Compositions and methods for generating lymphoid lineage cells

Abstract

The present disclosure relates to compositions and methods for modulating signaling in a cell and / or for differentiating / maturing a cell. Compositions of this disclosure, as may be used in associated methods, may include first and second segments that are connected to one another. When used in methods to differentiate and / or mature immune cells, compositions of this disclosure advantageously increase frequency and yields of arising cells in comparison to conditions deploying only first or second segments separately, or both individually (not connected).

Description

COMPOSITIONS AND METHODS FOR GENERATING LYMPHOID LINEAGE CELLSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority from United States Provisional Patent Application No. 63 / 660,249, filed June 14, 2024, the entire content of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates to cell culture applications, more specifically to cell culture applications using hematopoietic or hematopoietic-like cells, and still more specifically to compositions and methods relating to lymphoid lineages.BACKGROUND

[0003] T cells, B cells and NK cells are lymphocytes that provide defense against pathogens and tumors. T cells help maintain host tolerance and mediate immune responses. B cells are involved in humoral immunity and produce antibodies or may be stimulated to produce antibodies. Natural killer (NK) cells are involved in innate immune responses against infections and malignancies by secreting proinflammatory cytokines around virus-infected or tumor cells.

[0004] The Notch signaling pathway is evolutionarily well conserved and has been shown to play important role in cell-fate decisions in human T and NK cell development. Activation of Notch receptors on hematopoietic stem and / or progenitor cells (HSPCs) leads to inhibition of B cell and erythromyeloid lineage differentiation and drives T lineage induction.

[0005] Cell adhesion molecules (CAMs) are regulators of T cell and NK cell development, helping to control migration, homing, retention, proliferation, and lineage maturation. With respect to T cells, CAMs mediate both physical anchoring in the thymic microenvironment and transmission of signals that support survival and lineage commitment. In the context of NK cells, CAMs are essential for the retention, proliferation, maturation, and functional development of NK cell progenitors.

[0006] In conventional culture systems, Notch ligands and CAMs have been used separately, resulting in limited control over the CAM-to-Notch ligand ratio and inconsistent spatial organizations, leading to imprecise control over interaction of each with a target cell. However, with the emergence of cell and gene therapy technologies, efficient and scalable differentiation to lymphoid lineages is of greater importance.

[0007] Thus, there is a need for improved compositions and methods comprising a Notch ligand and CAMs to generate lymphoid cells, such as to serve the manufacture of cell therapy products and other clinical uses.SUMMARY

[0008] The present disclosure relates to compositions and methods for modulating signaling in a cell, and / or for generating / differentiating / maturing a population of lymphoid cells.

[0009] In one aspect, compositions are provided for modulating signaling in a cell, such as a cell comprising a Notch receptor. In another aspect, compositions are provided for generating, differentiating, and / or maturing a population of lymphoid cells.

[0010] In any aspect, a composition of this disclosure may comprise a first segment (e.g. a first polypeptide) and a second segment (e.g. a second polypeptide). The first segment may comprise or encode a Notch ligand. The second segment may comprise a cell adhesion molecule. The first segment and the second segment may be connected or linked, or connectable or linkable. The first segment and the second segment (when linked or connected) may be immobilized or passively adsorbed on a solid phase.

[0011] A first segment of a composition of this disclosure may comprise one or more EGF-like repeats. A first segment of a composition of this disclosure may comprise a DSL domain. A first segment of a composition of this disclosure may comprise a C2 domain. A first segment of a composition of this disclosure may comprise any combination of the foregoing. A first segment of a composition of this disclosure may comprise one or more of: one or more EGF-like repeats; a DSL domain; and a C2 domain. In a specific embodiment, a first segment does not comprise a transmembrane domain, such as of a Notch ligand.

[0012] If a first segment comprises a Notch ligand, in a specific embodiment the Notch ligand is a Delta-like protein or a fragment of a Delta-like protein. In a specific embodiment the Delta-like protein is DLL1 or DLL4, or a fragment thereof.

[0013] A second segment of a composition of this disclosure may comprise at least one Immunoglobulin (Ig)-like domain. A second segment of a composition of this disclosure may comprise at least one fibronectin type III (Fnlll) domain. A second segment of a composition of this disclosure may comprise an integrin binding domain or an integrin interacting motif. A second segment of a composition of this disclosure may comprise a heparin binding domain. A second segment of a composition of this disclosure may comprise any combination of this foregoing.

[0014] In a specific embodiment, an integrin binding domain or an integrin binding motif of a second segment of a composition of this disclosure may comprise an RGD motif, and LDV motif, or both an RGD motif and an LDV motif.

[0015] Specifically, a cell adhesion molecule may be an Ig-like domain containing superfamily member (e.g. ICAM-1 , VCAM-1), or a fragment thereof. Or, a cell adhesion molecule may be an extracellular matrix protein (e.g. fibronectin, laminin, etc.), or a fragment thereof.

[0016] A first segment or a second segment or both the first and the second segment may comprise a polypeptide. If a first segment or a second segment or both the first and the second segment comprise a polypeptide, the first segment and the second segment may be connected either directly through a single peptide bond or via peptide linker. Or, the first segment and the second segment may be comprised in a fusion protein, and the first segment and the second segment are in the same reading frame. Or, the first segment may encode or may be conjugated to a first member of a complementary binding pair, and the second segment may encode or may be conjugated to a second member of the complementary binding pair.

[0017] A composition of this disclosure may be immobilized, such as conjugated or passively adsorbed, on a solid phase, and thus in another aspect of this disclosure are provided compositions comprising a composition (having first and second segments, as described herein) immobilized on a solid phase, such as via conventional techniques. In a specific embodiment, the solid phase is a cell culture substrate or a bead or a particle. In addition, or in the alternative, the solid phase may be a porous growth matrix.

[0018] In another aspect of this disclosure are provided methods for modulating signaling in a cell. In another aspect of this disclosure are provided methods of generating, differentiating, and / or maturing a population of lymphoid cells.

[0019] Methods of this disclosure may comprise contacting a precursor of a population of lymphoid cells with a composition as described herein. Contacting (and subsequent culture, if necessary) may be performed for a sufficient time to modulate signaling in the precursor (or at least some cells of a population of precursors), and / or to generate a population of lymphoid cells.

[0020] Methods of this disclosure, and more particularly contacting a target cell (e.g. precursor of a population of lymphoid cells) with the composition, may be performed in static (e.g. 2D) or suspension (e.g. 3D) culture conditions.

[0021] A target cell (e.g. precursor of a population of lymphoid cells) is not particularly limited, but may correspond to a PSC-derived or a primary hematopoietic stem / progenitor cell (e.g. CD34+HSPC) or a lymphoid progenitor (e.g. CD5+, CD7+, or CD5+CD7+cell)

[0022] An output cell or a population thereof (e.g. a population of lymphoid cells) may comprise lymphoid / lymphocyte progenitors. An output cell or a population thereof (e.g. a population of lymphoid cells) may comprise NK cells or T cells. If an output cell or a population thereof comprises T cells, the T cells may be double positive CD4+CD8+T cells, single positive CD8+T cells, single positive CD4+T cells, CD3+TCRap+T cells, or CD3+TCRy6+T cells.

[0023] Methods of this disclosure may comprise preferentially generating CD3+TCRap+T cells over CD3+TCRy6+T cells at or below a threshold concentration of a composition of this disclosure. Methods of this disclosure may comprise preferentially generating CD3+TCRy6+T cells over CD3+TCRap+T cells at or above a threshold concentration of a composition of this disclosure.

[0024] Methods of this disclosure may further comprise generating, differentiating, or maturing between about 1 to 10 times more lymphoid progenitor cells from a precursor (of a population of lymphoid cells) in the presence of a composition of this disclosure, in comparison to a control condition where the precursor is contacted with a first segment alone, a second segment alone, or the first and the second segments that are not connected.

[0025] Methods of this disclosure may further comprise generating, differentiating, or maturing between about 1 to 20 times more output lymphocytes from a precursor (of a population of lymphoid cells) in the presence of a composition of this disclosure, in comparison to a control condition where the precursor is contacted with a first segment alone, a second segment alone, or the first and the second segments that are not connected.

[0026] Methods of this disclosure may be serum-free and / or feeder-free.

[0027] Methods of this disclosure may be scalable (e.g. in static or suspension culture, or from static to suspension culture), for greater output / generation / differentiation / maturation of a population of lymphoid cells (such as lymphoid progenitors and / or lymphocytes).BRIEF DESCRIPTION OF THE DRAWINGS

[0028] For a better understanding of the various embodiments described herein, and to show more clearly how these various embodiments may be carried into effect, reference will be made, by way of example, to the accompanying drawings which show at least one example embodiment,and which are now described. The drawings are not intended to limit the scope of the teachings described herein.

[0029] Figure 1 shows exemplary constructs / compositions of this disclosure. D4.4, D4.5, D4.8 and D4.9 each comprise an extracellular domain fragment of human Notch ligand DLL4 (hDLL4) connected / linked to a cell adhesion molecule (CAM). CAM1 corresponds to a fibronectin fragment comprising Fnlll 9-12; CAM2 corresponds to a fibronectin fragment comprising Fnlll 8-10 and Fnlll 12-14; CAM3 corresponds to a vascular cell adhesion molecule 1 (VCAM-1) fragment comprising Ig-like domains 1-7; and CAM4 corresponds to an intracellular adhesion molecule 1 (ICAM-1) fragment comprising Ig-like domains 1-5.

[0030] Figures 2A-F show bar graphs summarizing differentiation of lymphoid progenitors and lymphocytes using constructs / compositions of this disclosure. Frequency and yield of: day-14 cord blood (CB)-derived (Figure 2A) and day-7-10 pluripotent stem cell (PSC)-derived (Figure 2B) CD5+CD7+lymphoid progenitors; day-39 CB-derived (Figure 2C) and day-21-24 PSC-derived (Figure 2D) CD4+CD8+double positive T cells; and day-46 CB-derived (Figure 2E) and day-28- 31 PSC-derived (Figure 2F) CD8+single positive T cells gated on CD3+TCRap+T cells. Cells were generated in static culture (e.g. 2D) conditions using plate-immobilized D4.5 or a control where each of Fibronectin and DL4 were individually immobilized. For PSC cultures, CD34+HSPCs were generated from 4 PSC lines (H9 ESC, WLS-1 C iPSC, STi0003-A iPSC, or STi0004- A iPSC). For CB-derived cultures, bars represent mean ± SEM of 34 donors in (Figure 2A), and mean of 2 donors in (Figure 2C) and (Figure 2E). For PSC-derived cultures, bars represent mean ± SEM of 13 replicates in (Figure 2B), 14 replicates in (Figure 2D), and 4 replicates in (Figure 2E).

[0031] Figure 3 shows a bar graph summarizing the frequency and yield of day 21-24 PSC- derived CD56+NK cells. PSC-derived cells were generated from the lines indicated in Figure 2 in static culture (e.g. 2D) conditions using plate-immobilized D4.5 or a control where each of Fibronectin and DL4 were individually immobilized. Bars represent mean ± SEM of 9 replicates.

