribosome binding, RNA stability, or to affect RNA splicing) into a Gimap6-expressing element in the cell.

[0069] In embodiments of the present disclosure employing mRNA delivery to cells, known chemical modifications can be used to circumvent the innate immune response in cells. For example, synthetic RNAs containing only canonical nucleotides can bind to pattern recognition receptors and induce a strong immune response in cells. This response can result in translation block, secretion of inflammatory cytokines, and cell death. RNAs containing certain non-canonical nucleotides can avoid detection by the innate immune system and can be translated into proteins with high efficiency. See US Pat. No. 9,181,319, incorporated herein by reference, particularly for nucleotide modifications to circumvent the innate immune response.

[0070] In some embodiments, expression of Dnmt3b and / or Gimap6 is increased by introducing a transgene into cells, which can direct the desired level of overexpression (with varying promoter strength or other selection of expression control elements). Various viral vectors or transfection reagents (including lipid nanoparticles) known in the art can be used to introduce the transgene. In some embodiments, expression of Dnmt3b and / or Gimap6 is increased by transgene-free methods (e.g., episomal delivery). In some embodiments, expression or activity of Dnmt3b and / or Gimap6, or other genes disclosed herein, is increased using gene editing techniques, for example, to introduce one or more modifications to increase promoter strength, ribosome binding, or RNA stability.

[0071] In some embodiments, the method includes applying cyclic 2D, 3D, or 4D stretch to cells. In various embodiments, the cells subjected to cyclic 2D, 3D, or 4D stretch are selected from one or more of CD34-enriched cells, iPSCs, ECs, and HECs. For example, the cell population is introduced into a bioreactor that provides cyclic strain biomechanical stretch, as described in WO 2017 / 096215, which is incorporated herein by reference in its entirety. The cyclic strain biomechanical stretch can increase the activity or expression of Dnmt3b and / or Gimap6. In these embodiments, the mechanical means applies a stretching force to the cells or to a cell culture surface having cells (e.g., ECs or HECs) cultured thereon. For example, cyclic 2D, 3D, or 4D stretching can be applied to cells ex vivo under defined and controlled cyclic strain conditions using a computer-controlled vacuum pump system or other means for providing a stretching force (e.g., the FlexCell™ Tension System, CytoStretcher System) attached to a flexible, biocompatible and / or biomimetic surface. For example, the applied cyclic stretching can be about 1% to about 20% cyclic strain (e.g., about 6% cyclic strain) for several hours or days (e.g., about 7 days). In various embodiments, the cyclic strain is applied for at least about 1 hour, at least about 2 hours, at least about 6 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 120 hours, at least about 144 hours, or at least about 168 hours.

[0072] Alternatively, or in addition, EHT is stimulated by Trpv4 activation, which can be by contacting cells (e.g., CD34-enriched cells, ECs, or HECs) with one or more Trpv4 agonists, optionally selected from GSK1016790A, 4α-PDD, or analogs and / or derivatives thereof.

[0073] When a cell population is described herein as having a particular phenotype, it is understood that the phenotype represents a substantial portion of the cell population, such as at least 25%, at least 40%, or at least about 50%, or at least about 60%, or at least about 75%, or at least about 80%, or at least about 90% of the cell population. Furthermore, in various steps, the cell population can be enriched for cells of the desired phenotype and / or depleted of cells of an undesired phenotype, such that the cell population contains at least about 75%, or at least about 80%, or at least about 90% of the desired phenotype. Such positive and negative selection methods are known in the art. For example, cells can be sorted based on cell surface antigens (including those described herein) using a fluorescence-activated cell sorter or magnetic beads that bind to certain cell surface antigens. Negative selection columns can be used to remove cells expressing undesirable cell surface markers. In some embodiments, cells are enriched for CD34+ cells (before and / or after undergoing EHT). In some embodiments, the cell population is cultured under conditions that promote the proliferation of CD34+ cells, thereby producing an expanded population of stem cells. Additionally, subpopulations of B cell lineages can be enriched or isolated using these techniques.

[0074] In various embodiments, CD34+ cells (e.g., suspension and / or adherent cells) are harvested from cultures undergoing endothelial-hematopoietic transition between days 8 and 20 of iPSC differentiation (e.g., between days 10 and 17 of iPSC differentiation).

[0075] In various embodiments, the HSCs or CD34-enriched cells are further expanded. For example, the HSCs or CD34-enriched cells can be expanded according to the methods disclosed in US8,168,428, US9,028,811, US10,272,110, and US10,278,990, which are incorporated by reference in their entireties. In some embodiments, the ex vivo expansion of the HSCs or CD34-enriched cells employs prostaglandin E2 (PGE2) or a PGE2 derivative. In some embodiments of the present disclosure, the HSCs comprise at least about 0.01% LT-HSCs, or at least about 0.05% LT-HSCs, or at least about 0.1% LT-HSCs, or at least about 0.5% LT-HSCs, or at least about 1% LT-HSCs.

[0076] Hematopoietic stem cells (HSCs) that promote lymphopoiesis or give rise to lymphoid lineages can be identified based on the expression of CD34 and the absence of lineage-specific markers (referred to as Lin-). In some embodiments, a population of stem cells containing HSCs is enriched, for example, as described in US9,834,754, which is incorporated herein by reference in its entirety. For example, this process can include sorting the cell population based on the expression of one or more of CD34, CD90, CD38, CD43, CD45, CD19, CD20, CD138, or CD10. CD34 + , CD90 + , CD38 - , CD19, CD20, CD138, and / or CD43 - In some embodiments, a stem cell population for differentiation into hematopoietic lineages is selected for further differentiation from a fraction that is at least about 80% CD34 + , or at least about 90% CD34 + , or at least about 95% CD34 + is.

[0077] In some embodiments, stem cell populations, or CD34+ enriched cell or fraction thereof, or derivative populations, are expanded as described in US 2020 / 0308540, which is incorporated herein by reference in its entirety. For example, the cells are expanded by exposing them to an aryl hydrocarbon receptor antagonist, including, for example, SR1 or an SR1 derivative. See also Wagner et al., Cell Stem Cell 2016;18(1):144-55, and Boitano A., et al., Aryl Hydrocarbon Receptor Antagonists Promote the Expansion of Human Hematopoietic Stem Cells. Science 2010 Sep 10;329(5997):1345-1348.

[0078] In some embodiments, CD34 + Compounds that promote cell proliferation include pyrimidoindole derivatives, including, for example, UM171 or UM729 (see US2020 / 0308540, incorporated herein by reference).

[0079] In some embodiments, the stem cell population or CD34+ enriched cells are further enriched for or modified to express periostin and / or platelet-derived growth factor receptor alpha (pdgfra), as described in WO2020 / 205969 (incorporated herein by reference in its entirety). Such expression can be by transgene-free methods, including but not limited to, by delivering an encoding transcript to the cells, by introducing an encoding transgene, or by introducing a non-integrating episome into the cells. In some embodiments, gene editing is employed to introduce genetic modifications into expression elements in the cells, such as to increase promoter activity or strength, ribosome binding, RNA stability, or affect RNA splicing.

[0080] In yet another embodiment, the stem cell population or CD34+ enriched cells are cultured with an inhibitor of histone methyltransferase EZH1. Alternatively, EZH1 is partially or completely deleted or inactivated, or transiently silenced, in the stem cell population. Inhibition of EZH1 can direct myeloid progenitor cells (e.g., CD34+CD45+) to the lymphoid lineage. See WO2018 / 048828, which is incorporated herein by reference in its entirety. In yet another embodiment, EZH1 is overexpressed in the stem cell population.