[0032] Figures 4A-C show bar graphs summarizing the frequency and yield of day-14 CD5+CD7+lymphoid progenitors differentiated in static culture from CB-derived CD34+HSPCs. In panel (Figure 4A), CB-derived CD34+cells were cultured in plates coated as follows: individually Fibronectin and DL4 (control); DLL4 (DL4) (SinoBiological); individually DLL4 together with CAM1 , CAM2, CAM3, or CAM4 (DL4+CAM1 , DL4+CAM2, DL4+CAM3 or DL4+CAM4); or constructs / compositions of Figure 1 (D4.4, D4.5, D4.8 and D4.9). In panel (Figure 4B), CB-derived CD34+HSPCs were cultured either in the control of panel (Figure 4A), streptavidin (SAV)-pbeadsfunctionalized with immobilized DLL4 together with CAM1 , CAM2, or CAM3 (DL4+CAM1 beads, DL4+CAM2 beads or DL4+CAM3 beads), or SAV-pbeads functionalized with immobilized constructs / compositions of Figure 1 (D4.4 beads, D4.5 beads, or D4.8 beads). In panel (Figure 4C), CB-derived CD34+HSPCs were cultured either in the control of panels (Figure 4A) and (Figure 4B) or with carboxyl pbeads functionalized with immobilized D4.4 or D4.5. Bars represent mean ± SEM of 2 donors.

[0033] Figures 5A-F show bar graphs summarizing differentiation of lymphoid progenitors and lymphocytes using constructs / compositions of this disclosure. Frequency and yield of: day-14 CB-derived (Figure 5A) and day-7-10 PSC-derived (Figure 5B) CD5+CD7+lymphoid progenitors; day-42 CB-derived (Figure 5C) and day-21-24 PSC-derived (Figure 5D) CD4+CD8+double positive T cells; day-42 CB-derived CD3+TCRap+T cells gated on CD4+CD8+T cells (Figure 5E), and day-42 CB-derived CD4+CD8+double positive T cells under scaled up conditions (Figure 5F). The control corresponds to static culture conditions where each of Fibronectin and DL4 were individually immobilized on plates. In Figure 5A)-F) the “D4.5 beads” or “3D beads” conditions correspond to suspension (e.g. 3D) culture conditions, while in B) and D) the “D4.5” condition corresponds to bead-immobilized D4.5 in static (e.g. 2D) culture conditions. Bars represent the mean ±SEM of 4 donors (A), 3 donors (C and E), 1 donor (F), and mean ± SEM of 4 replicates (B and D).

[0034] Figure 6 shows a summary of the frequency and yield of day-14 CB-derived CD5+CD7+lymphoid progenitors cultured under static conditions in the presence of construct / composition variants. CB-derived CD34+cells were cultured in plates coated as follows: in the absence (no DL4) or presence of DLL4 (DL4); individually DLL4 and CAM2 (DL4+CAM2); D4.5 of Figure 1 (D4.5); a construct / composition comprising CAM2 having an ablated RGD motif (D4.10); a construct / composition comprising CAM2 having an ablated RGD motif together with non-ablated CAM2 separately (D4.10+CAM2); a construct / composition comprising CAM2 having an ablated LDV motif (D4.11); a construct / composition comprising CAM2 having an ablated LDV motif together with non-ablated CAM2 separately (D4.11 +CAM2); a construct / composition comprising CAM2 having an ablated RGD and LDV motif (D4.12); and a construct / composition comprising CAM2 having an ablated RGD and LDV motif together with non-ablated CAM2 separately (D4.12+CAM2). Data represents mean of 2 donors.

[0035] Figure 7 shows a summary of the frequency of day-42 CB-derived CD3+TCRap+and CD3+TCRy6+(gated on double positive T cells). CB-derived CD34+HSPCs were cultured under suspension (e.g. 3D) conditions in increasing concentrations (cm2 / mL) of D4.5-immobilized SAVpbeads (as indicated). Control conditions correspond to static (e.g. 2D) culture in a plate coated with either Fibronectin and DLL4 individually, or with D4.5. Data represents the mean of 1-3 donors.

[0036] Figures 8A and B show bar graphs summarizing differentiation of lymphoid progenitors and lymphocytes using constructs / compositions of this disclosure. Frequency and yield of day- 14 CB-derived (Figure 8A) and day-7-10 PSC-derived (Figure 8B) CD5+CD7+lymphoid progenitors. The control corresponds to static culture conditions where each of Fibronectin and DL4 were individually immobilized on plates. An exemplary construct / composition of this disclosure (e.g. D4.5) was passively adsorbed (or chemically conjugated, data not shown) on a porous growth matrix (Fibra-Cell® disks, Eppendorf) and cultured under either static (2D) or suspension (3D) conditions. Data is from one donor for (A) and one biological replicate for (B).DETAILED DESCRIPTION

[0037] This disclosure relates to compositions and to methods involving use of such compositions for modulating signaling in a cell. In one aspect, compositions and associated methods are used to generate a population of lymphoid cells, such as a population of lymphoid progenitors and / or lymphocytes, such as from a population of stem cells (e.g. hematopoietic stem or progenitor cells).

[0038] Compositions of this disclosure may be at least bifunctional, comprising a first segment and a second segment. The first segment may comprise or encode Notch ligand activity, and the second segment may comprise or encode cell adhesion properties. Compositions of this disclosure may comprise or encode one or more polypeptides, and whether in use or as such may be immobilized on a solid phase.

[0039] Where used in this disclosure, the term “Notch ligand” refers to an organic or inorganic entity / moiety capable of being bound by and activating / modulating signaling though a Notch receptor. Two types of canonical Notch ligands are known in mammals: Delta-type and Jagged- type. In mammals, there are three delta-like ligands (DLLs), DLL1 , DLL3 and DLL4, and two jagged (JAG) ligands, JAG1 and JAG2. A full-length Delta-like ligand typically comprises an extracellular domain (including a N-terminal C2 domain, a Delta / Serrate / LAG-2 (DSL) domain, and a plurality of epidermal growth factor (EGF) repeats), a transmembrane domain, and a post- synaptic density protein ligand (PDZ) domain (except DLL3). DLL3 comprises six EGF repeats whereas DLL1 and DLL4 comprise eight EGF repeats. JAG1 and JAG2 comprise 16 EGF repeats. In a particular embodiment, a Notch ligand of this disclosure is DLL4 (or DL4) or DLL1 (or DL1), whether full-length or fragmentary, and if a fragment then it may comprise an assemblyof non-contiguous sequences thereof. A Notch ligand or moiety having Notch ligand activity may be modified for enhanced function. Constructs / compositions of this disclosure may comprise only the extracellular domain of a Notch ligand or a portion(s) thereof, in which case it may not comprise the transmembrane domain. In a specific embodiment, a Notch ligand may comprise a signal peptide (e.g. ~25 N-terminal amino acids) that directs protein secretion.

[0040] Where used in this disclosure, the term “cell adhesion moiety”, “cell adhesion molecule”, or “cell adhesion polypeptide”, may be used interchangeably, and in either case refers to an entity / moiety that provides appropriate signals or attachment sites to cells of interest. A cell adhesion molecule (CAM) may comprise a protein or a polypeptide that participates in cell-cell and / or cell-substrate interactions. A CAM of this disclosure may be or comprise a cell adhesion property of an immunoglobulin (Ig) superfamily (IgSF) member; an integrin family member; a cadherin superfamily member; or a selectin family member. Proteins of the IgSF comprise an immunoglobulin loop domain that plays an important part in adhesion properties to cells, and nonlimiting examples include synaptic cell adhesion molecules, neural cell adhesion molecules (e.g. NCAMs), intercellular cell adhesion molecule (e.g. ICAM-1), vascular cell adhesion molecule (e.g. VCAM-1), a platelet-endothelial cell adhesion molecule (e.g. PECAM-1). Integrins mediate cell interactions with a variety of ligands on cells or the extracellular matrix, such as extracellular matrix protein(s). Many integrins bind to an RGD or LDV tripeptide motif. Thus, a CAM of this disclosure may be or may comprise an integrin binding domain or integrin interacting motif, such as an extracellular matrix protein (e.g. collagen, fibrinogen, fibronectin, laminin, and vitronectin). In the context of in vitro or ex vivo workflows, CAMs may be immobilized / coated (e.g. such as by passive adsorption, chemical conjugation, or otherwise) on a wall of a cell culture vessel or on a solid surface or phase (e.g. a particle, microcarrier, macrocarrier, disks, or bead).

[0041] Where used in this disclosure, the term “solid phase” refers to any surface or substrate (natural or synthetic) that may come into contact with a cell and support its culture, analysis, or processing. A solid phase may refer to a porous, non-porous, or a fibrous material which may be functionalized with a(n immobilized) construct / composition of this disclosure, whereby functionalization may facilitate attachment of cells thereto and / or provision of signals or cues to cells that promote growth / differentiation. Non-limiting examples of a solid phase include particles, beads (including pbeads or nanobeads), proteins, lipids, nucleic acid molecules, (hollow) fibers, culture receptacles / vessels (e.g. tube, dish, well, bottle, flask, Transwell™), filter, mesh, screen, membrane, macrocarriers, ceramics, porous growth matrix (e.g. disks), etc. Non-limitingexamples of a porous solid phase include Fibra-Cel® disks (Eppendorf™ AG, Germany), BioNOC™ II carriers (CESCO BioProducts, Atlanta, GA), or the like.

[0042] Where used in this disclosure, the term “functionalize” or “immobilized”, or derivatives thereof, refers to attachment or binding of a ligand (e.g. a Notch signaling ligand, a CAM, integrin ligand, fibronectin, collagen, and laminin, etc.), an antigen, a peptide or protein to a substrate or solid phase through covalent interactions, non-covalent interactions, specific affinity-based interactions as between complementary binding pairs, or other suitable means.

[0043] Where used in this disclosure, the term “passive adsorption” refers to functionalization of a substrate / solid phase primarily via non-covalent interactions with a ligand. Non-covalent interactions may occur via hydrogen bonding; Van der Waals forces (mainly London dispersion forces); dipole-dipole interactions, hydrophobic interactions, ionic interactions (such as ion-ion and ion-dipole attractions), and pi-pi stacking. For a complex ligand like a protein, passive adsorption onto a substrate can result in strong, persistent binding that may not reflect simple reversible equilibrium, potentially due to multiple contact points or conformational changes.

[0044] Where used in this disclosure the term “hematopoietic stem and progenitor cell” or “HSPC” refers to a cell of the hematopoietic lineage that is capable of self-renewal and / or differentiating into a more specialized cell of the hematopoietic lineage. HSPC may be obtained from bone marrow (BM), umbilical cord blood (CB), embryonic through to adult peripheral blood (PB), thymus, peripheral lymph nodes, gastrointestinal tract, tonsils, gravid uterus, liver, spleen or any other tissue having localized populations of HSPC. HSPC may also be differentiated from pluripotent stem cells (PSC) such as induced pluripotent stem cells (iPSC), embryonic stem cells (ESC), naive stem cells, extended stem cells, or the like. A hallmark of HSPC is the expression of the transmembrane phosphoglycoprotein CD34, thus HSPC may be referred to as CD34+cells. Human HSPCs may be further defined by expression of CD45 and CD34, and may be still further defined by combinations of markers such as CD38, CD43, CD45RO, CD45RA, CD10, CD49f, CD59, CD90, CD109, CD117, CD133, CD166, HLA-DR, CD201 , and integrin-alpha3 which may be used to distinguish HSPC subsets. HSPCs may lack expression, or have only low expression, of markers such as Glycophorin A, CD3, CD4, CD8, CD14, CD15, CD19, CD20 and CD56; such markers may be characteristic of more mature blood cells. The term “hematopoietic progenitor cell” may be used interchangeably with either “hematopoietic stem and progenitor cell” or “HSPC”.

[0045] Where used in this disclosure, the term “precursor”, such as in the context of a population of lymphoid cells, refers to a cell that is capable of differentiating into a cell of the hematopoieticlineage. A precursor may be primary- or PSC-derived. If primary, a precursor may correspond to any stem cell or progenitor cell along the hematopoietic lineage. In one no-limiting example, a precursor may correspond to a primary- or PSC-derived CD34+cell (e.g. a HSPC). In another non-limiting example, a precursor may correspond to a primary- or PSC-derived CD5+, CD7+, or CD5+CD7+cell (e.g. a lymphoid progenitor). A precursor may be comprised in a population of cells, which population may be >50% pure, >60% pure, >70% pure, >80% pure, or >90% pure, and such population may be purified by immunomagnetic separation or fluorescence activated cell sorting. A precursor may directly differentiate into a downstream lymphoid cell type, such as a T or a NK cell, or may undergo one or more further steps of differentiation before becoming a T or a NK cell.

[0046] Where used in this disclosure, the term “lymphoid progenitor” refers to a cell type that is more specialized than a HSPC and is capable of further differentiating into one or more lymphoid cell types, such as a lymphocyte (e.g. B cells, T cells, or NK cells). A lymphoid progenitor cell may be a direct descendant of a precursor (or population thereof), such as an HSPC or a cell type downstream of an HSPC. Relatedly, a lymphoid progenitor cell may directly differentiate into a downstream lymphoid cell type (e.g. lymphocytes) or may undergo one or more further steps of differentiation before becoming the lymphoid cell type. A lymphoid progenitor may be positive for the phenotypic markers CD7 and / or CD5. In another example, a lymphoid progenitor cell may be negative for both CD7 and CD5. Other phenotypic markers that may be expressed by lymphoid progenitor cells include CD10, CD45RA, CD34, CD38, CD161 , CD122, CD1 17, CD127, CD1 a and / or integrinB7. A lymphoid progenitor may differentiate into any type of lymphoid cell, or may be more restricted in its differentiation capacity.

[0047] T lymphocytes (e.g. T cells) may derive from multi-lymphoid progenitors (MLPs) or common lymphoid progenitors (CLPs). T cells are typically characterized by: the absence of NK- , B-, and erythromyeloid-specific markers; the expression of one or more of CD3, TCRap (or TCRy6), CD4 and / or CD8; and their effector functions. Although, some subsets of T cells may comprise characteristics of both T and NK cells, and may or may not express TCRap or TCRy6, and may or may not also express CD4, CD8, CD56, CD16 and NK1 .1. Effector functions of T cells may include the production of cytokines, killing of infected or tumor target cells, or the stimulation of other hematopoietic cell types. T cells may further be characterized by the expression of CD8a, CD8B, CD45RA and CD27. The differentiation of T cells from a PSC or HSPC (e.g. PSC-derived HSPC) is usually intermediated by one or more progenitor(s) (e.g. PSC- derived mesodermal precursor(s) and / or lymphoid progenitor(s)).

[0048] NK lymphocytes (e.g. NK cells) may derive from multi-lymphoid progenitors (MLPs) or common lymphoid progenitors (CLPs). NK cells are typically characterized by: the absence of T and B cell-specific markers; the expression of CD56 with or without CD16 (low affinity Fc gamma receptor 3A, expressed on a subset of NK cells); and their effector functions. Effector functions of NK cells may include cytotoxicity and / or the production of inflammatory cytokines such as IFNy and / or TNFa. NK cells may further be characterized by the expression of activating and inhibitory receptors referred to as killer immunoglobulin-like receptors (KIRs). Other activating receptors that NK cells may express include NKG2D, the CD94 / NKG2 receptors including NKG2CNKG2E and NKG2F and natural cytotoxicity receptors (NCRs) including NKp30, NKp44, and NKp46. Other inhibitory receptors include CD94 / NKG2 receptors including NKG2A, and NKG2B. The differentiation of NK cells from PSC or HSPC (e.g. PSC-derived HSPC) is usually intermediated by one or more progenitor(s) (e.g. PSC-derived mesodermal precursor(s) and / or lymphoid progenitor(s)).Compositions

[0049] In a general sense, this disclosure provides compositions for modulating signaling in a cell. A composition of this disclosure may comprise a first segment and a second segment, each of which may be independently selected from or comprise a ligand, a receptor, a small molecule, a synthetic polymer, and a macromolecule.

[0050] In one aspect, a composition for modulating signaling in a cell may stimulate proliferation / expansion of the cell. In the same or a different aspect, a composition for modulating signaling in a cell may stimulate differentiation / (re)programming of the cell.

[0051] Signal-modulating compositions of this disclosure are not particularly limited in terms of target cell type. In most cases, a target cell type of a composition bears on its extracellular surface a receptor or other feature that upon engagement with a first segment and / or a second segment of the composition transmits a signal intracellularly. In a specific example, a target cell type is a lymphoid cell, or a precursor / progenitor thereof. A target cell type may be comprised in a population of cells that has either been purified or is mixed.

[0052] In one aspect, this disclosure provides a composition for generating a population of lymphoid cells, such as lymphoid / lymphocyte progenitors and / or lymphocytes. Relatedly, aspects of this disclosure provide compositions for maturing and / or differentiating a population of lymphoid cells, such as lymphoid / lymphocyte progenitors and / or lymphocytes.

[0053] In one aspect, this disclosure provides a composition for generating a population of lymphoid cells (e.g. lymphoid / lymphocyte progenitors and / or lymphocytes), such as from a stem or progenitor cell (or population thereof), whether a PSC, HSPC, or otherwise. In one aspect, this disclosure provides a composition for maturing and / or differentiating a population of lymphoid cells (e.g. lymphoid / lymphocyte progenitors and / or lymphocytes), such as from a stem or progenitor cell (or population thereof), whether a PSC, HSPC, or otherwise.

[0054] Compositions may comprise a first segment having a first cell-engaging function / property, and a second segment having a second cell-engaging function / property. While compositions may comprise individual or plural first and / or second segments, and optionally additional segments, at least the first segment and the second segment may be connected to each other, directly or indirectly.

[0055] A first cell-engaging function / property of a first segment may comprise or encode a Notch ligand or a moiety having Notch ligand activity. A Notch ligand or a moiety having Notch ligand activity may be or may comprise any substance that binds to a receptor of Notch signaling (e.g. NOTCH1 , NOTCH2, NOTCH3, and NOTCH4). In addition to its role in embryonic, neural, cardiovascular, and endocrine development, and in various diseases and cancers, the Notch signaling pathway plays an important role in promoting T cell development and repressing concomitant B cell development.

[0056] The nature of the Notch ligand or moiety having Notch ligand activity comprised in the first segment of a composition of this disclosure is not particularly limited. Such ligand / moiety may be chemical and / or biological in nature. If chemical, the ligand / moiety may be a chemical compound, such as a small molecule or the like. If biological, the ligand / moiety may be a macromolecule, including but not limited to a peptide, polypeptide, or a protein. In some embodiments, the first segment comprises one or more biological elements and one or more chemical elements.

[0057] Preferably, a first segment of the described compositions is or comprises / encodes a polypeptide having Notch ligand activity (e.g. a Notch ligand). Such polypeptide may correspond to a naturally occurring protein or fragment thereof, or a non-naturally occurring protein / polypeptide (e.g. a recombinant or engineered protein / polypeptide). Regardless, a polypeptide of a first segment may encode a full-length Notch ligand or a fragment thereof. If a first segment corresponds to a full-length Notch ligand, then it may correspond to either of DLL- 1 , DLL-2, DLL-3, DLL-4, JAG1 , or JAG2, and preferably DLL1 or DLL4. In such case a firstsegment may comprise each of an extracellular domain (including a C2 domain, a DSL domain, and a plurality of EGF repeats), a transmembrane domain, and PDZ domain (except if DLL3).

[0058] If a first segment corresponds to a polypeptide fragment of a Notch ligand or a polypeptide having Notch ligand activity, it may include one or domains or motifs of DLL-1 , DLL-2, DLL-3, DLL-4, JAG1 , or JAG2, and preferably of DLL1 or DLL4. Specifically, a first segment comprising a polypeptide fragment of a Notch ligand or a polypeptide having Notch ligand activity, may comprise one or more EGF-like repeats, a DSL domain (or a portion thereof), and / or a C2 domain (or a portion thereof). More specifically, a first segment comprising a polypeptide fragment of a Notch ligand or a polypeptide having Notch ligand activity, may comprise one or more of the following: one or more EGF-like repeats; a DSL domain; and a C2 domain. In one embodiment, the first segment may further comprise a signal sequence or peptide. In some embodiments, a first segment comprising a polypeptide fragment of a Notch ligand or a polypeptide having Notch ligand activity, does not comprise a transmembrane domain and / or sequences / domains downstream of the transmembrane domain.

[0059] The Notch ligand or the fragment thereof, if a polypeptide or a corresponding polynucleotide, may correspond to its consensus sequence, or it may be modified / mutated by means known to a person skilled in the art, such as to facilitate expression, enhance / modify function, etc.

[0060] A second cell-engaging function / property of a second segment may comprise or encode a cell adhesion molecule or a moiety having such activity. A cell adhesion molecule or a moiety having such activity may be or may comprise any substance that interacts with and at least temporarily binds a cell in contact therewith. Thus, a cell adhesion molecule or a moiety having such activity may interact with a receptor, glycoprotein, integrin, or any other component of a cell (membrane) that when bound or associated initiates the transmission of a signal from an exterior to an interior of a cell.

[0061] The nature of a cell adhesion molecule or a moiety having such activity comprised in the second segment of a composition of this disclosure is not particularly limited. Such molecule / moiety may be chemical and / or biological in nature. If chemical, such molecule / moiety may be a chemical compound, such as a small molecule or the like. If biological, the molecule / moiety may be a macromolecule, including but not limited to a peptide, polypeptide, or a protein. In some embodiments, the second segment comprises one or more biological elements and one or more chemical elements.