[0081] In various embodiments, the HSC population or fractions thereof can be differentiated into B cell lineages or their precursors, such as multipotent progenitors (MPPs), common lymphoid progenitors (CLPs), common lymphoid 2 progenitors (LCA-2), early pre-B cells, late pre-B cells, pre-B cells, immature B cell lineages, and fractions thereof, from which mature B cells can be generated. B cells generated according to this process can have a phenotype consistent with transitional B cells, regulatory B cells, marginal zone B cells, follicular B cells, activated B cells, memory B cells, or plasma B cells, as previously described.

[0082] In some embodiments, HSC populations or fractions thereof are differentiated into B cells or their progenitor or derivative cells, with or without the use of an agonist of a mechanosensitive receptor or mechanosensitive channel, such as Yoda1. In some embodiments, the use of an agonist of a mechanosensitive receptor or mechanosensitive channel, such as Yoda1, is optional. Thus, in some embodiments, CD34+ cells are enriched from a differentiated pluripotent stem cell population to prepare a CD34+ enriched population. Endothelial-hematopoietic transformation of the CD34+ enriched cell population is induced for at least 2 days but not more than 12 days, optionally with the use of an agonist of a mechanosensitive receptor or mechanosensitive channel, such as Yoda1, jedi1, jedi2, or ssRNA40. HSCs and / or HSPCs are differentiated into a progenitor B cell population or a B cell population.

[0083] In some embodiments, endothelial-to-hematopoietic transformation of the CD34+ enriched cell population is induced for at least 2 days, optionally further for at least about 4 hours, or at least about 8 hours, or at least about 12 hours, or at least about 16 hours, or at least about 20 hours, or at least about 24 hours, or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days. Generally, EHT is not induced for more than 12 days. In various embodiments, EHT is induced for 4 days to about 8 days or 5 days to about 7 days.

[0084] In some embodiments, lymphocyte-hematopoietic lineages are cultured ex vivo with a partial or complete Notch ligand, SHH, extracellular matrix component(s), and / or a combination thereof to differentiate HSCs into lymphocyte-hematopoietic lineages. Furthermore, according to known processes, a feeder layer of heterologous OP9-DL1 or STO mouse fibroblasts, or blood-derived peripheral blood mononuclear cells (PBMCs), or cord blood-derived mesenchymal stem cells or lymphocyte-derived cancer cell line cells can be employed for differentiation of hematopoietic cells into lymphocyte-hematopoietic lineages, T cells, or NK cells, and optionally for differentiation of HSCs into other lineages. The OP9-DL1 coculture system uses a bone marrow stromal cell line (OP9) transduced with the Notch ligand Delta-like-1 (DLL1) to support T cell development from stem cell sources. The OP9-DL1 system limits the potential of the cells for clinical use. Therefore, in some embodiments, the method employs a feeder-free system for generating B cell lineages from hiPSCs for clinical use. In a non-limiting example, to generate cells from the lymphohematopoietic lineage utilizing Notch ligands, iPSC expansion is performed for 6 days, followed by embryoid body formation, which takes about 8 days. The cells are further cultured for about 5 days to allow the development of CD34+ hemogenic endothelial cells from which the HSCs are derived. The HSCs are then cultured in a medium that promotes differentiation into lymphohematopoietic cells, such as the B cell lineage, as described herein.

[0085] In some embodiments, the presence of cytokines and / or growth factors is desired, including, but not limited to, stem cell factor, Fms-like tyrosine kinase 3 ligand, VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, and optionally, a BMP activator to initiate definitive hemogenic endothelial differentiation into multipotent progenitor cells (MPPs), common lymphoid progenitors (CLPs), common lymphoid 2 progenitors (LCA-2s), early pre-B cells, late pre-B cells, pre-B cells, or immature B cell lineages, optionally comprising: (ii) contacting lymphohematopoietic lineage cells derived from pluripotent stem cells with a composition comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL3, IL7, and IL15, wherein the medium does not comprise one or more of VEGF, bFGF, TPO, a BMP activator, and a ROCK inhibitor to initiate lymphohematopoietic lineage differentiation.

[0086] As used herein, the term "Notch ligand" refers to a ligand capable of binding to a Notch receptor polypeptide present on the membrane of hematopoietic stem cells or progenitor T cells. Notch receptors include Notch-1, Notch-2, Notch-3, and Notch-4. Notch ligands typically have a DSL domain (D-Delta, S-Serrate, and L-Lag2) containing 20-22 amino acids at the amino terminus and 3-8 EGF repeats on the extracellular surface. In various embodiments, the Notch ligand comprises at least one of Delta-like-1 (DLL1), Delta-like-4 (DLL4), SFIP3 or a functional portion thereof, Jagged1 (JAG1), Jagged2 (JAG2), Delta-like ligand 3 (DLL3), and X-Delta2. In vivo, a key signal delivered by thymic stromal cells to invading lymphoid progenitors is mediated by DL4, which is expressed by cortical thymic epithelial cells.

[0087] As used herein, "Notch ligand" also includes intact (full-length), partial (truncated), or modified (containing one or more mutations, such as conservative mutations) Notch ligands, as well as Notch ligands or fragments thereof from any species that retain at least one activity or function of the full-length Notch ligand. Also included are peptides that mimic Notch ligands. Notch ligands can be "canonical Notch ligands" or "non-canonical Notch ligands." Canonical Notch ligands are typically characterized by an N-terminal (NT) domain followed by a Delta / Serrate / LAG-2 (DSL) domain and an extracellular domain containing multiple tandemly arranged epidermal growth factor (EGF)-like repeats. The DSL domain, along with the adjacent NT domain and the first two EGF repeats containing Delta and OSM-11-like (DOS) motifs, are typically required for canonical ligands to bind to Notch. The intracellular domains of some canonical ligands contain a carboxy-terminal PSD-95 / Dlg / ZO-1-ligand (PDZL) motif, which plays a role independent of Notch signaling. The C. elegans DSL ligand lacks the DOS motif but has been proposed to cooperate with ligands containing only DOS to activate Notch signaling.

[0088] In some embodiments, the Notch ligand is an anti-Notch (agonist) antibody that can bind to and participate in Notch signaling. In some embodiments, the antibody is a monoclonal antibody (including a human or humanized antibody), a single-chain antibody (scFv), a nanobody, or other antibody fragment or antigen-binding molecule that can activate the Notch signaling pathway.

[0089] In some embodiments, the Notch ligand is a Delta family Notch ligand. In some embodiments, the Delta family ligand is Delta-1 (Genbank Accession No. AF003522, Homo sapiens), Delta-like 1 (DLL1, Genbank Accession Nos. NM_005618 and NP_005609, Homo sapiens; Genbank Accession Nos. X80903, 148324, M. musculus), Delta-4 (Genbank Accession No. AF273454, BAB18580, Mus musculus; Genbank Accession Nos. AF279305, AAF81912, Homo sapiens), and / or Delta-like 4 (DLL4, Genbank Accession Nos. Q9NR61, AAF76427, AF253468, NM_019074, Homo sapiens; Genbank Accession No. NM 019454, Mus musculus). Notch ligands are commercially available or can be produced, for example, by recombinant DNA techniques.

[0090] In some embodiments, the Notch ligand comprises an amino acid sequence that is at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 97% identical (e.g., about 100% identical) to a human DLL1 or DLL4 Notch ligand. A functional derivative of a Notch ligand (including a fragment or portion thereof) will be capable of binding to and activating a Notch receptor. Binding to a Notch receptor can be determined by a variety of methods known in the art, including in vitro binding assays and receptor activation / cell signaling assays.

[0091] In various embodiments, the HSC / HSPC population is cultured in artificial thymic organoids (ATO). See Hagen, M. et al. (2019). ATO will involve culturing HSCs (or aggregates of HSCs) with a Notch ligand-expressing stromal cell line under serum-free conditions. Artificial thymic organoids are composed of naive CD3 + CD8 + and CD3 +CD4 + This is a 3D system that induces the differentiation of hematopoietic precursors into T cell or B cell lineages. In some embodiments, the artificial thymus organoid comprises DLL4 and BMP2, or functional fragments thereof.

[0092] In some embodiments, the Notch ligand is DLL4 with one or more affinity-enhancing mutations, such as one or more (or all) of the following: for hDLL4, G28S, F107L, I143F, H194Y, L206P, N257P, T271L, F280Y, S301R, and Q305P. See Gonzalez-Perez, et al., Affinity-matured DLL4 ligands as broad-spectrum modulators of Notch signaling, Nature Chemical Biology (2022).

[0093] In various embodiments, the Notch ligand is soluble and optionally immobilized on microparticles or nanoparticles that are optionally paramagnetic to enable magnetic enrichment or concentration processes. In yet other embodiments, the Notch ligand is immobilized on a 2D or 3D culture surface, optionally using other adhesion molecules such as VCAM-1. See US 2020 / 0399599, incorporated herein by reference in its entirety. In other embodiments, the beads or particles are constructed of biological materials, such as polymers (e.g., polystyrene or PLGA), gold, iron dextran, or particles formed from lipids and / or proteins. In various embodiments, the particles have a diameter or largest dimension of about 0.01 μm (10 nm) to about 500 μm (e.g., about 1 μm to about 7 μm). In still other embodiments, polymer scaffolds with conjugated ligands can be employed, as described in WO 2020 / 131582, incorporated herein by reference in its entirety. For example, the scaffold can be constructed from polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivatives, gelatin, collagen, agarose, hyaluronic acid, poly(lysine), polyhydroxybutyrate, poly-epsilon-caprolactone, polyphosphazine, poly(vinyl alcohol), poly(alkylene oxide), poly(ethylene oxide), poly(allylamine), poly(acrylate), poly(4-aminomethylstyrene), Pluronic® polyol, poloxamer, poly(uronic acid), poly(anhydride), poly(vinylpyrrolidone), and any combination thereof. In some embodiments, the scaffold comprises pores having diameters of about 1 pm to 100 pm.

[0094] In some embodiments, the C-terminus of the Notch ligand is conjugated to a support of choice. In some embodiments, this can involve adding a sequence to the C-terminus of the Notch ligand that can be enzymatically conjugated to the support, for example, via a biotin molecule. In another embodiment, the Notch ligand-Fc fusion is prepared such that the Fc segment can be immobilized by binding to Protein A or Protein G that has been conjugated to a support. Of course, any of the known protein conjugation methods can be employed.

[0095] Thus, in various embodiments, the Notch ligand can be immobilized, functionalized, and / or embedded in a 2D or 3D culture system. The Notch ligand can be incorporated with components of an extracellular matrix, such as one or more selected from fibronectin, retronectin, and laminin. In some embodiments, the Notch ligand and / or components of the extracellular matrix are embedded in an inert material that provides 3D culture conditions. Exemplary materials include, but are not limited to, cellulose, alginate, and combinations thereof. In some embodiments, the Notch ligand, components of the extracellular matrix, or combinations thereof, are in contact with culture conditions that provide cells with a topographical pattern and / or texture (e.g., roughness) that promotes differentiation and / or proliferation.

[0096] In various aspects and embodiments, the present disclosure provides a culture platform method for obtaining B cell lineages (as described) or progenitor cells thereof. In various embodiments, the method includes contacting cells (e.g., CD34+ cells from EBs generated as described herein) with an effective amount of an agonist of a mechanosensitive receptor or mechanosensitive channel that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezol. Exemplary Piezol agonists include Yoda1, single-stranded (ss)RNA (e.g., ssRNA40), Jedi1, and Jedi2. In some embodiments, the mechanosensitive receptor is Trpv4. An exemplary Trpv4 agonist is GSK1016790A. The culture medium may include one or more growth factors and cytokines selected from TPO, SCF, Flt3L, IL3, IL-6, IL7, IL-11, IGF, bFGF, and IL15. The culture medium may optionally contain one or more of VEGF, bFGF, a BMP activator, a Wnt pathway activator, or a ROCK inhibitor (e.g., thiazovivin or Y27632). The HSC population produced accordingly (and which may be enriched for CD34+ cells) is cultured in the presence of cytokines, growth factors, and / or small molecules that promote the formation of lymphocytes, including B cell lineages. For example, such cytokines and growth factors may include a combination of IL-3, IL-7, IL-15, SCF, and FLT-3L. IL-3 may be omitted in some embodiments, particularly at later stages of culture. For example, CD19+ cells may be cultured with IL-7, SCF, and Flt-3L. Other processes for generating B cell lineages are known in the art and may be employed. B cells may be activated ex vivo using cytokines and factors such as IL-4, IL-5, and IL-6, and CD40 ligand.

[0097] In some embodiments, the B cell lineage expresses a chimeric antigen receptor (CAR) based on gene editing of iPSCs, embryonic bodies, hCD34+ cells, or B cell progenitor cells or lines. In some embodiments, the B cell lineage expresses the CAR via mRNA expression. Additionally, or optionally, the B CAR can be engineered to express cytokines (e.g., IL-4, IL-6, IL-15, etc., or interferons) to make the B cell-CAR more potent in targeting tumor or infected cells.

[0098] In non-limiting examples, B cells or progenitor cells can be efficiently transduced with vectors such as, but not limited to, retroviral or non-integrating viral vectors (e.g., adenovirus, adeno-associated virus, integration-deficient retrolentivirus, poxvirus), or non-viral vectors (e.g., plasmid vectors, artificial chromosomes), or episomal or episomal hybrid vectors carrying first, second, third, fourth, or fifth generation CARs targeting tumor antigens (see, e.g., Sadelain et al., Cancer Discov. 3(4):388-398 (2013); Jensen et al., Immunol. Rev. 257:127-133 (2014); Sharpe et al., Dis. Model Meeh. 8(4):337-350 (2015); Brentjens et al., Clin. Cancer Res. 13:5426-5435 (2007); Gade et al. (See, e.g., E. et al., Cancer Res. 65:9080-9088 (2005); Maher et al., Nat. Biotechnol. 20:70-75 (2002); Kershaw et al., J. Immunol. 173:2143-2150 (2004); Sadelain et al., Curr. Opin. Immunol. (2009); Hollyman et al., J. Immunother. 32:169-180 (2009). Each of these aforementioned references is incorporated herein by reference in its entirety.

[0099] CARs are designed to enhance the ability of cells to recognize, bind to, and kill target cells, such as tumor cells or virus-infected cells or tissues. In some embodiments, CARs enhance the ability of B cells to recognize target cells. In some embodiments, CARs enhance B cell activity, for example, through antigen presentation, costimulation, and B cell activation via cytokine production. In some embodiments, CAR-B cells target the following tumors or tumor antigens: (i) human epidermal growth factor receptor 2 (HER2) - ovarian cancer, breast cancer, glioblastoma, colon cancer, osteosarcoma, and medulloblastoma; (ii) epidermal growth factor receptor (EGFR) - non-small cell lung cancer, epithelial carcinoma, and glioma; (iii) mesothelin-mesothelioma, ovarian cancer, and pancreatic adenocarcinoma; (iv) prostate-specific membrane antigen (PSMA)-prostate cancer; (v) Carcinoembryonic antigen (CEA) - pancreatic adenocarcinoma, breast cancer, and colorectal cancer; (vi) glypican-3-hepatocellular carcinoma; (vii) epidermal growth factor receptor variant III (EGFRvIII)-glioblastoma; (viii) Disialoganglioside 2 (GD2)—neuroblastoma and melanoma; (ix) Carbonic anhydrase IX (CAIX) - Renal cell carcinoma; (x) Interleukin-13Ra2-glioma, (xi) fibroblast activation protein (FAP) - malignant pleural mesothelioma; (xii) L1 cell adhesion molecule (L1-CAM)—neuroblastoma, melanoma, and ovarian; (xiii) Cancer antigen 125 (CA125)-epithelial ovarian cancer, (xiv) Cluster of differentiation antigen 133 (CD133) - glioblastoma, cholangiocarcinoma, adenocarcinoma, (xv) Cancer / Testis Antigen 1B (CTAG1B) - melanoma and ovarian cancer; (xvi) Mucin 1 - seminal vesicle cancer, (xvii) folate receptor-a (FR-a)-ovarian cancer; (xviii) a growth factor receptor selected from one or more of ErbB1, ErbB2, ErbB3, or ErbB4, IGF1R, IGF2R, TβR I-II, VEGFR1, VEGFR2, VEGFR3, PDGFR(α / β), or FGFR1-4.