[0062] Preferably, a second segment of the described compositions is or comprises / encodes a polypeptide having cell adhesion / binding activity. Such polypeptide may correspond to a naturally occurring protein or fragment thereof, or a non-naturally occurring protein / polypeptide (e.g. a recombinant or engineered protein / polypeptide). Regardless, a protein or fragment thereof of a second segment may correspond to an IgSF member (e.g. containing an Ig-like domain), an extracellular matrix protein, or any other protein comprising integrin binding activity. IgSF members are well known, particularly those of mammals, and may correspond to a VCAM, an NCAM, or an ICAM. Extracellular matrix proteins are well known, particularly those of mammals, and may correspond to fibronectin, vitronectin, laminin, or the like.

[0063] Regardless if a second segment comprises or encodes a naturally or non-naturally occurring full-length or fragmentary cell adhesion molecule, the second segment may comprise at least one immunoglobulin (Ig)-like domain (as may be found in VCAM, NCAM, or ICAM, for example) or a portion thereof having cell-cell or cell-substrate binding activity. A cell adhesion molecule (e.g. VCAM, NCAM, extracellular matrix protein, etc.) or a portion thereof having cellcell or cell-substrate binding activity may comprise at least one Ig-like domain, at least two Ig-like domains, at least three Ig-like domains, at least four Ig-like domains, at least five Ig-like domains, at least six Ig-like domains, or at least seven Ig-like domains.

[0064] In addition, or in the alternative, regardless if a second segment comprises or encodes a naturally or non-naturally occurring full-length or fragmentary cell adhesion molecule, the second segment may comprise at least one fibronectin type III (Fnlll) domain or a portion thereof having cell-cell or cell-substrate binding activity. At least one fibronectin type III (Fnlll) domain may correspond to a type III 8, III 9, III 10, III 11 , III 12, III 13, or III 14 domain. A second segment of this disclosure may comprise each of a type III 8, III 9, III 10, III 11 , III 12, III 13, and III 14 domain, or may comprise any two, three, four, five, or six of the foregoing.

[0065] In further addition or in the alternative, regardless if a second segment comprises or encodes a naturally or non-naturally occurring full-length or fragmentary cell adhesion molecule, the second segment may comprise an integrin binding domain or an integrin interacting motif. An exemplary integrin binding domain or integrin interacting motif includes a tripeptide Leu-Asp-Val (LDV) motifs, such as of fibronectin, laminin, ICAM and VCAM, among others. Another exemplary integrin binding domain or integrin interacting motif includes a tripeptide Arg-Gly-Asp (RGD) motif, such as of fibronectin, laminin, and VCAM-1 , among others. Fnlll domains may comprise one or more integrin binding domains or integrin interacting motifs, as may a connecting segment 1 (CS- 1) site of fibronectin.

[0066] In still further addition or in the alternative, regardless if a second segment comprises or encodes a naturally or non-naturally occurring full-length or fragmentary cell adhesion molecule, the second segment may comprise a heparin binding domain or a portion thereof having cell-cell or cell-substrate binding activity. By way of example, certain Fnlll domains, such as a III 12, III 13, or Illi 14, may function as a heparin binding domain.

[0067] A cell adhesion molecule or moiety having such activity, if a polypeptide or a corresponding polynucleotide, may correspond to its consensus sequence, or it may be modified / mutated by means known to a person skilled in the art, such as to facilitate expression, enhance / modify function, etc.

[0068] In some embodiments, one or more functional (e.g. non-mutated or -ablated) integrin binding domain or integrin interacting motif is required to modulate signaling in a target cell (and / or to generate a population of desired cells). For example, ablating an RGD motif (such as by RGD- >RGE substitution) of a second segment may lead to loss of or reduced function, such as by reducing a5p1 integrin binding. As another example, ablating an LDV motif (such as by LDV- >LEV substitution) of a second segment may lead to loss of or reduced function, such as by reducing a4p1 integrin binding. As another example, ablating both an RGD motif (RGD->RGE) and an LDV motif (LDV-<LEV) of a second segment may lead to an additive loss or reduction of function. Thus, in one embodiment, a composition of this disclosure comprises a Notch ligand (as described above) and at least one functional integrin binding domain or integrin interacting motif, or a Notch ligand (as described above) and more than one functional integrin binding domain or integrin interacting motif. In one embodiment, a composition of this disclosure comprises a Notch ligand (as described above) and at least one functional RGD or LDV motif, or a Notch ligand (as described above) and more than one functional RGD and / or LDV motif.

[0069] A first segment and a second segment of a composition of this disclosure may be connected (or connectable) or linked (or linkable) to one another. The first and second segments may be connected or linked in any way known in the art, whether in a 1 :1 stoichiometry or otherwise. In one embodiment, the first and the second segments as connected or linked are in a context that is not naturally occurring (e.g. not ordinarily found in nature). More specifically, if one or both of the first and second segments, as connected, comprise a polypeptide, a sequence or an open reading frame thereof may not be naturally occurring or known. Thus, a composition of this disclosure (for modulating signaling and / or generating a lymphoid cell population) may comprise a first segment comprising a Notch ligand or a moiety having Notch ligand activityconnected or linked to a second segment comprising a cell adhesion molecule or a moiety having such activity in a context that is not naturally occurring or ordinarily found in nature.

[0070] By way of non-limiting example, a first segment and a second segment may be connected directly through a single peptide bond, or may be connected via a peptide linker. A peptide linker is not particularly limited by size or sequence, provided it does not interfere with conformation of functional regions / portions of the first and second segments of the composition. Specifically, a peptide linker may comprise 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, or 5 or fewer amino acids amino acids. More specifically, a peptide linker may be a Gly-Ser linker, or the like. If the peptide linker is a Gly-Ser linker, the linker may comprise the amino acid sequence GGS, GGGS, or GGGGS.

[0071] Alternatively, a first segment may encode or be conjugated to a first member of a complementary binding pair, and a second segment may encode or be conjugated to a second member of the complementary binding pair (e.g. biotin:streptavidin; concanavalin A:dextran / glucose; antibody:antigen; complementary nucleotide sequences; etc.).

[0072] As another alternative way to link or connect first and second segments, in a composition where the first and second segments are polypeptides (e.g. a polypeptide composition) they may be comprised in a fusion protein. In such case, it may be important to preserve the reading frame of the fusion protein across first and second segments.

[0073] A composition of this disclosure comprising a first segment and a second segment may be or may in use be immobilized on a solid phase by various means known to a person skilled in the art. For example, the first and the second segments may be pre-connected before being immobilized, or one of the first or second segment may first be immobilized on a solid phase before the other of the first or second segment is connected or linked thereto. Incidentally, in a different aspect, this disclosure contemplates a composition (as described herein) that is immobilized on a solid phase.

[0074] A solid phase upon which a composition of this disclosure may be immobilized or functionalized is not particularly limited, but in non-limiting examples may correspond to a wall of a culture vessel, such as a well, plate, flask, bag, insert, disks, hollow fiber, bioreactor, or the like. Alternatively, a solid phase upon which a composition of this disclosure may be immobilized or functionalized is capable of being suspended in solution (e.g. culture medium), such as a bead, particle, microcarrier, microcarrier, porous matrix, or the like.

[0075] Means of immobilizing and / or functionalizing a composition of this disclosure on a solid phase are well known in the art, but in non-limiting examples may include (passive) adsorption, covalent conjugation, chemical or small molecule linker conjugation, hydrophobic interactions, ionic attachment, streptavidin-biotin mediated conjugation, or protein A or G mediated conjugation.

[0076] Where the solid phase is a bead, particle, or the like, they are not specifically limited but it is preferable that they are non-toxic to cells and may be functionalized as required in this disclosure. While beads of this disclosure may comprise a broad range of average diameters, typical average bead diameters may range between about 1-20 pm (± 1 pm), about 2-18 pm (± 2pm), about 5-15 pm (± 2pm), or about 8-12 pm (± 2pm). In one embodiment, an average bead diameter is 5 pm, or about 5 pm (± 2pm). Beads, particles, or the like of this disclosure may be magnetic or non-magnetic, polystyrene, or any other polymer that may be functionalized with an immobilized composition of this disclosure.

[0077] A concentration of a composition of this disclosure immobilized per cm2of a bead, particle or other sold phase may range between about 200-2000 ng, about 175-1750 ng, about 150-1500 ng, about 125-1250 ng, about 100-1000 ng, about 75-750 ng, about 50-500 ng, about 25-250 ng, or about 10-100 ng. Expressed differently, a concentration of a composition immobilized per cm2of a bead, particle or other solid phase may range between about 0.1 to about 35 picomoles, about 1 to about 30 picomoles, about 5 to about 25 picomoles, about 8 to about 20 picomoles, or about 10 to about 15 picomoles.

[0078] In the specific context where a solid phase is a wall / substrate of a cell culture vessel, receptacle or an insert, a concentration of the composition immobilized thereon (in a suitable medium or buffer used to coat the surface) may range between about 0.2 to 50 pg / mL, about 1 to about 40 pg / mL, about 5 to 30 pg / mL, about 10 to 25 pg / mL, or about 15 to 20 pg / mL. Expressed differently, a concentration of a composition of this disclosure loaded per area of a solid phase (e.g. culture vessel / receptacle wall, or a bead / particle) may range between about 50 ng / cm2-15 pg / cm2, between about 40 ng / cm2-12 pg / cm2, between about 25 ng / cm2-10 pg / cm2’ or between about 15 ng / cm2-8 pg / cm2.

[0079] In the specific context where a solid phase is a porous growth matrix (e.g. a porous / fibrous disk), an amount / quantity / concentration of the composition immobilized thereon may range between about 10-1700 ng, about 50-1500 ng, about 75-1200 ng, about 100-1000 ng, about 75-800 ng, about 50-500 ng, about 20-250 ng, or about 10-100 ng per cm2of the growth matrix.A porous growth matrix comprising the foregoing concentrations of immobilized composition may be used (in static or suspension culture) from 1 disk per culture volume to up to 10s, 100s, or 1000s of disks per culture volume. By way of example, if the culture volume is about 500 pL to 5 mL, then a quantity of disks may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, and can be scaled accordingly.

[0080] In the specific context of streptavidin-biotin mediated conjugation, a composition of this disclosure may be biotinylated and conjugated to a streptavidin-modified solid surface (or vice versa), such as a bead or particle. In certain embodiments, the composition (or beads) may be biotinylated at a range between 1-10, between 2-8, or 4-6 mole biotin per mole of first and / or second segment. In one embodiment, the composition (or beads) may be biotinylated at 1-2 mole of biotin per mole of first or second segment.

[0081] Immobilized compositions of this disclosure may be used in both static culture (e.g. 2D) or in suspension culture (e.g. 3D, such as under agitation).Methods

[0082] In a general sense, this disclosure also provides methods of modulating signaling in a cell using a composition, as described in any aspect or embodiment above.

[0083] In one aspect, methods of modulating signaling in a cell may comprise stimulating proliferation / expansion of the cell using a composition of this disclosure. In the same ora different aspect, methods of modulating signaling in a cell may comprise stimulating differentiation / (re)programming of the cell using a composition of this disclosure.