[0100] Thus, in some aspects and embodiments of the invention, genetically engineered B cell lines, or their precursors or progeny, are engineered to express a chimeric antigen receptor (CAR) on the cell surface, particularly a CAR that specifically binds to a growth factor receptor. Most typically, the CAR comprises the intracellular domain from Fcε receptor γ (FcεRIγ). However, in further contemplated embodiments, the CAR may also comprise the T cell receptor (TCR) CD3ζ (CD3ζ) intracellular domain, alone or in combination with additional components from second- or third-generation CAR constructs (e.g., CD28, CD134, CD137, and / or ICOS).

[0101] In some embodiments, the CAR comprises at least one domain that inhibits anti-autoimmune or phagocytic signaling in B cells (e.g., the extracellular domain, the transmembrane domain, and / or the intracellular domain). In some embodiments, the CAR improves the effector activity of B cells relative to cells of the same type that do not contain the CAR, for example, by inhibiting CD47 and / or SIRPα activity. In some embodiments, the CAR functions as a dominant negative receptor by binding to CD47 and inhibiting SIRPα activity (e.g., CD47 sink).

[0102] In some embodiments, CAR-modified B cells exhibit increased production of one or more inflammatory cytokines relative to unmodified B cells. The one or more inflammatory cytokines can be chosen from one or more of TNF-alpha, IL-6, IL-1a, IL-1b, IL-12, IL-18, IL-8, IL-2, IL-23, IFN-alpha, IFN-beta, IFN-gamma, IL-2, IL-8, IL33, CCL3, CXCL12, CCL22, CCL4, CXCL10, or CCL2.

[0103] In one aspect, the present disclosure provides a cell composition comprising a cell population (e.g., a B-cell lineage population). The cell composition of the present disclosure (e.g., prepared according to the present disclosure) may further comprise a pharmaceutically acceptable excipient or carrier. Such excipient or carrier solutions may also contain buffers, diluents, and other suitable additives. A buffer refers to a solution or liquid whose chemical composition neutralizes acids or bases without significantly changing the pH. Examples of buffers contemplated by the present invention include, but are not limited to, physiological saline (0.9% NaCl), 5% dextrose in water (D5W), Dulbecco's phosphate-buffered saline (PBS), and Ringer's solution. The composition may comprise a vehicle suitable for intravenous infusion or other administration routes, and the composition may include a suitable cryoprotectant. An exemplary carrier is DMSO (e.g., about 10% DMSO). Other carriers may include dimethoxyethane (DME), N,N-dimethylformamide (DMF), or dimethylacetamide, including mixtures or combinations thereof. The cell composition may be provided in an appropriate volume within an implantable device (eg, a scaffold), or within a bag, or within a vial, tube, or container, and stored frozen until use.

[0104] In various embodiments, the B cells are derived from the HLA-edited iPSCs described. For example, in some embodiments, the B cells are derived from HLA-A neg , homozygous for both HLA-B and HLA-C, HLA-DPB1 neg , and HLA-DQB1 neg In some embodiments, the B cells are further homozygous for HLA-DRB1.

[0105] In some embodiments, the cell population is a precursor B cell population capable of engraftment in the thymus, spleen, or secondary lymphoid organs upon administration to a subject in need thereof. In various embodiments, a composition for cell therapy is prepared comprising the cell population and a pharmaceutically acceptable vehicle. The pharmaceutical composition comprises at least about 10 2 cells, or at least about 103 or at least about 10 4 or at least about 10 5 or at least about 10 6 or at least about 10 7 or at least about 10 8 cells, or at least about 10 9 cells, or at least about 10 10 cells, or at least about 10 11 cells, or at least about 10 12 cells, or at least about 10 13 cells, or at least about 10 14 For example, in some embodiments, pharmaceutical compositions containing about 100,000 to about 400,000 cells per kilogram (e.g., about 200,000 cells / kg) are administered. In other embodiments, the cells are administered at a concentration of about 10 per kilogram of recipient body weight. 5 ~Approx. 5×10 5 cells (e.g., approximately 2.5 x 10 5 cells / kg), or approximately 10 per kilogram 6 ~Approx. 5×10 6 cells (e.g., approximately 2.5 x 10 6 cells / kg), or approximately 5 x 10 cells per kilogram 6 ~about 10 7 cells (e.g., approximately 5 x 10 6 cells / kg), or approximately 10 per kilogram 7 ~about 10 8 cells (e.g., approximately 5 x 10 7 cells / kg), or approximately 10 per kilogram 8 ~about 10 9 cells (e.g., approximately 5 x 10 8 cells / kg), or approximately 10 per kilogram 9 ~about 10 10 cells, or approximately 10 10 ~about 10 11 cells, or approximately 10 per kilogram 11 ~about 10 12 cells, or approximately 10 per kilogram 12 ~about 1013 cells, or approximately 10 per kilogram 13 ~about 10 14 It is administered in individual cells.

[0106] The pharmaceutical composition may also contain additional therapeutic agents for the treatment of a particular target disorder. For example, the pharmaceutical composition may also include cytokines and growth factors (e.g., interleukins, interferons, FGF, VEGF, PDGF, PIGF, STAT, etc.). Such additional factors and / or agents may be included in the pharmaceutical composition to provide the benefits of the therapeutic approaches disclosed herein, i.e., improved therapeutic efficacy with reduced systemic toxicity.

[0107] In another aspect, the present invention provides methods for cell therapy comprising administering a cell population described herein or a pharmaceutically acceptable composition thereof to a human subject in need thereof. In various embodiments, the methods described herein are used to treat hematological (malignant and non-malignant) disorders, bone marrow disorders, immune disorders, and infectious diseases. In various embodiments, the human subject has a condition comprising one or more of lymphopenia, cancer, immunodeficiency, and autoimmune disease. Examples of diseases include various autoimmune disorders, including, but not limited to, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), some forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, and granulomatosis with polyangiitis (Wegener's granulomatosis).Hematologic malignancies that can be treated include, but are not limited to, acute and chronic leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome. Infectious diseases that can be treated include, but are not limited to, HIV (human immunodeficiency virus), RSV (respiratory syncytial virus), EBV (Epstein-Barr virus), CMV (cytomegalovirus), adenovirus, and BK polyomavirus-related disorders. Other conditions include bone marrow failure syndromes and certain genetic disorders (e.g., genetic disorders affecting the immune system). In some embodiments, the subject has a cancer, such as a solid tumor, including, but not limited to, tumors of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testis, bladder, kidney, head, neck, stomach, cervix, rectum, larynx, or esophagus.

[0108] The cell populations can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disease or disorder being treated, the particular mammal (e.g., human) being treated, the clinical condition of the individual patient, the cause of the disease or disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to physicians. The therapeutically effective amount of cells administered will be governed by such considerations.

[0109] Other compounds may be administered along with the cells, such as cytotoxic agents, immunosuppressants, and / or cytokines or growth factors (e.g., stem cell factor, thrombopoietin, transforming growth factor (TGF)-α or β, fibroblast growth factor (FGF), the angiopoietin (Ang) family of growth factors, insulin-like growth factor, granulocyte-macrophage colony-stimulating factor, TNF-α or β, VEGF, interleukins (e.g., IL-2, 6, 7, 8, 10, 12, 15, etc.), and interferons (e.g., INF-alpha or gamma)). Coadministration includes simultaneous administration using separate formulations or a single pharmaceutical formulation, and sequential administration in either order, preferably with a period during which both (and all) active agents simultaneously exert their biological activities.

[0110] For the use of HLA-edited B cells (e.g., derived from gene-edited iPSCs), subjects can be matched at retained HLA loci, such as one or more (or all) of HLA-B, HLA-C, and HLA-DRB1.

[0111] In certain embodiments, prior to therapy, the patient may undergo lymphodepleting chemotherapy (or other known processes) using chemotherapeutic agents such as fludarabine or cyclophosphamide.

[0112] As used herein, the term "about" means ±10% of the associated numerical value.

[0113] Certain aspects and embodiments of the present disclosure are further illustrated with reference to the following examples. [Example]

[0114] Example 1 - ETV2 overexpression increases the yield of hemogenic endothelial cells and enhances CD34+ cell formation during iPSC differentiation, but does not affect pluripotency. method iPSCs were developed from hCD34+ cells by episomal reprogramming, as known in the art and essentially as described in Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells, Science 318, 1917-1920, (2007), and J. Yu, et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801, (2009). Embryoid bodies and hemogenic endothelial differentiation were performed essentially as described in R. Sugimura, et al., Hematopoietic stem and progenitor cells from human pluripotent stem cells. Nature 545, 432-438, (2017); C. M. Sturgeon, et al., Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat Biotechnol 32, 554-561, (2014); J. Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920, (2007); and J. Yu, et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801, (2009).

[0115] Briefly, hiPSCs were dissociated and resuspended in medium supplemented with L-glutamine, penicillin / streptomycin, ascorbic acid, human holo-transferrin, monothioglycerol, BMP4, and Y-27632. The cells were then seeded onto 10 cm dishes (EZSPHERE or low-attachment plates) for EB formation. On day 1, bFGF and BMP4 were added to the medium. On day 2, the medium was replaced with medium containing SB431542, CHIR99021, bFGF, and BMP4. On day 4, the cell medium was replaced with medium supplemented with VEGF and bFGF. On day 6, the cell medium was replaced with medium supplemented with bFGF, VEGF, interleukin (IL)-6, IGF-1, IL-11, SCF, and EPO. Cells were maintained in an incubator with 5% CO2, 5% O2, and 95% humidity. To harvest CD34+ cells, EBs were dissociated on day 8, cells were filtered through a 70 μm strainer, and CD34+ cells were isolated by CD34 magnetic bead staining.

[0116] result Induced pluripotent stem cells (iPSCs) were transduced using an adenoviral vector containing both ETV2 and GFP sequences under the control of the EF1A promoter. After transduction, approximately 45% of the iPSC cultures were observed to be GFP-positive, confirming ETV2 overexpression (ETV2-OE). It was further observed that ETV2-OE in iPSC cells retained the pluripotent properties of iPSCs, as indicated by the stemness marker expression TRA-1-60 (Figure 1). Figure 1 shows a FACS plot demonstrating the transduction efficiency of iPSCs using an adenoviral vector for overexpressing ETV2 and GFP sequences.

[0117] Next, ETV2-OE-iPSCs (along with control iPSCs transduced with a vector carrying the GFP sequence but without ETV2) were differentiated into embryoid bodies and then into hemogenic endothelial cells (Strugeon et al., 2014). The results showed that overexpression of ETV2 significantly increased the expression of CD235a. - CD34 in the population+ and CD31 + These results suggest that ETV2-OE enhances the formation of hemogenic endothelial cells, as evidenced by the expression of markers (Figure 2). Specifically, Figure 2 shows representative flow cytometry analysis and relative quantification of hemogenic endothelial cells (defined here as CD235a-CD34+CD31+), demonstrating that ETV2-OE enhances the formation of hemogenic endothelial cells compared to controls.

[0118] Furthermore, the results showed that ETV2-OE expresses CD34 + This suggests that ETV2-OE enhances CD34+ cell formation (Figure 3). Figure 3 shows representative flow cytometry analysis and relative quantification of CD34+ cells demonstrating that ETV2-OE enhances CD34+ cell formation.

[0119] Overall, these data indicate that ETV2 overexpression in iPSCs does not affect their pluripotent properties, but promotes their ability to undergo hemogenic endothelial and hematopoietic differentiation.

[0120] Example 2 - iPSC-derived HSCs generated by Piezo1 activation undergo T cell differentiation similar to bone marrow-derived HSCs. method To analyze EHT, EB-derived CD34+ cells were suspended in medium containing Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3. After the cells had adhered to the bottom of the well (by visual inspection) for approximately 4-18 hours, Yoda1 was added to the culture. After 4-7 days, cells were collected for analysis.

[0121] iPSCs were differentiated into embryoid bodies for 8 days. On day 8, CD34+ cells from the iPSC-derived embryoid bodies were harvested and cultured for an additional 5–7 days to induce endothelial-hematopoietic (EHT) transition. CD34+ cells were then harvested from the EHT cultures on days 5–7 for further hematopoietic lineage differentiation.

[0122] CD34+ cells harvested from EHT cultures on days 5–7 (or days 13–21 total of differentiation from iPSCs) were seeded onto 48-well plates pre-coated with rhDL4 and retronectin. T lineage differentiation was induced in medium containing aMEM, FBS, ITS-G, 2BME, ascorbic acid-2-phosphate, Glutamax, rhSCF, rhTPO, rhIL7, FLT3L, rhSDF-1α, and SB203580.

[0123] 80% of the medium was changed every other day from days 2 to 6. On day 7, cells were transferred to new coated plates and analyzed for the presence of pre-T cells (CD34+CD7+CD5+ / -).

[0124] 80% of the medium was changed every other day from days 8 to 13. On day 14, 100,000 cells / well were transferred to new coated plates, and cells were analyzed for the presence of pre-T cells (CD34-CD7+CD5+ / -).

[0125] 80% of the medium was changed every other day from days 15 to 20. Cells were harvested on day 21 and analyzed via FACS for CD3, CD8, CD5, CD7, TCRab expression on behalf of T cells, and / or activated using CD3 / CD28 beads to assess their functional properties.

[0126] After 21 days of differentiation, cells were harvested and approximately 80,000 cells were replated into new 96-well culture plates in RPMI 1640 (without L-glutamine, without phenol red) with FBS, L-glutamine, and IL-2, and then activated with 1:1 CD3 / CD28 beads. After 72 hours of activation with CD3 / CD28 beads, cells were analyzed for CD3, CD69, and CD25 expression by FACS and for IFN-γ expression using RT-qPCR. Supernatants were analyzed by ELISA.

[0127] result Figures 4A and 4B show that iPSC-derived HSCs derived by EHT of D8 CD34+ cells (using Piezol activation in this example) undergo pre-T cell differentiation, similar to bone marrow (BM)-HSCs. Furthermore, Figures 5A and 5B show that iPSC-derived HSCs generated by EHT of D8 CD34+ cells (using Piezol activation in this example) undergo T cell differentiation, similar to BM-HSCs, and can be activated with CD3 / CD28 beads. Figure 6 shows that iPSC-derived HSCs generated by EHT of D8 CD34+ cells (using Piezol activation in this example) can differentiate into functional T cells, as demonstrated by INFγ expression upon stimulation with CD3 / CD28 beads. Together, these results demonstrate that EHT of D8 CD34+ cells enhances their ability to further differentiate into hematopoietic lineages ex vivo.