[0084] Signal-modulating methods of this disclosure are not particularly limited in terms of target cell type contacted by a composition of the disclosure, provided that the cell type is receptive or responsive to the composition, and more particularly receptive or responsive to one or both of a first segment and a second segment thereof. Thus, a target cell type may bear on its extracellular surface, a receptor or other feature that upon engaging with a composition (e.g. a first and / or second segment thereof) transmits a signal intracellularly.

[0085] A target cell type may be a pluripotent stem cell (PSC), or PSC-derived. PSC lines are known and widely available, such as from STEMCELL Technologies, as are methods of and / or kits for generating PSC (and their downstream differentiation). Alternatively, a target cell type may be primary-derived, that is obtained from a subject (e.g. a mammal such as a human, rodent, or otherwise), or a cell line.

[0086] An exemplary target cell type contacted by a composition of this disclosure (regardless of whether it is a cell line or primary-derived) includes but is not limited to a precursor / progenitor of an output cell type. More specifically, an exemplary target cell type contacted by a composition of this disclosure includes but is not limited to a lymphoid cell, such as a lymphoid stem or progenitor cell (e.g. a CD34+HSPC). Or, the target cell type may be a cell type downstream of a CD34+HSPC, but nevertheless along the hematopoietic lineage, such as a precursor or lymphoid progenitor cell.

[0087] When the target cell type is a lymphoid cell, an output cell type (following contact with a composition of this disclosure) will depend on the nature of the target cell type. For example, if a target cell type corresponds to a CD34+HSPC, then an output cell type may correspond to a lymphoid / lymphocyte progenitor (e.g. a CD5+, a CD7+, or a CD5+CD7+lymphoid progenitor). As another example, if a target cell type corresponds to a lymphoid / lymphocyte progenitor (e.g. a CD5+, a CD7+, or a CD5+CD7+lymphoid progenitor), then an output cell may correspond to a more committed progenitor (e.g. pro-T cell) or a lymphocyte (e.g. a T or NK cell).

[0088] If an output cell corresponds to a (primary- or PSC-derived) T cell, the T cell may express one or more of CD3, TCRap, TCRy6, CD4 and CD8. In one embodiment, the T cells are CD4+CD8+double positive T cells. In one embodiment, the T cells are CD8+double positive T cells. In one embodiment, the T cells are CD4+double positive T cells. In one embodiment, the T cells are CD3+TCRap+T cells. In one embodiment, the T cells are CD3+TCRy6+T cells.

[0089] If an output cell corresponds to a (primary- or PSC-derived) NK cell, the NK cell may express one or more of CD56 and CD16 (low affinity Fc gamma receptor 3A, expressed on a subset of NK cells). Output NK cells may further be characterized by the expression of activating and inhibitory receptors referred to as killer immunoglobulin-like receptors (KIRs) and other activating receptors such as NKG2D and natural cytotoxicity receptors (NCRs) including NKp30, NKp44, and NKp46. In one embodiment, the NK cells are CD56+NK cells.

[0090] A target cell (e.g. a lymphoid cell), contacted with a composition, may be comprised in a population of cells that is either homogeneous or heterogeneous. If homogeneous, the population of cells may have been purified or enriched prior to contacting with a composition. If heterogeneous, the population of cells may be naturally mixed (e.g. as taken from a subject or a culture vessel) or may be mixed by a user by combining different cell types as desired. Further, a heterogeneous may nevertheless have been purified or enriched, but may comprise contaminating cells, due to imperfect purification, spontaneous differentiation, or otherwise.Various instruments and / or reagents for isolating / enriching cells are commercially available, such as from STEMCELL Technologies.

[0091] In one aspect, this disclosure provides methods of generating a population of lymphoid cells, such as lymphoid / lymphocyte progenitors, through contacting a precursor (of the population of lymphoid cells) with a composition as described herein. Relatedly, aspects of this disclosure may provide methods of maturing and / or differentiating a population of lymphoid cells, such as lymphoid / lymphocyte progenitors, through contacting a precursor (of the population of lymphoid cells) with a composition as described herein.

[0092] In one aspect, this disclosure provides methods of generating a population of lymphoid cells, such as lymphoid / lymphocyte progenitors, such as from a stem or progenitor cell (or population thereof), whether a PSC, HSPC, or otherwise, through contacting a precursor (of the population of lymphoid cells) with a composition as described herein. Relatedly, aspects of this disclosure may provide methods of maturing and / or differentiating a population of lymphoid cells, such as lymphoid / lymphocyte progenitors, such as from a stem or progenitor cell (or population thereof), whether a PSC, HSPC, or otherwise, through contacting a precursor (of the population of lymphoid cells) with a composition as described herein.

[0093] The nature of a precursor of a population of lymphoid cells may be as described herein. In one embodiment, a precursor of a population of lymphoid cells is a primary-derived hematopoietic stem or progenitor cell (e.g. CD34+HSPC). In one embodiment, a precursor of a population of lymphoid cells is a PSC-derived hematopoietic stem or progenitor cell (e.g. CD34+HSPC). If PSC-derived, a precursor of a population of lymphoid cells (e.g. PSC-derived CD34+cell, or a downstream lymphoid progenitor thereof) may be differentiated using known protocols or commercially available kits (e.g. STEMdiff™ Hematopoietic Kit or its components, STEMCELL Technologies). If primary- or PSC-derived, lymphoid cells may be generated under serum- and / or feeder / stroma cell-free conditions.

[0094] The nature of a lymphoid / lymphocyte progenitors may be as described above. In one embodiment, the population of lymphoid cell comprises CD5+cells, CD7+cells, or CD5+CD7+cells.

[0095] In one aspect, this disclosure provides methods of generating a population of lymphoid cells, such as lymphocytes, through contacting a precursor (of the population of lymphoid cells) with a composition as described herein. Relatedly, aspects of this disclosure may provide methods of maturing and / or differentiating a population of lymphoid cells, such as lymphocytes,through contacting a precursor (of the population of lymphoid cells) with a composition as described herein.

[0096] In one aspect, this disclosure provides methods of generating a population of lymphoid cells, such as lymphocytes, such as from a stem or progenitor cell (or population thereof), whether a PSC, HSPC, or otherwise, through contacting a precursor (of the population of lymphoid cells) with a composition as described herein. Relatedly, aspects of this disclosure may provide methods of maturing and / or differentiating a population of lymphoid cells, such as lymphocytes, such as from a stem or progenitor cell (or population thereof), whether a PSC, HSPC, or otherwise, through contacting a precursor (of the population of lymphoid cells) with a composition as described herein.

[0097] The nature of a precursor of a population of lymphoid cells may be as described above. If an output lymphoid cell corresponds to a lymphocyte, then a precursor of the population of lymphoid cells may be a primary-derived hematopoietic stem or progenitor cell (e.g. CD34+HSPC) or a primary-derived lymphoid / lymphocyte progenitor (e.g. CD5+cells, CD7+cells, or CD5+CD7+cell). If an output lymphoid cell corresponds to a lymphocyte, then a precursor of the population of lymphoid cells may be a PSC-derived hematopoietic stem or progenitor cell (e.g. CD34+HSPC) or a PSC-derived lymphoid / lymphocyte progenitor (e.g. CD5+cells, CD7+cells, orCD5+CD7+cell).

[0098] The nature of a lymphocyte may be as described above. In one embodiment, the lymphoid cells is an NK cell (e.g. CD56+NK cell). In one embodiment, the lymphoid cell is a T cell (e.g. CD4+CD8+T cell, CD8+T cell, CD3+TCRap+T cell; and / or CD3+TCRy6+T cell.

[0099] Methods of this disclosure may comprise contacting a target cell (e.g. a precursor of a population of lymphoid cells, or a population of precursors) with a composition of this disclosure for a sufficient period of time in a sufficient culture medium. Methods of any aspect of this disclosure may further comprise culturing a target cell (as contacted with a composition of this disclosure) for a sufficient time, such as to modulate signaling therein and / or to generate / differentiate / mature an output cell.

[0100] After coming into contact with a composition of this disclosure, an output population of cells may comprise cells that are differentiated or matured relative to a target cell (e.g. a cell not having been contacted with the composition). Or, after coming into contact with a composition of this disclosure, an output population of cells may comprise more differentiated or matured cells relative to a target cell (e.g. a cell not having been contacted with the composition).

[0101] A sufficient time of contacting a target cell type with a composition of this disclosure may vary depending on the target cell type. For example, if differentiating / maturing / generating lymphoid / lymphocyte progenitors, a duration of contacting with a composition (in a suitable culture medium) may be between about 1 and 28 days, between about 3 and 21 days, between about 5 and 14 days, or between about 1 and 7 days. As another example, if differentiating / maturing / generating lymphocytes, a duration of contacting (in a suitable culture medium) with a composition may be between about 1 and 28 days, between about 5 and 24 days, between about 7 and 21 days, between about 10 and 18 days, or between about 12 and 16 days.

[0102] The nature of a medium used in the disclosed methods may depend on the target cell type (e.g. starting or input cell, or population thereof) and / or the desired output, after contact with a composition of this disclosure. Thus, a suitable culture medium used in methods of this disclosure may be any medium that supports a target cell type as signaling thereof is modulated and / or as it is differentiated / matured (through contact with a composition of this disclosure).

[0103] Media may comprise serum or may be serum-free. Preferably, media of this disclosure are serum-free. If serum-free, it may be necessary to include in such medium a serum replacement supplement, such as BIT 9500 Serum Substitute (STEMCELL Technologies), or other commercially available serum replacement solutions. Alternatively, components ordinarily present in serum that are needed for culturing or differentiating cells of this disclosure may be individually added at an acceptable concentration to medium.

[0104] Media may require specific supplementation to support a target cell type as signaling thereof is modulated and / or as it is differentiated / matured (through contact with a composition of this disclosure). Exemplary supplementation includes but is not limited to one or more cytokines, one or more growth factors, other proteins, one or more antioxidants, one or more vitamins, and / or one or more small molecules.

[0105] In the context of generating lymphoid cells, common cytokines and growth factors include but are not limited to IL-2, IL-3, IL-6, IL-7, IL-11 , IL-15, SCF, FLT3L, TPO, EPO, VEGF, and IGF- 1 or IGF-2. Such cytokines and / or growth factors may respectively be present at concentrations between about 1-1000 ng / mL, or about 1-100 ng / mL, or about 5-50 ng / mL. Not all of the foregoing are typically required in a medium for differentiating / maturing lymphoid progenitors from a precursor (e.g. a CD34+cell) or lymphocytes from a precursor (e.g. a CD34+cell, or a CD5+, CD7+, or CD5+CD7+progenitor). A more limited subset may correspond to one or more of SCF, FLT3L, and TPO, and / or one or more of IL-2, IL-3, IL-7 and IL-15.

[0106] Media specifically used to culture / differentiate / mature hematopoietic lineage cells, such as lymphoid cells, are commercially available (e.g. StemSpan™, StemDiff™, or ImmunoCult™- branded media, STEMCELL Technologies).