[0128] Example 3 - Evaluation of off-target editing in HLA knockout HSCs HLA typing of HLA-edited (e.g., triple knockout) HSC clones was performed to check for unwanted editing and ensure that major editing events, e.g., deletion(s), did not occur within other regions of chromosome 6. Sequencing methods and analyses were performed to assess the extent of gRNA off-target activity and select gRNAs that represent a low risk of affecting non-target HLA genes.

[0129] Sequencing was performed using in situ cleavage labeling in fixed and permeabilized cells by ligating full-length P5 sequencing adapters to end-prepared DSBs. Genomic DNA was extracted, fragmented, end-prepared, and ligated using chemically modified semi-functional P7 adapters. The resulting DNA library contained a mixture of functional DSB-labeled fragments (P5:P7) and non-functional genomic DNA fragments (P7:P7). Subsequent DNA sequencing of the DNA library enriched for the DNA-labeled fragments, eliminating all irrelevant non-functional DNA. Because the library preparation was PCR-free, each resulting sequencing read was equivalent to a single labeled DSB end from the cell. This generated a DNA cleavage readout, enabling direct detection and quantification of genomic DSBs by sequencing without the need for error correction, and allowed for the unambiguous mapping of off-target mutations.

[0130] Table 1 below summarizes the results of the editing strategy in two representative clones relative to wild-type cells. [Table 1-1] [Table 1-2]

[0131] Table 2 provides non-limiting examples of gRNAs used in experiments that can be used to knock out expression of the indicated HLA genes and haplotypes. [Table 2]

[0132] The results show that the editing strategy was successful in selectively targeting the HLA-A, DPB1, and DQB1 genes without affecting other HLA genes or introducing major deletions elsewhere.

[0133] These results were confirmed by phenotypic analysis of the HLA-edited clones by FACS and immunofluorescence. As shown in Figures 9A and 9B, the HLA-edited cells tested positive for overall expression of HLA class I molecules, comparable to that of wild-type cells (gHSCs). Specific expression of HLA-A via immunofluorescence confirmed that HLA-A was not expressed in the HLA-edited cells, supporting the finding that the gene editing strategy successfully deleted only the HLA-A gene. Specifically, Figure 7A shows that all HLA-edited cells were positive for HLA-like class I molecules to the same extent as wild-type (WT) (i.e., non-HLA-edited) cells. This result indicates that despite the deletion of HLA-A, other class I molecules, such as HLA-B and HLA-C, were expressed and were not affected by the gene editing strategy.

[0134] To confirm that the HLA-A gene had been deleted, the specific expression of HLA-A was analyzed by immunofluorescence. As can be seen in Figure 7B, HLA-A was not expressed in the HLA-edited clones, indicating that the gene editing strategy was effective in specifically deleting only the HLA-A gene. Such preservation of overall class I expression with deletion of HLA-A would facilitate patient matching while avoiding NK cell-mediated rejection.

[0135] Example 4 - Evaluation of pluripotency and immune compatibility of HLA-edited HSCs The ability of HLA-edited cells to preserve pluripotency was assessed. As shown in Figure 8, immunofluorescence evaluation of HLA-edited iPSC clones demonstrated that they maintained tri-lineage differentiation, with ectodermal differentiation indicated by NESTIN-488 and PAX6-594 staining, mesodermal differentiation indicated by GATA-488 staining, and endodermal differentiation indicated by CXCR4-488 and FOX2A-594 staining.

[0136] HLA class I molecules are expressed on the surface of all nucleated cells, and if HLA class I molecules are mismatched between donor and recipient, the cells can be recognized and killed by CD8+ T cells. In addition, HLA mismatches can lead to cytokine release syndrome (CRS) and graft-versus-host disease (GVHD). Conversely, complete deletion of HLA-I molecules via B2M KO would render the cells targets for NK cell-mediated cytotoxicity. Preservation of overall class I expression with deletion of HLA-A could facilitate patient matching while avoiding NK cell-mediated rejection. Therefore, the immune compatibility of HLA-edited HSCs was tested by coculture with peripheral blood mononuclear cells (PBMCs) to assess whether immune cells would reject grafts of HLA-edited and wild-type HSCs (gHSCs).

[0137] Wild-type (i.e., gHSCs) and HLA-edited HSCs were cocultured with PBMCs bearing HLA-B and HLA-C markers but mismatched HLA-A. B2M KO HSCs, which lack expression of HLA class I molecules, and CIITA KO HSCs, which lack expression of class II molecules, were used as controls to compare the degree of PBMC-mediated cytotoxicity for HLA-null and HLA-mismatched HSCs, respectively. Figure 9 shows the results of a PBMC-mediated cytotoxicity assay in the coculture, measured by Annexin V staining. The results show that deletion of HLA-A in HLA-edited HSCs protected the cells from PBMC-mediated cytotoxicity, while WT, B2M KO, and CIITA KO HSCs were susceptible to PBMC-mediated cytotoxicity. Coculture of HSCs with sorted CD8+ T cells from the same PBMC donor protected HLA-edited and B2M KO HSCs from CD8+ T cytotoxicity. Conversely, HSCs co-cultured with sorted NK cells protected only WT and HLA-edited cells from NK cell-mediated cytotoxicity.

[0138] In summary, the immunocompatibility results indicate that CD8+ T cells present in the PBMC samples were involved in killing cells bearing mismatched HLA molecules (WT and CIITA KO), while NK cells present in the PBMCs were involved in killing HLA-null cells (B2M KO). However, HLA-edited HSCs were protected from CD8+ T cell-mediated cytotoxicity (because mismatched HLA-A had been knocked out) and from NK cell-mediated cytotoxicity (because HLA class I molecule expression was largely preserved).

[0139] Example 5 - Evaluation of in vivo engraftment potential of HLA-edited HSCs To evaluate the engraftment potential of HLA-edited HSCs, the cells' ability to engraft in vivo was assessed by competitive transplantation against wild-type HSCs. Equal proportions of mCherry HLA-edited HSCs and wild-type HSCs (gHSCs) were mixed and transplanted into mice. Bone marrow (BM) and peripheral blood samples were collected and evaluated by FACS to compare the relative amounts of each cell type present in the samples. As shown in Figure 10, both HLA-edited and wild-type HSCs contributed to approximately equal engraftment in BM and peripheral blood samples. These results confirm that HLA-edited HSCs (prepared according to the present disclosure) are comparable to wild-type HSCs in their engraftment and reconstitution potential. Therefore, the properties of wild-type (unedited, parental) HSCs are expected to match those of the HLA-edited HSCs of the present disclosure for generating T cell lineages.

[0140] Example 6 - Differentiation of HLA-edited HSCs into CD4+ / CD8+ T cells Antigen-presenting cells (APCs) present antigens to helper CD4+ T cells through HLA-II molecules. Activation of helper CD4+ T cells promotes the generation of antigen-specific CD8+ T cells, which further evolve into antigen-specific CTLs. Similarly, HLA class I molecules are expressed on the surface of all nucleated cells and present peptide fragments of proteins to CD8+ CTLs from within the cell. CTLs induce cytotoxic killing of target (infected) cells upon recognition of HLA-I-peptide complexes expressed on the cell surface. Therefore, studies were performed to determine whether deletion of HLA-A affects class I peptide presentation of edited HSCs. As shown in Figures 11A and 11B, immunopeptidome analysis indicates that deletion of HLA-A does not affect overall class I peptide presentation. HLA-A-edited cells exhibited comparable peptide and protein presentation compared to wild-type HSCs (gHSCs). Furthermore, as shown in Figures 12A and 12B, deletion of HLA-DQB1 and HLA-DPB1 does not affect overall class II peptide presentation by macrophages differentiated from HSCs. Together, these data suggest that despite the deletion of HLA-A, HLA-DQ, and HLA-DP molecules, the cells (and their derived lineages) retain the ability to present a wide range of class I and II peptides.

[0141] Example 7 - In vivo testing of antigen-mediated immune responses. Figure 13 is a schematic diagram of the delayed-type hypersensitivity reaction, showing the sensitization and elicitation stages of antigen presentation. Briefly, upon antigen injection, the antigen is processed by antigen-presenting cells (APCs) and presented by MHC class II molecules on the surface of the APCs. CD4+ T cells recognize peptide-MHC on the antigen-presenting cells (APCs). Upon antigen administration, CD4+ helper T cells are activated, and cytokines recruit macrophages and other immune cells that induce tissue swelling.

[0142] A delayed-type hypersensitivity assay was performed on transplanted mice. Specifically, mice were sensitized by subcutaneous injection of sheep red blood cells as an antigen. If the mouse has a functional immune system, APCs process the antigen and present the peptide antigen to CD4+ T cells. The mouse was then stimulated by subcutaneous injection of the same antigen in the left paw. At this point, T cells are activated and secrete cytokines that recruit macrophages and other immune cells to the antigen injection site, resulting in tissue swelling. In this assay, a functional immune system resulted in swelling of the left paw, as measured with a microcaliper.

[0143] As can be seen in Figures 14A and 14B, control (non-transplanted) mice, due to their immunodeficiency, did not show swelling in their left paws. Conversely, mice transplanted with cord blood CD34+ cells showed tissue swelling, doubling the diameter of their left paws. Similar immune system responses were seen in both mice transplanted with WT (non-edited HSCs) and mice transplanted with HLA-edited HSCs.

[0144] Example 8 - Evaluation of differentiation and maturation of HSC-derived T cells (pre-T cells) Next, we tested the ability of HSC-derived T cells (pre-T cells) to differentiate into mature T cells. After a 35-day differentiation period, pre-T cells were assessed by cell sorting for the presence of CD4+, CD8+, and AB+ T cell populations. As shown in Figure 15, pre-T cells differentiated into CD4+, CD8+, and αβ+ T cells more efficiently than bone marrow (BM)-derived CD34+ cells and embryonic body (EB)-derived CD34+ cells.

[0145] Next, to test their functional properties, each T cell population was cocultured with a CD19+ lymphoma cell line and an anti-CD3 / CD19 bispecific antibody. In this experimental model, the bispecific antibody engaged both the CD3 receptor on T cells and the CD19 cell surface receptor on lymphoma cells, thus inducing T cell activation. The degree of activation was assessed by measuring subsequent T cell-mediated cytotoxicity compared to pan-T cell controls. As shown in Figure 16, pre-T cells demonstrated statistically significant superiority in cytotoxicity compared to both BM CD34+ T cells and EB CD34+ T cells.

[0146] Example 9 - Evaluation of HSC properties evolving into pre-T cells. The ability of HSCs to evolve into pre-T cells was assessed by measuring the CD34-CD7+ marker on pre-T cells. As shown in Figure 17, FACS analysis demonstrated that HSCs produced according to the present disclosure successfully differentiated into CD34-CD7+ pre-T cells compared to bone marrow-derived CD34+ cells or EB-derived CD34+ cells.

[0147] Next, the expression of T cell-specific transcription factors and thymic engraftment molecules was measured. Figure 18A shows increased TCF7 expression in pre-T cells derived from HSCs of the present disclosure, and Figure 18B shows increased CCR7 expression. Figure 19A shows that pre-T cells derived from HSCs engraft and differentiate in the thymus. Figure 19B shows FACS analysis of the CD3+ cell population of cells gated on the CD45+ cell population, demonstrating the superior engraftment and differentiation potential of pre-T cells derived from HSCs in the thymus. Pre-T cells in this example were prepared from HSCs using Piezo1 activation, as previously described.

[0148] In vitro activation of HSC-derived T cells was also measured, as illustrated in Figure 20. The top panel of Figure 20 shows FACS analysis of activated T cells from different sources, including HSCs of the present disclosure (e.g., prepared using Piezol activation). T cells prepared from HSCs of the present disclosure showed comparable or superior activation, as measured by increased CD107 expression. The bottom panel shows Dynabeads activation, where activated T cells express inflammatory cytokines. T cells derived from HSCs according to the present disclosure (e.g., prepared using Piezol activation) expressed higher levels of inflammatory cytokines, as exemplified by TNF-α and interferon-gamma expression levels.

[0149] Example 10: Differentiation of HLA-edited HSCs into hematopoietic lineage, promonocyte / macrophage cells Experiments were conducted to determine whether HLA deletion affects the ability of HSCs to differentiate into different types of immune cells. HLA-edited HSCs were differentiated into promonocyte / macrophage cells using a process essentially as described in Example 2. It was determined that HLA-edited HSCs were capable of differentiating into the monocyte / macrophage lineage equivalent to wild-type (non-HLA-edited) HSCs, as measured by their CD11b+-CD14+ expression (Figure 21A). Furthermore, the CD11b+-CD14+ gated population showed equivalent HLA-I and HLA-II expression (Figure 21B), also indicating that HLA-edited HSCs preserved their overall expression of both class I and class II molecules.

[0150] The global expression of other class II molecules in HLA-DQB1 and HLA-DPB1 supported by edited HSCs was assessed by evaluating their expression in macrophages differentiated from HSCs. The design of this study is shown schematically in Figure 22A. It was found that deletion of HLA-DQB1 and HLA-DPB1 did not affect the expression of other HLA class II molecules (Figure 22B). For example, HLA-DR was equally expressed in both WT and HLA-edited cells (Figure 22C). In Figures 22B and 22C, CIITA-KO is the positive control.

[0151] Example 11: Differentiation of HLA-edited HSCs into proplatelets It was determined that HLA-edited HSCs can differentiate into megakaryocytes (MKs) and further differentiate into platelets. Differentiation was compared to bone marrow (BM)-derived CD34+ cells and iPSC-CD34+ cells. As shown in Figure 23, HLA-edited HSCs showed a statistically significant increase in platelet content compared to BM CD34+ and iPSC-CD34+ cell populations. Thus, HLA-edited HSCs can differentiate into megakaryocytes (MKs), which can further support differentiation into platelets.

[0152] Example 12 - Evaluation of degranulation and cytotoxicity of immunocompatible HSC-derived NK cells Next, we evaluated the ability of triple-knockout HSCs to differentiate into NK cells that maintained their degranulation and cytotoxicity. As shown in Figure 24, HSCs effectively differentiated into NK cells, as determined by fluorescence-activated cell sorting (FACS) experiments gated based on the expression of the known NK cell surface marker CD56. HSCs also demonstrated the ability to differentiate at least CD34+ BM and iPSC-EB CD34+ cell populations. To measure the ability of HSC-derived NK cells to effectively kill tumor cells, we performed the experimental protocol shown in Figure 25A. HSC-derived NK cells were cocultured for 3.5 hours with K562 HLA-null cells, a human erythroid myeloid blastoid leukemia cell line derived from the pleural effusion of a patient with chronic myeloid leukemia. These cells express ligands for aNKR, and their lack of HLA cell surface expression also contributes to NK cell activation by suppressing negative signaling through iNKR. Thus, these HLA-null cell lines have the potential to induce distinct functional profiles in NK cells and their subsets. After coculture, the degree of NK cell degranulation was measured using FACS and Annexin V staining, as well as a cytotoxicity assay. As shown in Figure 25B, Annexin V staining demonstrated that HSC-derived NK cells exhibited a higher degree of activation from HLA-null K562 cells than from CD34+ BM and iPSC-EB CD34+ cells. This was confirmed by the cytotoxicity assay results, as shown in Figure 25C.