[0107] Methods of this disclosure may be scalable from microwell volumes to flask and / or bioreactor volumes. To facilitate scaling, methods of this disclosure, at any scale, may be performed under static conditions (e.g. 2D) and / or agitation / suspension conditions (e.g. 3D), in the presence of a composition of this disclosure, whether or not immobilized / functionalized on a solid phase.

[0108] As disclosed herein, a composition may be immobilized on a solid phase, and related description herein is applicable to methods wherein a first segment and a second segment are immobilized (such as by conjugation, passive adsorption, or otherwise) on a solid phase.

[0109] In any aspect of this disclosure, loading of a composition on a solid phase may correlate with performance (e.g. generation / differentiation). For example, increasing a loading ratio of a composition per unit surface area of the solid phase may increase the frequency and / or yield of cells arising after contact with the composition.

[0110] If a composition is immobilized on a particle, bead, or the like, whether in static or suspension conditions, particle / bead:cell ratio may influence performance of the composition in methods of this disclosure (e.g. increase frequency and / or yield of lymphoid cells (e.g. lymphoid progenitors and lymphocytes). For example, a ratio of particle, beads, or the like to cells may be between about 0.5:1 and 30,000:1 , between about 2:1 and 20000:1 , between about 10:1 and 1000:1 , between about 100:1 and 8000:1 , between about 250:1 and 5000:1 , between about 500:1 and 2500:1 , between 1000:1 and 2000:1 , or about 1000 (± 100):1. In aspects, where a composition is described, and such composition comprises a composition / construct, as disclosed herein, immobilized on a solid phase that is a bead, particle, or the like, the foregoing ratios may likewise apply.

[0111] Use of compositions, such as in methods of this disclosure, may meet or exceed the frequency and / or yield of output (e.g. generated, differentiated, and / or matured) cells (e.g. lymphoid progenitors and / or lymphocytes) following contact of target cells with a composition of this disclosure, as compared to a (control) condition where a Notch ligand, a CAM, or both a Notch ligand and a CAM individually (e.g. not connected) contact(s) the target cells.

[0112] In any aspect of this disclosure, use of compositions, such as in the methods of this disclosure, may provide improved frequency of output (e.g. generated, differentiated, and / or matured) cells (e.g. lymphoid progenitors and / or lymphocytes) relative to conventional conditions where Notch ligand and / or CAM are used individually (e.g. not connected). Thus, methods of this disclosure may comprise generating (e.g. outputting / maturing / differentiating) about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or higher frequency of lymphoid progenitor cells or lymphocytes, in comparison to a control condition where a target cell (e.g. a precursor) is contacted with a first segment alone, a second segment alone, or the first and the second segments individual (that are not connected in a composition).

[0113] In any aspect of this disclosure, use of compositions, such as in the methods of this disclosure, may provide improved yield of output (e.g. generated, differentiated, and / or matured) cells (e.g. lymphoid progenitors and / or lymphocytes) relative to conventional conditions where Notch ligand and / or CAM are used individually (e.g. not connected). Thus, methods of this disclosure may comprise generating (e.g. outputting / maturing / differentiating) about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or higher yield of lymphoid progenitor cells or lymphocytes, in comparison to a control condition where a target cell (e.g. a precursor) is contacted with a first segment alone, a second segment alone, or the first and the second segments individual (that are not connected in a composition).

[0114] In any aspect of this disclosure, use of compositions such as in the methods of this disclosure, generates / produces a preferential output of a specific population of mature(d) cells (e.g. lymphocytes) depends on composition concentration (as may be immobilized on a solid phase). In one embodiment, CD3+TCRap+T cells are generated preferentially over CD3+TCRy6+T cells at or below a threshold concentration of a composition of this disclosure (as may be immobilized on a solid phase). In such embodiment, the threshold concentration may be about <2 cm2beads per mL culture volume. Stated differently, the threshold concentration may range between about 2-20 picomoles, or about 4-18 picomoles, or about 10-15 picomoles. More specifically, the threshold concentration may be about 17 picomoles ± 1 picomole.

[0115] In one embodiment, CD3+TCRy6+T cells are generated preferentially over CD3+TCRap+T cells at or above a threshold concentration of a composition of this disclosure (as may be immobilized on a solid phase). In such embodiment, the threshold concentration may be about >2 cm2beads per mL culture medium, or >2 cm2beads per mL culture volume (e.g. 4-8 cm2beadsper mL culture volume). Stated differently, the threshold concentration may be above about 2-20 picomoles, or above about 4-18 picomoles, or above about 10-15 picomoles. More specifically, the threshold concentration may be above about 17 picomoles ± 1 picomole. Still more specifically, the threshold concentration may range between about 20-100 picomoles, about 25- 90 picomoles, about 30-80 picomoles, about 35-70 picomoles.

[0116] In one embodiment, use of compositions such as in the methods of this disclosure, preferentially generates CD3+TCRap+T cells over CD3+TCRy6+T cells or preferential generation of CD3+TCRy6+T cells over CD3+TCRap+T cells in a concentration-dependent manner of a composition. In such embodiment the concentration dependency may be as described in the foregoing paragraphs.

[0117] Compositions and methods of this disclosure may generate, differentiate, and / or mature lymphoid cells in vitro and / or ex vivo with improved outputs compared to convention, by precisely providing first segment to second ratios, while ensuring uniform spatial organization of first and segments relative to one another in a culture system, thereby enhancing overall binding avidity through relatively higher affinity CAM interactions supporting relatively lower affinity Notch ligand binding. Thus, composition and methods of this disclosure may greatly facilitate downstream applications, including cell therapy manufacturing and other clinical / diagnostic applications.

[0118] The following non-limiting examples are illustrative of the present disclosure.ExamplesExample 1: Composition design and production

[0119] The recombinant D4.4, D4.5, D4.8, and D4.9 compositions depicted in Figure 1 were expressed using a mammalian expression vector system by positioning a coding sequence of the extracellular domain of human DLL4 (hDLL4; aa residues 1-524) separated by a glycine-serine linker from a coding sequence of a cell adhesion molecule (CAM). Exemplary CAMs linked to the extracellular domain of hDLL4 comprise: a polypeptide comprising fibronectin Type III repeats 9- 12 (CAM1); a polypeptide comprising fibronectin Type III repeats 8-10 and 12-14 (CAM2); a polypeptide comprising seven extracellular Ig-like (Ig-like 1 to Ig-like 7) domains of human VCAM- 1 (CAM3); and a polypeptide comprising five extracellular Ig-like (Ig-like 1 to Ig-like 5) domains of human ICAM-1 (CAM4). C-terminal uncleavable histidine (His6) tags and AviTag™ biotinylation sites may be included to facilitate downstream processes.

[0120] Ablated versions of D4.5 were produced by: aspartate substitution with glutamate of Fnlll 10 RGD motif (RGD RGE, referred to as construct D4.10); aspartate substitution with glutamate of CS-1 LDV motif (LDVLEV, referred to as construct D4.11); or by ablating both the Fnlll 10 RGD and CS-1 LDV motifs as described (referred to as construct D4.12).

[0121] Expression vectors were individually transfected into host cells, such as CHO cells, using a standard PEI transfection method, and secreted D4.4, D4.5, D4.8, D4.9, D4.10, D4.11 , and D4.12 were respectively purified, such as over nickel columns (Cytiva) as per manufacturer’s instructions. Purified polypeptides were dialyzed against PBS (with or without addition of 1 mM EDTA and 0.2% Pluronic F-68) and stored.Example 2: Procedure to immobilize compositions

[0122] D4.4, D4.5, D4.8, D4.9, D4.10, D4.11 , and D4.12, and a control composition were adsorbed to substrates (e.g. plates, beads, porous growth matrix, etc.) using conventional approaches. In the case of plates, various concentrations (0.194 ug / mL up to 47.25 ug / mL in PBS) were added to each well of a flat-bottom non-tissue culture treated 96-well plate and incubated at 4°C for at least 2 hours. Coating solutions were then aspirated, and wells rinsed with PBS prior to plating cells.

[0123] In the case of beads, polypeptide compositions could be immobilized as follows. For streptavidin-coated beads (Spherotech, ~5 pm), respective biotinylated polypeptide compositions of Example 1 were diluted in 1X PBS buffer followed by streptavidin bead addition. The bead polypeptide mixture was incubated on a rotary mixer for 1 hour at room temperature. The beads were then pelleted on a magnet and washed with 1X PBS buffer. The polypeptide conjugated beads were stored at 4°C until further use. For carboxyl magnetic particles (Spherotech, ~5 pm), covalent conjugation of respective compositions / constructs of Example 1 to beads was initiated by first activating the carboxyl group in a solution of EDC and sulfo-NHS in a suitable buffer. Thereafter, the beads were magnetically pelleted, washed in MES buffer and then incubated with ~100-500 pg of the polypeptide composition on a rotary mixer for 1 hr at room temperature. The beads were quenched by incubating in a solution of 35 mM Glycine for 20 minutes, magnetically pelleted, and washed in 1X PBS buffer. The conjugated carboxyl magnetic beads could be sterilized by a conventional method (e.g. gamma irradiation) before storage at 4°C until further use.Example 3: Differentiating lymphoid progenitors and lymphocytes from HSPCs in coated plates

[0124] Differentiation of CD34+hematopoietic progenitors to CD5+CD7+lymphoid progenitors (progenitor T cells; pro-T) and beyond to T-lineage cells was performed using plates coated as described in Example 2 or as a control individually coating Fibronectin and DLL4-Fc.

[0125] Briefly, 1000 (96-well plate), 5000 (24-well plate), 10000 (12-well plate) or 25000 (6-well plate) cord blood-derived HSPC / well were resuspended in StemSpan™ Lymphoid Progenitor Expansion Medium (STEMCELL Technologies). Where CD34+cells were derived from H9 ESC, WLS-1 C iPSC, STi0003-A, or STi0004-A using the STEMdiff™ Hematopoietic - EB Reagents (STEMCELL Technologies), the number of seeded PSC-derived CD34+cells was 2.5-5X higher.

[0126] The average frequency of viable day-14 CB-derived CD5+CD7+lymphoid progenitors was >80% in plates coated with D4.5 compared to ~60% for plates coated individually with fibronectin and DLL4-Fc (Control). Further, the average yield of CD7+CD5+lymphoid progenitors per input CD34+cell in plates coated with D4.5 was ~500 cells compared to ~230 cells in control plates (Figure 2A). For PSC-derived CD34+HSPCs, average frequency of viable day 7-10 CD7+CD5+lymphoid progenitors was ~40% in plates coated with D4.5, compared to ~25% for the control condition, and the average yield of viable day 7-10 CD7+CD5+lymphoid progenitors per input CD34+cell was ~40 cells in plates coated with D4.5 compared to ~10 cells for the control condition (Figure 2B). D4.4 composition of Example 1 performed similarly to D4.5 in terms of frequency and yield of CB-derived and / or PSC-derived CD5+CD7+lymphoid progenitors (data not shown).