[0153] References 1. Nianias, A. & Themeli, M. Induced Pluripotent Stem Cell (iPSC)-Derived Lymphocytes for Adoptive Cell Immunotherapy: Recent Advances and Challenges. Curr Hematol Malig Rep 14, 261-268 (2019). 2.Brauer,P.M.,Singh,J.,Xhiku,S.& Zuniga-Pfluecker,J.C.T Cell Genesis:In Vitro Veritas Est?Trends Immunol 37,889-901(2016). 3.Kennedy,M.et al.T Lymphocyte Potential Marks the Emergence of Definitive Hematopoietic Progenitors in Human Pluripotent Stem Cell Differentiation Cultures.Cell Reports 2,1722-1735(2012). 4.Sturgeon,C.M.,Ditadi,A.,Awong,G.,Kennedy,M.& Keller,G.Wnt Signaling Controls the Specification of Definitive and Primitive Hematopoiesis From Human Pluripotent Stem Cells.Nat Biotechnol 32,554-561(2014). 5.Chang,C.-W.,Lai,Y.-S.,Lamb,L.S.& Townes,T.M.Broad T-Cell Receptor Repertoire in T-Lymphocytes Derived from Human Induced Pluripotent Stem Cells.PLoS One 9,(2014). 6.Nishimura,T.et al.Generation of Rejuvenated Antigen-Specific T Cells by Reprogramming to Pluripotency and Redifferentiation.Cell Stem Cell 12,114-126(2013). 7.Themeli,M.et al.Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy.Nat Biotechnol 31,928-933(2013). 8.Vizcardo,R.et al.Regeneration of Human Tumor Antigen-Specific T Cells from iPSCs Derived from Mature CD8+T Cells.Cell Stem Cell 12,31-36(2013). 9.Montel-Hagen,A.et al.Organoid-induced differentiation of conventional T cells from human pluripotent stem cells.Cell Stem Cell 24,376-389.e8(2019). 10.Guo,R.et al.Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors.Cell Research 30,21-33(2020). 11.Nagano,S.et al.High Frequency Production of T Cell-Derived iPSC Clones Capable of Generating Potent Cytotoxic T Cells.Molecular Therapy-Methods & Clinical Development 16,126-135(2020). 12.Iriguchi,S.et al.A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy.Nature Communications 12,430(2021).

Claims

1. 1. A method for preparing a B cell population or a progenitor thereof, comprising: enriching CD34+ cells from the differentiated pluripotent stem cell (PSC) population to prepare a CD34+ enriched population; inducing endothelial-to-hematopoietic transformation of the CD34+ enriched cell population for at least 2 days but not more than 12 days to produce a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem progenitor cells (HSPCs); differentiating said population comprising HSCs and / or HSPCs into a progenitor B cell population or a B cell population.

2. 2. The method of claim 1, wherein the PSC population is a human iPSC population derived from lymphocytes, umbilical cord blood cells, peripheral blood mononuclear cells, CD34+ cells, or human primary tissue.

3. 3. The method of claim 2, wherein the iPSC population is derived from CD34+ cells isolated from peripheral blood.

4. 4. The method of claim 2 or 3, wherein the iPSCs are homozygous for one or more HLA class I and / or HLA class II genes.

5. The method of claim 4, wherein the iPSCs are homozygous for HLA-DRB1.

6. The method of claim 4, wherein the iPSCs are homozygous for both HLA-B and HLA-C.

7. 5. The method of any one of claims 2 to 4, wherein the iPSCs have been gene-edited to delete one or more HLA class I genes, one or more class II genes, and / or one or more genes that govern HLA or MHC expression or presentation capacity.

8. The method of claim 7, wherein the iPSCs comprise a deletion of HLA-A.

9. The method of claim 7 or 8, wherein the iPSCs comprise a deletion of HLA-DPB1 and / or HLA-DQB1.

10. The iPSCs are HLA-A ｎｅｇ , homozygous for both HLA-B and HLA-C, HLA-DPB1 ｎｅｇ , and HLA-DQB1 ｎｅｇ 10. The method of any one of claims 2 to 9, wherein the mouse is optionally further gene-edited to be homozygous for HLA-DRB1.

11. The method according to claim 7, wherein the one or more genes controlling the ability to express or present HLA or MHC are β2-microglobulin and / or CIITA.

12. The method of any one of claims 1 to 11, wherein CD34+ enrichment and endothelial-hematopoietic transition are induced between days 8 and 15 of iPSC differentiation.

13. 13. The method of any one of claims 1 to 12, wherein the CD34+ enriched population is cultured in a medium containing Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3.

14. 14. The method of claim 12 or 13, wherein the endothelial-to-hematopoietic conversion generates a HSC population comprising one or more of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells, and hematopoietic stem progenitor cells.

15. The method of any one of claims 12 to 14, wherein the CD34+ cells are harvested from a culture undergoing endothelial-hematopoietic transformation, including harvesting of CD34+ floating and / or adherent cells.

16. The method of any one of claims 1 to 15, wherein the HSC population comprises long-term hematopoietic stem cells (LT-HSCs).

17. The method of any one of claims 1 to 16, wherein the induction of endothelial-hematopoietic transition comprises increasing the expression or activity of dnmt3b.

18. 18. The method of claim 17, wherein the induction of endothelial-to-hematopoietic transition comprises applying cyclic stretch to the CD34-enriched cells.

19. 19. The method of claim 18, wherein the cyclic stretching is 2D, 3D, or 4D cyclic stretching.

20. The method according to any one of claims 1 to 16, wherein the induction of endothelial-hematopoietic transition comprises Piezo1 activation.

21. 21. The method of claim 20, wherein the Piezo1 activation is by contacting the CD34+ enriched cells or a fraction thereof, optionally with one or more Piezo1 agonists selected from Yoda1, ssRNA40, Jedi1, Jedi2, or analogs or derivatives thereof.

22. The method of any one of claims 1 to 16, wherein the induction of endothelial-hematopoietic transition comprises Trpv4 activation.

23. 23. The method of claim 22, wherein said Trpv4 activation is by contacting said CD34+ enriched cells, optionally with one or more Trpv4 agonists selected from GSK1016790A, 4α-PDD, or analogs or derivatives thereof.

24. 24. The method of any one of claims 1 to 23, wherein the B cell lineage is selected from multipotent progenitor cells (MPP), common lymphoid progenitor cells (CLP), common lymphoid 2 progenitor cells (LCA-2), early pre-B cells, late pre-B cells, pre-B cells, immature B cells.

25. 25. The method of any one of claims 1 to 24, wherein the B cell lineage is capable of differentiating into one or more lineages having a phenotype consistent with transitional B cells, regulatory B cells, marginal zone B cells, follicular B cells, activated B cells, memory B cells, or plasma B cells, or a combination thereof.

26. 26. The method of claim 24 or 25, wherein the B cell lineage expresses a chimeric antigen receptor (CAR).

27. 27. The method of claim 26, wherein the CAR-modified B-cell lineage of immune cells is selected from one or more of CAR-transitional B cells, CAR-regulatory B cells, CAR-marginal zone B cells, or CAR-follicular B cells, or CAR-activated B cells, CAR-memory B cells, or CAR-plasma B cells.

28. A B cell lineage cell population, or a pharmaceutically acceptable composition thereof, produced by the method of any one of claims 1 to 27.

29. A B cell population, or a pharmaceutically acceptable composition thereof, wherein the B cell population is HLA-A ｎｅｇ , homozygous for both HLA-B and HLA-C, HLA-DPB1 ｎｅｇ , and HLA-DQB1 ｎｅｇ , optionally further homozygous for HLA-DRB1, or a pharmaceutically acceptable composition thereof.

30. 30. A method for cell therapy comprising administering the B-cell lineage cell population of claim 28 or 29, or a pharmaceutically acceptable composition thereof, to a human subject in need thereof.

31. 31. The method of claim 30, wherein the human subject has a condition comprising one or more of lymphopenia, cancer, immunodeficiency, autoimmune disease, viral infection, skeletal dysplasia, and bone marrow failure syndrome.

32. 32. The method of claim 31 , wherein the subject has cancer, optionally a solid tumor.

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