[0127] After 14 days in culture, arising CD5+CD7+lymphoid progenitors were differentiated into CD4+CD8+double positive T cells. Briefly, day-14 cells were seeded in StemSpan™ T Cell Progenitor Maturation Medium (STEMCELL Technologies) at 1 x 104cells / mL (5 x 103cells / well) per well of a 24-well plate coated with either polypeptide or control compositions and cultured for 14-28 days. Day 28-42 CD4+CD8+double positive T cells were harvested into tubes for cell counting and phenotypic analysis.

[0128] The average frequency of viable day 39 CB-derived CD4+CD8+double positive T cells was >70% in plates coated with D4.5 compared to ~50% for the control, and the average yield of viable day-39 CB-derived CD4+CD8+double positive T cells was slightly higher in plates coated with D4.5 compared to the control (Figure 2C). For PSC-derived CD34+HSPCs, average frequency of viable day 21-24 CD4+CD8+double positive PSC-derived T cells was ~50% in plates coated with D4.5 compared to ~12% in control plates, and the average yield of viable day 21-24 CD4+CD8+double positive T cells per input CD34+cell was ~44 cells in plates coated with D4.5 compared to ~4 cells in the control condition (Figure 2D). D4.4 composition performed similarlyto D4.5 in terms of frequency and yield of CB-derived and / or PSC-derived CD4+CD8+double positive T cells (data not shown).

[0129] Further differentiation of day 28-42 CD4+CD8+double positive CB-derived T cells and day 21-24 CD4+CD8+double positive PSC-derived T cells to CD8+single positive T cells was performed. Briefly, 1x106cell / ml of double positive T cells were resuspended in 500 pL of CD8 SP T Cell Maturation Medium (STEMCELL Technologies) together with ImmunoCult™ Human T Cell Activator (STEMCELL Technologies) and seeded into tissue culture-treated plates coated as described above. After 3-4 days, wells were topped up with 500 pl of CD8 SP T Cell Maturation Medium and on the 7thday the cells were harvested into tubes for cell counting and phenotypic analysis.

[0130] The average frequency of viable day-49 CB-derived CD8+single positive T cells was >70% in plates coated with D4.5 compared to ~40% for the control condition, and the average yield of viable day-46 CD8+single positive T cells was comparable between the D4.5 coated condition and the control condition (Figure 2E). The average frequency of viable day 28-31 PSC- derived CD8+single positive T cells was ~40% in plates coated with D4.5 compared to ~30% in control plates (Figure 2F). D4.4 composition performed similarly to D4.5 in terms of frequency and yield of CB-derived and / or PSC-derived CD8+single positive T cells (data not shown)

[0131] In the case of PSC-derived CD56+NK cell generation, day 7-10 CD5+CD7+lymphoid progenitor cells were generated essentially as described above. Following, 1x105cells in 500 pL of StemSpan™ NK Cell Differentiation Medium (STEMCELL Technologies) were seeded into each well of a 24-well tissue-culture treated plate and cultured for 14 days in the absence of a polypeptide or control composition. Day 21 -24 cells were harvested for cell counting and phenotypic analysis.

[0132] The average frequency of viable day 21-24 PSC-derived CD56+NK cells was slightly higher for those NK cells differentiated from lymphoid progenitors generated on D4.5-coated plates compared to the control. Further, the average yield of such CD56+NK cells per input PSC >5400 cells when differentiated from lymphocyte progenitors generated in plates coated with D4.5 compared to ~2300 cells in the control (Figure 3).

[0133] Taken together, these results demonstrate that the polypeptide compositions of this disclosure capably differentiate / mature lymphocytes from CB-derived or PSC-derived CD34+cells at levels that are either comparable or greater relative to a control.Example 4: Differentiating lymphoid progenitors from HSPCs under static conditions

[0134] The compositions of Example 1 were evaluated against conditions where the components were not connected to one another prior to surface immobilization and against a control where Fibronectin and DLL4-Fc were individually immobilized on a plate.

[0135] CD34+hematopoietic progenitors were differentiated to CD5+CD7+lymphoid progenitors (progenitor T cells; pro-T), and beyond to T-lineage cells, as described in Examples 2 and 3. The average frequency of viable day-14 CB-derived CD7+CD5+lymphoid progenitors generated under static culture conditions in plates coated with the compositions of Example 1 (D4.4, D4.5, D4.8 and D4.9) was in every case higher than when the components of each composition were individually coated on the plates (D4.4>DL4+CAM1 ; D4.5>DL4+CAM2; D4.8>DL4+CAM3; and D4.9>DL4+CAM4). In addition, the average frequencies obtained in these experiments were comparable between DL4+CAM2, DL4+CAM3, and the control, while each of D4.4, D4.5, and D4.8 outperformed the control (Figure 4A). With regard to the average yield of viable day-14 CD5+CD7+lymphoid progenitors per input CD34+cell in the same experimental conditions as in the foregoing, the same trends of enhanced performance were observed when components were connected as opposed to unconnected (Figure 4A).

[0136] Essentially the same static culture experiments were carried out as in the foregoing except that the compositions of Example 1 or the respective components thereof were surface immobilized on beads / particles, as described in Example 2. Briefly, 1000 (96-well plate), 5000 (24-well plate), 10000 (12-well plate) or 25000 (6-well plate) cord blood-derived HSPC / well were resuspended in StemSpan™ Lymphoid Progenitor Expansion Medium (STEMCELL Technologies) and cultured under static conditions for 14 days in the presence of ~6.4x104pm2bead area per input cell (approximately equivalent to 900 5-pm diameter beads per input cell).

[0137] The average frequency of viable day-14 CB-derived CD7+CD5+lymphoid progenitors generated under static culture conditions in the presence of beads comprising immobilized compositions of Example 1 (D4.4, D4.5, or D4.8) was in every case higher than when the components of each composition were individually coated on the beads (D4.4>DL4+CAM1 ; D4.5>DL4+CAM2; and D4.8>DL4+CAM3), although for D4.5 and D4.8 it was only slightly higher. In addition, the average frequencies obtained in these experiments were comparable between DL4+CAM1 and the control, while the other conditions each outperformed the control (Figure 4B). With regard to the average yield of viable day-14 CD5+CD7+lymphoid progenitors per input CD34+cell in the same experimental conditions as in the foregoing, the same trends of enhancedperformance were observed when components were connected as opposed to unconnected, and in every case outperformed the control (Figure 4B).

[0138] These results demonstrate that various cell adhesion molecules of Example 1 capably differentiate CD34+HSPCs into lymphoid progenitors to the same or higher extent than a control. Furthermore, the results show that the compositions of Example 1 (whether immobilized on a plate or a bead) outperformed counterparts when respective components of each composition were individually immobilized.

[0139] Also in static (2D) culture, the average frequency of viable day-14 CB-derived CD5+CD7+lymphoid progenitors generated in the presence of conjugated carboxyl pbeads was ~60% for pbeads conjugated with D4.4 and ~90% for pbeads conjugated with D4.5, compared to ~70% for the control comprising individually plate-immobilized Fibronectin and DLL4-Fc. Further, the average yield of CD5+CD7+lymphoid progenitors per input CD34+cell in the presence of D4.4 conjugated pbeads was ~75 cells and ~250 cells for D4.5 conjugated pbeads, compared to <200 cells for the control (Figure 4C).

[0140] Together with the data in Figure 4B, these results demonstrate that polypeptide compositions of this disclosure when immobilized on different types of pbeads can capably differentiate CD34+cells into lymphoid progenitors in a 2D static condition.Example 5: Differentiating lymphoid progenitors from HSPCs under suspension conditions

[0141] Differentiation of CB-derived CD34+hematopoietic progenitors to CD5+CD7+lymphoid progenitors and beyond was performed essentially as described in Examples 3 and 4 but in suspension (e.g. 3D) culture. Compositions of Example 1 were surface immobilized as in Examples 2 and ~1-4 cm2 / mL of SAV beads were added to non-tissue culture treated 6-well plates and cultured on an orbital shaker platform (in an incubator) at 70 rpm. At the end of the culture period, cells and beads were transferred to a tube and fractionated by incubating the tubes in the presence of a magnetic field (e.g. EasyEights™ EasySep™ Magnet).

[0142] The average frequency of arising viable day-14 CB-derived CD7+CD5+lymphoid progenitors was ~85% in the presence of bead-immobilized D4.5 as compared to ~70% for a static control where plates were coated individually with Fibronectin and DLL4-Fc (control), and the average yield of progenitors per input CD34+cell in the presence of bead-immobilized D4.5 was comparable to the control (Figure 5A). The average frequency of arising viable day 7-10 PSC-derived CD7+CD5+lymphoid progenitors was ~75% and nearly 2 / 3 higher than the control,but slightly lower than a separate control where D4.5 was plate-immobilized (Figure 5B). Further, the average yield of arising progenitors per input PSC-derived CD34+cell was likewise higher (~30) relative to the control (<20), but roughly half the separate control where D4.5 was plate- immobilized (Figure 5B).

[0143] These results demonstrate that the compositions of this disclosure when immobilized on beads capably differentiate lymphoid progenitor cells from CB- or PSC-derived CD34+cells when cultured in suspension (e.g. 3D).

[0144] After 14 days in culture, 5 x 105lymphoid progenitors were resuspended into StemSpan™ T Cell Progenitor Maturation Medium and seeded into wells of tissue culture-treated plates together with beads as coated in Example 2 and cultured as described in Example 3. The average frequency of arising viable day 42 CB-derived CD4+CD8+double positive T cells was ~70% in the presence of bead-immobilized D4.5 as compared to ~30% for a static control where plates were coated individually with Fibronectin and DLL4-Fc (control), and the average yield of double positive T cells per input CD34+cell was slightly higher than the control when differentiated in the presence of bead-immobilized D4.5 (Figure 5C). Interestingly, among CB-derived double positive T cells (such as of Figure 5C), the average frequency of CD3+TCRap+cells was ~35% and about 3 times higher in the bead-immobilized D4.5 condition compared to the control (~10%) (Figure 5E).

[0145] Regarding PSC-derived progenitors, 1.25 x 106cells / mL of day 7-10 CD5+CD7+lymphoid progenitors were differentiated into CD4+CD8+double positive T cells as described in Example 3. The average frequency of arising viable day 21 -24 CD4+CD8+double positive T cells was comparable between conditions where D4.5 was plate- or bead-immobilized, and in both cases much greater (e.g. 4-5X) than a control where plates were coated individually with Fibronectin and DLL4-Fc (Control) (Figure 5D). In the same conditions, the average yield of viable day 21-24 CD4+CD8+double positive T cells was ~5X greater for bead-immobilized D4.5 compared to the control, but lower than for plate-immobilized D4.5 (Figure 5D).

[0146] These results demonstrate that the compositions of this disclosure when immobilized on beads capably differentiate / mature lymphocytes from CB- or PSC-derived progenitors when cultured in suspension (e.g. 3D).Example 6: Scalability of cultures with immobilized beads in suspension

[0147] Scalability of suspension cultures using immobilized beads to produce larger numbers of CD4+CD8+double positive T cells from CD5+CD7+lymphoid progenitors was explored. Briefly, essentially as described in Examples 3 and 5, 1 .25x105cells / mL of a starting population of CB- derived CD5+CD7+lymphoid progenitors were added to a 2 mL of culture medium in a 6-well plate or to a 30 mL of culture medium in a 125-mL baffled shaker flask, and the cells were cultured for 28 days on an orbital shaker at 70 RPM with a 100% medium exchanges every 3-4 days. Average frequency and yield of day-42 double positive T cells were calculated.

[0148] The average frequency of arising viable day-42 CD4+CD8+double positive T cells in both the 6-well plate and the shaker flask were much higher (~4X) than the control condition (e.g. individually plate-immobilized Fibronectin and DLL4-Fc), and the average frequencies between the two suspension culture conditions were similar (~80% vs ~70%). Likewise, the average yield of viable day-42 CD4+CD8+double positive T cells per input CD34+cell for both suspension culture conditions were much higher (~5X and ~10X for 6-well and shaker conditions, respectively) than the control condition, and the average frequency of such cells per input cell were nearly 33% higher for shaker flask cultured cells compared to 6-well plate cultured cells (Figure 5F). As proof-of-concept, the magnetic beads were removed from the double positive T cells using a commercially available Easy250 magnet (STEMCELL Technologies). Using this process, cells were obtained with less than one percent frequency of residual beads (data not shown).

[0149] These results demonstrate that the compositions of this disclosure are amenable for large- scale differentiation of lymphoid progenitors to double positive T cells in suspension.Example 7: Identifying important functional domains of the compositions

[0150] Interaction between the DL4 segment and the domains of the CAM segments, of the compositions of Example 1 , were investigated and the results shown in Figure 6. As described in Example 1 , mutations to integrin binding motifs of the CAM segment were expressed (D4.10, D4.11 , and D4.12) and each tested in static culture assays differentiating CB-derived CD34+cells to lymphoid progenitors, essentially as described in Example 3.

[0151] The average frequency of viable day-14 CD7+CD5+lymphoid progenitors decreased by 50% or more using the RGD ablated (D4.10) and LDV ablated (D4.1 1) variants relative to the D4.5 control. In both cases, respectively combining the ablated compositions with exogenous non-ablated CAM molecules somewhat restored function, but not to the levels of the D4.5 control. For the dual ablated (D4.12) variant, the function of this composition was almost completelydestroyed, and its function was somewhat restored by combination with exogenous non-ablated CAM molecules, but not to the levels of the D4.5 control. Similarly, the average yield of day-14 CD7+CD5+lymphoid progenitors per input CD34+cell decreased by roughly 10-fold using the RGD ablated (D4.10) and LDV ablated (D4.1 1) variants relative to the D4.5 control. Combining the ablated compositions with exogenous non-ablated CAM molecules somewhat restored function for only D4.11 , but not to the levels of the D4.5 control (or the individually plate-bound Fibronectin and DL4-Fc control. For the dual ablated (D4.12) variant, the function of this composition was almost completely destroyed yielding few or no lymphoid progenitors, and its function was somewhat restored by combination with exogenous non-ablated CAM molecules, but not to the levels of the D4.5 control or the individually plate-bound Fibronectin and DLL4-Fc control.

[0152] These results demonstrate that integrin binding motifs (e.g. RGD and / or LDV motifs) in combination with DLL4, or a fragment thereof, are important for differentiating CD34+cells to lymphoid progenitors and further to lymphocytes, and the close association (such as in a single polypeptide) provides improvement over use of each segment non-connectedly.Example 8: Concentration dependent skewing to T cell subtypes

[0153] Titration experiments of bead-immobilized D4.5 during day 14-42 suspension culture of the T cell differentiation workflow, as described in Example 5, revealed a surprising concentrationdependent skewing to either CD3+TCRap+or CD3+TCRy6+T cells.

[0154] At lower concentrations (e.g. about 2.0 cm2 / mL bead concentration or lower, corresponding to ~15-20 picomole or lower D4.5 concentration), a higher frequency of CB-derived CD3+TCRap+T cells emerged, while lowerthan control frequencies of such cells emerged at bead concentrations above 2.0 cm2 / mL (Figure 7). In contrast, at higher concentrations (e.g. >2.0 cm2 / mL bead concentration, corresponding to higher than ~15-20 picomole D4.5 concentration), a higher frequency of CB-derived CD3+TCRy6+T cells emerged, while lower than control frequencies of such cells emerged at bead concentrations of about 2.0 cm2 / mL or lower (Figure 7). A similar dependency on bead concentration was shown for differentiation of PSC-derived CD3+TCRap+T cells or CD3+TCRy6+T cells from progenitors (data not shown).Example 9: Differentiating lymphocyte progenitors with porous growth matrix-immobilized compositions

[0155] A composition of this disclosure may be immobilized on a porous growth matrix through passive adsorption and / or covalent linkage. Briefly, a 40 pg / mL solution of D4.5 was combinedwith ~150 Fibra-Cel® disks (Eppendorf™ AG, Germany) in a 50-mL tube and incubated overnight at 4 °C on a rotating tube mixer to passively adsorb the protein on the disks. The disks were rinsed with PBS, resuspended in a small volume of culture medium, and using sterile forceps transferred into 1 mL StemSpan™ Lymphoid Progenitor Expansion Medium in a 24-well plate (4 disks per well) or into 2 mL culture medium in a 6-well plate (20 disks per well). If cultured in a 24-well plate, each well contained 5000 CB-derived HSPCs or 25000 PSC-derived HSPCs and cultured under static conditions with 50% medium exchange every 3-4 days for 14 days, with daily manual mixing by pipette. If cultured in a 6-well plate, each well contained 25000 CB-derived HSPCs or 125000 PSC-derived HSPCs, and cultured on an orbital shaker at 70 RPM under suspension conditions with 100% medium exchanges every 3-4 days for 14 days.

[0156] The frequency and yield of day-14 CB-derived CD5+CD7+lymphoid progenitors generated under static culture was low (<20% and ~20 / input CD34+cell, respectively) for both D4.5 passively adsorbed Fibra-Cel® disks and the plate individually coated with fibronectin and DLL4-Fc (control); however, an ~80% frequency and ~80 / input CD34+cell yield of CB-derived day-14 CD5+CD7+lymphoid progenitors was observed when D4.5 passively adsorbed Fibra-Cel® disks were cultured under suspension conditions (Figure 8A). For PSC-derived cells, the frequency and yield of day-14 CB-derived CD5+CD7+lymphoid progenitors generated under static culture was low (~20% and ~5 / input CD34+cell, respectively) for both D4.5 passively adsorbed Fibra-Cel® disks and the plate-coated control; however, for D4.5 passively adsorbed Fibra-Cel® disks cultured under suspension conditions, while a frequency of only ~30% day-14 CD5+CD7+lymphoid progenitors was observed a more than two-fold increase in yield / per input CD34+cell was observed (~12 cells / input) (Figure 8B).

[0157] These results demonstrate that the polypeptide compositions of this disclosure are compatible with an alternate solid support material such as a porous growth matrix for generating, differentiating, and / or maturing CD5+CD7+lymphoid progenitors.

Claims

Claims1 . A composition for modulating signaling in a cell or for generating a population of lymphoid cells, comprising: a first segment comprising or encoding a Notch ligand connected to a second segment comprising a cell adhesion molecule, wherein the first segment and the second segment are immobilized on a solid phase.

2. The composition of claim 1 , wherein the first segment: a) comprises one or more of: one or more EGF-like repeats; a DSL domain; and a C2 domain; and / or b) does not comprise a transmembrane domain.

3. The composition of claim 1 or 2, wherein the Notch ligand is a Delta-like protein or a fragment of a Delta-like protein.

4. The composition of claim 3, wherein the Delta-like protein is DLL1 or DLL4.

5. The composition of any one of claims 1 to 4, wherein the second segment comprises a) at least one Immunoglobulin (Ig)-like domain; and / or b) at least one fibronectin type III (Fnlll) domain; and / or c) an integrin binding domain or an integrin interacting motif; and / or d) a heparin binding domain.

6. The composition of claim 5, wherein the integrin binding domain or the integrin interacting motif comprises an RGD motif and / or a LDV motif.

7. The composition of claim 5, wherein the cell adhesion molecule is an immunoglobulin (Ig)-like domain containing superfamily member or an extracellular matrix protein.

8. The composition of claim 7, wherein: a) the Ig-like domain containing superfamily member is ICAM-1 , VCAM-1 , or a fragment thereof; and b) the extracellular matrix protein is fibronectin or a fragment thereof.

9. The composition of any one of claims 1 to 8, wherein the first segment, the second segment, or both the first and the second segments comprise a polypeptide, and a) the first segment and the second segment are connected either directly through a single peptide bond or via a peptide linker;b) the first segment encodes or is conjugated to a first member of a complementary binding pair, and the second segment encodes or is conjugated to a second member of the complementary binding pair; or c) the first segment and the second segment are comprised in a fusion protein, and the first segment and the second segment are in the same reading frame.

10. The composition of any one of claims 1 to 9, wherein the solid phase is a) a cell culture substrate or a bead or a particle; or b) a porous growth matrix.

11. A method of generating a population of lymphoid cells, the method comprising: contacting a precursor of the population of lymphoid cells with a composition according to any one of claims 1 to 10 for a sufficient time to generate the population of lymphoid cells.

12. The method of claim 11 , wherein contacting the precursor of the population of lymphoid cells with the composition is in a static or a suspension culture environment.

13. The method of claim 11 or 12, wherein the precursor is a PSC-derived or a primary hematopoietic stem / progenitor cell (HSPC) or a lymphoid progenitor.

14. The method of any one of claims 11 -13, wherein the population of lymphoid cells comprises lymphoid progenitors.

15. The method of any one of claims 11 -14, wherein the population of lymphoid cells comprises lymphocytes which are: a) NK cells; or b) T cells.

16. The method of claim 15, wherein the T cells are: a) CD4+CD8+double positive T cells; b) CD8+single positive cells; c) CD4+ single positive T cells; d) CD3+TCRap+T cells; and / or e) CD3+TCRy6+T cells.

17. The method of claim 16, wherein a) CD3+TCRap+T cells are generated preferentially to CD3+TCRy6+T cells at or below a threshold concentration of the composition; and / orb) CD3+TCRy6+T cells are generated preferentially to CD3+TCRap+T cells above a threshold concentration of the composition.

18. The method of claim 14, further comprising generating between about 1 to 10 times more lymphoid progenitor cells in comparison to a control condition where the precursor is contacted with a first segment alone, a second segment alone, or the first and the second segments that are not connected.

19. The method of claim 14 or 15, further comprising generating between about 1 to 20 times more lymphocytes in comparison to a control condition where the precursor is contacted with a first segment alone, a second segment alone, or the first and the second segments that are not connected.

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